Molecular control of the bidirectional transition between stem cells and skeletal muscles

Funding Type: 
Basic Biology II
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
iPS Cell
Public Abstract: 
The understanding of the biological basis of adult somatic cell reprogramming to a pluripotent state is opening new horizons in regenerative medicine and is a key step toward “personalized” stem cell-based therapies to treat diseases for which there is currently no cure. Genetic neuromuscular disorders include a large number of diseases (e.g. muscular dystrophies) that invariably lead children to the wheelchair and eventually death. Likewise, the age-related decline in muscle mass and function has detrimental effect on the overall performance and life span in elderly populations. Stem cell-mediated regeneration of muscles in these patients is the most awaited therapeutic approach, but effective applications are limited by technical and ethical issues. Thus, the potential to reprogram diseased muscles back to early embryonic, stem cell-like state is a promising avenue to cure fatal muscular diseases. Quite paradoxically, no evidence exists that skeletal muscle cells could be reprogrammed into pluripotent stem cells with the existing technologies, suggesting that skeletal muscle cells are resistant to induced pluripotency. Our preliminary evidence indeed indicates that skeletal muscles are refractory to induced pluripotency and points to an “epigenetic restriction” of pluripotency that is established during the commitment toward a terminal differentiated phenotype. This proposal will shed light on the mechanism underpinning the antagonism between the cellular factors that induce pluripotency and those factors and events that restrict pluripotency, promote differentiation into skeletal muscles and maintain the terminally differentiated phenotype typical of skeletal muscles. This study will fill an important gap existing in our current knowledge on the basic mechanism that governs the transition between the pluripotency typical of stem cells and the cellular specialization of differentiated cells, and viceversa. The knowledge generated by our proposal will create the molecular rationale for the generation of muscle stem cells from hESCs and from skeletal muscles. As such, it will have a strong impact in the regenerative medicine for neuromuscular diseases, as it will provide the molecular insight to devise optimal strategies for stem-cell mediated regeneration in the treatment of muscular disorders.
Statement of Benefit to California: 
The increased life span in the population of developing countries and states, such as California, poses a number of new issues related to the health control and social assistance in elderly population. For instance, the age-associated muscle atrophy (sarcopenia), and the muscle catabolism occurring as a consequence of chronic diseases (i.e. cancer cachexia, AIDS or chronic infections, terminal stages of cardiovascular diseases) or prolonged pharmacological treatments (i.e. chemotherapy) lead to a reduced performance, increased morbidity, and request for medical and social assistance for an increasing percentage of the Californian population. Thus, the identification of pharmacological strategies toward regenerating aged or diseased skeletal muscles is a critical task for the development of future health strategies in California. Furthermore, the identification of stem cell-mediated strategies in regenerative medicine will fuel hopes for the treatment of genetic neuromuscular diseases, such as muscular dystrophies, and will reduce the emotional, social and economic impact that patients confined to wheel chair have on public opinion and health. More in general, the discovery of pharmacological applications for stem cell employment in neuromuscular diseases will help to establish a leadership in regenerative medicine, will inspire new technologies and will give the impetus to new initiatives attracting financial resources and a new generation of stem cell scientists in California. The generation of stem cell scientists is particularly important to create and propagate in the future a productive environment fueling the research in regenerative medicine. This proposal will also be instrumental to train and commit to the stem cell research new MD and PhD scientists that will provide a valuable resource to propel the advances in regenerative medicine in California.
Progress Report: 
  • Public Summary of Scientific Progress
  • The discovery of induced pluripotent stem (iPS) cells has generated much excitement, because, like embryonic stem (ES) cells, they have the potential to differentiate into every cell type in the body, yet their derivation is straightforward technically and does not require the use of human embryos. There are three related challenges that must be met before iPS cells can be applied to regenerative therapy: 1) Identify methods that improve the efficiency of iPS cell generation and the quality of derived iPS cells, 2) determine whether iPS cells can function identically to ES cells, which remain the gold standard, and 3) identify methods that can control the differentiation of iPS cells into the cell types desired for regenerative therapy. Current methods for iPS cell derivation are highly inefficient: only a small percentage of any human adult cell population can be successfully de-differentiated to an ES-like state. Directing the differentiation of iPS or ES cells is also highly inefficient: in every directed-differentiation method, only a small percentage of cells differentiate into the desired cell type. Therefore, understanding the behavior of individual cells within a mixed population is the level of detail required to understand the processes that will ultimately improve the methodologies utilized for their application to regenerative medicine.
  • A new proteomic technology called mass cytometry, developed by Dr. Scott Tanner at the University of Toronto, was recently applied in the Nolan lab to measure over 40 (eventually 100) parameters simultaneously at the single cell level. Mass cytometry uses rare earth metal-tagged antibodies to label cells both on the surface and inside the cell, then vaporizes those cells at 13,000 degrees Fahrenheit and counts how many of each of the different tags a cell had at the time of its demise. During this year a critical aspect of this study was the development of new software tools to organize and interpret the high-dimensional data sets generated by mass cytometry. An effective and informative means for displaying this multi-dimensional data was the application of Spanning-tree Progression Analysis of Density normalized Event (SPADE analysis), which creates an intuitive 2D representation of multi-dimensional data. SPADE recovers the underlying branching and continual structure of high-dimensional cytometry data by recognizing the subtle differences between cellular phenotypes in n-dimensional space (where n is the number of markers acquired) and tracing them in a straightforward manner. The entire n-dimensional hierarchy is visualized in two dimensions as SPADE trees. Furthermore, in this past year we developed “Time-SPADE” analysis in which trees are built sequentially by time point, allowing for time course analysis of the changing cell populations that occur during the highly dynamic processes of iPS cell derivation and ES/iPS cell differentiation.
  • In our continuing collaboration with Dr. Marius Wernig at Stanford, a recognized leader in the biology and methodologies of iPS cell reprogramming, we applied mass cytometry and SPADE analysis to study the behavior of single cells during reprogramming. Using specific metal-tagged antibody panels that we developed, we were able to trace the molecular path of de-differentiation to the ES-like state, and we showed that the co-expression of the four Yamanaka factors at both the mRNA and protein level is critical for successful reprogramming. To study differentiation in addition to de-differentiation, we applied mass cytometry and Time/SPADE methodologies to trace the behaviors of single cells within an entire sample as they undergo differentiation into defined cell types. For these experiments we were able to trace ES cells as they underwent changes into mesoderm, ectoderm and endoderm, the three cell lineages that give rise to all of our organs. These experiments set the stage for using protocols to induce differentiation into a specific cell type such as neuronal, hematopoietic, cardiac, and pancreatic as just some of the possibilities. Studying these processes at the single cell level with unprecedented detail will facilitate our optimization of iPS cell derivation and differentiation for human regenerative therapy.
  • In the past year, we have leveraged the power of high-dimensional single cell analysis to map the transitions that occur during iPS cell reprogramming, a novel method that allows for the creation of patient-specific pluripotent stem cells with great potential for regenerative medicine. We have also mapped the transitions that occur during differentiation of these iPS cells to cell types such as neural and cardiovascular, a critical step for their eventual application to regenerative medicine therapies. Our step-by-step mapping of these long, multi-step processes at the single cell level reveals multiple new points of control that allow for manipulation and optimization tailored to each patient’s specific therapy needs.
  • Our research is focused on iPS cell reprogramming, a method that allows for the creation of patient-specific pluripotent stem cells with great potential for regenerative medicine. The process of iPS cell reprogramming is long (> 2 weeks) and inefficient (<1%), and therefore time course analysis at the single cell level single cell level is required to identify and characterize the minority population of successfully reprogramming cells. In the past year, we have leveraged the power of high-dimensional single cell analysis to map the transitions that occur during cellular reprogramming. We have identified a key intermediate stage of cellular reprogramming, and we have isolated and characterized this key intermediate population. We have also identified a late-stage non-productive reprogramming trajectory, and characterized the cellular signaling requirements for successful reprogramming to the pluripotent state. We anticipate that this work will lead to improved reprogramming protocols that produce enriched, higher-quality pluripotent stem cells for use in regenerative therapy.

© 2013 California Institute for Regenerative Medicine