Reprogramming Transcription Factors Research Articles

MEK and TGF-beta Inhibition Promotes Reprogramming without the Use of Transcription Factor.

The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages in vitro nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells.

Critical role of the miR-200 family in regulating differentiation and proliferation of neurons.

The generation of differentiated and functional neurons is a complex process, which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200, miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells, induction of miR-200 family was observed in differentiating neural stem cells, demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However, inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells, an inverse relationship was observed between expression of reprogramming transcription factors (SOX2, KLF4, NANOG, OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover, we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion, our results indicate that increased expression of the miR-200 family promotes neuronal differentiation, while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4.

The cancer stem cell phenotype: You can't win until you learn how to lose it

Cancer stem cells and their relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. We recently showed that reprogramming transcription factors induce glioblastoma cells to become stem-like and tumor propagating via a mechanism involving changes in global DNA methylation and downregulation of miRNAs.

A Place to Call Home: Bioengineering Pluripotential Stem Cell Cultures

Pluripotent stem cells (PSCs) have the power to revolutionize the future of cell-based therapies and regenerative medicine. However, stem/progenitor cell use in the clinical arsenal has been hampered by discrepancies resulting from stem cell engineering and expansion, as well as in their (mass) differentiation in culture. Moreover, the manner in which external conditions affect PSC and induced-pluripotent stem cell lineage establishment as well as maturation remains controversial. In this review, we examine novel methods of cell engineering and the role of reprogramming transcription factors in PSC development. In addition, we explore the effect of external environmental signals on PSC cultivation and differentiation by elucidating key components of the primordial stem cell microenvironment, the blastocyst. Furthermore, we assess the effects of hypoxic conditions on DNA editing, gene expression, and protein function in PSC self-renewal and growth. Finally, we speculate on the principal use of gap junction subunit expression as relevant biomarkers of PSC fate. Improving bioreactor design and pertinent cell biomarker classification could vastly enhance manufactured stem cell yield and quality, thereby increasing the potency and safety of therapeutic cells to be used in regenerative medicine.

Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs.

Pioneer transcription factors in cell reprogramming.

A subset of eukaryotic transcription factors possesses the remarkable ability to reprogram one type of cell into another. The transcription factors that reprogram cell fate are invariably those that are crucial for the initial cell programming in embryonic development. To elicit cell programming or reprogramming, transcription factors must be able to engage genes that are developmentally silenced and inappropriate for expression in the original cell. Developmentally silenced genes are typically embedded in "closed" chromatin that is covered by nucleosomes and not hypersensitive to nuclease probes such as DNase I. Biochemical and genomic studies have shown that transcription factors with the highest reprogramming activity often have the special ability to engage their target sites on nucleosomal DNA, thus behaving as "pioneer factors" to initiate events in closed chromatin. Other reprogramming factors appear dependent on pioneer factors for engaging nucleosomes and closed chromatin. However, certain genomic domains in which nucleosomes are occluded by higher-order chromatin structures, such as in heterochromatin, are resistant to pioneer factor binding. Understanding the means by which pioneer factors can engage closed chromatin and how heterochromatin can prevent such binding promises to advance our ability to reprogram cell fates at will and is the topic of this review.

Sphere formation permits Oct4 reprogramming of ciliary body epithelial cells into induced pluripotent stem cells.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by defined sets of transcription factors. We previously described reprogramming of monolayer-cultured adult mouse ciliary body epithelial (CE) cells by Oct4 and Klf4, but not with Oct4 alone. In this study, we report that Oct4 alone is sufficient to reprogram CE cells to iPS cells through sphere formation. Furthermore, we demonstrate that sphere formation induces a partial reprogramming state characterized by expression of retinal progenitor markers, upregulation of reprogramming transcription factors, such as Sall4 and Nanog, demethylation in the promoter regions of pluripotency associated genes, and mesenchymal to epithelial transition. The Oct4-iPS cells maintained normal karyotypes, expressed markers for pluripotent stem cells, and were capable of differentiating into derivatives of all three embryonic germ layers in vivo and in vitro. These findings suggest that sphere formation may render somatic cells more susceptible to reprogramming.

Genome-wide characterization of the routes to pluripotency.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

337 PROMOTER-DEPENDENT SILENCING OF REPROGRAMMING TRANSCRIPTION FACTORS IN MOUSE INDUCED PLURIPOTENT STEM CELLS PRODUCED WITH SLEEPING BEAUTY TRANSPOSON VECTORS

Epigenetic silencing of the transgenes has been considered a prerequisite for complete reprogramming of mouse somatic cells to induced pluripotent stem cells (miPSC). Here, we examined the activity status of the reprogramming transcription factors in miPSC produced with Sleeping Beauty (SB) transposon vectors carrying expression cassettes with the porcine OCT4, SOX2, c-MYC, and KLF4 (pOSMK) under the control of doxycycline (DOX)-inducible (TetO) or constitutive (CAG) promoters. Mouse embryo fibroblasts (MEF) were electroporated with SB-TetO-rTA-SV40pA-TetO-pOSMK-IRES-tdTomato-bGHpA (TetO group) or with SB-loxP-CAG-pOSMK-IRES-tdTomato-SV40pA-loxP (CAG group) together with SB100x (SB transposase). The cells were cultured on mitotically inactivated MEF feeders with DMEM supplemented with 20% knockout serum replacement, 2 mM l-glutamine, penicillin-streptomycin, nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1000 U mL–1 of ESGRO, and 5 µg mL–1 of DOX. The miPSC colonies were individually picked, disaggregated to single cells, and propagated further under the same culture conditions. Three cell lines from each experimental group were examined for pluripotency characteristics, and the activity of the transgenes was monitored by the presence of tdTomato fluorescence and by RT-PCR. The miPSC produced with TetO vector silenced the transgene expression within 11 days post-transfection (in the presence of DOX) and upregulated the endogenous pluripotency genes Oct4, Sox2, Nanog, Rex1, and Utf1. These cells showed typical miPSC morphology and ability to differentiate into cells from the 3 primary germ layers in vitro and in vivo (teratomas). At the same time, the miPSC from the CAG group did not silence the transgenes even after 20 passages of continuous propagation, although they upregulated the endogenous pluripotency genes similarly to the TetO group. Moreover, these cells also showed ability to differentiate in vitro into cells from the 3 germ layers (contracting cardiac myocytes, neurons, epithelia) expressing differentiation markers Afp, Sox17, Gata4, Gata6, cardiac troponin, nestin, and PGP 9.5. Following Cre-mediated excision of the reprogramming cassette, the miPSC from the CAG group continued to self-renew and the expression of pluripotency markers Oct4, Sox2, Nanog, and Rex1 did not change significantly, as evidenced by real-time RT PCR (all P > 0.1), showing that these cells were not dependent on the transgenes for maintaining their pluripotency characteristics. Currently, we are investigating the ability of the miPSC from the CAG group to differentiate in vivo by producing teratomas and chimeras. The results from our preliminary investigations suggest that porcine transcription factors can be used for production of miPSC and that the silencing of the reprogramming transcription factors in miPSC is promoter-dependent, but may not be absolutely necessary for complete reprogramming to pluripotency.

Identification of regulatory factors for mesenchymal stem cell-derived salivary epithelial cells in a co-culture system.

Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.

DNMT-dependent suppression of microRNA regulates the induction of GBM tumor-propagating phenotype by Oct4 and Sox2.

Cancer stem-like cells represent poorly differentiated multipotent tumor-propagating cells that contribute disproportionately to therapeutic resistance and tumor recurrence. Transcriptional mechanisms that control the phenotypic conversion of tumor cells lacking tumor-propagating potential to tumor-propagating stem-like cells remain obscure. Here we show that the reprogramming transcription factors Oct4 and Sox2 induce glioblastoma cells to become stem-like and tumor-propagating via a mechanism involving direct DNA methyl transferase (DNMT) promoter transactivation, resulting in global DNA methylation- and DNMT-dependent downregulation of multiple microRNAs (miRNAs). We show that one such downregulated miRNA, miRNA-148a, inhibits glioblastoma cell stem-like properties and tumor-propagating potential. This study identifies a novel and targetable molecular circuit by which glioma cell stemness and tumor-propagating capacity are regulated.

Generation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are generated from somatic cells that have been reprogrammed by the ectopic expression of defined embryonic transcription factors. This technology has provided investigators with a powerful tool for modelling disease and developing treatments for human disorders. This chapter provides the researcher with some background on iPSCs and details on how to produce MEF-conditioned medium, prepare mitotically arrested mouse embryonic fibroblasts (MEFs), create iPSCs using viral vectors, passage iPSCs, and cryopreserve iPSCs. The methods offered here have been used in many laboratories around the world and the reader can initially follow these methods. However, not all cell types are easily transduced using viral vectors and other methods of delivering the reprogramming transcription factors may need to be tested.

MYC activates stem-like cell potential in hepatocarcinoma by a p53-dependent mechanism.

Activation of c-MYC is an oncogenic hallmark of many cancers, including liver cancer, and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c-MYC activation for the biology of hepatic cancer stem cells (CSC) are undefined. Here, distinct levels of c-MYC overexpression were established by using two dose-dependent tetracycline-inducible systems in four hepatoma cell lines with different p53 mutational status. The CSCs were evaluated using side population (SP) approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g., NANOG, OCT4, and EpCAM), expansion of SP, and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a proapoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC-induced self-renewal capacity of liver cancer cells was exerted in a p53-dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of c-MYC overexpression. Together, our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC-induced CSC phenotype in liver cancer cells.

EPIGENETIC REGULATION OF GBM CELL STEMNESS AND TUMOR PROPAGATING CAPACITY BY OCT4 AND SOX2

BACKGROUND: (blind field). METHODS: Experiments utilized human glioblastoma neurosphere lines and low passage (<5) primary neurospheres derived from clinical GBM specimens enriched in GBM sphere-forming stem cells. The Adherent GBM cell lines that lack stem-like tumor-propagating cells were used to test phenotype reprogramming by Oct4 and Sox2. Lentiviral and retroviral systems were used for Oct4/Sox2 and micro-RNA gene transfer, respectively. Brain cancer miRNA PCR Arrays (SABiosciences) and quantitative RT-PCR were used to evaluate miRNA expression. OCT4 and Sox2 transcriptional targets were identified and validated by ChIP-PCR and luciferase reporter assays. Micro-RNA promoter methylation was evaluated using bisulfite sequencing. GBM cell stemness was evaluated by sphere-forming assays and analyses of stem cell marker expression by qRT-PCR, immunoblotting and flow cytometry. Tumor xenograft propagating capacity was assayed in immune-deficient mice. RESULTS: In this study, we identify a novel molecular circuit linking reprogramming transcription factors, DNA methylation and microRNA that regulates glioblastoma cell stemness and tumor-propagating capacity. We show that the Oct4 and Sox2 transcription factors induce human glioma cells to transition to a stem-like tumor-propagating state through the direct induction of DNA methyl transferases leading to the methylation-dependent transcriptional regulation of microRNAs. Specifically, miR-148a expression is shown to correlate inversely with GBM cell stemness, to be repressed by Oct4/Sox2-driven DNA methylation, and to inhibit GBM cell stemness and tumor propagation by GBM sphere-forming cells. CONCLUSIONS: These results identify a new methylation-dependent transcriptional axis by which reprogramming transcription factors regulate tumor cell stemness and tumor propagating capacity. Our results also identify miR-148a as a novel tumor-suppressing inhibitor of glioma cell stemness and suggest new approaches for treating GBM by targeting stem-like tumor propagating cell populations. SECONDARY CATEGORY: n/a.

Concise Review: A Population Shift View of Cellular Reprogramming

Cellular reprogramming can offer valuable insight into disease mechanism and has the potential to provide novel tools for regenerative medicine. Yet it remains an inefficient and often incomplete process. However, experiments show that almost all somatic cells eventually give rise to the pluripotent state, albeit at different latencies, as long as expression of reprogramming transcription factors is maintained. Furthermore, it appears that specific subpopulations of cells can be identified that show enhanced propensities to be reprogrammed to the pluripotent state. It has been proposed that an initial stochastic process is responsible for this initial priming that is followed by a deterministic process that directs the primed cells into the pluripotent state. Here, we propose a population shift view of cellular reprogramming, which explains these observations and reconciles the stochastic and deterministic nature of this process. According to this view, a small population of cells, whose states are closer to the pluripotent state and reside in pre-existing energetically favorable trajectories, will be initially selected for reprogramming. Moreover, by maintaining ectopic expression of reprogramming factors, other cells enter these pathways as a result of transcriptional and epigenetic stochastic variations. Consequently, increasing numbers of cells reach the pluripotent state, and the cell population distribution shifts toward this state. Importantly, additional perturbations can change the epigenetic landscape, allowing cells more access to the reprogramming trajectories, thereby increasing reprogramming efficiency. Knowledge of the initial cellular subpopulations and pathways of states that lead to the final cellular state should allow us to design alternative perturbation strategies to improve reprogramming efficiency and fidelity.

The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro.

Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.

Fine-Tuning of iPSC Derivation by an Inducible Reprogramming System at the Protein Level

SummaryInduced pluripotent stem cells (iPSCs) generated from somatic cells by ectopic expression of reprogramming factors, e.g., POU5F1 (OCT4), KLF4, and SOX2, have great potential for regenerative medicine. However, before they can be used in a clinical setting, the mechanism of reprogramming needs to be better understood. Here, by engineering reprogramming factors to a destabilizing protein domain, we achieved inducible generation of mouse and pig iPSCs. Stability of the fusion protein was precisely regulated by the addition of the cell-permeable small molecule trimethoprim (TMP) in a dose-dependent manner. With these tools, we found that during the early and middle stages of reprogramming, exogenous OCT4 or KLF4 could be omitted, whereas exogenous SOX2 expression at early and middle stages was required for successful reprogramming. Our TMP reprogramming system is useful for defining the stoichiometry and temporal requirements of transcription factors for reprogramming.

Patient-Specific Induced Pluripotent Stem Cells for SOD1-Associated Amyotrophic Lateral Sclerosis Pathogenesis Studies

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells, called induced pluripotent stem cells (iPSCs), can be an unlimited source of specialized cell types for the body. Thus, autologous somatic cell replacement therapy becomes possible, as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited, and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons.

Promise of Human Induced Pluripotent Stem Cells in Skin Regeneration and Investigation

Induced pluripotent stem cell (iPSC) technology has initiated a new era in biomedical science. The skin has been realized as an ideal platform for iPSC applications; unlike other organs, the skin is easily accessible, highly proliferative, and reconstitutable. Currently, skin equivalents can be generated from iPSCs not only from healthy individuals but also from patients with genodermatoses, providing novel platforms for dissecting disease pathophysiology and establishing cell-based therapy. With their developmental plasticity, iPSCs may also enable the regeneration of skin appendages. The iPSC technology may provide novel remedies for intractable disorders, once key issues particularly, safety concerns, are cleared.

Exogenous Mesp1 expression induces a cardiac progenitor fate

Objectives: Direct conversion of fibroblasts into cardiomyocyte-like cells was reported by using Gata4, Mef2c and Tbx5 (GMT) as reprogramming transcription factors. Beside us, many other investigators reported the insufficient reprogramming capacity of GMT to induce a complete phenotype conversion into cardiomyocyte like cells.