Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways

Abstract Background Stem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells. Methods In the present study, we used the previously constructed luciferase reporters driven by the promoter and 3’-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways. Results We screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways. Conclusions These findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

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391 A COMPARATIVE GENE EXPRESSION ANALYSIS BETWEEN SHEEP EARLY EMBRYONIC DEVELOPMENT AND PUTATIVE EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab391 ◽

2010 ◽

Vol 22 (1) ◽

pp. 352

Author(s):

B. M. Murray ◽

S. Schmoelzl ◽

N. M. Andronicos ◽

J. R. Hill ◽

P. J. Verma ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

In Vitro Culture ◽

Culture Media ◽

Embryonic Stem ◽

Oct4 Expression ◽

Pluripotency Markers

The optimization of culture media to support the isolation of embryonic stem cells relies on methods to monitor whether the pluripotent state of the cultured cells has been maintained. We developed a panel of gene expression assays that allowed us to correlate molecular measures of pluripotency or lineage differentiation with a developmental time course. By conducting quantitative PCR analysis of sheep embryos over Day 6.5 to 24 and sheep inner cell mass (ICM) cells cultured over 25 days, we tested whether culture media designed to inhibit differentiation are able to maintain sheep ICM cells in a pluripotent state. Briefly, embryos were collected from Merino ewes (n = 50, 3 years) at Day 6.5, 12, 16, 20, and 24 post-AI. Embryos were collected from the dissected uterine tracts of slaughtered ewes, excluding Day 6.5 blastocysts, which were surgically recovered from superovulated ewes. For the in vitro culture, Day 6.5 ICM cells were isolated by immunosurgery and cultured on mitomycin-C-treated mouse embryonic fibroblasts in an inhibitor-based medium (3i, based on Ying Q-L et al. 2008 Nature 453, 519-523). Real-time PCR assays for pluripotency (OCT4, SOX2, NANOG) and differentiation (ectodermal: FGF5, PAX6; endodermal: GATA4, GATA6, Somatostatin; mesodermal: BMP4, Connexin40) of sheep candidate genes were conducted on cDNA prepared from these samples and normalized against the reference genes RPL19 and RPS26. In in vivo embryos, pluripotency markers OCT4, SOX2, and NANOG all decreased between Day 6.5 and Day 20, although OCT4 expression spiked around Day 16. More interestingly, pluripotency expression decreased during in vitro culture, with NANOG expression completely lost by passage 2 at Day 11 and OCT4 expression at an equivalent Day 24 embryo basal level by Day 14. The endodermal markers GATA6 and GATA4 decreased between Day 6.5 and Day 12, respectively, although in vitro GATA4 was only expressed once at Day 7. In vivo FGF5 and both PAX6 and Somatostatin displayed a delayed onset, increasing expression from Day 16 and 20, respectively, whereas the ectodermal markers were already expressed by Day 7 in vitro. Both mesodermal markers Connexin40 and BMP4 presented minor fold changes in both data sets. In conclusion, this study has verified the primer sets and described a sheep in vivo embryo gene expression profile comprising both pluripotent and differentiation candidates. Furthermore, the decrease of pluripotency markers together with the appearance of differentiation markers during in vitro culture of ICM cells suggest that culturing ICM cells in 3i media is not sufficient to maintain a sheep-specific pluripotent population of cells. Therefore, future studies will be aimed at manipulating the current in vitro system to focus on maintaining pluripotent genes such as NANOG and OCT4 in culture.

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DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition

International Journal of Molecular Sciences ◽

10.3390/ijms22115954 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5954

Author(s):

Julee Kim ◽

Sangki Baek ◽

Yean-Ju Hong ◽

Michelle Novais de Paula ◽

Musharrat Jahan Prima ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem ◽

Mrna Levels ◽

Self Renewal ◽

Free Condition ◽

Long Term Culture ◽

Defined Media ◽

Pluripotency Markers

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

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Impact of OCT4 and Its Related Signaling Pathways on Gastrointestinal Cancers: Focusing on Targeted Therapy

Iranian Journal of Allergy Asthma and Immunology ◽

10.18502/ijaai.v19i3.3451 ◽

2020 ◽

Author(s):

Hamid Cheshomi ◽

Omid Gholami ◽

Babak Peyroshabani ◽

Abolfazl Rad

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Cancer Stem Cells ◽

Signaling Pathways ◽

Cancer Progression ◽

Gastrointestinal Cancers ◽

Oct4 Expression ◽

Pou Domain ◽

Transcription Factor 4 ◽

Transcription Factor 1

There are many pieces of evidence support the effect of cancer stem cells on the initiation and progression of cancer. However, related mechanisms involved in these phenomena are far more complicated to understand. The function of different stemness factorsin cancer stem cells (CSCs) and their complex associations at different levels of cancer have been reported. Therefore, it seems that focusing on one master factor would be more helpful to complete the puzzle of singling pathways in these cells. Octamer-binding transcription factor 4 (OCT4) also known as POU domain, class 5, transcription factor 1(POU5F1), one of these key pluripotency factors, has important roles in both embryogenesis and tumorigenesis. In this review, we gathered information about the association of different markers with OCT4 expression in three types of gastrointestinal cancers including esophageal, gastric and colorectal cancers. OCT4 through different signaling pathways has an impact on different processes of gastrointestinal cancers such as proliferation, invasion, and metastasis. Based on the literature, OCT4 has great effects on cancer progression at different stages, therefore we suggested it has potential implications in therapeutic options.

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Faculty Opinions recommendation of Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1677956.1501058 ◽

2010 ◽

Author(s):

Richard Gilbertson

Keyword(s):

Stem Cells ◽

Self Renewal

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Cancer Stem Cell Microenvironment Models with Biomaterial Scaffolds In Vitro

Processes ◽

10.3390/pr9010045 ◽

2020 ◽

Vol 9 (1) ◽

pp. 45

Author(s):

Ghmkin Hassan ◽

Said M. Afify ◽

Shiro Kitano ◽

Akimasa Seno ◽

Hiroko Ishii ◽

...

Keyword(s):

Stem Cells ◽

Cancer Biology ◽

Advanced Technology ◽

Cancer Microenvironment ◽

Critical Factors ◽

Self Renewal ◽

Cell Microenvironment ◽

Biological Phenomena ◽

Biomaterial Scaffolds

Defined by its potential for self-renewal, differentiation and tumorigenicity, cancer stem cells (CSCs) are considered responsible for drug resistance and relapse. To understand the behavior of CSC, the effects of the microenvironment in each tissue are a matter of great concerns for scientists in cancer biology. However, there are many complicated obstacles in the mimicking the microenvironment of CSCs even with current advanced technology. In this context, novel biomaterials have widely been assessed as in vitro platforms for their ability to mimic cancer microenvironment. These efforts should be successful to identify and characterize various CSCs specific in each type of cancer. Therefore, extracellular matrix scaffolds made of biomaterial will modulate the interactions and facilitate the investigation of CSC associated with biological phenomena simplifying the complexity of the microenvironment. In this review, we summarize latest advances in biomaterial scaffolds, which are exploited to mimic CSC microenvironment, and their chemical and biological requirements with discussion. The discussion includes the possible effects on both cells in tumors and microenvironment to propose what the critical factors are in controlling the CSC microenvironment focusing the future investigation. Our insights on their availability in drug screening will also follow the discussion.

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Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽

10.3390/cells10040876 ◽

2021 ◽

Vol 10 (4) ◽

pp. 876

Author(s):

Raquel Bernad ◽

Cian J. Lynch ◽

Rocio G. Urdinguio ◽

Camille Stephan-Otto Attolini ◽

Mario F. Fraga ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Primordial Germ Cell ◽

Normal Karyotype ◽

Cell Types ◽

Dna Demethylation ◽

Starting State ◽

Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

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Isolation, Culture and Identification of Bovine Skeletal Stem Cells

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2816 ◽

2021 ◽

Vol 11 (12) ◽

pp. 2337-2345

Author(s):

Junhui Lai ◽

Qin Yang ◽

Ruining Liang ◽

Weijun Guan ◽

Xiuxia Li

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Bone Formation ◽

Growth Plate ◽

Bone Development ◽

Long Bone ◽

Biological Characterization ◽

Self Renewal ◽

And Cartilage

The growth plate is essential in long bone formation and contains a wealth of skeletal stem cells (SSCs). Though the origin and the mechanism for SSCs generation remain uncertain, recent studies demonstrate the transition from cartilage to bone that in the lineage for bone development. SSCs possesses the ability to differentiate into bone and cartilage in vitro. In this research, we aimed to isolate and culture the skeletal stem cells from bovine cattle and then studied its biological characterization. The results showed that these bovine SSCs are positive for PDPN+CD73+CD164+CD90+CD44+ cell surface bio-markers, they are capable of self-renewal and differentiation. Our dates proved that SSCs exists in bovine’s long bone.

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Abstract 129: Embryonic Stem Cell Derived Exosomes Revive Endogenous Repair Mechanisms In Failing Heart

Circulation Research ◽

10.1161/res.115.suppl_1.129 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Mohsin Khan ◽

Suresh K Verma ◽

Alexander R Mackie ◽

Erin Vaughan ◽

Srikanth Garikipati ◽

...

Keyword(s):

Stem Cells ◽

Therapeutic Potential ◽

Embryonic Stem ◽

Cardiac Regeneration ◽

Great Promise ◽

Target Cells ◽

Free System ◽

Teratoma Formation

Rationale: Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to ethical concerns, lack of autologous donors and teratoma formation. Recently, it has been observed that beneficial effects of stem cells are mediated by exosomes secreted out under various physiological conditions. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown. Objective: Determine the effect of ESC derived exosomes for cardiac repair and modulation of CPCs functions in the heart following myocardial infarction. Methods and Results: Exosomes were isolated from murine ESCs (mES Ex) or embryonic fibroblasts (MEFs) by ultracentrifugation and verified by Flotillin-1 immunoblot analysis. Induction of pluripotent markers, survival and in vitro tube formation was enhanced in target cells receiving ESC exosomes indicating therapeutic potential of mES Ex. mES Ex administration resulted in enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex mediated considerable enhancement of cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 4 weeks after mES Ex transfer. miRNA Array analysis of ESC and MEF exosomes revealed significantly high expression of miR290-295 cluster in the ESC exosomes compared to MEF exosomes. The underlying beneficial effect of mES Ex was tied to delivery of ESC miR-294 to the heart and in particular CPCs thereby promoting CPC survival and proliferation as analyzed by FACS based cell death analysis and CyQuant assay respectively. Interestingly, enhanced G1/S transition was observed in CPCs treated with miR-294 in conjunction with significant reduction of G1 phase. Conclusion: In conclusion, mES Ex provide a novel cell free system for cardiac regeneration with the ability to modulate both cardiomyocyte and CPC based repair programs in the heart thereby avoiding the risk of teratoma formation associated with ESCs.

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