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  1. Fulltext Zyxin regulates embryonic stem cell fate by modulating mechanical and biochemical signaling interface
    Zhang S, Chong LH, Woon JYX, Chua TX, Cheruba E, Yip AK, et al.
    Commun Biol, 2023 Jan 18;6(1):62.
    PMID: 36653484 DOI: 10.1038/s42003-023-04421-0
    Biochemical signaling and mechano-transduction are both critical in regulating stem cell fate. How crosstalk between mechanical and biochemical cues influences embryonic development, however, is not extensively investigated. Using a comparative study of focal adhesion constituents between mouse embryonic stem cell (mESC) and their differentiated counterparts, we find while zyxin is lowly expressed in mESCs, its levels increase dramatically during early differentiation. Interestingly, overexpression of zyxin in mESCs suppresses Oct4 and Nanog. Using an integrative biochemical and biophysical approach, we demonstrate involvement of zyxin in regulating pluripotency through actin stress fibres and focal adhesions which are known to modulate cellular traction stress and facilitate substrate rigidity-sensing. YAP signaling is identified as an important biochemical effector of zyxin-induced mechanotransduction. These results provide insights into the role of zyxin in the integration of mechanical and biochemical cues for the regulation of embryonic stem cell fate.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  2. The Promises and Pitfalls of CRISPR-Mediated Base Editing in Stem Cells
    Wong PK, Mohamad Zamberi NN, Syafruddin SE, Cheah FC, Azmi N, Law JX, et al.
    CRISPR J, 2023 Jun;6(3):196-215.
    PMID: 37219623 DOI: 10.1089/crispr.2023.0013
    Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic disorders. Precise base edits can be achieved using a variety of CRISPR-centric approaches, particularly homology-directed repair and the recently developed base editors and prime editors. Despite its much-touted potential, editing single DNA bases is technically challenging. In this review, we discuss the strategies for achieving exact base edits in the creation of various stem cell-based models for use in elucidating disease mechanisms and assessing drug efficacy, and the unique characteristics of stem cells that warrant special considerations.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  3. Fulltext Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition
    Ho SY, Goh CW, Gan JY, Lee YS, Lam MK, Hong N, et al.
    Zebrafish, 2014 Oct;11(5):407-20.
    PMID: 24967707 DOI: 10.1089/zeb.2013.0879
    Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.
    Matched MeSH terms: Embryonic Stem Cells/metabolism*
  4. Fulltext Frequent co-expression of miRNA-5p and -3p species and cross-targeting in induced pluripotent stem cells
    Huang CJ, Nguyen PN, Choo KB, Sugii S, Wee K, Cheong SK, et al.
    Int J Med Sci, 2014;11(8):824-33.
    PMID: 24936146 DOI: 10.7150/ijms.8358
    A miRNA precursor generally gives rise to one major miRNA species derived from the 5' arm, and are called miRNA-5p. However, more recent studies have shown co-expression of miRNA-5p and -3p, albeit in different concentrations, in cancer cells targeting different sets of transcripts. Co-expression and regulation of the -5p and -3p miRNA species in stem cells, particularly in the reprogramming process, have not been studied.
    Matched MeSH terms: Embryonic Stem Cells/metabolism*
  5. Application of multiplex PCR for characterization of human embryonic stem cells (hESCs) and its differentiated progenies
    Mamidi MK, Pal R, Bhonde R, Zakaria Z, Totey S
    J Biomol Screen, 2010 Jul;15(6):630-43.
    PMID: 20530724 DOI: 10.1177/1087057110370211
    Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.
    Matched MeSH terms: Embryonic Stem Cells/metabolism*
  6. Fulltext MicroRNA-124a inhibits endoderm lineage commitment by targeting Sox17 and Gata6 in mouse embryonic stem cells
    Liew LC, Gailhouste L, Tan GC, Yamamoto Y, Takeshita F, Nakagama H, et al.
    Stem Cells, 2020 04;38(4):504-515.
    PMID: 31828873 DOI: 10.1002/stem.3136
    The role of microRNAs (miRNAs) during mouse early development, especially in endoderm germ layer formation, is largely unknown. Here, via miRNA profiling during endoderm differentiation, we discovered that miR-124a negatively regulates endoderm lineage commitment in mouse embryonic stem cells (mESCs). To further investigate the functional role of miR-124a in early stages of differentiation, transfection of embryoid bodies with miR-124a mimic was performed. We showed that overexpression of miR-124a inhibits endoderm differentiation in vitro through targeting the 3'-untranslated region (UTR) of Sox17 and Gata6, revealing the existence of interplay between miR-124a and the Sox17/Gata6 transcription factors in hepato-specific gene regulation. In addition, we presented a feasible in vivo system that utilizes teratoma and gene expression profiling from microarray to quantitatively evaluate the functional role of miRNA in lineage specification. We demonstrated that ectopic expression of miR-124a in teratomas by intratumor delivery of miR-124a mimic and Atelocollagen, significantly suppressed endoderm and mesoderm lineage differentiation while augmenting the differentiation into ectoderm lineage. Collectively, our findings suggest that miR-124a plays a significant role in mESCs lineage commitment.
    Matched MeSH terms: Mouse Embryonic Stem Cells/metabolism*
  7. Fulltext Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs
    Chen YM, Chen LH, Li MP, Li HF, Higuchi A, Kumar SS, et al.
    Sci Rep, 2017 03 23;7:45146.
    PMID: 28332572 DOI: 10.1038/srep45146
    Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  8. Fulltext A concise review on epigenetic regulation: insight into molecular mechanisms
    Golbabapour S, Abdulla MA, Hajrezaei M
    Int J Mol Sci, 2011;12(12):8661-94.
    PMID: 22272098 DOI: 10.3390/ijms12128661
    Epigenetic mechanisms are responsible for the regulation of transcription of imprinted genes and those that induce a totipotent state. Starting just after fertilization, DNA methylation pattern undergoes establishment, reestablishment and maintenance. These modifications are important for normal embryo and placental developments. Throughout life and passing to the next generation, epigenetic events establish, maintain, erase and reestablish. In the context of differentiated cell reprogramming, demethylation and activation of genes whose expressions contribute to the pluripotent state is the crux of the matter. In this review, firstly, regulatory epigenetic mechanisms related to somatic cell nuclear transfer (SCNT) reprogramming are discussed, followed by embryonic development, and placental epigenetic issues.
    Matched MeSH terms: Embryonic Stem Cells/metabolism*
  9. Fulltext Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming
    Choong PF, Teh HX, Teoh HK, Ong HK, Choo KB, Sugii S, et al.
    Int J Med Sci, 2014;11(11):1154-60.
    PMID: 25170299 DOI: 10.7150/ijms.8281
    Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  10. Fulltext 5-Azacytidine is insufficient for cardiogenesis in human adipose-derived stem cells
    Wan Safwani WK, Makpol S, Sathapan S, Chua KH
    J Negat Results Biomed, 2012;11:3.
    PMID: 22221649 DOI: 10.1186/1477-5751-11-3
    Adipose tissue is a source of multipotent adult stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic cells and adipogenic cells. Several reports have shown adipose-derived stem cells (ASCs) have the ability to undergo cardiomyogenesis. Studies have shown 5-azacytidine can successfully drive stem cells such as bone marrow derived stem cells to differentiate into cardiomyogenic cells. Therefore, in this study, we investigated the effect 5-azacytidine on the cardiogenic ability of ASCs.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  11. Human chorion-derived stem cells: changes in stem cell properties during serial passage
    Fariha MM, Chua KH, Tan GC, Tan AE, Hayati AR
    Cytotherapy, 2011 May;13(5):582-93.
    PMID: 21231803 DOI: 10.3109/14653249.2010.549121
    BACKGROUND AIMS: Fetal membrane from human placenta tissue has been described as a potential source of stem cells. Despite abundant literature on amnion stem cells, there are limited studies on the stem cell properties of chorion-derived stem cells.

    METHODS: The main aim was to determine the stemness properties of serial-passaged human chorion-derived stem cells (hCDSC). Quantitative polymerase chain reaction (PCR) was performed to reveal the following stemness gene expression in serial-passaged hCDSC: Oct-4, Sox-2, FGF-4, Rex-1, TERT, Nanog (3), Nestin, FZD-9, ABCG-2 and BST-1. Cell growth rate was evaluated from passage (P) 1 until P5. The colony-forming unit-fibroblast (CFU-F) frequency of P3 and P5 cells and multilineage differentiation potential of P5 cells were determined. The immunophenotype of hCDSC was compared using the surface markers CD9, CD31, CD34, CD44, CD45, CD73, CD90, CD117, HLA-ABC and HLA-DR, -DP and -DQ. Immunostaining for trophoblast markers was done on P0, P1, P3 and P5 cells to detect the contamination of trophoblasts in culture, while chromosomal abnormality was screened by cytogenetic analysis of P5 cells.

    RESULTS: The surface markers for mesenchymal lineage in hCDSC were more highly expressed at P5 compared with P3 and P0, indicating the increased purity of these stem cells after serial passage. Indeed, all the stemness genes except TERT were expressed at P1, P3 and P5 hCDSC. Furthermore, human chorion contained high clonogenic precursors with a 1:30 CFU-F frequency. Successful adipogenic, chondrogenic and osteogenic differentiation demonstrated the multilineage potential of hCDSC. The karyotyping analysis showed hCDSC maintained chromosomal stability after serial passage.

    CONCLUSIONS: hCDSC retain multipotent potential even at later passages, hence are a promising source for cell therapy in the future.

    Matched MeSH terms: Embryonic Stem Cells/metabolism
  12. Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells
    Alhaji SY, Nordin N, Ngai SC, Al Abbar A, Mei L, Abdullah S
    Gene, 2020 Oct 20;758:144958.
    PMID: 32683073 DOI: 10.1016/j.gene.2020.144958
    Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.
    Matched MeSH terms: Embryonic Stem Cells/metabolism*
  13. A plethora of human pluripotent stem cells
    Gao L, Thilakavathy K, Nordin N
    Cell Biol Int, 2013 Sep;37(9):875-87.
    PMID: 23619972 DOI: 10.1002/cbin.10120
    At the early stages of mammalian development, a number of developmentally plastic cells appear that possess the ability to give rise to all of the differentiated cell types normally derived from the three primary germ layers - unique character known as pluripotency. To date, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been shown to be truly pluripotent. However, recent studies have revealed a variety of other cells that demonstrate pluripotentiality, including very small embryonic-like stem cells (VSELs), amniotic fluid stem cells (AFSCs), marrow-isolated adult multilineage inducible cells (MIAMI) and multipotent adult precursor cells (MAPCs). This review summarises key features of these six kinds of pluripotent and potentially pluripotent stem cells (ESCs, iPSCs, VSELs, AFSCs, MIAMI and MAPCs) and the evidence for their pluripotency properties.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  14. Fulltext Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro
    Ghazalli N, Wu X, Walker S, Trieu N, Hsin LY, Choe J, et al.
    Stem Cells Dev, 2018 07 01;27(13):898-909.
    PMID: 29717618 DOI: 10.1089/scd.2017.0160
    Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.
    Matched MeSH terms: Embryonic Stem Cells/metabolism*
  15. Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells
    Mamidi MK, Pal R, Mori NA, Arumugam G, Thrichelvam ST, Noor PJ, et al.
    J Cell Biochem, 2011 May;112(5):1353-63.
    PMID: 21337383 DOI: 10.1002/jcb.23052
    Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
  16. Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells
    Soo JS, Ng CH, Tan SH, Malik RA, Teh YC, Tan BS, et al.
    Apoptosis, 2015 Oct;20(10):1373-87.
    PMID: 26276035 DOI: 10.1007/s10495-015-1158-5
    Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FEC-induced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers.
    Matched MeSH terms: Embryonic Stem Cells/metabolism
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