The Efficiency of Neurospheres Derived from Human Wharton's Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats.

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and ß-tubulin 3 through the Wnt3A signaling pathway regulation markers (ß-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells.

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of ß-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-ß signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.

Mesenchymal stem cells for restoring endometrial function: An infertility perspective.

BACKGROUND: Mesenchymal stem cells (MSCs) can be derived from several tissues such as bone marrow, placenta, adipose tissue, or endometrial tissue. MSCs gain a lot of attention for cell-based therapy due to their characteristics including differentiation ability and immunomodulatory effect. Preclinical and clinical studies demonstrated that MSCs can be applied to treat female infertility by improving of the functions of ovary and uterus. This mini- review focuses on the current study of treatment of endometrial infertility by using MSCs. METHODS: The present study performed a literature review focusing on the effect of MSCs for treatment of women infertility caused by endometrial dysfunction. RESULTS: Bone marrow-, umbilical cord-, adipose-, amniotic-, and menstruation-derived MSCs enhance endometrial cell proliferation, injury repairs as well as reducing scar formation. The beneficial mechanism probably via immunomodulatory, cell differentiation, stimulates endometrial cell proliferation and down-regulation of fibrosis genes. The major advantage of using MSCs is to improve endometrial functions resulting in increased implantation and pregnancy. CONCLUSIONS: MSCs exhibit a potential for endometrial infertility treatment. Adipose- and menstruation-derived stem cells show advantages over other sources because the cells can be derived easily and do not causes graft rejection after autologous transplantation.

Human Caesarean scar-derived feeder cells: a novel feeder cell type for culturing human pluripotent stem cells without exogenous basic fibroblast growth factor supplementation.

In a feeder-dependent culture system of human pluripotent stem cells (hPSCs), coculture with mouse embryonic fibroblasts may limit the clinical use of hPSCs. The aim of this study was to determine the feasibility of using human Caesarean scar fibroblasts (HSFs) as feeder cells for the culture of hPSCs. HSFs were isolated and characterised and cocultured with hPSCs, and the pluripotency, differentiation ability and karyotypic stability of hPSCs were determined. Inactivated HSFs expressed genes (including inhibin subunit beta A (INHBA), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), transforming growth factor-ß1 (TGFB1), collagen alpha-1(I) (COL1A1) and fibronectin-1 (FN1) that have been implicated in the maintenance of hPSC pluripotency. When HSFs were used as feeder cells, the pluripotency and karyotypic stability of hPSC lines did not change after prolonged coculture. Interestingly, exogenous FGF2 could be omitted from the culture medium when HSFs were used as feeder cells for hESCs but not hiPSCs. hESCs cocultured with HSF feeder cells in medium without FGF2 supplementation maintained their pluripotency (as confirmed by the expression of pluripotency markers and genes), differentiated invitro into embryonic germ layers and maintained their normal karyotype. The present study demonstrates that HSFs are a novel feeder cell type for culturing hPSCs and that supplementation of exogenous FGF2 is not necessary for the Chula2.hES line.

High ambient temperature directly decreases milk synthesis in the mammary gland in Saanen goats.

The mammary gland is a privileged organ for mammals. Because of their high capacity for milk synthesis, dairy ruminants have been distributed throughout the world. In tropical areas, dairy animals face high ambient temperatures (HTa). The indirect effect of HTa on milk synthesis is mediated in part by a reduction in feed intake. The current experiment focused on the direct natural effect of HTa on mammary function. Multiparous Saanen goats were used in this study. The physiological responses for HTa were evaluated from the control period during the winter and from the natural HTa during the summer. Milk samples were collected for isolation of the goat milk cells to study the expression of the ß-1,4 galactosyltransferase (ß-GALT1), Akt, and heat shock protein 70 (HSP70) genes. Although goats in the summer maintained rectal temperature and plasma cortisol levels similar to those observed in the winter, the higher respiratory rate and lower feed intake and milk yield (MY) from the goats in the summer indicated that the goats in the summer were exposed to a higher degree of HTa. This was supported by the significantly higher level of plasma glutathione peroxidase (GPX) activity. Moreover, the relative expression levels of ß-GALT1 and Akt were not different. The relative expression of HSP70 during the summer was significantly higher than what was observed in cells isolated in the winter. In conclusion, the HTa effect on MY during the summer was related to its indirect effect on feed intake. The direct HTa effect might be related to HSP70 gene expression in goat milk cells and to plasma GPX activity. However, the natural HTa did not affect the expression of Akt or ß-GALT1.

Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon).

To achieve in creating permanent shrimp cell lines, cellular arrest of primary cells in the culture is needed to be firstly solved. Considering the insertion of some markers affecting cellular proliferation into primary haemocytes in order to produce the black tiger shrimp cell line and the very low percent of transduced cells previously reported in penaeid shrimps, these paved us the way to set up suitable gene delivery protocols to increase percent of transduced cells in the shrimp as our primary aim. In this study, electroporation and lipofection were used to transfer construct plasmids (pLL3.7 plasmids containing CMV promoters and pGL-IE1-126(A)-EGFP plasmids carrying WSSV IE1 promoters) into primary haemocytes. As it was difficult to distinguish between cells expressing EGFP signal and auto-fluorescence of many dead cells occurred by electroporation during the first 72 h of experiment; so, only lipofection was managed to deliver plasmids into primary cells. Surprisingly, numbers of suspected proliferative cells were derived after electroporation with no insertion of immortalising markers. These cells survived in vitro for up to 45 days with high rate of cell viability, but the number of viable cells decreased throughout the experiment. In addition, these cells expressed genes and proteins closely related to hyaline cells determined using RT-PCR and western blot. For the lipofection experiment, no green fluorescence signal was detected in any primary cell introduced with these plasmids, suggesting that plasmids were not successfully inserted into cells. Also, a number of primary haemocytes had the apoptotic cell death characteristic within 5 days after lipofection. These possibly result from using inappropriate lipofection protocol and chemical substances. In summary, finding out suitable protocols to elevate the percent of transduced cells is still necessary. Additionally, continuous shrimp cell lines would be possibly established by transforming suspected proliferative cells derived from electroporation in this study.

Triploid human embryonic stem cells derived from tripronuclear zygotes displayed pluripotency and trophoblast differentiation ability similar to the diploid human embryonic stem cells.

Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line.

The ROCK inhibitor Y-26732 enhances the survival and proliferation of human embryonic stem cell-derived neural progenitor cells upon dissociation.

Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 µM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation.

Proline-based solution maintains cell viability and stemness of canine adipose-derived mesenchymal stem cells after hypothermic storage.

Transportation of mesenchymal stem cells (MSCs) under hypothermic conditions in 0.9% normal saline solution (NSS) might increase cell death and alter the stemness of MSCs. The present study aimed to evaluate the effect of proline-based solution (PL-BS) on cell viability and the stemness of newly established canine adipose-derived mesenchymal stem cells (cAD-MSCs) under hypothermic conditions. Characterized cAD-MSCs were stored in 1, 10, and 100 mM PL-BS or NSS at 4°C for 6, 9, and 12 hours prior to an evaluation. The results demonstrated that storage in 1 mM PL-BS for 6 hours decreased cell apoptosis and proliferation ability, but improved cell viability and mitochondrial membrane potential. cAD-MSCs maintained their high expression of CD44 and CD90, but had a low expression of CD34 and MHC class II. Trilineage differentiation ability of cAD-MSCs was not affected by storage in 1 mM PL-BS. Gene expression analysis demonstrated that immunomodulatory genes, including IDO, HGF, PGE-2, and IL-6, were upregulated in cAD-MSCs stored in 1 mM PL-BS. In conclusion, PL-BS can be effectively applied for storing cAD-MSCs under hypothermic conditions. These findings provide a new solution for effective handling of cAD-MSCs which might be promising for clinical applications.

Melatonin attenuates dimethyl sulfoxide- and Zika virus-induced degeneration of porcine induced neural stem cells.

Domestic pigs have become increasingly popular as a model for human diseases such as neurological diseases. Drug discovery platforms have increasingly been used to identify novel compounds that combat neurodegeneration. Currently, bioactive molecules such as melatonin have been demonstrated to offer a neuroprotective effect in several studies. However, a neurodegenerative platform to study novel compounds in a porcine model has not been fully established. In this study, characterized porcine induced neural stem cells (iNSCs) were used for evaluation of the protective effect of melatonin against chemical and pathogenic stimulation. First, the effects of different concentrations of melatonin on the proliferation of porcine iNSCs were studied. Second, porcine iNSCs were treated with the appropriate concentration of melatonin prior to induced degeneration with dimethyl sulfoxide or Zika virus (ZIKV). The results demonstrated that the percentages of Ki67 expression in porcine iNSCs cultured in 0.1, 1, and 10 nM melatonin were not significantly different from that in the control groups. Melatonin at 1 nM protected porcine iNSCs from DMSO-induced degeneration, as confirmed by a dead cell exclusion assay and mitochondrial membrane potential (ΔΨm) analysis. In addition, pretreatment with melatonin reduced the percentage of dead porcine iNSCs after ZIKV infection. Melatonin increased the ΔΨm, resulting in a decrease in cell degeneration. However, pretreatment with melatonin was unable to suppress ZIKV replication in porcine iNSCs. In conclusion, the present study demonstrated the anti-degenerative effect of melatonin against DMSO- and ZIKV-induced degeneration in porcine iNSCs.

Characterization of stem cells from human ovarian follicular fluid; a potential source of autologous stem cell for cell-based therapy.

Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24 h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2 weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, ß-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluid exhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus.

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.

Effect of incubation temperature on lactogenic function of goat milk-derived mammary epithelial cells.

In general, goat mammary epithelial cells (MECs) are cultured in vitro under 37 °C. We demonstrated previously that goat MECs differentiate under 37 °C although their body temperature is approximately 39 °C. This study aimed to investigate the influence of 39 °C on lactogenic differentiation of goat milk-derived MECs. The results revealed that HSP70 gene was significantly elevated at 1 h after an exposure to 39 °C but declined at 48 h thereafter. Oxidative stress status was not significantly affected by 39 °C. Expressions of CSN2, ß-GALT1, α-LA, and Akt genes tended to increase after the differentiation under 39 °C. Secretion of lactose under 39 °C was not significantly lower than 37 °C. In conclusion, incubation temperature at 39 °C does not dramatically affect lactogenic function of goat milk-derived MECs.

Development of human embryonic stem cell derivation.

OBJECTIVE: To establish human embryonic stem (hES) cells from human embryos. DESIGN: Experimental study. SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University. MATERIAL AND METHOD: Abnormal and normal fertilization embryos were cultured in vitro until reaching blastocyst stage. Four different methods for isolation of ICMs were used. Immunosurgery, mechanical isolation, laser assists, and whole blastocyst culture were performed. The feeder layers used in the present study were fibroblasts, isolated from either mouse or human. Mechanical splitting of ICM outgrowths or hES-like cells was performed for propagation of cells. Characterization of hES-like cells was conducted by morphology, detection of immunostaining of Oct-4, and enzymatic activity of alkaline phosphatase (AP). HES-like cells were spontaneously differentiated through suspension culture of embryoid body (EB). Subsequent differentiation was done on gelatin-coated dishes. MAIN OUTCOME MEASURE: Establishment of hES cells. RESULTS: By using abnormal fertilization embryos, 80.0% (8/10) of blastocysts were able to attach on the feeder layers, 50% (4/8) formed ICM outgrowths, but no hES-like cells were established. By using normal fertilization embryos, 84.6% (22/26) of blastocysts were able to attach on feeder layers, 18.2% (4/22) formed ICM outgrowths. One hES-like cell line was successfully established by using mechanical isolation of ICMs and human adult skin fibroblasts as feeder layers. This hES-like cells exhibited typical morphology of hES cells, positive staining for Oct-4 and AP. hES-like cells were able to form EB and differentiated into neural-like cells. CONCLUSION: This is the first report in Thailand that hES-like cells can be isolated from normal development human embryos at blastocysts-stage using mechanical isolation of ICM and culture with human adult skin fibroblast as feeder layers.

Generation of two human iPSC lines (MDCUi001-A and MDCUi001-B) from dermal fibroblasts of a Thai patient with X-linked osteogenesis imperfecta using integration-free Sendai virus.

Two clones of human induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts isolated from a one-year-old Thai patient with X-linked osteogenesis imperfecta. The patient harbored a mutation, p.N459S, in the MBTPS2 gene. The cells were reprogrammed using an integration-free Sendai virus containing KLF4, c-MYC, OCT4 and SOX2. Both of the established iPSC lines (MDCUi001-A and MDCUi001-B) maintained normal karyotype, expressed pluripotent markers and differentiated into all three germ layers.

Effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocysts and subsequent derivation of embryonic stem (ES) cells.

OBJECTIVE: To determine the effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocyst and the efficiency of embryonic stem (ES) cell derivation. DESIGN: Experimental study SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University MATERIAL AND METHOD: In vivo fertilized zygotes were collected and subjected to in vitro culture in potassium simplex optimized medium (KSOM) containing 1,000 unit/ml LIF. The developmental ability of the zygote to blastocyst-stage and the cell numbers in blastocysts were evaluated Expanded blastocysts developed in different culture media were subsequently subjected to ES cell derivation. MAIN OUTCOME MEASURE (s): The influence of LIF on the quality of and the total cell numbers of blastocyst developed in vitro. RESULTS: Supplementation of LIF in KSOM increased the rate of hatching blastocysts (63.8% vs. 53.7%; p < 0.05) and total cell numbers (91.4 +/- 15.0 vs. 85.1 +/- 7.7; p < 0.05) compared to KSOM alone. ES cells were obtained 66.7% from blastocysts developed in KSOM-LIF versus 41.7% in KSOM (p > 0.05). Established ES cell lines showed typical colony and characteristics of pluripotent murine ES cells. CONCLUSION: LIF improved the quality of in vitro produced blastocysts but not enhanced ES cell derivation.

Modulated expression of the HIV-1 2LTR zinc finger efficiently interferes with the HIV integration process.

Lentiviral vectors have emerged as the most efficient system to stably transfer and insert genes into cells. By adding a tetracycline (Tet)-inducible promoter, transgene expression delivered by a lentiviral vector can be expressed whenever needed and halted when necessary. Here we have constructed a doxycycline (Dox)-inducible lentiviral vector which efficiently introduces a designed zinc finger protein, 2-long terminal repeat zinc-finger protein (2LTRZFP), into hematopoietic cell lines and evaluated its expression in pluripotent stem cells. As a result this lentiviral inducible system can regulate 2LTRZFP expression in the SupT1 T-cell line and in pluripotent stem cells. Using this vector, no basal expression was detected in the T-cell line and its induction was achieved with low Dox concentrations. Remarkably, the intracellular regulatory expression of 2LTRZFP significantly inhibited HIV-1 integration and replication in HIV-inoculated SupT1 cells. This approach could provide a potential tool for gene therapy applications, which efficiently control and reduce the side effect of therapeutic genes expression.

Report of the International Stem Cell Banking Initiative Workshop Activity: Current Hurdles and Progress in Seed-Stock Banking of Human Pluripotent Stem Cells.

This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Translational Medicine 2017;6:1956-1962.

Conventional and novel methods for embryonic stem cell line derivation.

Cell therapy is the promising therapeutic tool for the next decade. "Regenerative Medicine" based on cell and tissue replacement therapy is proposed as a revolutionary approach to various chronic and incurable conditions. The first key step for successful cell therapy is the establishment of clinical grade human Embryonic Stem Cell (hESC) lines. This article provides a concise summary on conventional and novel methods for hESC line derivation. There is also discussion on progression, future direction and problems in hESC line development. In Thailand, more advance knowledge, skill, and technology are required to develop the first human embryonic stem cell line and step forward to make cell therapy a reality.

Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines.

Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFß1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.