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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/cytology; Embryonic Stem Cells/metabolism*
Fulltext Neural Commitment of Embryonic Stem Cells through the Formation of Embryoid Bodies (EBs)

Liyang G, Abdullah S, Rosli R, Nordin N

Malays J Med Sci, 2014 Sep-Oct;21(5):8-16.
PMID: 25977628 MyJurnal

An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4-/4+ protocol.

Matched MeSH terms: Embryonic Stem Cells
Treatment viability of stem cells in ophthalmology

Jeganathan VS, Palanisamy M

Curr Opin Ophthalmol, 2010 May;21(3):213-7.
PMID: 20393292 DOI: 10.1097/ICU.0b013e32833867ad

Adult ocular stem cells have the potential to restore vision in patients previously deemed incurable. This review summarizes strides in stem cell research and stumbling blocks that must be overcome to enable treatment viability in ophthalmology.

Matched MeSH terms: Embryonic Stem Cells*
Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments

Kamei KI, Mashimo Y, Yoshioka M, Tokunaga Y, Fockenberg C, Terada S, et al.

Small, 2017 05;13(18).
PMID: 28272774 DOI: 10.1002/smll.201603104

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Matched MeSH terms: Embryonic Stem Cells/cytology*
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
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
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/cytology*; Embryonic Stem Cells/metabolism*
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/cytology; Embryonic Stem Cells/metabolism
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/cytology; Embryonic Stem Cells/metabolism*
Scalable production of pancreatic islet progenitors for diabetes cell therapies

Lock LT, Tzanakakis ES

Med J Malaysia, 2008 Jul;63 Suppl A:5-6.
PMID: 19024957

Embryonic stem cells (ESCs) can be an inexhaustible source of islet cells for transplantation. Previously published protocols have been characterized by low differentiation efficiency. In this study, we developed a scalable system for the growth and differentiation of hESCs towards pancreatic islets. Our results showed that hESCs can be grown on microcarriers to a larger scale and directed to differentiate into pancreatic progenitor endoderm cells. This culture system would represent an economical differentiation protocol that can be scaled-up to meet the demand in islet transplantation.

Matched MeSH terms: Embryonic Stem Cells/cytology*; Embryonic Stem Cells/transplantation
Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening

Vinoth KJ, Manikandan J, Sethu S, Balakrishnan L, Heng A, Lu K, et al.

J Biotechnol, 2014 Aug 20;184:154-68.
PMID: 24862194 DOI: 10.1016/j.jbiotec.2014.05.009

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.

Matched MeSH terms: Embryonic Stem Cells/drug effects*; Embryonic Stem Cells/radiation effects
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/cytology; Embryonic Stem Cells/drug effects; Embryonic Stem Cells/metabolism
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/classification; Embryonic Stem Cells/cytology*; Embryonic Stem Cells/metabolism
Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces

Peng IC, Yeh CC, Lu YT, Muduli S, Ling QD, Alarfaj AA, et al.

Biomaterials, 2016 Jan;76:76-86.
PMID: 26519650 DOI: 10.1016/j.biomaterials.2015.10.039

Stem cell culture is typically based on batch-type culture, which is laborious and expensive. Here, we propose a continuous harvest method for stem cells cultured on thermoresponsive nanobrush surfaces. In this method, stem cells are partially detached from the nanobrush surface by reducing the temperature of the culture medium below the critical solution temperature needed for thermoresponse. The detached stem cells are harvested by exchange into fresh culture medium. Following this, the remaining cells are continuously cultured by expansion in fresh culture medium at 37 °C. Thermoresponsive nanobrush surfaces were prepared by coating block copolymers containing polystyrene (for hydrophobic anchoring onto culture dishes) with three types of polymers: (a) polyacrylic acid with cell-binding oligopeptides, (b) thermoresponsive poly-N-isopropylacrylamide, and (c) hydrophilic poly(ethyleneglycol)methacrylate. The optimal coating durations and compositions for these copolymers to facilitate adequate attachment and detachment of human adipose-derived stem cells (hADSCs) and embryonic stem cells (hESCs) were determined. hADSCs and hESCs were continuously harvested for 5 and 3 cycles, respectively, via the partial detachment of cells from thermoresponsive nanobrush surfaces.

Matched MeSH terms: Human Embryonic Stem Cells
Thermoresponsive surfaces designed for the proliferation and differentiation of human pluripotent stem cells

Higuchi A, Hirad AH, Kumar SS, Munusamy MA, Alarfaj AA

Acta Biomater, 2020 10 15;116:162-173.
PMID: 32911107 DOI: 10.1016/j.actbio.2020.09.010

Thermoresponsive surfaces enable the detachment of cells or cell sheets by decreasing the temperature of the surface when harvesting the cells. However, human pluripotent stem cells (hPSCs), such as embryonic stem cells and induced pluripotent stem cells, cannot be directly cultured on a thermoresponsive surface; hPSCs need a specific extracellular matrix to bind to the integrin receptors on their surfaces. We prepared a thermoresponsive surface by using poly(N-isopropylacrylamide-co-butylacrylate) and recombinant vitronectin to provide an optimal coating concentration for the hPSC culture. hPSCs can be cultured on the same thermoresponsive surface for 5 passages by partial detachment of the cells from the surface by decreasing the temperature for 30 min; then, the remaining hPSCs were subsequently cultured on the same dishes following the addition of new cultivation media. The detached cells, even after continual culture for five passages, showed high pluripotency, the ability to differentiate into cells derived from the 3 germ layers and the ability to undergo cardiac differentiation.

Matched MeSH terms: Embryonic Stem Cells
Fulltext A Comparison of Culture Characteristics between Human Amniotic Mesenchymal Stem Cells and Dental Stem Cells

Yusoff NH, Alshehadat SA, Azlina A, Kannan TP, Hamid SS

Trop Life Sci Res, 2015 Apr;26(1):21-9.
PMID: 26868590 MyJurnal

In the past decade, the field of stem cell biology is of major interest among researchers due to its broad therapeutic potential. Stem cells are a class of undifferentiated cells that are able to differentiate into specialised cell types. Stem cells can be classified into two main types: adult stem cells (adult tissues) and embryonic stem cells (embryos formed during the blastocyst phase of embryological development). This review will discuss two types of adult mesenchymal stem cells, dental stem cells and amniotic stem cells, with respect to their differentiation lineages, passage numbers and animal model studies. Amniotic stem cells have a greater number of differentiation lineages than dental stem cells. On the contrary, dental stem cells showed the highest number of passages compared to amniotic stem cells. For tissue regeneration based on animal studies, amniotic stem cells showed the shortest time to regenerate in comparison with dental stem cells.

Matched MeSH terms: Embryonic Stem Cells
Fulltext MiR-3099 is Overexpressed in Differentiating 46c Mouse Embryonic Stem Cells upon Neural Induction

Zainal Abidin S, Abbaspourbabaei M, Ntimi CM, Siew WH, Pike-See C, Rosli R, et al.

Malays J Med Sci, 2014 Dec;21(Spec Issue):27-33.
PMID: 25941460 MyJurnal

MicroRNAs (miRNAs) have a crucial role in gene expression regulation and protein synthesis, especially in the central nervous system. In developing mouse embryos a novel miRNA, miR-3099, is highly expressed, particularly in the central nervous system. This study aims to determine the expression of miR-3099 during cellular differentiation of 46C mouse embryonic stem cells after neural induction with N2/B27 medium.

Matched MeSH terms: Mouse Embryonic Stem Cells
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*
Inducible transgene expression in undifferentiated mouse embryonic stem cells and embryoid bodies

Nordin N, Nathan S, Li M, Mason JO

Med J Malaysia, 2008 Jul;63 Suppl A:59-60.
PMID: 19024983

Obtaining pure population of neural cells from embryonic stem (ES) cells remains a challenge as little is known about the genes that govern embryonic stem cell differentiation. Using mouse ES cells, we aim to uncover the mechanisms that regulate neural differentiation of ES cells by focusing on roles played by Wnt family genes. Combining two techniques, Cre/loxP-based genetic recombination and ligand-dependent activation of Cre, we have generated transgenic ES cell lines that allow for the temporal control of expression and activity of Wnt gene (Wnt1-Ha) and Wnt antagonist (Dkk1). The ability of these cell lines in inducing the expression of transgene in undifferentiated ES cells and, more importantly, in differentiated derivatives of ES cells in vitro is evaluated.

Matched MeSH terms: Embryonic Stem Cells/cytology*
Human Embryonic Stem Cell Research: Ethical Views of Buddhist, Hindu and Catholic Leaders in Malaysia

Sivaraman MA, Noor SN

Sci Eng Ethics, 2016 Apr;22(2):467-85.
PMID: 26049934 DOI: 10.1007/s11948-015-9666-9

Embryonic Stem Cell Research (ESCR) raises ethical issues. In the process of research, embryos may be destroyed and, to some, such an act entails the 'killing of human life'. Past studies have sought the views of scientists and the general public on the ethics of ESCR. This study, however, explores multi-faith ethical viewpoints, in particular, those of Buddhists, Hindus and Catholics in Malaysia, on ESCR. Responses were gathered via semi-structured, face-to-face interviews. Three main ethical quandaries emerged from the data: (1) sanctity of life, (2) do no harm, and (3) 'intention' of the research. Concerns regarding the sanctity of life are directed at particular research protocols which interfere with religious notions of human ensoulment and early consciousness. The principle of 'do no harm' which is closely related to ahimsa prohibits all acts of violence. Responses obtained indicate that respondents either discourage research that inflicts harm on living entities or allow ESCR with reservations. 'Intention' of the research seems to be an interesting and viable rationale that would permit ESCR for the Buddhists and Hindus. Research that is intended for the purpose of alleviating human suffering is seen as being ethical. This study also notes that Catholics oppose ESCR on the basis of the inviolability of human life.

Matched MeSH terms: Embryonic Stem Cells*

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