Identification of hESC-mediated molecular mechanism that positively regulates the regenerative capacity of post-natal tissues
The tissue regenerative capacity deteriorates with age in animals and in humans, leading to the loss of organ function, which is well exemplified in skeletal muscle, but is poorly understood in molecular terms. Our recent work uncovered that factors produced by human embryonic stem cells have a unique ability to enhance the regenerative responses of organ stem cells, dedicated for tissue maintenance and repair, be they young or old and located in young or old organism. This proposal seeks to understand the molecular mechanism of this novel phenomenon, which is two-fold important: in expanding our knowledge of the stem cell biology and in developing entirely novel embryonic stem cell-based therapeutic applications that do not have the side-effects associated with immune rejection. Importantly, this uncovered enhancement of tissue repair is conserved between mice and humans , which allows use of an animal model for identifying these therapeutically-relevant human factors and greatly facilitates the pre-clinical data collection, interpretation and translation to clinic.
The main goals of this Proposal are to identify the embryonic pro-regenerative factors, to understand their mode of action and to validate their efficiency for enhancing and rejuvenating repair of injured and pathological tissues in an animal model. Notably, using the infrastructure of [REDACTED] the data generated by this work will be quickly disseminated to [REDACTED] clinicians and will be applied through Clinical Affiliates Program for clinical studies and human trials.
Identifying these embryonic stem cell-produced pro-regenerative factors will help counter the loss of tissue maintenance and repair in the old, generally and not just in skeletal muscle, and will be of immediate therapeutic value without a need for “humanization” and without the risk of immune rejection. Additionally, for muscle wasting caused by diabetes and immobility, and in Duchenne/Becker and Limb-Girdle myopathies, these factors will boost the performance of satellite cells struggling to repair continuous myofiber deterioration, thus countering degeneration and improving organ function.
Degenerative diseases in which the bodies capacity to regenerate new tissue can no longer keep up with tissue death is a major problem for society in general and for State of California in particular. The lack of tissue repair that eventually leads to the loss of organ function is undeniable and devastating trait of aging that causes many degenerative disorders, exemplified by Parkinson’s, Alzheimer’s and muscle atrophy. Therefore, Californians with life-long skills, expertise and invaluable knowledge can no longer contribute to society and do not enjoy life fully. In recent years biologists and clinicians realized that practical therapies would only emerge when the balance between the regenerative and the degenerative processes were properly understood in biomedical terms. Comprehensively, the proposed research seeks to uncover novel evolutionary conserved molecular regulation that is mediated by human embryonic stem cells and promotes regenerative capacity of postnatal stem cells (likely, generally and not just in skeletal muscle). Qualified scientists from underrepresented minorities will be involved with this academic and translational stem cell project, hence allowing expand the representation of all Californians in the cutting-edge biomedical research. This proposal describes steps to rejuvenate stem cell responses in the old and to rescue tissue repair in people suffering from debilitating degenerative diseases. The outcomes of this work will insure that the health prognosis is significantly improved for older Californians, especially those afflicted with degenerative disorders, and that the results of these studies are translated as rapidly as possible to the clinical setting where their practical benefit can be fully utilized. Thus this work seeks not only to improve the quality of life for our older citizens, but also to reduce the health-cost associated with treating currently incurable degenerative diseases. The developing therapies will be immediately applicable for all Californians irregardless of their ethnic background, gender or age.
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.
Although functional organ stem cells persist in the old, tissue damage invariably overwhelms tissue repair, ultimately causing the demise of an organism. The poor performance of stem cells in an aged organ, such as skeletal muscle, is caused by the changes in regulatory pathways such as Notch, MAPK and TGF‐β, where old differentiated tissues and blood circulation inhibit the regenerative performance of organ stem cells. While responses of adult stem cells are regulated extrinsically and age‐specifically,
our work recently published puts forward experimental evidence suggesting that embryonic cells have an intrinsic youthful barrier to aging and produce soluble pro‐regenerative proteins that signal the MAPK pathway for rejuvenating myogenesis. Future identification of this activity will improve our understanding of embryonic versus adult regulation of tissue regeneration suggesting novel strategies for organ rejuvenation. Comprehensively, our progress of the last year indicates that if the age‐imposed decline in the regenerative capacity of stem cells was understood, the debilitating lack of organ maintenance in the old could be ameliorated and perhaps, even reversed.The same understanding is also required for successful transplantation of stem and progenitor cells into older individuals and for combatting many tissue degenerative disorders: namely, productive performance of transplanted cells is dependent on the niche into which they are placed and the inhibitory factors of the aged and pathological niches need to be identified and neutralized. Additional recently published work was focused on developing new strategies for providing new source of regenerative cells to people who suffer from genetic myopaties (where their own muscle stem cells become exhausted due to the progression of the disease). Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. We utilized small molecule inhibitors to dedifferentiate muscle into dividing myogenic cells, without gene over expression, which is clinically adaptable. The reprogrammed muscle precursor cells contributed to muscle regeneration in vitro and in vivo and were unequivocally distinguished because of the lineage marking method. These findings enhance understanding of cell-fate determination and create novel therapeutic approaches for improved muscle repair. Moreover, one more of our recently published papers has identified new ways of making muscle progenitor cells to fuse more robustly into muscle fibers, hence enabling deliberate control of muscle tissue formation. We are at the latest stages in our work on the design of novel biomimetic stem cell niches, which based on our current results make easy to expand in culture progenitor cells (e.g. derived from paints) akin to muscle stem cells and enhancing the efficiency of cell transplantation to such an extent that progressive muscle loss in genetic myopathies is predicted to be averted. We have also deciphered some of the fundamental properties of embryonic stem cells, which would enable deliberate control of their self-renewal and tissue specific differentiation and the manuscript describing these findings has been submitted to Cell.
Since the last progress report we have confirmed and extrapolated our studies and, as proposed last year, we have identified specific proteins that are secreted by human embryonic stem cells and that enhance muscle regeneration. We have extrapolated the mouse findings and see that these therapeutic embryonic proteins have the pro-regenerative activity on human muscle cells, and excitingly, show that these factors also enhance proliferation of neuronal stem cells and even combat the Alzheimer’s disease (modeled in human cortical neurons derived from embryonic stem cells). We are starting to understand how these pro-regenerative proteins act (which will help to optimally harness their therapeutic potential). We are also in the process of attempting rejuvenation of tissue repair in live aged animals and the preliminary results are encouraging. Notably, the manuscripts, which were listed in the last progress report as in preparation or submitted have been published.
The work on the hESC-secreted pro-regenerative factors has been published in two papers and the third manuscript is under review. Additionally, an invention disclosure has been filed with UC Berkeley on the enrichment of the pro-regenerative activity in proteins with heparin-containing domains and thus, our ability to enrich these therapeutic factors by heparin-coated beads.
- Nat Commun (2014) Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration. (PubMed: 24915299)
- Aging (2014) Mechanisms of action of hESC-secreted proteins that enhance human and mouse myogenesis
- Integr Biol (Camb) (2013) Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. (PubMed: 23407661)
- PLoS One (2013) Regenerative Capacity of Old Muscle Stem Cells Declines without Significant Accumulation of DNA Damage. (PubMed: 23704914)
- J Cell Biol (2013) DNA methyltransferase-3-dependent nonrandom template segregation in differentiating embryonic stem cells. (PubMed: 24127215)
- Aging (Albany NY) (2013) hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding. (PubMed: 23793469)
- Cell Cycle (2012) Heterochronic parabiosis for the study of the effects of aging on stem cells and their niches. (PubMed: 22617385)
- Aging (Albany NY) (2012) Age dependent increase in the levels of osteopontin inhibits skeletal muscle regeneration. (PubMed: 22915705)
- Aging (Albany NY) (2011) Embryonic anti-aging niche. (PubMed: 21666284)
- Biochem Biophys Res Commun (2011) Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes. (PubMed: 21910971)
- Chem Biol (2011) Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells. (PubMed: 21944754)
- Methods Mol Biol (2010) Preparation of adult muscle fiber-associated stem/precursor cells. (PubMed: 20405365)
- Aging Cell (2009) Relative roles of TGF-beta1 and Wnt in the systemic regulation and aging of satellite cell responses. (PubMed: 19732043)
- EMBO Mol Med (2009) Molecular aging and rejuvenation of human muscle stem cells. (PubMed: 20049743)