Expression Of Pluripotency-associated Genes Research Articles

Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a post-implantation, epiblast stem cell state back to a pre-implantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

Neural, adipocyte and hepatic differentiation potential of primary and secondary hair follicle stem cells isolated from Arbas Cashmere goats

BackgroundArbas Cashmere goats are excellent domestic breeds with high yields of wool and cashmere. Their wool and cashmere can bring huge benefits to the livestock industry. Our studies intend to more fully understand the biological characteristics of hair follicle stem cells (HFSCs) in order to further explore the mechanisms of wool and cashmere regular regeneration. And they have been increasingly considered as promising multipotent cells in regenerative medicine because of their capacity to self-renew and differentiate. However, many aspects of the specific growth characteristics and differentiation ability of HFSCs remain unknown. This study aimed to further explore the growth characteristics and pluripotency of primary hair follicle stem cells (PHFSCs) and secondary hair follicle stem cells (SHFCs).ResultsWe obtained PHFSCs and SHFSCs from Arbas Cashmere goats using combined isolation and purification methods. The proliferation and vitality of the two types of HFSCs, as well as the growth patterns, were examined. HFSC-specific markers and genes related to pluripotency, were subsequently identified. The PHFSCs and SHFSCs of Arbas Cashmere goat have a typical cobblestone morphology. Moreover, the PHFSCs and SHFSCs express HFSC surface markers, including CD34, K14, K15, K19 and LGR5. We also identified pluripotency-associated gene expression, including SOX2, OCT4 and SOX9, in PHFSCs and SHFSCs. Finally, PHFSCs and SHFSCs displayed multipotent abilities. PHFSCs and SHFSCs can be directed to differentiate into adipocyte-like, neural-like, and hepatocyte-like cells.ConclusionsIn conclusion, this study confirmed that the biological characteristics and differentiation potential of PHFSCs and SHFSCs from Arbas Cashmere goats. These findings broaden and refine our knowledge of types and characteristics of adult stem cells.

Abstract 144: Nuclear Smooth Muscle α-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease

Missense pathogenic variants in ACTA2, encoding α-smooth muscle actin (SMA), predispose to thoracic aortic aneurysms. A subset of these ACTA2 pathogenic variants also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific ACTA2 pathogenic variants predispose to cerebrovascular disease, we confirmed that SMA is localized to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Pluripotent stem cell-derived SMCs from patients with ACTA2 -associated cerebrovascular disease ( ACTA2 R179C) as well as SMCs explanted from a knockin mouse model ( Acta2 SMC-R179C/+ ) confirm that R179 variants disrupt SMA nuclear localization. Both mouse and human cells harboring the R179C pathogenic variant proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the variant is heterozygous, these cells show a dominant negative impact on nuclear SMA function through dramatically reduced levels of SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, single cell RNA sequencing analysis of aortic tissue from an ACTA2 R179H patient confirms dedifferentiation of SMCs as a key phenotype associated with this mutation. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with ACTA2 R179 pathogenic variants.

Generation and Gene Expression Profiles of Grevy's Zebra Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) can serve as a biological resource for functional and conservation research for various species. This realization has led to the generation of iPSCs from many species, including those identified as endangered. However, the understanding of species variation in mammalian iPSCs remains largely unknown. To gain insight into species variation in iPSCs, we generated iPSCs from a new species Grevy's zebra (Equus grevyi; gz-iPSCs), which has been listed as endangered in the IUCN (International Union for Conservation of Nature) Red List. We isolated primary fibroblast cells from an individual and successfully reprogrammed them into iPSCs. The generated gz-iPSCs continued to grow under primed-type culture condition and showed pluripotency and differentiation potential. To describe the molecular characteristics of gz-iPSCs, we performed RNA sequencing analysis. The gz-iPSC transcriptome showed robust expression of pluripotency-associated genes reported in human and mouse, suggesting evolutionary conservation among the species. This study provides insight into the iPSCs from a rare species and helps the understanding of the gene expression basis underlying mammalian pluripotent stem cells.

Expression of pluripotency-related genes in human glioblastoma.

BackgroundCancer is a group of heterogeneous diseases characterized by several disruptions of the genetic and epigenetic components of cell biology. Some types of cancer have been shown to be constituted by a mosaic of cells with variable differentiation states, with more aggressive tumors being more undifferentiated. In most cases, undifferentiated tumor cells express associated embryonic markers such as the OCT4, NANOG, SOX2, and CARM1 genes. The ectopic or reminiscent expression of some master regulator genes of pluripotency has been indicated as the cause of the poorly differentiated state of tumors, and based on the evidence of some reports, can be used as a possible therapeutic target. Considering this information, a more detailed investigation of the expression of pluripotency-associated genes is necessary to evaluate the roles of these genes in the etiology of some tumors and their use targets of therapy.MethodsThe expression of four pluripotency-related genes was investigated (OCT4, NANOG, SOX2, and CARM1) in the most malignant primary human brain tumor, glioblastoma (GBM).Results and ConclusionThe results demonstrated a signature of OCT4/SOX2/CARM1 genes and a significant increase of CARM1 expression in GBM cases.

β-catenin links cell seeding density to global gene expression during mouse embryonic stem cell differentiation

SummaryAlthough cell density is known to affect numerous biological processes including gene expression and cell fate specification, mechanistic understanding of what factors link cell density to global gene regulation is lacking. Here, we reveal that the expression of thousands of genes in mouse embryonic stem cells (mESCs) is affected by cell seeding density and that low cell density enhances the efficiency of differentiation. Mechanistically, β-catenin is localized primarily to adherens junctions during both self-renewal and differentiation at high density. However, when mESCs differentiate at low density, β-catenin translocates to the nucleus and associates with Tcf7l1, inducing co-occupied lineage markers. Meanwhile, Esrrb sustains the expression of pluripotency-associated genes while repressing lineage markers at high density, and its association with DNA decreases at low density. Our results provide new insights into the previously neglected but pervasive phenomenon of density-dependent gene regulation.

Abstract P174: Nuclear Smooth Muscle α-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease

Missense mutations in ACTA2, encoding α-smooth muscle actin (α-SMA), predispose to thoracic aortic aneurysms. A subset of these ACTA2 mutations also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific ACTA2 mutations predispose to cerebrovascular disease, we confirmed that α-SMA translocates to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Expression of individual mutant α-SMAs in 293 cells determined that missense mutations associated with cerebrovascular disease inhibit this nuclear translocation. To further examine the effects of reduced nuclear α-SMA with ACTA2 mutations, we used two model systems: pluripotent stem cell-derived SMCs from patients with ACTA2-associated cerebrovascular disease (ACTA2 R179C) as well as SMCs explanted from a knockin mouse model (Acta2SMC-R179C/+). Both mouse and human cells harboring the R179C mutation proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the mutation is heterozygous, these cells show a dominant negative impact on nuclear α-SMA function through dramatically reduced levels of α-SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, forced nuclear localization of α-SMA in WT SMCs increases levels of SMC contractile proteins. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with ACTA2 R179 mutations.

Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

4-phenylbutyrate exerts stage-specific effects on cardiac differentiation via HDAC inhibition.

4-phenylbutyrate (4-PBA), a terminal aromatic substituted fatty acid, is used widely to specifically attenuate endoplasmic reticulum (ER) stress and inhibit histone deacetylases (HDACs). In this study, we investigated the effect of 4-PBA on cardiac differentiation of mouse embryonic stem (ES) cells. Herein, we found that 4-PBA regulated cardiac differentiation in a stage-specific manner just like trichostatin A (TSA), a well-known HDAC inhibitor. 4-PBA and TSA favored the early-stage differentiation, but inhibited the late-stage cardiac differentiation via acetylation. Mechanistic studies suggested that HDACs exhibited a temporal expression profiling during cardiomyogenesis. Hdac1 expression underwent a decrease at the early stage, while was upregulated at the late stage of cardiac induction. During the early stage of cardiac differentiation, acetylation favored the induction of Isl1 and Nkx2.5, two transcription factors of cardiac progenitors. During the late stage, histone acetylation induced by 4-PBA or TSA interrupted the gene silence of Oct4, a key determinant of self-renewal and pluripotency. Thereby, 4-PBA and TSA at the late stage hindered the exit from pluripotency, and attenuated the expression of cardiac-specific contractile proteins. Overexpression of HDAC1 and p300 exerted different effects at the distinct stages of cardiac induction. Collectively, our study shows that timely manipulation of HDACs exhibits distinct effects on cardiac differentiation. And the context-dependent effects of HDAC inhibitors depend on cell differentiation states marked by the temporal expression of pluripotency-associated genes.

Induction of autophagy promotes porcine parthenogenetic embryo development under low oxygen conditions.

Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.

Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report.

BackgroundCell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.ObjectiveTo derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.Materials and MethodsHuman ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.ResultsThe pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.ConclusionCryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.

CRISPR-Cas9-mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes.

Prkdc encodes for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) playing a key role in nonhomologous end joining pathway during DNA double-strand break repair and also influencing the homologous recombination (HR) repair system by phosphorylation of proteins involved in HR. In addition, Prkdc has other critical functions in biological processes, such as transcriptional regulation, telomere stability, apoptosis, and metabolism. DNA-PKcs upregulates during in vitro differentiation of mouse embryonic stem cells (mESCs). To address the potential role of Prkdc in mESCs pluripotency and in vitro differentiation into ectoderm, mesoderm, and endoderm germ layers under normal physiological conditions, a bi-allelic Prkdc-knockout cell line was generated in the present study by employing CRISPR/Cas9 system, and subsequently, its potential role in stemness and development was studied. The results of the study showed that the expression of pluripotency-associated genes, including Nanog and Sox-2 were overexpressed in the bi-allelic Prkdc-knockout cell line. Also, bi-allelic Prkdc-knockout cell line was shown to have typical mESCs cell morphology, cell cycle distribution, and alkaline phosphatase activity. Furthermore, the results of the study revealed that the expression of several germ layer markers is modulated in Prkdc-knockout lines. In conclusion, the findings of our study demonstrated the role of Prkdc during differentiation and development of ESCs.

RNA Targets Ribogenesis Factor WDR43 to Chromatin for Transcription and Pluripotency Control.

Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as achromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of theelongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects inPol II release and pluripotency-associated geneexpression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.

The Anthrax Toxin Receptor 1 (ANTXR1) Is Enriched in Pancreatic Cancer Stem Cells Derived from Primary Tumor Cultures

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related mortality. Cancer stem cells (CSCs) have been shown to be the drivers of pancreatic tumor growth, metastasis, and chemoresistance, but our understanding of these cells is still limited by our inability to efficiently identify and isolate them. While a number of markers capable of identifying pancreatic CSCs (PaCSCs) have been discovered since 2007, there is no doubt that more markers are still needed. The anthrax toxin receptor 1 (ANTXR1) was identified as a functional biomarker of triple-negative breast CSCs, and PDAC patients stratified based on ANTXR1 expression levels showed increased mortality and enrichment of pathways known to be necessary for CSC biology, including TGF-β, NOTCH, Wnt/β-catenin, and IL-6/JAK/STAT3 signaling and epithelial to mesenchymal transition, suggesting that ANTXR1 may represent a putative PaCSC marker. In this study, we show that ANTXR1+ cells are not only detectable across a panel of 7 PDAC patient-derived xenograft primary cultures but ANTXR1 expression significantly increased in CSC-enriched 3D sphere cultures. Importantly, ANTXR1+ cells also coexpressed other known PaCSC markers such as CD44, CD133, and autofluorescence, and ANTXR1+ cells displayed enhanced CSC functional and molecular properties, including increased self-renewal and expression of pluripotency-associated genes, compared to ANTXR1− cells. Thus, this study validates ANTXR1 as a new PaCSC marker and we propose its use in identifying CSCs in this tumor type and its exploitation in the development of CSC-targeted therapies for PDAC.

Understanding the mechanism underlying the acquisition of radioresistance in human prostate cancer cells.

Acquisition of radioresistance (RR) has been reported during cancer treatment with fractionated irradiation. However, RR is poorly understood in the prognosis of radiotherapy. Although radiotherapy is important in the treatment of prostate cancer (PCa), acquisition of RR has been reported in PCa with an increased number of cancer stem cells (CSCs), neuroendocrine differentiation (NED) and epithelial-mesenchymal transition. However, to the best of our knowledge, the mechanism underlying RR acquisition during fractionated irradiation remains unclear. In the present study, human PCa cell lines were subjected to fractionated irradiation according to a fixed schedule as follows: Irradiation (IR)1, 2 Gy/day with a total of 20 Gy; IR2, 4 Gy/day with a total of 20 Gy; and IR3, 4 Gy/day with a total of 56 Gy. The expression of cluster of differentiation (CD)44, a CSC marker, was identified to be increased by fractionated irradiation, particularly in DU145 cells. The expression levels of CD133 and CD138 were increased compared with those in parental cells following a single irradiation or multiple irradiations; however, the expression levels decreased with subsequent irradiation. RR was evidently acquired by exposure to 56 Gy radiation, which resulted in increased expression of the NED markers CD133 and CD138, and increased mRNA expression levels of the pluripotency-associated genes octamer-binding transcription factor 4 and Nanog homeobox. These data indicate that radiation-induced CSCs emerge due to the exposure of cells to fractionated irradiation. In addition, the consequent increase in the expression of NED markers is possibly induced by the increased expression of pluripotency-associated genes. Therefore, it can be suggested that cancer cells acquire RR due to increased expression of pluripotency-associated genes following exposure to fractionated irradiation.

Peroxisome Proliferator-Activated Receptor α Agonist and Its Target Nanog Cooperate to Induce Pluripotency.

The pharmaceutical compounds that modulate pluripotent stem cell (PSC) identity and function are increasingly adopted to generate qualified PSCs and their derivatives, which have promising potential in regenerative medicine, in pursuit of more accuracy and safety and less cost. Here, we demonstrate the peroxisome proliferator-activated receptor α (PPARα) agonist as a novel enhancer of pluripotency acquisition and induced pluripotent stem cell (iPSC) generation. We found that PPARα agonist, examined and selected Food and Drug Administration (FDA) -approved compound libraries, increase the expression of pluripotency-associated genes, such as Nanog, Nr5A2, Oct4, and Rex1, during the reprogramming process and facilitate iPSC generation by enhancing their reprogramming efficiency. A reprogramming-promoting effect of PPARα occurred via the upregulation of Nanog, which is essential for the induction and maintenance of pluripotency. Through bioinformatic analysis, we identified putative peroxisome proliferator responsive elements (PPREs) located within the promoter region of the Nanog gene. We also determined that PPARα can activate Nanog transcription by specific binding to putative PPREs. Taken together, our findings suggest that PPARα is an important regulator of PSC pluripotency and reprogramming, and PPARα agonists can be used to improve PSC technology and regenerative medicine.

Effects of ten-eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells.

Ten-eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5-E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.

Optimization of the treatment conditions with glycogen synthase kinase-3 inhibitor towards enhancing the proliferation of human induced pluripotent stem cells while maintaining an undifferentiated state under feeder-free conditions

The small molecule inhibitor CHIR99021 (CHIR) is well known as a selective glycogen synthase kinase-3 inhibitor. The purpose of our study was to optimize the conditions of CHIR supplementation that will enhance the proliferation of human induced pluripotent stem cells (hiPSCs) while maintaining their undifferentiated state under feeder-free conditions in adherent cultures for 4 days. Our results revealed that both of the timing and concentration of CHIR affected cell behaviors of hiPSCs, such as colony formation, cell proliferation, and differentiation. The addition of 1-3μM CHIR to hiPSCs cultures in the late 2-day period of a 4-day cultivation was effective in enhancing cell proliferation. Treatment with 3μM CHIR significantly enhanced cell proliferation, but led to differentiation of hiPSCs when the treatment was carried out over 4 days. Treatment with higher concentration of CHIR was also conducive to deviating hiPSCs from their undifferentiated state. Treatment with 10μM CHIR led to decreased expression of pluripotency-associated genes and increased level of mesoderm marker genes, but failed to provided any growth-promoting effect. Interestingly, when treatment with 1μM CHIR was confined to the late 2-day period of a 4-day cultivation, cell proliferation was enhanced without detectable deviation from the undifferentiated state as evidenced by the expression levels of pluripotency-associated genes, e.g., OCT3/4, NANOG, SOX2, and REX1. Repeated use of 1μM CHIR in subcultures provided no adverse effect on the proliferation of hiPSCs. Our results indicated that carefully designed CHIR treatment allows for enhanced proliferation of hiPSCs.

The in vitro biological assessment of the stability of cigarette smoke aqueous extracts

The small molecule inhibitor CHIR99021 (CHIR) is well known as a selective glycogen synthase kinase-3 inhibitor. The purpose of our study was to optimize the conditions of CHIR supplementation that will enhance the proliferation of human induced pluripotent stem cells (hiPSCs) while maintaining their undifferentiated state under feeder-free conditions in adherent cultures for 4 days. Our results revealed that both of the timing and concentration of CHIR affected cell behaviors of hiPSCs, such as colony formation, cell proliferation, and differentiation. The addition of 1–3 μM CHIR to hiPSCs cultures in the late 2-day period of a 4-day cultivation was effective in enhancing cell proliferation. Treatment with 3 μM CHIR significantly enhanced cell proliferation, but led to differentiation of hiPSCs when the treatment was carried out over 4 days. Treatment with higher concentration of CHIR was also conducive to deviating hiPSCs from their undifferentiated state. Treatment with 10 μM CHIR led to decreased expression of pluripotency-associated genes and increased level of mesoderm marker genes, but failed to provided any growth-promoting effect. Interestingly, when treatment with 1 μM CHIR was confined to the late 2-day period of a 4-day cultivation, cell proliferation was enhanced without detectable deviation from the undifferentiated state as evidenced by the expression levels of pluripotency-associated genes, e.g., OCT3/4, NANOG, SOX2, and REX1. Repeated use of 1 μM CHIR in subcultures provided no adverse effect on the proliferation of hiPSCs. Our results indicated that carefully designed CHIR treatment allows for enhanced proliferation of hiPSCs.

Regulation of DNA demethylation by the XPC DNA repair complex in somatic and pluripotent stem cells.

Faithful resetting of the epigenetic memory of a somatic cell to a pluripotent state during cellular reprogramming requires DNA methylation to silence somatic gene expression and dynamic DNA demethylation to activate pluripotency gene transcription. The removal of methylated cytosines requires the base excision repair enzyme TDG, but the mechanism by which TDG-dependent DNA demethylation occurs in a rapid and site-specific manner remains unclear. Here we show that the XPC DNA repair complex is a potent accelerator of global and locus-specific DNA demethylation in somatic and pluripotent stem cells. XPC cooperates with TDG genome-wide to stimulate the turnover of essential intermediates by overcoming slow TDG-abasic product dissociation during active DNA demethylation. We further establish that DNA demethylation induced by XPC expression in somatic cells overcomes an early epigenetic barrier in cellular reprogramming and facilitates the generation of more robust induced pluripotent stem cells, characterized by enhanced pluripotency-associated gene expression and self-renewal capacity. Taken together with our previous studies establishing the XPC complex as a transcriptional coactivator, our findings underscore two distinct but complementary mechanisms by which XPC influences gene regulation by coordinating efficient TDG-mediated DNA demethylation along with active transcription during somatic cell reprogramming.