We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines.
Delivery of bone marrow cells (BMCs) to the heart has substantially improved cardiac function in most rodent models of myocardial infarction (MI), but clinical trials of BMC therapy have led to only modest improvements. Rodent models typically involve intra-myocardial injection of BMCs from distinct donor individuals that are healthy, unlike autologous BMCs used for clinical trials that are from post-MI individuals. Using BMCs from post-MI donor mice, we discovered that recent MI impaired BMC therapeutic efficacy. MI led to myocardial inflammation and an increased inflammatory state in the bone marrow, changing the BMC composition and reducing their efficacy. Injection of a general anti-inflammatory drug or a specific interleukin-1 inhibitor to post-MI donor mice prevented this impairment. Our findings offer an explanation of why human trials have not matched the success of rodent experiments, and suggest potential strategies to improve the success of clinical autologous BMC therapy.
Our iPSC-CM platform provides novel mechanistic and therapeutic insights into the contribution of random X chromosome inactivation to disease phenotype in X-linked Danon disease.
Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.
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