Mechanobiological control of human pluripotent stem cell neurogenesis

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01328
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
Directly Reprogrammed Cell
Public Abstract: 
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 long-term care. 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.
Progress Report: 
  • During this year, we have demonstrated that hematopoietic stem cells are originated from the cells that line the inside of blood vessels, named endothelial cells. Budding of hematopoietic stem cells from endothelial cells occurs during a specific and restricted time window during development and progress has been made to elucidate the regulatory genetic networks involved in this process. We have also demonstrated that hemogenic endothelium is derived from one specific embryonic tissue (lateral plate mesoderm). This information will be used to recapitulate similar conditions in vitro and induce the growth of hematopoietic stem cells outside the body from adult endothelial cells.
  • The objective of this proposal was to identify factors that allow blood vessels to generate hematopoietic stem cells early in the embryonic stage. The process of blood generation from vessels is a normal step in development, but it is poorly understood. We predicted that precise information related to the operational factors in the embryo would allow us to reproduce this process in a petri dish and generate hematopoietic stem cells when needed (situations associated with blood transplantation or cancer).
  • In the second year of this proposal, we have made significant progress and identified critical factors that are responsible for the generation of hematopoietic stem cells from the endothelium (inner layer of blood vessels). These experiments were performed in mouse embryos, as it would be impossible do achieve this goal in human samples. The genes identified are not novel, but have not been associated with this capacity previously. To verify our findings we have independently performed additional experiments and validated the information obtained from sequencing the transcripts.
  • In addition, we developed a series of novel tools to test the biological relevance of the genes identified in vivo (using mouse embryos). Specifically, we have tested whether forced expression of these genes could induce the generation of hematopoietic stem cells. Interestingly, we found that a single manipulation was not sufficient, but multiple and specific manipulations resulted in the generation of blood from endothelium. This was a very exciting result as indicated that we are in the right track and identified factors that can reprogram blood vessels to bud blood stem cells. With this information at hand, we moved into human cells (in petri dishes).
  • The first step was to test whether human endothelial cells could offer a supportive niche for the growth of hematopoietic cells. To our surprise, we found that in the absence of any manipulation, endothelial cells could direct differentiation and support the expansion of CD34+ cells (progenitor blood cells) to a very specific blood cell type, named macrophages. These were rather unexpected results that indicated the ability of endothelial cells to offer a niche for a selective group of blood cells. The final question in the proposal was to test whether the modification of endothelial cells with the identified factors could induce the formation of blood from these cells. For this, we have generated specific reagents and are currently performing the final series of experiments.
  • In this grant application we have been able to investigate the mechanisms by which endothelial cells, the cells that line the inner aspects of the entire circulatory system, produce blood cells. This capacity, called “hemogenic” (giving rise to blood) can be extremely advantageous in pathological situations when generation of new blood cells are needed, such as during leukemia or in organ-transplantation. Although the hemogenic capacity of the endothelium is, under normal conditions, restricted development we have been able to “reprogram” this ability in endothelial cells. For this, we first investigated the genes that responsible for this hemogenic activity during development using mouse models and tissue culture cells. Using this strategy we found key transcription factors in hemogenic endothelium not present in other (non-hemogenic) endothelial cells. Subsequently, we validated that these genes were able to convey hemogenic capacity when expressed in non-hemogenic sites. Finally, using human endothelial cells, we have been able to impose expression of these key transcription factors in endothelial cells. Our data indicates that the forced expression of these factors is able to initiate a program that is likely to result in blood cell generation. The progress achieved through this grant place us in a remarkable position to carry out pre-clinical trials to evaluate the potential of this technology.

© 2013 California Institute for Regenerative Medicine