International Journal of Molecular SciencesArticleEnhanced Osteogenic Differentiation of Pluripotent Stem Cellsvia γ-Secretase InhibitionSummer A. Helmi 1,2, Leili Rohani 3, Ahmed R. Zaher 2, Youssry M. El Hawary 2 and Derrick E. Rancourt 1,*



Citation: Helmi, S.A.; Rohani, L.;Zaher, A.R.; El Hawary, Y.M.;Rancourt, D.E. Enhanced OsteogenicDifferentiation of Pluripotent StemCells via γ-Secretase Inhibition. Int. J.Mol. Sci. 2021, 22, 5215. https://doi.org/10.3390/ijms22105215Academic Editor: Rivka OfirReceived: 19 March 2021Accepted: 10 May 2021Published: 14 May 2021Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2021 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).1 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada;summer.helmi@ucalgary.ca2 Department of Oral Biology, Faculty of Dentistry, Mansoura University, Mansoura 35516, Egypt;ahmedrz2016@hotmail.com (A.R.Z.); Yhawary2007@mans.edu.eg (Y.M.E.H.)3 Department of Medicine, School of Biomedical Engineering, The University of British Columbia,Vancouver, BC V6T 1Z3, Canada; leili.rohani@ubc.ca* Correspondence: rancourt@ucalgary.ca; Tel.: +1-403-220-2888Abstract: Bone healing is a complex, well-organized process. Multiple factors regulate this process,including growth factors, hormones, cytokines, mechanical stimulation, and aging. One of the mostimportant signaling pathways that affect bone healing is the Notch signaling pathway. It has asignificant role in controlling the differentiation of bone mesenchymal stem cells and forming newbone. Interventions to enhance the healing of critical-sized bone defects are of great importance, andstem cell transplantations are eminent candidates for treating such defects. Understanding how Notchsignaling impacts pluripotent stem cell differentiation can significantly enhance osteogenesis andimprove the overall healing process upon transplantation. In Rancourt’s lab, mouse embryonic stemcells (ESC) have been successfully differentiated to the osteogenic cell lineage. This study investigatesthe role of Notch signaling inhibition in the osteogenic differentiation of mouse embryonic andinduced pluripotent stem cells (iPS). Our data showed that Notch inhibition greatly enhanced thedifferentiation of both mouse embryonic and induced pluripotent stem cells.Keywords: embryonic stem cells; iPS; notch signaling; osteogenic differentiation; regenerative medicine1. IntroductionStem cell therapy is an attractive alternative approach used for bone repair. The in-volvement of stem cells in the healing process is critical for complicated non-union fracturesresulting from trauma, blood insufficiency, and defects associated with chronic diseasesthat significantly impact the healing process, such as diabetes and osteoporosis [1,2].Loading scaffolds with bone marrow-derived mesenchymal stem cells (MSC) en-hanced bone defect healing compared to using the scaffold without MSC [3–5]. Whilesuccessful, the MSC harvesting process, the number of cells harvested, and the tendencyof this type of cells to senesce after a limited number of passages made the search foralternative regenerative cells necessary [6].Embryonic stem cells (ESC) can differentiate into all three germ layers. The highproliferation potential and the tremendous self-renewal ability give these cells an advantageover MSC. ESC have been applied for bone regeneration and proved to be an ideal cellsource for bone repair [7,8]. However, the derivation of human ESC and the ethical debatearound the destruction of human embryos make these cells’ clinical use impossible [9].Accordingly, induced pluripotent stem cells (iPS) became an alternative to overcome thedisadvantages of ESC. Takahashi and Yamanaka used four transcription factors (Oct4, Sox2,Klf4, and c-Myc) to reprogram fibroblasts back to pluripotency. The produced cell line hadthe same enormous differentiation capacity as ESC [10]. Moreover, iPS bypass the ethicaldilemma of destroying human embryos and can be derived from patients to overcomegraft rejection. All the criteria mentioned above make iPS a superior cell transplant choiceto enrich scaffolds used for bone repair [11,12].Int. J. Mol. Sci. 2021, 22, 5215. https://doi.org/10.3390/ijms22105215 https://www.mdpi.com/journal/ijmsInt. J. Mol. Sci. 2021, 22, 5215 2 of 18The osteogenic differentiation of bone progenitor cells and bone marrow mesenchymalstem cells (MSC) results from the interaction between different types of cell signaling. Forexample, endocrine signaling like hormones, such as parathyroid hormone (PTH) [13], sexhormones [14,15], and glucocorticoids [16,17]. Besides, paracrine and autocrine signalingare also involved in the differentiation process through local growth factors like bonemorphogenetic proteins (BMP), transforming growth factor-beta (TGF-β), and fibroblastgrowth factor-2 (FGF-2) [1,18,19]. The type of interaction causes a cascade of intercellularand intracellular events that result in the differentiation of MSC and osteogenic progenitorsto become bone [20].The Notch signaling pathway has fundamental roles in several developmental pro-cesses. This signaling pathway acts in a paracrine manner wherein a ligand-bearing cellsignals a receptor-bearing neighbor. This way, Notch signaling maintains and regulatesstem cells, cell differentiation, and cellular homeostasis. It contributes to the regulationof tissue balance and the maintenance of stem cells in adults. It also regulates cell dif-ferentiation, proliferation, survival, and apoptosis. These essential functions take placethrough “lateral inhibition” and “boundary induction” [21,22]. In “lateral inhibition”,Notch signaling contributes to binary cell fate choices in cells, which are developmentallyequivalent. This mechanism occurs by the inhibition of one fate in some cells and allowingthem to adopt another different fate, which means that the cell that adopts the alternativefate blocks this choice in its neighbors [23,24]. “In boundary induction”, Notch signalinginduces new cell fates rather than selecting from two alternative ones [21,25].Canonical Notch signaling consists of the Notch receptors Notch1, Notch2, Notch3,and Notch4. These receptors are transmembrane proteins that bind to Notch ligandsJagged1, Jagged2, Delta-like1, Delta-like3, and Delta-like 4 of the neighboring cells. Ligand-receptor binding promotes a proteolytic reaction where a TACE/ADAM10 and γ-secretasepresenilins cleave the Notch intracellular domains (NICD) [26]. NICD then translocates tothe nucleus to bind to DNA-binding protein CSL (CBF1/Suppressor of Hairless/LAG-1)to regulate the target genes. The most popular of these target genes are the basic helix-loop-helix (bHLH) transcriptional repressors of the Hairy enhancer of split (HES) andHairy-related (Hrt) protein families (HEY) [27]. While Notch activation promotes stem cellself-renewal [28], Notch function depends on the stage of development, cell type, and cellstate [22,29].Several studies described the role of Notch signaling in osteoblast fate determination ofMSC in the literature with opposing results. Some studies concluded that Notch signalingwas essential for stem cell renewal and osteoblast progenitor pool maintenance. Osteoblastdifferentiation increased upon inhibition of some or all of the Notch signaling cascade.Moreover, Notch activation impaired the differentiation, maturation, and matrix productionof osteoblasts [30–32]. On the other hand, the results of various in vitro and in vivo studiesindicated that activation of Notch signaling enhanced the differentiation of MSC andprecursor cell lines [33]. Notch activation also improved matrix mineralization and boneremodeling in animal models [34–36].These studies proposed that the effects of Notch signaling in bone regeneration are cellcontext-dependent. The Notch signaling pathway has different outcomes depending onthe differentiation status of target cells and the niche's nature wherein the cells self-renewand differentiate in terms of bone health or disease. However, how Notch carries out suchdiverse roles in bone cells remains elusive. In the current study, we used DAPT γ-secretaseinhibitor to inhibit the Notch signaling pathway and observed the consequent effects onthe differentiation of embryonic and induced pluripotent stem cells to osteoblasts.2. Results2.1. Notch Inhibition Synergizes Dexamethasone Mediated More Than Vitamin D MediatedOsteogenic DifferentiationWe compared two osteogenic differentiation media combined with DAPT Notchinhibitor to optimize the differentiation protocol used in this study. DAPT efficiently blocksthe presenilin–γ-secretase complex [37] and prevents Notch signaling activation [38,39].Int. J. Mol. Sci. 2021, 22, 5215 3 of 18On day 10 of osteogenic differentiation, both differentiation media showed a significantincrease in bone marker gene expression compared to controls and cell cultures whereDAPT was absent. D3 embryoid bodies (EBs) cultured in Dexamethasone (DEX) combinedwith DAPT showed higher expression of the early osteogenic marker RUNX2 (p < 0.0001)and the late osteogenic markers OCN (p < 0.0001) and SPARC (Osteonectin) (p < 0.0001)compared to cultures that received both vitamin D (VITD) and vitamin D combined withDAPT (Figure 1A). To confirm these results, on day 30 of osteogenic differentiation, cellscultured in both types of osteogenic media combined with DAPT were fixed and stainedwith Alizarin Red stain to confirm the differentiation of D3 cells to active osteoblastscapable of the formation of calcified bone matrix. The calcified matrix was stained in red.We captured images of the stained cultures, and the stained surface area was calculatedusing ImageJ software. Cells differentiated under the effect of Dexamethasone and DAPTshowed wider stained surface area than cells differentiated under the impact of vitamin Dcombined with DAPT and control cultures that did not receive the Notch inhibitor (p = 0.03)(Figure 1B,C). Based on the previous results, we continued to use dexamethasone-basedmedia for the rest of this study.Figure 1. Evaluation of differentiation with Dexamethasone (DEX) and Vitamin D (VITD) based osteogenic media combinedwith DAPT Notch inhibitor of D3 ES cells. (A) Osteogenic gene expression with different media types at 10 days ofosteogenic differentiation for the genes RUNX2 (p < 0.0001), Osteocalcin (OCN) (p < 0.0001), and SPARC (Osteonectin)(p < 0.0001) (n = 3). Dexamethasone with DAPT (D10-DEX.DAPT), Dexamethasone without DAPT (D10-DEX), vitamin D3with DAPT (D10-VITD.DAPT), and vitamin D3 without DAPT (D10-VITD). (B) Alizarin Red staining (A.R) for cell cultureson different types of osteogenic media at day 30 of osteogenic differentiation, scale bar 100 µm. (C) Quantification of surfacearea positive for Alizarin Red staining (p < 0.05). * = p < 0.05, **** = p < 0.0001.Int. J. Mol. Sci. 2021, 22, 5215 4 of 182.2. Effect of Notch Inhibition on the Bone Marker Gene Expression during the OsteogenicDifferentiation of Embryonic and Induced Pluripotent Stem CellsTo demonstrate the effect of Notch inhibition on the various time points duringosteogenic differentiation of embryonic and induced pluripotent stem cells, we performedqRT-PCR starting on day 5 and up to day 30 with five-day intervals in between. Geneexpression fold changes of the early marker RUNX2 and the late markers OCN and SPARCwere quantified. On day 5 of differentiation, Notch inhibition increased the expression ofRUNX2 (p = 0.02) and SPARC (p = 0.03) for embryonic and induced pluripotent stem cells,while this effect was not demonstrated for the late marker OCN (p = 0.1). On day 10, thedifference in expression of RUNX2 and SPARC increased in the Notch inhibition groupsin both D3 ESC and iPS (p = 0.007), compared to the groups with no inhibition. At thistime point, OCN gene expression was significantly increased in both cell lines with Notchinhibition compared to the groups where no inhibitor was added (p = 0.002) (Figure 2A,B).As the differentiation process progressed, on days 15 and 25, gene expression foldchange continued to be higher in the D3 and iPS samples where the Notch inhibitor wasadded for the genes RUNX2 (p < 0.0001 on day 15, p < 0.0001 on day 25), SPARC (p < 0.0001on day 15, p < 0.0001on day 25), and OCN (p < 0.0001 on 15 days, p = 0.0003 on day 25 )when compared to the samples without the Notch inhibitor (Figure 2C,D). Moving forward,and on day 30 of osteogenesis and as the differentiation process reached a late stage, theD3 and iPS samples where the Notch inhibitor was added showed higher expression ofRUNX2 (p = 0.01) and SPARC (p < 0.0001) when compared to the samples where no Notchinhibition took place. This was not the case for OCN; Notch inhibition did not increaseOCN expression at this point for the cell lines under investigation (p < 0.0001) (Figure 2E).2.3. Detection of the Effect of Notch Inhibition on Bone Protein Expression during the OsteogenicDifferentiation ProcessTo identify the effect of Notch inhibition on bone protein expression and to furtherconfirm the previous results, immunofluorescent staining and confocal microscopy wereapplied to the early differentiated ESC and iPS cell cultures on day 10 and the late differen-tiated cell cultures on day 30. The staining was against the early osteogenic marker RUNX2(Figure 3) and the late markers SPARC (Osteonectin) (Figure 4), and Osteocalcin (OCN)(Figure 5). For early cultures, fluorescence quantification showed increased expression ofthe RUNX2 (p < 0.0001 for ESC, p < 0.0001 for iPS), SPARC (p = 0.0004 for ESC, p < 0.0001 foriPS), and OCN (p = 0.02 for ESC, p = 0.0002 for iPS) when compared to cultures where Notchinhibition was absent for ESC and iPS. On day 30, fluorescence quantification revealed thesame augmented effect regarding the expression of the three proteins RUNX2 (p < 0.0001for ESC, p < 0.0001 for iPS), OCN (p = 0.01 for ESC, p < 0.0001 for iPS), and SPARC (p = 0.002for ESC, p = 0.03 for iPS) in the cell cultures that received the Notch inhibitor. This outcomewas observed in both ESCs and iPS.Int. J. Mol. Sci. 2021, 22, 5215 5 of 18Figure 2. Gene expression on days 5 to 30 of osteogenesis. (A) Gene expression on day 5 of osteogenesis, Dexamethasone incombination with DAPT in D3 ESC cells (D5-DAPT-D3) and iPS (D5-DAPT-iPS), Dexamethasone alone (D5-D3), (D5-iPS)for RUNX2 (p < 0.05), SPARC (p < 0.05), OCN (p > 0.05). (B) Gene expression on day 10 of osteogenesis. Dexamethasone incombination with DAPT in D3 cells (D10-DAPT-D3) and iPS (D10-DAPT_iPS), Dexamethasone alone (D10-D3), (D10-iPS)for RUNX2 (p < 0.01), SPARC (p < 0.01), OCN (p < 0.01). (C) Gene expression on day 15, Dexamethasone in combination withDAPT in D3 (D15-DAPT-D3) and iPS (D15-DAPT-iPS), Dexamethasone alone (D15-D3), (D15-iPS) for RUNX2-(p < 0.0001),SPARC (p < 0.0001), OCN (p < 0.0001). (D) Gene expression on day 25: Dexamethasone in combination with DAPT D3(D25-DAPT-D3) and iPS (D25-DAPT-iPS), Dexamethasone alone (D25-D3), (D25-iPS) for RUNX2-(p < 0.0001), SPARC(p < 0.0001), OCN (p < 0.001). (E) Gene expression on day 30 of osteogenesis. Dexamethasone in combination with DAPTin D3 (D30-DAPT-D3) and iPS (D30-DAPT-iPS), Dexamethasone alone (D30-D3), (D30-iPS) for RUNX2-(p < 0.05), SPARC(p < 0.0001), OCN (p < 0.0001) (n = 3). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.Int. J. Mol. Sci. 2021, 22, 5215 6 of 18Figure 3. Immunofluorescent staining against the early bone marker RUNX2. (A) RUNX2 expression on day 10 of osteogenicdifferentiation in ESC. (B) RUNX2 expression on day 30 of osteogenic differentiation in ESC. (C) RUNX2 expression onday 10 of osteogenesis in iPS. (D) RUNX2 expression on day 30 of osteogenesis in iPS. In all the previous figures: (a) Cellcultures where DAPT Notch inhibitor is added, (b) cell cultures where DAPT Notch inhibitor is absent, (c) Quantification offluorescence intensity and comparison between the two cell culture conditions (with and without DAPT); (A-c) (p < 0.0001),(B-c) (p < 0.0001), (C-c) (p < 0.0001), (D-c) (p < 0.0001); Scale bar 20 µm. **** = p < 0.0001.Int. J. Mol. Sci. 2021, 22, 5215 7 of 18Figure 4. Immunofluorescent staining against the late bone marker SPARC. (A) SPARC expression on day 10 of osteogenicdifferentiation in ESC. (B) SPARC expression on day 30 of osteogenic differentiation in ESC. (C) SPARC expression onday 10 of osteogenesis in iPS. (D) SPARC expression on day 30 of osteogenesis in iPS. In all the previous figures: (a) Cellcultures where DAPT Notch inhibitor is added, (b) cell cultures where DAPT Notch inhibitor is absent. (c) Quantification offluorescence intensity and comparison between the two cell culture conditions (with and without DAPT); (A-c) (p < 0.001),(B-c) (p < 0.01), (C-c) (p < 0.0001), (D-c) (p < 0.05); Scale bar 20 µm. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.Int. J. Mol. Sci. 2021, 22, 5215 8 of 18Figure 5. Immunofluorescent staining against the late bone marker OCN. (A) OCN expression on day 10 of osteogenicdifferentiation in ESC and IPS. (B) OCN expression on day 30 of osteogenic differentiation in ESC. (C) OCN expressionon day 10 of osteogenesis in IPS. (D) OCN expression on day 30 of osteogenesis in iPS. In all the previous figures: (a) Cellcultures where DAPT Notch inhibitor is added. (b) cell cultures where DAPT Notch inhibitor is absent. (c) Quantificationof fluorescence intensity and comparison between the two cell culture conditions (with Notch inhibitor and the absenceof Notch inhibitor), (A-c) (p < 0.05), (B-c) (p < 0.05), (C-c) (p < 0.001), (D-c) (p < 0.0001); Scale bar 20 µm. * = p < 0.05,*** = p < 0.001, **** = p < 0.0001.Int. J. Mol. Sci. 2021, 22, 5215 9 of 182.4. Detection of Mesodermal Differentiation of ESC and iPSAs bone is mesodermal in origin, we detected mesoderm formation during the os-teogenic differentiation process (Figure 6). qRT-PCR showed the highest expression of theearly mesodermal marker Brachyury during the embryoid body stage. Expression levelsdecreased as differentiation progressed to the osteogenic fate. We found that in both ESC(p < 0.0001) and iPS (p < 0.0001), Notch inhibition heightened the expression of Brachyuryon days 5, 15, and 25.Figure 6. Detection of the mesodermal marker Brachyury. (A) expression of Brachyury in differenti-ating ESC; Embryoid bodies at 5 days (D5-Ebs), 5 days of osteogenic differentiation without DAPT(D5-D3), 5 days with DAPT (D5-DAPT-D3), 15 days without DAPT (D-15-D3), 15 days with DAPT(D15-DAPT-D3), 25 days without DAPT (D25-D3), 25 days with DAPT (D25-DAPT-D3) (p < 0.0001).(B) expression of Brachyury in differentiating iPS; Embryoid bodies at 5 days (D5-Ebs), 5 days of os-teogenic differentiation without DAPT (D5-iPS), 5 days with DAPT (D5-DAPT-iPS), 15 days withoutDAPT (D-15-iPS), 15 days with DAPT (D15-DAPT-iPS), 25 days without DAPT (D25-iPS), 25 dayswith DAPT (D25-DAPT-iPS) (p < 0.0001) (n = 3). **** = p < 0.0001.2.5. Expression of HES1 and HEY1 as Notch Target Genes during OsteogenesisqRT-PCR detected the expression changes of HES1 and HEY1 during the variousdifferentiation stages (Figure 7). HES1 (Figure 7A) and HEY1(Figure 7B) were expressed inundifferentiated cells and EBs of ESC and iPS. On day 5, the expression of HES1 and HEY1did not seem to be affected by γ-secretase inhibition, and the expression levels were nearlysimilar in both ESC and iPS. At later time points on days 15 and 25, expression levels weresignificantly decreased by Notch inhibition. This observation was attained in ESC (p=0.02)and iPS (p = 0.02).Int. J. Mol. Sci. 2021, 22, 5215 10 of 18Figure 7. Effect of DAPT on the expression of Notch target genes HES1 and HEY1 in ESC and iPS cells on days 5, 15, and 25of differentiation. (A) (a) HES1 Expression in D3 cells, (b) HES1 Expression in iPS. On day 5 (D5-DAPT-D3), (D5-DAPT-iPS),on day 15 (D15-DAPT-D3), (D15-DAPT-iPS), and day 25 (D25-DAPT-D3), (D25-DAPT-iPS) (p < 0.05) (n = 3). (B); (a) HEY1expression in D3 cells. (b) HEY1 expression in iPS at day 5 (D5-DAPT-D3), (D5-DAPT-iPS), at ay 15 (D15-DAPT-D3),(D15-DAPT-iPS), and day 25 (D25-DAPT-D3), (D25-DAPT-iPS) (p < 0.05) (n = 3). * = p < 0.05.3. DiscussionImproving the differentiation process of pluripotent stem cells through understandingthe signaling cues that control and contribute to the differentiation process to the desiredcell type, in addition to the correct choice of the transplantation scaffold, can yield asuccessful nearby clinical therapy for a wide variety of tissue and organ defects [40–42].The Notch signaling pathway plays a substantial role in the differentiation of iPS toseveral adult cell types. Notch inhibition during the early stage of iPS differentiation toblood cells caused a significant decrease in mature erythrocytes in cultures [43]. Notchactivation during the differentiation of induced pluripotent stem cells derived from patientswith hypoplastic left heart syndrome (HLHS) restored their cardiomyocyte differentiationcapacity and beating rate. It suppressed smooth muscle cell formation [44]. On theother hand, Notch inhibition improved the differentiation of iPS to neural stem cells [45].Moreover, Notch inhibition accelerated the neuronal generation of pluripotent stem cellsin cell cultures and after transplantation to treat spinal cord injury [46]. Moreover, timelyinhibition of Notch signaling in synergy with ascorbic acid promoted cardiomyocytes'differentiation from induced pluripotent stem cells [47].Notch signaling proved to play an essential role in skeletal development and boneremodeling [48]; furthermore, this signaling pathway is critical for skeletal stem celldifferentiation and renewal [16]. In the current study, we investigated the role of Notchinhibition on the osteogenic differentiation of embryonic and induced pluripotent stemcells. Our data revealed that the knockdown of the Notch signaling pathway via γ-secretaseinhibition enhanced mouse embryonic and induced pluripotent stem cell differentiationand commitment to the osteogenic fate.Consistent with our results, several reports have indicated that the inhibition ofNotch signaling regulated in vitro osteogenic differentiation from various progenitor andInt. J. Mol. Sci. 2021, 22, 5215 11 of 18immature cell types. A study conducted in vitro revealed that Notch1 decreased osteoblastprecursor cell differentiation [49]. Notch1 activation in mesenchymal stem cells and matureosteoblasts caused severe osteopenia and resulted in defective bone structure formation.Besides, the deletion of Notch1 and Notch2 in osteoblast progenitor cells resulted inincreased osteoblast number and cancellous bone formation [31]. Notch1 inhibition alsodecreased the proliferation yet promoted the osteogenic differentiation of bone marrowmesenchymal stem cells [50]. Other studies reported how γ-secretase inhibition restoredthe osteogenic differentiation capacity of aged bone marrow stem cells in mice [51] which issimilar to the reinstated effect γ-secretase inhibition had on human skeletal mesenchymalstem cells used for ectopic bone formation in mice [52].Dexamethasone and 1α,25-dihydroxy vitamin D3 (VITD) are the principal and stan-dard components of osteogenic cell culture media [53]. Our results showed that combiningDAPT Notch inhibitor with Dexamethasone resulted in a synergistic effect on ESC andiPS osteogenic differentiation compared with combining VITD and DAPT (Figure 1). Dex-amethasone was demonstrated to stimulate the differentiation of stem cells to osteoblaststhrough multiple mechanisms. These mechanisms include the activation of RUNX2 expres-sion through wnt/β-catenin pathway activation. Dexamethasone also increases RUNX2phosphorylation by the mitogen-activated protein kinase (MAPK).Moreover, Dexamethasone activates RUNX2 transcription through TAZ activation(transcriptional coactivator with PDZ-binding motif) [54]. On the other hand, some studieshave demonstrated that VITD suppresses RUNX2 expression in mouse cells by binding tovitamin D receptors in the nucleus [55]. While both components proved to decrease Notchreceptor expression in differentiating cells [56], which in turn enhances RUNX2 expressionthrough the inactivation of the Notch target gene HEY-1, Dexamethasone appears to havethe most potent effect on the expression of RUNX2, which in turn, activates the expressionof many other osteogenic genes. These findings seem to explain its powerful impact on theosteogenic differentiation process when combined with DAPT.qPCR results showed significantly increased expression of the osteogenic gene RUNX2in ESC and iPS at various time points in the cell cultures that received DAPT compared tothe cultures where DAPT was absent. These results came in agreement with the fact that theNotch1 and Notch2 receptor inhibition suppressed Notch target genes HES1, HEY1 whichare major inhibitors of RUNX2 activation [31,57]. Similarly, OCN expression levels wereenhanced by Notch inhibition as differentiation progressed. We suggest that this improve-ment is subsequent to RUNX2 improved expression [58]. Another mechanism contributingto OCN expression's advancement might be glycolysis stimulation by inhibiting the Notchsignaling pathway since OCN expression is highly dependent on glycolysis [32,59,60].SPARC, also known as Osteonectin, is a protein produced by mature osteoblasts andsome unmineralized tissue cells [61]. In bone, this protein is associated with the productionof type I collagen; SPARC contains a collagen-binding domain and a hydroxyapatitebinding region, which allows this protein to bind collagen and hydroxyapatite crystalsand release calcium ions, which is essential for the mineralization of the collagen matrixin bones [62]. Our results demonstrated that Notch inhibition maintained a stimulatoryeffect on the gene expression of SPARC during the course of the experiment. To ourknowledge, the mechanism wherein Notch signaling enhances SPARC expression duringbone formation is unknown. However, our results are aligned with other studies suggestingthat Notch inhibition increased SPARC expression in neuroblastoma, astrocytoma, andmedulloblastoma. Our studies indicate that the stimulatory effect that Notch inhibitionhad on SPARC gene expression results from suppressing HES1 and HEY1 [63–65]. Furtherinvestigation is required to determine if enhancing SPARC expression by inhibiting Notchduring the osteogenic differentiation occurs via the exact mechanism.As bone is mesodermal in origin, we tested the mesodermal marker Brachyury ex-pression [66] and investigated how Notch inhibition affected its expression. The resultsdemonstrated a higher expression level in pluripotent stem cell-derived embryoid bodies.During stochastic differentiation to all three germ layers, Brachyury expression levelsInt. J. Mol. Sci. 2021, 22, 5215 12 of 18decreased as differentiation progressed [67]. Our observation is corroborated by a previousstudy indicating that Notch's activation led to disruption in some mesodermal precursors’differentiation [68]. Likewise, Other studies suggested that Notch 1 inhibition upregulatedBrachyury expression and improved cardiac differentiation of embryonic stem cells [69,70].This improvement in expression can be another contributing factor to the overall aug-menting effect that Notch inhibition had on osteogenic differentiation. Immunofluorescentimaging revealed results that strengthened the qPCR results. Fluorescence quantificationshowed increased expression of the early marker RUNX2 and the late markers SPARC andOCN at early differentiation (10 days) and late differentiation (30 days).For further confirmation of the above results, we tested the expression of HES1 andHEY1 with DAPT application. We observed a decreased expression of HES1 and HEY1 inESC and iPS on days 15 and 25 in response to DAPT application. Surprisingly, we noticedan increased expression of both genes on day 5 of application. Other studies reported thatthe expression levels of the Notch target Genes HES1, HEY1, HEY2 increased despite DAPTapplication at the early stages of inhibition [71,72]. They suggested that the BMP-SMAD1/5pathway had synergistic action on the Notch signaling pathway independent of γ-secretaseat the early differentiation stage [73].To our knowledge, there are very few studies that investigate how Notch signalingaffects the differentiation of embryonic and induced pluripotent stem cells to the osteogeniclineage. Our results demonstrated the augmenting effect that Notch inhibition had onthe osteogenic differentiation of embryonic and induced pluripotent stem cells on thetranscription and translation levels. Here, we shed light on the synergistic effect that thecombination of Dexamethasone and DAPT had on the differentiation process. Moreover,our results demonstrated that enhanced mesodermal differentiation might be anotherelement to consider contributing to improving the differentiation process's outcome. Wesuggest that additional studies are needed to fully understand the crosstalk among thedifferent signaling pathways that control stem cell differentiation to bone cells. Furtherstudies need to be accomplished to promote the differentiation outcome of embryonic andinduced pluripotent stem cells to bring stem cell-based therapies to fruition.4. Materials and Methods4.1. Cell Lines and Cultures4.1.1. Pluripotent Stem CellsMouse embryonic (D3 line) [74] and induced pluripotent (miPS line) [75] stem cellswere used for this study. D3 and miPS cells were maintained in the pluripotent state inhigh glucose DMEM (Gibco, Burlington, ON, Canada) supplemented with 15% FBS, 1%non-essential amino acids (Invitrogen, Burlington, ON, Canada), 50 U/ mL Penicillin, and50 µg/mL Streptomycin (Invitrogen, Burlington, ON, Canada), 0.1 mM β-mercaptoethanol(Gibco) and 1000 U/mL Leukemia Inhibitory Factor (LIF). Pluripotent cultures were sub-cultured every second day on murine embryonic fibroblast feeder cells (MEFs).4.1.2. Embryoid Body (EB) FormationD3 and miPS embryoid bodies were formed using the hanging drop method. [76] Day 2EBs were transferred to differentiation media composed of DMEM (Gibco, Burlington, ON,Canada), supplemented with 15% FBS (Gibco), 1% non-essential amino acids (Invitrogen,Burlington, ON, Canada), 50 U/mL Penicillin and 50 µg/mL Streptomycin (Invitrogen,Burlington, ON, Canada), 0.1 mM β-mercaptoethanol (Gibco, Burlington, ON, Canada) forthree days before transitioning to osteogenic differentiation [77].4.1.3. Osteogenic DifferentiationFor vitamin D3 and dexamethasone-based differentiation protocols, day 5 EBs weretransferred to gelatin-coated cell culture dishes containing osteoblast differentiation media.The media composed of β-glycerophosphate (10 mM) (Sigma, High River, AB, Canada),ascorbic acid (50 µg/mL) (Sigma, High River, AB, Canada), and either 1,25-OH2 vitaminInt. J. Mol. Sci. 2021, 22, 5215 13 of 18D3 or Dexamethasone (5 × 10−8M) (Sigma, High River, AB, Canada) starting at day 5 andup to day 30.4.2. Notch Pathway InhibitionNotch γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) (10 µM) (Roche, High River, AB, Canada) was added to the osteogenicmedia for both D3 and miPS cells from day 5 to 30 of differentiation [78].4.3. Evaluation of Osteogenic Differentiation4.3.1. Alizarin Red StainingDay 30 cell cultures were fixed overnight at 4 ◦C in 4% paraformaldehyde (PFA). Thenext day, cell cultures were washed 3 times in PBS and placed in 1% KOH solution for48 hours at 4 ◦C and subsequently stained with Alizarin Red (1 mg Alizarin Red, 100 mL of1% KOH) for 48 h at 4 ◦C. The cultures were rinsed with 1% KOH several times. Afterward,PBS was added for visualization. The stained surface area was measured and analyzedusing ImageJ software (National Institutes of Health and the Laboratory for Optical andComputational Instrumentation (LOCI, University of Wisconsin), Bethesda, MD, USA).4.3.2. qPCRmRNA Isolation and cDNA SynthesisCell cultures containing differentiated EBs were isolated at multiple time points(days 5, 10, 15, 25, and 30 of osteogenesis). Cells were isolated using TrypLE Expressdissociation reagent (Gibco, Burlington, ON, Canada). mRNA Isolation was performedusing PureLink™ RNA Mini Kit (Thermo Fisher Scientific, Calgary, AB, Canada) accordingto the manufacturer's instructions. mRNA was measured using a Nano Photometer P-Class(IMPLEN, Munich Germany). cDNA synthesis was performed using SuperScript™ IVReverse Transcriptase (Thermo Fisher Scientific, Calgary, AB, Canada) according to themanufacturer’s instructions.RT-qPCR AnalysisRT-qPCR was employed to quantify the gene expression levels using TaqMan Gene Ex-pression Assays. For osteogenesis, we used the early maker Runx2 (Assay ID Mm00501584-m1) in addition to late osteogenic markers Osteocalcin (OCN) (Assay ID Mm 03413826-mH)and SPARC (Osteonectin) (Assay ID Mm00486332-m1). For Notch target gene expression,we used HES1 (Probe ID Mm01342805-m1) and HEY1 (Assay ID Mm00468865-m1). Forearly mesodermal differentiation, we used Brachyury (Assay ID Mm00496699-m1). AllTaqMan Gene Expression Assays were obtained from Thermo Fisher Scientific. TaqManUniversal PCR MasterMix No AmpErase (Applied Biosystems) was used according to themanufacturer's instructions. StepOnePlus™ Real-Time PCR System was used for runningall the samples with the following program: UNG incubation at 50 ◦C for 2 min; enzymeactivation at 95 ◦C for 20 s; denaturation at 95 ◦C for 3 s; annealing was performed for40 cycles at 60 ◦C for 30 s (40 cycles). The resulting threshold (Ct) values were analyzedwith the ∆∆Ct method. GAPDH was used as the reference gene, and undifferentiatedD3/miPS cells were used as reference samples. 3 biological replicas and 3 technical replicasof each sample were used for the analysis of this test.4.4. Immunofluorescence4.4.1. Immunofluorescent StainingEBs were generated according to the previously described method. On day 5, EBswere seeded on gelatin-coated glass-bottom cell culture dishes Fluorodish (World PrecisionInstruments, Sarasota, FL, USA). On days 10 and 30 of osteogenic differentiation, cellswere washed in PBS and fixed 4% PFA in PBS for 45 min. Cells were washed in PBS andpermeabilized using 0.25% Triton X-100 (Sigma, High River, AB, Canada) in PBS for 45 min.Blocking was done using 5% filtered BSA (Thermo Fisher Scientific, Calgary, AB, Canada)Int. J. Mol. Sci. 2021, 22, 5215 14 of 18in PBS for 4 hours. Primary antibodies to the osteogenic markers Runx2, OCN, and SPARC(Santa Cruz Biotechnology, Dallas, TX, USA) were added, and dishes were kept at 4 ◦Covernight. The next day, cells were washed in PBS, and the secondary antibody Alexa 568(Millipore, Etobicoke, ON, Canada) was added to the cells for 10 min at room temperature.After washing the cells in PBS, Hoechst (Millipore, Etobicoke, ON, Canada), in PBS, wasadded for 10 min at room temperature to stain the nuclei. Cells were washed in PBS thenenough fresh PBS was added to keep the cells from drying.4.4.2. Fluorescence Intensity and DistributionTen areas were selected at random in the pictures chosen for analysis, and bothintensity and distribution of positive staining were analyzed by ImageJ software. The datawere normalized to control measurements (Figure 8).Figure 8. An example of the selection of fluorescent area stained by secondary antibody and representation of fluorescencequantification and distribution.5. Statistical AnalysisFor qPCR and quantification of surface area stained with Alizarin Red, one-wayANOVA test was used to compare sample groups. p values < 0.05 were consideredsignificant.For fluorescent intensity analysis, Two-sided paired student's t-test was used to com-pare sample groups. p values < 0.05 were considered significant.* = p < 0.05.** = p < 0.01.*** = p < 0.001.**** = p < 0.0001.ns = non-significant (p > 0.05)Author Contributions: Conceptualization, S.A.H., and D.E.R.; methodology, S.A.H., L.R., and D.E.R.;software, S.A.H. and L.R.; formal analysis, S.A.H. and L.R.; resources, D.E.R., A.R.Z., Y.M.E.H.;Funding Acquisition, D.E.R., A.R.Z., Y.M.E.H.; data curation, S.A.H. and D.E.R.; original draftpreparation, S.A.H.; writing—review and editing, D.E.R.; supervision, D.E.R. All authors have readand agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: Not applicable.Int. J. Mol. Sci. 2021, 22, 5215 15 of 18Informed Consent Statement: Not applicable.Data Availability Statement: Not applicable.Acknowledgments: We acknowledge the Egyptian Ministry of Higher Educations' mission sectorfor funding the first author for the first two years of her Ph.D. and the University of Calgary EyesHigh for funding the second author. We additionally acknowledge Anne Vaahtokari at the ArnieCharbonneau Cancer Institute, University of Calgary, for her support with confocal imaging.Conflicts of Interest: The authors declare no conflict of interest.References1. Chen, C.Y.; Rao, S.S.; Tan, Y.J.; Luo, M.J.; Hu, X.K.; Yin, H.; Huang, J.; Hu, Y.; Luo, Z.W.; Liu, Z.Z.; et al. Extracellular vesicles fromhuman urine-derived stem cells prevent osteoporosis by transferring CTHRC1 and OPG. Bone Res. 2019, 7, 1–14. [CrossRef]2. Hernigou, P.; Guissou, I.; Homma, Y.; Poignard, A.; Chevallier, N.; Rouard, H.; Flouzat Lachaniette, C.H. 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