Egg-sharing in return for subsidized fertility treatment: a possible solution for therapeutic cloning?
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Cloning, Organism↗
Biomedical subjects
Boon Chin Heng
Publications and source records attributed to Boon Chin Heng.
Directing stem cells into the keratinocyte lineage in vitro.
A major area of research in regenerative medicine is the potential application of stem cells in skin grafting and tissue engineering. This would require well defined and efficient protocols for directing the commitment and differentiation of stem cells into the keratinocyte lineage, together with their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying skin tissue biology, as well as facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for skin-related biomaterials and drugs could also utilize protocols developed for the commitment and differentiation of stem cells into the keratinocyte lineage. Hence, this review critically examines the various strategies that could be employed to direct the commitment and differentiation of stem cells into the keratinocyte lineage in vitro.
Cell Culture Techniques↗
Factors influencing stem cell differentiation into the hepatic lineage in vitro.
A major area of research in transplantation medicine is the potential application of stem cells in liver regeneration. This would require well-defined and efficient protocols for directing the differentiation of stem cells into the hepatic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying hepatogenesis and liver metabolism. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for newly developed biomaterials and drugs, could also utilize protocols developed for the hepatic differentiation of stem cells. Hence, this review critically examines the various strategies that could be employed to direct the differentiation of stem cells into the hepatic lineage in vitro.
Animals↗
Comments about possible use of human embryonic stem cell-derived cardiomyocytes to direct autologous adult stem cells into the cardiomyogenic lineage.
Several studies have shown that cell-transplantation therapy following myocardial infarction has some efficacy in aiding myocardial repair and subsequent recovery of heart function. Large-scale production of human embryonic stem cell-derived cardiomyocytes can potentially provide an abundant supply of donor cells for myocardial transplantation. There are, however, immunological barriers to their use in human clinical therapy.A novel approach would be to look at utilizing human embryonic stem cell-derived cardiomyocytes to reprogram autologous adult stem cells to express cardiomyogenic function, instead of using these directly for transplantation. This could be achieved through a number of novel techniques. Enucleated cytoplasts generated from human embryonic stem cell-derived cardiomyocytes could be fused with autologous adult stem cells to generate cytoplasmic hybrids or cybrids. Adult stem cells could also be temporarily permeabilized and exposed to cytoplasmic extracts derived from these cardiomyocytes. Alternatively, intact cells or enucleated cytoplasts from human embryonic stem cell-derived cardiomyocytes could be co-cultured with adult stem cells in vitro, to provide the cellular contacts and electrical coupling that might enable some degree of trans-differentiation to take place. This review would therefore examine the potential advantages and disadvantages of such a novel approach, in comparison to other more conventional techniques such as the use of exogenous cytokines/growth factors or the use of genetic modulation.
Adult↗
Potential benefits of co-transplanting autologous adult stem cells together with human embryonic stem cells or their differentiated derivatives.
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Adult↗
Intracellular antibodies (intrabodies) versus RNA interference for therapeutic applications.
Currently there are two technology platforms for the ablation of protein function: (a) RNAi-mediated gene-silencing at the post-transcriptional level, and (b) intrabody knockout of protein function at the post-translational level. Both approaches hold much promise for therapeutic applications. The pertinent question is how to choose between these alternative approaches. This commentary examines the advantages and disadvantages of these newly-emerging technology platforms. The RNAi approach is much less technically challenging than the intrabody-mediated knockout of protein function, but its major limitation is non-specificity. Although it is time-consuming and labor-intensive to generate intrabodies for specific intracellular protein targets, a much higher level of specificity can be attained. Ultimately, the choice between these strategies depend on the specific application in question, as well as on further technical advances in both technology platforms.
Antibodies↗
Late-adhering human embryonic stem cell clumps during serial passage can yield morphologically 'normal-looking' colonies.
During serial passage of human embryonic stem (hES) cells, freshly-seeded hES cell clumps that do not adhere to the newly-plated murine embryonic fibroblast (MEF) feeder layer after culturing overnight, are routinely discarded with the changing of fresh culture media. Preliminary observations revealed that non-adhered hES cell clumps that were still viable possessed a distinct, rounded morphology with 'sharp,' clearly-defined edges. Typically >90% of 'viable' hES cell clumps with such morphology adhered after a further 24 hr of culture on newly-plated MEF. The remaining non-adhered cell clumps that had ill-defined jagged edges could represent either non-viable hES cells or senescent MEF carried over from the previous sub-culture. Virtually none of these adhered to the newly-plated MEF feeder layer. The overwhelming majority of 'viable,' late-adhering hES cell clumps (typically >90%) gave rise to morphologically 'normal-looking' hES colonies, which appeared to remain undifferentiated. Additionally, hES cell clumps that were attached together with the majority of the passaged colonies, but were somewhat delayed in the adhesion process, compared to neighbouring colonies, also gave rise to morphologically 'normal-looking' undifferentiated colonies that were virtually indistinguishable from neighbouring colonies. Hence, it may be useful to retain late-adhering hES cell clumps during serial passage of hES cells, rather than discarding them.
Cell Adhesion↗
Effects of granulosa coculture on in-vitro oocyte meiotic maturation within a putatively less competent murine model.
A less competent murine in vitro maturation (IVM) model was achieved by shortening the standard duration of in vivo PMSG stimulation from 48 to 24 h and selecting only naked/partially naked GV oocytes from a mixture of large and small follicles. Porcine granulosa coculture enhanced meiotic maturation within such a less competent model (37.3% versus 23.1%, P<0.05), while no significant enhancement was observed with macaque and murine granulosa coculture. Culture of porcine granulosa on extracellular matrix (ECM) gel resulted in a more differentiated morphology, but did not significantly further enhance the beneficial effects it already had on meiotic maturation. Increased concentrations of serum as well as the supplementation of gonadotrophins and follicular fluid within the culture milieu did not enhance IVM under both cell-free and coculture conditions. Porcine granulosa-conditioned medium also enhanced meiotic maturation (36.5% versus 26.7%, P<0.05), which was not diminished upon freeze-thawing (35.8% versus 22.6%, P<0.05). Enhancement of meiotic maturation by porcine granulosa coculture did not however translate to significant improvements in developmental competence, as assessed by in vitro fertilization (IVF) and embryo culture to the blastocyst stage, followed by total cell counts. ECM gel had a detrimental effect on fertilization and developmental competence, even though it had no detrimental effect on meiotic maturation itself.
Animals↗
Can RNA interference be used to expand the plasticity of autologous adult stem cells?
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Animals↗
Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro.
A major area in regenerative medicine is the application of stem cells in bone reconstruction and bone tissue engineering. This will require well-defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages on transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying osteogenesis and bone development, and facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for bone-related biomaterials and drugs could also use protocols developed for the osteogenic differentiation of stem cells. This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the osteogenic lineage in vitro.
Animals↗
Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro.
Most studies on stem cell transplantation therapy on myocardially infarcted animal models and phase-I human clinical trials have focused on the use of undifferentiated stem cells. There is a strong possibility that some degree of cardiomyogenic differentiation of stem cells in vitro prior to transplantation would result in higher engraftment efficiency, as well as enhanced myocardial regeneration and recovery of heart function. Additionally, this may also alleviate the probability of spontaneous differentiation of stem cells into undesired lineages and reduces the risk of teratoma formation, in the case of embryonic stem cells. The development of efficient protocols for directing the cardiomyogenic differentiation of stem cells in vitro will also provide a useful model for molecular studies and genetic manipulation. This review therefore critically examines the various techniques that could possibly be used to direct and control the cardiomyogenic differentiation of stem cells in vitro.
Animals↗
An overview and synopsis of techniques for directing stem cell differentiation in vitro.
The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.
Animals↗
Strategies for the cryopreservation of microencapsulated cells.
The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.
Animals↗
Potential utility of cell-permeable transcription factors to direct stem cell differentiation.
Application of embryonic and adult stem cells in regenerative medicine will require efficient protocols for directing stem cell differentiation into well-defined lineages. Differentiation induced by exogenous cytokines, growth factors, or extracellular matrix components will require extended in vitro culture that would delay autologous transplantation and may well alter the immunogenicity of cultured cells. Genetic modulation to direct stem cell differentiation may obviate prolonged culture, but safety concerns preclude clinical application of genetically altered cells in the foreseeable future A novel alternative would be to incorporate protein transduction domains (PTDs) into recombinant transcription factors that play important roles in somatic differentiation. Such protein-engineered transcription factors would then have the ability to translocate across the cell membrane and be internalized within the cytosol, where they would act as paracrine signaling molecules. Upon internalization, the recombinant transcription factors would only have a limited active half-life, so that their effects may only be transient. However, this could provide sufficient stimulus for initiating stem cell differentiation into a required lineage.
Cell Differentiation↗
Directing stem cell differentiation into the chondrogenic lineage in vitro.
A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery. This requires well-defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage-related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells. Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.
Animals↗
Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.
This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.
Animals↗
Culture media conditioned by heat-shocked osteoblasts enhances the osteogenesis of bone marrow-derived mesenchymal stromal cells.
Osteogenic cells differentiated from bone marrow-derived mesenchymal stromal cells (MSC) hold much promise in bone tissue engineering and reconstructive surgery. There is a dire need for well-defined and efficient protocols to promote the osteogenesis of ex vivo cultured MSC. Hence, this study investigated whether a combination of chemical stimuli (ascorbic acid, beta-glycerophosphate and dexamethasone) and culture media conditioned by a human foetal osteoblast cell line (hFOB) had any synergistic effect on the osteogenesis of MSC. Conditioned media with or without prior heat shock treatment (42 degrees C for 1 h) of the hFOB cell line, were collected and tested on rabbit MSC cultures, in the presence and absence of chemical stimuli. Osteogenic differentiation of MSC was assessed on both day 14 and 21 of ex vivo culture. The results showed conclusively that conditioned media promoted osteogenesis of MSC, which was further enhanced by prior heat shock-treatment of the hFOB cells, as well as by the presence of chemical stimuli. Among all experimental groups, the combination of culture medium conditioned by heat shocked hFOB cells together with chemical stimuli, exhibited the highest level of calcium mineralization, as assessed by Von Kossa staining. This provides clear evidence of a synergistic effect of conditioned media, heat shock and chemical stimuli. It is hoped that the data may contribute to the development of a more well-defined and efficient in vitro culture protocol to promote the osteogenesis of MSC for both clinical and non-clinical applications.
Animals↗
Comparison of different test models for the assessment of cytotoxicity of composite resins.
This study compared the use of different test models to assess the cytotoxicity of a dental composite. The cytotoxicity of a composite polymerized using two halogen-based light-curing units (LCUs) (Max LC and Astralis) and two light-emitting diode LCUs (E-light and Freelight) served as the basis of comparison. Disk-shaped specimens (7 mm diameter, 2 mm high) were fabricated using the four different light sources. The specimens were used in several cytotoxicity test models: direct and indirect contact tests as well as an extract test with an established cell line L-929. The cells were stained with neutral red after cell-material contact for 48 h. Neutral red-stained areas (in mm2, for direct and indirect tests) and absorbance readings (for extract tests) were analysed statistically using ANOVA and the Tukey post hoc test, with P < 0.05 considered to be significantly different. Good correlation between direct and indirect contact tests (r = 0.903) was found. The extract test was the least correlated among the three tests. It was found that the E-light + Freelight-cured composite elicited cytotoxicity from the correlated studies. Uncured specimens were most detrimental to the cells in all tests. Our data demonstrated that composite cured with light-emitting diode LCUs were cytotoxic to L-929 cells. Different test models were found to give rise to different findings. Thus, a good cell-material contact method would replicate more closely the physiological situation in vivo. This in turn would give more clinically relevant results.
Animals↗