Basic Biology III
Stem Cell Use:
Induced pluripotent stem cells (iPSCs) have tremendous potential for patient-specific cell therapies, which bypasses immune rejection issues and ethical concerns for embryonic stem cells (ESCs). However, to fully harness the therapeutic potential of iPSCs, many fundamental issues of cell transplantation remain to be addressed, e.g., how iPSC-derived cells participate in tissue regeneration, which type of cells should be derived for specific therapy, and what kind of matrix is more effective for cell therapies. The goal of this project is to use iPSC-derived neural crest stem cells (NCSCs) and nerve regeneration as a model to address these fundamental issues of stem cell therapies. NCSCs are multipotent and can differentiate into cell types in all three germ layers (including neural, vascular, osteogenic and chondrogenic cells), which makes NCSC a valuable model to study stem cell differentiation and tissue regeneration. Peripheral nerve injuries and demyelinating diseases (e.g., multiple sclerosis, familial dysautonomia) affect millions of people. Stem cell therapy is a promising approach to cure these diseases, which will have broad impact on healthcare. This project will advance our understanding of how extracellular microenvironment (native or engineered) regulates stem cell fate and behavior during tissue regeneration, and whether stem cells such as iPSC-NCSCs and differentiated cells such as iPSC-Schwann cells have different therapeutic effects. The results from this project will provide insights that will facilitate the translation of stem cell technologies into therapies for nerve injuries, demyelinating diseases and many other disorders that may be treated with iPSC-NCSCs.
Statement of Benefit to California:
Induced pluripotent stem cells (iPSCs), especially iPSCs without the integration of reprogramming factors into the genome, are valuable to model disease and to generate autologous cells for therapies. Understanding the role and differentiation of iPSC-derived cells in tissue regeneration will facilitate the translation of stem cell technologies into clinical applications. iPSC-derived neural crest stem cells (NCSCs) can differentiate into a variety of cell types, and hold promise for the therapies of diseases such as nerve injuries, demyelinating diseases, spina bifida, vascular diseases, osteoporosis and arthritis. The isolation and characterization of iPSC-NCSCs will provide a basis for their broad applications in tissue regeneration and disease modeling. This project will use peripheral nerve regeneration as a model to address the fundamental issues of using iPSC-NCSCs for therapies. Peripheral nerve injuries (over 800,000 cases in the United States every year) are very common following traumatic injuries and major surgeries (e.g., removing tumor), which often require surgical repair. Stem cell therapies can accelerate nerve regeneration and avoid the degeneration of muscle and other tissues lack of innervation. Since iPSC-NCSCs can promote the myelination of axons, the therapies for nerve injuries could also be adopted to treat demyelinating diseases. In many cases of stem cell therapies, matrix and scaffold materials are needed to enhance cell survival and achieve local delivery. The studies on appropriate matrix for stem cell delivery will provide a rational basis for designing and optimizing materials for stem cell therapies. The fundamental issues addressed in this project, such as the differentiation and signaling of transplanted cells, the therapeutic effects of cells at the different stages of differentiation and the roles of delivery matrix/materials, will have implications for stem cell therapies in many other tissues. Overall, the results from this project will advance our knowledge on stem cell differentiation and function during tissue regeneration, help us translate the knowledge into clinical applications, and benefit the health care in California and our society.
Induced pluripotent stem cells (iPSCs) have tremendous potential for regenerative medicine applications. Here we use peripheral nerve regeneration as a model to address the fundamental issues of using iPSCs and their derivatives for therapies. Specifically, we used integration-free iPSCs for our studies because this type of iPSCs has potential for clinical applications. We derived and characterized neural crest stem cells (NCSCs) from integration-free iPSCs, and demonstrated that these NCSCs can differentiate into a variety of cell types, including Schwann cells. We delivered NCSCs into nerve conduits to treat peripheral nerve injuries, and performed functional studies, electrophysiology analysis and histological analysis. Ongoing studies suggest that the transplantation of iPSC-NCSCs accelerate nerve regeneration. To investigate the interactions of transplanted stem cells with endogenous neural progenitors, we isolated and characterized endogenous progenitors from injured nerves, which will be used for mechanistic studies. In addition, we engineered the chemical components and the structure of nerve conduits, and developed and characterized hydrogels that could be used to deliver neurotrophic factors and minimize scar formation. The roles of neurotrophic factors, transplanted/endogenous stem cells and matrix for stem cell delivery will be investigated.
We use peripheral nerve regeneration as a model to address the critical issues of using induced pluripotent stem cells (iPSCs) and their derivatives for tissue regeneration. In the past year, we have made progress in all three Specific Aims. We generated 5 new integration-free IPSC lines by using episomal reprogramming. We also tested the methods of using biomaterials and chemical compounds to reprogram cells, in the presence or absence of transcriptional factors. We have derived and characterized additional neural crest stem cell (NCSC) lines from these new iPSC lines, and demonstrated that these NCSCs are multipotent in their differentiation potential. To investigate the mechanisms of how NCSCs enhanced the functional recovery of transected sciatic nerves, we examined the effects of paracrine signaling, cell differentiation and matrix stiffness. In vivo experiments showed that transplanted cells secreted neurotrophic factors to promote axon regeneration. In addition, NCSCs differentiated into Schwann cells to enhance myelination. The stiffness of extracellular matrix (ECM) indeed has effect on NCSC differentiation.
Here we use peripheral nerve regeneration as a model to address the critical issues of using induced pluripotent stem cells (iPSCs) and their derivatives for tissue regeneration. In the past year, we have made progress in all three Specific Aims, as detailed below. In Specific Aim 1, we generated 5 new integration-free IPSC lines by using episomal reprogramming. We also optimized the protocol to derive neural crest stem cells (NCSCs) from integration-free human iPSCs, and fully characterized the derived cells. Transplantation of selected NCSC lines significantly improved the functional recovery of peripheral nerve following injury. In addition, transplanted NCSCs differentiated into Schwann cells around regenerated axons. Nerve growth factor (NGF) appeared to be a major neurotrophic factor expressed by NCSCs, which was involved in nerve regeneration. In Specific Aim 2, we derived and characterized Schwann cells from NCSCs. Transplantation of NCSCs or Schwann cells showed that NCSC transplantation had better functional recovery than Schwann cell transplantation, suggesting that the differentiation stage of transplanted cells is critical for stem cell therapies. In Specific Aim 3, we demonstrated that the soft matrix worked much better than stiffer matrix for NCSC delivery and the functional recovery of damaged nerve. A new direction for this Specific Aim is a ground-breaking finding that matrix stiffness regulates the direct reprograming of fibroblasts into neurons, which has applications in generating neurons for drug discovery and disease modeling. Overall, our findings underline the importance of stem cell differentiation stage and biomaterials property in stem cell therapies, and will have broad impact on using stem cells for nerve regeneration and many other regenerative medicine applications.