A Computational Model for Understanding Stem Cell, Trophectoderm and Endoderm Lineage Determination

Background: Recent studies have associated the transcription factors, Oct4, Sox2 and Nanog as parts of a self-regulating network which is responsible for maintaining embryonic stem cell properties: self renewal and pluripotency. In addition, mutual antagonism between two of these and other master regulators have been shown to regulate lineage determination. In particular, an excess of Cdx2 over Oct4 determines the trophectoderm lineage whereas an excess of Gata-6 over Nanog determines differentiation into the endoderm lineage. Also, under/over-expression studies of the master regulator Oct4 have revealed that some self-renewal/pluripotency as well as differentiation genes are expressed in a biphasic manner with respect to the concentration of Oct4. Methodology/Principal Findings: We construct a dynamical model of a minimalistic network, extracted from ChIP-on-chip and microarray data as well as literature studies. The model is based upon differential equations and makes two plausible assumptions; activation of Gata-6 by Oct4 and repression of Nanog by an Oct4–Gata-6 heterodimer. With these assumptions, the results of simulations successfully describe the biphasic behavior as well as lineage commitment. The model also predicts that reprogramming the network from a differentiated state, in particular the endoderm state, into a stem cell state, is best achieved by over-expressing Nanog, rather than by suppression of differentiation genes such as Gata-6. Conclusions: The computational model provides a mechanistic understanding of how different lineages arise from the dynamics of the underlying regulatory network. It provides a framework to explore strategies of reprogramming a cell from a differentiated state to a stem cell state through directed perturbations. Such an approach is highly relevant to regenerative medicine since it allows for a rapid search over the host of possibilities for reprogramming to a stem cell state

The key role of micrornas in self-renewal and differentiation of embryonic stem cells

Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinctive developmental stages, mimicking the pre-and the post-implantation events during the embryo development, respectively. The complex molecular mechanisms governing the transition from ESCs into EpiSCs are orchestrated by fluctuating levels of pluripotency transcription factors (Nanog, Oct4, etc.) and wide-ranging remodeling of the epigenetic landscape. Recent studies highlighted the pivotal role of microRNAs (miRNAs) in balancing the switch from self-renewal to differentiation of ESCs. Of note, evidence deriving from miRNA-based reprogramming strategies underscores the role of the non-coding RNAs in the induction and maintenance of the stemness properties. In this review, we revised recent studies concerning the functions mediated by miRNAs in ESCs, with the aim of giving a comprehensive view of the highly dynamic miRNA-mediated tuning, essential to guarantee cell cycle progression, pluripotency maintenance and the proper commitment of ESCs

Rolling ES cells down the Waddington landscape with Oct4 and Sox2

Embryonic stem cell (ESC) pluripotency is maintained by core transcriptional circuits whereby critical factors sustain their own expression while preventing the expression of genes required for differentiation. Thomson et al. (2011) now show that two core components of the pluripotency circuit, Oct4 and Sox2, are also critical for germ layer fate choice

Rationale and Methodology of Reprogramming for Generation of Induced Pluripotent Stem Cells and Induced Neural Progenitor Cells.

Great progress has been made regarding the capabilities to modify somatic cell fate ever since the technology for generation of induced pluripotent stem cells (iPSCs) was discovered in 2006. Later, induced neural progenitor cells (iNPCs) were generated from mouse and human cells, bypassing some of the concerns and risks of using iPSCs in neuroscience applications. To overcome the limitation of viral vector induced reprogramming, bioactive small molecules (SM) have been explored to enhance the efficiency of reprogramming or even replace transcription factors (TFs), making the reprogrammed cells more amenable to clinical application. The chemical induced reprogramming process is a simple process from a technical perspective, but the choice of SM at each step is vital during the procedure. The mechanisms underlying cell transdifferentiation are still poorly understood, although, several experimental data and insights have indicated the rationale of cell reprogramming. The process begins with the forced expression of specific TFs or activation/inhibition of cell signaling pathways by bioactive chemicals in defined culture condition, which initiates the further reactivation of endogenous gene program and an optimal stoichiometric expression of the endogenous pluri- or multi-potency genes, and finally leads to the birth of reprogrammed cells such as iPSCs and iNPCs. In this review, we first outline the rationale and discuss the methodology of iPSCs and iNPCs in a stepwise manner; and then we also discuss the chemical-based reprogramming of iPSCs and iNPCs

Mechanisms and models of somatic cell reprogramming

Whitehead Institute for Biomedical Research (Jerome and Florence Brill Graduate Student Fellowship)National Institutes of Health (U.S.) (US NIH grant RO1-CA087869)National Institutes of Health (U.S.) (US NIH grant R37-CA084198)National Science Foundation (U.S.) (NSF Graduate Research Fellowship)National Institutes of Health (U.S.) ((NIH) Kirschstein National Research Service Award,1 F32 GM099153-01A1)Vertex Pharmaceuticals Incorporated (Vertex Scholar

Defining the Mechanism by which Synthetic Polymer Surfaces Support Human Pluripotent Stem Cell Self-Renewal.

Human pluripotent stem cells (hPSCs), which include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have become a promising resource for regenerative medicine and research into early development because these cells are able to indefinitely self-renew and are capable of differentiation into specialized cell types of all three germ layers and trophoectoderm. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. Synthetic polymers, such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel for hPSC culture. The main purpose of this dissertation is to define the mechanisms by which hPSCs are propagated on synthetic polymers. By physical modifications of PMEDSAH, we found that modifying substrate thickness changed the physical properties, and thus altered pluripotent stem cell behavior. Our data suggest that the 105 nm thick atom transfer radical polymerization (ATRP) PMEDSAH possesses the optimal gel architecture for hPSC expansion with its intermediate thickness, hydrophilicity, surface charge, and a moderate degree of inter-chain association. Our findings demonstrate the importance of polymer physical properties in hPSC expansion. The 105 nm thick ATRP PMEDSAH and similar modifications may be used to obtain scalable populations of clinical-grade hPSCs for regenerative medicine. Although a specific group of transcription factors, such as OCT4, SOX2 and NANOG, are known to play critical roles in hPSC pluripotency and reprogramming, other factors and the key signaling pathways regulating these important properties are not completely understood. In this dissertation, we also investigated the role of the PSC marker Developmental Pluripotency Associated 5 (DPPA5) in hPSCs. Our data demonstrate higher expression of DPPA5 in hPSCs under PMEDSAH and other feeder-free conditions, compared to MEFs. DPPA5 stabilizes protein levels and enhances the function of NANOG. Finally, DPPA5 increases the hiPSC-reprogramming efficiency. These results provide new molecular insight into the function of the DPPA5 in hPSCs. Our findings extend our understanding of the mechanism by which PMEDSAH and other feeder-free conditions support hPSC self-renewal, and offers improvements to current protocols in hPSC maintenance and reprogramming.PhDOral Health SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113368/1/xuqian_1.pd

Krüppel-like factors in cancer progression: three fingers on the steering wheel

Kruppel-like factors (KLFs) comprise a highly conserved family of zinc finger transcription factors, that are involved in a plethora of cellular processes, ranging from proliferation and apoptosis to differentiation, migration and pluripotency. During the last few years, evidence on their role and deregulation in different human cancers has been emerging. This review will discuss current knowledge on Kruppel-like transcription in the epithelial-mesenchymal transition (EMT), invasion and metastasis, with a focus on epithelial cancer biology and the extensive interface with pluripotency. Furthermore, as KLFs are able to mediate different outcomes, important influences of the cellular and microenvironmental context will be highlighted. Finally, we attempt to integrate diverse findings on KLF functions in EMT and stem cell biology to fit in the current model of cellular plasticity as a tool for successful metastatic dissemination

Identification and Characterization of a LIF-STAT3/Activin-Smad2/3 Dual Responsive Pluripotent Stem Cell State

The medical and bioindustrial applications of pluripotent stem cells rely on our understanding of their biology. Pluripotent stem cell lines derived from embryos in different stages depend on distinct signalling pathways. Embryonic stem cells (ESCs), derived from the inner cell mass (ICM) of preimplantation embryos, are dependent on LIF/STAT3 signalling, while epiblast stem cells (EpiSCs), established from the postimplantation embryos, require Activin A/Smad2/3 signalling. Recent studies revealed the presence of intermediate pluripotent stem cell populations. Their growth factor responsiveness, gene expression pattern and associated chromatic signatures, are compatible with the state intermediate between ESCs and EpiSCs. However, it remains unknown whether such cell populations represent a stable clonally intermediate cell state. In this thesis, I describe the discovery and characterization of novel stem cell lines displaying gene expression pattern intermediate between ESCs and EpiSCs. These cells respond to LIF/STAT3 as well as Activin/Smad2/3 signalling at single cell level. They can integrate into the ICM and generate chimeric embryos. In keeping with a more advanced differentiation stage than that of ESCs, the LIF/Activin dual responsive stem cells showed accelerated temporal gene expression kinetics during in vitro differentiation in embryo bodies. I found that these properties are shared by some induced pluripotent stem cell (iPSC) lines. The notion of an intermediate state was consolidated by a genome-wide microarray profiling. The hierarchical clustering analysis grouped LIF/Activin dual responsive stem cells together into a cluster intermediate between ESCs and EpiSCs. These findings advanced our understanding of the regulation of pluripotency. A better understanding of distinct differentiation state of pluripotent stem cells and their signalling responsiveness is crucial for developing tailored strategies for lineage/cell type differentiation