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.

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.

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.

The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.

Human embryonic stem cells (hESC) present a potentially unlimited supply of hematopoietic progenitors for cell-based therapies. However, current protocols for generating these progenitors typically also generate undesired cell types due to imprecise control of the hESC microenvironment and poor understanding of the signalling networks regulating mesoderm differentiation (the germ layer from which hematopoietic cells emerge). This report demonstrates that activation of the downstream effectors of Activin/Nodal and bone morphogenetic protein (BMP) signalling (Smad2 (composite of Sma (smaller) and Mad (mothers against decapentaplegic) and Smad1, respectively) are both required for mesoderm differentiation. It is further shown that microcontact printing-mediated control of hESC colony size creates local microenvironments that guide differentiation, via a Smad1-dependent mechanism, preferentially towards the mesoderm lineage. These findings demonstrate the need for precise control of the microenvironment in order to effectively guide hESC differentiation to produce specific cell types for potential therapeutic applications.

Human embryonic stem cells (hESC) present a potentially unlimited supply of hematopoietic progenitors for cell-based therapies. However, current protocols for generating these progenitors typically also generate undesired cell types due to imprecise control of the hESC microenvironment and poor understanding of the signalling networks regulating mesoderm differentiation (the germ layer from which hematopoietic cells emerge). This report demonstrates that activation of the downstream effectors of Activin/Nodal and bone morphogenetic protein (BMP) signalling (Smad2 (composite of Sma (smaller) and Mad (mothers against decapentaplegic) and Smad1, respectively) are both required for mesoderm differentiation. It is further shown that microcontact printing-mediated control of hESC colony size creates local microenvironments that guide differentiation, via a Smad1-dependent mechanism, preferentially towards the mesoderm lineage. These findings demonstrate the need for precise control of the microenvironment in order to effectively guide hESC differentiation to produce specific cell types for potential therapeutic applications.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18893号 / 医博第4004号 / 新制||医||1009(附属図書館) / 31844 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 羽賀 博典, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Human pluripotent stem cells (hPSCs) such as, human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS) are a valuable resource to generate bespoke cell types for a number of therapeutic applications involving cell therapy, drug screening and disease modelling. The overarching goal of this project was to generate a set of transgenic tools by gene targeting and genetic modification of hESCs for applications in stem cell biology such as the in vitro isolation, analyses and derivation of lineage specific cell types. The transgenic tools generated in this study were designed and tested in particular for the human 3rd pharyngeal pouch epithelium (3PPE) like cells and its derivatives, namely the thymus and parathyroid, which are key organs involved in T-cell development and calcium homeostasis respectively. The forkhead transcription factor FOXN1 is considered a master regulator of the development of the thymic epithelium (TEC), the major functional component of the thymic stroma, which is intimately involved in T-cell differentiation. So, to facilitate the prospective isolation of FOXN1 expressing TECs, gene targeting was employed to place a fluorescent reporter and a lineage selection antibiotic resistance gene under the direct control of the endogenous FOXN1 promoter. To date, I have not been able to detect either the fluorescent reporter, or FOXN1 expression using published directed differentiation protocols, but only what can be deemed as precursors expressing the cytokeratin K5 and other markers associated with the development of the thymus and parthyroid from 3PPE. The lack of endogenous FOXN1 activation was observed in both the unmodified parent and the targeted FOXN1 knock-in human ES lines. Further, over-expression of FOXN1 cDNA during the differentiation protocol did not result in the activation of endogenous FOXN1. So, the results evinced in this study could be due to a number of reasons such as, technical issues associated with transference of the published protocols to the cell lines used in this study, differences in hESC lines, and effects of different hESC culture methods and practices. The homeobox gene HOXA3 is expressed in the 3PPE during development. So, a HOXA3 transgenic reporter hESC line could be an invaluable tool for prospective isolation of in vitro derived 3PPE like cells. The reporter was generated by Piggy Bac transposase mediated transposition of a HOXA3 containing Bacterial Artificial Chromsome (BAC) in the FOXN1 knock-in human ES line. To date, this is biggest reported cargo that has been successfully transposed in human ESCs. Moreover, this is the first lineage specific double reporter transgenic hESC line that has been reported for this lineage. This HOXA3 reporter line was then used to isolate and enrich for HOXA3 expressing 3PPE like cells with very high efficiencies during the directed differentiation of hESCs, thus demonstrating the key objective of this transgenic hESC line for this study. In a novel parallel approach, I have conceived, designed and generated transgenic hESCs lines capable of inducible and constitutive over-expression of key transcription factors involved in the development of 3PPE and its derivatives, the thymus and parathyroid. The objective of the said over-expression hESC lines was to interrogate if such a system could elicit morphological and gene expression changes in hESCs following over-expression. By testing the chosen panel of transcription factors in hESCs, I was able to detect cells expressing FOXN1 and GCMB, which are key markers of TECs and PTECs. Further, I have isolated an expandable population of cells expressing markers analogous to their in vivo counterpart found in the 3PPE of a developing mouse embryo around E9.0. The in vivo potency of these in vitro derived 3PPE like cells is yet to be ascertained. Nevertheless, transgenic constructs generated in this experiment could also be tested during future attempts at the differentiation of hESCs to TECs and PTECs, and also used as a basis for future studies involving the direct conversion of patient specific fibroblasts to 3PPE like cells and its derivatives. In summary, several transgenic tools developed in this project, namely the FOXN1 knock-in transgenic hESC line, FOXN1-HOXA3 double transgenic hESC line, over-expression 3PPE transgenes and hESC transgenic lines, and results from the deployment of these tools provide a foundation, from which protocols to generate functional TECs and PTECs can be refined and optimised. These transgenic hESC lines also provide a tractable model, which could be used to interrogate the development of human TECs and PTECs from human 3PPE, and identify hitherto unknown early events in their development in an in vitro reductionist setting.

Electrospun biomaterial scaffolds can be engineered to support the neural differentiation of induced pluripotent stem cells. As electrospinning produces scaffolds consisting of nano or microfibers, these topographical features can be used as cues to direct stem cell differentiation. These nano and microscale scaffolds can also be used to deliver chemical cues, such as small molecules and growth factors, to direct the differentiation of induced pluripotent stem cells into neural phenotypes. Induced pluripotent stem cells can become any cell type found in the body, making them a powerful tool for engineering tissues. Therefore, a combination of an engineered biomaterial scaffold with induced pluripotent stem cells is a promising approach for neural tissue engineering applications. As detailed in this thesis, electrospun scaffolds support the neuronal differentiation of induced pluripotent stem cells through delivering the appropriate chemical cues and also presenting physical cues, specifically topography to enhance neuronal regeneration. This thesis seeks to evaluate the following topics: multifunctional electrospun scaffolds for promoting neuronal differentiation of induced pluripotent stem cells, neuronal differentiation of human induced pluripotent stem cells seeded on electrospun scaffolds with varied topographies, and controlled release of glial cell-derived neurotrophic factor from random and aligned electrospun nanofibers. / Graduate / nkhadem@uvic.ca