UC Berkeley

One of the goals of tissue engineering is to create technologies that will improve or replace biological function of diseased or damaged cells and tissues. The purpose of my thesis work is to determine how the mechanical properties of the murine microenvironment, specifically matrix stiffness, can affect the function and behavior of cells and tissues. Previous research has shown that stiffness is a powerful mechanical property; it is associated with breast and liver cancer, and can also be used to control stem cell differentiation. In order to create an optimal microenvironment for the generation of new cells and tissues, it is essential that we further investigate how matrix stiffness controls cells and tissue behavior. The motivation for this thesis work is twofold; the first is to find an improved system and cell source for the generation of hematopoietic progenitors and stem cells, and the second is to characterize better technologies for tissue-engineered vascular grafts.

The first chapter of this thesis work investigates how to create an in vitro microenvironment that can be used to study matrix stiffness. I then examine how to use the system to create mESC-derived Flk-1⁺ hemangioblasts, which are precursor cells for the hematopoietic, cardiac and vascular lineages. Both natural and synthetic hydrogel systems are examined using three different techniques for measuring substrate elastic modulus: a comparator, a rheometer and an atomic force microscope (AFM). The data shows that polyacrylamide and AFM are the most appropriate systems for this work. Cell spreading studies demonstrate the morphological differences of cells on various stiffnesses. Short-term studies show that mESC differentiation is dependent on both matrix stiffness and the extracellular matrix protein that is used to attach the cells to the surface, but is not affected by the addition of certain growth factors (i.e. TGF-β, PDGF-BB) in culture media. In addition, the data shows that the effect of matrix stiffness on cell fate may be determined within 3 days of seeding on a particular surface, suggesting that mESCs can "remember" the effects of their mechanical microenvironment for a short period of time.

The second part of this work looks at how matrix stiffness affects the differentiation of mESC-derived Flk-1⁺ hemangioblasts into the hematopoietic, vascular and cardiac lineages. The results demonstrate that matrix stiffness has a bimodal effect on the derivation of Flk-1⁺ hemangioblasts from mESCs, with a medium stiffness (~43kPa) generating the highest quantity. Data from long-term studies shows that cells grown on softer surfaces (~0.4kPa) generate more hematopoietic progenitor cells, whereas cells on stiffer surfaces (~43kPa to 1GPa) derive more vascular-like cells. The study also suggests that softer surfaces may also be more appropriate for cardiac cell generation, though this may be because softer surfaces encourage cells to grow more in a three-dimensional conformation, which is known to contribute to cardiac cell development.

The last part of the work focuses on the effects of matrix stiffness on a more macro scale, by examining the stiffness of embryonic and adult tissues. For the embryonic tissues, the goal is to measure and correlate the matrix stiffness of the blood-deriving organs to those used in the in vitro studies in order to create a better system for studying the generation of hematopoietic stem cells and progenitors. For the adult tissue studies, tissue-engineering vascular grafts supported with either collagen I hydrogels supplemented with or without TGF- β, or Matrigel hydrogels are implanted into rats for 3 months so as to better simulate the mechanical properties of healthy vascular tissue. The results show that TGF- β increases the stiffness of the tissue surrounding the vascular graft, resulting in elastic moduli similar to that of native healthy tissue. The Matrigel hydrogels only moderately increase the stiffness of the surrounding tissue and may be too weak to be a viable option for tissue-engineered vascular grafts.

This work demonstrates the importance of matrix stiffness when designing systems to control cell fate and for tissue regenerative purposes. On a cellular level, although chemical factors can regulate stem cell differentiation to a certain extent, the surrounding physical environment can also influence stem cell differentiation, specifically into the hemangioblast-related fate. On a tissue level, researchers may be able to better mimic vascular tissue by manipulating the stiffness and composition of tissue-engineered grafts, thus helping the patency of these grafts. Future considerations for this work would include the addition of in vivo analyses for the mESC differentiation studies, as well as using other types of hydrogel systems, such as those made from biocompatible materials. Research should also be conducted using induced pluripotent stem cells to eliminate ethical concerns and the possibility of rejection by the host. For the tissue mechanics studies, there is still a need for an accurate, comprehensive system for measuring embryonic tissue in vivo. These mechanotransduction experiments are important for the foundation of creating better microenvironments for tissue engineering applications.