Regenerative therapies require effective differentiation of stem cells to cell types that are functionally identical to those found in vivo. Many current differentiation protocols merely involve optimization of proteins added to the culture media, but do not consider the microenvironmental context in which cells differentiate during development or tissue repair. When we include the biophysical parameter of substrate stiffness, we are able to enhance differentiation of human embryonic stem cells to multipotent mesodermal progenitors, cells that can go on to form muscles, cartilage, and bone. We observe that this differentiation is accompanied by colony-wide organization and coordinated movements. Mesoderm forms during the developmental process known as gastrulation, and we also see aspects of this complex process recapitulated in our system. For example, we observe cells migrate and ingress into a region with similarities to the gastrulation-initiating structure called the primitive streak. We can therefore use this system to optimize directed differentiation protocols, to characterize and manipulate the forces and mechanisms required for coordinated differentiation, and to identify signals involved in primitive streak formation. Together, these studies will allows us to answer questions about the signals required for cell type specification and migration during spontaneous self-organization in the developing embryo.
Regenerative medicine requires efficient generation of cells that are identical to their respective population in the human body, but traditional protocols often lead to inefficient or incomplete directed differentiation. By optimizing biophysical parameters such as substrate stiffness and colony geometry, we show that we are able to efficiently differentiate human embryonic stem cells to mesoderm progenitors, indicating that we can use engineering principles to design more efficient directed differentiation strategies. In addition to providing tunable parameters for any type of differentiation, this system also allows us to probe the molecular basis of early mesoderm differentiation. This will enable us to gain valuable insight into how physical parameters regulate this process in vivo, which is crucial for establishing robust tissue regeneration techniques. In our system, mesoderm commitment is accompanied by cell movements and colony-wide organization representative of some aspects of early embryogenesis, which we will study in more detail to understand how biophysical forces initiate and reinforce key signaling pathways required during morphogenesis. By tracking and manipulating cells during these gastrulation-like movements, we will be able to identify relevant proteins and knock them down to mimic disease states with the ultimate goal of rectifying human embryologic defects and informing the future of regenerative medicine.
This Fundamental Mechanisms Award proposal is focused on a very early stage of embryonic development called gastrulation. At that stage, the epiblast, a uniform cell sheet, undergoes extensive cell movements and signaling interactions between cells to form the 3 germ layers; endoderm, mesoderm and ectoderm. The applicant has demonstrated that differentiation of human embryonic stem cells (hESC) into mesoderm is enhanced, and that cells undergo movements resembling those during gastrulation, when the stiffness of the surface they grow on is optimized. Using this system, the applicant proposes to investigate the relationship between these self-organizing movements and mesoderm formation and to interrogate whether and how the tensile forces exerted during cell movement promote mesoderm differentiation. This will be achieved by measuring and manipulating the forces and by identifying the molecular signals that mediate the effects of surface stiffness and tensile forces. The goal is to shed light on gastrulation events in human, and thereby help optimize methods for generating developmentally faithful cell types from hESC.
Significance and Innovation
- While a role for mechanical forces in differentiation has been appreciated in the field, this is an innovative and significant study to test how these forces regulate hESC differentiation.
- Reviewers appreciated the ability to model gastrulation using hESC and felt the proposed studies could therefore lead to new insights that can help improve differentiation protocols.
Feasibility and Experimental Design
- Overall, the proposed studies are logically designed; reviewers noted that the experiments to interrogate self-organizing movements were particularly well thought out.
- Reviewers pointed out technical limitations of some of the experiments, e.g. the ability to obtain meaningful and accurate mechanistic insights could be limited by the heterogeneous differentiation of the cell populations to be examined.
- The preliminary data presented establish motivation for the project and suggest feasibility.
- This project is technically very challenging and some of the required tools, while very interesting, still need to be generated. This raised concern regarding feasibility, but reviewers expressed confidence that useful information will be obtained in the course of the proposed studies.
Principal Investigator (PI) and Research Team
- The PI is a world leader in the field of mechanobiology and the cell microenvironment, and has assembled a group of highly skilled co-investigators that fully complement the PI’s expertise.
Responsiveness to the RFA
- The studies outlined in the proposal are responsive to the RFA.