Funding opportunities

Force, Dimensionality and Stem Cell Fate

Funding Type: 
SEED Grant
Grant Number: 
RS1-00449
Principle Investigator: 
Funds requested: 
$561 082
Funding Recommendations: 
Recommended if funds allow
Grant approved: 
Yes
Public Abstract: 
Human embryonic stem cells (hESCs) are cells derived from human embryos early in development before their fate has been sealed. These cells grow and differentiate in response to a variety of stimuli to eventually give rise to all of the differentiated tissues in the body. By exploiting the remarkable potential of hESCs to differentiate into multiple cell lineages, medicine stands to benefit enormously. To do so requires a comprehensive understanding of the optimal conditions to grow and differentiate these cells. What is known is that the physical environment in which hESCs reside plays an important role in regulating their tissue-specific differentiation. Recent work has highlighted the importance of the composition and structure of the extracellular matrix (ECM), within which hESCs exist in vivo, in directing hESC differentiation during embryonic development. In an embryo, hESCs differentiate in a dynamic and structurally distinct three-dimensional (3D) ECM, rich in nutrients and exogenous stimuli (force). Mechanical stimulation (via matrix compliance and externally applied force) dramatically influences the formation and development of the embryo. Despite these compelling observations, information regarding the mechanisms whereby matrix compliance and external force regulate hESC differentiation in 3D is extremely limited. Instead, the majority of the research on hESCs has been in two-dimensional (2D) culture on stiff plastic substrates, despite the lack of physiological relevancy. To address this issue, we will investigate the role of the ECM in 2D and 3D on hESC behavior using biomaterials with well-defined compositional and physical properties. We will assess the role of exogenous force by building a bioreactor designed to impart oscillatory compressive loading on hESCs cultured in 3D ECMs. We will test whether force modulates hESC fate by altering the function of the small RhoGTPase Rac. We will achieve this goal by: determining whether matrix compliance influences hESC differentiation in 2D and 3D and exploring the role of force on Rac activity and function, building a bioreactor capable of imparting controlled cyclic compressive loading to 3D hESC embedded in engineered biomaterial constructs, and by characterizing the effects of dynamic compression on hESC fate by manipulating the loading system. Because our appreciation and understanding of the mechanisms whereby matrix compliance and external force regulate hESC fate is extremely limited, this work would not likely be federally funded. These studies are essential to illustrate the critical role of matrix force in hESC fate and lay the foundation for future studies aimed at clarifying molecular mechanisms. The work will also assist in establishing defined, in vitro systems that more closely recapitulate the in vivo behavior of hESCs to permit their pluripotent propagation, and ensure their correct specification thereby ensuring the safe application of hESCs for human therapy.
Statement of Benefit to California: 
The growing worlds of human embryonic stem cell (hESC) science, bioengineering and regenerative medicine offer hope in the treatment of many diseases ranging from breast cancer to diabetes to Parkinson’s. Integral to the stem cell therapy treatment of these diseases is a fundamental understanding of the intricate mechanisms that govern stem cell growth, differentiation, maintenance and commitment. Much effort has concentrated on the role of exogenous biochemical supplementation to direct hESC differentiation and commitment. We seek to bridge the gap between the worlds of stem cell biology and bioengineering, adding an innovative approach to direct hESC lineage specification through the three-dimensional (3D) modulation of the extracellular matrix (ECM) microenvironment. We propose the building of a novel bioreactor to elucidate the roles that mechanical stimulation and the structure-function relationship of the ECM environment play in hESC differentiation and commitment in 3D. Through the development of this novel bioreactor, we will clarify and optimize parameters and mechanisms governing the growth, differentiation, maintenance and stability of hESCs that might otherwise go unnoticed with biochemical stimulation alone. These objectives are particularly relevant given the critical importance of establishing defined in vitro conditions in which embryos and ESCs can be derived and propagated with minimal contamination. Our studies also will have significance with regards to assisting investigators to improve their ability to rigorously maintain non-differentiated hESCs under conditions that more accurately recapitulate the in vivo situation, and thereafter aid in the generation of directed lineage specification of hESC differentiation. The latter point is particularly relevant because directed cell lineage specification should greatly reduce the potential for transplanted hESC to spawn terato-carcinomas upon in vivo transplantation. We envision that once our prototype Force Bioreactor has been generated and validated, the Scientific community will have access to our facilities and experimental approaches so that they will be able to apply similar methods to their basic and translational stem cell studies. We will facilitate this information and technology transfer through the active dissemination of our research findings, as well as via the establishment of the {REDACTED} of which the P.I. {REDACTED} has been appointed as Director. The approach in this proposal is both innovative and multidisciplinary, bridging together multiple genres of science and engineering. In today’s rapidly evolving world of research, the greatest impacts in stem cell research will be made by those willing to break out of traditional scientific paradigms, merging fields that will ultimately contribute solutions to the diseases that we face.
Review Summary: 
The underlying hypothesis of the work is that mechanical matrix force is a potent regulator of stem cell fate, specifically through small RhoGTPase Rac signaling. In this proposal the specific aims are to (1) Examine the growth and differentiation of HES into mesoderm, endoderm, ectoderm as a function of matrix compliance (stiffening) of the laminin substrate in two dimensions (2D); (2) Examine the role of matrix compliance in 3D on the RhoGTPase Rac signaling pathway using laminin-conjugated PEG gels; and (3) Build a bioreactor which can be used to precisely regulate loading and mechanical forces on contained HES cultures, to determine how these forces regulate cell fate. SIGNIFICANCE AND INNOVATION: The importance of ECM on hESC renewal and differentiation is well known. The importance of the 3D environment in some aspects of hESC culture (e.g. in EB) is also established. There is also no doubt, both microscopic (at cellular level) and macroscopic (at scale much greater than single cell) mechanical force will affect hESC behavior. However, the evidence is scant that macroscopic mechanical force (the subject of the proposed study) plays an important role in hESC proliferation, renewal or in lineage commitment aspect of differentiation. The innovation is high since the group has technical skills that are unique for building the bioreactor, and analyzing the outcome based on precisely defined imposed mechanical forces. The bioreactor will apparently be based on one constructed in the past by one of the co-investigators. STRENGTHS: -Strong expertise in biomechanical force and in employing 3D system as a model ECM -Aided by expert in hECS to the PI’s expertise -The results of investigation will definitely answer the question whether mechanical forces (in the physiological range) affect hESC behavior or not. The strength of the proposal is the team that has been brought together. Since the most important part of the proposal for the HES cell field is the construction of the bioreactor, that work should start earlier than outlined in the experimental timeline. -The bioreactor may find other applications. WEAKNESSES: The list of assays to be done is huge (and without paragraphs nearly impossible to read) and the grant needs a bit more focus. The idea of defining basic lineage determination and focusing on one or a few signaling pathways should help to guide the research. For the endodermal lineage identification the proposal is to stain for AFP and alpha-fetoprotein (?). It would have helped the proposal if the PI could state why the many bioreactors built to date are insufficient for the studies proposed. The dependence on novel biomaterials can have many unexpected downsides. Tissue engineering has been stymied by the unpredictable effects of materials used in easily manufactured environments. Nonetheless as a leader in this area, the choice of materials will be dictated by the local experience as well as that in the literature. • No prior experience in hESC. • PA gel, even conjugated with laminin, may not be appropriate for hECS. However, that’s the premise of the proposal. • The selection of force parameter range appears to be arbitrary. • The 3D culture method is sketchy. Will hESC survive the preparation condition? • Although the dynamic compression is interesting, the conditions to be tested are not necessarily physiologically relevant. DISCUSSION: One discussant pointed out that there was real mathematical analyses to be employed and that the investigator had picked the right system to use to apply such mathematical analysis. It was noted that the preliminary data from the lab is compelling and that force is likely mediated through RhoGtPase Rac, given compelling data from other labs. Also noted that laminin in 2d, 3D matrices is extremely morphogenic. In sum, this proposal represents mathematical strength, coupled with good matrix cell biology with proven effect on hESCs.
Conflicts: 

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