Burhan Gharaibeh

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.

Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle-derived stem cells expressing bone morphogenetic protein 4 and soluble Flt-1

OBJECTIVE The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt-1 (sFlt-1), on the chondrogenesis of skeletal muscle-derived stem cells (MDSCs) in a rat model of osteoarthritis (OA). METHODS We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP-4) in combination with MDSCs expressing VEGF or sFlt-1. RESULTS In vivo, a combination of sFlt-1- and BMP-4-transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt-1- and BMP-4-transduced MDSCs compared with a combination of VEGF- and BMP-4-transduced MDSCs or with BMP-4-transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP-4-transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes. CONCLUSION Our results demonstrate that MDSC-based therapy involving sFlt-1 and BMP-4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

Pericyte-based human tissue engineered vascular grafts.

The success of small-diameter tissue engineered vascular grafts (TEVGs) greatly relies on an appropriate cell source and an efficient cellular delivery and carrier system. Pericytes have recently been shown to express mesenchymal stem cell features. Their relative availability and multipotentiality make them a promising candidate for TEVG applications. The objective of this study was to incorporate pericytes into a biodegradable scaffold rapidly, densely and efficiently, and to assess the efficacy of the pericyte-seeded scaffold in vivo. Bi-layered elastomeric poly(ester-urethane)urea scaffolds (length = 10 mm; inner diameter = 1.3 mm) were bulk seeded with 3 x 10(6) pericytes using a customized rotational vacuum seeding device in less than 2 min (seeding efficiency > 90%). The seeded scaffolds were cultured in spinner flasks for 2 days and then implanted into Lewis rats as aortic interposition grafts for 8 weeks. Results showed pericytes populated the porous layer of the scaffolds evenly and maintained their original phenotype after the dynamic culture. After implantation, pericyte-seeded TEVGs showed a significant higher patency rate than the unseeded control: 100% versus 38% (p < 0.05). Patent pericyte-seeded TEVGs revealed extensive tissue remodeling with collagen and elastin present. The remodeled tissue consisted of multiple layers of alpha-smooth muscle actin- and calponin-positive cells, and a von Willebrand factor-positive monolayer in the lumen. These results demonstrate the feasibility of a pericyte-based TEVG and suggest that the pericytes play a role in maintaining patency of the TEVG as an arterial conduit.

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells.

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative nonmyocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft–host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.

The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair

OBJECTIVE Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

Biologic approaches to enhance rotator cuff healing after injury.

BACKGROUND Despite the advances in surgical procedures to repair the rotator cuff, there is a high incidence of failure. Biologic approaches, such as growth factor delivery and stem cell and gene therapy, are potential targets for optimization to improve the outcome of rotator cuff therapies and reduce rates of reinjury. This article outlines the current evidence for growth factor and stem cell therapy in tendon healing and the augmentation of rotator cuff repair. METHODS Literature on the PubMed-National Center for Biotechnology Information database was searched using the keywords growth factor, factor, gene therapy, stem cell, mesenchymal, or bone marrow in combination with rotator cuff, supraspinatus, or infraspinatus. Articles that studied growth factors or stem cells alone in rotator cuff repair were selected. Only 3 records showed use of stem cells in rotator cuff repair; thus, we expanded our search to include selected studies on stem cells and Achilles or patellar tendon repairs. Bibliographies and proceedings of meetings were searched to include additional applicable studies. We also included hitherto unpublished data by our group on the use of stem cell transplantation for rotator cuff therapy. RESULTS More than 70 articles are summarized, with focus on recent original research papers and significant reviews that summarized earlier records. CONCLUSIONS Use of growth factors, stem cell therapy, and other tissue-engineering means serve to augment classical surgical rotator cuff repair procedures. The combination of stem cells and growth factors resulted in enhanced repair that emulated uninjured tissue, but the literature search reflected paucity of research in this field. Preclinical evidence from gene therapy and stem cell studies can be used as a start to move therapy from the experimental phase to clinical translation in patients.

Follistatin Improves Skeletal Muscle Healing after Injury and Disease through an Interaction with Muscle Regeneration, Angiogenesis, and Fibrosis

Recovery from skeletal muscle injury is often incomplete because of the formation of fibrosis and inadequate myofiber regeneration; therefore, injured muscle could benefit significantly from therapies that both stimulate muscle regeneration and inhibit fibrosis. To this end, we focused on blocking myostatin, a member of the transforming growth factor-β superfamily and a negative regulator of muscle regeneration, with the myostatin antagonist follistatin. In vivo, follistatin-overexpressing transgenic mice underwent significantly greater myofiber regeneration and had less fibrosis formation compared with wild-type mice after skeletal muscle injury. Follistatins mode of action is likely due to its ability to block myostatin and enhance neovacularization. Furthermore, muscle progenitor cells isolated from follistatin-overexpressing mice were significantly superior to muscle progenitors isolated from wild-type mice at regenerating dystrophin-positive myofibers when transplanted into the skeletal muscle of dystrophic mdx/severe combined immunodeficiency mice. In vitro, follistatin stimulated myoblasts to express MyoD, Myf5, and myogenin, which are myogenic transcription factors that promote myogenic differentiation. Moreover, follistatins ability to enhance muscle differentiation is at least partially due to its ability to block myostatin, activin A, and transforming growth factor-β1, all of which are negative regulators of muscle cell differentiation. The findings of this study suggest that follistatin is a promising agent for improving skeletal muscle healing after injury and muscle diseases, such as the muscular dystrophies.

Biological approaches to improve skeletal muscle healing after injury and disease

Skeletal muscle injury and repair are complex processes, including well-coordinated steps of degeneration, inflammation, regeneration, and fibrosis. We have reviewed the recent literature including studies by our group that describe how to modulate the processes of skeletal muscle repair and regeneration. Antiinflammatory drugs that target cyclooxygenase-2 were found to hamper the skeletal muscle repair process. Muscle regeneration phase can be aided by growth factors, including insulin-like growth factor-1 and nerve growth factor, but these factors are typically short-lived, and thus more effective methods of delivery are needed. Skeletal muscle damage caused by traumatic injury or genetic diseases can benefit from cell therapy; however, the majority of transplanted muscle cells (myoblasts) are unable to survive the immune response and hypoxic conditions. Our group has isolated neonatal skeletal muscle derived stem cells (MDSCs) that appear to repair muscle tissue in a more effective manner than myoblasts, most likely due to their better resistance to oxidative stress. Enhancing antioxidant levels of MDSCs led to improved regenerative potential. It is becoming increasingly clear that stem cells tissue repair by direct differentiation and paracrine effects leading to neovascularization of injured site and chemoattraction of host cells. The factors invoked in paracrine action are still under investigation. Our group has found that angiotensin II receptor blocker (losartan) significantly reduces fibrotic tissue formation and improves repair of murine injured muscle. Based on these data, we have conducted a case study on two hamstring injury patients and found that losartan treatment was well tolerated and possibly improved recovery time. We believe this medication holds great promise to optimize muscle repair in humans.

The use of blood vessel-derived stem cells for meniscal regeneration and repair.

PURPOSE Surgical repairs of tears in the vascular region of the meniscus usually heal better than repairs performed in the avascular region; thus, we hypothesized that this region might possess a richer supply of vascular-derived stem cells than the avascular region. METHODS In this study, we analyzed 6 menisci extracted from aborted human fetuses and 12 human lateral menisci extracted from adult human subjects undergoing total knee arthroplasty. Menisci were immunostained for CD34 (a stem cell marker) and CD146 (a pericyte marker) in situ, whereas other menisci were dissected into two regions (peripheral and inner) and used to isolate meniscus-derived cells by flow cytometry. Cell populations expressing CD34 and CD146 were tested for their multilineage differentiation potentials, including chondrogenic, osteogenic, and adipogenic lineages. Fetal peripheral meniscus cells were transplanted by intracapsular injection into the knee joints of an athymic rat meniscal tear model. Rat menisci were extracted and histologically evaluated after 4 wk posttransplantation. RESULTS Immunohistochemistry and flow cytometric analyses demonstrated that a higher number of CD34- and CD146-positive cells were found in the peripheral region compared with the inner region. The CD34- and CD146-positive cells isolated from the vascular region of both fetal and adult menisci demonstrated multilineage differentiation capacities and were more potent than cells isolated from the inner (avascular) region. Fetal CD34- and CD146-positive cells transplanted into the athymic rat knee joint were recruited into the meniscal tear sites and contributed to meniscus repair. CONCLUSIONS The vascularized region of the meniscus contains more stem cells than the avascular region. These meniscal-derived stem cells were multipotent and contributed to meniscal regeneration.