Publications by authors named "Kamthorn Pruksananonda"

24 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

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

Activation of adenosine monophosphate-activated protein kinase (AMPK) enhances energy metabolism, motility, and fertilizing ability of cryopreserved spermatozoa in domestic cat model.

J Assist Reprod Genet 2019 Jul 11;36(7):1401-1412. Epub 2019 May 11.

Smithsonian Conservation Biology Institute, Front Royal, Virginia and Washington DC, USA.

Purpose: Increasing intracellular energy storage by chemically activating adenosine monophosphate-activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5'-aminoimidasole-4-carboxamide-1-β-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation.

Methods: In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60 min in the presence of 0 (control), 0.5, 2.0, and 5.0 mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels.

Results: AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5 mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0 mM AICAR for 30 min.

Conclusions: Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men.
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http://dx.doi.org/10.1007/s10815-019-01470-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642251PMC
July 2019

Epigenetic modification of long interspersed elements-1 in cumulus cells of mature and immature oocytes from patients with polycystic ovary syndrome.

Clin Exp Reprod Med 2016 Jun 23;43(2):82-9. Epub 2016 Jun 23.

Center of Excellence in Molecular Genetics of Cancer and Human Disease, Department of Anatomy, King Chulalongkorn Memorial Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Objective: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls.

Methods: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1.

Results: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients.

Conclusion: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.
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http://dx.doi.org/10.5653/cerm.2016.43.2.82DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4925871PMC
June 2016

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

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

Assessment of medical ethics of fourth-year medical students.

J Med Assoc Thai 2010 Sep;93(9):1115-8

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

From 2005 to 2008, the authors assessed the medical ethics of 779 medical students in the Department of Obstetrics and Gynecology at the Faculty of Medicine, Chulalongkorn University by using the Chula Method. This was conducted through a written examination asking the students to express their opinions about ethical issues. Their answers were rated as either Satisfactory (S) or Unsatisfactory (U). It was found that 750 students (96.3%) earned S while 29 students (3.7%) earned U. The results from this study can not be compared with the results from studies published in medical journals. Thus, knowledge about medical ethics is not complete even though it is intensively taught in medical schools and has been practiced for a long time. The authors would like to propose a new assessment of the medical ethics so that it can be systematically applied.
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September 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

Risk factors for ovarian hyperstimulation syndrome in Thai patients using gonadotropins for in vitro fertilization.

Am J Health Syst Pharm 2008 Jun;65(12):1148-53

Department of Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

Purpose: A prospective observational study was conducted to identify risk factors for ovarian hyperstimulation syndrome (OHSS) in Thai patients using gonadotropins for in vitro fertilization.

Methods: Outpatients receiving a short and a long protocol of ovarian stimulation with a recombinant follicle-stimulating hormone (rec FSH) at a Bangkok hospital were enrolled. Patients in the short protocol received rec FSH and the gonadotropin-releasing-hormone agonist buserelin acetate by subcutaneous injection after blood sampling on day 2 of the patient's cycle. When one or more follicles reached a diameter of 18 mm as determined by ultrasonography and the serum estradiol (E(2)) concentration exceeded 400 pg/mL, patients received human chorionic gonadotropin (hCG); oocytes were retrieved 34-36 hours later. The long protocol differed only in that patients received nasal buserelin acetate and rec FSH 10 days before the cycle started and on day 2 of the cycle. Serum levels of E(2), luteinizing hormone, and FSH were monitored at baseline. In addition to E(2) concentration and follicle size and number, patients' weight, waist circumference, complete blood counts, and C-reactive protein (CRP) levels were monitored at intervals. Patients were instructed to notify the hospital of any symptoms of OHSS.

Results: Of 117 patients enrolled, 13 had moderate OHSS and 1 had severe OHSS; all recovered. Patients with OHSS were significantly younger, had significantly lower body mass index, and had significantly higher E(2) before hCG injection, total number of follicles, total number of oocytes retrieved, and white blood cell and neutrophil counts after hCG injection. Patients with polycystic ovary syndrome were significantly more likely to have OHSS. CRP levels were higher in OHSS patients, but the difference was not significant. Multiple logistic regression showed that the combination of serum E(2) peak concentration of > or =4500 pg/mL and total number of oocytes retrieved of > or =15 predicted the occurrence of moderate-to-severe OHSS; 12 of 14 study patients who met these criteria had OHSS.

Conclusion: Serum E(2) peak concentrations of > or =4500 pg/mL and total number of oocytes > or =15 may be useful indicators for identifying patients at high risk for moderate-to-severe OHSS.
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http://dx.doi.org/10.2146/ajhp070566DOI Listing
June 2008

International trends in bioethics for embryonic stem cell research.

J Med Assoc Thai 2006 Sep;89(9):1542-4

Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Rama 4 Rd, Pathumwan, Bangkok 10330, Thailand.

Embryonic stem cell (ESC) has been recognized as one of the most promising therapeutic tools for the next decade. However, there are many controversial issues in bioethics for this challenging research area. Each country has its own distinct regulations and policies for ESC research due to their differences in cultural background, religious belief and political influence. These differences will eventually play an important role on international ESC research collaboration. The present article will provide a concise summary of the different policies and regulations regarding bioethical points for ESC research worldwide and show current progress towards establishing standard bioethical guidelines for ESC research on the international level.
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September 2006

Cell therapy: hype or hope.

J Med Assoc Thai 2006 Apr;89(4):550-7

Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Rama IV Rd, Pathumwan, Bangkok 10330, Thailand.

Cell therapy is a promising therapeutic tool for the next decade. It has a potential to cure a number of chronic diseases and conditions related to aging processes or degenerative changes. In addition, it could be used to replace cells and tissues in injured organs. Furthermore, it may provide a novel approach to congenital anomalies and genetic disorders where current therapeutic options are limited However, many crucial questions need answers to ensure a safe, effective and successful solution in the field of cell therapy. In Thailand, innovative knowledge and expertise in stem cell biology and technology are required as the key elements to make cell therapy a "real" hope.
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April 2006

Comparison of two different fixed doses of follitropin-beta in controlled ovarian hyperstimulation: A prospective randomized, double blind clinical trial.

J Med Assoc Thai 2004 Oct;87(10):1151-5

Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.

A prospective randomized, double blind, single centre study was conducted to compare the efficacy, efficiency and clinical side effects of daily fixed dose regimen of either 100 IU or 200 IU of recombinant follicle stimulating hormone(rFSH) Follitropin beta in down-regulated women undergoing controlled ovarian hyperstimulation(COH) for either conventional in vitro fertilization(IVF) or intracytoplasmic sperm injection(ICSI). A total of sixty women were randomly allocated according to the criteria for the treatment by either 100 IU(n = 30) or 200 IU (n = 30) of FSH. Although more cycle cancellations due to low response were observed in the 100 IU group (n = 9 vs n = 2), two cases of mild and moderate ovarian hyperstimulation syndrome were noted in the higher dose group. Subjects in the group treated with 200 IU appeared to yield more follicles > 17 mm (4.4 vs 3.3, p = 0.05) and more oocytes compared to the group treated with 100 IU (9.2 versus 6.0 oocytes, NS). The total dosage required to develop at least three follicles according to the protocol was significantly lower in the group treated with 100 IU (1203.33 versus 2106. 67, P < 0.0001). In conclusion, a fixed daily dose of 200 IU of rFSH Follitropin beta compared to a fixed daily dose of 100 IU is more effective in terms of follicles > 17 mm development and the number of oocytes retrieved along with a lower cancellation rate, but less efficient as indicated by a higher total rFSH dose needed
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October 2004

Correlation between human follicular diameter and oocyte outcomes in an ICSI program.

J Assist Reprod Genet 2003 Apr;20(4):143-7

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

Purpose: To determine the correlation between the follicular sizes and oocyte recovery, metaphase II oocyte recovery, fertilization rate and good embryo quality from mature and immature oocytes in an intracytoplasmic sperm injection (ICSI) program.

Methods: 991 follicles obtained from 72 ICSI cycles were classified into three groups according to their diameters as measured by transvaginal ultrasound including group A (< 10 mm), group B (10-14 mm), and group C (> 14 mm). All obtained oocytes were classified according to their nuclear maturation: germinal vesicle (GV), metaphase I (MI) and metaphase II (MII). Mature oocytes underwent ICSI while immature oocytes were further cultured until maturity before ICSI was performed. The rates of fertilization and good quality embryos at day 3 were evaluated.

Results: A progressive and significant increase in the rates of oocyte recovery and MII oocyte recovery were observed from group A follicles compared to the other groups (p < 0.001). The fertilization rate of mature and in vitro matured oocytes, as well as the rate of good quality embryos showed a tendency to increase from group A to group C follicles, but not significantly. The corresponding fertilization rates were 78 and 55.3% (p < 0.001) for mature and in vitro matured oocytes, respectively.

Conclusion: Collection of oocytes from small follicles, especially with a mean diameter less than 10 mm, and in vitro maturation of immature oocytes before fertilization may allow the total number of good quality and transferable embryos to be increased.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3455636PMC
http://dx.doi.org/10.1023/a:1022977002954DOI Listing
April 2003

The effect of growth hormone on the development of in vitro matured unstimulated human oocytes.

J Med Assoc Thai 2002 Aug;85(8):907-14

Deparanent of Obstetrics & Gynaecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Objective: To investigate the effect of growth hormone on the development of in vitro matured unstimulated human oocytes.

Design: Randomized controlled study.

Setting: Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn university.

Material And Method: 108 germinal vesicle-stage oocytes were retrieved from 47 patients undergoing gynecologic surgery. They were aspirated either during gynecologic surgery or from excised ovaries. The oocytes were then cultured in vitro with or without growth hormone (1,000 ng/ml) in medium199 supplemented with sodium pyruvate, FSH, LH, antibiotic and synthetic serum. Incubation was done at 37 degree C with 5 per cent CO2 in air and nuclear stage was assessed after 18, 42, 66 and 90 h of incubation.

Main Outcome Measure: Attainment of metaphase II and GVBD RESULTS: After in vitro culture, there were no significant differences in maturation and GVBD rate. 27 of 52 (51.9%) oocytes (GV) in growth hormone group matured to metaphase II compared with 25 of 53 (47.2%) GV in control group. GVBD rate for germinal vesicle-stage in growth hormone group was 76.9 per cent compared with 79.2 per cent in control group.

Conclusion: Culture of immature oocytes in vitro with growth hormone results in similar maturation rate as that without GH.
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August 2002

Predictive value of human chorionic gonadotropin in the outcome of early pregnancy achieved by assisted reproductive technology.

J Med Assoc Thai 2002 Jun;85 Suppl 1:S447-54

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

This study was undertaken to evaluate the predictive value of the serum human chorionic gonadotropin (hCG) in pregnancies achieved by assisted reproductive techonology (ART). Two hundred and eighty-six pregnancies were studied retrospectively from September 1989 to June 1998. The serum hCG samples at 2-6 weeks after embryo transfer (ET) were analysed by fluoroimmunoassay. Pregnancy status was followed by ultrasonography. There were 100 nonviable pregnancies (NP), 140 viable single pregnancies (VSP) and 46 viable multiple pregnancies (VMP). The sensitivity, specificity, positive and negative predictive value of the D14 hCG (<160 mIU/ml) in distinguishing NP from VSP were 79 per cent, 75 per cent, 68 per cent and 84 per cent, respectively and of the D14 hCG (>350 mIU/ml) in distinguishing VMP from VSP were 82 per cent, 75 per cent, 56 per cent and 91 per cent, respectively. In conclusion, the serum hCG may be used to predict the outcome of early pregnancy achieved by ART.
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June 2002

Effects of initiation day of clomiphene citrate on the endometrium of women with regular menstrual cycles.

Fertil Steril 2002 Jul;78(1):102-7

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

Objective: To investigate the effects of initiation time of clomiphene citrate (CC) on the endometrium of women with regular menstrual cycles.

Design: Randomized, double-blind, cross-over study.

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

Patient(s): Thirty-three healthy female volunteers with regular menstrual cycles.

Intervention(s): The volunteers were randomized to receive either 100 mg of CC on days 1-5 and placebo on days 5-9 (study group) or placebo on days 1-5 and CC on days 5-9 (control group). After a wash-out period of 1 month of CC treatment, the medication was switched in each group. Ultrasonography was performed daily after day 10 of the cycle to detect ovulation. Ultrasonography for endometrial appearance and thickness, endometrial sampling, and blood samples obtained for determination of E(2) and P levels were performed 7 days after ovulation in both groups.

Main Outcome Measure(s): Morphometric analysis, histologic dating, and ultrasonographic appearance and thickness of the endometrium.

Result(s): Morphometric parameters, histologic dating, and ultrasonographic appearance and thickness of the endometrium were similar in both groups.

Conclusion(s): Starting CC on either day 1 or day 5 of the menstrual cycle did not have any differential effects on the endometrium of women with regular menstrual cycles, particularly regarding the morphometric analysis, histologic dating, or ultrasonographic appearance.
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http://dx.doi.org/10.1016/s0015-0282(02)03192-8DOI Listing
July 2002