scholarly journals Pumilio Proteins Exert Distinct Biological Functions and Multiple Modes of Post-Transcriptional Regulation in Embryonic Stem Cell Pluripotency and Early Embryogenesis

2019 ◽  
Author(s):  
Katherine E. Uyhazi ◽  
Yiying Yang ◽  
Na Liu ◽  
Hongying Qi ◽  
Xiao A. Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Feedback Loop ◽  
Embryonic Stem ◽  
Early Embryogenesis ◽  
Self Renewal ◽  
Target Mrnas ◽  
Regulatory Feedback Loop ◽  
Post Transcriptional Regulation ◽  
Pluripotency Genes

ABSTRACTGene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional levels, but not at the post-transcriptional levels. Pumilio (Pum) proteins are among the few known translational regulators required for stem cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double mutants are developmentally delayed at the morula stage and lethal by embryonic day 8.5 (e8.5). Correspondingly, Pum1/2 double mutant ESCs display severely reduced self-renewal and differentiation, revealing the combined function of Pum1 and Pum2 in ESC pluripotency. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, indicating that Pum1 mainly promote differentiation; whereas Pum2-deficient ESCs show decreased expression of pluripotency genes and accelerated differentiation, indicating that Pum2 promotes self-renewal. Thus, Pum1 and Pum2 each uniquely contributes to one of the two complementary aspects of pluripotency. Furthermore, we show that Pum1 and Pum2 achieve ESC functions by forming a negative auto- and inter-regulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation as expected but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal the distinct roles of individual mammalian Pum proteins in ESCs and their collectively essential functions in ESC pluripotency and embryogenesis. Moreover, they demonstrate three novel modes of regulation of Pum proteins towards target mRNAs.SIGNIFICANCE STATEMENTThis report demonstrates the essential functions of mammalian Pumilio (Pum) proteins for embryonic stem cells (ESCs) pluripotency and embryogenesis. Moreover, it reveals the contrasting but complementary function of individual Pum proteins in regulating distinct aspects of ESC pluripotency, despite their largely overlapping expression and extremely high homology. Furthermore, it unravels a complex regulatory network in which Pum1 and Pum2 form a negative auto- and inter-regulatory feedback loop that regulates 1,486 mRNAs not only by translational repression as expected but also by promoting translation and enhancing or reducing stability of different target mRNAs, which reveals novel modes of post-transcriptional regulation mediated by Pum.

scholarly journals Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs)

2017 ◽  
Vol 44 (4) ◽  
pp. 1435-1444 ◽  
Author(s):  
Hong-Xi Zhao ◽  
Feng Jiang ◽  
Ya-Jing Zhu ◽  
Li Wang ◽  
Ke Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Human Embryonic Stem Cells ◽  
Immune Tolerance ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Immunological Tolerance ◽  
Self Renewal ◽  
Hesc Lines

Background: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. Methods: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. Results: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. Conclusions: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

scholarly journals Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Sergio Mora-Castilla ◽  
Juan R. Tejedo ◽  
Rafael Tapia-Limonchi ◽  
Irene Díaz ◽  
Ana B. Hitos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Directed Differentiation ◽  
Lineage Differentiation ◽  
Induction Of Differentiation ◽  
Differentiating Agent ◽  
Pluripotency Genes

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

HMGA1, a Factor Enriched in Hematopoietic Stem Cells, Embryonic Stem Cells, and Hematologic Malignancy, Enhances Cellular Reprogramming to a Pluripotent Stem-Like Cell.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2323-2323
Author(s):  
Linda Resar ◽  
Sandeep N Shah ◽  
Candace Kerr ◽  
Leslie Cope ◽  
Elias Zambidis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cancer Cells ◽  
Embryonic Stem ◽  
Cellular Reprogramming ◽  
Transcriptional Networks ◽  
Hematopoietic Stem ◽  
Stem Cell State ◽  
Pluripotency Genes

Abstract Abstract 2323 Although recent studies have identified genes important in hematopoietic stem cells (HSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), the molecular underpinnings of normal stem cell function are unclear. A better understanding of key stem cell pathways will be essential for the safe use of stem cells in regenerative medicine and should also uncover novel therapeutic targets in aggressive hematologic malignancies and other stem-like cancer cells. To elucidate the molecular underpinnings of “stemness”, we investigated transcriptional networks in pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes the HMGA1a and HMGA1b chromatin remodeling proteins. These proteins bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. HMGA1 is highly expressed during embryogenesis with low or undetectable levels in adult, differentiated tissues. HMGA1 is also enriched in HSCs, hESCs, iPSCs, refractory leukemia, and poorly differentiated solid tumors. Our group discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive leukemia in transgenic mice. We also found that high levels of HMGA1 expression correlate with relapse in acute lymphoblastic leukemia. Together, these findings suggest that HMGA1 drives a stem cell phenotype during normal development, hematopoiesis, and malignant transformation. To further investigate the role of HMGA1 in a stem cell state, we compared its expression in iPSCs, hESCs, HSCs, and cancer cells. HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in HSCs and cancer cells, and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation in hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells (mesenchymal stem cells, HSCs, and fetal lung fibroblasts) to an iPSC together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC or OSKM). HMGA1 results in an increase in the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign, teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In summary, our findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in hematologic and other malignancies or exploited in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ZMYM2 inhibits NANOG-mediated reprogramming

2019 ◽  
Vol 4 ◽  
pp. 88 ◽  
Author(s):  
Moyra Lawrence ◽  
Thorold W. Theunissen ◽  
Patrick Lombard ◽  
David J. Adams ◽  
José C. R. Silva
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
The Self ◽  
Negative Regulator ◽  
Leukaemia Inhibitory Factor ◽  
Self Renewal ◽  
Epiblast Stem Cells

Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency.  Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process. Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3β inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF). Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG’s actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.

339 INFLUENCE OF bFGF AND ACTIVIN A ON CAT FEEDER AND EMBRYONIC STEM CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 258
Author(s):  
M. Duque ◽  
M. N. Biancardi ◽  
J. H. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Embryonic Stem Cells ◽  
Culture Medium ◽  
Glycogen Synthase ◽  
Embryonic Stem ◽  
Activin A ◽  
Self Renewal ◽  
Embryonic Fibroblast

Different feeder cells (FC) influence the isolation, proliferation, and self-renewal of cat embryonic stem cells (cat ESC; Gómez et al. 2010 Theriogenology 74, 498–515) possibly by secretion of growth factors that affect intracellular signalling pathways involved in self-renewal. Supplementation of the culture medium with fibroblast growth factor (FGF) stimulates the secretion of Activin A in mouse and human FC, which enhances undifferentiation in human ESC (Eiselleova et al. 2008 Int. J. Dev. Biol. 52, 353-363). Moreover, the Activin/Nodal pathway plays an important role in maintaining pluripotency of hESC through mechanism(s) in which FGF acts as a competence factor (Vallier et al. 2005 J. Cell Sci. 118, 4495–4509). Little is known about secretion of growth factors by cat FC and whether cat ESC use the activin/nodal pathway for their self-renewal. Our previous work has indicated that culturing cat ESC with bFGF enhances the stem cell replication and self-renewal (Gómez et al. 2010 Theriogenology 74, 498–515). Here we evaluated the effect of bFGF supplementation in the culture medium on the abilities of cat embryonic fibroblast (CEF) and mouse embryonic fibroblast (MEF) FC to: (1) secrete Activin A and (2) support undifferentiated growth of cat ESC. For experiment 1, mitomycin-C-treated CEF (n = 2) and MEF (n = 2) were, respectively, cultured with ESC medium supplemented with (1) LIF (1000 IU), (2) bFGF (10 ng mL–1), (3) LIF + bFGF, or (4) no factors. The medium for each condition was collected at 24 h after culture and Activin A protein concentration was detected with a feline Activin A-ELISA kit. Results showed that supplementation of ESC medium with bFGF with or without LIF significantly increased the secretion of Activin A in MEF (5256 and 7048 ng mL–1, respectively; P < 0.001), but not in CEF (150 and 131 ng mL–1, respectively). Moreover, differences in Activin A secretion were observed between both MEF cell lines (10 269 v. 2034 ng mL–1; P < 0.001). For experiment 2, cat ESC were cultured in CEF or MEF in the ESC medium supplemented with bFGF (10 ng mL–1), LIF (1000 UI), and an inhibitor of glycogen synthase kinase-3 β (GSK3-b), SB 216763 (2.1 µM mL–1). Results showed differences in morphology of cat ESC cultured in CEF or MEF, where colonies cultured in CEF had clearly defined borders and a tightly domed shape, with a high nucleus to cytoplasm ratio and prominent nucleoli. In comparison, ESC cultured in MEF had poorly defined borders and a flattened shape. In addition, the mean cell size of colonies at passage 8 (P8) cultured on CEF was larger (612 ± 0.9 µm) than that of those cultured on MEF (360 ± 0.5 µm; P < 0.001). Colonies cultured on MEF differentiated into fibroblast-like cells and other noncharacterised cell types after P8. These results clearly indicated that CEF do not secrete Activin A. The negative effect of Activin A on the morphology of cat ESC cultured on MEF may suggest a synergism between GSK3b inhibitor and Activin A that may induce differentiation, possibly into mesoendodermal cells (Teo et al. 2014 Stem Cell Rep. 3, 5–14). Studies that evaluate the effects of supplementing ESC medium with a lower concentration of Activin A may help to elucidate the importance of the Activin/Nodal pathway in cat ESC.

scholarly journals Oct4 Gene Expression in Primary Colorectal Cancer Promotes Liver Metastasis

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Shiki Fujino ◽  
Norikatsu Miyoshi
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Stem Cells ◽  
Stem Cell ◽  
Liver Metastasis ◽  
Embryonic Stem ◽  
Clinical Samples ◽  
Self Renewal ◽  
Oct4 Expression

Purpose. The Oct4 gene plays an important role in undifferentiated embryonic stem cells and regulates stem cell pluripotency. The aim of this study was to examine the relationship between Oct4 expression and liver metastasis of colorectal cancer (CRC) in clinical samples and investigate the role and abilities of Oct4-positive CRC cells. Methods. The study included 158 patients who underwent surgery for CRC between 2009 and 2011. The correlations between the Oct4 gene expression and the clinical parameters were assessed, and liver metastasis-free survival (LMFS) was evaluated in these patients. Oct4-EGFP-positive cells were established to examine their subpopulation and ability. The capacity to form liver metastasis in vivo was examined using CRC cell lines and primary cultured CRC cells. Results. LMFS was significantly poor in the Oct4 high-expression group compared with the low-expression group (P=0.008). Multivariate analyses showed that Oct4 expression (P=0.015) and TNM stage (P<0.001) were significantly correlated with LMFS. Oct4-EGFP-positive cells highly expressed stem cell-associated markers and had self-renewal and differentiation abilities. Oct4-high cells actively formed liver metastasis. Conclusion. The Oct4 expression was correlated with liver metastasis in CRC patients. Oct4 expression cells have self-renewal and differentiation abilities like those of cancer stem cells. Oct4 contributed to forming liver metastasis in CRC.

scholarly journals Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells

2015 ◽  
Vol 112 (44) ◽  
pp. E5936-E5943 ◽  
Author(s):  
Haixia Chen ◽  
Renpeng Guo ◽  
Qian Zhang ◽  
Hongchao Guo ◽  
Meng Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Genomic Stability ◽  
Mouse Embryonic Stem Cells ◽  
Telomere Maintenance ◽  
Erk Signaling ◽  
Self Renewal ◽  
Cycle Arrest ◽  
Pluripotency Genes

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs.

scholarly journals Distinct SoxB1 networks are required for naïve and primed pluripotency

2017 ◽  
Author(s):  
Andrea Corsinotti ◽  
Frederick C. K. Wong ◽  
Tülin Tatar ◽  
Iwona Szczerbinska ◽  
Florian Halbritter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Functional Redundancy ◽  
Self Renewal ◽  
Cell Fate Decisions ◽  
Pluripotent State

AbstractDeletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naïve pluripotency.

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