Kinase signaling analysis of iPS cell reprogramming and differentiation
Like embryonic stem (ES) cells, induced pluripotent stem (iPS) cells can differentiate into every cell type in the body, providing enormous potential for regenerative medicine. Unlike ES cells, the derivation of iPS cells is more straightforward technically, and can be performed on human adult cells. This potentially obviates the need for donated eggs or embryos, and permits the ability to generate patient-specific stem cells for disease research, drug development, and new cell-based therapies - generating great excitement in the scientific community as well as with the public.
iPS cells hold great promise for regenerative medicine, but the cellular signaling that controls their derivation and function remains poorly understood. We are developing methods to measure protein phosphorylation (the most common mechanism of cellular signaling) in iPS cells, and we will use the key signaling events we identify to improve the speed and efficiency of iPS derivation, as well as the safety and utility of iPS cells for regenerative medicine. In addition to improved iPS cell protocols that will benefit basic science and clinical therapy, the methods we develop to measure protein phosphorylation in will make a valuable diagnostic test for iPS and iPS-derived cells to determine their safety and functionality before use in patient-specific regenerative therapy.
Our proposal will benefit California in three important ways:
First, we will advance the field of stem cell biology as demanded by the people of California when they voted for Proposition 71, the California Stem Cell Research and Cures Initiative, on November 2, 2004 to establish The California Institute for Regenerative Medicine (CIRM). The mission of CIRM is to support and advance stem cell research and regenerative medicine under the highest ethical and medical standards for the discovery and development of cures, therapies, diagnostics and research technologies to relieve human suffering from chronic disease and injury. Our proposal will add new, essential knowledge concerning the function and molecular mechanisms of induced pluripotent stem (iPS) cells. The recent discovery of iPS cells has opened new frontiers in patient-specific regenerative therapy, the study of embryonic development and cellular differentiation, and the ability to create disease-specific cell lines for drug testing and the study of disease mechanism. Our study of the kinase signaling networks that control their derivation and function will add new, essential knowledge to the field of stem cell biology that will be published in a timely manner and readily available.
Second, the improved methods, protocols, and techniques we identify to control iPS cell derivation and function will be of great utility for the translation of iPS cells to the clinic, which will provide health benefits to all Californians. Our studies will identify new methods for iPS cell derivation that improve their safety for regenerative therapy by reducing their oncogenic potential. We will also develop new methods to control their differentiation into specified lineages or cell types for use in regenerative medicine. Additionally, the high-dimensional flow cytometry techniques we develop to analyze stem cell biology will be a useful quality control test to use on human stem and stem-derived cells before their use in regenerative therapy.
Third, our research will benefit California’s robust biotechnology industry, not only by improving regenerative therapy, but also by identifying improved methods for the generation of disease-specific cell lines for drug testing and the study of disease mechanism. Strengthening the California biotechnology industry benefits all Californians, not only through improved drugs and therapies that benefit their health, but also by bringing more business to the state, increasing tax revenues, and providing much-needed employment opportunities.
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.
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