Growth and Development Lab

Please see the sub-sites of the Growth and Development Lab above for more information.

Intro to the Lab

Dr. Johnny Huard became the director of the Growth & Development Laboratory in 1996. Since that time, the laboratory has more than tripled in size and now employs more than 45 diverse professionals, including basic scientists, residents, post-doctoral fellows, graduate students, and administrative staff. The laboratory generated more than 10 peer-reviewed manuscripts, and presented 26 scientific meeting abstracts during the year of 2006. Most recently, at the Orthopaedic Research Society’s Annual meeting, members of the SCRC presented 23 posters, 6 of which were New Investigator Research Award (NIRA) nominees, and two of which were NIRA winners. Dr. Huard’s work is currently supported by 7 National Institutes of Health grants, 2 Department of Defense grants, 2 Foundation grants and 3 endowed chairs which total more than $2.4 million annually.

Major Research Interests

• Stem cell isolation and characterization of cells from skeletal muscle, fat and blood vessels.
• Alleviation of the muscular degeneration associated with DMD through stem cell transplantation and genetic engineering.
• Bone and cartilage growth, repair, regeneration, and regulation through stem cell transplantation.
• Cardiac and skeletal muscle injury repair, regeneration, and fibrosis prevention.
• Alleviation of urological dysfunction through stem cell transplantation (done in collaboration with Dr. Michael Chancellor, Department of Urology).
• Stem cell manipulation via genetic and tissue engineering techniques with the aim of enhancing proficiency for regenerating nerve and spinal cord following injury.

One of Dr. Huard’s primary areas of interest involves gene therapy for skeletal muscle injury, and, especially, how such research relates to DMD therapies—an area for which his discoveries have received national and international recognition.

Current Research

Muscle regeneration through stem cell transplantation:

(Johnny Huard, Ph.D.: Principal Investigator)
Intensive efforts have been made to develop an effective therapy for DMD. Although myoblast transplantation has been found capable of transiently delivering dystrophin and improving the strength of injected dystrophic muscle, the approach has been hindered by numerous limitations, including immune rejection, poor cellular survival rates, and limited dissemination of the injected cells. Using the pre-plate technique, we have isolated 3 populations of myogenic cells from normal mouse skeletal muscle on the basis of cellular adhesion characteristics and proliferation behavior. Although 2 of these populations displayed satellite cell characteristics, the third population contained long-term proliferating (LTP) cells expressing hematopoietic stem cell markers. These LTP cells retain their phenotype for more than 30 passages and can differentiate into various lineages, including muscle, neural, osteogenic, and endothelial. More importantly, the transplantation of the LTP cells, in contrast to satellite cell transplantation, significantly improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. We therefore believe that the LTP cells represent a novel population of muscle-derived stem cells (MDSCs) that will significantly improve muscle cell mediated therapies. The overall goal of this research is to determine whether transplanted MDSCs can deliver dystrophin and lead to improvements in both the structure and function of dystrophic muscle. The present research seeks to further investigate the ability of these MDSCs to ameliorate the function of dystrophic muscle. We are also performing experiments to determine whether the isolation method or the age and condition of the skeletal muscle source influence the cells’ biological properties. In addition, we are working to evaluate the relative importance of MDSC survival, self-renewal, and pluripotent behavior on the improved transplantation capacity of these cells. Finally, we are investigating the use of systemic delivery of MDSCs to achieve dystrophin delivery and improve function in dystrophic muscle that is inaccessible via intramuscular injection. This project continues to increase our understanding of myogenic cell populations that display stem cell characteristics and may open new alternatives to improve muscle regeneration in dystrophic muscle via transplantation of MDSCs.

This study was initially funded through the Muscular Dystrophy Association of the USA in 2000 for a period of two years. This study has been funded since 2002 by a five-year grant from the National Institutes of Health (1 R01 AR049684-02). A competitive renewal was sent for this grant February 2006.

Muscle-based tissue engineering to improve bone healing:

(Johnny Huard, Ph.D.: Principal Investigator; Bruno Péault, Ph.D.: Co-Investigator)
Segmental bone defects and non-unions are relatively common in the craniofacial skeleton. Osteogenic proteins, including bone morphogenetic protein-2 and 4 (BMP-2,4), can promote bone healing in segmental bone defects, but the osteogenic proteins’ short half-lives and rapid clearance by the bloodstream limit the success of this technique. Gene therapy and tissue engineering approaches that result in high expression levels of these proteins may help to further improve craniofacial bone healing. Our recently isolated clonal population of muscle-derived stem cells (mc13 cells) that express stem cell markers, differentiate into both myogenic and osteogenic lineages, and, more importantly, improve bone healing in a calvarial bone defect. This may make them ideal as a cell source with which to mediate gene transfer of osteogenic proteins. The long-term goal of this research is the development of gene therapy approaches based on this novel population of muscle-derived stem cells to efficiently deliver osteogenic proteins and improve craniofacial bone healing. We are testing and comparing the mechanisms by which these muscle-derived stem cells differentiate into osteogenic lineages upon stimulation with BMP-2 and BMP-4. In addition, we are characterizing various approaches to muscle-based tissue engineering that combine the use of muscle-derived stem cells for ex vivo gene transfer of osteogenic proteins with a scaffold to improve bone healing in a mouse calvarial defect. We continue to investigate the persistence of osteogenic protein expression, the presence of immune responses or undesirable side effects related to the overexpression of these proteins, and the biological effects of the proteins on fracture healing. We also are examining the use of vascular endothelial growth factor (VEGF), a well-known angiogenic factor, to improve bone healing.Although this research is focused on muscle-based tissue engineering to regenerate a calvarial defect, this technique will ultimately be applied to other craniofacial sites and to appendicular bones. This research has enhanced and expanded our knowledge of bone healing and will prove useful to researchers working to develop a clinically relevant molecular therapy approach to treat osseous deficiencies.

This study has been funded since 2001 by a five-year grant from the National Institutes of Health (5 R01 DE13420-03, and 2 R01 DE13420-06). The competitive renewal for this grant has been awarded and will begin July 1, 2006 for an additional 5 years.

Gene and cell therapy of Duchenne muscular dystrophy – Muscle stem cell therapies for cardiac repair:

(Joseph C. Glorioso III, Ph.D.: Principal Investigator; Johnny Huard, Ph.D.: Co-PI)
The long-term goal of this project is to investigate the use of muscle-derived stem cells as a novel cell source for cardiac cell transplantation in a cardiomyopathic murine model of muscular dystrophy.

Cardiomyopathy is a serious heart disease that often leads to congestive heart failure, a condition in which the heart muscle can no longer effectively pump blood. Patients that suffer from various muscle diseases, including DMD, develop progressive cardiomyopathy. Cellular cardiomyoplasty (CCM), a procedure that involves the transplantation of exogenous cells into damaged myocardium, has been proposed as a possible therapy to regenerate diseased myocardium and deliver therapeutic genes. Although a wide variety of cell types has been used for CCM, various limitations (including ethical, biological, or technical challenges) have impeded their suitability for use in human patients. We have used the modified pre-plate technique to isolate a novel population of muscle-derived stem cells (MDSCs) that display improved transplantation capacity in skeletal muscle when compared to satellite cells. The MDSCs’ ability to proliferate in vivo for an extended period of time—combined with their strong capacity for self-renewal, multipotent differentiation, and immune-privileged behavior—reveals at least a partial basis for the benefits associated with their use in cell transplantation in skeletal muscle. This project is designed to investigate the use of MDSCs as a novel cell source for cardiac cell transplantation in a cardiomyopathic murine model of muscular dystrophy. We already have observed that MDSCs delivered by intracardiac injection display good cell survival and can deliver dystrophin within the dystrophic myocardium. We are investigating whether MDSCs implanted into the hearts of dystrophic mice display an improved transplantation capacity when compared with conventional satellite cell implantation. We also plan to explore the relative contribution of the MDSCs’ capacity for long-term proliferation and self-renewal to the increased regeneration capacity of these cells after transplantation into heart muscle. Finally, we are working to determine the degree to which development of approaches to prevent fibrosis and improve angiogenesis would further enhance the regenerative capacity of muscle-derived cells in the heart. This project will increase our understanding of the basic biology of myogenic cell populations that display stem cell characteristics. This information may, in turn, reveal new techniques to improve heart regeneration and repair via the transplantation of muscle-derived stem cells.

This study has been funded since September, 2003 by a five-year grant from the National Institutes of Health (1 U54 AR050733-03).

Biological approaches to improve muscle healing:

(Johnny Huard, Ph.D.: Principal Investigator)
Muscle injuries are common and disabling. The inefficiency of the regeneration initiated in muscle shortly after injury is largely due to fibrosis. Our research has shown that transforming growth factor beta-1 (TGF-β1), plays a key role in skeletal muscle fibrosis, and that the use of antifibrotic agents that inactivate TGF-β1 can reduce muscle fibrosis and significantly improve muscle healing after injury. We have identified several agents that block the effect of TGF-β1. Based on our preliminary data, however, we suspect that decorin also directly enhances muscle regeneration via other mechanisms. Therefore, this grant application outlines gain- and loss-of-function experiments designed to further evaluate the beneficial effect of decorin on muscle regeneration after laceration injury. We will determine whether decorin promotes muscle regeneration by inducing up-regulation of myogenic genes p21, and PGC-1 a ; molecules involved in the muscle regeneration process. Research has shown that decorin influences macrophages, and that anti-inflammatory drugs delay muscle healing; therefore, we will also test whether decorin promotes muscle regeneration/healing by influencing the inflammatory phase (Specific Aim 1). Our recent findings indicate that decorin blocks the inhibitory effects of myostatin (MSTN), a well-known muscle growth regulator, on muscle cell differentiation through a potential down-regulation of MSTN and/or up-regulation of follistatin (FLST), a well-known antagonist of MSTN. We therefore posit that the beneficial effect of decorin on muscle regeneration/healing is mediated through MSTN and/or FLST. We propose to conduct a series of experiments involving various transgenic/knockout mouse models that express different levels of decorin, MSTN, and FLST, to determine whether any interaction exists between these molecules during muscle regeneration and repair after injury (Specific Aim 2). Because it has been observed that decorin deficiency leads to impaired angiogenesis in other tissues, we will investigate the influence of decorin on the vascular supply in injured skeletal muscle (Specific Aim 3). If angiogenesis is involved in decorin’s beneficial effect on muscle healing, we will test whether exposure to neuromuscular electrical stimulation, a clinically available therapeutic modality, can be used as a strategy to enhance decorin expression and angiogenesis in the injured skeletal muscle. The experiments described in this application should not only reveal the mechanism(s) by which decorin regulates different phases of muscle healing, but could also lead to the development of new therapeutic approaches and rehabilitation strategies for promoting the healing of injured or diseased skeletal muscles.

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