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.
Reporting Period:
Year 2
N/A
Reporting Period:
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.
Reporting Period:
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.
Reporting Period:
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.
Reporting Period:
NCE Year 6
The financial support from the CIRM has enabled us to develop an effective protocol for deriving progenitor cells from humane embryonic stem cells. A manuscript describing these results have been published in PLoS One . The ESC-derived cells wee find to undergo myogenic differentiation both in vitro and in vivo. The differentiated cells were positive for Pax3, Myf5, design, and MyoG. We also demonstrated that the presence of WNT3A in culture significantly promoted the myogenic commitment of these hESC-derived progenitor cells. In vivo transplantation of these committed cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice reveals survival and engraftment of the donor cells. The cells contributed to the regeneration of damaged muscle fibers and the satellite cell compartment. In lieu of the limited cell source for treating skeletal muscle defects, the hESC-derived PDGFRA(+) cells exhibit significant in vitro expansion while maintaining their myogenic potential. The results described in this study provide a proof-of-principle that myogenic progenitor cells with in vivo engraftment potential can be derived from hESCs without genetic manipulation. A manuscript based on these results is published in Nature Scientific Reports. During the course of these studies we have also developed a biomaterials assisted cell delivery approach to improve the survival, continued differentiation, and contribution of the transplanted cells to muscle tissue repair. Our findings show that the biomimetic material-assisted delivery of hESC-derived myogenic progenitor cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice significantly promotes survival and engraftment of transplanted cells in a dose-dependent manner (>200 fold improvement). A manuscript describing the results have been published recently in ACS Biomaterials.
Grant Application Details
Application Title:
A Novel Microenvironment-Mediated Functional Skeletal Muscle from Human Embryonic Stem Cells and their In Vivo Engraftment
Public Abstract:
Muscle wasting is a serious clinical problem associated with a number of diseases and health conditions, affecting individuals of all ages. Muscular dystrophy (MD) is a form of muscle wasting disease resulting from genetic mutations. Duchenne muscular dystrophy (DMD) is the most common form of MD that limits motility and life expectancy of children. It is characterized by progressive skeletal muscle degeneration, and occurs in 1 out of every 3,500 male births. Currently, there is no effective treatment to stop or reverse DMD. One of the potential clinical solutions for treating DMD is cell transplantation where the implanted cells contribute to the functional muscle regeneration. Adult stem cells such as mesoangioblasts and AC133 cells have already been shown to ameliorate dystrophic muscle pathology in animals. Although embryonic stem cells (ESCs) can provide unlimited numbers of progenitor cells with the ability to contribute to the formations of functional skeletal muscle, their ability to reverse/repair wasting muscle has not been explored in detail. To this end, we seek to develop an ESC-based cell transplantation therapy for treating muscle-wasting pathology, focusing on DMD. Three specific aims are proposed to achieve this overall goal. (i) Optimize the microenvironment factors comprising of cell-matrix interaction, cytokines, and cell-secreted morphogens on differentiation of ESC into muscle cells using a combinatorial array technology. (ii) Derive and characterize a population of clinically viable ESC-derived muscle progenitor cells using a dynamic, three-dimensional (3D), bioengineered niche. The tailored niche would be comprised of a multifunctional hydrogel that exhibits “excitation-contraction” dynamics. We will then functionalize this hydrogel with optimized ECM components and use it to investigate myogenic differentiation of ESCs in a 3D environment, and to derive engraftable muscle progenitor cells from ESCs. (iii) Investigate the therapeutic efficacy of ESC-derived progenitors for treating DMD pathology using animal models. The molecular pathways involved in myogenic differentiation of ESCs will be analyzed using proteomics and transcriptomics tools. Successful completion of this study could offer broad applicability of stem-cell-based therapies for treating all types of muscle wasting diseases and other pathologies associated with dysfunctional muscles. Unraveling the microenvironment factors and molecular events determining the commitment of ESCs into specific lineages will significantly increase our understanding of stem cell biology and their application in regenerative medicine. In addition, the multifunctional hydrogel (mechanical actuator) developed here could find many applications in the field of artificial functional implants where a muscle-like response is desirable, cardiac tissue repair, drug delivery, and biomimetic devices.
Statement of Benefit to California:
The proposed study seeks to develop a clinically viable cell transplantation therapy based on pluripotent embryonic stem cells (ESCs) for treating muscular dystrophies (MDs), especially Duchenne muscular dystrophy (DMD). DMD is the most common form of MD that limits motility and shortens life expectancy of children, and is estimated to occur 1 in 3500 male births. DMD is characterized by progressive muscle wasting, limited ambulation, compromised lung or cardiac function, paralysis, and ultimately premature death. DMDfund (http://www.dmdfund.org), a California-based forum dedicated to families of children affected by DMD, emphasizes the devastating nature of this disease “... in contrast to cancer, DMD currently is uniformly fatal. As a result there is only a 3-fold difference in the number of childhood deaths due to cancer vs. DMD, but DMD research is relatively scarce. The paucity of research may be the reason that there is no cure for this devastating neuromuscular disease. Without research, there is no hope. Shouldn't all people at least been given the chance to hope?” Clearly, there is a need to develop new paradigms for treating DMD. The PI has proposed one possible approach to address this need and it will contribute to the State of California and its citizens in the many ways: Clinical benefits: Successful completion of the project will help develop new stem-cell-based therapies for treating DMD and other muscle wasting diseases. Unraveling the microenvironment factors and molecular events that determine the commitment of ESCs will significantly increase their treatment potential. Concepts and knowledge gained from the study can translate into other areas of regenerative medicine. Economic benefits: There are two ways the proposed work could contribute to the economy of California. First, improvements in DMD treatment could bring down the healthcare costs. Second, tools and technologies developed during the course of this study (soft mechanical actuators, smart biomimetic hydrogels, etc.) can find various other applications in biomedical fields such as artificial functional implants, cardiac tissue repair, drug delivery, and biomimetic devices. Such devices could be of great interest to California-based biotechnology companies. The PI’s past work involving musculoskeletal tissues has contributed to a start-up biotech company located in California, and the PI hopes to contribute similarly through the proposed studies. Education, training, and awareness: One of the most important components of the PI’s laboratory is training future leaders in the biomedical/stem cell field. We expect to train 10-12 researchers within the course of this project (clinical fellows, postdoctoral fellows, graduate students, undergraduate students, and high school students). These very students and trainees will, in the future, lead to the development of newer and more advanced strategies for regenerative medicine.
