A Novel Microenvironment-Mediated Functional Skeletal Muscle from Human Embryonic Stem Cells and their In Vivo Engraftment

A Novel Microenvironment-Mediated Functional Skeletal Muscle from Human Embryonic Stem Cells and their In Vivo Engraftment

Funding Type: 
New Faculty II
Grant Number: 
RN2-00945
Award Value: 
$2,300,569
Disease Focus: 
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The overarching goal of this proposal is to develop robust clinically viable stem cell based therapeutics for muscle wasting focusing on Duchenne Muscular Dystrophy. Three specific aims were proposed to achieve this goal, and during the past year we have made significant progress towards this goal. The focus of aim one is to optimize the microenvironment factors comprising of cell-matrix interaction, cytokines, and cell-secreted morphogens on differentiation of stem cells into muscle cells. Stem cells require complex signals for differentiation and many of which are unknown, making it a challenge to direct tissue specific differentiation. We have adopted a stepwise differentiation strategy to promote myogenic differentiation of pluripotent stem cells (embryonic stem cells, ESCs and induced pluripotent stem cells, iPSCs). This involves differentiating the pluripotent stem cells into mesoderm progenitor cells followed by differentiating them into muscle cells. Our experimental findings show that our approach promotes efficient mesoderm differentiation of pluripotent stem cells. These extensively expandable ESC/iPSC-derived mesoderm progenitor cells are now being subjected to myogenic differentiation. We have also identified and optimized the combination of soluble factors that promote myogenic differentiation of muscle progenitor cells and stem cells. These soluble factors were found to promote myotube formation of muscle progenitor cells, in addition to promoting myogenic differentiation of stem cells. In an effort to understand the role of cell-matrix interactions, we have developed a novel method to decellularize the native skeletal muscle while maintaining their structural, biochemical composition, and mechanical properties unaltered. This approach has much more implications than supporting myogenic differentiation because it can be used as a model system to examine the changes ECM undergoes with pathological changes and how these changes affect stem cell myogenic differentiation and engraftment. The second aim of the proposed study is to test the hypothesis that a bioengineered niche exhibiting “excitation-contraction” dynamics encoded with biochemical cues can be used as a 3D microenvironment to promote myogenic differentiation of stem cells. The first goal of this aim is to develop a bioengineered synthetic niche recapitulating the biophysical cues of native skeletal muscle. To this end, we have developed synthetic hydrogel based biocompatible electro-mechanical matrices, which not only provide three-dimensional structural support to the embedded cells but also can simultaneously provide dynamic mechanical and electrical cues to the cells. A unique aspect of these matrices is that they can undergo reversible, anisotropic bending dynamics in an electric field and functions like multi-functional mechanical actuators. The direction and magnitude of this bending can be tuned through the hydrogel crosslink density while maintaining the same electric potential gradient, allowing precise control over the mechanical strain imparted to the cells in a three-dimensional environment. Our results showed that these bioengineered electro-mechanical niches not only support stem cell culture but also promotes their proliferation and differentiation. The third aim of the proposed study is on evaluating the engraftment and differentiation of in vitro conditioned stem cells using animal models. Based on the progress made in aims 1 and 2, we are certain that we will be able to initiate the animal studies soon. In sum, we have made significant progress in aims 1 and 2 of the proposed study. We have submitted one manuscript and three more manuscripts are under preparation. Another important achievement is training the researchers in stem cells. In addition to the postdoctoral and graduate fellows, we also trained undergraduate and high school students; Jomkuan Theprungsirikul from United World College, Montezuma, NM, had spent the summer in our lab to learn more about regenerative medicine and its potential in irradiating the public health problems. The PI visited Francis Parker School and gave a talk on stem cells on September 23 (Stem Cell Awareness Day). The PI also gave an invited talk on stem cell and regenerative medicine at the 3rd International Congress of NanoBiotechnology & Nanomedicine NanoBio 2009 held in San Francisco, CA.

Year 2

N/A

Year 3

During the reporting period, we successfully developed a protocol for deriving progenitor cells from human embryonic stem cells, which is being written up for a publication. The ESC-derived progenitor cells were found to undergo both myogenesis in vitro and in vivo. We were able to significantly expand these cells in vitro and the in vitro cultured cells expressed a number of muscle markers Pax3, Myf5, desmin, and MyoG. A muscle injury model was then used to investigate the in vivo viability and engraftment efficiency of these cells. A significant fraction of the transplanted cells were found to be engrafted. Additionally, we observed localization of a large number of transplanted cells in the centers of muscle fibers indicating the contribution of the transplanted cells towards the muscle regeneration. We have also developed a muscle-like synthetic material (termed as electromechanical material) that simultaneously provides mechanical and electrical cues to the embedded cells. A manuscript based on these results is published in Advanced Functional Materials, a highly regarded journal in interdisciplinary materials science. This work also received significant press coverage. The above-developed system will allow us determine the effect of various physicochemical cues of the matrix on myogenic commitment and maturation of progenitor cells.

Year 4

During the reporting period, we successfully developed a protocol for deriving progenitor cells from human embryonic stem cells, which is being written up for a publication. The ESC-derived progenitor cells were found to undergo both myogenesis in vitro and in vivo. We were able to significantly expand these cells in vitro and the in vitro cultured cells expressed a number of muscle markers Pax3, Myf5, desmin, and MyoG. A muscle injury model was then used to investigate the in vivo viability and engraftment efficiency of these cells. A significant fraction of the transplanted cells were found to be engrafted. Additionally, we observed localization of a large number of transplanted cells in the centers of muscle fibers indicating the contribution of the transplanted cells towards the muscle regeneration. We have also developed a muscle-like synthetic material (termed as electromechanical material) that simultaneously provides mechanical and electrical cues to the embedded cells. A manuscript based on these results is published in Advanced Functional Materials, a highly regarded journal in interdisciplinary materials science. This work also received significant press coverage. The above-developed system will allow us determine the effect of various physicochemical cues of the matrix on myogenic commitment and maturation of progenitor cells.

Year 5

We have developed a protocol devoid of genetic manipulations to derived progenitor cells with myogenic differentiation potential from human embryonic stem cells (hESCs). These hESC-derived cells underwent myogenic differentiation in vitro both in the presence and absence of serum in culture medium. When transplanted in vivo into an injured muscle, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. A manuscript describing these results have been published (Hwang Y, Suk S,Lin S, Tierney M, Du B, Seo T, Mitchell A, Sacco A, Varghese S. Directed in vitro myogenesis of human embryonic stem cells and their in vivo engraftment. PLoS One, 8, e72023 (2013). We have further modified the culture conditions to improve myogenic differentiation. When transplanted into injured muscles of immune-deficient NOD/SCID mice, a significant portion of fraction of the transplanted cells were found to be engrafted. Additionally, we observed localization of a large number of transplanted cells in the centers of muscle fibers indicating the contribution of the transplanted cells towards the muscle regeneration. Additionally, we also observed contribution of the transplanted donor cells towards the satellite compartment. The high proliferative capacity of hESCs along with their ability to differentiate into functional skeletal myogenic progenitor cells and engraft in vivo without teratoma formation highlights their potential therapeutic applications to ameliorate skeletal muscle defects and injuries.

Publications

© 2013 California Institute for Regenerative Medicine