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Improved in vitro methods for differentiating induced pluripotent stem cells to cardiomyocytes Chu, Jun Ming Axel 2018

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 IMPROVED IN VITRO METHODS FOR DIFFERENTIATING  INDUCED PLURIPOTENT STEM CELLS TO CARDIOMYOCYTES by  Jun Ming Axel Chu  B.Eng., The National University of Singapore, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biomedical Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2018  © Jun Ming Axel Chu, 2018  ii Abstract  Human adult cardiomyocytes are hard to obtain and difficult to culture. This has led to patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iCMs) becoming an attractive alternative, serving as a platform for cardiovascular disease modeling, drug toxicity screening, regenerative therapies, and the understanding of human physiology. Unfortunately, current protocols of generating iCMs remain unpredictable, yielding cellular subtypes (ventricular, atrial, nodal cells, cardiac fibroblasts and others) that vary in proportion and levels of maturity, depending primarily on the methodology employed. This led to the scope of my research, which was to understand the minimal biological and physical parameters governing cardiac differentiation from iPS cells, so as to improve current cardiac differentiation systems. My experiments into the effects of substrate stiffness on differentiation revealed that cardiac maturity could be accelerated for iPS cells differentiated on hard substrates (~1.72MPa) compared to cells differentiated on soft (~5kPa) or intermediate (~130kPa) substrates. Experiments investigating the effects of topography also showed that freshly differentiated iCMs could be directed to form uniaxial contracting cardiac tissues by seeding them into millimeter-sized linear grooved channels. Finally, proof-of-principle experiments into inductive co-cultures revealed that previously differentiated iCMs co-cultivated with iPS cells constituted a sufficient stimulatory system to induce cardiac differentiation – as determined by the exhibition of spontaneous self-contractions, expression of cardiac specific markers, and structural organization of sarcomeres. This process was achieved without the exogenous addition of pathway inhibitors and morphogens, suggesting that ‘older’ iCMs serves as an adequate stimulatory source capable of recapitulating the necessary culture environment for cardiac differentiation.  iii Lay Summary  Developments in science and technology have made the creation of human stem cells from other adult cells, such as skin cells, a reality. This discovery not only sidestepped the ethical dilemma of having to sacrifice preimplantation human embryos, but also opened up numerous avenues for biomedical applications. One such application is the converting of these stem cells into functional heart cells for the treatment of heart failures. To do this, scientists use various soluble factors to ‘reprogram’ the stem cells into these ‘new’ heart cells. However, heart cells generated this way are typically less developed compared to hearts cells of an actual adult human heart. As such, in order to improve the process for converting stem cells into heart cells, this body of work studied various factors that might influence this process. First, the effect of stiffness was explored. Here, it was revealed that stem cells grown on harder surfaces showed greater signs of cardiac maturity compared to those on softer surfaces. Next, the effect of topography was investigated. Freshly generated heart cells were placed into grooved linear channels and allowed to grow. It was discovered that these heart cells took the shape of the channel, forming heart muscle strips resembling that of actual muscle fibers. Lastly, an original approach to convert stem cells to heart cells was also explored. Here, ‘older’ previously generated heart cells were added to ‘new’ stem cells and by way of ‘cross-talk’ between cells, it was demonstrated that ‘new’ stem cells could be ‘coaxed’ into becoming functional heart cells.   iv Preface  This thesis is original, unpublished, independent work by the author, J. M. Axel Chu.   v Table of Contents  Abstract .......................................................................................................................................... ii	Lay Summary ............................................................................................................................... iii	Preface ........................................................................................................................................... iv	Table of Contents ...........................................................................................................................v	List of Tables ................................................................................................................................ ix	List of Figures ................................................................................................................................. x	Glossary ...................................................................................................................................... xiii	Acknowledgements .................................................................................................................... xvi	Dedication .................................................................................................................................. xvii	Chapter 1: Introduction ................................................................................................................1	1.1	 Building the heart in a dish ................................................................................................ 1	1.2	 Stem cells: A glimpse of the past, present, and future ....................................................... 2	1.3	 Current strategies for cardiac cell differentiation .............................................................. 3	1.4	 Defining characteristics of cardiomyocytes ....................................................................... 6	1.5	 Substrate stiffness and cardiac differentiation ................................................................... 7	1.6	 Engineered three-dimensional heart tissues ....................................................................... 8	1.7	 Inductive co-cultures and cardiac differentiation .............................................................. 9	1.8	 Thesis aim and hypotheses ............................................................................................... 12	Chapter 2: Materials and Methods ............................................................................................14	2.1	 Tissue culture ................................................................................................................... 14	2.1.1	 Induced pluripotent stem cell line: IMR90-4 ............................................................ 14	 vi 2.1.2	 Fluorescently tagged human induced pluripotent stem cell line: AICS-0016 .......... 14	2.1.3	 Matrigel coatings ...................................................................................................... 15	2.1.4	 Standard culture of iPS cells ..................................................................................... 16	2.1.5	 Cardiac differentiation with GSK3 inhibitor and Wnt inhibitor (GiWi Protocol) .... 18	2.2	 Cell labeling with fluorescent dye ................................................................................... 21	2.3	 Microscope systems ......................................................................................................... 21	2.4	 Kymograph generation and analysis ................................................................................ 22	2.5	 Immunocytochemistry ..................................................................................................... 23	2.6	 Flow cytometry ................................................................................................................ 24	2.7	 Fabrication of polydimethylsiloxane (PDMS) substrates with varying stiffness ............ 25	2.8	 Statistical analysis ............................................................................................................ 28	2.9	 Fabrication of PDMS linear cardiac channel ................................................................... 28	Chapter 3: Substrate Stiffness and Topography ......................................................................30	3.1	 Effects of substrate stiffness on cardiac differentiation ................................................... 30	3.2	 Topographical manipulation of iPS cell-derived cardiomyocytes ................................... 36	3.3	 Chapter discussion ........................................................................................................... 38	3.3.1	 Effects of substrate stiffness on cardiac differentiation ............................................ 38	3.3.2	 Topographical manipulation of iPS cell-derived cardiomyocytes ............................ 40	Chapter 4: Co-Culture Cardiac Differentiation .......................................................................41	4.1	 Co-culture cardiac differentiation of fluorescent dye labeled-IMR90-4 iPS cells .......... 41	4.1.1	 Pre-culture method for co-culture differentiation of iPS cells .................................. 42	4.1.2	 Bi-culture method for co-culture differentiation of iPS cells ................................... 46	4.2	 Co-culture cardiac differentiation of GFP-tagged AICS-0016 iPS cells ......................... 50	 vii 4.2.1	 Validation of fluorescence in AICS-0016 iPS cell line ............................................ 51	4.2.2	 Co-culture of AICS-0016 iPS cells with IMR90-4 iCMs via pre-culture method ... 52	4.2.3	 Verification of spontaneous self-contraction in co-cultured AICS-0016 cells ......... 53	4.2.4	 Characterization of maturity via immunocytochemistry .......................................... 56	4.2.5	 Characterization of differentiation efficiency via flow cytometry ........................... 59	4.2.6	 Effect of varying iCM seeding density on differentiation outcome ......................... 64	4.2.7	 Role of cell-to-cell contact in co-culture based cardiac differentiation .................... 69	4.3	 Chapter discussion ........................................................................................................... 73	Chapter 5: Conclusions and Future Directions ........................................................................76	5.1	 What’s new? ..................................................................................................................... 77	5.1.1	 Substrate stiffness and cardiac differentiation of iPS cells ....................................... 77	5.1.2	 Topographical manipulation of iPS cell-derived cardiomyocytes (iCMs) ............... 78	5.1.3	 Novel co-culture method of cardiac differentiation of iPS cells using previously differentiated iPS cell-derived cardiomyocytes (iCMs) ....................................................... 78	5.2	 Big picture contribution ................................................................................................... 79	5.3	 Clinical perspective .......................................................................................................... 79	5.3.1	 Modeling of cardiomyopathies with iPS cell-derived cardiomyocytes .................... 80	5.3.2	 Drug-induced cardiotoxicity testing using iPS cell-derived cardiomyocytes ........... 80	5.3.3	 Cell-based regenerative therapy ................................................................................ 81	5.4	 Future directions .............................................................................................................. 81	5.5	 Conclusion ....................................................................................................................... 86	Bibliography .................................................................................................................................87	Appendix A ............................................................................................................................... 96	 viii A.1	 Characterization of differentiation efficiency of novel co-culture method by flow cytometry – independent replicate ........................................................................................ 96	Appendix B ............................................................................................................................... 98	B.1	 iPS cell maintenance, expansion, and differentiation protocols .................................. 98	B.2	 Labeling iPS cells with CellTracker red dye ............................................................. 105	B.3	 Immunocytochemistry staining of cells adhered on coverslip .................................. 106	B.4	 Intracellular staining for flow cytometry .................................................................. 107	  ix List of Tables  Table 1.1     Methods for monolayer differentiation of hPSC-CMs ............................................... 5	Table 1.2     List of inductive co-culture assays ............................................................................ 11	Table 4.1     Cardiac differentiation efficiency of various conditions quantified via flow cytometry for cTnT and α-actinin. ................................................................................................ 63 Table 4.2     Cardiac differentiation efficiency of various conditions quantified via flow cytometry for cTnT, α-actinin, and GFP+ve cells. ......................................................................... 63 Table 4.3     Cardiac differentiation efficiency of various iCM seeding densities quantified via flow cytometry for cTnT and α-actinin. ........................................................................................ 69 Table A.1     Cardiac differentiation efficiency of various conditions quantified via flow cytometry for α-actinin ................................................................................................................. 97	Table A.2     Cardiac differentiation efficiency of various conditions quantified via flow cytometry for α-actinin and GFP+ve cells ...................................................................................... 97	     x List of Figures  Figure 2.1     Images of live Human AICS-0016 iPS cells colonies expressing mEGFP tagged          beta-actin ....................................................................................................................................... 15 Figure 2.2     Morphology of Human IMR90-4 iPS cells ............................................................. 18 Figure 2.3     Schematic of GiWi protocol .................................................................................... 20 Figure 2.4     Typical appearance of IMR90-4 iPS and iCM cell colonies ................................... 20 Figure 2.5     Microscope systems used ........................................................................................ 22 Figure 2.6     Fabrication of PDMS substrates of tunable elastic modulus ................................... 27 Figure 2.7     3D cardiac channel device ....................................................................................... 29 Figure 3.1     Morphological characterization of cardiomyocytes derived from iPS cells cultured and differentiated on various substrates ........................................................................................ 33 Figure 3.2     Flow cytometry analysis for cTnT expression of cardiomyocytes differentiated from iPS cells on substrates of variable stiffness .......................................................................... 34 Figure 3.3     Flow cytometry analysis for HCN4 expression of cardiomyocytes differentiated from iPS cells on substrates of variable stiffness .......................................................................... 35 Figure 3.4     Microscopy images of 12 day-old iCMs cultured in 1mm × 10mm linear PDMS channel for 4 days ......................................................................................................................... 37 Figure 4.1     Graphic of cell tracking procedure with CellTracker red dye ................................. 42 Figure 4.2     Workflow schematic of pre-culture experiments .................................................... 43 Figure 4.3     Fluorescent images and kymographs of pre-cultures .............................................. 45 Figure 4.4     Workflow schematic of bi-culture experiments ...................................................... 47 Figure 4.5     Fluorescent images and kymographs of bi-cultures ................................................ 49  xi Figure 4.6     Flow cytometry analysis of IMR90-4 iPSCs and AICS-0016 iPSCs ...................... 51 Figure 4.7     Schematic of pre-culture method used to initiate cardiac differentiation of AICS-0016 iPS cells ................................................................................................................................ 52 Figure 4.8     Fluorescent images and kymographs of AICS-0016 + IMR90-4 pre-cultures ....... 55 Figure 4.9     Fluorescent images of cells stained for α-actinin via immunocytochemistry ......... 57 Figure 4.10    Immunocytochemistry images of GiWi differentiated AICS-0016 cells ............... 58 Figure 4.11    Flow cytometric analysis for cTnT cardiac marker (GFP β-actin vs. cTnT) ......... 61 Figure 4.12    Flow cytometric analysis for α-actinin cardiac marker (GFP β-actin vs. α-actinin)....................................................................................................................................................... 62 Figure 4.13    Illustration of IMR90-4 iCMs seeded at varying densities to AICS-0016 iPS cells in pre-culture ................................................................................................................................. 65 Figure 4.14    Differentiation efficiency for pre-cultures with varying IMR90-iCM seeding densities - assessed by cTnT expression ....................................................................................... 66 Figure 4.15    Differentiation efficiency for pre-cultures with varying IMR90-iCM seeding densities - assessed by α-actinin expression ................................................................................. 67 Figure 4.16    Immunohistochemistry images of pre-cultured AICS-0016 cells seeded with varying IMR90-iCM seeding densities ......................................................................................... 68 Figure 4.17    Illustration of non-contact cell culture hanging insert setup .................................. 70 Figure 4.18    Representative microscopy images validating hanging culture insert system ....... 71 Figure 4.19    Flow cytometry density plots (cTnT vs. GFP) for AICS-0016 iPS cells cultured with iCM hanging insert vs. AICS-0016 iPS cells cultured with only basal medium + supplement (negative control) .......................................................................................................................... 71 Figure 4.20    Fluorescent images from non-contact co-culture assay, stained for α-actinin ....... 72  xii Figure A.1     Flow cytometric analysis of various culture conditions for α-actinin cardiac marker (GFP β-actin vs. α-actinin) ........................................................................................................... 96   xiii Glossary  ACTB  actin-beta	BMPs	 	 bone morphogenetic proteins, a group of growth factors that play key roles in    during development  BMP4  bone morphogenetic protein 4 BSA  bovine serum albumin Cas9  CRISPR associated protein 9 CCD  charge-coupled device CDM1	  cardiomyocyte differentiation medium 1 CDM2  cardiomyocyte differentiation medium 2 CMs  cardiomyocytes CMTs  cardiac microtissues  CIM  cardiovascular progenitor cell induction medium CRISPR clustered regularly interspaced short palindromic repeats  cTnT  cardiac troponin T Cy5  indodicarbocyanine, a near-infrared fluorescence-emitting dye that has excitation    and emission peak wavelengths of approximately 649nm/666nm DAPI  4’, 6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to A-T rich   regions in DNA DIC	 	 differential interference contrast DMEM		 Dulbecco’s Modified Eagle’s Medium DMSO	  dimethyl sulfoxide  xiv ECM	  extracellular matrix EDTA  ethylenediaminetetraacetic acid EHS  Engelberth-Holm-Swarm EHTs  engineered heart tissues ES	cell  embryonic stem cell FACS  fluorescence-activated cell sorting FAK  focal adhesion kinase  FBS	  fetal bovine serum FGFs	  fibroblasts growth factors FITC	  fluoresceine isothiocyanate, a fluorescent molecule that has excitation and    emission peak wavelengths of approximately 490nm/525nm, giving it a green    color GFP  green fluorescent protein GFR	 	 growth factor reduced  GSK3  glycogen synthase kinase 3 HCN4  potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 hES	cell human embryonic stem cell hPSC-CM human pluripotent stem cell-derived cardiomyocyte iCM  induced pluripotent stem cell-derived cardiomyocyte IgG  immunoglobulin G iPS	cell  induced pluripotent stem cell JNK  c-Jun N-terminal kinase  KLF4   Kruppel-like factor 4  xv LMYC	  myc-related gene from lung cancer LIN28  Lin-28 homolog A MEF-CM  mouse embryonic fibroblast-conditioned medium mEGFP	 monomeric enhanced green fluorescent protein	MHC-α myosin heavy chain-α isoform MHC-β  myosin heavy chain-β isoform MYC  v-myc avian myelocytomatosis viral oncogene homolog NANOG homeobox protein NANOG OCT4	  octamer-binding transcription factor 4  PBS	  phosphate-buffered saline  PDMS	  polydimethylsiloxane ROCK	  rho-associated protein kinase RPMI	1640 Roswell Park Memorial Institute medium shp53  transformation-related protein 53 SOX2  sex determining region Y – box 2 TCPS	  tissue culture polystyrene THY1  thymocyte differentiation antigen 1 TMRM  tetramethylrhodamine, methyl ester TRITC	  tetramethylrhodamine isothiocyanate, a bright orange-fluorescent dye with    excitation and emission peak wavelengths of approximately 557nm/576nm VE  visceral endoderm Wnt  wingless-type mouse mammary tumor virus integration site  xvi Acknowledgements  I would first like to thank my supervisors, Dr. Mu Chiao and Dr. Chinten James Lim, whose doors were always open to me when I ran into trouble with my research. I will be forever grateful for their invaluable insights and advice that have contributed immensely to my development.    Conversations with Sanam Shafaattalab from the Tibbits lab of Simon Fraser University were also particularly insightful, leading to many new and exciting ideas.   My lab mates, Eric Zhao, Pascal Leclair, Lakshana Sreenivasan, Jack Liu, Foujan Pedari, Vicky Li, Hongbin Zhang, Guangyi Cao, and Ali Shademani, were exceedingly generous with their knowledge and were instrumental in my training as a scientist and engineer. I would especially like to thank Eric Zhao, who eased my transition from mechanical engineering to biomedical sciences and showed me the ropes to many experimental techniques.   I would also like to thank the Natural Sciences and Engineering Research Council of Canada for providing funding through the Engineers in Scrubs training program.  Finally, I like to express my profound gratitude to my parents for providing me unwavering support and encouragement throughout my years of education and research. This body of work would not have been possible without them. Thank you.   xvii Dedication  To my late mother. I couldn’t have done this without you. Thank you for your love and support.  1 Chapter 1: Introduction  1.1 Building the heart in a dish Animals like the zebrafish and other teleost fishes can regenerate large portions of their heart after injury. The human heart on the other hand does not enjoy the same kind of regenerative capacity, forming scar tissues instead of making new functional muscle after myocardial infarction – often leading to fatal arrhythmias and heart failures. For decades, the bulk of scientists were convinced that the heart was a post-mitotic organ, characterized by a predetermined number of myocytes at birth [1]. Although recent studies have indicated that adult human cardiomyocytes are replaced at detectable rates [2], this native capacity is low and insufficient to be exploited for large-scale in vitro purposes. This conundrum led to the rise of human induced pluripotent stem cell-derived cardiomyocytes (iCMs) as a prospective alternative, to generate large quantities of patient-specific, functionally relevant cardiomyocytes for therapeutics and research. While current strategies for differentiating induced pluripotent stem (iPS) cells into cardiomyocytes yields high efficiencies (>80%) [3], unpublished anecdotal evidence suggests reproducibility and robustness of protocols needs to be improved to achieve greater consistency between lines and laboratories. Moreover, protocols remain unpredictable, yielding a mixed population of cardiomyocyte subtypes that also vary in terms of maturity [4]. This gap in literature motivated my undertaking of this exploratory project, to discover the physical and biology parameters governing cardiac differentiation and enable improvements to current differentiation systems.    2 1.2 Stem cells: A glimpse of the past, present, and future  To attempt cardiac differentiation, one must first understand the nature stem cells. Stem cells are undifferentiated cells capable of giving rise to other specialized cell types. Regardless of their source, they exhibit two defining properties: 1) Self-renewal – able to go through numerous cycles of cell division while maintaining undifferentiated state. 2) Pluripotency – capable of becoming cells in all three germ layers i.e. any cell type in the body. In 1981, two groups led by Martin Evans and Matthew Kaufman, from the University of Cambridge, independently derived embryonic stem (ES) cells from mouse embryos [5, 6]. However, it was not until 1998 that the first human ES cells lines were first reported [7]. Soon after its discovery, controversy ensued, centered on the moral implication of destroying human embryos.   This ethical conundrum was finally circumvented in 2006 when Takahashi and Yamanaka first created induced pluripotent stem (iPS) cells from adult dermal fibroblasts using four transcription factors, OCT4 (octamer-binding transcription factor 4), SOX2 (sex determining region Y – box 2), KLF4 (Kruppel-like factor 4), and MYC (v-myc avian myelocytomatosis viral oncogene homolog) [8, 9]. This ability to stimulate a person’s own cells to a stem-like state reduces the need for human embryos in research while also opening new exciting possibilities for personalized medicine [10].  In principle, iPS cells can be differentiated to all cell types, limited only by our current procedural know how.   This seminal work rapidly catalyzed a flurry of effort to generate functional cardiomyocytes in vitro from these iPS cells. Motivated by the prospect of generating patient / disease specific cardiomyocytes for both basic and clinical applications like disease modeling, drug toxicity screening / discovery, and cell-based regenerative therapies, researchers have been tremendously productive in developing numerous cardiac differentiation strategies for iPS cells.  3 1.3 Current strategies for cardiac cell differentiation  One of the first reports of successful in vitro stem cell to cardiomyocyte differentiation came from Joseph Itskovitz-Eldor’s team when they documented the production of spontaneously contracting structures generated from human embryonic stem (ES) cells via three-dimensional embryoid bodies [11]. Subsequent research showed that these cardiomyocytes derived from human ES and iPS cells exhibited many of the structural and functional properties of native heart cells [12], prompting further investigation and development of more robust cardiomyocyte differentiation strategies.   To date, there have been a number of differentiation protocols reported for differentiating stem cells to cardiomyocytes and these protocols can be categorized into three general approaches: 1) embryoid bodies 2) co-cultures with an inducer 3) two-dimensional monolayers. In the embryoid bodies (EBs) method, stem cells are grown suspended in the culture media where the cells form spherical conglomerates resembling the morula. Cells in the EBs differentiate into derivatives of the three primary germ layers, with a portion of the EBs forming spontaneously contracting regions containing cardiomyocytes [13]. Derivation of cardiomyocytes via co-culture is achieved by incubating appropriate inducers (for example, END-2 cell line [14]) with pluripotent stem cells to initiate cardiac differentiation. Lastly, in the monolayer method, stem cells are grown adhered to the culture plate to a high density before cells are treated with various differentiation factors.   The monolayer method is by far the most popular among the three approaches, mainly due to its ease of handling and its relatively larger scale production of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) [15]. One of the earliest monolayer methods were first described by Laflamme et al., employing timed treatments of protein-based morphogens activin  4 A and bone morphogenetic protein 4 (BMP4) to induce cardiac differentiation of hES cells [16]. Activin A and BMP4 both belong to the transforming growth factor beta (TGF- β) superfamily of signaling proteins, previously identified in animal models to be key players involved in cardiogenesis [4]. Since Laflamme’s pioneering monolayer method, others have developed more robust and reliable procedures based on Wnt pathway modulation using chemical inhibitors of GSK3 (CHIR99021) and Wnt (IWP-2) [17]. This method was termed ‘GiWi protocol’ by authors Lian and Palecek who were the first to develop the technique in 2012 [18]. Relative to recombinant growth factors, the use of chemically synthesized small molecules has the advantage of being cheaper in cost and less variable in terms of reproducibility. Since the discovery of this so-called GiWi protocol, others have also made incremental improvements / modifications to the method.  Table 1.1 provides a summary of some of the main hPSC-CMs monolayer cardiogenic protocols reported in literature.             5    Table 1.1: Methods for monolayer differentiation of hPSC-CMs. References: Laflamme et al. [12]. Lian et al. [14]. Cao et al. [15]. Burridge et al. [16]. Abbreviations: MEF-CM, mouse embryonic fibroblast-conditioned medium; CIM, cardiovascular progenitor cell induction medium; CDM1 and CDM2, cardiomyocyte differentiation medium 1 and 2.    Culture	condi-ons	Laflamme	et	al.	(2007)	Cells: 	 	ESCs	Substrate: 	Matrigel	Medium:	 	MEF-CM	Differen-a-on	protocol	 Efficiency	d0	 d1	 d5	 d9	Ac7vin	A	 BMP4	RPMI	1640	+	B27	(-insulin)	 RPMI	1640	+	B27	(+insulin)	~30%	Lian	et	al.	(2012)	Cells: 	 	ESCs	or	iPSCs	Substrate: 	Matrigel	Medium:	 	mTeSR1	d0	 d1	 d5	 d9	GSK3	In.	 Wnt	In.	RPMI	1640	+	B27	(-insulin)	 RPMI	1640	+	B27	(+insulin)	~85%	d7	Cao	et	al.	(2013)	Cells: 	 	ESCs	or	iPSCs	Substrate: 	Matrigel	Medium:	 	mTeSR1	d0	 d3	 d15	GSK3	In.	+	BMP4	+	Asc.	acid	BMP4	+	Wnt/β-catenin	in.	Basal	CIM	 Basal	CDM1		(-insulin)	~80%	d6	Basal	CDM2		(+insulin)	Burridge	et	al.	(2014)	Cells: 	 	ESCs	or	iPSCs	Substrate: 	Matrigel	Medium:	 	Essen7al	8			 	 	medium	d0	 d2	 d7	GSK3	In.	 Wnt	In.	RPMI	1640	+	albumin	+	L-ascorbic	acid	2-phosphate	~85%	d4	 6 1.4 Defining characteristics of cardiomyocytes Currently, experts in the field of cardiac differentiation have yet to reach an agreement on where to draw the line between a cardiac progenitor and a fully differentiated cardiomyocyte. Naturally, the cells generated from differentiation and reprogramming fall into a spectrum of varying maturity, with the starting cell population at one end of the spectrum and fully differentiated mature cardiomyocyte at the other end. Often, in such cardiac differentiation and reprogramming experiments, the population of cells would express basic baseline features, such as the expression of a cardiac specific marker like cardiac troponin T (cTnT), and a decreasing number of cells in the population meeting stricter criteria such as, exhibition of spontaneous contractions and calcium oscillations.   As proposed by Addis et al., a cell is considered successfully reprogrammed to a cardiomyocyte if it meets the following three criteria [21]: I. The cell has a gene expression pattern that resembles that of cardiomyocytes more than any other cell type.  II. The cell expresses structural proteins, such as cardiac troponin T, α-myosin heavy chain, α-actinin, and they are arranged into sarcomeres.  III. The cell exhibits at least one functional characteristic, such as spontaneous contractions, action potentials, and calcium oscillations.    7 1.5 Substrate stiffness and cardiac differentiation In recent years, there has been growing consensus among scientists that the extracellular environment of cells may play a crucial role in providing signaling cues necessary for cells being differentiated to cardiomyocytes [22]. This conjuncture is based on results showing cardiomyocytes generated in vivo generally yielding higher rates of conversion compared to similar in vitro experiments [23, 24]. This led researchers to suspect that the key to improving in vitro cardiomyocyte reprogramming lies in the recreation of the physical microenvironment of the native heart. Some examples of such ‘non-soluble’ factors that may affect cellular differentiation include cyclic mechanical stretching [25, 26], substrate stiffness [27, 28], and fluid shear forces [29].    In a seminal paper published in 2006, Engler et al. showed that it was possible to direct differentiation of stem cells to different lineages by simply varying the stiffness of substrate on which the cells grew [27].  Engler differentiated mesenchymal stem cells to neuron-like, myoblast-like, and osteoblast-like lineages by way of modulating only the stiffness of substrates that cells grew on. With this discovery, Engler’s team hypothesized that the cells can sense the stiffness of the substrate by ‘pulling’ on it and with the aid of force transduction proteins on its membrane. They can sense the force required to deform the substrate and hence sense the stiffness of the extracellular environment. The process of a cell sensing and responding to mechanical stimuli through biochemical signals that provokes certain cellular response is known as mechanotransduction. Since then, there has also been reported works achieving similar results using iPS cells [28].   8 1.6 Engineered three-dimensional heart tissues  In addition to the development of two-dimensional (2D) cardiac systems, there have been various attempts at creating hPSC-CM based three-dimensional (3D) engineered heart tissues. Researchers postulated that 3D systems are better suited to recapitulate the physiology and function of the native heart, making them a more representative and preferred in vitro models than their 2D counterparts [4]. Examples of 3D systems that have been validated independently to a high degree include three main approaches: 1) engineered heart tissues (EHTs [30, 31]) systems 2) cardiac microtissues (CMTs [32, 33]) systems 3) cardiac biowires [32] systems. EHTs and CMTs both rely on the use of elastic anchors to align the cardiac tissue (i.e. cardiomyocytes self-align by contracting against anchors). EHT fabrication involves encapsulating cardiomyocytes between two silicon posts whereas CMT fabrication involves tethering cardiomyocytes to polydimethylsiloxane (PDMS) cantilevers. Cardiac biowires on the other hand consists of casting cardiomyocyte-collagen gel mixture around a surgical suture in a PDMS mold.   The above-described 3D systems each have pros and cons. The main advantage of 3D platforms is that they mimic cellular structure and native heart function to a greater degree than 2D systems, which suffers from ‘unnatural’ geometric constrains. This gives 3D platforms the edge of being more physiologically relevant as an in vitro model for disease modeling and testing of candidate drugs. However, despite its clear advantage, the adoption of hPSC-CM based 3D systems for commercial and clinical use is still relatively limited, owing primarily to the cost, complexity, and reliability of these systems [4].   9 1.7 Inductive co-cultures and cardiac differentiation As mentioned earlier, one of the strategies of generating cardiomyocytes from stem cells is by co-culture with an appropriate inducer. This approach stem from the logical reasoning that one can overcome the inherent limitation of precisely recapitulating the biochemical signaling events associated with cardiac organogenesis by handing the task over to already differentiated cardiomyocytes. However, this approach is not without theoretical flaws. Cardiomyocytes that have been terminally differentiated or are not stimulated by ischemia / injury may not produce the necessary signaling cues essential to cardiac differentiation [33]. Moreover, the perceived plasticity of cultured stem cells in in vivo transplantation may be attributed to an entirely different set of milieu dependent differentiation mechanisms that may be impossible to recreate in an in vitro setting [34]. Despite this, it has not stopped researchers from attempting to derive cardiomyocytes from other cell types (stem cells or otherwise) by inductive co-cultures.   One of the first reported successes of creating cardiomyocytes from human pluripotent stem cells via co-culture induction came from Mummery et al. [14]. Through their groundbreaking work, they showed that co-culture of human embryonic stem (hES) cells with mouse visceral-endoderm-like (VE-like) cells initiated differentiation into beating cells. Although the exact mechanism is still unknown, bone morphogenetic proteins (BMPs), fibroblasts growth factors (FGFs), and inhibitors of Wnt produced by endoderm are likely players involved in the process. Similarly, when Rudy-Reil et al. co-cultured murine embryonic stem cells with a bilayer of avian precardiac endoderm / mesoderm, the number of contractile embryoid bodies was significantly increased compared to cells cultured alone [35]. More recently, Dong-Bo Ou’s team demonstrated that the long-term differentiation of mouse embryonic stem cells (ESCs) into cardiomyocytes (CMs) proved more efficient when co- 10 cultured with mouse neonatal cardiomyocytes compared to sole treatment with ascorbic acid to induce cardiac differentiation [36]. In a direct follow-up study published in 2016, the authors concluded that co-culture of stem cells with neonatal cardiomyocytes induces genes to be expressed in a mature pattern and stimulated the proliferation of stem cell-derived CMs by activating FAK/JNK signaling [37].   Although rare, examples of transdifferentiation of already differentiated cells (non stem cells) into cardiomyocytes have also been documented in literature. Most notable were findings reported by Condoelli et al. showing rat endothelial cells, freshly isolated from embryonic vessels, changing fate and transdifferentiating into beating cardiomyocytes after being co-cultured with neonatal rat CMs [38]. Badorff et al. sustained this finding by observing the transdifferentiation of endothelial progenitor cells, obtained from peripheral blood mononuclear cells of healthy adults and coronary heart disease patients, into cardiomyocytes after co-cultivation with rat cardiomyocytes [39]. The fact that endothelial cells, a type of non stem cell, can produce cardiomyocytes sheds additional light on the plasticity of cell fates and also on the robustness of co-culture inductive assays as a potential system of generating cardiomyocytes.  Table 1.2 outlines a compiled list of inductive co-culture assays that have been reported in literature.    11 Table 1.2: List of inductive co-culture assays. Column 1 shows a list of cell types successfully reprogrammed into functional cardiomyocytes via inductive co-culture.  Column 2 shows the list of corresponding cell types that were used as stimulatory source. Column 3 indicates the reprogramming mechanism – differentiation or transdifferentiation.  Cell type Co-cultured with (Stimulatory source) Reprogramming mechanism References Human mesenchymal stem cells Human cardiomyocytes Differentiation Rangappa et al. [30] Human embryonic stem cells Mouse visceral-endoderm-like cells  Differentiation  Mummery et al. [10] Human induced-pluripotent stem cells Mouse visceral-endoderm-like cells Differentiation Freund et al. [31] Human endothelial progenitor cells Rat cardiomyocytes Transdifferentiation Badorff et al. [29] Mouse induced-pluripotent stem cells Mouse neonatal cardiomyocytes Differentiation Ou et al. [27]  Rat mesenchymal stem cells Rat neonatal cardiomyocytes Differentiation  Yoon et al. [32] Rat skeletal muscle-derived cells Rat neonatal cardiomyocytes Transdifferentiation Iijima et al. [33] Murine embryonic stem cells  Avian precardiac endoderm/mesoderm Differentiation Rudy-Reil et al. [25] Rat endothelial cells Rat neonatal cardiomyocytes Transdifferentiation Condorelli et al. [28] Human adult circulating endothelial progenitor cells  Rat neonatal ventricular cardiomyocytes Transdifferentiation Koranagi et al. [34] Human embryonic stem cells Mouse visceral-endoderm-like cells  Differentiation  Passier et al. [35] Human embryonic stem cells Mouse visceral-endoderm-like cells  Differentiation  Mummery et al. [36] Mouse induced pluripotent stem cells Mouse stroma cells Differentiation Narazaki et al. [37] Human embryonic stem cells Mouse visceral-endoderm-like cells  Differentiation  Xu et al. [38] Mouse embryonic stem cells Mouse stroma cells  Differentiation Yamashita et al. [39] Rat mesenchymal stem cells Rat neonatal ventricular cardiomyocytes Differentiation  Zeng et al. [40] Mouse embryonic stem cells Mouse neonatal cardiomyocytes Differentiation Ou et al. [26] Rat bone marrow mesenchymal stromal cells Rat neonatal cardiomyocytes Transdifferentiation He et al. [41] Mouse embryonic stem cells Mouse bone marrow stromal cells  Differentiation Yue et al. [42] Human mesenchymal stem cells Rat cardiomyocytes Differentiation Plotnikov et al. [43]  12 1.8 Thesis aim and hypotheses The aim of my thesis is to address current methodological gaps in pluripotent stem cell-to-cardiomyocyte differentiation strategies, by investigating various physical and biological parameters governing the process. Based on rationales described below, I explored three areas of interest, formulating specific hypothesis for each:  1. The effects of substrate stiffness on cardiac differentiation outcome of iPS cells – (Chapter 3). Rationale:  Accumulating evidence has linked physical parameters, like substrate stiffness, to cardiac differentiation outcomes. This phenomenon could be exploited to assess if substrates of tunable stiffness may be an efficient method to accelerate maturation and/or enhance cellular subtype specification of iPS cell-derived cardiomyocytes.  Hypothesis: I hypothesize that substrate stiffness modulation in conjunction with treatment of traditional soluble factors, will accelerate maturation of iPS cell-derived cardiomyocytes and also enhance subtype specification, as measured by the expression of various cardiac specific and subtype specific markers.  2. Generation of three-dimensional heart tissue using topographical manipulation – (Chapter 3). Rationale: 3D heart models are more physiologically representative than their 2D counterparts. However, the cost and complexity of current leading 3D  13 hPSC-CM platforms are limiting factors in its adoption for pre-clinical research applications. A simpler and cost effective method for generating functionally relevant engineered cardiac tissues may be preferable.  Hypothesis: I postulate that freshly differentiated iCMs can be topographically manipulated by way of a straightforward process of seeding cells in grooved linear ‘cardiac channels’ to form uniaxially contracting, millimeter-sized three-dimensional heart tissues.  3. Derivation of cardiomyocytes from iPS cells using inductive co-culture with previously differentiated iCMs – (Chapter 4). Rationale:  Noticeably lacking from existing literature is a co-culture inductive platform utilizing stem cell-derived cardiomyocytes as a stimulatory source for cardiac reprogramming (of stem cells or otherwise). The ability to do so may represent a ‘near natural’ process for differentiating iPS cells to iCMs that is not limited by the use of defined pathway inhibitors or morphogens.  Hypothesis: I hypothesize that non-differentiated iPS cells, co-cultured with ‘older’ iCMs, can be differentiated into functional cardiomyocytes without the exogenous addition of pathway inhibitors or morphogens.   14 Chapter 2: Materials and Methods  2.1  Tissue culture  2.1.1 Induced pluripotent stem cell line: IMR90-4 The iPS cell line, IMR90-4, from WiCell Research Institute, Inc., used in this body of work was gifted to us by Sanam Shafaattalab and Dr. Glen Tibbits of BC Children’s Hospital Research Institute and Simon Fraser University. The IMR90-4 iPS cell line was generated from lung fibroblasts obtained from a 16-week old human female fetus using viral transduction of the OCT4, NANOG, SOX2, and Lin-28 homolog A (LIN28) genes. After reprogramming, the iPS cells were passaged 41 times, the last 16 of which were maintained in mTeSR1 medium on Matrigel coated dishes. Upon receipt of the IMR90-4 iPS cells, they were exclusively cultured in mTeSR1 medium and on Matrigel coated surfaces.  2.1.2 Fluorescently tagged human induced pluripotent stem cell line: AICS-0016 The AICS-0016 iPS cell line is a human clonal line in which the actin-beta (ACTB) gene has been endogenously tagged with the monomeric enhanced green fluorescent protein (mEGFP) sequence using CRISPR/Cas9 technology. As such, beta-actin proteins in cells are fluorescently tagged with mEGFP, allowing visualization of cells’ actin filament structures under fluorescent microscopy. This cell line was developed by the Allen Institute for Cell Science (Seattle, WA) and distributed by the Coriell Institute for Medical Research. An example of AICS-0016 iPS cells imaged using fluorescent microscopy is shown in Figure 2.1.   15  The AICS-0016 iPS cell line was used primarily in my co-culture assays to track cardiac differentiation outcomes of iPS cells co-cultured with iPS cell-derived cardiomyocytes (iCMs). Prior to its receipt, the AICS-0016 cell line was maintained in mTeSR1 medium, cultured on Matrigel, and passaged 34 times. It is derived from its parental human iPS cell line, GM25256, which was reprogrammed using episomal vectors OCT3/4, shp53, SOX2, KLF4, LMYC, and LIN28 from human fibroblasts of a 30-year-old human male.   Culture and freezing methods used for the AICS-0016 iPS cell line were identical to those used for the IMR90-4 iPS cell line described in the preceding section.  Figure 2.1: Images of live Human AICS-0016 iPS cells colonies expressing mEGFP tagged beta-actin.  (A) GFP fluorescence image.  (B) Corresponding DIC image.   2.1.3  Matrigel coatings  Basement membranes are thin extracellular matrices that underlay cells in vivo. To facilitate the adhesion of iPS cells to tissue culture plates, I utilize a thin coating of Corning® Matrigel® Matrix Growth Factor Reduced (GFR) (Corning #354230) – a solubilized basement membrane protein mix– to pre-coat culture plates prior to iPS cell seeding. This Matrigel is extracted from 100μm	 100μm	(A)	 (B)	 16 the Engelberth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix (ECM) proteins. ECM proteins such as laminin, collagen IV, heparin sulfate proteoglycans, and entactin/nidogen, are major components found in Matrigel and aid in cell adhesion. Other components include various growth factors like transforming growth factor-beta, epidermal growth factor, insulin-like growth factor, and fibroblast growth factor, which occur naturally in EHS tumors.   Matrigel coating of tissue culture plates were done by dissolving Matrigel aliquots in cold DMEM/F-12 medium (GE Healthcare Life Sciences #SH30023.01) and dispensing the dissolved solution onto the well surface of tissue culture plates. Culturewares, like pipette tips, pipettes, and tissue culture plates that comes into contact with Matrigel were pre-chilled at 4oC to prevent Matrigel from forming a gel prematurely, which might result in poor surface coating. Matrigel solution and culture plates were kept on ice throughout the preparation process. Upon immersing well surface with Matrigel solution, plates were left on a flat surface in a biosafety cabinet and allowed to cure at room temperature for 1 hour. 0.5mg of Matrigel was used to coat a single 6-well plate. After 1 hour, Matrigel solution together with all unbound material was aspirated and the well gently rinsed with mTeSR™1 medium (Stemcell Technologies #05850).   2.1.4 Standard culture of iPS cells  Unless stated otherwise, the following iPS cell manipulation and reagent volumes are for a single well (culture area 9.5cm2) of a 6-well plate.   Complete mTeSR1 medium was prepared by adding the mTeSR1 5X supplement to the mTeSR1 basal medium. Complete mTeSR1 medium was stored frozen at -20oC in aliquots of 50mL and thawed at 4oC overnight when needed. Once seeded, the iPS cells were maintained in  17 2mL mTeSR1 medium with daily medium renewal until the cells reached ideal confluency for passaging, initiation of differentiation to cardiomyocytes, or harvesting for experiments. The optimal time to reach ideal confluency (~80%) depends primarily on the initial cell seeding density. Typically, a seeding density of 1×105 cells in a single well of a 6-well plate would enable cells to reach optimal confluency in 3 – 4 days. In this time, cell colonies were visually examined for spontaneous and unspecific differentiation. If spontaneous differentiation was detected to be greater than 20%, differentiated colonies were physically removed by suction using a 2mL-aspirating pipette. An example of a spontaneous differentiated unspecified colony is illustrated in Figure 2.2.  To dissociate iPS cells for passaging, the spent mTeSR1 medium is removed by aspiration, the cells rinsed once with Versene® (EDTA) 0.02% (Lonza #17-711E), and then treated with fresh 1mL Versene for 7 minutes at room temperature. The Versene solution was then carefully aspirated to not disturb the cell layer and 2mL fresh mTeSR1 medium was added. Repeated pipetting with a P1000 pipetman (5 – 10 times) was used to wash the loosely attached iPS cells off the culture surface and to fully dissociate the cells from its larger cell aggregates. Care was taken to ensure minimal fluid shear forces on cells by pipetting gently. The split ratio when passaging varied from 1:3 to 1:12, depending on the downstream application for the cells. Typically, cells were split at a larger ratio for expansion purposes and at a lower ratio for use in differentiation experiments. Immediately after cells were added to Matrigel-coated well surfaces, the small molecule, Y-27632 (Stemcell Technologies #72302), a highly potent and selective inhibitor of Rho-associated protein kinase (ROCK) was added at a concentration of 10µM to each well. The use of ROCK inhibitor is known to increase cell viability of stem cells by  18 reducing anoikis, a form of programmed cell death that occurs due to cell detachment from extracellular matrix [54].  To prepare iPS cells for cryopreservation, cells at 80% confluency were treated with Versene like before, and mFeSR®1 (Stemcell Technologies #05855) cryofreeze medium used to dislodge the cells and prepare a homogenous cell suspension. Typically, cells form a 6-well plate were pooled together as a resuspension in 5mL of mFeSR1, and 1mL aliquots transferred to each cryofreeze storage vial. The vials were placed into isopropanol-bathed freezing containers (Mr Frosty™) at room temperature and the container placed at -80oC overnight. The next day, the cryo-vials are transferred to liquid nitrogen for long-term storage.   Figure 2.2: Morphology of Human IMR90-4 iPS cells.  (A) Undifferentiated monolayer of human iPS cells colonies.  (B) Colony of spontaneous differentiation (red arrow).   2.1.5 Cardiac differentiation with GSK3 inhibitor and Wnt inhibitor (GiWi Protocol) To generate cardiomyocytes from iPS cells via traditional biochemical means, I used small molecule inhibitors of glycogen synthase kinase 3 (GSK3) and Wnt, commonly referred to as the GiWi protocol [3], and schematically outlined in Figure 2.3. iPS cells were seeded on Matrigel-(A)	 (B)	 19 coated 6-well plates at a density of 1×105 cells per well and maintained in mTeSR1 medium. After 3 – 4 days of growth with daily medium renewal, cells should reach 80 – 90% confluency. Figure 2.4 (A) and (B) shows the typical appearance of iPS cell colonies 1 day after seeding and immediately prior to the initiation of differentiation.   On occasion, cells may appear to not grow as expected. Poor or uneven Matrigel coating usually causes this. It should be noted that simply extending the iPS cell growth period beyond the stipulated time is unlikely to yield the desired confluency. Instead, iPS cells generally tend to proliferate and crowd out each other when left in culture for prolonged periods, resulting in multi-layered colonies and increased chance of spontaneous differentiation.  When iPS cells reached its optimal confluency, mTeSR1 medium was aspirated out and changed to RPMI 1640 basal medium (Sigma-Aldrich #R8758) with B-27 supplement (minus insulin) (Life Technologies #A1895601). CHIR-99021 (Selleck Chemicals #S2924), a GSK-3α/β inhibitor was added to the medium to a final concentration of 12µM. After 24 hours of CHIR-99021 treatment, the medium was replaced with RPMI 1640 basal medium with B-27 supplement (minus insulin) and left for 48 hours. After that time, the medium was refreshed with RPMI 1640 basal medium with B-27 supplement (minus insulin) and IWP-2 (Stemcell Technologies #72122), a Wnt pathway inhibitor, to a final concentration of 5µM. After another 48 hours, the medium was aspirated and replaced with RPMI 1640 basal medium with B-27 supplement (complete with insulin) (Life Technologies #17504-044). This medium was renewed every 3 days thereafter. When differentiated on traditional polystyrene culture plates, spontaneous contractions are typically observed 7 days after the onset of CHIR-99021 treatment. With continued maintenance, the iPS cell-derived cardiomyocytes remodel and recruit more cells  20 to form larger contracting networks. Figure 2.4 (C) and (D) illustrate iCM clusters at day 7 and day 14 respectively after the onset of initiation of differentiation.    Figure 2.3: Schematic of GiWi protocol.  Four days prior to differentiation (d-4), iPS cells are seeded onto Matrigel-coated wells.  On day 0 (d0), GSK3 inhibitor (CHIR), is added.  On day 1 (d1), medium is renewed.  On day 3 (d3), Wnt inhibitor (IWP-2) is added.  On day 5 (d5), medium is renewed.   On day 7 (d7), medium is changed to RPMI / B27 supplement + insulin.               Figure 2.4: Typical appearance of IMR90-4 iPS and iCM cell colonies.  (A) iPS cell colonies 1 day after seeding.  (B) iPS cell colonies immediately prior to differentiation, at ~90% confluency.  (C) iCMs on day 7 of differentiation.  (D) iCMs on day 14 of differentiation. d-4	Cell	seed	d0	CHIR	d1	Basal	medium	d3	IWP-2	d5	Basal	medium	 d7		mTeSR1		RPMI	with	B27	minus	insulin		RPMI	with	B27	+	insulin		Contrac@ng	cells		Figure B.2: The appearance of iPSC colonies 1 day after plating (left) and immediately priorto passaging (right).B.1.6 Feeding iPSCsiPSC medium must be renewed daily until the cells are ready for freezing, passaging, or experimen-tation.1. Observe the pluripotent stem cells using a microscope. If they require passaging, follow theprocedures in Section B.1.7.2. Warm a tube of mTeSR1 medium to room temperature in the biosafety cabinet for 15 minutes.3. Replace the aspiration Pasteur pipette with a new sterile pipette.4. Aspirate the spent medium with the sterile Pasteur pipette and a P200 pipette tip. Use adifferent pipette tip for each well to reduce the risk of contamination.5. Add 2mL of fresh mTeSR1 medium to each well. Pipette against the side wall of the culturewell to reduce fluid shear forces on the cells.6. Return the plate to the 37C incubator.B.1.7 Passaging iPSCsiPSCs are ready for passaging when continguous colonies of cells reach a size of greater than 1mmin its widest dimension, as shown in Figure B.2. Passaging is also required when cells show vis-ible morphological changes indicative of differentiation. We use EDTA (Versene, 0.02%) for forpassaging, which is a gentle method that gives clusters of cell aggregate instead of fully dissociatedcells. The passage ratio can vary between 1:8 and 1:20, depending on the density of cell coloniesafter plating.1. Warm a tube of mTeSR1 medium to room temperature in the biosafety cabinet for 15 minutes.78Figure B.2: The appearance of iPSC colonies 1 day after plating (left) and immediately priorto passagin (right).B.1.6 Feeding iPSCsiPSC medium must be renewed daily until the cells are ready for freezing, passaging, or experimen-tation.1. Observ the pluripotent stem cells using a mi roscope. If th y require passaging, follow theproc dures in Section B.1.7.2. W rm a tube of mTeSR1 medium t roo t mpe ature in the biosafety cabinet for 15 minutes.3. Replace the aspiration Pasteur pipette with a new sterile pipette.4. Aspirat th spent medium with th sterile Pasteur pipette and a P200 pipette tip. Use adifferent pipette tip for each well to reduce the risk f contamination.5. Add 2mL of fresh mTeSR1 medium to each well. Pipette against the side wall of the culturewell to reduce fluid shear forces on the cells.6. Return the plate to the 37C incubator.B.1.7 Passa ing iPSCsiPSCs are ready for passaging whe continguous colonies of cells re ch a size of greater than 1mmin its widest dime sion, as shown n Figure B.2. Passag ng is also required wh n cells show vis-ible morphologi al changes indicative of differentiation. W use EDTA (Versene, 0.02%) f r forpassaging, which is a g ntle method that gives clusters of cell a gr gate instead of fully dissociatedcells. The passage ratio c n vary between 1:8 and 1:20, dependi g on the density of ell coloniesafter plating.1. W rm a tube of mTeSR1 medium t roo t mpe ature in the biosafety cabinet for 15 minutes.78(A)	 (B)	(C)	 (D)	100μm	100μm	 100μm	 21 2.2 Cell labeling with fluorescent dye The CellTracker™ Red CMTPX fluorescent dye (ThermoFisher Scientific #C34552) was used for monitoring and for tracking individual live cells. When loaded, the dye passes through cell membranes into cells, where it is retained through several generations (typically three to six divisions). The dye transfers only to daughter cells, but not to adjacent cells in a population, making it ideal for my co-culture applications, where the fate of a mixture of labeled and unlabeled cells could be tracked for up to 11 days. Additionally, the CellTracker™ Red CMTPX fluorescent dye was designed to be stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH.   To prepare stocks of the CellTracker dye, the lyophilized product was warmed to room temperature and dissolved in cell culture grade DMSO to a final concentration of 10mM. Aliquots of 1mL were prepared and stored at 4oC. To stain cells, in a 6-well dish, the culture medium is removed and replaced with 2mL of working dye solution diluted with serum free culture medium to 5µM (pre-warmed to 37oC), and incubated for 30 minutes in an incubator. Finally, the dye solution was removed and appropriate culture medium of choice was added to cells. Freshly labeled cells were incubated for at 2 days prior to co-culture experiments to allow cells to recover from mechanical stresses caused by the dye treatment. Cells were imaged using the Olympus IX81 inverted fluorescence microscope system using 572nm excitation filter (for red staining) and single band pass emission filter for detection of fluorescent dye.   2.3 Microscope systems Fluorescent images were acquired using the Olympus IX81 microscope system (Figure 2.5 (A)); an advanced motorized inverted fluorescent microscope system equipped with a range of  22 objectives (4× - 60×) and a cooled monochrome CCD camera (CoolSNAP HQ2 from Photometrics®). The system is equipped with a temperature and CO2-controlled chamber that allowed both live and fixed cell imaging to be performed. The X-Cite® exacte illuminator is used as a light source and the system has 360nm (for blue staining), 492nm (for green staining), and 572nm (for red staining) excitation filters. When visualizing blue, green, and red fluorescent markers, the microscope uses the triple pass dichroic and used a single band pass filter when imaging each channel. MetaMorph software (v. 7.8) was used for data acquisition and post-acquisition analysis was performed using the ImageJ processing program. The Olympus Relief Contrast system also allows for DIC contrast imaging on the system.  For quick and convenient phase contrast imaging, the Olympus CKX31 inverted microscope equipped with a digital camera (Novel Optics #HDCE-20C) connected via USB to a personal computer was used (Figure 2.5 (B)).           Figure 2.5: Microscope systems used. (A) Olympus IX81 fluorescent microscope system.  (B) Olympus CKX31 microscope with digital camera connected to a personal computer.   2.4 Kymograph generation and analysis Kymographs are graphical representation of spatial position over time in which the spatial axis represents time. It allows rhythmic motion, such as spontaneous self-contractions of (A)	 (B)	 23 cardiomyocytes, to be computed by performing a selected line scan at every frame of a video. From the periodic motions of spontaneously beating cardiomyocytes, peaks of contraction could be rendered. The beat frequency (beats per unit time) of contracting cardiomyocytes and their clusters were calculated by dividing the total number of peaks by total time.   Kymographs were generated using the image-processing tool, ImageJ.   2.5 Immunocytochemistry Immunocytochemistry is a common laboratory technique that allows the visualization of specific target proteins within cells and tissues by virtue of its specific binding to antibodies. For the purpose of visualizing α-actinin in cells, cells needed to be harvested and reseeded at low density onto glass coverslips.   For dissociation, cells were rinsed with phosphate-buffered saline (PBS) (Sigma-Aldrich #D8537) before treatment with 1mL trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.05% trypsin, GE Healthcare #SH30236.01) for 5 – 9 minutes at 37oC. For reference, treatment with 0.05% trypsin will dissociate iPS cells in ~5 minutes, while iCMs may require up to ~9 minutes. Next, 2mL of RPMI 1640 basal medium with 10% fetal bovine serum (FBS) was added as a neutralizing medium to trypsin. Using a P1000 pipetman, remaining cells were washed off and transferred to a 15mL conical tube. Cells were pelleted by centrifugation at 300g for 5 minutes and subsequently resuspended in appropriate medium (mTeSR1 medium for iPS cells and RPMI 1640 basal medium with B-27 supplement complete with insulin for iCMs).   To seed cells at low density onto coverslips, cells were counted using an automatic cell counter (Bio-Rad TC20™) and 5×105 cells were seeded onto Matrigel-coated 25mm circular coverslips (Fisher Scientific #12-545-102) and placed in 6-well plates. Cells were incubated at  24 37oC with 5% CO2 for 24 hours and allowed to adhere. Subsequently, cells were washed with PBS to remove dead cells before fixation and permeabilization steps.   To fix and permeabilize cells, cells were treated with 3.7% formaldehyde in PBS for 15 minutes (Fisher Scientific #BP531-500), followed by 0.1% triton X-100 (Sigma-Aldrich #9002-93-1) for 5 minutes, both steps performed at room temperature. BSA diluted with PBS to 1% concentration, was added to cells for 20 minutes as a blocking buffer for unspecific antibody binding. Next, anti-α-actinin antibody (Sigma-Aldrich #A7811) was incubated with cells at a 1:800 dilution in PBS overnight at 4oC. Bound primary antibodies were visualized by applying cells with goat anti-mouse IgG, DyLight 633 conjugate (ThermoFisher Scientific #35513) diluted at 1:250 for 30 minutes at 4 oC. Cells were washed 3 times with PBS at every step. Finally, the samples were inverted, mounted onto glass slides using mounting reagent, ProLong Gold with 4’, 6-diamidino-2-phenylindole (DAPI) (Life Technologies #P-36931), and imaged using a fluorescent microscope (Olympus IX81 microscope system).   2.6 Flow cytometry Flow cytometry is a biotechnology that uses lasers for cell counting, cell sorting, and biomarker detection. Dissociated single cells are suspended in a fluid sheath and passed through an electronic detector that enables the simultaneous analysis of multiple physical (cell size and granularity) and biological (expression level of fluorescently labeled proteins) properties of cells.   Cells were harvested for flow cytometry using 0.05% trypsin as described in section 2.2 and transferred into disposable culture tubes. Fixation and permeabilization of cells were done by resuspending cells thoroughly in 250µL BD Cytofix/Cytoperm™ solution (BD Biosciences #554722) and left on a shaker for 20 minutes at 4oC. To avoid large cell aggregates, samples  25 were vortexed prior to and during addition of BD Cytofix/Cytoperm solution. Cells were washed 2 times with 1mL BD Perm/Wash™ buffer (BD Biosciences #554723) and centrifuged to obtain pellets. For short-term storage and use within 3 days, cell pellets were resuspended in 1mL of BD Perm/Wash buffer and stored at 4oC.  Samples were stained individually with primary antibodies, anti-cardiac troponin T (anti-cTnT) (ThermoFisher Scientific #MA5-12960), anti-α-actinin (Sigma-Aldrich #A7811), and anti-HCN4 cyclic nucleotide-gated channel (NeuroMab #73-150), at dilutions of 1:100, 1:800, 1:2 respectively for 30 minutes at 4oC. Secondary antibody, goat anti-mouse IgG, DyLight 633 conjugate (ThermoFisher Scientific #35513), was applied at a dilution of 1:250 and also incubated for 30 minutes at 4oC. Between each antibody-staining step, the cells were washed 2 times with BD Perm/Wash buffer. Cells in 200µL of BD Perm/Wash buffer, data were acquired automatically using the BD Accuri™ C6 flow cytometer, with acquisition criteria of 30,000 events set to a ‘slow’ flow rate. The data was saved onto a computer and analyzed using single-cell flow analysis package (FlowJo v10.2).  2.7 Fabrication of polydimethylsiloxane (PDMS) substrates with varying stiffness To create substrates of tunable elastic modulus, two blends of commercially available PDMS subtypes, Sylgard 527 and Sylgard 184 (Dow Corning) were used. This technique enabled us to fabricate substrates with predicted elastic modulus between 5kPa – 1.72MPa [55]. Sylgard 527 (soft substrate) was prepared as per manufacturer’s directions by mixing equal parts (by mass) of part A and part B in a plastic polypropylene cylinder under sterile condition. A plastic stirrer was used to ensure thorough mixing by stirring in a figure-eight motion for 5 – 7 minutes. Sylgard  26 184 (hard substrate) was also prepared as per manufacturer’s directions by mixing 10 parts base to 1 part of curing agent, using the same mixing technique. To fabricate substrates of intermediate stiffness, Sylgard 184 and 527 blends were first prepared as described above and then combined at mass ratio 1:5 – mixing was performed using the same technique. Once mixed, the PDMS was poured into 6-well plates to create ~1mm thick films (Figure 2.6 (A)). All PDMS substrates were placed into a vacuum chamber and degased for 30 minutes to create bubble-free samples. Subsequently, substrates were cured at room temperature for at least 48 hours prior to any experiments. Previous experiments have shown that this cure time and temperature was sufficient to cure PDMS fully and give consistent mechanical property [56]. Before cells were seeded onto PDMS substrates, they were pre-coated with Matrigel according to protocol described in section 2.1.1 (Matrigel coatings). Figure 2.6 (B) illustrate steps of the fabrication process and Figure 2.6 (C) shows an image of PDMS strips of varying stiffness clamped and freely suspended from a retort stand.            27                    Figure 2.6: Fabrication of PDMS substrates of tunable elastic modulus. (A) Graphic showing PDMS substrate fabricated at bottom of 6-wells. Soft, intermediate, and hard PDMS have predicted elastic modulus of ~5kPa, 130kPa, and 1.72MPa respectively. (B) Illustration of steps involved in PDMS fabrication process. (C) Image of PDMS substrates clamped and freely suspended.    (A)	PDMS	Substrate	(~1mm	thickness)	6-well	plate	So>:	~5kPa	Intermediate:	~130kPa	Hard:	~1.72MPa	BN4404 - BioMEMS, Yong Zhang, NUSPDMSWeighSylgard 184 is a two-component heat-curing system, i.e. it consist of a base part and a curing agent part. Take a common plastic cup and fill it with one part curing agent and ten parts of base (by weight). Start with the curing agent, since it is harder to pour the right amount of it! 7-10 g material will be sufficient for covering one template, e.g. 0.7 g curing agent and 7 g base. A small error in the amounts will not effect the final result though. MixUse a plastic spoon to mix the base and the curing agent. Mix it rather carefully for at least a few minutes, depending on the amount of material. When you mix it you will incorporate a lot of air in the solution. Don’t worry –it will be removed in the next step. Use a plastic spoon to mix it. DegasAfter the mixing the silicone mixture will be full of air bubbles and needs degassing. This is done in an exsiccator using vacuum. During the degassing the silicone expand and start to look like foam, this means that you can only have a small (<5 g) amount in each plastic cup else it will overflow. Also remove the spoon. When the silicone is completely clear and transparent it is finished. BN4404 - BioMEMS, Yong Zhang, NUSPDMSWeighSylgard 184 is a two-component heat-curing system, i.e. it consist of a base part and a curing agent part. Take a common plastic cup and fill it with one part curing agent and ten parts of base (by weight). Start with the curing agent, since it is harder to pour the right amount of it! 7-10 g material will be sufficient for covering one template, e.g. 0.7 g curing agent and 7 g base. A small error in the amounts will not effect the final result though. MixUse a plastic spoon to mix the base and the curing agent. Mix it rather carefully for at least a few minutes, epending n the amount of material. When you mix it ou will incorporate a lot of air in the solution. Don’t worry –it will be removed in the next step. Use a pl stic spoon to mix it. D gasAfter the mixing the silicone mixture will be full of air bubbles and needs degassing. This is done in an exsiccator using vacuum. During the degassing the silicone exp nd and start to look like foam, this means that you can only have a small (<5 g) amount in each plastic cup else it will overflow. Also remove the spoon. When the silicone is completely clear and transparent it is finished. BN4404 - BioMEMS, Yong Zhang, NUSPDMSWeighSylgard 184 is a two-component heat-curing system, i.e. it consist of a base part and a curing agent part. Take a common plastic cup and fill it with one part curing agent and ten parts of base (by weight). Start with the curing agent, since it is harder to pour the right amount of it! 7-10 g material will be sufficient for covering one template, e.g. 0.7 g curing agent and 7 g base. A small error in the amounts will not effect the final result though. MixUse a plastic spoon to mix the base and the curing agent. Mix it rather carefully for at least a few minutes, depending on the amount of material. When you mix it you will incorporate a lot of air in the solution. Don’t worry –it will be removed in the next step. Use a plastic spoon to mix it. DegasAfter the mixing the silicone mixture will be full of air bubbles and needs degassing. This is done in an exsiccator using vacuum. During the degassing the silicone expand and start to look like foam, this means that you can only have a small (<5 g) amount in each plastic cup else it will overflow. Also remove the spoon. When the silicone is completely clear and transparent it is finished. BN4404 - BioMEMS, Yong Zhang, NUSPDMSDispenseDispensing the silicone on to the template can be a bit tricky, as you do not want to trap air in the process. Sylgard 184 has relatively low viscosity, so the flow is no problem. It is also possible to make the viscosity even lower by mixing in silicone oil or h xane, but this is seldom necessary. Dispensing the materi l at the ce ter of the template from a low altitude minimizes the risk of trapped air. K ep the template horizontal duri g dispense. SpreadPick up the template with a pair of flat tweezers and start tilting it at  low angle. The material will now start to spread. By tilting it in different directions it is possibl  to cover the whole template by silicone. Try to make the shape as circular as possible. When you think it is ok, leave it for a minute in order to get a flatter top surface. Stamps thickness can range from ~0.1 to 5 mm or more. CuringSylgard 184 is heat curing. It is curable from less than room temperature to over 150°C. Sylgard 184 also has temperature dep ndant shrinkage as seen below. Curing in 140°C (~15 min) will make the st mp shrink almost exactly 3 %. Take the template and place it in a pre-heated oven. The time is not that critical, it is almost impossible to cure it a too long time. BN4404 - BioMEMS, Yong Zhang, NUSPDMSDispenseDispensing the silicone on to the template can be a bit tricky, as you do not want to trap air in the process. Sylgard 184 has relatively low viscosity, so the flow is no problem. It is also possible to make the viscosity even lower by mixing in silicone oil or hexane, but this is seldom necessary. Dispensing the material at the center of the template from a low altitude minimizes the risk of trapped air. Keep the template horizontal during dispense. SpreadPick up the template with a pair of flat tweezers and start tilting it at a low angle. The material will now start to spread. By tilting it in different directions it is possible to cover the whole template by silicone. Try to make the shape as circular as possible. When you think it is ok, leave it for a minute in order to get a flatter top surface. Stamps thickness can range from ~0.1 to 5 mm or more. CuringSylgard 184 is heat curing. It is curable from less than room temperature to over 150°C. Sylgard 184 also has temperature dependant shrinkage as seen below. Curing in 140°C (~15 min) will make the stamp shrink almost exactly 3 %. Take the template and place it in a pre-heated oven. The time is not that critical, it is almost impossible to cure it a too long time. BN4404 - BioMEMS, Yong Zhang, NUSPDMSDispenseDispensing the silicone on to the template can be a bit tricky, as you do not want to trap air in the process. Sylgard 184 has relatively low viscosity, so the flow is no problem. It is also possible to make the viscosity even lower by mixing in silicone oil or hexane, but this is seldom necessary. Dispensing the material at the center of the template from a low altitude minimizes the risk of trapped air. Keep the template horizontal during dispens . SpreadPick up the template with a pair of flat tweezers and start tilting it at a low angle. The material will now start to spread. By tilting it in different directions it is possible to cover the whole template by silicone. Try to make the shape as circular as possible. When you think it is ok, leave it for a minute in order to get a flatter top surface. Stamps thickness c n range from ~0.1 to 5 mm or more. CuringSylgard 184 is heat curing. It is cur ble from less than room temperature to over 150°C. Sylgard 184 also has temperature d p ndant shrinkage as seen below. Curing in 140°C (~15 min) will make the stamp shrink almost exactly 3 %. Take the template and place it in a pre-heated oven. The time is not that critical, it is almost impossible to cure it a too long time. Weigh		 Mix	 Degas	Di pense	 Spread	 Cure	(B)	(C)	Hard	PDMS	substrate	Intermediate	PDMS	substrate	So>	PDMS	substrate	Cells	 28 2.8 Statistical analysis Statistical significance between means was determined using independent samples t-tests followed by Levene’s test for equality of variances. IBM SPSS statistics software package (v. 22) was used for purpose of statistical analysis. Comparisons with P < 0.05 (*), P < 0.01 (**), and P < 0.001(***) were determined to be significant.   2.9 Fabrication of PDMS linear cardiac channel Linear rectangular cardiac channels of various dimensions were fabricated by casting PDMS into a 3D printed mold (see Figure 2.7 for design details). Rapid prototyping of 3D mold was performed using Asiga Pico 3D printer and designed using computer aided design (CAD) software SolidWorks (2016 student edition). Sylgard 184 PDMS was prepared as per manufacturer’s instructions by combining 10 parts base component with 1 part curing component. Subsequently, PDMS (still in liquid form) was poured into the 3D printed mold and then placed in a vacuum chamber for 30 minutes to eliminate air bubbles. PDMS samples were left to cure on a flat surface at room temperature for at least 48 hours before samples were removed from the mold.   The PDMS cardiac channel device consists of rectangular grooved linear channels 2mm in depth, 1mm in width, and variable lengths of 1, 2, 3, 5, 7, and 10mm.    29   Figure 2.7:  3D cardiac channel device. (A) 3D printed mold design. (B) 3D printed mold. (C) PDMS cardiac channel device. Channels have dimensions of 2mm depth, 1mm width, and variable lengths (1, 2, 3, 5, 7, and 10mm).  (B)	3D	printed	mold	   1      3      5      2      7      1      10   A AB BC CD DE EF F8877665544332211DRAWNCHK'DAPPV'DMFGQ.AUNLESS OTHERWISE SPECIFIED:DIMENSIONS ARE IN MILLIMETERSSURFACE FINISH:TOLERANCES:   LINEAR:   ANGULAR:FINISH: DEBURR AND BREAK SHARP EDGESNAME SIGNATURE DATEMATERIAL:DO NOT SCALE DRAWING REVISIONTITLE:DWG NO.SCALE:2:1 SHEET 1 OF 1A3WEIGHT: Cardiac Strip Channel Master Mold V3SOLIDWORKS Educational Product. For Instructional Use Only   1      3      5      2      7      1      10   A AB BC CD DE EF F8877665544332211DRAWNCHK'DAPPV'DMFGQ.AUNLESS OTHERWISE SPECIFIED:DIMENSIONS ARE IN MILLIMETERSSURFACE FINISH:TOLERANCES:   LINEAR:   ANGULAR:FINISH: DEBURR AND BREAK SHARP EDGESNAME SIGNATURE DATEMATERIAL:DO NOT SCALE DRAWING REVISIONTITLE:DWG NO.SCALE:2:1 SHEET 1 OF 1A3WEIGHT: Cardiac Strip Ch nnel Master Mold V3SOLIDWORKS Educational Product. For Instructional Use Only(A)	Mold	design	 Channel	length:	1mm	–	10mm	Channel	width:	1mm	Channel	depth:	2mm	(C)	PDMS	cardiac	channel	device	Top	view	 3D	view	 30 Chapter 3: Substrate Stiffness and Topography Accumulating evidence has linked physical parameters to various differentiation outcomes observed in stem cells [4, 27]. In this chapter, I sought to shed further light on this phenomenon in order to exploit potentially relevant mechanotransductive effects to accelerate maturation and / or enhance cellular subtype specification of iPS cell-derived cardiomyocytes. The effects of two specific forms of physical stimuli were explored: 1) substrate stiffness and 2) topography. Section 3.1 investigates the effects of substrate stiffness modulation applied with traditional ‘soluble factors’ on cardiac differentiation outcomes of iPS cells and section 3.2 evaluates the ability of freshly differentiated iPS cell-derived cardiomyocytes to be topographically manipulated to form three-dimensional cardiac tissues in millimeter-sized PDMS grooved linear channels.  3.1 Effects of substrate stiffness on cardiac differentiation To investigate the effects of substrate stiffness on iPS cell differentiation to cardiomyocytes, IMR90-4 iPS cells were seeded onto three PDMS substrates with variable elastic moduli, defined in this body of work into 3 categories: soft, intermediate, and hard. The PDMS substrates have predicted elastic modulus of 5kPa, 130kPa, and 1.72MPa respectively, based on previously published data [55]. Cells were also seeded onto unmodified tissue culture polystyrene (TCPS) plates for comparison.   Prior to iPS cell seeding, all substrate surfaces were pre-coated with Matrigel. iPS cells were maintained in mTeSR1 medium and grown on substrates to ~80% confluency (typically 3 – 4 days of culturing) before cardiac differentiation was initiated. Differentiation was carried out using the GSK inhibitor-Wnt inhibitor (GiWi) protocol, as described in section 2.1.5.  31 Spontaneous beating were observed on all substrates 8 days after the onset of differentiation. Figure 3.1 shows representative microscopy images and corresponding kymographs of beating clusters acquired. Visual inspection of clusters showed no obvious morphological differences between cardiomyocyte clusters generated on the various substrates. However, analysis of kymographs performed at five randomly selected clusters on each well revealed that iPS cells differentiated on soft, intermediate, hard PDMS, and TCPS have different average beat frequencies of (0.6 ± 0.1) Hz, (0.4 ± 0.1) Hz, (0.9 ± 0.2) Hz, and (0.6 ± 0.1) Hz respectively.  Nine days after the onset of differentiation, cells were harvested for flow cytometry assessment of cardiac troponin T (cTnT) and potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) expression. cTnT is a part of the contractile apparatus of cardiomyocytes and a known cardiac-specific marker indicative of cardiomyocyte maturity [59, 60]. HCN4 is used as a specific marker for the nodal cardiac cell subtype [57].   Flow cytometric analysis of iPS cells differentiated on substrates of variable stiffness revealed that the proportion of cells expressing high levels of cTnT (cTnT+ve cells) is increased for cells differentiated on hard PDMS substrates compared to those differentiated on soft and intermediate substrates (Figure 3.2). However, no statistical difference was found between all three PDMS substrates and the mechanically rigid TCPS. cTnT expression was quantified by averaging the proportion of cTnT+ve cells from triplicate experiments (n=3). Gating for cTnT+ve cells was set up using non-differentiated iPS cells as negative control.  The enhanced proportion of cells expressing high levels of cTnT on iPS cells differentiated on hard PDMS substrates compared to soft and intermediate substrates suggest a possible stiffness-dependency of in vitro cardiac differentiation of stem cells. Inferring a step  32 further, the perceived increased expression of cTnT may be indicative of accelerated maturation of iCMs generated on stiffer substrates compared to softer substrates of the same material.   Finally, my investigation into the effect substrate stiffness on cardiomyocyte subtype generation (i.e. nodal myocytes vs. working myocytes) suggested that the proportion of cells expressing high levels of HCN4 (HCN4+ve cells), decreased with increasing PDMS stiffness (Figure 3.3). However, these results were not statistically significant for all the substrate conditions compared.    33  Figure 3.1: Morphological characterization of cardiomyocytes derived from iPS cells cultured and differentiated on various substrates. As shown are representative microscopy images of the time-lapse videos acquired on day 8. The dotted red line and arrowhead indicates the kymograph tracings of the spontaneously beating clusters for cells cultured on (A) Soft PDMS, (B) Intermediate PDMS, (C) Hard PDMS and (D) unmodified TCPS. The calculated average beat frequencies (Hz) and scale bars are as indicated. (A)	 					So'	PDMS	substrate	0	10	5	(B)	 					Int.	PDMS	substrate	 ~0.4Hz	(C)	 					Hard	PDMS	substrate	0	10	5	~0.6Hz	0	10	5	~0.9Hz	(D)	 					TCPS	substrate	0	10	5	~0.6Hz	 34                      Figure 3.2: Flow cytometry analysis for cTnT expression of cardiomyocytes differentiated from iPS cells on substrates of variable stiffness. (A) Representative flow cytometry density plots (side scatter vs. cTnT) for cells harvested 9 days following onset of GiWi differentiation on the indicated substrates. Non-differentiated iPS cells is included as a negative control. (B) Proportion of cells that are cTnT+ve following GiWi differentiation on indicated substrates. As plotted is the averaged data of triplicate experiments conducted at the same time. Error bars denote the standard deviation.  PDMS	substrates	***	*	n.s.	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS										Propor%on	of	cTnT+	Cells	(%)	i 			 t l		 I t.	 	 	PDMS	substrates	*	**	n.s.	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS	**	cTnT	iPS	cells	 So,	PDMS	 Int.	PDMS	Hard	PDMS	 TCPS	cTnT	cTnT	(A)	(B)	*     p<0.05 **   p<0.01  *** p<0.001  n.s. non significant   35                                 Figure 3.3: Flow cytometry analysis for HCN4 expression of cardiomyocytes differentiated from iPS cells on substrates of variable stiffness. (A) Representative flow cytometry density plots (side scatter vs. HCN4) for cells harvested 9 days following onset of GiWi differentiation on the indicated substrates. Non-differentiated iPS cells is included as a negative control. (B) Proportion of cells that are HCN4+ve following GiWi differentiation on indicated substrates. As plotted is the averaged data of triplicate experiments conducted at the same time. Error bars denote the standard deviation. HCN4	iPS	cells	 So.	PDMS	 Int.	PDMS	Hard	PDMS	 TCPS	HCN4	HCN4	PDMS	substrates	n.s.	PDMS	substrates	n.s.	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS	0	10	20	30	40	50	60	70	80	Propor%on	of	cTnT+	Cells	(%)	iPSC		Neg	Ctrl	So6	 Int.	 Hard	 TCPS										Propor%on	of	cTnT+	Cells	(%)	i 			 t l		 I t.	 	 	0	10	20	30	40	50	60	Propor%on	of	HCN4+ve	Cells	iPSC		Neg	Ctrl	So4 Int.	 Hard	 TCPS	0	10	20	30	40	50	60	Propor%on	of	HCN4+ve	Cells	l	So4	 Int.	 Hard	 TCPS	0	10	20	30	40	50	60	Propor%on	of	HCN4+ve	Cells	iPSC		Neg	Ctrl	So4	 In S								Propor%on	of	HCN4+ve	Cells	iPSCeg	Ctrl	4	 Int.	 Hard TCPS	(A)	(B)	n.s. non significant   36 3.2 Topographical manipulation of iPS cell-derived cardiomyocytes I tested the ability of freshly differentiated iPS cell-derived cardiomyocytes (iCMs) to be topographically manipulated to form uniaxial contracting 3-dimensional (3D) cardiac tissues. First, linear PDMS channels (see section 2.8 for channel design and dimensions) were pre-coated with a thin layer of Matrigel (see section 2.1.1 for detailed Matrigel coating procedure). Subsequently, twelve-day-old iCMs generated using the GiWi protocol were dissociated and re-seeded into the PDMS channel using a P200 pipetman. Due to limitations in the fabrication process (i.e. damage to other channels), only the 1mm × 10mm channel was used in experiments. One million iCMs were seeded into the millimeter-sized (width=1mm, length=10mm, depth=2mm) PDMS linear channel and maintained for 4 days in RPMI / B-27 supplement (complete with insulin) medium for cells to adhere and remodel.  Figure 3.4 shows the result of iCMs cultured in the channel after 4 days. In that time, iCMs remodeled, restructured, and reorganized to form a single 3D cardiac tissue, exhibiting spontaneous uniaxial self-contractions along the longitudinal axis of the channel. Kymograph analysis indicated a beat frequency of ~0.9Hz. Results in this section revealed remarkable plasticity of freshly differentiated iCMs to be manipulated topographically and also provided a relatively simple method for generating millimeter-sized 3D cardiac tissues in vitro.  37                  Figure 3.4: Microscopy images of 12 day-old iCMs cultured in 1mm × 10mm linear PDMS channel for 4 days.  (A) Image showing single 3D cardiac tissue exhibiting spontaneous uniaxial contraction along longitudinal axis of channel. (B) Tail end of 3D cardiac tissue at increased magnification. Green dotted box indicates area of analysis presented in (C). (C) Area of analysis indicating cardiac tissue in relaxed state and contracted state. Annotations shows reference line (red dotted line) and maximum displacement of the cardiac tissue (orange arrow). (A)	(B)	Uniaxial		contrac,on	(C)			Ref	line	Relaxed	state	 Contracted	state	50μm	 50μm	Displacement	 38 3.3 Chapter discussion In this section, the significance of the above-described findings are interpreted and discussed.  3.3.1 Effects of substrate stiffness on cardiac differentiation  Organogenesis is a delicate process that is sensitive to physical parameters such as substrate stiffness [58]. In this chapter, I showed that iPS cells differentiated on PDMS substrates of varying elastic moduli yield different phenotypic outcomes. Most striking was the statistically significant increase in cTnT expression, a cardiac specific marker; in cells differentiated on hard PDMS substrates (~1.72MPa) compared to those differentiated on soft (~5kPa) and intermediate (~130kPa) PDMS substrates. This finding suggests a synergistic effect between substrate stiffness and traditional biochemical cues in accelerating the maturation of iPS cell-generated cardiomyocytes. This dependency of stem cell differentiation on substrate stiffness could potentially be exploited to yield functionally mature cardiomyocytes in a shorter timeframe, increasing throughput and scalability of existing human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) platforms. However, with no statistical significance between PDMS substrates and conventional polystyrene material, it remains to be seen whether a substrate with controlled stiffness has indeed an added advantage over traditional tissue cultureware. That said, it should be noted that only three variations of substrate stiffness were considered in this body of work and results could potentially yield statistical significance after optimization.    Sustaining the above notion that iCMs generated on hard PDMS enhances maturity compared to soft and intermediate stiffness substrates, results from kymograph analysis showed clusters on hard substrates contracting at rates of (0.9±0.2) Hz, which is in the range similar to human adult cardiomyocytes (~1.0 Hz) [4]. Clusters on soft and intermediate PDMS substrates  39 on the other hand were contracting at (0.6±0.1) Hz and (0.4±0.1) Hz respectively, which are marginally slower than that of adult native cardiomyocytes. Although, due to numerous confounding factors, beat frequency is often regarded as an inapt indicator of cardiomyocyte maturity when considered in isolation [59]. That said, it should be noted that when analyzed in conjunction with other measures of maturity, like the expression of cardiac specific markers (e.g. cTnT), beat frequency gives a meaningful quantification of functional property of cardiomyocyte clusters.  Finally, the lack of existing hPSC-CM systems capable of generating subtype specific cardiomyocytes (nodal myocytes vs. working myocytes) also motivated my investigation to explore a possible link between substrate stiffness and cardiomyocyte subtype specification. Flow cytometry analysis of iPS cells differentiated on substrates of various stiffness in a 9-day timespan revealed no significant change in HCN4 expression (nodal cell specific marker) between samples. As such, despite the clear inverse trend between substrate stiffness and HCN4 expression (i.e. proportion of cells that were HCN4+ve decreased with increasing substrate stiffness), no definitive conclusion was reached with regards to the effects of substrate stiffness on cardiomyocyte subtype specification. That said, 9 days might be too short a timeframe for freshly differentiated iCMs to adequately diverge CM subtype lineage and develop detectable differences in subtype specific markers (the differentiation protocol itself takes 7 days to complete). On that note, future experiments would certainly benefit from some level of optimization in order to determine an ideal time point for cell harvest and analysis.   40 3.3.2 Topographical manipulation of iPS cell-derived cardiomyocytes Despite the recent developments of hPSC-CM based engineered heart tissue platforms for drug development and disease modeling studies, cost and complexity of these systems are still of paramount concern [4]. In this chapter, I sought to address the complexity issue of current systems by proposing a straightforward PDMS-based ‘cardiac channel’ platform for generating functionally relevant engineered cardiac tissues. My investigation revealed that iCMs seeded in linear channels could be topographically manipulated to form millimeter-sized, uniaxially contracting, single cardiac strips. Compared to existing hPSC-CM engineered heart tissue strategies, my method is rapid (cardiac strips form after 4 days from iCM seeding), does not require the use of structural anchors / support, mechanical or electrical stimulation, perfusion bioreactors, or other conditions that might complicate future clinical translation. Instead, I utilized only the topography provided by channel walls to direct tissue formation, and optimized seeding density to develop a highly efficient iCM-based in vitro 3D heart tissue system.  With this proof-of-concept PDMS-fabricated device, I have demonstrated a scalable methodology to generate functional human cardiac tissues with clinical relevance (millimeter-sized). The simplicity of the system and its ability to rapidly generate functionally matured tissues in vitro certainly provides grounds for further development and investigation of this technology.      41 Chapter 4: Co-Culture Cardiac Differentiation Various types of stem cells and non-stem cells have been shown to differentiate / transdifferentiate into cardiomyocytes by way of co-culture with appropriate inducer cells (see section 1.7 for more background detail on inductive co-cultures). However, distinctively lacking from current literature is a co-culture induction system utilizing stem cell-derived cardiomyocytes as a stimulatory source for cardiac reprogramming (of stem cells or otherwise).  In this chapter, I sought to address this knowledge gap by investigating the potential of a novel cardiac differentiation method based exclusively on co-culture with previously differentiated iCMs as a cardiac inducer for iPS cells.  4.1 Co-culture cardiac differentiation of fluorescent dye labeled-IMR90-4 iPS cells To test my hypothesis that non-differentiated iPS cells can be differentiated into cardiomyocytes through co-culture incubation with iCMs, I co-cultured non-differentiated IMR90-4 iPS cells with iCMs derived from 30 – 45 days old GiWi-differentiated IMR90-4 (age of iCMs is calculated from onset of CHIR99021 treatment). To do this, I seeded cells either sequentially (Pre-culture) or simultaneously (Bi-culture) in Matrigel-coated 6-wells. To enable tracking of cells differentiated as a consequence of the co-culture, non-differentiated iPS cells were labeled with CellTracker™ Red, a fluorescent dye that facilitates tracking of the labeled cell through three to six divisions for up to eleven days. Figure 4.1 shows a graphic illustrating the cell tracking procedures used. Successful reprograming of iPS cells to cardiomyocytes was assessed on the basis of fluorescent-labeled cells exhibiting spontaneous self-contractions.   42   Figure 4.1: Graphic of cell tracking procedure with CellTracker red dye.  Non-differentiated iPS cells were incubated in red dye for 30 minutes at a dye concentration of 5µM while previously GiWi-differentiated iCMs were unlabeled. For simultaneous bi-cultures, the two cell types were mixed and seeded at the same time while for pre-cultures; the fluorescent-labeled iPS cells were seeded two days prior to the unlabeled GiWi-differentiated iCMs.  4.1.1 Pre-culture method for co-culture differentiation of iPS cells As illustrated in Figure 4.2 (pre-culture workflow schematic), pre-cultures were prepared by first labeling iPS cells in Matrigel-coated 6-wells with the CellTracker red fluorescent dye, 2 days prior to adding 3×106  iCMs to the culture. iCMs were then harvested using 0.05% trypsin and neutralized using RPMI 1640 basal medium with 10%. FBS. Pre-cultures were then maintained in RPMI 1640 basal medium with B-27 supplement (minus insulin) for 7 days with medium renewed on days 1, 3, and 5 respectively. Day 0 was defined as the day iCMs were added to iPS cell culture. On day 7, the medium was changed to RPMI 1640 basal medium with B-27 Figure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after s eding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptStep	1:	Label	non-differen3ated	iPS	cells	with	CellTracker	and	generate	iCMs	from	non-labeled	iPS	cells	iPS	Cells	 iCMs	(GiWi-differen3ated	from	iPS	cells)	Unlabeled	Figure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoi s were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in P C 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptStep	2:	Mix	2	cell	types	(Bi-culture)	 or	 Seed	iPS	cells	followed	by		iCMs	2	days	later	(Pre-culture)	Figure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript+	Figure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue n clear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dy , respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye con entration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dil tion of DAPI. For Si ultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after see ing the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in gr en cytoplasmic dy , redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluoresc nce microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for , cells we e i cuba ed for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes we e seededtwo days prior to the myocytes. Organoids were im ged by fluorescence microscopy odays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCar iac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded o to microchannels, while for Pre-culture, the non-m ocytes were see edtwo days prior to the myoc tes. Organoids were imag d by fluore cence microscopy ondays 1 and 4 after se ding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear dye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-cultures, the three cell types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes were seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscop  ndays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Auth  manuscript; available in PMC 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Cell Tracking ProcedureCardiac FB, D4T EC, and neonatal rat CM were incubated in green cytoplasmic dye, redcytoplasmic dye, and blue nuclear ye, respectively. For FB and EC, cells were incubatedfor 30 minutes at a dye concentration of 5 µM, while for CM, cells were incubated for 1hour in a 1:5 dilution of DAPI. For Simultaneous Tri-c ltures, h  thr e c ll types weremixed and seeded onto microchannels, while for Pre-culture, the non-myocytes ere seededtwo days prior to the myocytes. Organoids were imaged by fluorescence microscopy ondays 1 and 4 after seeding the CM.Iyer et al. Page 13J Tissue Eng Regen Med. Author manuscript; available in P C 2010 March 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript 43 supplement (complete with insulin) and pre-cultures were imaged a day later under a fluorescent microscope. On that same day, pre-cultures were harvested and reseeded at low density (5×105 cells) onto 25mm Matrigel-coated glass cover slips placed in 6-wells. Cells were incubated for 24 hours to give cells sufficient time to adhere. The next day, cells on the glass slips were imaged.    Figure 4.2: Workflow schematic of pre-culture experiments. iPS cells were labeled with CellTracker red in culture two days before onset of co-culture. The non-labeled iCMs are derived from 30 – 45 day old GiWi-differentiated iPS cells. iCMs are dissociated and added to labeled-iPS cells still in culture at d0 to initiate the co-culture.   Microscopy images of pre-cultures on day 8 revealed large spontaneously contracting clusters (Figure 4.3A) with fluorescently labeled cells integrated into the cardiac construct (Figure 4.3B). A kymograph analysis was performed on the corresponding contracting clusters (Figure 4.3C). The periodic perturbations (‘spikes’) in the kymograph are indicative of rhythmic motion, characteristic of spontaneous cellular contractions.   As alluded to in preceding paragraph, since fluorescently labeled cells were observed to be integrated into the contracting tissue, it could not be determined if those cells have been mTeSR1	 RPMI	with	B27	minus	insulin	 RPMI	with	B27	+	insulin	d-4	iPSC	seed	d-2	Red	dye	d0	Add	iCMs	d1	Basal	medium	 d7	d3	Basal	medium	d5	Basal	medium	Day 0 Day 4 Day 5 Day 9 Day ??Activin A BMP4 Begin cyclic strainFigure 4.7: A schematic of the differentiation protocol used to generate cardiomy cytes fromiPSCs. Cells are seeded on Matrigel on Day 0 and reach 90% conflue ce by Day 4.Activin A (100ng/mL) is applied for 24 hours, followed by 4 days of BMP4 trea ment(10ng/mL) without medium change. After BMP4 treatment, 5% cyclic strain is appliedcontinously at 1Hz using the HexCycler.Figure 4.8: A: A spontaneously beating cluster of cardiomyocytes. The yellow line indicatesthe region used for kymograph analysis. B: Kymograph analysis gives a beat frequencyof 0.35Hz.tus, as shown by immunocytochemistry. Figure 4.9 shows staining of iPSC-derived cardiomyocytes.Cell nuclei are stained blue by DAPI, which binds to regions of DNA rich in adenine and thymine.The proteins of interest are stained green. Those nuclei not surrounded by green belong to non-cardiomyocytes, most likely cells that have differentiated to a cardiac fibroblast lineage. Both theunstrained and strained cells stained positively for cTnT, but the strained cells exhibited punctatestaining while the unstrained cells had more filamentous staining. Sarcomeric striations revealedby ↵-actinin staining were common in the unstrained specimen, but much rarer in the unstrainedspecimen. This suggests that the strain regimen hindered the maturation of these cardiomyocytes.46iCMs	added	to	iPSC	culture	d8	Pre-culture	wells	imaged	d9	Low	density	glass	slips	imaged	 44 successfully differentiated to iCMs or were just being tugged along by the contracting cluster. As such, to obtain definitive visual confirmation that iPS cells have been successfully reprogrammed to functional cardiomyocytes, single cell spontaneous self-contractions needed to be observed in fluorescently labeled cells. To do this, cells were harvested and reseeded at low-density onto glass slips and then imaged again the following day. This imaging at low cell density allowed the visualization of single cells and revealed the presence of fluorescently labeled self-contracting isolated single cells. Figure 4.3D shows an example of a fluorescent labeled cell spontaneously contracting at a beat frequency of ~0.5Hz (cell 1). This documentation had provided observational evidence that co-culture iPS cells with iCMs in a pre-culture format were sufficient as a stimulatory system to initiate cardiac differentiation in some iPS cells. Importantly, this differentiation occurred without the use of exogenously added inhibitors or morphogens, other than presumed ‘natural’ factors produced by the iCMs or by the iPS cells in response to the interaction.   Imaging at multiple locations also revealed non-fluorescent contracting cells, presumably iCMs that were added to the culture to initiate differentiation on day 0. Figure 4.3E shows an example of a non-fluorescent cell contracting at a beat frequency of ~1Hz (cell 3). Cells 2 and 4 were included in the analysis as negative controls and show examples of non-contracting cells with straight lined kymograph profiles.    45                   Figure 4.3: Fluorescent images and kymographs of pre-cultures. (A) DIC image of pre-culture on day 8 (scale bar: 500µm). (B) Inset of red box from image (A) - overlay of DIC and TRITC images (scale bar: 100µm). (C) Kymograph of contracting cluster in image (A) - arrow indicates direction of corresponding line scan (scale bar: 50µm). (D) Overlay image of pre-culture at low density showing fluorescently labeled cell exhibiting spontaneous self contraction – cell 1 (scale bar: 50µm). (E) Overlay image of pre-culture at low density showing non-fluorescently labeled cell exhibiting spontaneous self contraction – cell 3 (scale bar: 50µm).     (F) Corresponding kymographs of cells 1, 2, 3, and 4 (scale bar: 5µm). (A)	 (B)	0	20	10	5	15	Pre-culture	wells	imaged	on	day	8	(D)	 (F)	(E)	0	20	10	5	15	Pre-culture	seeded	at	low-density	on	glass	slips	imaged	on	day	9	0	20	10	5	15	(C)	0	20	10	5	15	0	20	10	5	15	1	 2	3	4	1	 2	3	 4	~0.7Hz	~0.5Hz	~1.0Hz	Non	contrac4ng	Non	contrac4ng	 46 4.1.2 Bi-culture method for co-culture differentiation of iPS cells Similar to the pre-culture method, bi-cultures were prepared by labeling non-differentiated iPS cells with the CellTracker fluorescent red dye, 2 days prior to mixing the iPS cells with 3×106 non-labeled iCMs. However, unlike pre-cultures, where iCMs were added sequentially to iPS cells, bi-cultures were prepared by mixing iCM and non-differentiated iPS cell suspensions together, before seeding the combined suspension to culture wells.   To prepare bi-cultures, iPS cells and iCMs were harvested at the same time using 0.05% trypsin. Next, cells were neutralized with RPMI basal medium + 10% FBS, and centrifuged at 300g to obtain pellets. Subsequently, individual pellets were resuspended in RPMI basal medium + B-27 supplement (minus insulin) and then counted using an automatic cell counter. 3×106  iCMs were mixed with iPS cells to form a combined cell suspension consisting of fluorescently labeled iPS cells and non-fluorescently labeled iCMs. Rock inhibitor was added to the suspension to a final concentration of 10µM and the combined cell suspension was mixed by vortexing on medium speed for 30 – 45 seconds. This vortexing step was critical in ensuring thorough mixing of the two cell types. The combined cell suspension was then dispensed onto Matrigel-coated 6-wells.    Bi-cultures were maintained in RPMI/B-27 supplement (minus insulin) medium for 7 days and then switched to RPMI/B-27 supplement (complete with insulin) medium. Medium renewals were carried out at the same intervals as pre-culture experiments. Microscopy analysis of bi-cultures was also performed according to protocols described in the pre-culture section. Figure 4.4 illustrates the workflow of bi-culture experiments.   47  Figure 4.4: Workflow schematic of bi-culture experiments. iPS cells were labeled with CellTracker red in culture two days before onset of co-culture. The non-labeled iCMs are derived from 30 – 45 day old GiWi-differentiated iPS cells. Both iCMs and labeled-iPS cells are dissociated, mixed and reseeded onto freshly prepared Matrigel coating at d0 to initiate the co-culture.   Microscopy analysis of bi-culture on day 8 revealed large spontaneously contracting cell clusters (Figure 4.5A). Kymograph analysis of these clusters indicated an average beat frequency of ~1.9Hz (Figure 4.5C). Overlay images (DIC + TRITC) of these contracting clusters at higher magnification showed fluorescently labeled cells integrated into cardiac constructs (Figure 4.5B). As such, in order to verify if iPS cells were successfully differentiated to iCMs, cells were harvested and reseeded at low-density (5×105 cells per 6-well) onto Matrigel-coated glass cover slips for observation of cells in their isolated state.   Upon thorough visual scanning of low cell density glass slips, no fluorescently labeled contracting cells were found. Instead, all clearly fluorescent cells were either non-contracting (Figure 4.5D – cell 1) or attached to a non-fluorescent contracting cell and being tugged along (Figure 4.5E – cell 2). As such, it was unclear if differentiation of iPS cells via bi-culture incubation with iCMs had successfully led to iPS cells being reprogrammed to cardiomyocytes. mTeSR1	 RPMI	with	B27	minus	insulin	 RPMI	with	B27	+	insulin	d-4	iPSC	seed	d-2	Red	dye	d0	Mix		iCM	+	iPSC	d1	Basal	medium	 d7	d3	Basal	medium	d5	Basal	medium	iCMs	and	iPSCs	mixed,	centrifuged	and	seeded	+	mix	d8	Bi-culture	wells	imaged	d9	Low	density	glass	slips	imaged	 48 Instead, all spontaneously self-contracting cells observed in the bi-cultures were non-fluorescently labeled (Figure 4.5F – cell 3). Figure 4.5F included a kymograph analysis of a non-fluorescent non-contracting cell (cell 4) as a negative control.   49                   Figure 4.5: Fluorescent images and kymographs of bi-cultures. (A) DIC image of bi-culture on day 8 (scale bar: 500µm). (B) Inset of red box from image (A) - overlay of DIC and TRITC images (scale bar: 100µm). (C) Kymograph of contracting cluster in image (A) - arrow indicates direction of corresponding line scan (scale bar: 50µm). (D) Overlay image of bi-culture at low density showing example of non-contracting fluorescent labeled cell (cell 1) (scale bar: 50µm). (E) Overlay image of bi-culture at low density showing fluorescently labeled cell (cell 2) tugged along by contracting cell (scale bar: 50µm). (F) Overlay image of bi-culture at low density showing non-fluorescently labeled cell exhibiting spontaneous self-contraction (cell 3) (scale bar: 50µm). (G) Corresponding kymographs of cells 1, 3, and 4 (scale bar: 5µm). Non	contrac)ng	(A)	 (B)	0	20	10	5	15	Bi-culture	wells	imaged	on	day	8	(D)	(G)	(F)	0	20	10	5	15	Bi-culture	seeded	at	low-density	on	glass	slips	imaged	on	day	9	0	20	10	5	15	(C)	0	20	10	5	15	0	20	10	5	15	1	3	4	1	2	3	 4	~1.9Hz	~1.7Hz	Non	contrac)ng	Non	contrac)ng	(E)	2	 50 4.2 Co-culture cardiac differentiation of GFP-tagged AICS-0016 iPS cells As documented in section 4.1 (Co-culture cardiac differentiation of fluorescent dye labeled-IMR90-4 iPS cells), incubation of iPS cells with iCMs in a pre-culture format was sufficient as a stimulatory system to initiate cardiac differentiation in iPS cells. As such, to further investigate the robustness of this novel differentiation method, I extended its application to include a different iPS cell line (AICS-0016).  The AICS-0016 iPS cell line is a human clonal line in which the beta-actin proteins in cells are GFP tagged, allowing the visualization and tracking of AICS-0016 cells both spatially and temporally. The inclusion of AICS-0016 into my co-culture experiments was intended to address two potential issues from the previous fluorescent dye labeling system. 1) Fluorescent dyes degrade and its brightness diminishes over time due to cell metabolic breakdown. This limits how long co-culture assays could be conducted (i.e. assays limited to within an 11 day time period as fluorescent output from the dye becomes undetectable after that timeframe). 2) Fluorescent dyes might ‘leak’ from cells (although very unlikely) and be unintentionally incorporated into adjacent non-stained cells. This might compromise the validity and quality of data obtained. As such, the use of AICS-0016 cells, an endogenously GFP-tagged cell line, in my co-culture studies serves not only to answer the question of whether my novel co-culture differentiation method works on multiple iPS cell lines, but also serves to supplement and support the findings from the previous section.     51 4.2.1 Validation of fluorescence in AICS-0016 iPS cell line To determine if the GFP signal from AICS-0016 cell line can be accurately measured by flow cytometry, AICS-0016 iPS cells were harvested with 0.05% trypsin, fixed and permeabilized with BD Cytofix / Cytoperm kit, and then analyzed alongside IMR90-4 iPS cells. Figure 4.6 illustrates the gating scheme used for the 2 cell types (Figure 4.6A and B) and their corresponding GFP expression in a histogram plot (Figure 4.6C). Flow analysis revealed ~99.8% of AICS-0016 iPS cells were GFP positive (GFP+ve) with respect to the IMR90-4 iPS cells, with histograms exhibiting a distinct peak shift.              Figure 4.6: Flow cytometry analysis of IMR90-4 iPSCs and AICS-0016 iPSCs. (A) Gating scheme for IMR90-4 iPS cells. (B) Gating scheme for AICS-0016 iPS cells. (C) Histogram plot for GFP expression (IMR90-4 vs. AICS-0016).  Cells30.90 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-HCells25.80 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-HIMR90-4 iPS cells AICS-0016 iPS cellsGFP +99.8100 102 104 106FL1-A :: FL1-A03006009001.2KCountSample Name Subset Name CountA01 IMR90 iPSC.fcs Cells 18089 A02 AICS16 b actin iPSC.fcs Cells 19330 Forward	sca*er	area	(FS-A)	(A)	 (B)	IMR90-4	iPS	cells	 AICS-0016	iPS	cells	GFP	Sample	IMR90-4	iPSCs	AICS-0016	iPSCs	(C)	Cells30.90 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-HCells25.80 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-H100 102 104 106FL1-A :: FL1-A03006009001.2KCountSample Nam Subset Name CountA02 AICS16 b actin iPSC.fcs Cells 19330 A01 IMR90 iPSC.fcs Cells 18089 GFP+0100 102 104 106FL1-A :: FL1-A01.0M2.0M3.0M4.0MSSC-A :: SSC-AGFP+99.8100 102 104 106FL1-A :: FL1-A01.0M2.0M3.0M4.0MSSC-A :: SSC-AIMR90 iPSC AICS16 iPSCIMR90 iPSC AICS16 iPSCCells30.90 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-HCells25.80 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-H100 102 104 106FL1-A :: FL1-A03006009001.2KCountSample Name Subset Name CountA02 AICS16 b actin iPSC.fcs Cells 19330 0  IMR90 iPS .fcs Cells 18089 GFP+0100 102 104 106FL1-A :: FL1-A01.0M2.0M3.0M4.0MSSC-A :: SSC-AGFP+99.8100 102 104 106FL1-A :: FL1-A01.0M2.0M3.0M4.0MSSC-A :: SSC-AIMR90 iPSC AICS16 iPSCIMR90 iPSC AICS16 iPSCCells30.90 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-HC lls25.80 5.0M 10M 15MFSC-A :: FSC-A05.0M10M15MFSC-H :: FSC-H100 102 104 106FL1-A :: FL1-A03006009001.2KCountSample Name Subset Name CountA02 AICS16 b actin iPSC.fcs Cells 19330 A01 IMR90 iPSC.fcs Cell  18089 GFP+0100 102 104 106FL1-A :: FL1-A01.0M2.0M3.0M4.0MSSC-A :: SSC-AGFP+99.8100 102 104 106FL1-A :: FL1-A01.0M2.0M3.0M4.0MSSC-A :: SSC-AIMR90 iPSC AICS16 iPSCIMR90 iPSC AICS16 iPSCGFP+ve	99.8	 52 4.2.2 Co-culture of AICS-0016 iPS cells with IMR90-4 iCMs via pre-culture method As concluded from co-culture experiments with fluorescent dye-labeled iPS cells, incubation of iPS cells with iCMs using a pre-culture design was successful in inducing cardiac differentiation. However, the same could not be said for iPS cells co-cultured with iCMs in a bi-culture manner. As such, the pre-culture option was chosen as the co-culture method to initiate cardiac differentiation of AICS-0016 iPS cells.  Pre-cultures of AICS-0016 iPS cells with IMR90-4 iCMs were carried out as follows. AICS-0016 iPS cells were seeded onto Matrigel-coated wells and maintained in mTeSR1 medium for 4 days to ~80 – 90% confluence. Next, 30 – 45 days old IMR90-4 iCMs were harvested and added to the AICS-0016 iPS cell culture in a pre-culture manner. The combined pre-culture was then maintained in RPMI + B-27 (minus insulin) medium for 7 days with medium renewal carried out on days 1, 3, and 5. On day 7, the medium was switched to RPMI + B-27 (complete with insulin) and maintained by renewing the medium every 3 days. Figure 4.7 outlines a schematic of the pre-culture incubation scheme used to initiate cardiac differentiation of AICS-0016 iPS cells.   Figure 4.7: Schematic of pre-culture method used to initiate cardiac differentiation of AICS-0016 iPS cells.  mTeSR1	 RPMI	with	B27	minus	insulin	 RPMI	with	B27	+	insulin	d-4	AICS-16	iPSC	seed	d0	Add	IMR-90	iCMs	d1	Basal	medium	 d7	d3	Basal	medium	d5	Basal	medium	Day 0 Day 4 Day 5 Day 9 Day ??Activin A BMP4 Begin cyclic strainFigure 4.7: A schematic of the differentiation protocol used to generate cardiomyocytes fromiPSCs. Cells are seeded on Matrigel on Day 0 and reach 90% confluence by Day 4.Activin A (100ng/mL) is applied for 24 hours, followed by 4 days of BMP4 treatment(10ng/mL) without medium change. After BMP4 treatment, 5% cyclic strain is appliedcontinously at 1Hz using the HexCycler.Figure 4.8: A: A spontaneously beating cluster of cardiomyocytes. The yellow line indicatesthe region used for kymograph analysis. B: Kymograph analysis gives a beat frequencyof 0.35Hz.tus, as shown by immunocytochemistry. Figure 4.9 shows staining of iPSC-derived cardiomyocytes.Cell nuclei are stained blue by DAPI, which binds to regions of DNA rich in adenine and thymine.The proteins of interest are stained green. Those nuclei not surrounded by green belong to non-cardiomyocytes, most likely cells that have differentiated to a cardiac fibroblast lineage. Both theunstrained and strained cells stained positively for cTnT, but the strained cells exhibited punctatestaining while the unstrained cells had more filamentous staining. Sarcomeric striations revealedby ↵-actinin staining were common in the unstrained specimen, but much rarer in the unstrainedspecimen. This suggests that the strain regimen hindered the maturation of these cardiomyocytes.46IMR-90	iCMs	added	to		AICS-16	iPSC	culture	Medium	renewed	every	3	days	 53 4.2.3 Verification of spontaneous self-contraction in co-cultured AICS-0016 cells Cells exhibiting spontaneous self-contraction are considered by researchers as one of the main functional attributes indicating successful differentiation of stem cells into cardiomyocytes [21]. As such, to obtain observational evidence of spontaneous beating in AICS-0016 cells, a thorough microscopy analysis was performed on the co-cultures. Images were acquired using the Olympus IX81 inverted fluorescence microscope system with exposures for DIC and GFP images set at 50ms and 500ms respectively. Images were analyzed using image analysis software, ImageJ.   Image analysis of co-cultures carried out on day 23 revealed extensive networks of spontaneously contracting clusters (Figure 4.8A) with GFP+ve AICS-0016 cells integrated into and concentrated at contracting nodes (Figure 4.8B). A kymograph analysis performed on one of these beating clusters indicated a beat frequency of 1.7Hz (Figure 4.8C). In the interest of detecting spontaneous self-contractions of AICS-0016 cells in an isolated single cell manner, co-cultures were harvested on day 25 using trypsin and reseeded at low density (5×105 cells per 6-well) onto 25mm Matrigel-coated glass slips. On day 26, glass slips were imaged again at higher magnification.   Images acquired of glass slips revealed the presence of GFP+ve isolated single cells exhibiting spontaneous self-contraction (Figure 4.8D). Kymograph analysis shows a beat frequency of 1.4Hz for one such cell (Figure 4.8F – cell 1). This observation of GFP+ve cells initiating spontaneous self-contraction provides supporting evidence for the hypothesis that incubation of iPS cells in a pre-culture manner with iCMs constitutes a sufficient system to induce cardiac differentiation in iPS cells. This novel pre-culturing system also exhibits robustness in accommodating cells of different cell lines.   54  Figure 4.8E – cell 3 was included in the analysis as a control and shows a GFP-ve contracting cell (presumably an IMR90-4 iCM that was added to the culture to initiate differentiation on day 0). Cells 2 and 4 were also included as negative controls and show two non-contracting cells with straight lined kymograph profiles.     55                    Figure 4.8: Fluorescent images and kymographs of AICS-0016 + IMR90-4 pre-cultures.  (A) DIC image on day 23 (scale bar: 500µm). (B) Inset of red box from image (A) - overlay of DIC and GFP images (scale bar: 100µm). (C) Kymograph of contracting cluster in image (A) - arrow indicates direction of corresponding line scan (scale bar: 50µm). (D) Overlay image at low cell density showing example of GFP+ve self-contracting cell (cell 1) (scale bar: 50µm).            (E) Overlay image at low cell density showing GFP-ve self-contracting cell (cell 3) (scale bar: 50µm). (F) Corresponding kymographs of cells 1, 2, 3, and 4 (scale bar: 5µm). (A)	 (B)	AICS-16	+	IMR90	pre-culture	wells	imaged	on	day	23	(D)	AICS-16	+	IMR90	pre-culture	seeded	at	low-density	on	glass	slips	imaged	on	day	26	0	20	10	5	15	(C)	 ~1.7Hz	Non	contrac/ng	Non	contrac/ng	(d)	1	 2	3	0	10	5	4	 0	10	5	0	10	5	0	10	5	1	2	4	3	(E)	(F)	~1.0Hz	~1.4Hz	 56 4.2.4 Characterization of maturity via immunocytochemistry Another defining characteristic of cardiomyocytes is the expression of structural proteins, such as α-actinin, and the organization of these proteins into sarcomeres [21]. To characterize the structural maturity of resulting iCMs generated via pre-culture incubation method, immunocytochemistry staining for α-actinin protein was performed and maturity was assessed by the presence of sarcomeric striations. Three groups of AICS-0016 iPS cells treated with different conditions were compared. Group A: AICS-0016 iPS cells differentiated using the GiWi protocol (positive control). Group B: AICS-0016 iPS cells differentiated using novel pre-culture method (treatment group). Group C: AICS-0016 iPS cells that were subjected to only basal medium + supplement changes with no differentiation factors added (negative control).  Analysis of images revealed the distinct formation of sarcomeric striations in GFP+ve cells that were treated with the GiWi protocol (Figure 4.9A) and those that were incubated using the pre-culture method (Figure 4.9B). The presence of sarcomeric striation formation in co-cultured AICS-0016 cells is indicative of cells having mature contractile apparatus, a hallmark of functional cardiomyocytes. This provides another metric supporting the validity of the proposed novel pre-culture differentiation method.   Figure 4.9C was included into the experimental design as a negative control and shows AICS-0016 cells subjected to only basal medium and supplement changes without addition of any differentiation factors. Medium renewals for these cells were performed in parallel to pre-cultures and GiWi methods, minus only their differentiation cues (i.e. no GSK / Wnt inhibitors or co-culture with iCMs). In these cells, no sarcomeric striations were found and α-actinin was dispersed arbitrarily throughout cells.   57                   Figure 4.9: Fluorescent images of cells stained for α-actinin via immunocytochemistry.  (A) AICS-0016 cells differentiated using GiWi protocol - positive control.  (B) AICS-0016 cells differentiated using novel pre-culture method – treatment group.  (C) AICS-0016 cells treated with only basal medium + supplement change (no differentiation factors added) – negative control. Distinct sarcomeric striations found in cells treated with GiWi and pre-culture method but none found in medium change control condition.  Medium	change	GiWi	protocol	 Pre-culture	method	5μm	5μm	 5μm	Inset	Inset	 Inset	(C)	(A)	 (B)	α-ac3nin	GFP	DAPI	Merged	α-ac3nin	GFP	DAPI	Merged	α-ac3nin	GFP	DAPI	Merged	GFP	GFP	 GFP	α-ac3nin	α-ac3nin	 α-ac3nin	 58  Immunocytochemistry studies also revealed another noteworthy phenomenon. As AICS-0016 iPS cells differentiate into cardiomyocytes, their GFP fluorescence diminishes significantly but not altogether lost. Figure 4.10 shows examples of fluorescent images of AICS-0016 cells treated with GiWi protocol and imaged 26 days later. From analysis of these images, it was observed that iPS cells that differentiated into cardiomyocytes (indicated by displays of sarcomeric striation), cells 1 and 2, have diminished GFP fluorescence compared to iPS cells that differentiated into a non-cardiac lineage (indicated by the lack of sarcomeric striation), cell 3. This phenomenon of reduced GFP expression in AICS-0016 cells was observed among all cells successfully differentiated into cardiomyocytes; although the extent of this signal reduction varied from cell to cell.   The reduced GFP fluorescence readout in AICS-0016 iPSC-derived cardiomyocytes would have an eventual impact on the quantification of differentiation efficiency in co-cultured cells using flow cytometry, which relies on the GFP fluorescence signal from the AICS-0016 cells to distinguish them from the non-fluorescent IMR90 iCM cells. This limitation would be discussed in more detail in the next section.        Figure 4.10: Immunocytochemistry images of GiWi differentiated AICS-0016 cells. (A) Overlay image showing α-actinin (red), GFP (green), and cell nuclei (blue). (B) Fluorescent image showing only GFP (green). GFP fluorescent signal diminished in cells differentiated into cardiomyocytes (cells 1 and 2) but did not diminish when differentiated into non-cardiac cells (cell 3).  (A)	 (B)	α-ac*nin	GFP	DAPI	 GFP	1	2	3	 1	2	3	Diminished	GFP	 59 4.2.5 Characterization of differentiation efficiency via flow cytometry To obtain quantitative data assessing the efficacy of the pre-culture method as a viable cardiac differentiation system, differentiation efficiency was analyzed using flow cytometry and compared to relevant control groups. Differentiation efficiency was defined as the proportion of cells in a given population expressing specific cardiac markers (i.e. either cardiac troponin T (cTnT) or α-actinin). GFP fluorescence from the AICS-0016 cells allowed them to be separated from the co-culture, which consisted of both non-fluorescent IMR90-4 iCMs and GFP-tagged AICS-0016 cells. IMR90-4 iPS cells, AICS-0016 iPS cells, and AICS-0016 cells subjected to only basal medium + supplement changes, were used as negative controls and gating scheme was set up according to those three cases. For the example shown in Figure 4.11, IMR90-4 iPS cells were gated in Q4 (GFP-ve and cTnT-ve), while AICS-0016 iPS cells and AICS-0016 medium change cells were gated in Q3 (GFP+ve and cTnT-ve).  Using the controls stated above, and gating only for GFP+ve cells, flow cytometry analysis of 26 days old pre-cultured samples revealed that ~1.1% of AICS-0016 cells were positive for cTnT (Figure 4.11B) and ~1.5% were positive for α-actinin (Figure 4.12B). This differentiation efficiency was lower compared to AICS-0016 cells differentiated using the standard GiWi protocol in the same timeframe, where ~5.3% of cells were cTnT+ve (Figure 4.11A) and ~3.0% were α-actinin+ve (Figure 4.12A). The results for differentiation efficiencies of AICS-0016 cells are conservatively reported due to the restriction that only cells in Q2 (GFP+ve and cTnT+ve / α-actinin+ve) were considered true positives.   As alluded to in the preceding section, AICS-0016 iPS cells differentiated into cardiomyocytes suffer from diminished GFP fluorescence, as indicated visually via immunocytochemistry. This phenomenon of reduced GFP fluorescence in AICS-0016 iPS cell- 60 derived cardiomyocytes is also observed in the flow cytometry analysis – leftward shift in the cTnT+ve and α-actinin+ve population of AICS-0016 GiWi-differentiated cells (Figure 4.11A) and Figure 4.12A) indicates reduced GFP fluorescent intensity. The diminished GFP signal in cardiac cells limited my ability to cleanly gate GFP+ve cells from GFP-ve cells – i.e. some AICS-0016 cells that were successfully differentiated to cardiomyocytes had significantly reduced GFP and overlapped with non-GFP IMR90-4 iCMs. As such, the current methodology does not facilitate an accurate accounting of the differentiation efficiencies for both GiWi and pre-culture methods.  However, despite this limitation in gating that prevented a complete valuation of the actual differentiation efficiency, flow data presented remains relevant in providing a relative comparison between the differentiation efficiencies of the novel pre-culture method and established methods like the GiWi protocol.   Table 4.1 and 4.2 summarizes the differentiation efficiencies of the various conditions quantified by flow cytometry for cardiac specific markers, cTnT and α-actinin. Table 4.1 is not gated for GFP+ve cells and reports the total proportion of cTnT+ve and α-actinin+ve cells in both Q1 and Q2. Table 4.2, is gated for GFP+ve cells and shows the proportion of cTnT+ve and α-actinin+ve cells in Q2. Due to the diminished GFP fluorescence in AICS-0016 differentiated cells, the above-described gating scheme is needed in order to meaningfully interpret the flow cytometry data. Table 4.1 (not gated for GFP+ve cells) compares the differentiation efficiencies of GiWi differentiation IMR90-4 and AICS-0016 cells and shows comparable efficiencies between the two cells lines – i.e. 50.2% vs. 54.2% and 58.0% vs. 44.5%, for cTnT and α-actinin respectively. Table 4.2 (gated for GFP+ve cells – i.e. only cells in Q2), reports a conservative estimate of differentiation efficiency for the novel pre-culture method (1.1% cTnT+ve and 1.5%  61 α-actinin+ve) and compares that to a similarly gated GiWi-differentiated AICS-0016 cell population (5.3% cTnT+ve and 3.0% α-actinin+ve).                    Figure 4.11: Flow cytometric analysis for cTnT cardiac marker (GFP β-actin vs. cTnT).  (A) Positive controls – from left to right: IMR90-4 GiWi differentiated, and AICS-0016 GiWi differentiated.  (B) Pre-cultured cells showing ~1.00% of cTnT+ve AICS-0016 cells (conservative assessment). (C) Negative controls – from left to right: IMR90-4 iPSCs, AICS-0016 iPSCs, and AICS-0016 cells treated with only basal medium + supplement change (no differentiation factors added). Gating	legend:	Q1:	(GFP-ve,	cTnT+ve);	 Q2:	(GFP+ve,	cTnT+ve);		 Q3:	(GFP+ve,	cTnT-ve);		 Q4:	(GFP-ve,	cTnT-ve)	(C)	Nega)ve	controls	GFP	β-ac)n	IMR90	-	iPSC	 AICS16	-	iPSC	 AICS16	–	Medium	change	(A)	Posi)ve	controls	IMR90	-	GiWi	 AICS16	-	GiWi	GFP	β-ac)n	(B)	Pre-culture	method	AICS16	–	Pre-cultured	GFP	β-ac)n	Q10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% S eding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A023456107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 10 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding Density 62                    Figure 4.12: Flow cytometric analysis for α-actinin cardiac marker (GFP β-actin vs. α-actinin).  (A) Positive controls – from left to right: IMR90-4 GiWi differentiated, and AICS-0016 GiWi differentiated. (B) Pre-cultured cells showing ~1.3% of α-actinin+ve AICS-0016 cells (conservative assessment). (C) Negative controls – from left to right: IMR90-4 iPSCs, AICS-0016 iPSCs, and AICS-0016 cells treated with only basal medium + supplement change (no differentiation factors added).(C)	Nega)ve	controls	GFP	β-ac)n	IMR90	-	iPSC	 AICS16	-	iPSC	 AICS16	–	Medium	change	(A)	Posi)ve	controls	IMR90	-	GiWi	 AICS16	-	GiWi	GFP	β-ac)n	(B)	Pre-culture	method	AICS16	–	Pre-cultured	GFP	β-ac)n	Q10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding De sity 25% Seeding De sity 50% Seeding De sity 100% Seeding De sityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10 59Q21. 1Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-A1.28 0.4973.4.80 103 104 105 106 107-  :: -0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Se ding DensityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding De sity 25% Seeding De sity 50% Seeding De sity 100% Seeding De sityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityGating	legend:	Q1:	(GFP-ve,	α-actinin+ve);					Q2:	(GFP+ve,	α-actinin+ve);					Q3:	(GFP+ve,	α-actinin-ve);					Q4:	(GFP-ve,	α-actinin-ve)	 63 Table 4.1: Cardiac differentiation efficiency of various conditions quantified via flow cytometry for cTnT and α-actinin. The reported percentages are the sum of Q1 and Q2, which includes both GFP+ve and GFP-ve cells.   * Not applicable (n/a) because ungated population (Q1 + Q2) of pre-cultured cells consists  of a mixture of IMR90-4 and AICS-0016. Thus, differentiation efficiency is not  assessable.   Table 4.2: Cardiac differentiation efficiency of various conditions quantified via flow cytometry for cTnT, α-actinin, and GFP+ve cells. The reported percentages are Q2 gated cells – i.e. only cells that are GFP+ve. Novel pre-culture method gave 1.1% and 1.5% cTnT+ve and α-actinin+ve cells respectively after 26 days in culture. Standard GiWi protocol yielded 5.3% and 3.0% cTnT+ve and α-actinin+ve in the same time frame. Assessment was performed in a conservative manner and only cells in Q2 (GFP+ve and cTnT+ve / α-actinin+ve) were considered as true positives.   ** Experiment independently replicated (see appendix section A.1 for details).  Cardiac	troponin	T	(cTnT)	α-ac1nin	IMR90-4	GiWi	 iPSCs	AICS-0016	GiWi	 iPSCs	 Medium	change	Pre-culture	50.2%	58.0%	0.0%	0.0%	54.2%	44.5%	0.0%	0.0%	0.2%	0.2%	n/a	*	n/a	*	Cardiac	troponin	T	(cTnT)	α-ac1nin	IMR90-4	GiWi	 iPSCs	AICS-0016	GiWi	 iPSCs	 Medium	change	Pre-culture	0.0%	0.0%	0.0%	0.0%	5.3%	3.0%	0.0%	0.0%	0.1%	0.1%	1.1%	1.5%	**	 64 4.2.6 Effect of varying iCM seeding density on differentiation outcome  All co-culture assays discussed thus far used a seeding density of 3 million iCMs seeded into 1 well of a 6-well multidish to initiate differentiation of iPS cells. (I.e. 3 million iCMs were added to ~90% confluent iPS cells in 6-wells). As shown in previous sections, this method constituted a sufficient method to induce cardiac differentiation of iPS cells. However, a follow-up question that I sought to address was whether varying the initial iCM seeding density to iPS cells had an effect on the differentiation outcome. To that end, an assay was set up as illustrated in Figure 4.13.  31 days old IMR90-iCMs were harvested and seeded in a pre-culture manner at the following densities, 0.3, 0.75, 1.5, and 3.0 million cells onto ~90% confluent AICS16-iPS cells to initiate the co-culture. On day 26, cells were harvested for flow cytometry and immunocytochemistry analysis. Flow cytometric results revealed a positive correlation between initial IMR90-iCM seeding density and the eventual proportion of AICS16 cells expressing cardiac markers (Figure 4.14 and Figure 4.15) after 26 days of incubation. Table 4.3 summarizes the cardiac differentiation efficiency of the four seeding density conditions investigated. This positive trend between iCM seeding density and subsequent number of cells expressing cardiac markers is indicative of differentiation stimulatory effects originating from the seeded iCMs within the co-culture. It is also suggestive that direct iCM-iPS cell-cell contact is a requisite factor, which I investigated in section 4.2.7.  As described before, the pre-cultured cells were also stained for sarcomeric α-actinin to further validate successful iCM differentiation revealed by the flow cytometry-based assay. Figure 4.16 shows the results of this immunocytochemistry stain. All four seeding density  65 conditions shows α-actinin striations in GFP+ve cells, which is indicative of successful cardiac reprogramming in those AICS-0016 iPS cells.                     Figure 4.13: Illustration of IMR90-4 iCMs seeded at varying densities to AICS-0016 iPS cells in pre-culture.  Negative controls (IMR90-iPSCs, AICS16-iPSCs, and AICS16-medium change) and positive controls (IMR90-GiWi and AICS16-GiWi) were set up in parallel but not shown in the illustration.   Day 0 Day 4 Day 5 Day 9 Day ??Activin A BMP4 Begin cyclic strainFigure 4.7: A schematic of the differentiation protocol used to generate cardiomyocytes fromiPSCs. Cells are seeded on Matrigel on Day 0 and reach 90% confluence by Day 4.Activin A (100ng/mL) is applied for 24 hours, followed by 4 days of BMP4 treatment(10ng/mL) without medium change. After BMP4 treatment, 5% cyclic strain is appliedcontinously at 1Hz using the HexCycler.Figure 4.8: A: A spontaneously beating cluster of cardiomyocytes. The yellow line indicatesthe region used for kymograph analysis. B: Kymograph analysis gives a beat frequencyof 0.35Hz.tus, as shown by immunocytochemistry. Figure 4.9 shows staining of iPSC-derived cardiomyocytes.Cell nuclei are stained blue by DAPI, which binds to regions of DNA rich in adenine and thymine.The proteins of interest are stained green. Those nuclei not surrounded by green belong to non-cardiomyocytes, most likely cells that have differentiated to a cardiac fibroblast lineage. Both theunstrained and strained cells stained positively for cTnT, but the strained cells exhibited punctatestaining while the unstrained cells had more filamentous staining. Sarcomeric striations revealedby ↵-actinin staining were common in the unstrained specimen, but much rarer in the unstrainedspecimen. This suggests that the strain regimen hindered the maturation of these cardiomyocytes.46Day 0 Day 4 Day 5 Day 9 Day ??Activin A BMP4 Begin cyclic strainFigure 4.7: A schematic of the differe tiation protocol us d to generate cardiomyocytes fromiPSCs. Cells are seeded on Matrigel on Day 0 and reach 90% confluence by Day 4.Activin A (100ng/mL) is applied for 24 hours, followed by 4 days of BMP4 treatment(10ng/mL) without medium change. Af r BMP4 treat ent, 5% cyclic strain is appliedcontinously at 1Hz using the HexCycler.Figure 4.8: A: A spontaneously beating cluster of cardiomyocytes. The yellow line indicatesthe region used for kymograph analysis. B: Kymograph analysis gives a beat frequencyof 0.35Hz.tus, as shown by immunocytochemistry. Figure 4.9 shows staining f iPSC-derived cardiomy cytes.Cell nuclei are stained blue by DAPI, which binds to regions of DNA rich in adeni e and thymine.The proteins of interest are stained green. Those nuclei not surrounded by green belong to non-cardiomyocytes, most likely cells t at have differentiated to a cardiac fibroblast lineage. Both theunstrained and strained cells stained positively for cTnT, but the strained cells exhibited punctatestaining while the unstrained cells had more filamentous staining. Sarc meric striati ns revealedby ↵-actinin staining were common in the unstrained specimen, but much rarer in the unstrainedspecimen. This suggests that the strain regimen hindered the maturation of these cardiomyocytes.46Day 0Day 4Day 5ay 9ay ??Activin ABMP4Begin cyclic strainFigure4.7:AscheaticofthedifferentitioprtocolusedtogenerecardiomyocytesfromiPSCs.CellsareseededonMatrigelonDay0andreach90%confluencebyDay4.ActivinA(100ng/mL)isappliedfor24hours,fllowedby4daysofBMP4treatment(10ng/mL)withoutmediuhnge.AfterBMP4treatment,5%cyclicstrainisapplidcontinouslyat1HzusingtheHexCycler.Figure4.8:A:Apontaneouslybeatingclutrofcardiomyocyte.Theyellowlineindicatestheregionusedforkymogrphanalysis.B:Kymgraphanalysisgivesabeatfrequencyof0.35Hz.tus,asshownbyimmuncytchemistry.Figure4.9showstainingofiPSC-derivedcardiomyocytes.CellnucleiarestainedbluebyDAPI,whichbindstoreginsofDNArichinadenineandthymie.Theproteinsofinterestarestainedgreen.Thosencleinotsrroundedbygreenbelngtono-cardiomyocytes,mostlikelycellsthathavedifferentitedtocardiacfibroblastlineage.BohtheunstrainedandstrainedcellsstainedpositivelyforcTnT,butthestrainedcellsxhibitedpuncttestainingwhiletheunstrainedcellshadmorefilamentoutainig.Sarcmerictriationsrevealedby↵-actininstainingwerecommonintheustrainedspecimen,butmucrarerintheunstraiedspecimen.Thissuggeststhatthestrainregimenhinderedthematurationfthesecardiomyocytes.46Day 0a 4Day 5ay 9ay ??Activin ABMP4Begin cyclic strainFigure4.7:AscheaticofthedifferetitioprtocolusdtogenerecardiomyocytesfromiPSCs.CellsareseededonMatrigelonDay0andreach90%confluencebyDay4.ActivinA(100ng/mL)isappliedfor24hurs,fllowedby4daysofBMP4treatment(10ng/mL)withoutmediumhange.AfterBMP4treatment,5%cyclicstrainisappliedcontinouslyat1HzusingtheHexCycler.Figure4.8:A:Apontaneouslybeatingclutrofcardiomyocyte.Theyellowlineindicatestheregionusedforkymogrphanalysis.B:Kymgraphanalysisgivesabeatfrequencyof0.35Hz.tus,asshownbyimmuncytchemistry.Figure4.9showstainingfiPSC-derivedcardiomycytes.CellnucleiarestainedbluebyDAPI,whichbindstoreginsofDNArichinadenieandthymie.Theproteinsofinterestarestainedgreen.Thosencleinotsrrundedbygreebelongtoo-cardiomyocytes,mostlikelycellstathavedifferetitedtocardiacfibroblastlineage.BohtheunstrainedandstrainedcellsstaiedpositivelyforcTnT,butthestrainedcellsxhibitedpuncttestainingwhiletheunstrainedcellshadmorefilamentoutaining.Sarcmerictriatinsrevealedby↵-actininstainingwerecommonintheunstrainedspecimen,butmucrarerintheunstraiedspecimen.Thissuggeststhatthestrainregimenhinderedthematurationofthesecardiomyocytes.460.3×106	IMR-90	iCMs	added	to	AICS-16	iPS	cells	0.75×106	IMR-90	iCMs	added	to	AICS-16	iPS	cells	1.5×106	IMR-90	iCMs	added	to	AICS-16	iPS	cells	3.0×106	IMR-90	iCMs	added	to	AICS-16	iPS	cells	*Relevant	controls	set	up	in	pa all l	(not	shown)	 66                   Figure 4.14: Differentiation efficiency for pre-cultures with varying IMR90-iCM seeding densities - assessed by cTnT expression. Negative controls (IMR90-iPSCs, AICS16-iPSCs, and AICS16-medium change) and positive controls (IMR90-GiWi and AICS16-GiWi) were prepared in parallel but not shown.       GFP	 GFP	GFP	 GFP	*Gated	rela,ve	to	relevant	controls	(not	shown)	0.3×106	iCM	seeded		 0.75×106	iCM	seeded		1.5×106	iCM	seeded		 3.0×106	iCM	seeded		Q10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Medi  ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10Q20.046Q399.8Q40.190 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.039Q20Q30.013Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ148.9Q25.34Q328.9Q417.00 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.051Q20.14Q393.9Q45.990 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.67Q20.17Q388.4Q410.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.01Q20.41Q391.8Q46.800 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.38Q20.51Q391.4Q46.710 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ13.29Q21.05Q391.3Q44.370 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ150.2Q20.032Q30.10Q449.70 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AiPSC ControlsIMR90 AICS16GiWiIMR90 AICS16Media ChangeCoculture10% Seedi g Density 25% Seedi g Density 50% Seedi g Density 100% Seedi g Density 67                   Figure 4.15: Differentiation efficiency for pre-cultures with varying IMR90-iCM seeding densities - assessed by α-actinin expression. Negative controls (IMR90-iPSCs, AICS16-iPSCs, and AICS16-medium change) and positive controls (IMR90-GiWi and AICS16-GiWi) were prepared in parallel but not shown.     GFP	 GFP	GFP	 GFP	*Gated	rela,ve	to	relevant	controls	(not	shown)	0.3×106	iCM	seeded		 0.75×106	iCM	seeded		1.5×106	iCM	seeded		 3.0×106	iCM	seeded		Q10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media ChangeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding DensityQ10.046Q20Q30.025Q499.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10Q20.015Q398.9Q41.070 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ158.0Q20.042Q38.40E-3Q441.90 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ141.5Q22.95Q326.4Q429.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.012Q20.14Q377.0Q422.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.66Q20.17Q366.4Q432.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ10.59Q21.21Q376.1Q422.10 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ11.28Q20.49Q373.4Q424.80 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AQ12.23Q21.45Q380.2Q416.20 103 104 105 106 107FL1-A :: FL1-A0102103104105106107FL4-A :: FL4-AIMR90 AICS16iPSC Controls GiWiIMR90 AICS16Media Ch ngeCoculture10% Seeding Density 25% Seeding Density 50% Seeding Density 100% Seeding Density 68                 Figure 4.16: Immunohistochemistry images of pre-cultured AICS-0016 cells seeded with varying IMR90-iCM seeding densities.  All four conditions show α-actinin striations in GFP+ve cells, which is indicative of successful cardiac reprogramming of AICS-0016 iPS cells.     0.3×106	iCM	seeded		 0.75×106	iCM	seeded		1.5×106	iCM	seeded		 3.0×106	iCM	seeded		 69 Table 4.3: Cardiac differentiation efficiency of various iCM seeding densities quantified via flow cytometry for cTnT and α-actinin. The reported percentages are Q2 gated cells – only cells that are GFP+ve. Results show a positive trend between initial IMR90-iCM seeding density and proportion of AICS-16 expressing cardiac markers (cTnT or α-actinin) after 26 days.    4.2.7 Role of cell-to-cell contact in co-culture based cardiac differentiation To investigate the role of iCM-iPS cell-cell contact in co-culture cardiac differentiation, a non-contact co-culture assay was performed using polyethylene terephthalate (PET) culture inserts (Greiner Bio-One #657610). In this experiment, 30 – 45 days old IMR90-iCMs were harvested, reseeded onto Matrigel-coated PET culture inserts, and placed into 6-wells containing monolayer of AICS16-iPS cells (as shown in Figure 4.17). The PET inserts have a porous membrane (pore size 1µm) that allows the exchange of soluble biochemical factors between cells while providing physical separation between them.  This non-contact co-culture was prepared in parallel with AICS-0016 cells subjected to only basal medium + supplement changes (negative control). Differentiation outcome was assessed using flow cytometry for cardiac marker (cTnT) and immunocytochemistry for the presence of α-actinin sarcomeric striations.     Cardiac	troponin	T	(cTnT)	α-ac1nin	GiWi	AICS-0016	0.3×106	iCM	seeded		Pre-culture	5.3%	3.0%	0.2%	0.2%	0.75×106	iCM	seeded		1.5×106	iCM	seeded		3.0×106	iCM	seeded		0.4%	1.2%	0.5%	0.5%	1.1%	1.5%	Medium	change	0.1%	0.1%	 70  Figure 4.17: Illustration of non-contact cell culture hanging insert setup.    To validate the hanging culture insert as an adequate system for the non-contact co-culture assay, I verified two aspects of the setup. The first was the verification that functional properties of cardiomyocytes were preserved after reseeding (i.e. checked if spontaneous beating was reinitiated after reseeding onto membranes). The second verification was that iCMs on the membrane were confined and did not fall through the mesh to the bottom well. This was performed using visual inspection for the presence of cells in a medium-filled empty well incubated for 4 days with the iCM culture insert. Figure 4.18 shows representative microscopy images illustrating positive validation of the hanging culture insert setup.   After its validation, the IMR90-iCMs hanging culture insert was incubated with AICS16-iPS cells for 26 days. After which time, the AICS16 cells were harvested, with some seeded at low-density onto glass slips for immunocytochemical analysis and the rest fixed / permeabilized, and stained for cTnT cardiac marker for flow cytometry. Flow cytometric results showed low expression levels of cTnT in non-contact co-cultured AICS16 cells (i.e. expression comparable to negative control case) (Figure 4.19). Flow cytometric results were also supported by immunocytochemical analysis, which showed an absence of α-actinin sarcomeric striations in the Cell	culture	insert	Medium	AICS16-iPS	cells		monolayer	IMR90-iCMs	Porous	PET	membrane	 71 non-contact AICS16 cells (Figure 4.20). Taken together, these results suggest that direct cell-to-cell contact is required for cardiac co-culture differentiation of stem cells.            Figure 4.18: Representative microscopy images validating hanging culture insert system. (A) Image and kymograph analysis showing iCM reinitiating spontaneous contraction on PET membrane beating at ~1.2Hz. (B) Image showing complete absence of cells in a medium-filled 6-well after 4 days of incubation with hanging culture insert (i.e. no iCMs fell through the PET membrane).    Figure 4.19: Flow cytometry density plots (cTnT vs. GFP) for AICS-0016 iPS cells cultured with iCM hanging insert vs. AICS-0016 iPS cells cultured with only basal medium + supplement (negative control). Flow cytometric results shows AICS-0016 cells co-cultured with iCM in a hanging insert format expressing low levels of cTnT (<0.1% of cells cTnT+ve), comparable to AICS-0016 cells that were subjected to only basal medium + supplement change.  Forward	sca*er	area	(FS-A)	(A)	Forward	and	side	sca0er	ga2ng	Hanging	insert	 Medium	change	GFP	(B)	cTnT	expression	Cells41.40 5.0M 10M 15MFSC-A :: FSC-A0300K600K900K1.2MSSC-A :: SSC-AA01 Basket_AICS16_cTnT.fcsUngated30000Cells34.40 5.0M 10M 15MFSC-A :: FSC-A0300K600K900K1.2MSSC-A :: SSC-AB01 Media change_AICS16_cTnT.fcsUngated30000cTnT+0.097100 102 104 106FL4-A :: FL4-A0100200300400500CountA01 Basket_AICS16_cTnT.fcsCells12422cTnT+0.097100 102 104 106FL4-A :: FL4-A0100200300CountB01 Media change_AICS16_cTnT.fcsCells10314Cells41.40 5.0M 10M 15MFSC-A :: FSC-A0300K600K900K1.2MSSC-A :: SSC-AA01 Basket_AICS16_cTnT.fcsUngated30000Cells34.40 5.0M 10M 15MFSC-A :: FSC-A0300K600K900K1.2MSSC-A :: SSC-AB01 Media change_AICS16_cTnT.fcsUngated30000cTnT+0.097100 102 104 106FL4-A :: FL4-A0100200300400500CountA01 Basket_AICS16_cTnT.fcsCells12422cTnT+0.097100 102 104 106FL4-A :: FL4-A0100200300CountB01 Media change_AICS16_cTnT.fcsCells10314Hanging	insert	 Medium	change	cTnT+ve	0.038	cTnT+ve0.038100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA01 Basket_AICS16_cTnT.fcsCells2653cTnT+ve0.067100 102 104 106FL1-A :: FL1-A1001 11021 31041 5106107FL4-A :: FL4-AB01 Media change_AICS16_cTnT.fcsCells2993cTnT+ve0.038100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA01 Basket_AICS16_cTnT.fcsCells2653cTnT+ve0.067100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB01 Media change_AICS16_cTnT.fcsCells2993(A)	 (B)	0	10	5	~1.2Hz	 72  Figure 4.20: Fluorescent images from non-contact co-culture assay, stained for α-actinin. (A) AICS-0016 cells incubated with IMR90-iCM non-contact hanging insert. (B) AICS-0016 cells treated with only basal medium + supplement change (no differentiation factors added) - negative control.  No α-actinin sarcomeric striations found in either non-contact co-culture or medium change case. Medium	change	Non-contact	insert	5μm	 5μm	 5μm	Inset	Inset	(B)	(A)	α-ac2nin	GFP	DAPI	α-ac2nin	GFP	DAPI	Merged	α-ac2nin	GFDAPI	Merged	α-ac2nin	α-ac2nin	 73 4.3 Chapter discussion Based on the results presented in this chapter, it was revealed that non-differentiated iPS cells which have been co-cultured with iCMs in a pre-culture manner, were successfully differentiated into cells exhibiting self-contractility and other distinguishing features of functional cardiomyocytes – formation of sarcomeric striations and expression of cardiac specific markers. This successful reprogramming of iPS cells into functional iCMs was achieved without the addition of exogenous pathway inhibitors or morphogens into the co-culture, suggesting that the ‘older’ seeded iCMs constituted a sufficient system that recapitulates the stimulatory environment for cardiac differentiation of iPS cells into relevant cardiomyocyte subtypes. The proposed co-culture cardiac differentiation method also demonstrated robustness in terms of being able to accommodate iPS cells derived from different hosts / sources (i.e. two different iPS cell lines were investigated, both showing successful differentiation outcomes). While previous co-culture studies have showed the derivation of cardiomyocytes from other cell types (stem cells or otherwise), none have demonstrated the process using iCM as a cardiac differentiation inducer.   Three mixing strategies were considered during the optimization of the co-culture method. The first was a pre-culture approach, where iCMs were added to adhered iPS cells sequentially. The second was a variation of the pre-culture method, where the two cell types were seeded in reverse order (i.e. iPS cells added to adhered iCMs). The third strategy considered was the bi-culture approach, where both iPS cells and iCMs were harvested, combined, and reseeded simultaneously. Of the three strategies considered, only the first yielded successfully differentiated cells. Though not further explored, a likely explanation for this could be a limitation of substrate surface area for cell adhesion, where both iPS cells and iCMs  74 compete to adhere to the limited well surface. In the second and third co-culture approaches, only few iPS cells might have actually made it to the well surface as it competes with iCMs to adhere. This might result in low initial iPS cell number that could translate to the poor differentiation outcome observed. This ‘race to the surface’ phenomenon has also been documented in other co-cultures experiments [64, 65], though not in the context of stem cells and stem cell differentiation.   To optimize the co-culture method further, the number of input iCMs applied to iPS cells in the starting co-culture was varied. This was done to assess if less input iCMs could be used, while maintaining the same level of cardiac differentiation efficiency. Differentiation efficiency was evaluated by flow cytometry for cardiac specific markers, cTnT and α-actinin, and gated for GFP+ve cells (initially non-differentiated cells). Differentiation efficiency was directly correlated with quantity of input iCMs. This positive trend suggests that the number of input iCMs limits co-culture cardiac differentiation efficiency (i.e. to increase differentiation efficiency, more input iCMs have to be used). Even though only four iCM input seeding densities were investigated in this assay, the clear trend provides compelling evidence for its validity.  Further, I wondered if the positive trend between input iCMs and differentiation efficiency could be an indication of the essentiality of direct cell-to-cell contact to the whole process of co-culture differentiation. Since more iCMs seeded results in more iCM-to-iPSC contact, it stands to argue that because of the increased cell-to-cell contact, differentiation efficiency was thus increased. To investigate this, a non-contact co-culture assay, utilizing hanging culture inserts, was set up and compared to a relevant negative control. Assessed using microscopy, immunocytochemistry, and flow cytometry, it was determined that the non-contact co-culture did not result in cardiac differentiation (i.e. no functional (spontaneous beating) or  75 structural (no sarcomeric striations) properties were detected). This finding supports the above-mentioned postulate that direct cell-to-cell contact is essential in co-culture based cardiac differentiation. Previous works have also reported similar results [41, 42, 66], although not with iPS cells or with iCMs as a stimulatory source. Studies by Iijima et al. went a step further and demonstrated that not only is cell-to-cell contact essential to cardiac differentiation, but the cyclic contraction of neighboring cardiomyocytes is also necessary for cardiac reprogramming of adjacent cells [43]. With regards to molecular pathway, researchers are now trying to unravel the players involved in the intercellular coupling and communication governing this process. Cell adhesion molecules, like E-cadherin [44], have since been implicated as a key mediator of the communication.    In summary, while highly efficient and convenient, the standard GiWi method for cardiomyocyte generation may fail for iPS cells derived from certain genetic backgrounds. Since GiWi method targets the Wnt signaling pathway, it may not work for individuals that have pathway specific mutations. In such circumstance, a benefit of using my novel ‘near natural’ co-culture method for programming stem cell fate can serve to circumvent this challenge and generate physiologically relevant patient-specific cardiomyocytes.  76 Chapter 5: Conclusions and Future Directions Methods for differentiating iPS cells to cardiomyocytes continue to improve and evolve; none is decidedly more convenient than with the monolayer method using differentiation factors. One such method, the so-called ‘GiWi’ protocol has been widely adopted due to its speed (7 days treatment period) and efficiency (~85% efficiency) in generating functional cardiomyocytes from stem cells in vitro [18]. However, despite its popularity among researchers, achieving optimal benefits for this technology is still very much hampered by the poor differentiation outcomes associated with the method [60] – i.e. GiWi-differentiated cardiomyocytes are immature in nature and remains a poorly defined mixture of cellular subtypes (ventricular, atrial, nodal, cardiac fibroblasts and others) [59]. This lack of control over the differentiation outcome has significantly stifled its potential for eventual downstream commercial and clinical applications [61]. Other in vitro cardiac differentiation strategies, such as those developed by Cao et al. [19], Burridge et al. [20] and Kadari et al. [62], also suffer from a similar lack of control despite the added complexity of using additional morphogens and inhibitors in their differentiation cocktail. This clear methodological limitation of current pluripotent stem cell-to-cardiomyocyte differentiation strategies motivated the undertaking of this exploratory project aimed at improving existing differentiation systems by investigating various physical and biological parameters governing the stem cell cardiac differentiation process.  Based on rationales described in the thesis’ introduction, three specific areas were identified and investigated in this body of work: 1. Effects of substrate stiffness on cardiac differentiation of iPS cells. 2. Topographical manipulation of iCMs to form three-dimensional heart tissues.  77 3. Derivation of cardiomyocytes from iPS cells using inductive co-culture with previously differentiated iCMs.   5.1 What’s new?  Even though tremendous strides have been made to hPSC-CM technologies in recent years, this highly multidisciplinary field is still at an early stage of development [63]. Many questions need to be answered and improvements need to be made before clinical applications, like personalized therapies and drug toxicity testing, become a reality. Here, I reiterate some of the main findings of the thesis (for full discussion, see ‘Chapter discussion’ sections 3.3 and 4.3) and highlight specific areas of novelty that serves to supplement our current state of knowledge.  5.1.1 Substrate stiffness and cardiac differentiation of iPS cells Here, I investigated the effects of substrate stiffness modulation applied in combination with traditional ‘soluble factors’ on cardiac differentiation outcomes of iPS cells. It was revealed that substrate stiffness modulation promoted maturation of the molecular and functional properties of human iPS cell-derived cardiac tissues. IPS cells differentiated on hard PDMS substrates (~1.72MPa) yielded increased proportions of cTnT+ve cells (a cardiac specific structural protein) compared to iPS cells differentiated on soft (~5kPa) and intermediate (~130kPa) PDMS substrates. Resulting cardiac clusters on hard substrates also displayed contraction rates of (0.9±0.2) Hz, comparable to human adult native cardiomyocytes (~1.0 Hz), while cardiac clusters on soft and intermediate PDMS substrates contracted at marginally slower rates.  78 5.1.2 Topographical manipulation of iPS cell-derived cardiomyocytes (iCMs) To investigate the effects of topography on differentiated iCMs, freshly GiWi-differentiated iCMs were re-seeded into millimeter-sized grooved linear PDMS channels. It was demonstrated that these freshly differentiated iCMs exhibited remarkable plasticity in being manipulated topographically to form single 3D cardiac tissues. Compared to existing hPSC-CM engineered heart tissue strategies, this proposed method is rapid (cardiac strips form after 4 days from iCM seeding), does not require the use of structural anchors / support, mechanical or electrical stimulation, perfusion bioreactors, or other conditions that might complicate future clinical translation.  5.1.3 Novel co-culture method of cardiac differentiation of iPS cells using previously differentiated iPS cell-derived cardiomyocytes (iCMs) Various types of stem cells have been shown to differentiate into cardiomyocytes by way of co-culture with appropriate inducer cells. However, a thorough search of relevant literature (see section 1.7) revealed that no study has demonstrated the utility of a co-culture induction system using stem cell-derived cardiomyocytes as a stimulatory source for cardiac reprogramming. Here, I demonstrated for the first time that non-differentiated iPS cells which have been co-cultured with iCMs in a pre-culture manner, could be differentiated into cells exhibiting self-contractility and other distinguishing features of functional cardiomyocytes – formation of sarcomeric striations and expression of cardiac specific markers. This novel co-culture cardiac differentiation method also proved robust when applied to iPS cells derived from different hosts / sources (i.e. two different iPS cell lines were investigated in my studies). Exploring more, this study further showed that direct cell-to-cell contact is essential in co-culture based cardiac  79 differentiation. In conclusion, this novel ‘near natural’ co-culture method demonstrated for the first time that existing ‘older’ iCMs within a iPS cell-iCM co-culture constituted a sufficient system capable of recapitulating the stimulatory environment necessary for the differentiation of iPS cells into the relevant cellular subtypes.  5.2 Big picture contribution This body of work has contributed to the building of a bioengineering toolbox for the development of more clinically relevant engineered heart tissues. In this thesis, links between biophysical cues (e.g. substrate stiffness, topography, cell-cell communication) and the functional response of developing hPSC-CMs were made. This has in turn contributed to the improved understanding of maturation cues associated with the developing human myocardium.  5.3 Clinical perspective A systematic analysis of the global burden of disease revealed that mortality due to cardiomyopathy and myocarditis in 2013 alone reached 400,000 cases, an increase of 50% since 1990. The study also puts the number of deaths attributed to heart-related diseases, including atrial fibrillation, rheumatic heart disease, coronary heart disease, hypertensive heart disease, and endocarditis, at over 10 million [64]. The creation of a reliable in vitro source of cardiomyocyte can be used for a wide range of clinically relevant applications that could help potentially help in the fight against heart disease. In this section, I discuss some of the clinical applications of in vitro cardiomyocytes.  80 5.3.1 Modeling of cardiomyopathies with iPS cell-derived cardiomyocytes Induced pluripotent stem cells derived from patients with known cardiomyopathies can be used generate disease-specific cardiomyocytes for in vitro modeling purposes. Itzhaki et al. reported one of the first demonstrations of such an application, when they used iPS cells derived from dermal fibroblasts of patients with long QT syndrome and then differentiated those iPS cells into cardiomyocytes [65]. Long QT syndrome (LQTS) is a condition affecting repolarization of the heart after a heartbeat, increasing the risk of irregular heartbeat that can result in fainting or sudden death [66]. By generating these patient-specific cardiomyocytes, Itzhaki and colleagues were able to study the disease in vitro and also test potential pharmacological agents for treatment of the disease.   5.3.2 Drug-induced cardiotoxicity testing using iPS cell-derived cardiomyocytes Cardiotoxicity remains a concern in drug screening. Lacking a reliable in vitro model, current drug safety testing methodologies primarily involve preclinical and clinical trials, in which human volunteers are subjected to candidate drugs and monitored for potential side effects. This testing strategy is not only costly, but it puts healthy human volunteers at risk. Despite some limitations, human iPS cell-derived cardiomyocytes is an attractive alternative capable of recapitulating many attributes of human cardiomyocytes and their drug responses [67]. However, the accuracy of these models depends significantly on the ability to generate mature cardiomyocytes as many of the ion channels targeted by cardiotoxic drugs are expressed exclusively in mature cardiomyocytes. As such, future iPS cell-derived cardiomyocyte screening platforms have to effectively recapitulate this electrophysiological and functional maturity of cardiomyocytes.  81 5.3.3 Cell-based regenerative therapy Human iPS cells are a promising cell source for regenerative therapies for heart repair because they can be expanded readily in vitro and differentiated into functional cardiomyocytes [68]. It is estimated that approximately 1 billion new cardiomyocytes are needed for heart repair after a myocardial infarction [69]. Several studies have reported moderate improvements to cardiac heart function in mice treated with factors to induce cardiac fibroblasts to transdifferentiate into cardiomyocytes in situ following a myocardial infarction [23, 24, 77]. Such in situ methods do not face problems with engraftment but runs the risk of generating teratomas [70].   Another approach to attempt cardiac regenerative therapy is to generate iPS cells from patient’s own somatic cells, differentiating those cells into cardiomyocytes, and then forming tissues with those cells. Since these cardiac tissues are derived from the patient’s own cells, issues concerning immunological rejection are avoided. Several methods of cell delivery, such as ‘cell-sheet technique’ [71] and ‘myocardial needle injection’ [72], have been explored but are still in early stages of development. In sum, the optimal cell delivery method or ideal cell type for effective heart repair is still unknown [81, 82].  5.4 Future directions Despite the seemingly well-characterized protocols used to expand and maintain iPS cells, problems associated with variability leading to poor reproducibility were a recurring issue in experiments. Identifying factors and processes that cause such inter and intra experimental variability would be of paramount importance, particularly in the stem cell research field, where experiments are expensive and time-consuming. A recent commentary in Nature Cell Biology has raised significant issue with the current standard of maintaining the quality of pluripotent  82 stem cells and identified two potential sources of variability: 1) the quality of starting population and 2) reagents used [73]. The mTeSR1 medium used in my experiments for example contains bovine serum albumin, which is known to exhibit considerable batch-to-batch variability [74]. Even though the creators of the mTeSR1 medium have made clear of the rigorous quality control steps put in place (particularly for this reason) [75], future experiments would no doubt benefit from the use of a chemically defined, albumin-free medium for stem cell culturing. The TeSR™-E8™ medium, developed by Chen et al. [76], is a feeder-free and animal component-free culture medium suitable for human iPS cell maintenance and when used in combination with improved starting iPS cell characterization, could potentially reduce variability seen in experiments.   Another aspect that warrants further improvement is the way in which specific cardiac protein expressions are quantified and studied. In this thesis, flow cytometry was the lone tool used to quantify maturity by correlating the fluorescence intensity to the amount of cardiac specific protein expressed. Indeed, flow cytometry is a powerful analytical tool capable of providing simultaneous measurements of multiple parameters of cells (e.g. cell size, granularity, and degree of fluorescence of individual cells). However, it remains an end-point analysis, where cells have to be harvested, then fixed and permeabilized prior to intracellular staining. Thus, flow cytometry-based analysis only accommodates single time point quantification of cardiac maturity. On this note, future assays assessing cardiac maturity will certainly benefit from the use of mitochondrial dyes, such as tetramethylrhodamine methyl ester (TMRM), in which cardiac maturation of cells can be inferred from the increasing accumulation of mitochondria per cell. In this manner, resulting cardiomyocyte maturity can be acquired from live cells and in situ, without having to harvest and sacrifice cells. Additionally, using a CRISPR-edited iPS cell line  83 expressing GFP-tagged mitochondria marker (such as TOM20), may also serve to complement this measurement.   Conventional GiWi-differentiated cardiomyocytes are structurally and functionally immature – more comparable to fetal cardiomyocytes than adult [4, 87]. Chapter 3 of my thesis revealed that substrate stiffness modulation promoted the maturation of iPS cell-derived cardiomyocytes – i.e. iPS cells differentiated on hard PDMS substrates yielded significant increase in cTnT expression compared to iPS cells differentiated on soft and intermediate substrates. However, it remains unclear if substrates with specified stiffness have an added advantage over traditional tissue cultureware since no statistical significance was found between iPS cells differentiated on PDMS substrates and conventional polystyrene material. With that in mind, it should be noted that only three variations of substrate stiffness were considered. Thus, future experiments would need to be optimized further to include more stiffness conditions, so as to potentially tease out valuable substrate stiffness induced maturation effects on stem cell differentiation.  A possible link between substrate stiffness and cardiomyocyte subtype specification was also investigated (nodal myocytes vs. working myocytes). Results revealed an inverse trend between substrate stiffness and the proportion of nodal cells generated, assessed by flow cytometry using HCN4 (a nodal cell specific marker). Even though these results did not yield statistical significance, the strong trend observed warrants further investigation. Similar to the preceding paragraph, future experiments will benefit from further optimization to include more stiffness conditions.  Proof-of-concept experiments using PDMS-fabricated millimeter-sized linear channels further demonstrated the profound impact mechanical stimuli have on iPS cell differentiation and  84 development. Freshly GiWi-differentiated iCMs seeded into the linear channels exhibited remarkable plasticity to be manipulated topographically to form single, uniaxially contracting, 3D cardiac tissues. Through this work, a scalable methodology of generating functional human cardiac tissues with clinical relevance (millimeter-sized) was established. The relative simplicity and efficiency of the system, compared to other hPSC-CM engineered heart tissue platforms (see section 1.6 for background on current in vitro 3D heart systems), provides grounds for further investigation of this technology. However, various methodological intricacies (such as harvesting of cardiac strips, optimized iCM seeding density, and optimized channel dimensions etc.) still need to be ironed out.  Apart from the two physical stimuli explored in this thesis (substrate stiffness and topography), previous studies have also implicated other forms of mechanical stimulation, such as fluid stress [29] and mechanical force [25], as factors affecting cardiac maturation of stem cells. Research has also shown that these mechanical stress conditions have a potentially synergistic effect on cellular behavior – i.e. combined multiple stress conditions has an added effect compared to cells individually stimulated [88, 89]. However, most mechanotransduction studies presented in literature (and also of those explored in this thesis) only investigate isolated mechanical stimulation on cells and neglects potentially cumulative effects from multiple stimulatory sources. Therefore, the next appropriate step is to develop a platform capable of delivering multimodal stress stimuli to cells.  Pivoting away from mechanotransduction, chapter 4 describes the development of a novel co-culture method of generating cardiomyocytes from GFP+ve/iPS cells by using previously GiWi-differentiated GFP-ve/iCMs as cardiac inducer cells. To assess the efficiency of this novel system, flow cytometry was used to gate for GFP+ve cells stained for cardiomyocyte  85 markers, cTnT and α-actinin. However, visual inspection by immunocytochemistry (see section 4.2.4) and quantitative analysis by flow cytometry (see section 4.2.5) revealed that the GFP signal from the fluorescently labeled AICS16-iPS cells diminishes upon differentiation to a cardiac lineage. This diminished GFP signal in AICS16-iCMs limits the ability to cleanly gate GFP+ve cells from GFP-ve cells, effectively preventing the assessment of the true differentiation efficiency of the proposed method. Thus, to tackle this issue, one potential strategy is to use an anti-GFP antibody conjugate that binds to the native GFP to boost the diminished GFP signal intensity. This might work to restore the signal of GFP+ve/iCMs to levels high enough for the complete separation of GFP+ve and GFP-ve populations. Another potential solution, that does not require the use of additional antibodies (which may increase the risk of antibody cross reactivity), is to use a different GFP-tagged iPS cell line – one where GFP is labeled on a different protein and intensity unaffected by differentiation. A potential candidate is the AICS11 iPS cell line, which is a human iPS cell line with outer mitochondrial membrane receptors (TOM20) fluorescently tagged with mEGFP.  Future investigation of this novel co-culture method would also include sorting GFP-ve/iCMs (‘older’ seeded cardiomyocytes) into its individual subpopulations (ventricular, atrial, nodal, and cardiac fibroblasts); to determine which cell populations facilitates differentiation. This can be achieved using fluorescence-activated cell sorting (FACS). Myosin heavy chain, α isoform (MHC-α) and myosin heavy chain, β isoform (MHC-β) can be used as markers to separate atrial and ventricular myocytes, respectively [77], while HCN4 can be used as a nodal cell marker [57]. Thymocyte differentiation antigen 1 (THY1) may be used as a surface marker for cardiac fibroblasts [78]. It should be noted that if the co-culture differentiation method fails to initiate cardiac differentiation in iPS cells when seeded with these individually sorted cells, it  86 would suggest that a heterogeneous mix of cell types is not only sufficient, but also necessary, for cardiac differentiation of iPS cells.    5.5 Conclusion Results of my work have showed that iPS cells and its derivatives are sensitive to substrate stiffness, topography, and co-culture with appropriate inducer cells. 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J Mol Cell Cardiol, 42, 5 (May 2007), 991-1000.    96 Appendix A    Supplementary Experiments and Results  A.1 Characterization of differentiation efficiency of novel co-culture method by flow cytometry – independent replicate    Figure A.1: Flow cytometric analysis of various culture conditions for α-actinin cardiac marker (GFP β-actin vs. α-actinin). (A) Positive controls – from left to right: IMR90-4 GiWi differentiated, and AICS-0016 GiWi differentiated. (B) Pre-cultured cells showing ~1.0% of α-actinin+ve AICS-0016 cells (conservative assessment). (C) Negative controls – from left to right: IMR90-4 iPSCs, AICS-0016 iPSCs, and AICS-0016 cells treated with only basal medium + supplement change (no differentiation factors added). (C)	Nega)ve	controls	GFP	β-ac)n	IMR90	-	iPSC	 AICS16	-	iPSC	 AICS16	–	Medium	change	(A)	Posi)ve	controls	IMR90	-	GiWi	 AICS16	-	GiWi	GFP	β-ac)n	(B)	Pre-culture	method	AICS16	–	Pre-cultured	GFP	β-ac)n	Quandrant 10.20Quandrant 20Quandrant 30.18Quandrant 499.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA03 IMR90-iPSC-a act.fcsCells16480Quandrant 10.020Quandrant 20.20Quandrant 398.3Quandrant 41.40100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA06 AICS16-iPSC-a act.fcsCells24578Quandrant 146.3Quandrant 20Quandrant 30.064Quandrant 454.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB03 IMR90-GiWI iCM-a act.fcsCells18764Quandrant 120.1Quandrant 22.20Quandrant 369.5Quandrant 48.76100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB06 AICS16-GiWI iCM-a act.fcsCells15891Quandrant 11.57Quandrant 20.95Quandrant 389.3Quandrant 48.22100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AC06 Pre culture day 40-a act.fcsCells18751Quandrant 10Quandrant 20Quandrant 397.9Quandrant 41.96100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AD03 Media change-a act.fcsCells6384Negative ControlsPositive ControlsPre-cultureQuandrant 10.20Quandrant 20Quandrant 30.18Quandrant 499.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA03 IMR90-iPSC-a act.fcsCells16480Quandrant 10.020Quandrant 20.20Quandrant 398.3Quandrant 41.40100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA06 AICS16-iPSC-a act.fcsCells24578Quandrant 146.3Quandrant 20Quandrant 30.064Quandrant 454.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB03 IMR90-GiWI iCM-a act.fcsCells18764Quandrant 120.1Quandrant 22.20Quandrant 369.5Quandrant 48.76100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB06 AICS16-GiWI iCM-a act.fcsCells15891Quandrant 11.57Quandrant 20.95Quandrant 389.3Quandrant 48.22100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AC06 Pre culture day 40-a act.fcsCells18751Quandrant 10Quandrant 20Quandrant 397.9Quandrant 41.96100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AD03 Media change-a act.fcsCells6384Negative ControlsPositive ControlsPre-cultureQuandrant 10.20Quandrant 20Quandrant 30.18Quandrant 499.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA03 IMR90-iPSC-a act.fcsCells16480Quandrant 10.020Quandrant 20.20Quandrant 398.3Quandrant 41.40100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA06 AICS16-iPSC-a act.fcsCells24578Quandrant 146.3Quandrant 20Quandrant 30.064Quandrant 454.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB03 IMR90-GiWI iCM-a act.fcsCells18764Quandrant 120.1Quandrant 22.20Quandrant 369.5Quandrant 48.76100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB06 AICS16-GiWI iCM-a act.fcsCells15891Quandrant 11.57Quandrant 20.95Quandrant 389.3Quandrant 48.22100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AC06 Pre culture day 40-a act.fcsCells18751Quandrant 10Quandrant 20Quandrant 397.9Quandrant 41.96100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AD03 Media change-a act.fcsCells6384Negative ControlsPositive ControlsPre-cultureQuandrant 10.20Quandrant 20Quandrant 30.18Quandrant 499.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA03 IMR90-iPSC-a act.fcsCells16480Quandrant 10.020Quandrant 20.20Quandrant 398.3Quandrant 41.40100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA06 AICS16-iPSC-a act.fcsCells24578Quandrant 146.3Quandrant 20Quandrant 30.064Quandrant 454.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB03 IMR90-GiWI iCM-a act.fcsCells18764Quandrant 120.1Quandrant 22.20Quandrant 369.5Quandrant 48.76100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB06 AICS16-GiWI iCM-a act.fcsCells15891Quandrant 11.57Quandrant 20.95Quandrant 389.3Quandrant 48.22100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AC06 Pre culture day 40-a act.fcsCells18751Quandrant 10Quandrant 20Quandrant 397.9Quandrant 41.96100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AD03 Media change-a act.fcsCells6384Negative ControlsPositive ControlsPre-cultureQuandrant 10.20Quandrant 20Quandrant 30.18Quandrant 499.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA03 IMR90-iPSC-a act.fcsCells16480Quandrant 10.020Quandrant 20.20Quandrant 398.3Quandrant 41.40100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA06 AICS16-iP C-a act.fcsCells24578Quandrant 146.3Quandrant 20Quandrant 30.064Quandrant 454.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB03 IMR90-GiWI iCM-a act.fcsCells18764Quandrant 120.1Quandrant 22.20Quandrant 369.5Quandrant 48.76100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB06 AICS16-GiWI iCM-a act.fcsCells15891Quandrant 11.57Quandrant 20.95Quandrant 389.3Quandrant 48.22100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AC06 Pre culture day 40-  act.fcsCells18751Quandrant 10Quandrant 20Quandrant 397.9Quandrant 41.96100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AD03 Media change-a act.fcsCells6384Negativ  ControlsPositive ControlsPre-cultureQuandrant 10.20Quandrant 20Quandrant 30.18Quandrant 499.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA03 IMR90-iPSC-a act.fcsCells16480Quandrant 10.020Quandrant 20.20Quandrant 398.3Quandrant 41.40100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AA06 AICS16-iPSC-a act.fcsCells24578Quandrant 146.3Quandrant 20Quandrant 30.064Quandrant 454.4100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB03 IMR90-GiWI iCM-a act.fcsCells18764Quandrant 120.1Quandrant 22.20Quandrant 369.5Quandrant 48.76100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AB06 AICS16-GiWI iCM-a act.fcsCells15891Quandrant 11.57Quandrant 20.95Quandrant 389.3Quandrant 48.22100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AC06 Pre culture day 40-a act.fcsCells18751Quandrant 10Quandrant 20Quandrant 397.9Quandrant 41.96100 102 104 106FL1-A :: FL1-A100101102103104105106107FL4-A :: FL4-AD03 Media change-a act.fcsCells6384Negative ControlsPositive ControlsPre-culture 97 Table A.1: Cardiac differentiation efficiency of various conditions quantified via flow cytometry for α-actinin. The reported percentages are the sum of Q1 and Q2, which includes both GFP+ve and GFP-ve cells.   * Not applicable (n/a) because ungated population (Q1 + Q2) of pre-cultured cells consists  of a mixture of IMR90-4 and AICS-0016. Thus, differentiation efficiency is not  assessable.   Table A.2: Cardiac differentiation efficiency of various conditions quantified via flow cytometry for α-actinin and GFP+ve cells. The reported percentages are Q2 gated cells – i.e. only cells that are GFP+ve. Novel pre-culture method gave 1.0% α-actinin+ve cells respectively after 26 days in culture. Standard GiWi protocol yielded 2.2% α-actinin+ve in the same time frame. Assessment was performed in a conservative manner and only cells in Q2 (GFP+ve and α-actinin+ve) were considered as true positives.   ** Experiment independently replicated (see appendix section 4.2.5 for details).α-ac%nin	IMR90-4	GiWi	 iPSCs	AICS-0016	GiWi	 iPSCs	 Medium	change	Pre-culture	46.3%	 0.2%	 22.3%	 0.2%	 0.0%	 n/a	*	α-ac%nin	IMR90-4	GiWi	 iPSCs	AICS-0016	GiWi	 iPSCs	 Medium	change	Pre-culture	0.0%	 0.0%	 2.2%	 0.2%	 0.0%	 1.0%	**	 98 Appendix B    Cell Culture Protocols  B.1 iPS cell maintenance, expansion, and differentiation protocols Without the use of a non-pluripotent feeder cell layer, a thin coating of Matrigel was used to facilitate the attachment of iPS cells to well surface. Matrigel gels rapidly above 10oC and care must be taken to ensure its proper coating.  B.1.1 Matrigel aliquoting and storage 1. One day prior to aliquoting, place Matrigel in a 4oC refrigerator to thaw. Additionally, place any cultureware that may come into contact with Matrigel into a 4oC refrigerator to be pre-chilled. 2. Calculate the volume of Matrigel to be aliquoted per tube. Based on manufacturer’s recommendations, for thin Matrigel coat, use 0.5mg Matrigel per 6-well plate. Matrigel concentration varies by lot and thus, aliquot volume should be calculated for every new lot obtained. 3. On the day of aliquoting, place all pre-chilled cultureware and Matrigel into a biosafety cabinet. Matrigel should be placed on ice to keep it chilled. 4. Aliquot Matrigel into tubes at appropriate volumes calculated above and ensure pipette tips remain cold during the process by switching after every 4 – 5 tubes. 5. Placed aliquoted tubes into -20oC freezer.   99 B.1.2 Matrigel aliquoting and storage Do not thaw and refreeze Matrigel. As such each aliquot must be used all at one time. 1. One day prior plating Matrigel, pre-chill 6-well plates and any cultureware that may come into contact with Matrigel at 4oC. 2. On the day of Matrigel plating, bring Matrigel aliquot, blank DMEM/F-12 medium, and P1000 pipetman into a biosafety cabinet. 3. Using the P1000 pipetman, add 1mL blank DMEM/F-12 medium to Matirgel aliquot to dissolve it. 4. Transfer the mixture to a 15mL conical tube and bring volume to 1mL for each well to be coated with blank DMEM/F-12 medium. 5. Add diluted Matrigel solution to each well and rock plate in orthogonal direction to ensure even coating. 6. Allow Matrigel to set for 1 hour at room temperature in the biosafety cabinet before cell seeding.  B.1.3 Preparing mTeSR1 medium Mixing the basal component with its 5X supplement component makes complete mTeSR1 medium. The 5X supplement contains recombinant proteins that degrade rapidly at high temperatures and repeated freeze-thaw cycles. As such, the complete mTeSR1 medium should be kept frozen and thawed only when ready for use.  1. Thaw the 5X supplement at 4oC overnight and mix by inverting it 3 – 4 times. 2. Add the 5X supplement to the basal mTeSR1 medium and mix thoroughly by inverting 5 – 10 times.   100 3. Aliquot complete mTeSR1 medium into 50mL conical tubes and store at -20oC freezer. 4. When needed, thaw mTeSR1 aliquots at 4oC overnight. 5. Unused complete mTeSR1 medium can be stored at 4oC for up to 2 weeks.  B.1.4 Preparing ROCK inhibitor aliquots Inhibitor of ROCK (Y-27632) was used to reduce anoikis and improve cell viability when plating iPS cells. 1. When received, centrifuge the Y-27632 powder at 9,000g for 30 seconds.  2. Under sterile conditions, add 624µL PBS to Y-27632 powder. 3. Pipette up and down to ensure powder is fully dissolved. 4. Aliquot stock solution into 8 microtubes of equal volume. 5. Store aliquots at -20oC freezer.  B.1.5 Thawing and plating iPS cells For long-term storage, iPS cells are stored in liquid nitrogen. To prevent potential injury, eye protection should be used when removing iPS cell vials from liquid nitrogen storage. 1. Pre-warm complete mTeSR1 medium at room temperature for at least 15 minutes. 2. Remove iPS cell vial from liquid nitrogen storage and roll between gloved hands to remove frost.  3. Place iPS cell vial in a 37oC water bath and thaw cell solution until a single ice crystal remains.  4. Clean vial dry with a dry towel and spray vial down with 70% ethanol to sterilize. 5. In the biosafety cabinet, transfer cells into a 15mL conical tube.  101 6. Gently add 9mL room temperature mTeSR1 medium drop wise. 7. Spin down the tube at 300g for 5 minutes to obtain cell pellet. 8. Aspirate supernatant and resuspend cells with 6mL of mTeSR1 medium. 9. Add 12µL of 5mM Rock inhibitor stock solution to a final concentration of 10µM. 10. Vortex gently to mix and dispense cell solution onto Matrigel coated 6-wells. (Note: DMEM/F-12 medium must be aspirated prior to seeding of iPS cells). 11. Place plate into an incubator and gently rock plate in orthogonal direction to ensure even distribution of cells.  B.1.6 Maintaining iPS cells In order to ensure undifferentiated state of iPS cells, mTeSR1 medium must be renewed daily. 1. Prior to feeding iPS cells, pre-warm mTeSR1 medium at room temperature for at least 15 minutes. 2. Aspirate spend mTeSR1 medium from well and gently add 2mL of fresh mTeSR1 medium to each 6-well. 3. Return plate to 37oC incubator.  B.1.7 Passaging iPS cells When iPS cells reach ~70 – 80% confluence, cells should be passaged to prevent unspecific differentiation. Passage ratio varies from 1:3 – 1:12, depending on application. Typically, cells were split at a larger ratio for expansion purposes and at a lower ratio for use in differentiation experiments. 1. Prior to passage, pre-warm mTeSR1 medium at room temperature for at least 15 minutes.  102 2. Aspirate spent mTeSR1 medium from 6-wells and rinse each well with 1mL EDTA. 3. Aspirate EDTA and add an addition 1mL EDTA and treat iPS cells for 7 minutes at room temperature. 4. Gently aspirate EDTA without disturbing cell layer. If cells become free floating, collect cells by pipetting, spin down, remove EDTA, and resuspend with mTeSR1 medium. 5. Using 3mL of mTeSR1 medium per well, wash cells off of well surface. 6. Resuspend cells and transfer into a 15mL conical tube.  7. Using additional mTeSR1 medium, bring volume up to appropriate level for passaging – i.e. 2mL per 6-well. 8. Add ROCK inhibitor to a final concentration of 10µM. 9. Gently mix cell suspension by vortexing.  10. Add cells drop wise to Matrigel coated 6-wells (Note: DMEM/F-12 medium should be aspirated prior to adding cells). 11. Place plate in incubator and rock orthogonally to ensure even distribution of cells.  B.1.8 Freezing iPS cells When iPS cells reach ~80% confluence, cells can be cryofrozen. 1. Place cryovials in a biosafety cabinet and label them with the cell line, passage number, and freeze date. 2. Thaw iPS cell cryofreeze medium, mFeSR1, at room temperature in biosafety cabinet.  3. Remove iPS cell plates from incubator and rinse wells to be frozen down with EDTA. 4. Aspirate and add 1mL of EDTA for 7 minutes at room temperature. 5. Aspirate EDTA carefully without disturbing cell layer.  103 6. Gently wash cells off with 3mL of mFeSR1 medium for each well and pool cells together in a 15mL conical tube. 7. Add mFeSR1 medium to desired freezing density. Typically, one 6-well plate can be frozen down to 5 – 10 vials.  8. Using a P1000 pipetman, transfer 1mL of cell suspension into each cryovial.  9. Place cryovials into an isopropanol freezing container and place the container in -80oC freezer overnight.  10. The following day, transfer the vials into liquid nitrogen for long-term storage.   B.1.9 Preparing CHIR99021 (Wnt pathway activator, inhibits GSK3) The small molecule CHIR99021 is used as the first of two Wnt signaling molecules to initiate differentiation of iPS cells to iCMs. 1. Spin down CHIR99021 powder at 10,000rpm for 1 minute. 2. Proceed to biosafety cabinet. 3. Add 298µL DMSO to 5mg CHIR99021 to get a stock concentration of 36mM. 4. Aliquot 30µL samples into 1.5mL tubes and store at -20oC freezer. Product is stable for up to 1 year.  B.1.10 Preparing IWP-2 (Wnt pathway inhibitor, inhibits porcupine) The small molecule IWP-2 is used as the second of two Wnt signaling molecules to initiate differentiation of iPS cells to iCMs. 1. Spin down IWP-2 powder at 10,000rpm for 1 minute. 2. Proceed to biosafety cabinet.  104 3. Add 428µL DMSO to 1mg IWP-2 to get a stock concentration of 5mM. 4. Incubate the mixture at 37oC for 10 minutes to dissolve IWP-2 completely. 5. Aliquot 50µL samples into 1.5mL tubes and store at -20oC freezer. Product is stable for up to 1 year.  B.1.11 Preparing B-27 supplements The GiWi differentiation protocol requires RPMI-1640 medium supplemented with B-27, with or without insulin at different steps. Section 2.1.5 details when each supplement is required. B-27 supplement is light sensitive and thus, should be protected accordingly.  1. Upon receipt, aliquot B-27 supplements into 400µL aliquots and store at -20oC freezer. 2. When needed, thaw 400µL B-27 aliquots at room temperature in a biosafety cabinet. 3. To make RPMI/B-27 supplemented medium, add the 400µL B-27 aliquot to 19.6mL of RPMI-1640 medium in a 50mL conical tube. 4. Use an aluminum foil to wrap the conical tube and protect from light. 5. Invert conical tube gently 5 – 10 times to mix. 6. Unused complete RPMI/B-27 supplemented medium can be stored at 4oC for up to 2 weeks. 7. Prior to addition of RPMI/B-27 supplemented medium to cells, the medium should be pre-warmed for at least 15 minutes at room temperature. Do not warm medium in a 37oC water bath.   105 B.2 Labeling iPS cells with CellTracker red dye The CellTracker™ Red CMTPX fluorescent dye (ThermoFisher Scientific #C34552) was used for monitoring and tracking of individual IMR90-4 iPS cells in co-culture experiments. Below outlines the labeling protocol used: 1. Before opening the dye vial, allow the product to warm to room temperature. 2. To prepare the stock solution, dissolve the lyophilized product in cell culture grade DMSO to a concentration of 10mM (i.e. 50µg of dye product in 7.28µL DMSO). 3. To prepare working solution, dilute the stock solution in mTeSR1 medium to a final concentration of 5µM (i.e. add 2.5µL of 10mM stock solution in 5mL of mTeSR1 medium). 4. Remove medium from iPS cell well and wash once with room temperature PBS. 5. Remove PBS and add 1mL of CellTracker working solution per 1 million cells (~3mL per 6-well of iPS cells at 80 – 90% confluence).  6. Incubate cells for 30 minutes at 37oC.  7. Wash cells with room temperature or pre-warmed PBS. 8. Remove PBS and add 1mL room temperature mTeSR1 medium per 1 million cells (~3mL per 6-well of iPS cells at 80 – 90% confluence). Incubate cells for another 30 minutes at 37oC. 9. Wash cells with room temperature or pre-warmed PBS. 10. Remove PBS and add 1mL room temperature mTeSR1 medium per 1 million cells (~3mL per 6-well of iPS cells at 80 – 90% confluence).  11. Incubate cells for at least 24 hours at 37oC. 12. iPS cells are now fluorescently labeled and ready for use.  106  B.3 Immunocytochemistry staining of cells adhered on coverslip To visualize specific target proteins within cells, immunocytochemistry was used. Protocol outlined below: 1. Prepare 3.7% formaldehyde by diluting 37% formaldehyde with PBS – e.g. 1mL 37% formaldehyde in 9mL PBS. Note: MUST prepare fresh.  2. Add 1mL of PBS coverslip to be stained and rinse gently by moving PBS orthogonally and then aspirating.  3. Add 2mL of 3.7% formaldehyde to well for 15 minutes at room temperature.  4. Drain and wash twice in PBS. 5. Prepare 0.1% TX-100/PBS by diluting 10% TX-100 with PBS – e.g. 100µL 10% TX-100 in 10mL PBS. 6. Treat cells with 0.1% TX-100 for 5 minutes at room temperature. 7. Aspirate TX-100 and wash cells 3 times with 1mL PBS. 8. Attach an appropriately sized parafilm to a flat plastic plate. 9. Add 40µL 1%BSA/PBS to parafilm and place glass cover slip on it (this step ensures glass is properly adhered and does not move around). 10. Add 200µL 1%BSA/PBS blocking buffer to cells on coverslip for 20 minutes at room temperature (blocking step). 11. Prepare primary antibody by diluting antibody with 1%BSA/PBS to appropriate concentration – e.g. cTnT 1:100, HCN4 1:2, α-actinin 1:800. 12. Aspirate 1%BSA/PBS solution from glass coverslips by aspirating at the edge of the coverslips with 200µL pipet tips.  107 13. Add primary antibody, 200µL to each coverslip for 1 hour at room temperature or overnight at 4oC.  14. Remove primary antibody and wash coverslip with PBS 3 times with 0.5mL PBS. Note: Note: Primary antibody can be reused and pipetted into an eppendorf tube and stored. 15. Dilute secondary antibody at 1:250 dilution with 1%BSA/PBS. Note: other dyes like phalloidin can be added at this step as well. 16. Add 200µL secondary antibody onto coverslips for 1 hour at room temperature, placed in the dark.  17. Aspirate secondary antibody and wash with 0.5mL PBS 3 times. 18. Add 60µL mounting media (Prolong Gold) onto glass slide.  19. Dip coverslip in 10mM Tris to remove excess salts. 20. Drain tris well by tapping edge of coverslip on a piece of dry towel. 21. Flip cell side down onto mounting media and place gently. Note: do not press coverslip and let capillary action to the job.  22. Leave slide and coverslip to cure overnight at room temperature in the dark before imaging.  B.4 Intracellular staining for flow cytometry To quantify specific target proteins within cells, intracellular staining of cells was performed and analyzed using flow cytometry. The protocol used is outlined below:  1. Warm complete RPMI, 0.05% trypsin, and PBS in 37oC water bath for 20 minutes. 2. Prepare BD Perm/Wash™ buffer by diluting with strile deionized water. 3. Remove spent medium from well of cells and rinse with 2mL PBS.  108 4. Add 1mL 0.05% trypsin and place well in incubator (Note: for iPS cells, 5 minutes is enough time to dissociate cells. For iCMs, may take up to 9 minutes or longer for cells to fully dissociate).  5. Remove well from incubator and add 2mL neutralizing medium (complete RPMI). 6. Pipette up and down with P1000 pipettman to wash cells off the well. 7. Pipette cell suspension into a 15mL conical tube. 8. Centrifuge for 5 minutes at 300g and aspirate supernatant.  9. Resuspend pellet with 2mL PBS and centrifuge for 5 minutes at 300g.  10. Remove supernatant and add 250µL BD Cytofix/Cytoperm to resuspend cells. 11. Place tube at 4oC cold room in the dark for 20 minutes.  12. Add 1mL Perm/Wash solution and centrifuge for 5 minutes at 300g. 13. Aspirate supernatant and add Perm/Wash solution and aliquot into desired number of FACS tubes. 14. Prepare primary antibody by diluting antibody with Perm/Wash solution to appropriate concentration – e.g. cTnT 1:100, HCN4 1:2, α-actinin 1:800. 15. Centrifuge FACS tubes for 5 minutes at 300g and aspirate supernatant. 23. Resuspend cells with 100µL primary antibody solution and place at 4oC cold room for 30 minutes. 24. Add 1mL of Perm/Wash solution and centrifuge for 5 minutes at 300g.  25. Prepare secondary antibody by duliting at 1:250 with Perm/Wash solution. 26. Aspirate supernatant and resuspend cells with 100µL primary antibody solution and place at 4oC cold room for 30 minutes.   109 27. Add 1mL of Perm/Wash solution and centrifuge for 5 minutes at 300g (wash cells twice for optimal flow cytometry results). 28. Resuspend cell pellet in 150µL Perm/Wash and proceed to flow cytometer.  

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