PubMed Health⌕ Search

Biomedical subjects

Boon Chin Heng

Publications and source records attributed to Boon Chin Heng.

At least 19 recordsLinked to original sources
PubMed2007

Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells.

The study of human embryonic stem cells (hESCs) can provide invaluable insights into the development of numerous human cell and tissue types in vitro. In this study, we addressed the potential of hESCs to undergo chondrogenesis and demonstrated the potential of hESC-derived embryoid bodies (EBs) to undergo a well-defined full-span chondrogenesis from chondrogenic induction to hypertrophic maturation. We compared chondrogenic differentiation of hESCs through EB direct-plating outgrowth system and EB-derived high-density micromass systems under defined serumfree chondrogenic conditions and demonstrated that cell-to-cell contact and bone morphogenetic protein 2 (BMP2) treatment enhanced chondrocyte differentiation, resulting in the formation of cartilaginous matrix rich in collagens and proteoglycans. Provision of a high-density three-dimensional (3D) microenvironment at the beginning of differentiation is critical in driving chondrogenesis because increasing EB seeding numbers in the EB-outgrowth system was unable to enhance chondrogenesis. Temporal order of chondrogenic differentiation and hypertrophic maturation indicated by the gene expression profiles of Col 1, Col 2, and Col 10, and the deposition of extracellular matrix (ECM) proteins, proteoglycans, and collagen II and X demonstrated that the in vivo progression of chondrocyte maturation is recapitulated in the hESC-derived EB model system established in this study. Furthermore, we also showed that BMP2 can influence EB differentiation to multiple cell fates, including that of extraembryonic endodermal and mesenchymal lineages in the EB-outgrowth system, but was more committed to driving the chondrogenic cell fate in the EB micromass system. Overall, our findings provide a potential 3D model system using hESCs to delineate gene function in lineage commitment and restriction of chondrogenesis during embryonic cartilage development.

Bone Morphogenetic Protein 2↗
PubMed2007

Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting.

Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.

Cell Culture Techniques↗
PubMed2006

Exposure of mouse cumulus cell nuclei to porcine ooplasmic extract eliminates TATA box protein binding to chromatin, but has no effect on DNA methylation.

PURPOSE: The low cloning efficiency with SCNT is due to incomplete or partial reprogramming of the donor somatic cell nuclei after microinjection into the enucleated oocyte. A possible solution may be to initiate nuclear reprogramming prior to SCNT. METHODS: Pre-exposure of donor somatic cell nuclei to a novel porcine ooplasmic extract prior to microinjection could possibly extend the duration of exposure to ooplamic nuclear reprogramming factors. The effects of the porcine ooplamic extract on two major markers of nuclear preprogramming: (1) TATA box protein binding to chromation and (2) DNA methylation was investigated. RESULTS: The results showed that pre-exposure of mouse cumulus cell nuclei to porcine ooplamic extract drastically reduced TATA box protein binding to chromatin, but had no effect on DNA methylation. CONCLUSIONS: Pre-exposure to the porcine ooplasmic extract had some limited effects on nuclear reprogramming. Whether this can lead to enhanced cloning efficiency needs to be further investigated.

Animals↗
PubMed2006

Donation of surplus frozen embryos for stem cell research or fertility treatment-should medical professionals and healthcare institutions be allowed to exercise undue influence on the informed decision of their former patients?

The increasing availability of clinical assisted reproduction has led to an accumulated surplus of frozen embryos within fertility clinics worldwide. Couples that have attained success in clinical assisted reproduction, and have no further desire to reproduce; are often faced with an agonizing dilemma on what to do with their surplus frozen embryos-whether to simply discard them, or donate either for scientific research or to other infertile couples. There is a risk that persons or institutions directly involved in procuring donated embryos will prioritize their own interests over the informed choice of the patient to donate either for scientific research or to other infertile couples. Very often, formerly infertile couples who have attained reproductive success feel an overwhelming sense of gratitude to the fertility doctor handling their treatment. Hence, there is a risk of medical professionals exercising undue influence on their former patients, to sway the final decision to their preferred outcome. In the private practice setting, the preferred outcome would likely be donation for the treatment of other infertile couples; whilst in the case of medical professionals affiliated with research or academic institutions, the preferred outcome would likely be donation for stem cell research.

Decision Making↗
PubMed2006

Mesenchymal stem cell sheets revitalize nonviable dense grafts: implications for repair of large-bone and tendon defects.

BACKGROUND: Large musculoskeletal defects are commonly reconstructed with allogeneic grafts. As cryopreserved allogeneic grafts lack viable cells, this often results in poorer clinical outcome. Current technology can not incorporate large number of cells to the dense grafts. This study aimed to investigate the feasibility of fabricating sheets of mesenchymal stem cells (MSCs) to revitalize cryopreserved grafts. METHODS: Human MSCs were isolated, characterized, and cultured to form a cell sheet in the presence of ascorbic acid. Once a sheet of MSCs was obtained, it was assembled onto the demineralized bone grafts or frozen tendon grafts by a wrapping technique. Then the assembled structure was cultured for 3 weeks. The macro morphology, histology, and immunohistochemistry of the grafts were evaluated. RESULTS: It was found that MSCs were able to form coherent cellular sheets within 3 weeks. When assembled with demineralized bone matrix, MSC sheets were similar to in situ periosteum and were able to differentiate into the osteochondral lineage. When assembled with frozen tendon graft, MSCs sheets were well-incorporated within the tissue sheath (peritenon) around the tendon, and adopted the characteristic spindle-shaped morphology of tenocyte-like cells. CONCLUSIONS: The results therefore demonstrated that MSCs sheets are easily fabricated and can maintain their differentiation potential within particular scaffolds, which would suggest a novel and convenient strategy for revitalizing large tissue grafts to improve clinical outcome.

Adolescent↗
PubMed2006

Low temperature tolerance of human embryonic stem cells.

This study investigated the effects of exposing human embryonic stem cells (hESC) to 4 degrees C and 25 degrees C for extended durations of 24h and 48h respectively. Cell survivability after low temperature exposure was assessed through the MTT assay. The results showed that hESC survivability after exposure to 25 degrees C and 4 degrees C for 24h was 77.3 +/- 4.8 % and 64.4 +/- 4.4 % respectively (significantly different, P < 0.05). The corresponding survival rates after 48h exposure to 25 degrees C and 4 degrees C was 71.0 +/- 0.5 % and 69.0 +/- 2.3 % respectively (not significantly different, P > 0.05). Spontaneous differentiation of hESC after low temperature exposure was assessed by morphological observations under bright-field and phase-contrast microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. hESC colonies were assigned into 3 grades according to their degree of spontaneous differentiation: (1) Grade A which was completely or mostly undifferentiated, (2) Grade B which was partially differentiated, and (3) Grade C which was mostly differentiated. In all low temperature exposed groups, about 95% of colonies remain undifferentiated (Grade A), which was not significantly different (P > 0.05) from the unexposed control group maintained at 37 degrees C. Additionally, normal karyotype was maintained in all low temperature-exposed groups, as assessed by fluorescence in situ hybridization (FISH) of metaphase spreads with telomere and centromere-specific PNA probes. Further analysis with m-FISH showed that chromosomal translocations were absent in all experimental groups. Hence, hESC possess relatively high-tolerance to extended durations of low temperature exposure, which could have useful implications for the salvage of hESC culture during infrequent occurrences of incubator break-down and power failure.

Journal Article↗
PubMed2006

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei.

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.

Animals↗
PubMed2006

The immunogenicity and immunomodulatory function of osteogenic cells differentiated from mesenchymal stem cells.

Multipotent mesenchymal stem cells (MSC) are reported to be immunoprivileged as well as immunosuppressive. Hence, they are ideal candidates for allogeneic transplantation to induce regeneration of diseased tissues and organs. However, it is not known whether MSC would retain their immunoprivileged and immunomodulatory properties after differentiating into the local cell types of the transplantation site. This study sought to investigate this question with a novel New Zealand White rabbit osteogenesis model. Results showed that osteogenic cells differentiated from MSC (DOC) in vitro did not express the MHC class II molecule, were incapable of inducing allogeneic lymphocyte proliferation in mixed lymphocyte culture or generating CTL, were inhibitory in ongoing lymphocyte proliferation, and secreted anti-inflammatory cytokines (IL-10 and TGF-beta). There was a significantly higher secretion of IL-10 by DOC than that by MSC, while there was no significant difference between the TGF-beta secretion of MSC and DOC in vitro. However, after IFN-gamma treatment, TGF-beta secretion by DOC significantly decreased despite the increased production by MSC. Four weeks after local DOC implantation, despite MHC class II expression, second-set allogeneic skin rejection showed similar survival to first-set allogeneic skin rejection and DOC appeared to function as osteoblasts. In conclusion, DOC retained their immunoprivileged and immunomodulatory properties in vitro, but the latter was lost following transplantation.

Animals↗
PubMed2006

Advanced maternal age as an indication for preimplantation genetic diagnosis (PGD)--the need for more judicious application in clinically assisted reproduction.

BACKGROUND: Pre-implantation genetic diagnosis (PGD) is often recommended for routine screening of chromosomal aneuploidy in older women undergoing clinical assisted reproduction, even though its prognostic value on this group of patients is rather ambiguous. What is needed is a clear set of ethical guidelines in the marketing of this technically complex and expensive procedure to patients who are neither suffers nor carriers of serious genetic diseases. METHODS: Professional counseling would be required to enable an informed decision from the patient. Differences in the prognostic value of PGD for different medical indications must be clearly presented to the patient and they must also be told that PGD is not a completely 'fool-proof' means of preventing birth defects. What may be detected are some genetic syndromes and conditions associated with cytogenetic abnormalities. RESULTS: The patient must be made aware that the results of previous studies have demonstrated that the overwhelming majority of chromosomally abnormal oocytes and embryos either fail to fertilize or implant, and therefore have a relatively low chance of resulting in a live birth with chromosomal defects. Statistical data, particularly the concept of relative risk must clearly be presented to the patient without exaggerating the risk of birth defects. The patient must also be rightfully informed that PGD carries a small risk of damaging the embryo, which could compromise chances of conception. CONCLUSIONS: PGD should be judiciously indicated for advanced maternal age in clinically assisted reproduction. Other diagnostic options that may be cheaper, and which also possess a high level of accuracy should be presented first to the patient i.e. amniocentesis, chorionic villus biopsy and ultrasonargraphy.

Aneuploidy↗
PubMed2006

Advances in oocyte cryopreservation technology will eventually blur the ethical and moral boundaries between compensated egg sharing and commercialized oocyte donation.

Compensated egg sharing was originally conceived as a patient self-help co-operative scheme to avoid overt 'commodification' of donor oocytes and prevent doctors and medical institutions from acting as 'brokers' of donated human material. As such, egg sharing is an ethically justifiable and much-preferred alternative to commercialized oocyte donation. However, recent advances in oocyte cryopreservation technology are likely to blur the ethical and moral boundaries between compensated egg sharing and commercialized oocyte donation. The banking of cryopreserved oocytes would negate the requirement for donors and recipients in egg sharing to have co-ordinated and synchronized treatment cycles. Instead, fertility doctors and medical institutions can now offer subsidized fertility treatment upfront to any patient willing to donate a portion of her retrieved cohort of oocytes for banking and subsequent donation. Thus, more opportunity is now open for them to act as 'middle-man' to broker the transaction of oocytes between donor and recipient, which would inevitably result in overt 'commodification' of donated human material. Administrative and processing fees will de finitely be billed to prospective recipients, for banking and storage of the cryopreserved oocytes, which would mean that a direct profit can now be made from the transaction of oocytes between donor and recipient.

Cryopreservation↗