Mimicking growth control in regenerative organisms to permit mammalian regeneration

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01592
ICOC Funds Committed: 
$0
Stem Cell Use: 
iPS Cell
Public Abstract: 
Humans and other mammals are unable to regenerate significant portions of muscles of the body or heart lost to trauma or ischemic injury, for example from motor vehicle accidents, surgery or heart attacks. Despite the fact that we know there are stem cells in our muscles, these do not suffice to replace the large amounts of tissue that can be lost in many types of injuries. Even more dramatically, in the heart, there is no significant ability to regenerate damaged tissue. Whereas humans may be said to have poor regenerative capacity, some animals have an extraordinary ability to replace damaged or lost body parts such as a limb, a jaw or a large part of the heart. Such regenerative vertebrates include urodele amphibians (salamanders and newts) or fish such as zebrafish. These animals excel at regeneration by utilizing mechanism that is distinct from classical stem cell based regeneration as we currently understand it in mammals. Regeneration in newts and zebrafish is based at least in part on dedifferentiation and proliferation of lineage-committed cells to regenerate skeletal muscle and the heart. Dedifferentiation is thought to provide large numbers of progenitor cells that proliferate at the site of an injury or an amputation and contribute to the regenerating structure. By contrast, resident stem cells that exist in humans and other mammals represent a small proportion of the total cells in a given tissue and it is easy to imagine how much proliferation would be required for these scarce cells to rebuild a complete organ such as the heart or a structure such as a limb. But what if we could recruit all of the cells at the site of an injury as our regenerative counterparts do? The overall goal of this proposal is to mimic the "regeneration by dedifferentiation" paradigm in human muscle cells and to enhance regeneration in vivo. While the basal growth control mechanisms in mammals such as ourselves are similar to those in regenerative organisms, we have added levels of complexity that, it is our hypothesis, contribute to suppressing regeneration. The goal of this proposal is to transiently target growth regulators that are present in mammals but conspicuously absent in regenerative organisms to induce differentiated cells such as cardiomyocytes to become progenitors that can expand at the site of an injury and then replace damaged tissue. This approach, if successful has the distinct advantage of mimicking a process that already exists in nature and as such may be readily transitioned to medical approaches in humans.
Statement of Benefit to California: 
Humans are unable to regenerate significant portions of muscles of the body or heart lost to trauma or ischemic injury, for example from motor vehicle accidents and surgery or from heart attacks. Despite the fact that we know there are stem cells in our muscles, these do not suffice to replace the large amounts of tissue that can be lost from in many types of injuries. Even more dramatically, in the heart, there is no significant ability to regenerate damaged tissue. In California, as elsewhere, debilitating injuries and heart disease take a staggering toll on patients, their families and on health care spending. The research described in this proposal has significant potential to benefit Californians. First, and foremost, the experiments are aimed at improving our ability to regenerate skeletal muscle and heart. If this research is successful, Californians will directly benefit from the translation to therapy. The people of California will be the first benefactors of new solutions generated by this CIRM funded research.
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