Public Abstract:
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and recently developed human induced pluripotent stem cells (iPSCs), hold great promise as attractive cell sources for tissue regeneration. Unlike other types of cells, hPSCs can self-renew indefinitely and possess the potential to differentiate into any type of cells in our body. Before hPSCs can be used for therapeutic purposes, methods must be developed to control their differentiation into functional mature cell types. Stem cells reside in a highly complex niche in vivo where they constantly respond to microenvironmental cues including soluble factors, extracellular matrix, adjacent cells and mechanical signals. To fully realize the therapeutic potential of hPSCs, it is critical to understand the mechanisms by which they receive information from microenvironment and how such interactions alter hPSC functions.
While the effect of individual type of microenvironmental cues on stem cell behavior has been studied in great depth, little is known about how the complex interplay of multiple types of microenvironmental cues would influence stem cell behavior. In addition, conventional iterative approach typically requires large amounts of cells and materials, and is slow an inefficient in discovery. To address these limitations, high-throughput screening has emerged as a novel approach to achieve rapid discovery with reduced materials and costs. However, most high-throughput studies on cell-material interactions to date have been performed on two-dimensional environments, while the architecture of the stem cell niche itself is three-dimensional. Recent research have clearly emphasized dimensionality as a critical determinant for regulating cell behavior, and systematic evaluation of stem cell responses to complex 3D signaling environment remains challenging.
Through working at the interface of biology, material science, and engineering, here we propose to develop novel 3D combinatorial systems to understand how microenvironmental signals influences stem cells fate decision in 3D, and to rapidly optimize stem cell niche using high-throughput strategies. Such studies can greatly accelerate the clinical applications of hPSCs by elucidating the mechanisms underlying the control of hPSC differentiation. The outcome of proposed work can also aid in the synthesis of culture microenvironments that emulate stem cell niche in vivo, and would have broad applications in areas such as tissue regeneration and drug delivery.
Statement of Benefit to California:
California is the most populated State in the US and many Californians suffer from diseases and injuries that lead to tissue loss and organ failure. With the rise of average life expectancy in our population, the number of Californians that suffer from devastating diseases will continue to increase. Human pluripotent stem cells (hPSCs) represent a promising candidate as cell sources for tissue repair and regenerative medicine. However, before they can be broadly used for therapeutic purposes, methods must be developed to control their differentiation into functional mature cell types. This proposal aims to elucidate the fundamental mechanisms by which hPSCs respond to the complex microenvironmental cues, and the outcomes of the proposed work will greatly accelerate the clinical translation of hPSCs for treating many Californian patients. Furthermore, the discovery from the proposed work will strengthen the leadership role of California in stem cell research. Our findings could also provide outstanding opportunities to stimulate the growth of biotechnology and pharmaceutical industries within the State as well as creating new job opportunities.
Review Summary:
EXECUTIVE SUMMARY
Stem cells reside in a highly complex niche in vivo where they must respond to cues from the microenvironment. This application proposes the development of a high-throughput approach to examine the effects of artificially constructed, three dimensional (3D) microenvironments on human pluripotent stem cells (hPSCs). Aim 1 develops a 3D combinatorial hydrogel microarray for hPSC encapsulation with a broad range of biochemical compositions and mechanical properties. Aim 2 identifies optimized combinatorial signaling environments for hPSC lineage-specific differentiation using a high-throughput assay. Aim 3 examines the interactive signaling of integrin and soluble growth factors on hPSC differentiation in a 3D microenvironment.
The reviewers acknowledged the potential significance and innovation of this proposal. The challenge of transitioning studies from a two-dimensional to a three-dimensional assessment of microenvironments was recognized as extremely important for understanding properties of the stem cell niche. Furthermore, the proposed study focuses on the interface of biology, materials science, and engineering and could provide new insights into the complex interplay of microenvironmental signals with hPSC fate. The investigation could have a major impact on potential applications of stem cell research and regenerative medicine.
Reviewers raised significant concerns about the proposal’s feasibility and experimental design. None of the aims were based on specific hypotheses, and the project was viewed as primarily an optimization study, not rooted in fundamental biological principles or addressing key scientific questions. Although reviewers were impressed by the preliminary data and felt that some experiments would successfully yield results, the relevance of predicted findings to real biological systems and the potential for these studies to yield novel information useful in future mechanistic studies were viewed as severely limited. Prospects for the project’s feasibility were further diminished by the lack of adequate information about planned computational analysis and the omission of plans for non-linear analysis of results. Additional concerns were raised about the rationale for modulating the delivery of soluble growth factors and the ability to recover cells from the 3D matrix, a prerequisite for appropriate cellular analysis.
Although the PI is a newly independent investigator, the review panel praised the applicant’s solid publication record, excellent training, and experience with biomaterials research. There was some concern as to whether the research team had sufficient members and depth to accomplish the proposed project. The research environment was viewed as exceptional.
In summary, this application uses a high-throughput approach to examine the effects of 3D in vitro microenvironments on hPSCs. Strengths of the proposal include the significance and innovation of the study and the PI’s impressive qualifications. Weaknesses include serious concerns regarding the lack of a unifying hypothesis, the project’s apparent prematurity, and deficiencies in feasibility and experimental design.
