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Biomedical subjects

Tong Cao

Publications and source records attributed to Tong Cao.

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

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

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

[Heavy metals contents in Haplocladium and their relationships with Shanghai City environment].

This paper measured the Cu, Pb, Cd, Zn and Cr contents in Haplocladium plants and corresponding soils at 13 monitoring sites of Shanghai City, and analyzed the relationships between plant heavy metals contents and environment. The results indicated that these 13 monitoring sites could be divided into three groups of different degree heavy metals pollution, i. e., 1) seriously polluted, including Guilin, Guangqi, and Fuxing Park near the most heavy-traffic areas with intense pollution and disturbance, and the plant pollutants contents (mg x kg(-1)) being Cu 35.25 to approximately 50.36, Pb 55.50 to approximately 65.00, Cd 1.68 to approximately 2.3, Zn 829.63 to approximately 1140.13, and Cr 7.41 to approximately 16.41, 2) polluted, including Changfeng, Guzhong, Zhongshan, and Heping Park as well as the campus of Shanghai Normal University at Xuhui District in the outskirts with less heavy metals pollution, and the plant pollutants contents (mg x kg(-1)) being Cu 18.51 to approximately 62.50, Pb 14.38 to approximately 34.25, Cd 0.81 to approximately 1.40, Zn 354.25 to approximately 671.75, and Cr 3.62 to approximately 25.08, and 3) basically unpolluted, including Sheshan Mt., Shanghai Botanic Garden, Chongming National Forest Park, Daguanyuan Park, and Luojing at the suburbs of Shanghai City, with the plant pollutants contents (mg x kg(-1)) being Cu 11.13 to approximately16.41, Pb 4.63 to approximately 27.25, Cd 0.93 to approximately 1.28, Zn 489.25 to approximately1086.75, and Cr 1.53 to approximately 7. 62. The heavy metals contents in Haplocladium plants had some correlations with corresponding soils, and there were some other factors besides soil affecting the heavy metals contents in Haplocladium plants. It was suggested that bryophytes as biological indicators could be used to indicate the heavy metals pollution in Shanghai City.

Bryopsida↗
PubMed2005

Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis.

A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 degrees C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 degrees C, with >90% of cells remaining viable after 90 min of incubation at 4 degrees C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 degrees C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen-thawed hES cells after incubation at 37 degrees C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze-thawing with conventional slow-cooling protocols.

Apoptosis↗
PubMed2005

Modulating gene expression in stem cells without recombinant DNA and permanent genetic modification.

Future therapeutic applications of stem cells in regenerative medicine require efficient techniques for modulating gene expression. Conventionally, this is achieved through the use of recombinant DNA, which invariably leads to permanent genetic alteration to the cell. Overwhelming safety and ethical concerns are likely to preclude the application of genetically modified stem cells in human clinical therapy for the foreseeable near future. An alternative may be to adopt a "milieu-based" approach to influence gene expression, by exposing stem cells to a cocktail of exogenous cytokines, growth factors, and extracellular matrix. Nevertheless, the non-specific pleiotropic effects exerted by various cytokines, growth factors, and extracellular matrix would make this a relatively inefficient approach. Moreover, a "milieu-based" approach is likely to require extended durations of in vitro culture, which might delay autologous transplantation of adult stem cells to the patient and might alter their immunogenicity through prolonged exposure to xenogenic proteins within the culture milieu. The obvious solution would be to deliver proteins, RNA, or their synthetic analogs, such as peptide nucleic acid, directly into the cell to modulate gene expression. Currently, two promising delivery platforms are available: (1) protein transduction domains, and (2) immunoliposomes. Because such molecules have a limited active half-life in the cytosol and are obviously not incorporated into the genetic code of the cell, these would only exert a transient modulatory effect on gene expression. Nevertheless, a transient effect may be preferable for clinical therapy, since this would ultimately avoid permanent genetic alteration to the cell.

Animals↗
PubMed2005

Reprogramming autologous skeletal myoblasts to express cardiomyogenic function. Challenges and possible approaches.

Cell transplantation therapy is emerging as a promising mode of treatment following myocardial infarction. Of the various cell types that can potentially be used for transplantation, autologous skeletal myoblasts appear particularly attractive, because this would avoid issues of immunogenicity, tumorigenesis, ethics and donor availability. Additionally, skeletal myoblasts display much higher levels of ischemic tolerance and graft survival compared to other cell types. There is some evidence for improvement in heart function with skeletal myoblast transplantation. However, histological analysis revealed that transplanted myoblasts do not transdifferentiate into functional cardiomyocytes in situ. This is evident by the lack of expression of cardiac-specific antigens, and the absence of intercalated disc formation. Instead, there is differentiation into myotubes that are not electromechanically coupled to neighboring cardiomyocytes. This could in turn limit the clinical efficacy of treatment. This review would therefore examine the various challenges faced in attempting to reprogram autologous skeletal myoblast to express cardiomyogenic function, together with the various possible strategies that could be employed to achieve this objective.

Cell Differentiation↗
PubMed2005

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. Genetic modulation to direct stem cell differentiation would obviate prolonged durations of in vitro culture; but there are overwhelming safety concerns with regards to the application of recombinant DNA technology in human clinical therapy. A novel alternative would be to incorporate protein transduction domains (PTD) into recombinant transcription factors that play important roles in somatic differentiation. Such protein-engineered transcription factors will then have the ability to translocate across the cell membrane and be internalized within the cytosol, thereby acting as paracrine signaling molecules. Upon internalization, the recombinant transcription factors would only have a limited active half-life, so that their effects are only transient. However, this could provide sufficient stimulus for initiating stem cell differentiation into a required lineage.

Cell Differentiation↗
PubMed2005

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. Overwhelming safety concerns would preclude the direct application of recombinant DNA technology in genetic modulation. Moreover, constitutive expression of 'stemness' genes would prevent adult stem cells from participating in tissue/organ regeneration upon transplantation. A novel alternative would be to incorporate protein transduction domains within intracellular proteins (i.e. transcription factors) that are associated with the 'stemness' phenotype. Such recombinant fusion proteins would then have the ability to translocate across the cell membrane and be internalized within the cytosol, thereby enabling them to exert a gene-modulatory effect on the cell, without any permanent genetic alteration. This would be particularly useful for maintaining the 'stemness' of adult stem cell populations during extensive ex vivo proliferation, to generate adequate cell numbers for transplantation therapy.

Adult↗
PubMed2005

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. This therefore, introduces the exciting prospect of utilizing human embryonic stem cells as biological 'catalysts' to promote tissue repair and regeneration in transplantation therapy. However, the immunological barrier against allogenic transplantation, as well as the teratogenic potential of human embryonic stem cells poses major technical challenges. A possible strategy to overcome the immunological barrier may be to impose a temporary regimen of immunosuppressive drugs followed by their gradual withdrawal, once adequate tissue regeneration has been achieved. Other more novel alternatives include the use of microencapsulation to block interaction with the transplant recipient's immune system, and co-transplantation with bone marrow-derived mesenchymal stem cells, which have been demonstrated to possess immuno-suppressive properties. The teratogenic potential of human embryonic stem cells could possibly be alleviated by directing the differentiation of these cells to specific lineages prior to transplantation, or through mitotic inactivation (gamma irradiation or mitomycin C exposure). Co-transplantation with autologous adult stem cells may represent a novel strategy to further enhance the 'catalytic' effects of human embryonic stem cells. The various factors secreted by human embryonic stem cells could then have a concentrated localized effect on relatively large numbers of co-transplanted autologous adult stem cells, which may in turn lead to enhanced repair and regeneration of the damaged tissue or organ. Moreover, there is also a possibility that synergistic interactions between the co-transplanted human embryonic stem cells and autologous adult stem cells, may somehow produce signals for the recruitment and migration of additional endogenous adult stem cells within the recipient (i.e. peripheral blood circulation, bone marrow), which could further enhance organ/tissue regeneration. Hence, the potential use of human embryonic stem cells as biological 'catalysts' to stimulate tissue repair and regeneration, appears to hold tremendous promise in the field of regenerative medicine. This new therapeutic strategy needs to thoroughly investigated, in view of its potentially important clinical applications.

Adult↗