Husnain Khawaja Haider

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.

Directing endothelial differentiation of human embryonic stem cells via transduction with an adenoviral vector expressing the VEGF165 gene

Endothelial progenitors derived from human embryonic stem cells (hESCs) hold much promise in clinical therapy. Conventionally, lineage‐specific differentiation of hESCs is achieved through supplementation of various cytokines and chemical factors within the culture milieu. Nevertheless, this is a highly inefficient approach that is often limited by poor replicability. An alternative is through genetic modulation with recombinant DNA. Hence, this study investigated whether transduction of hESCs with an adenoviral vector expressing the human VEGF165 gene (Ad‐hVEGF165) can enhance endothelial‐lineage differentiation. The hESCs were induced to form embryoid bodies (EBs) by culturing them within low‐attachment plates for 7 days, and were subsequently trypsinized into single cells, prior to transduction with Ad‐hVEGF165. Optimal transduction efficiency with high cell viability was achieved by 4‐h exposure of the differentiating hESCs to viral particles at a ratio of 1 : 500 for three consecutive days. ELISA results showed that Ad‐hVEGF165‐transduced cells secreted human vascular endothelial growth factor (hVEGF) for more than 30 days post‐transduction, peaking on day 8, and the conditioned medium from the transduced cells stimulated extensive proliferation of HUVEC. Real‐time PCR analysis showed positive upregulation of VEGF, Ang‐1, Flt‐1, Tie‐2, CD34, CD31, CD133 and Flk‐1 gene expression in Ad‐hVEGF165‐transduced cells. Additionally, flow cytometric analysis of CD133 cell surface marker revealed an approximately 5‐fold increase in CD133 marker expression in Ad‐hVEGF165‐transduced cells compared to the non‐transduced control. Hence, this study demonstrated that transduction of differentiating hESCs with Ad‐hVEGF165 facilitated expression of the VEGF transgene, which in turn significantly enhanced endothelial differentiation of hESCs. Copyright

Therapeutic angiogenesis by transplantation of human embryonic stem cell-derived CD133+ endothelial progenitor cells for cardiac repair

OBJECTIVE This study aim to enhance endothelial differentiation of human embryonic stem cells (hESCs) by transduction of an adenovirus (Ad) vector expressing hVEGF(165) gene (Ad-hVEGF(165) ). Purified hESC-derived CD133(+) endothelial progenitors were transplanted into a rat myocardial infarct model to assess their ability to contribute to heart regeneration. METHODS Optimal transduction efficiency with high cell viability was achieved by exposing differentiating hESCs to viral particles at a ratio of 1:500 for 4 h on three consecutive days. RESULTS Reverse transcription-PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF(165) -transduced cells. Additionally, flow cytometric analysis of CD133, a cell surface marker, revealed an approximately fivefold increase of CD133 marker expression in Ad-hVEGF(165)-transduced cells compared with the nontransduced control. Within a rat myocardial infarct model, transplanted CD133(+) endothelial progenitor cells survived and participated, both actively and passively, in the regeneration of the infarcted myocardium, as seen by an approximately threefold increase in mature blood vessel density (13.62 +/- 1.56 vs 5.11 +/- 1.23; p < 0.01), as well as significantly reduced infarct size (28% +/- 8.2% vs 76% +/- 5.6%; p < 0.01) in the transplanted group compared with the culture medium-injected control. There was significant improvement in heart function 6 weeks post-transplantation, as confirmed by regional blood-flow analysis (1.72 +/- 0.612 ml/min/g vs 0.8 +/- 0.256 ml/min/g; p < 0.05), as well as echocardiography assessment of left ventricular ejection fraction (60.855% +/- 7.7% vs 38.22 +/- 8.6%; p < 0.05) and fractional shortening (38.63% +/- 9.3% vs 25.2% +/- 7.11%; p < 0.05). CONCLUSION hESC-derived CD133(+) endothelial progenitor cells can be utilized to regenerate the infarcted heart.

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.

Utilizing stem cells for myocardial repair – to differentiate or not to differentiate prior to transplantation?

In recent years, tremendous interest has been aroused over the potential application of both embryonic (1) and adult (2) stem cells in the treatment of ischemic heart disease, which remains a major cause of morbidity and mortality in the developed world (3). This is undoubtedly due to the limited efficacy of current treatment modalities, which very often lead to inadequate recovery of heart function. For example, routine clinical techniques involving revascularization (i.e. angioplasty, thrombolysis, coronary artery bypass grafting) or surgical removal of diseased myocardium are often not able to fully alleviate ischemic injury and consequent myocardial fibrosis (4 /7). Other modes of treatment that are currently being developed such as xenotransplantation (8) and artificial mechanical heart (9), have not yet advanced significantly to impact on the clinical management of myocardial infarction. Hence, stem cell transplantation to repair the damaged myocardium appears to be a much more promising treatment option for the infarcted heart. Indeed, this has been confirmed by several recent studies in animal models and phase I human clinical trials, which have invariably demonstrated much efficacy in aiding myocardial regeneration and preventing fibrosis, with consequent enhancement in the recovery of ventricular function (10,11). Nevertheless, a key issue that has largely been ignored in the majority of these studies, is the differentiation status of the stem cells being transplanted into the damaged myocardium. The differentiation status of transplanted stem cells or their derivatives is likely to profoundly influence several factors that determine their ability to contribute to myocardial regeneration within the pathological heart model. These various factors will each be critically examined in turn. It is a well-established fact that undifferentiated human embryonic stem (hES) cells form teratomas upon transplantation into immunologically compromised animal models (12). Teratoma formation would obviously be detrimental to the transplant recipient. Hence for human clinical therapy, it is imperative that hES cells are differentiated to some degree prior to transplantation into the damaged myocardium, so as to reduce the likelihood of teratoma formation in situ (13). Another major concern is the possibility of hES cell differentiation into mononuclear cells, which could in turn initiate a graft vs host reaction. Again, this risk may be alleviated through some degree of commitment or differentiation of hES cells to specific lineages, prior to transplantation. Undifferentiated adult stem cells lack the ability to form teratomas, but could instead undergo non-specific multi-lineage differentiation in vitro (14,15). This is likely to reduce the clinical efficacy of transplantation therapy, since only a sub-fraction of the transplanted stem cells would have differentiated into lineages that can potentially contribute to myocardial repair, i.e. cardiomyocytes, vascular endothelium. Further complications may arise from the pathological state of the damaged myocardium providing an ‘‘abnormal’’ milieu for undifferentiated stem cells. The presence of necrotic/apoptotic cells, free radicals and inflammatory cytokines within the