Biomedical subjects

J Huard

Publications and source records attributed to J Huard.

At least 19 recordsLinked to original sources

PubMed2005

Injection of skeletal muscle-derived cells into the penis improves erectile function.

We investigated the effect of intrapenile injection of muscle-derived cells (MDC) on the erectile function in rats with bilateral cavernous nerve injury. Rat MDC were harvested and transduced with a retrovirus expressing the lacZ gene. Hanks' balanced salt solution (HBSS) (20 microl) or MDC (1 x 10(6) cells/side) were injected in each corpora cavernosa immediately before bilateral cavernous nerve transection. Intracavernous pressures (ICP) were measured 2 or 4 weeks after surgery with electrical stimulation of the pelvic nerves. Mean maximal ICP of sham group was significantly lower than that of control group both at 2 and 4 weeks after surgery. When MDC were injected into the penis, ICP improved over the sham-injected group at both 2 and 4 weeks after surgery. Percent area of PGP 9.5 staining was significantly greater in MDC-injected penis than in sham-injected at 2 and 4 weeks. Penile MDC injection can facilitate recovery of injured penile innervation and improve erectile function.

Animals↗

PubMed2005

Long-term self-renewal of postnatal muscle-derived stem cells.

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.

Aging↗

PubMed2005

[Ex-vivo gene therapy with BMP-4 for critically sized defects and enhancement of fracture healing in an osteoporotic animal model].

Fractures in osteoporotic bones or segment defects are problematic bone lesions with a reduced biological capability of regeneration. We tested the hypothesis that cell-mediated ex vivo gene therapy to deliver BMP4 can heal critically sized defects and improve bone healing in osteoporotic rats. Primary muscle-derived cells were isolated from the hindlimb muscle of rats and retrovirally transduced to express bone morphogenic protein 4 (BMP4). The bone formation was evaluated following local application of these cells in critically sized defects and in fractures of osteoporotic bones. Radiographic analysis revealed bridging callus formation in a critically sized defect in all specimens using muscle-derived cells expressing BMP4 at 12 weeks. These findings were confirmed by histological evaluation, which revealed callus bone formation with good integration to the distal and proximal bone. Following treatment with muscle-derived cells expressing BMP4, the bone healing process in the osteoporotic bone was improved to the level similar to that of normal bone. The ex vivo gene therapy could be a promising tool for the treatment of osteoporotic fractures and critically sized defects. The reduced number of complications (nonunions, loss of reduction, and fragment dislocation), shortening of hospitalization period, and improvement of bone strength are decisive advocates for this treatment option.

Animals↗

PubMed2005

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 non-myocytes 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.

Animals↗

PubMed2005

The use of relaxin improves healing in injured muscle.

To improve the functional recovery of injured skeletal muscle, we have focused our efforts on both enhancement of muscle regeneration and prevention of fibrosis. The polypeptide cytokine/growth factor relaxin can inhibit fibrous tissue formation in many tissues. As a member of the insulin-like growth factor family, relaxin also is a potential stimulator of muscle regeneration. In the current experiment, we examined the antifibrotic effect of relaxin in injured skeletal muscle. We also investigated if the injection of relaxin would influence muscle regeneration after injury. Our results demonstrate that relaxin treatment improved histologic and physiologic healing of muscles subjected to traumatic injury.

Animals↗

PubMed2004

Muscle stem cells can act as antigen-presenting cells: implication for gene therapy.

Research has shown that the use of a muscle-specific promoter can reduce immune response and improve gene transfer to muscle fibers. We investigated the efficiency of direct and ex vivo gene transfer to the skeletal muscles of 6- to 8-week-old mdx mice by using two adenoviral vectors: adenovirus (AD) encoding the luciferase gene under the cytomegalovirus (CMV) promoter (ADCMV) and AD encoding the same gene under the muscle creatine kinase (MCK) promoter (ADMCK). Direct intramuscular injection of ADMCK triggered a lower immune response that enabled more efficient delivery and more persistent expression of the transgene than did ADCMV injection. Similarly, ex vivo gene transfer using ADCMV-transduced muscle-derived stem cells (MDSCs) induced a stronger immune response and led to shorter transgene expression than did ex vivo gene transfer using ADMCK-transduced MDSCs. This immune response was due to the release of the antigen after MDSC death or to the ADCMV-transduced MDSCs acting as antigen-presenting cells (APCs) by expressing the transgene and rapidly initiating an immune response against subsequent viral inoculation. The use of a muscle-specific promoter that restricts transgene expression to differentiated muscle cells could prevent MDSCs from becoming APCs, and thereby could improve the efficiency of ex vivo gene transfer to skeletal muscle.

Adenoviridae↗

PubMed2004

Myogenic cellular transplantation and regeneration: sorting through progenitor heterogeneity.

Growth and regeneration of skeletal muscle fibers in response to injury are made possible by the presence of resident myogenic progenitor cells. Researchers have attempted to isolate and transplant these cells to regenerate new muscle in cases involving injury, disease, or genetic deficiencies. Reports from such experiments underscore the functional diversity of progenitors obtained from skeletal muscle; however, currently there is no reliable means by which to positively identify and isolate the most desirable muscle progenitor populations. Taking a cue from the hematopoietic community, researchers in this area have begun to investigate cell surface protein expression in progenitor populations. Previous findings in cultured myogenic cells and our results in cells obtained directly from dissociated muscle suspensions indicate that cells sorted based on their expression of the commonly-studied myogenic cell surface proteins Sca-1 and CD34 exhibit differing regenerative abilities. However, results obtained to date are insufficient to clearly delineate whether the expression of either of these proteins is an exclusive characteristic of efficient myogenic progenitors. Nonetheless, observations from these studies clearly suggest that progenitor heterogeneity should be an important consideration during the development and implementation of muscle regeneration strategies. Additional research is necessary to establish reliable selection criteria for the isolation of efficient progenitors, which will facilitate therapeutic discoveries and enhance our understanding of factors affecting regeneration outcomes.

Animals↗

PubMed2003

The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence.

Our goal was to determine whether periurethral injection of allogenic muscle-derived stem cells (MDSC) could increase the leak point pressure (LPP) in a denervated female rat model of stress urinary incontinence. Cells isolated from the gastrocnemius muscle of normal female rats were purified for a myogenic population by the preplate technique. Three experimental groups were established: a control group (C) had a sham operation without injections; a sciatic nerve transection group (D) had periurethral saline injections; and a sciatic nerve transsection group had periurethral MDSC injections (M). One week following treatment the LPP of groups C, D and M were 25.2+/-1.9 cmH(2)O, 28.6+/-0.8 cmH(2)O and 36.7+/-2.3 cmH(2)O, respectively. At 4 weeks the LPP of groups C, D and M were 25.8+/-2.5 cmH(2)O, 18.6+/-5.2 cmH(2)O and 44.1+/-6.6 cmH(2)O, respectively. Allogenic MDSC significantly improved the LPP in sciatic nerve-transected animals after both 1 and 4 weeks compared to denervated animals injected with saline.

Animals↗

PubMed2003

Nitric oxide synthase gene transfer for erectile dysfunction in a rat model.

OBJECTIVE: To determine whether over-expression of nitric oxide synthase (NOS) in the corpus cavernosum of the penis improves erectile function, as NO is an important transmitter for genitourinary tract function, mediating smooth muscle relaxation and being essential for penile erection. MATERIALS AND METHODS: The inducible form of the enzyme NOS (iNOS) was introduced into the corpus cavernosum of adult Sprague-Dawley rats (250-300 g) by injecting a solution of plasmid, adenovirus or adenovirus-transduced myoblast cells (adeno-myoblasts). Plasmid, adenovirus and adeno-myoblasts encoding the expression of the beta-galactosidase reporter gene were also injected into rats. RESULTS: Throughout the corpora cavernosum there was expression of beta-galactosidase after injecting each of the three solutions. Maximum staining was greatest for adeno-myoblast, then adenovirus and then plasmid. The mean (sd) basal intracavernosal pressure (ICP) of iNOS-treated animals (adenovirus and adeno-myoblast) increased to 55 (23) cmH2O, compared with naive animals with a basal ICP of 5 (6) cmH2O (P = 0.001). Stimulating the cavernosal nerve (15 Hz, 1.5 ms, 10-40 V, 1 min) resulted in a doubling of the ICP (adenovirus and adeno-myoblast) from the basal level of the iNOS-treated animals. Direct in situ measurement of NO showed the release of 1-1.3 micro mol/L in the adeno-myoblast penis. CONCLUSION: Myoblast-mediated gene therapy was more successful for delivering iNOS into the corpus cavernosum than direct adenovirus injection or plasmid transfection. Surprisingly, implanting muscle cells into the penis is not only feasible but also beneficial. Gene therapy for NOS may open new avenues of treatment for erectile dysfunction. Control of NOS expression would be necessary to prevent priapism.

Adenoviridae↗

PubMed2003

Analysis of rabbit articular cartilage repair after chondrocyte implantation using optical coherence tomography.

OBJECTIVE: To evaluate the utility and limitations of optical coherence tomography (OCT) for immediate, high-resolution structural analysis of rabbit articular repair tissue following chondrocyte implantation without excising or sectioning the specimen. METHODS: Full thickness articular cartilage defects were created in the patellar grooves of 30 adult rabbit knee joints. Allogenic cultured chondrocytes embedded in collagen gels were implanted into the surgical defects. A periosteal patch was then sutured over the chondrocyte-collagen composites. Six animals per time point were sacrificed at 2, 4, 8, 12 and 24 weeks after surgery. The repair tissues were sequentially analysed by arthroscopic surface imaging, OCT, and histology. The resulting images were compared to determine qualitative and quantitative features of surface roughness, repair tissue integration, and micro-architecture. Statistical analysis was performed using Student's t -testing and linear regression. RESULTS: OCT was able to identify the bone and cartilage interface in normal rabbit articular cartilage and regenerated cartilage at 24 weeks post chondrocyte implantation. OCT was able to identify hypertrophy at 4 and 8 weeks, and subtle surface fibrillations at 24 weeks, comparable with histological analysis at low magnification (20x). More importantly, OCT was able to detect embedded gaps between the repair tissue and surrounding host cartilage. CONCLUSION: Close correlation was observed between OCT and histological analysis of morphological features important to the assessment of articular cartilage repair. These results demonstrate that OCT is capable of providing immediate 'optical biopsy' of the rabbit articular cartilage repair tissue without damaging the specimen, and suggest that this new technique, if integrated with an arthroscope, can potentially be used in longitudinal studies of articular cartilage repair in vivo.

Animals↗

PubMed2003

Muscle cell-mediated gene delivery to the rotator cuff.

Rotator cuff lesions are one of the most common causes of upper extremity disability. Surgical therapy addresses mostly the extrinsic etiology, but not intrinsic factors such as aging, structural changes, low vascularity, and inflammatory processes. In this study, genetically engineered, highly purified muscle-derived cells (MDCs) were characterized and injected into the supraspinatus tendons of nude rats. The injected cells were monitored for 3 weeks. In vitro, the engineered, highly purified MDCs do not express vimentin; 98% of them are positive for the beta-galactosidase marker gene, and 99% hybridize with the specific pancentromeric mouse probe. beta-Galactosidase marker gene expression of the injected cells was detected up to 21 days. From day 7 after injection, the cell nuclei became spindle shaped, cells were integrated into the tendon collagen bundles, and the cells showed differentiation into vimentin-expressing fibroblastic cells. The results indicate that the rotator cuff tendon matrix and its original cellular components modulated the injected MDCs toward a fibroblastic phenotype. The compatibility and ability of MDCs to differentiate into other cell lineages, such as fibroblasts, might have high potential utility in tissue-engineering applications for tendon healing. This approach facilitates the application of muscle-derived progenitor cells and ex vivo gene therapy for the treatment of rotator cuff lesions.

Animals↗

PubMed2003

Muscle injuries and repair: the role of prostaglandins and inflammation.

Skeletal muscle injuries are a common problem in trauma and orthopaedic surgery. Muscle injuries undergo the healing phases of degeneration, inflammation, regeneration, and fibrosis. Current and experimental therapies to improve muscle regeneration and limit muscle fibrosis include conservative and surgical principles with the adjuvant use of non-steroidal anti-inflammatory drugs (NSAIDs) and growth factor manipulation. NSAIDs appear to have a paradoxical effect on the healing of muscle injuries with early signs of improvement and subsequent late impairment in functional capacity and histology. In vitro and in vivo studies have explored the role of the cyclooxygenases and prostaglandins in the biological processes of healing muscle, including precursor cell activation, myoblast proliferation, myoblast fusion, and muscle protein synthesis. Through use of more specific cyclooxygenase inhibitors, we may be able to better understand the role of inflammation in muscle healing.

Animals↗

PubMed2003

Modeling stem cell population growth: incorporating terms for proliferative heterogeneity.

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle-derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Apoptosis↗

PubMed2002

Muscle-derived stem cells.

The existence of cells with stem cell-like abilities derived from various tissues can now be extended to include the skeletal muscle compartment. Although researchers have focused on the utilization of these cells with regard to their myogenic capacity, initially exploring more efficient cellular therapy treatments for muscular dystrophy, it is becoming increasingly apparent that such cells may one day be used in the treatment of non-myogenic disorders. Evidence regarding the existence and differentiation capacity of muscle-derived stem cells is discussed, along with current theories regarding their proposed position within the myogenic hierarchy.

Adult↗

PubMed2002

Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction.

We have tested the feasibility of muscle-based gene therapy and tissue engineering for urological dysfunction using highly purified muscle-derived cells (MDC) that display stem cell characteristics. We then explored the potential use of these MDC as an alternative therapy for the treatment of impaired detrusor contractility. The MDC were genetically engineered to express the gene encoding beta-galactosidase and injected into the bladder walls of SCID mice. The injected bladders were harvested at various time-points after injection and assayed for beta-galactosidase activity; the presence of myofibers within the injected tissue was determined by detection of fast myosin heavy chain isoform (MyHCs). We have demonstrated that the injected MDC are capable of not only surviving in the lower urinary tract, but also improving the contractility of the bladder following an induced injury. Two potential mechanisms can be used to explain this finding. First, we have observed that some of the beta-galactosidase-expressing cells expressed alpha-smooth muscle actin, suggesting a differentiation into smooth muscle. Second, a stain for acetylcholine receptors (AChRs), which identifies the location of neuromuscular junctions, revealed that the myofibers derived from the doner cells became innervated into the bladder as early as 2 weeks after injection. These results suggest that gene therapy and tissue engineering based on MDC potentially can be used for urological dysfunction.

Actins↗

PubMed2002

Human skeletal muscle cells in ex vivo gene therapy to deliver bone morphogenetic protein-2.

We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised beta-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised beta-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches.

Adenoviridae↗

PubMed2002

Mechanisms of muscle stem cell expansion with cytokines.

Stem cell expansion and proliferation are important for cell transplantation and stem cell-mediated applications. While we have demonstrated that muscle stem cells can be obtained from adult skeletal muscle tissue, these cells represent only a small percentage of the muscle-derived cells and require in vitro expansion for successful stem cell-mediated therapies. In this study, we have examined the potential of several cytokines to stimulate stem cell growth by combining a non-exponential mathematical model with a unique cell culture system. The growth kinetics of two populations of muscle stem cells were characterized in culture medium supplemented with epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), FLT-3 ligand, hepatocyte growth factor, or stem cell factor (SCF). The division time (DT) and fraction of mitotically active cells were investigated as key parameters to further understand the mechanism of the expansion of the stem cell populations. Our results show that expansion of the freshly isolated, muscle-derived stem cells (MDSC) occurred by recruiting cells into the cell cycle in the presence of EGF, IGF-1, and SCF. However, expansion of the cultured stem cell clone, MC13, is attributed to a reduction of the length of the cell cycle in the presence of FGF-2, EGF, IGF-1, and SCF. Both MDSC and MC13 growth were inhibited in the presence of FLT-3 ligand by increasing the length of the cell cycle. Our results suggest that EGF, IGF-1, FGF-2, and SCF are important cytokines for stimulating the proliferation of MDSC. In addition, this study illustrates that expansion of stem cells occurs through different mechanisms, which consequently demonstrates the importance of monitoring several parameters of cell growth, such as DT and dividing fraction, following stimulation with growth factors.

Animals↗

PubMed2001

New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.

STUDY DESIGN: Human intervertebral disc cells were cultured in a new three-dimensional "pellet culture" system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed. OBJECTIVES: To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies. SUMMARY OF BACKGROUND DATA: The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the "pellet culture" method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes. METHODS: Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-beta1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice. RESULTS: Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-beta1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the beta-galactosidase marker gene in vivo for up to 2 weeks. CONCLUSIONS: The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebral disc cells retained their native phenotype in this three dimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.

Adult↗