Year 2 & 3
In 2009 beginning of 2010 we have focused on investigating what factors human embryonic stem cells (hESCs) may produce that enhance regeneration and if those factors have any effects by themselves on regeneration. We have published three papers and four book chapters funded at least in part by this award. One patent application has been filed with our University. We have used a proteomic antibody array to examine over 500 common signaling proteins at once to see if any are produced in much higher or lower levels by hESCs. We found that hESCs produce both positive growth factors and negative regulators of the TGF-beta family. We confirmed that typical growth factor signaling was in fact occurring in muscle cells exposed to hESC produced factors, and that hESCs produce a TGF-beta antagonist. This fits with our recently published work showing that young muscle regenerates well from strong growth factor signaling and low TGF-beta signals while old muscle regenerated poorly due to weak growth factor signaling and high TGF-beta signaling. Our current running hypothesis is that the positive growth factors produced by hESCs trigger injured muscle to initiate and maintain regeneration, the TGF-beta inhibitors produced by hESCs reduce the TGF-beta signaling, and the combination assures the robust regeneration of muscle. We also found a surprising increase in insulin production by hESCs and are integrating that result with ongoing regeneration experiments. In the next reporting period we will re-confirm that the levels of candidate proteins from the 500 antibody array actually are very highly produced by hESCs and that the signals from these proteins are perceived by regenerating muscle cells. For Aim 4 we have examined the effects on live regenerating muscle of administering the TGF-beta inhibitors that we found in Aim 2. Preliminary data indicates the effects on regeneration of old muscle look very promising. What was surprising is that administering these inhibitors to the whole animal appears to reduce TGF-beta levels in the whole animal, suggesting some kind of feed-back and perhaps effects on other tissues as well as muscle. For the next reporting period we will confirm these results. In addition we will analyze the effect on regeneration of administering the growth factors that we found in Aim 2, both alone and in combination with the inhibitors of TGF-beta.
In 2010 beginning of 2011, we have approached the identification and characterization of the proteins that are produced by hESCs and have the rejuvenating and pro-regenerative activity on adult muscle. Specifically, our data suggest that several other ligands of MAPK pathway secreted by hESCs are likely to enhance and rejuvenate the regeneration of old muscle tissue. Our work is at the stage of understanding the molecular mechanisms by which the aging of the regenerative potential of organ stem cells can be reversed by particular human embryonic factors that are capable of neutralizing the affects of aged niches on tissue regenerative capacity. We have submitted the several manuscripts on topics of enhanced tissue regeneration and we are preparing the manuscript that identifies hESC-based novel strategies for restoring high regenerative capacity to old muscle. Additionally, our data in progress suggest that muscle and brain age by similar molecular mechanisms and thus, therapeutic strategies for rejuvenating muscle repair might be applicable to the restoration of neurogenesis in aged brain. Finally, our data suggest that muscle stem cells either do not accumulate DNA damage with age or can efficiently repair such damage, when activated for tissue regeneration. Thus, the use of hESC-produced pro-regenerative factors for boosting the regenerative capacity of organ stem cells is likely to yield healthy, young tissue. Our plan is to develop further these projects that cross-fertilize each other and have a main theme of enhancing and rejuvenating tissue regeneration. In the next funding period we also plan to accomplish transition from mouse model to human cells and studies.