Basic Biology III
One big problem with stem cell therapies is that when the stem cells are transplanted, injected or otherwise administered to the patient, most of those cells simply die. The reasons they die are still not quite understood, but it seems they suffer a "culture shock" from their new environment. Remember that most therapeutic cells are grown in a lab on a plastic surface in a chemical nutrient medium, which certainly is not the same as in whatever tissue they are destined to try to repair. To a stem cell, this environment is its niche, just as an animal fits into its ecological niche. A big current gap in the utility of stem cell therapy is coordinating the therapeutic cell with a stem cell niche. In order to better prepare the therapeutic stem cell in the lab for its ultimate home in the body, we have recently reverse-engineered the muscle regenerative stem cell niche. We form a 3 dimensional, biochemically defined matrix and infuse that with biochemically defined growth and differentiation factors. In our niche, both mouse and human adult muscle cells self-assemble into muscle fibers, while some cells de-differentiate into myofiber-associated muscle stem cells. This project first will begin to translate our results from mouse muscle to human cells, demonstrate that these cells are functional in the niche, and then investigate how this niche works on the molecular level to instruct cells. 1. To determine if human muscle cells in the bioengineered niche make functional muscle in mice, with associated, functional, renewed muscle stem cells. 2. To determine if human healthy muscle cells, marked with the firefly luciferase gene will grow in and repair muscle in mice with muscular dystrophy, by imaging the glowing muscle in live mice using the Xenogen camera. 3. To define the molecular mechanisms by which the engineered niche is appropriate for muscle stem cells. We will focus on telomerase activity and signaling pathways that we have found important in regenerating muscle, comparing among the cells in the bioengineered niche, native regenerating muscle cells and cultured muscle cells. Significance: These experiments will uncover the properties of how the stem cell interacts with the niche, especially in muscle. This work translates our powerful findings in mouse to a human cell model, taking steps toward a long sought-after therapy for genetic/acquired myopathies, such as Duchenne Muscular Dystrophy, where traditional myoblast transplantation has failed due to massive cell death, poor engraftment and lack of self-renewal, and where traditional muscle stem cell transplantation is not practical since these cells do not grow well in the lab.
Statement of Benefit to California:
This research proposed herein benefits Californians, California as a society and the State of California. As it is applicable to all of humanity, Californians will ultimately see medical benefits from this basic research on the stem cell niche in muscle: improvements to their musculo-skeletal health and quality of life, a longer productive lifespan and enjoyment from a more active lifestyle. Californians suffering muscular dystrophies will benefit even sooner as this class of diseases will be targeted first. California as a society benefits from the aggregate medical benefits above, plus the economic benefits from fostering cutting edge research in the state at [REDACTED]. This proposal seeks to improve stem cell responses in the old and to rescue tissue repair in people suffering from debilitating degenerative diseases. The accumulated life-long skills, expertise and invaluable knowledge older or infirm Californians have to contribute will not be lost to our society as rapidly since we will be healthier and more able later in life. That this work is conducted in California benefits our local academic and high-tech cultures and economy. Qualified scientists from underrepresented minorities will be involved with this translational research, expanding the education and representation of all Californians in the cutting-edge biomedical research. That most of the research dollars are spent here in California puts more qualified people at work and boosts the living standard for all Californians. In the longer term, this research also reduces the health-costs associated with treating currently incurable degenerative diseases. The great state of California ultimately benefits from this work, in the short term at [REDACTED] and in the longer term through the enduring legacy of CIRM. The success of this project adds some small measure to the prestige of [REDACTED]. Success, with time, will justify CIRM’s mission of independent state sponsored biomedical research and lend support to future institutes of its kind.
Project Synopsis: This application focuses on the problem of cell death following transplantation. The applicant proposes that the primary reason cells die is the “shock” of being transplanted from the cell culture dish to a very different in vivo environment. The applicant hypothesizes that culturing muscle stem cells in a niche engineered to mimic the physiological niche will improve their survival following transplantation. The applicant has successfully demonstrated enhanced myogenic potential of cultured mouse muscle stem cells using this approach, and now proposes to extend the findings to human cells and investigate potential mechanisms. Three specific aims are proposed: (1) to determine if human myoblasts cultured in a bioengineered niche differentiate into functional muscle in vivo in immunodeficient mice; (2) to determine if healthy human muscle cell transplants grow in vivo in dystrophic mice; and (3) to define the molecular mechanisms by which the bioengineered niche regulates cell fate. Significance and Innovation: - The reviewers expressed mixed opinions regarding the significance and innovation of this proposal. - One view was that this proposal represents a potentially important advance for muscle regeneration and the findings could easily translate into other areas of cellular repair. Some reviewers found the reverse engineering approach to be a strength and noted that, if successful, it could allow expansion of human muscle stem cells from muscle biopsies. - The alternate view was that the project is only an incremental advance over the mouse cell work reported in the preliminary data and not particularly innovative. In addition, any therapy resulting from this work would be extremely complex from a regulatory perspective, making it difficult to achieve a significant impact. - The proposed longitudinal study of the fate of the transplanted human muscle cells in vivo over time using non-invasive imaging is novel and innovative. Feasibility and Experimental Design: - Insufficient evidence was provided to support the central hypothesis that cell death following transplantation is due is to the shock of cells moving to a new niche environment. The applicant further hypothesizes that growing cells in a more niche-like environment in vitro will reduce the shock to cells at the time of implantation and reduce cell death. The preliminary data only partially supports this hypothesis. - Aims 2 and 3 are highly dependent on Aim 1. - Reviewers expressed mixed opinions regarding the strength of the preliminary data. There was agreement that the mouse cell data are convincing but the human cell data were questioned. - One view was that the applicant presents convincing data that their designed niche can promote myofiber assembly from mouse and human muscle progenitors, and that these data demonstrate the feasibility of the proposed studies. - The alternate view was that the preliminary data support the interpretation that mouse myoblasts cultured in a niche culture system lead to the assembly of myofibril, but do not support the conclusion that human cells behave the same way. Unless human cells can generate myofibrils in vitro there will be no human model in which to undertake the mechanistic studies in Aim 3. - Reviewers also had mixed opinions regarding Aim 3. One view was that the proposed experiments, particularly the focus on specific signaling pathways that regulate satellite cell fate, are novel and a strength of the proposal. Other reviewers felt that Aim 3 lacks focus and investigates too many potential molecular mechanisms. Principal Investigator (PI) and Research Team: - Reviewers were not convinced that the team has the necessary expertise to undertake the proposed study. The PI is relatively inexperienced and has a modest publication record following his/her postdoctoral fellowship. - The research team consists of the PI at 90% effort and three personnel to be named (two master’s students and one technician). - The PI’s relative lack of experience could have been mitigated by the formation of a collaborative team. Specifically, collaborators with expertise in muscle function would have strengthened the proposal. - The budget is appropriate for the described research although there is no detailed breakdown of budgeted materials and supplies and it is unclear why a clinical grade inverted microscope is required. Responsiveness to the RFA: There were mixed opinions among reviewers about responsiveness. One view was that this application will address an important basic mechanism and is therefore responsive to the RFA. The alternate view was that the project does not satisfy the RFA criteria well, because it is more translational than basic and the most mechanistic part, Aim 3, includes significant work with mouse cells.