The discovery of human pluripotent stem cells (hPSCs) has opened a new field called regenerative medicine that offers new strategies for curing diseases and drug discovery. It also provides the means of regenerating disease-relevant cells in vitro for disease modeling, and the possibility of cell replacement therapy. Among the most promising applications of hPSCs technology is the generation of blood cells that can be used for engraftment or transfusion in the clinic. Generating engraftable hematopoietic stem cells from hPSCs in vitro can fulfill the promise of using hPSCs to cure human diseases. Making functional HSCs in vitro from hPSCs remains an elusive goal. There are key pathways that are misregulated during hPSCs differentiation, which could impair the engraftment potential of hPSCs. WNT signaling is needed in the early phase of differentiation. However, evidence from mouse models and human development show that WNT signaling is downregulated during the maturation of HSCs. Therefore, we hypothesize that mimicking the dynamics of WNT signaling temporally during the differentiation could improve the functional maturation of differentiated HPCs. To this end, we have established an inducible gene activation system based on dCas9-VPR that can activate endogenous loci. We performed targeted activation of negative regulators of WNT. The system has shown promise in specific activation of WNT negative regulators, AXIN2 and APC2, but it needs further optimization to be able to steer cell fate and obtain functional HSCs.

The generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts has revolutionized cell therapy by providing a source of autologous cells for transplantation. Several studies have demonstrated that transplantation of hiPSC-derived neural stem cells (hiPSC-NSCs) increases regeneration and recovery following stroke, supporting their therapeutic potential. However, major concerns for translating hiPSC transplantation therapy to the clinic are efficacy and safety. Therefore, there is demand to develop an optimal strategy to enhance the engraftment and regenerative capacity of transplanted hiPSC-NSCs. The recent published work shows that metformin, an FDA approved drug, is an optimal neuroregenerative agent that not only promotes the proliferation of neural stem cells but also enhances their neuronal differentiation. In this regard, we hypothesize that preconditioning of hiPSC-NSCs with metformin before transplantation into the stroke-damaged brain will improve engraftment and regenerative capabilities of hiPSC-NSCs, further enhancing cell-mediated functional recovery. Here we show that treatment of hiPSC-NSCs with metformin enhances the proliferation and differentiation of hiPSC-NSCs in culture even after withdrawal of metformin treatment, showing its promise as a novel preconditioning strategy. Furthermore, transplantation of preconditioned hiPSC-NSCs into a rat endothelin-1 ischemic stroke model showed an improved engraftment capability 1-week post-transplant. In addition, metformin preconditioned grafts survived longer compared to naïve grafts and were detectable at 8 weeks post-stroke. However, cell transplantation did not result in improve functional recovery when compared to sham group in this model. These studies represent a vital step in the optimization of hiPSC-NSC based transplantation to promote post-stroke recovery.

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Undifferentiated human embryonic stem cells (hESCs) grow on the extracellular matrix (ECM) substrate fibronectin (FN) in defined feeder-free conditions. The ECM is part of the hESCs pluripotent niche and supports their maintenance, but the contribution to survival remains to be elucidated. Understanding the mechanism of survival is particularly crucial in hESCs, since it affects their expansion in cell culture and ultimately translation of research to the clinic. HESCs bind to FN mainly via alpha5β1- integrin, known to be upstream of important survival cascades in other cell types. However, it is not understood if and how FN/integrin binding supports those molecular pathways in the context of pluripotent hESCs. The aim of this work was to elucidate the survival cascade downstream of the FN/integrin interaction in hESCs. Initially, when hESCs were cultured on a non-integrin activating substrate they initiated an apoptotic response that also occurred when β1-integrin was selectively blocked with antibody, leading the cells to detach from FN. Integrin activation is generally transduced within cells via a complex adhesome of scaffold and kinase proteins, among which the focal adhesion kinase (FAK) plays a key role. Indeed, blocking β1-integrin resulted in dephosphorylation of endogenous FAK in hESCs. When FAK kinase activity was directly inhibited (with small molecule inhibitors), hESCs responded by detaching from FN and activating caspase-3, leading to an increase in apoptosis. Furthermore, flow cytometry analysis showed that the population of hESCs that underwent apoptosis still retained the pluripotency-associated marker NANOG. FAK is a convergent point between growth factor signaling and the PI3K/Akt pathway, with a well-reported role in the maintenance of hESCs. Consistently, FN activated both AKT and its target the ubiquitin ligase MDM2 at the protein levels, while pAkt was reduced after β1-integrin blocking and FAK inhibition. Cell imaging showed that MDM2, which regulates p53 degradation in the nucleus, displayed reduced nuclear localisation after FAK inhibition, opening the possibility for a change in the p53 balance in hESCs. In fact, p53 protein increases after FAK inhibition corresponding also to caspase activation. Further investigation explored if FAK-dependent pathways are also implicated in the maintenance of hESC pluripotency. Inhibition of FAK led the cells that survived apoptosis to lose stem cell morphology, decrease pluripotency-associated markers and change nuclear shape. Moreover, a small pool of FAK was found in the nucleus of hESCs cultured on FN, but decreased after FAK inhibition. FAK was also co- immunoprecipitated with NANOG protein in standard hESC culture while NANOG decreased after sustained FAK inhibition. This data suggests that nuclear roles of FAK could support, together with the cytoplasmic activation of the PI3K cascade, both survival and pluripotency pathways requiring further investigation. In conclusion, the original contribution of this work is to identify in FAK the downstream survival effector of the FN/β1-integrin interaction in hESCs. HESCs survival is maintained by the binding of β1-integrin to FN and activation of FAK kinase and downstream PI3K/Akt, leading to the suppression of p53 and caspase activation. In parallel, promotion of these pathways by FAK is suggested also to support the key pluripotency circuitry, feeding into NANOG. Overall, FAK is proposed here as an important regulator of hESC survival and fate.

Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Human Genetics. Title from resource description page. Includes bibliographical references.

During mouse embryo gastrulation, the pluripotent epiblast gives rise to the three embryonic germ layers, the ectoderm, mesoderm and endoderm. After somitogenesis begins and pluripotency disappears from the epiblast, bipotent neuromesodermal progenitors (NMPs) drive axis elongation, contributing to the formation of the posterior nervous system, as well as the axial and paraxial mesoderm. Early NMPs arise in the E8.5 mouse embryo, in and near the primitive streak, while late NMPs are found in the tail bud (E9.5 - E13.5). NMP regions are characterized by coexpression of Tbra (Brachyury) and Sox2. Sox1, another neural related transcription factor, has also been detected in NMP regions. Importantly, it has been shown that Sox1 expression increases as NMPs transit from the primitive streak to the tail bud stages. Mouse epiblast derived stem cells (EpiSCs) recapitulate the properties of the post-implantation epiblast and therefore serve as a good in vitro system for the study of early lineage specification events. EpiSCs express pluripotency factors and early differentiation markers, including Sox2, Sox1 and Tbra. Based on studies reporting that EpiSC cultures contain distinct subpopulations that have progressed further into lineage specification, I analyzed the properties of the Tbra expressing EpiSCs and by dissecting their expression profile, I assess whether these cells are pluripotent or they have progressed further into lineage specification, possibly into an NM fate. I show that EpiSC cultures include a large fraction of Tbra/Sox2 double positive cells; however, Nanog expression was detected in the vast majority of Tbra+/Sox2+ EpiSCs suggesting that most of the Tbra+ cells are pluripotent rather than bipotent NMPs. Using a previously published Tbra-GFP reporter cell line, I present that Tbra-GFP+ cells constitute a dynamic fraction of the culture that has not exited pluripotency (as shown by expression of the pluripotency markers), but have adopted an early primitive streak-like character. Similar to the cells of the posterior epiblast, these EpiSCs are in a reversible state and they retain their ability to undergo neural differentiation. In contrast to the overlap of Tbra and Sox2 positivity in self-renewing EpiSCs, it has been shown that Tbra expression is mutually exclusive with expression of Sox1-GFP, that seems to mark a distinct subpopulation with neural-like characteristics. In vitro NMPs can be generated from EpiSCs upon treatment with Fgf2 and the Gsk- 3 antagonist/Wnt agonist CHIRON99021 (FGF/CHI). In these conditions, 80% of the culture becomes Tbra+/Sox2+. Given that Sox1 is present in NMP regions in vivo, I hypothesized that the NMP cultures could contain Tbra+Sox1+ NM bipotent cells. Most importantly, the upregulation of Sox1 at the tail bud stages drove the hypothesis that Sox1 expression could mark the transition from an early- to a late-like NMP state in vitro. In this study, using a Sox1-GFP reporter cell line, I show that Tbra/Sox2/Sox1-GFP triple positive cells emerge in FGF/CHI treated EpiSCs. Importantly, Sox1-GFP+ cells express NMP markers and are enriched in transcripts of Hox genes. The expression profile of Sox1-GFP+ cells resembles the alteration of Hox gene activation that takes place in the caudal progenitor regions during the transition from early NMPs (E8.5) to late NMPs (E9.5-10.5) and hence supports the hypothesis that Sox1-GFP marks NMPs that correspond to the axial progenitors found at tail bud stages. Although the gene activity observed in the Sox1-GFP+ subpopulation correlates with the NM developmental potential, these cells exhibit strong neurogenic capacity, while evidence for their ability to give rise to mesoderm differentiation products is still lacking. Since Tbra and Sox1/Sox2 are not expressed in NMP regions exclusively, but also in mesoderm and neural fated tissues respectively, double rather than single reporter cell lines would be more suitable tools for tracking and isolating bipotent NM progenitors in vivo and in vitro. Here, I present the CRISPR/Cas9-mediated generation of a reliable Tbra-GFP reporter ES cell line that in contrast to the one published before, contains both endogenous Tbra loci intact. By targeting the Sox2 locus in the Tbra-GFP ES cells, I generated a Tbra-GFP/Sox2-tdTomato double reporter ES cell line, that in the future, could help us to dissect the molecular mechanisms underlying the self-renewal and differentiation of NMPs.

During embryo development, extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) and promotes downstream cell specifications. Pluripotent stem cell (PSC) aggregates can recapitulate various aspects of embryogenesis in vitro, and incorporation of biomaterial microparticles also provides an ideal platform to study cell-biomaterial interactions. Stem cell interactions with ECM-based biomaterials can impact tissue remodeling and differentiation propensity via modulation of MMP activity. This work investigated the MMP activity and subsequent mesenchymal differentiation of embryonic stem cell (ESC) aggregates with incorporated gelatin methacrylate (GMA) MPs with either low (20%) or high (90%) cross-linking densities, corresponding to faster or slower degradation rate, respectively. GMA MP incorporation increased total MMP and MMP-2 levels within 3D ESC aggregates in a substrate-dependent manner. GMA MP-incorporated aggregates also expressed higher levels of epithelial-to-mesenchymal transition markers and displayed enhanced mesenchymal morphogenesis than aggregates without MPs, and the MP-mediated effects were completely abrogated with MMP inhibitor treatment. This work predicts that control of proteolytic responses via introducing ECM-based MPs may offer a novel avenue to engineer the ECM microenvironment to modulate stem cell differentiation.

In human pluripotent stem cell (hPSC) research and applications, the need for a culture system devoid of non-human components is crucial. Such a system should exhibit characteristics observed in conventional culture systems that have used mouse embryonic fibroblast feeders for hPSC self renewal without the requirement of excessive supplementation with growth factors. To achieve this, we focused on the identification and characterization of extracellular matrix (ECM) substrates for hPSC propagation. ECM substrates derived from mouse and human fibroblasts were assessed for their ability to support self-renewal of hPSCs. Characterization of hPSCs on ECM-based substrates demonstrated maintenance of pluripotent characteristics based on a) high nuclear-cytoplasmic ratio b) immunocytochemical analyses for pluripotent markers (Alkaline phosphatase, AP, Octamer Binding Transcription Factor-4, OCT4 and Specific surface embryonic antigen-4, SSEA4) c) in vitro differentiation potential by embryoid body formation d) Real time RT-PCR analysis for pluripotent and germ-layer specific markers and e) karyotype analysis for chromosome number. Compositional characterization of the ECM substrates using proteomic analysis identified some of the major constituents of the matrix that might contribute to hPSC self-renewal. Based on results from the proteomic analysis, combinatorial ECM substrates were formulated using commercially available proteins and evaluated for applicability in hPSC propagation. Extensive characterization of hPSC propagated on the ECM substrates suggest that a combination of heparan sulfate proteoglycan and fibronectin was sufficient for the promoting hPSC sef-renewal. Finally, an in-direct co-culture system utilizing microporous membranes coated with acellular substrates and a physically separated feeder layer was developed as a microenvironment for hPSC propagation. Real time conditioning of the growth medium and an ECM-based substrate for hPSC adhesion provides a synergy of the biochemical and biophysical cues necessary for hPSC self-renewal. hPSCs cultured in this system demonstrated equivalent pluripotent characteristics as those propagated in conventional culture systems, and provided opportunities for scale up without cell mixing. Overall, these studies could prove to be useful in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications.

Pluripotent embryonic stem (ES) cells are heterogeneous mixtures of naïve and lineage-primed states defined by distinct transcription factor expression profiles. However, the events that prime pluripotent cells for differentiation are not well understood. Id proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factors, contribute to pluripotency by blocking differentiation. Using Yeast-Two-Hybrid screening, our lab identified Tcf15 as an Id-regulated transcription factor. In this study, I first examined the expression of Tcf15 during differentiation in vitro and during early development in vivo in the mouse. Tcf15 expression is higher in primed pluripotent embryonic stem (ES) cells than in naïve ES cells or epiblast stem cells (EpiSCs). In addition, Tcf15 is expressed heterogeneously in ES cells and is also detected in the inner cell mass (ICM) of E4.5 mouse embryos. Expression of Tcf15 was upregulated during early stages of differentiation and downregulated before cells committed to any specific lineage. Using Tcf15-Venus reporter cells, I found that expression of Tcf15 is specifically associated with a novel subpopulation of ES cells primed for somatic lineages. Gain of function and loss of function studies were then performed to perturb Tcf15 expression in ES cells in order to assess the function of Tcf15 in self-renewal and during differentiation. An inducible Id-resistant form of Tcf15 accelerates somatic lineage commitment by maturating naïve pluripotent ES cells transit toward primed epiblast and later on epiblastderived somatic lineages whilst suppressing differentiation towards extraembryonic endoderm. Preliminary loss of function studies also suggest that down-regulation of Tcf15 may promote a naïve state within pluripotent cells. I investigated the mechanism by which Tcf15 expression becomes associated with the epiblast-primed state by identifying the upstream regulators and downstream targets of Tcf15. Tcf15 expression is dependent on FGF signalling. Microarray analysis identified that Tcf15 downregulates the naïve pluripotency determinant Nanog and upregulates the epiblast determinant Otx2. Taken together, our results suggest that Tcf15 acts in opposition to the pluripotency network to prime pluripotent cells towards differentiation.

In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.