Publications by authors named "Ruttachuk Rungsiwiwut"

22 Publications

  • Page 1 of 1

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

Hum Cell 2021 Mar 4;34(2):300-309. Epub 2021 Feb 4.

Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

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, β-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.
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http://dx.doi.org/10.1007/s13577-020-00439-2DOI Listing
March 2021

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

Reprod Med Biol 2021 Jan 20;20(1):13-19. Epub 2020 Jul 20.

Department of Obstetrics and Gynecology Faculty of Medicine Chulalongkorn University Bangkok Thailand.

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.
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http://dx.doi.org/10.1002/rmb2.12339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812475PMC
January 2021

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

J Therm Biol 2020 Dec 13;94:102783. Epub 2020 Nov 13.

Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand. Electronic address:

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 β-1,4 galactosyltransferase (β-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 β-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 β-GALT1.
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http://dx.doi.org/10.1016/j.jtherbio.2020.102783DOI Listing
December 2020

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

In Vitro Cell Dev Biol Anim 2020 Dec 16;56(10):842-846. Epub 2020 Nov 16.

Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand.

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, β-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.
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http://dx.doi.org/10.1007/s11626-020-00529-3DOI Listing
December 2020

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

Reprod Fertil Dev 2020 Jun;32(9):822-834

Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4, Bangkok 10330, Thailand; and Corresponding author. Email:

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-β1 (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.
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http://dx.doi.org/10.1071/RD19128DOI Listing
June 2020

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.

Stem Cell Res 2019 08 29;39:101493. Epub 2019 Jun 29.

Center of Excellence for Medical Genomics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, The Thai Red Cross Society, Bangkok 10330, Thailand.

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.
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http://dx.doi.org/10.1016/j.scr.2019.101493DOI Listing
August 2019

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

Biosci Rep 2018 10 7;38(5). Epub 2018 Sep 7.

Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand

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.
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http://dx.doi.org/10.1042/BSR20181109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127673PMC
October 2018

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

Fish Shellfish Immunol 2018 Oct 17;81:204-213. Epub 2018 Jul 17.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fsi.2018.07.030DOI Listing
October 2018

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

Stem Cells Transl Med 2017 11 24;6(11):1956-1962. Epub 2017 Oct 24.

UK Stem Cell Bank, Division of Advanced Therapies, NIBSC, South Mimms, UK.

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.
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http://dx.doi.org/10.1002/sctm.17-0144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430055PMC
November 2017

Transgene-free human induced pluripotent stem cell line (HS5-SV.hiPS) generated from cesarean scar-derived fibroblasts.

Stem Cell Res 2016 Jan 27;16(1):10-3. Epub 2015 Nov 27.

Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Chulalongkorn University, Bangkok, Thailand.

Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts.
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http://dx.doi.org/10.1016/j.scr.2015.11.006DOI Listing
January 2016

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

Stem Cells Int 2016 29;2016:4626048. Epub 2015 Dec 29.

Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.

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 TGFβ1, 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.
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http://dx.doi.org/10.1155/2016/4626048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709772PMC
February 2016

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

J Reprod Dev 2016 Apr 28;62(2):167-76. Epub 2016 Jan 28.

Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

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.
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http://dx.doi.org/10.1262/jrd.2015-113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848574PMC
April 2016

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

J Mol Microbiol Biotechnol 2015 20;25(6):372-80. Epub 2015 Nov 20.

Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.
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http://dx.doi.org/10.1159/000441453DOI Listing
December 2016

Tropism and Induction of Cytokines in Human Embryonic-Stem Cells-Derived Neural Progenitors upon Inoculation with Highly- Pathogenic Avian H5N1 Influenza Virus.

PLoS One 2015 14;10(8):e0135850. Epub 2015 Aug 14.

Department of Pathology, Faculty of Veterinary Sciences, Chulalongkorn University, Bangkok, Thailand.

Central nervous system (CNS) dysfunction caused by neurovirulent influenza viruses is a dreaded complication of infection, and may play a role in some neurodegenerative conditions, such as Parkinson-like diseases and encephalitis lethargica. Although CNS infection by highly pathogenic H5N1 virus has been demonstrated, it is unknown whether H5N1 infects neural progenitor cells, nor whether such infection plays a role in the neuroinflammation and neurodegeneration. To pursue this question, we infected human neural progenitor cells (hNPCs) differentiated from human embryonic stem cells in vitro with H5N1 virus, and studied the resulting cytopathology, cytokine expression, and genes involved in the differentiation. Human embryonic stem cells (BG01) were maintained and differentiated into the neural progenitors, and then infected by H5N1 virus (A/Chicken/Thailand/CUK2/04) at a multiplicity of infection of 1. At 6, 24, 48, and 72 hours post-infection (hpi), cytopathic effects were observed. Then cells were characterized by immunofluorescence and electron microscopy, supernatants quantified for virus titers, and sampled cells studied for candidate genes.The hNPCs were susceptible to H5N1 virus infection as determined by morphological observation and immunofluorescence. The infection was characterized by a significant up-regulation of TNF-α gene expression, while expressions of IFN-α2, IFN-β1, IFN-γ and IL-6 remained unchanged compared to mock-infected controls. Moreover, H5N1 infection did not appear to alter expression of neuronal and astrocytic markers of hNPCs, such as β-III tubulin and GFAP, respectively. The results indicate that hNPCs support H5N1 virus infection and may play a role in the neuroinflammation during acute viral encephalitis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135850PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537284PMC
May 2016

Wiskott-Aldrich syndrome iPS cells produce megakaryocytes with defects in cytoskeletal rearrangement and proplatelet formation.

Thromb Haemost 2015 Apr 18;113(4):792-805. Epub 2014 Dec 18.

Nipan Israsena, MD, PhD, Head, Stem Cell and Cell Therapy, Research Unit, Chulalongkorn University, Bangkok 10330, Thailand, Tel.: +662 256 4000 Ext 3589, Fax: +662 256 4911, E-mail:

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterised by microthrombocytopenia, complex immunodeficiency, autoimmunity, and haematologic malignancies. It is caused by mutations in the gene encoding WAS protein (WASP), a regulator of actin cytoskeleton and chromatin structure in various blood cell lineages. The molecular mechanisms underlying microthrombocytopenia caused by WASP mutations remain elusive. Murine models of WASP deficiency exhibited only mild thrombocytopenia with normal-sized platelets. Here we report on the successful generation of induced pluripotent stem cell (iPSC) lines from two patients with different mutations in WASP (c.1507T>A and c.55C>T). When differentiated into early CD34+ haematopoietic and megakaryocyte progenitors, the WAS-iPSC lines were indistinguishable from the wild-type iPSCs. However, all WAS-iPSC lines exhibited defects in platelet productionin vitro. WAS-iPSCs produced platelets with more irregular shapes and smaller sizes. Immunofluorescence and electron micrograph showed defects in cytoskeletal rearrangement, F-actin distribution, and proplatelet formation. Proplatelet defects were more pronounced when using culture systems with stromal feeders comparing to feeder-free culture condition. Overexpression of WASP in the WAS-iPSCs using a lentiviral vector improved proplatelet structures and increased the platelet size. Our findings substantiate the use of iPSC technology to elucidate the disease mechanisms of WAS in thrombopoiesis.
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http://dx.doi.org/10.1160/TH14-06-0503DOI Listing
April 2015

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

Cells Tissues Organs 2013 19;198(2):127-38. Epub 2013 Oct 19.

Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

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.
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http://dx.doi.org/10.1159/000354031DOI Listing
June 2014

Eighteen-year cryopreservation does not negatively affect the pluripotency of human embryos: evidence from embryonic stem cell derivation.

Biores Open Access 2012 Aug;1(4):166-73

Human Embryonic Stem Cell Research Center, Chulalongkorn University , Bangkok, Thailand . ; Department of Obstetrics and Gynecology (Reproductive Medicine Unit), Chulalongkorn University , Bangkok, Thailand .

Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research.
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http://dx.doi.org/10.1089/biores.2012.0242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559204PMC
August 2012

Comparative analysis of nuclear transfer embryo-derived mouse embryonic stem cells. Part I: cellular characterization.

Cell Reprogram 2012 Feb 28;14(1):56-67. Epub 2011 Dec 28.

Genetic Reprogramming Group, Agricultural Biotechnology Center, Gödöllő, Hungary.

Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. Karyotype analyses and cell growth studies did not reveal any significant variations. Despite some differences observed, the present study revealed that ntESC lines had similar differentiation competences compared to other ESCs. The results indicate that the observed differences may be related to the genotype rather than to the nuclear transfer technology.
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http://dx.doi.org/10.1089/cell.2011.0056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3275094PMC
February 2012

Generation of mouse embryonic stem cell lines from zona-free nuclear transfer embryos.

Cell Reprogram 2010 Feb;12(1):105-13

Micromanipulation and Genetic Reprogramming Group, Agricultural Biotechnology Center, Gödöllo, Hungary.

Pluripotent stem cells would have great potential in cell therapies and drug development when genetically matched with the patient; thus, histocompatible cells could be used in transplantation therapy or as a source of patient-specific cells for drug testing. Pluripotent embryonic stem cells (ESCs)-generated via somatic cell nuclear transfer (SCNT) or parthenogenesis (pESC)-are potential sources of histocompatible cells and tissues for transplantation. Earlier studies used the piezoelectric microinjection (PEM) technique for nuclear transfer (NT) in mouse. No specific studies examined zona-free (ZF) NT as an alternative NT method to generate genetically matched ESCs of a nuclear donor. In this study, we compared the efficiency of nuclear transfer-derived ESC (ntESC) line establishment from ZF-NT, ZF-parthenogenetic (PGA), and ZF-fertilized embryos with that of the PEM-NT method. Different nuclei donor cells [cumulus, ESC, and mouse embryonic fibroblast (MEF)] were used and the efficiency of ntESC derivation was investigated, along with their in vitro characterization. The ZF-NT method's efficiency was higher than that of the PEM-NT using cumulus cells. When ESCs and cumulus cells were used as nuclear donor cells, they resulted in significantly higher ZF-NT-derived ntESC line establishment rates compared to MEF cells. In conclusion, the nuclear donor cell type significantly affected the efficiency of ntESC line establishment, and the ZF-NT method was efficient to establish pluripotent ntESC lines.
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http://dx.doi.org/10.1089/cell.2009.0040DOI Listing
February 2010

Development of human embryonic stem cell derivation.

J Med Assoc Thai 2009 Apr;92(4):443-50

Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

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.
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April 2009

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

J Med Assoc Thai 2008 May;91(5):608-14

Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.

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.
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May 2008

Conventional and novel methods for embryonic stem cell line derivation.

J Med Assoc Thai 2006 Jun;89(6):896-903

Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

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.
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June 2006