Neurodegenerative disorders such as Alzheimer’s Disease and Parkinson’s Disease are devastating illnesses that result from the
death of specific populations of cells in the central nervous system (CNS), and there is a dire need for new therapies to treat
them. Stem cells, including adult neural stem cells (aNSCs) and neural progenitor cells derived from human pluripotent stem
cells such as embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have significant promise as the bases for
cell replacement therapies and for enhancing the development of traditional therapies such as small molecules.
Developing therapies based on these cells, however, requires the precise ability to control their differentiation into specific
neuronal subtypes, which is currently limited by numerous challenges. For example, while there has been progress in identifying
soluble signals that regulate neuronal differentiation, the field lacks a clear understanding of how cues from the solid-state
components of the stem cell microenvironment influence progression of these cell types towards neural lineages. In particular,
mechanical signals may represent a potentially untapped set of design tools that can be used to optimize both differentiation
into neurons in general and into specific neuronal subtypes from hESCs, iPSC, and aNSCs, results that would also have
considerable implications for mechanisms of human development and disease. Furthermore, it is unclear whether common
processes and principles drive neurogenesis from adult NSCs, hESC, and iPSCs, and a comparative analysis may therefore benefit
each of these fields.
Recent work from our laboratories has shown that aNSC self-renewal and differentiation may be regulated by controlling either
ECM stiffness (elasticity) or the activities of specific cellular proteins that enable cells to sense elasticity and generate force. We
now propose to investigate whether mechanobiological mechanisms also control hESC and human iPSC neurogenesis. First, we
will investigate whether microenvironmental stiffness can regulate neurogenesis and neuronal subtype distributions in adult as
well as human pluripotent stem cell derived NSCs. Second, we will determine whether neuronal differentiation may also be
regulated by controlling the cell's ability to sense and process mechanical information from its environment. Third, we will ask
whether these same approaches can be used to control neurogeneis in living organisms, i.e., in vivo. This work will advance our
understanding of mechanoregulation of stem cells, offer key comparative insights into NSC-, hESC-, and iPSC-based
neurogenesis with fundamental implications for human development and disease, and improve control of stem cell
differentiation for therapeutic applications.
Statement of Benefit to California:
We will develop novel strategies for controlling the generation of neuronal cells from stem cell sources including embryonic
stem cells and induced pluripotent stem cells. Given the tremendous implication of these approaches to the treatment of
neurologic disease, this work will benefit individual citizens of California and contribute to the state’s infrastructure by
advancing stem cell engineering and health care.
Many devastating neurodegenerative diseases, including Parkinson’s Disease and Alzheimer’s Disease, result directly from the
death of specific neuronal populations the brain. The incidence and prevalence of each of these diseases continues to rise as the
population of California ages, placing a terrible burden on both individual families and our health care system as a whole. The
ability to replace or regenerate these neurons from stem cells holds great promise for the treatment of these diseases.
Furthermore, the development of in principle limitless quantities of human neurons, including ones from patients afflicted with
neurodegenerative disorders via iPSC approaches, offers the potential for high throughput screening methods that can advance
the development of traditional small molecule and protein therapies. Our proposal seeks to develop completely new ways of
controlling stem cell neurogenesis by manipulating both mechanical force-based inputs of the stem cell microenvironment and
the cellular signaling systems that enable stem cells to sense, process, and respond to these inputs. Thus, in the long term, the
results of this work will directly benefit patients and their families, as well as the health care system charged with delivering their
Importantly, this proposal directly addresses several research targets of this RFA – the role of the endogenous microenvironment
in stem cell fate regulation, molecular mechanisms that enable engineered microenvironments to control stem cell fate,
characterization of molecular determinants of stem cell fate decisions during hPSC differentiation, and differentiation of hPSCs
into fully mature, metabolically functional cell types, tissues, and mini-organs – indicating that CIRM believes that the proposed
research program is a priority for California’s stem cell effort.
In addition, the principal investigator and co-investigator combine strong expertise in stem cell biology, cellular bioengineering,
biomaterials science, and medicine that uniquely positions them to take on this multidisciplinary challenge. Both have been
inventors on pending or issued patents, and the co-investigator has extensive experience working with California biotechnology
and stem cell technology companies. Finally, this collaborative project will focus diverse research groups with many students on
an important interdisciplinary project at the interface of science and engineering, thereby training valuable future employees
and contributing to the technological and economic development of California.
This proposal focuses on studying how mechanotransductive signals from the extracellular matrix (ECM) and other solid state components of the stem cell microenvironment affect neural differentiation. In Aim 1, the applicant proposes to test the effects of microenvironmental stiffness on the differentiation of adult neural stem cells and neural progenitor cells (NPCs) derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In Aim 2, the applicant will investigate the roles of specific mechanotransductive signaling proteins on neural differentiation using viral vector mediated inducible expression of constitutively active and dominant negative forms of these proteins in NPCs. Finally, in Aim 3, the applicant proposes to examine the in vivo roles of the proteins studied in Aim 2 on neural differentiation by transplanting the hESC-derived NPCs from Aim 2 into mice as well as by studying cell-type specific mouse knockout models of the same proteins.
Reviewers agreed that this proposal addresses an important problem and, if successful, could have an impact on the development of regenerative medical therapies. However, one reviewer cautioned that the significance of matrix mechanics in differentiation could be minimal relative to other factors. Some reviewers found the proposal to be novel and innovative while another disagreed, noting that many groups are studying the effects of mechanotransduction on cell differentiation, although perhaps not as well as this applicant's group. Another reviewer would have appreciated greater justification for the use of human stem cells. This reviewer noted that the applicant has extensive experience with rodent stem cells, which are easier to genetically manipulate and avoid the immune rejection issues associated with xenograft transplantation in Aim 3.
The reviewers raised a number of issues with the research plan that they felt could limit its impact. They found Aim 1 to be the strongest of the three and expressed significant concerns about Aim 2 & 3. With regard to Aim 2, reviewers worried that because the mechanotransductive targets are multi-factorial proteins, any modulation of their function may be independent of their mechanotransductive properties. The applicant acknowledges this problem but does not address it experimentally. One discussant noted that although the mechanotransductive signaling targets are pleiotrophic, it has been possible to get clean answers in other systems when appropriately constrained. Reviewers felt that Aim 3 is unlikely to answer questions about mechanotransduction and thus does not contribute to the rest of the proposal. One reviewer commented that Aim 3 lacks a clear, testable hypothesis and is likely to yield inconclusive results. Another reviewer did not understand the rationale for transplanting NPCs from Aim 2, which might be motor neuron precursors for example, into the striatum or dentate gyrus. This reviewer also noted that the genes of interest are pleiotropic and so it will be impossible to ascribe any differences in neural differentiation in vivo to effects on mechanotransduction. Reviewers described the proposal as well-written and organized and appreciated the wealth of preliminary data presented. As a minor criticism, one reviewer wondered why the constitutively active proteins expressed in Figure 2 enhanced neuronal differentiation while suppressing it in Figure 4.
The reviewers praised the applicant as a highly qualified young scientist and felt that the collaboration with a co-investigator was a strength of the proposal. One reviewer felt that the inclusion of three post-docs at 100% effort was too many, but in general the reviewers were impressed by the assembled research team.
Overall, while reviewers appreciated the novelty of the proposal and the importance of the scientific problem it addresses, they raised some concerns about the research plan, particularly Aims 2 & 3, which they felt could limit its ability to unambiguously address the role of mechanotransductive control mechanisms in neural differentiation.
A motion was made to move this application up into Tier 1 in order to add bioengineering to the portfolio. Reviewers summarized their critiques and the motion failed.