Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (Intrabody).

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA).

Transplanted human embryonic stem cells as biological 'catalysts' for tissue repair and regeneration.

Human embryonic stem cells have tremendous potential in the newly emerging field of regenerative medicine. Recently, it was demonstrated that the rescue of lethal cardiac defects in Id knockout mutant mouse embryos was not due to the transplanted cells giving rise to functional new tissues within the defective embryonic heart. Instead, there is indirect evidence that the observed therapeutic effect was due to various secreted factors emanating from the transplanted cells.

Incorporating protein transduction domains (PTD) within recombinant 'fusion' transcription factors. A novel strategy for directing 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. The use of exogenous cytokines, growth factors, or extracellular matrix substratum, will obviously require extended durations of in vitro culture. With autologous adult stem cells, this could delay transplantation to the patient, as well as alter the immunogenicity of the cultured autologous cells.

Incorporating protein transduction domains (PTD) within intracellular proteins associated with the 'stemness' phenotype. Novel use of such recombinant 'fusion' proteins to overcome current limitations of applying autologous adult stem cells in regenerative medicine?

Adult stem cells originating from post-natal tissues hold tremendous promise in regenerative medicine. Nevertheless, there are several deficiencies of adult stem cells that would limit their application in transplantation therapy, in particular their relative scarcity, restricted multi-potency and limited proliferative capacity in vitro. A possible approach to overcome these limitations would be to genetically modulate adult stem cells to strongly express genes that are closely associated with the 'stemness' phenotype.

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.

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.

"Waste" follicular aspirate from fertility treatment--a potential source of human germline stem cells?

Fertility clinics worldwide routinely produce a large volume of 'waste' follicular aspirate, which is potentially an abundant source of immature ovarian follicles. Current attempts to cultivate these further in vitro to yield viable mature oocytes for fertility treatment have not yet achieved much success. Instead, recent lines of evidence have emerged that are suggestive of a potential stem cell niche within such immature ovarian follicles.

Feeder cell density--a key parameter in human embryonic stem cell culture.

A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging.

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.

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.

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.

The cryopreservation of human embryonic stem cells.

hES (human embryonic stem) cells hold tremendous potential in the newly emerging field of regenerative medicine, in addition to being a useful tool in basic scientific research and for pharmacological and cytotoxicity screening. However, an essential prerequisite for the future widespread application of hES cells are the development of efficient cryopreservation protocols to facilitate their storage and transportation. This review summarizes the current state of progress in the field of hES cell cryopreservation, by critically examining and comparing the various cryopreservation protocols that have been developed.

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.

Effects of elevated temperature in vivo on the maturational and developmental competence of porcine germinal vesicle stage oocytes.

Postslaughter processing of sow carcasses results in the ovaries being exposed to temperatures of 41.3 to 42.1 degrees C within a 30-min time frame.

Slow-cooling protocols for microcapsule cryopreservation.

The relatively large size (300-400 microm) and fragile semi-permeable membrane of microcapsules makes them particularly prone to cryodamage. This study investigated slow-cooling protocols for the cryopreservation of microcapsules. Instead of a programmable freezing-machine, slow cooling was carried out directly within a -80 degrees C refrigerator.

Factors associated with transmission of severe acute respiratory syndrome among health-care workers in Singapore.

Between 1 and 22 March 2003, a nosocomial outbreak of Severe Acute Respiratory Syndrome (SARS) occurred at the Communicable Disease Centre in Tan Tock Seng Hospital, Singapore, the national treatment and isolation facility for patients with SARS. A case-control study with 36 cases and 50 controls was conducted of factors associated with the transmission of SARS within the hospital. In univariate analysis, contact with respiratory secretions elevated the odds ratio to 6.

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.

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.