Intestinal epithelial dopamine receptor signaling drives sex-specific disease exacerbation in a mouse model of multiple sclerosis.

Although the gut microbiota can influence central nervous system (CNS) autoimmune diseases, the contribution of the intestinal epithelium to CNS autoimmunity is less clear. Here, we showed that intestinal epithelial dopamine D2 receptors (IEC DRD2) promoted sex-specific disease progression in an animal model of multiple sclerosis. Female mice lacking Drd2 selectively in intestinal epithelial cells showed a blunted inflammatory response in the CNS and reduced disease progression. In contrast, overexpression or activation of IEC DRD2 by phenylethylamine administration exacerbated disease severity. This was accompanied by altered lysozyme expression and gut microbiota composition, including reduced abundance of Lactobacillus species. Furthermore, treatment with N2-acetyl-L-lysine, a metabolite derived from Lactobacillus, suppressed microglial activation and neurodegeneration. Taken together, our study indicates that IEC DRD2 hyperactivity impacts gut microbial abundances and increases susceptibility to CNS autoimmune diseases in a female-biased manner, opening up future avenues for sex-specific interventions of CNS autoimmune diseases.

The Structure and Function of Glial Networks: Beyond the Neuronal Connections.

Glial cells, consisting of astrocytes, oligodendrocyte lineage cells, and microglia, account for >50% of the total number of cells in the mammalian brain. They play key roles in the modulation of various brain activities under physiological and pathological conditions. Although the typical morphological features and characteristic functions of these cells are well described, the organization of interconnections of the different glial cell populations and their impact on the healthy and diseased brain is not completely understood. Understanding these processes remains a profound challenge. Accumulating evidence suggests that glial cells can form highly complex interconnections with each other. The astroglial network has been well described. Oligodendrocytes and microglia may also contribute to the formation of glial networks under various circumstances. In this review, we discuss the structure and function of glial networks and their pathological relevance to central nervous system diseases. We also highlight opportunities for future research on the glial connectome.

Intestinal Dopamine Receptor D2 is Required for Neuroprotection Against 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Dopaminergic Neurodegeneration.

A wealth of evidence has suggested that gastrointestinal dysfunction is associated with the onset and progression of Parkinson's disease (PD). However, the mechanisms underlying these links remain to be defined. Here, we investigated the impact of deregulation of intestinal dopamine D2 receptor (DRD2) signaling in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration. Dopamine/dopamine signaling in the mouse colon decreased with ageing. Selective ablation of Drd2, but not Drd4, in the intestinal epithelium, caused a more severe loss of dopaminergic neurons in the substantia nigra following MPTP challenge, and this was accompanied by a reduced abundance of succinate-producing Alleoprevotella in the gut microbiota. Administration of succinate markedly attenuated dopaminergic neuronal loss in MPTP-treated mice by elevating the mitochondrial membrane potential. This study suggests that intestinal epithelial DRD2 activity and succinate from the gut microbiome contribute to the maintenance of nigral DA neuron survival. These findings provide a potential strategy targeting neuroinflammation-related neurological disorders such as PD.

Central dopaminergic control of cell proliferation in the colonic epithelium.

Parkinson's disease (PD) is an age-related neurodegenerative disease, mainly characterized by the loss of dopaminergic (DA) neurons in the substantia nigra. Several non-motor symptoms, including those associated with gastrointestinal dysfunction, precede the classical motor symptoms in PD. However, the mechanisms underlying gastrointestinal dysfunction in the prodromal phase of PD remain elusive. Here, we investigated the contribution of the central DA system to cell proliferation in the colonic epithelium. Degeneration of nigrostriatal DA pathway induced by striatal 6-hydroxydopamine (6-OHDA) injection resulted in a marked reduction in cell proliferation in the colonic epithelium as assessed by Ki-67 and bromodeoxyuridine labeling assays. RNA-sequencing analysis confirmed the suppression of cell cycle-related gene expression in the colonic epithelium of 6-OHDA-lesioned mice. Mesencephalic DA neuron degeneration also caused the gut microbiota dysbiosis. Moreover, 6-OHDA-lesioned mice showed profoundly increased vulnerability to dextran sulfate sodium-induced colitis. Together, our study uncovers a crucial role for the integrity of nigral DA neurons in the maintenance of colonic epithelial cell homeostasis. Our data also provide a new strategy for protecting intestinal homeostasis in PD.

Highly-efficient and easy separation of γ-Fe2O3 selectively adsorbs U(⠥) in waters.

The migration and transformation of uranyl [U (Ⅵ)] ions in the environment are quite dependent on the geological condition in particular with the site enriched in Fe. In this study, the interfacial interaction of U (Ⅵ) ions with maghemite (γ-Fe2O3) particles was studied and the interaction mechanism was explored as well. Batch experiments confirm that γ-Fe2O3 can effectively remove U (Ⅵ) from an aqueous solution within a relatively short reaction time (R% > 92.01% within 3 min) and has a considerable capacity for U (Ⅵ) uptake (qt: 87.35 mg/g). γ-Fe2O3 displays an excellent selectivity for U (Ⅵ) elimination. Results on the effects of natural organic matter such as humic acid (HA) indicated that HA could promote the interfacial interaction between γ-Fe2O3 and U (Ⅵ) under acidic conditions. Compared with other radionuclides (e.g., Sr(Ⅱ) and Cs(Ⅰ)), U (Ⅵ) was more effectively removed by γ-Fe2O3. The U (Ⅵ) removal by γ-Fe2O3 is primarily due to electrostatic interactions and precipitation that result in the long-term retardation of uranium. γ-Fe2O3 not only can fast and selectively adsorb U (Ⅵ) but also can be magnetically recycled, demonstrating that γ-Fe2O3 is a cost-effective and promising material for the clean-up of uranyl ions from radioactive wastewater.

Assessment of heavy metals mobility and correlative recovery and decontamination from MSWI fly ash: Mechanism and hydrometallurgical process evaluation.

Fly ash from municipal solid waste incineration (MSWI) enriches many leachable toxic metals which readily migrate into the environment, posing serious risks to the ecosystem and human. In this study, the elements mobility, leaching availability as well as the potential maximum amounts of heavy metals in fly ash were thoroughly evaluated. To decontaminate the toxic elements from resulting fly ash leachates, The aqueous zinc (Zn) was recovered using Cyanex 572, cadmium (Cd) and copper (Cu) were effectively removed through adsorption process by a self-assembled hierarchical hydroxyapatite (HAP) nanostructure. The removal mechanism of Cd, Cu and Zn by leaching, extraction and adsorption was revealed with the results from XRD, ICP-MS and SEM. The results showed that fly ash has a high mobility under maximum availability leaching test (95% of fly ash was dissolved), a recovery rate of 91% for Zn can be obtained using Cyanex 572, and a high adsorption rate (> 95% for both Cu and Cd) was reached using HAP for the pristine fly ash leachate. The outcomes from isothermal and kinetic study revealed that Langmuir isotherm and pseudo-second order model can well describe the Cd and Cu adsorption behavior. Economic assessment suggested that the application of HAP for the removal of Cd and Cu is a technically sound and economically feasible approach. The findings of this study demonstrated that this comprehensive process integrated leaching, solvent extraction and consequential decontamination can be a practical strategy for MSWI fly ash treatment.

Graphene oxide functionalized with nano hydroxyapatite for the efficient removal of U(VI) from aqueous solution.

Water contamination caused by radionuclides is a major environmental issue. Uranium (U) belongs to the actinide group of elements. Hexavalent uranium (U(VI)) is radioactively and chemically harmful and highly mobile in the environment and wastewater stream. Therefore, developing highly efficient materials for minimizing the environmental impact of U(VI) is essential. To achieve this goal, we successfully synthesized a novel material, namely graphene oxide (GO)/hydroxyapatite (HAP), by directly assembling GO and HAP through a facile hydrothermal method, which exhibits effective U(VI) removal and immobilization. The GO/HAP composite has an outstanding sorption capacity for U(VI) (i.e., 373.00 mg/g) within 5 min at a pH of 3.0. The parameters from thermodynamic analysis indicated that the GO/HAP composite absorbed U(VI) through a process of spontaneous and exothermic adsorption. XPS, XRD, and FT-IR results revealed that the composite's phosphate group was mainly responsible for U(VI) retention and incorporation. The GO/HAP composite's enhanced U(VI) sorption capacity is most likely ascribed to the synergistic effect after functionalizing with nano HAP. The current findings may greatly facilitate the creation of rational design strategies to develop highly efficient materials that can treat radioactive wastewater.

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

BACKGROUND: Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, we created a Cre/Lox-ß-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, ß-galactosidase. By using this system in an adult mouse model, we found that ß-galactosidase positive mononuclear cells were generated from ß-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. CONCLUSIONS/SIGNIFICANCE: These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

The dose of growth factors influences the synergistic effect of vascular endothelial growth factor on bone morphogenetic protein 4-induced ectopic bone formation.

Although vascular endothelial growth factor (VEGF) has been shown to act synergistically with bone morphogenetic protein (BMP)2 and BMP4 to promote ectopic endochondral bone formation via cell-based BMP gene therapy, the optimal ratio of VEGF to either of the BMPs required to obtain this beneficial effect remains unclear. In the current study, two cell types (C2C12, NIH/3T3) were retrovirally transduced to express BMP4 only or both BMP4 and VEGF. The resulting groups of cells were tested for their cellular proliferation, in vitro mineralization capacity, survival potential, and ability to undergo ectopic bone formation when implanted into a gluteofemoral muscle pocket created in severe combined immunodeficient mice. Results showed that VEGF inhibited the in vitro calcification of C2C12 and NIH/3T3 cells transduced to express BMP4. In vivo, C2C12 and NIH/3T3 cells expressing BMP4 and VEGF displayed significantly less bone formation than the same cells expressing only BMP4. In vivo, our results indicated that, when the ratio of VEGF to BMP4 is high, a detrimental effect on ectopic bone formation is observed; however, when the ratio is kept low and constant over time, the detrimental effect that VEGF has on ectopic bone formation is lost. Our studies revealed that VEGF's synergistic role in BMP4 induced ectopic bone formation is dose and cell-type dependent, which is an important consideration for cell-based gene therapy and tissue engineering for bone healing.

Bone regeneration mediated by BMP4-expressing muscle-derived stem cells is affected by delivery system.

This study investigated the delivery of bone morphogenetic protein (BMP)4-secreting muscle-derived stem cells (MDSC-B4) capable of inducing bone formation in mice using collagen gel (CG), fibrin sealant (FS), and gelatin sponge carriers. After implanting these various cell-loaded scaffolds intramuscularly or into critical-size skull defects, we measured the extent of heterotopic ossification and calvarial defect healing over a 6-week period via radiographic, radiomorphometric, histological, and micro-computed tomography analyses. As expected, in the absence of MDSC-B4, there was no ectopic ossification and only minimal calvarial regeneration using each type of scaffold. Although CG and gelatin sponges loaded with BMP4-secreting cells produced the most ectopic bone, FS constructs produced bone with comparably less mineralization. In the mouse calvaria, we observed MDSC-B4-loaded scaffolds able to promote bone defect healing to a variable degree, but there were differences between these implants in the volume, shape, and morphology of regenerated bone. MDSC-B4 delivery in a gelatin sponge produced hypertrophic bone, whereas delivery in a CG and FS healed the defect with bone that closely resembled the quantity and configuration of native calvarium. In summary, hydrogels are suitable carriers for osteocompetent MDSCs in promoting bone regeneration, especially at craniofacial injury sites.

Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation.

BACKGROUND: Over a hundred years ago, Wolff originally observed that bone growth and remodeling are exquisitely sensitive to mechanical forces acting on the skeleton. Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse. Although the processes of mechanotransduction and functional response of bone to mechanical strain have been extensively studied, the molecular signaling mechanisms that mediate the response of bone cells to mechanical stimulation remain unclear. RESULTS: Here, we identify a novel germline mutation at the mouse Bone morphogenetic protein 5 (Bmp5) locus. Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5. Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo. Biomechanical studies of osteoblasts from these anatomic sites show that the mutation inhibits the proper response of bone cells to mechanical stimulation. CONCLUSION: The results from these genetic, biochemical, and biomechanical studies suggest that BMPs are required not only for skeletal patterning during embryonic development, but also for bone response and remodeling to mechanical stimulation at specific anatomic sites in the skeleton.

A relationship between vascular endothelial growth factor, angiogenesis, and cardiac repair after muscle stem cell transplantation into ischemic hearts.

OBJECTIVES: We investigated whether vascular endothelial growth factor (VEGF) was associated with the angiogenic and therapeutic effects induced after transplantation of skeletal muscle-derived stem cells (MDSCs) into a myocardial infarction (MI). BACKGROUND: Because very few MDSCs were found to differentiate into new blood vessels when injected into the heart, the mechanism underlying the occurrence of angiogenesis after MDSC transplantation is currently unknown. In the present study, we used a gain- or loss-of-VEGF function approach with skeletal MDSCs engineered to express VEGF or soluble Flt1, a VEGF-specific antagonist, to identify the involvement of VEGF in MDSC transplantation-induced neoangiogenesis. METHODS: Vascular endothelial growth factor- and soluble Flt1-engineered MDSCs were injected into an acute MI. Angiogenesis and cardiac function were evaluated by immunohistochemistry and echocardiography. RESULTS: Both control and VEGF-overexpressing MDSCs induced angiogenesis, prevented adverse cardiac remodeling, and improved function compared with saline-injected hearts. However, these therapeutic effects were diminished in hearts transplanted with MDSCs expressing soluble Flt1 despite successful cell engraftment. In vitro experiments demonstrated that MDSCs increased secretion of VEGF in response to hypoxia and cyclic stretch (likely conditions in ischemic hearts), suggesting that transplanted MDSCs release VEGF in vivo. CONCLUSIONS: Our findings suggest that VEGF is essential for the induction of angiogenesis and functional improvements observed after MDSC transplantation for infarct repair.

Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells.

OBJECTIVE: Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle exhibit long-time proliferation, high self-renewal, and multipotent differentiation. This study was undertaken to investigate the ability of MDSCs that were retrovirally transduced to express bone morphogenetic protein 4 (BMP-4) to differentiate into chondrocytes in vitro and in vivo and enhance articular cartilage repair. METHODS: Using monolayer and micromass pellet culture systems, we evaluated the in vitro chondrogenic differentiation of LacZ- and BMP-4-transduced MDSCs with or without transforming growth factor beta1 (TGFbeta1) stimulation. We used a nude rat model of a full-thickness articular cartilage defect to assess the duration of LacZ transgene expression and evaluate the ability of transplanted cells to acquire a chondrocytic phenotype. We evaluated cartilage repair macroscopically and histologically 4, 8, 12, and 24 weeks after surgery, and performed histologic grading of the repaired tissues. RESULTS: BMP-4-expressing MDSCs acquired a chondrocytic phenotype in vitro more effectively than did MDSCs expressing only LacZ; the addition of TGFbeta1 did not alter chondrogenic differentiation of the BMP-4-transduced MDSCs. LacZ expression within the repaired tissue continued for up to 12 weeks. Four weeks after surgery, we detected donor cells that coexpressed beta-galactosidase and type II collagen. Histologic scoring of the defect sites 24 weeks after transplantation revealed significantly better cartilage repair in animals that received BMP-4-transduced MDSCs than in those that received MDSCs expressing only LacZ. CONCLUSION: Local delivery of BMP-4 by genetically engineered MDSCs enhanced chondrogenesis and significantly improved articular cartilage repair in rats.

Nerve growth factor improves the muscle regeneration capacity of muscle stem cells in dystrophic muscle.

Researchers have attempted to use gene- and cell-based therapies to restore dystrophin and alleviate the muscle weakness that results from Duchenne muscular dystrophy (DMD). Our research group has isolated populations of muscle-derived stem cells (MDSCs) from the postnatal skeletal muscle of mice. In comparison with satellite cells, MDSCs display an improved transplantation capacity in dystrophic mdx muscle that we attribute to their ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages. Here we tested whether the use of nerve growth factor (NGF) improves the transplantation efficiency of MDSCs. We used two methods of in vitro NGF stimulation: retroviral transduction of MDSCs with a CL-NGF vector and direct stimulation of MDSCs with NGF protein. Neither method of NGF treatment changed the marker profile or proliferation behavior of the MDSCs, but direct stimulation with NGF protein significantly reduced the in vitro differentiation ability of the cells. NGF stimulation also significantly enhanced the engraftment efficiency of MDSCs transplanted within the dystrophic muscle of mdx mice, resulting in the regeneration of numerous dystrophin-positive muscle fibers. These findings highlight the importance of NGF as a modulatory molecule, the study of which will broaden our understanding of its biologic role in the regeneration and repair of skeletal muscle by musclederived cells.

VEGF improves, whereas sFlt1 inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis.

UNLABELLED: We studied the interaction between VEGF and BMP2 during bone formation and bone healing. Results indicate that VEGF antagonist inhibited BMP2-elicited bone formation, whereas the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing through modulation of angiogenesis. INTRODUCTION: Angiogenesis is closely associated with bone formation during normal bone development and is important for the bone formation elicited by BMP4. However, it remains unknown whether vascular endothelial growth factor (VEGF) also interacts with other BMPs, especially BMP2, in bone formation and bone healing. MATERIALS AND METHODS: For this study, mouse muscle-derived stem cells were transduced to express BMP2, VEGF, or VEGF antagonist (sFlt1). We studied the angiogenic process during endochondral bone formation elicited by BMP2, a prototypical osteogenic BMP. Using radiographic and histologic analyses, we also evaluated the interaction between VEGF and BMP2 during bone formation and bone healing. RESULTS: Our results indicate that BMP2-elicited bone formation comprises two phases of angiogenesis, with an early phase occurring before the appearance of hypertrophic cartilage, followed by a late phase coupled with the appearance of hypertrophic cartilage. Our finding that the administration of sFlt1, a specific antagonist of VEGF, significantly inhibited BMP2-induced bone formation and the associated angiogenesis indicates that endogenous VEGF activity is important for bone formation. Furthermore, we found that the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing by improving angiogenesis, which in turn led to accelerated cartilage resorption and enhanced mineralized bone formation. Our findings also indicate that the ratio between VEGF and BMP2 influences their synergistic interaction, with a higher proportion of VEGF leading to decreased synergism. Our study also revealed unique VEGF-BMP2 interactions that differ from the VEGF-BMP4 interactions that we have described previously. CONCLUSIONS: This study, along with previously published work, shows that VEGF interacts synergistically with both BMP4 and BMP2 but elicits substantially different effects with these two BMPs.

Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation.

UNLABELLED: After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage. INTRODUCTION: The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation. MATERIALS AND METHODS: We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle. RESULTS: Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation. CONCLUSIONS: Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.

Noggin improves bone healing elicited by muscle stem cells expressing inducible BMP4.

The aim of this study was to test the hypothesis that a specific antagonist may enhance the efficacy of its corresponding growth factor in a regulated tissue engineering strategy. Our prior research has led to the development of a retroviral vector that enables optimal regulated bone morphogenetic protein 4 (BMP4) expression in vitro and regulated bone formation in vivo with transduced muscle stem cells. However, when implanted in critical-sized calvarial defects, these cells led to residual bone formation without induction or bone overgrowth with induction, even at reduced cell doses. We thus co-implanted the aforementioned cells with stem cells engineered to express Noggin, a specific BMP antagonist. This approach, while preserving our ability to regulate bone regeneration closely, prevented both the basal level bone regeneration and the bone overgrowth and, more importantly, led to the regeneration of bone that more closely resembled normal bone. We believe that this regulatable tissue engineering strategy, enhanced by utilizing a specific antagonist, constitutes a new paradigm for tissue engineering and regenerative medicine.

Structural and functional healing of critical-size segmental bone defects by transduced muscle-derived cells expressing BMP4.

BACKGROUND: Our previous studies have shown that muscle-derived cells, including a population of muscle stem cells, transduced with a retroviral vector expressing bone morphogenetic protein 4 (BMP4) can improve the healing of critical-size calvarial defects. However, we did not evaluate the functionality of the healed bone. The purpose of this study was to determine whether primary muscle-derived cells transduced with retroBMP4 can heal a long bone defect both structurally and functionally. METHODS: Primary muscle-derived cells were genetically engineered to express BMP4 and were implanted into 7-mm femoral defects created in syngeneic rats. Muscle-derived cells transduced with retroLacZ were used in the control group. Bone healing was monitored by radiography, histology, and biomechanical testing at designated time points. RESULTS: Most of the defects treated with muscle-derived cells expressing BMP4 formed bridging callous by 6 weeks after surgery, and exhibited radiographically evident union at 12 weeks after cell implantation. Histological analysis at 12 weeks revealed that the medullary canal of the femur was restored and the cortex was remodeled between the proximal and distal ends of each BMP4-treated defect. In contrast, the defects treated with muscle-derived cells expressing beta-galactosidase displayed nonunion at all tested time points. An evaluation of the maximum torque-to-failure in the treatment group indicated that the healed bones possessed 77 +/- 28% of the strength of the contralateral intact femora. Torsional stiffness and energy-to-failure were not significantly different between the treated and intact limbs. CONCLUSIONS: This study demonstrated that primary muscle-derived cells transduced with retroBMP4 can elicit both structural and functional healing of critical-size segmental long bone defects created in rats.

Ex vivo gene therapy-induced endochondral bone formation: comparison of muscle-derived stem cells and different subpopulations of primary muscle-derived cells.

Muscle-based gene therapy and tissue engineering hold great promise for improving bone healing. However, the relative advantage of muscle-derived stem cells (MDSCs) or primary muscle-derived cells (MDCs) remains to be defined. We compared the ability of MDSCs and different subpopulations of MDCs (PP1 and PP3) to induce bone formation via ex vivo gene therapy. We were able to efficiently transduce the MDSCs and all the other evaluated populations of MDCs (efficiency of transduction = approximately 80%) by using a retroviral vector expressing human bone morphogenetic protein 4 (BMP4). All the transduced cell populations secreted high levels of BMP4 (140-300 ng/10(6) cells/24 h), but the MDSCs differentiated toward the osteogenic lineage more effectively than did the other muscle cell populations, as indicated by the expression of alkaline phosphatase, an early osteogenic marker. von Kossa staining indicated that mineralized bone formed as early as 7 days after implantation of any of the BMP4-expressing cell populations into immunocompetent syngeneic mice; however, MDSCs expressing BMP4 produced significantly more bone than did the other MDC populations, as evidenced by both histomorphometry and biochemical analysis. Further investigation revealed that MDSCs expressing BMP4 persisted for a significantly longer period of time at the bone forming sites than did the other BMP4-expressing MDC populations. Additionally, MDSCs expressing BMP4 triggered a smaller infiltration of CD4 lymphocytes within the bone forming areas than did the other MDC populations expressing BMP4. Finally, we demonstrated that MDSCs expressing BMP4 can heal a critical-sized skull bone defect in immunocompetent mice. In summary, this study shows that MDSCs are better than primary MDCs for use as cellular vehicles in BMP4-based ex vivo gene therapy to improve bone healing. The advantage of MDSCs may be attributable, at least in part, to their lower immunogenicity and higher capacity for in vivo survival.

Development of a self-inactivating tet-on retroviral vector expressing bone morphogenetic protein 4 to achieve regulated bone formation.

The aims of this study were to explore the possibility of improving the design of self-inactivating (SI) retroviral vectors and to develop an SI vector that would allow optimal tet-on-regulated therapeutic gene expression. To minimize any interference between the viral promoter and the inducible promoter, we deleted different regulatory elements in the 3'LTR and examined their effects on transgene expression in transfected or transduced cells. In transfected cells, such deletions reduced the transgene expression. The insertion of a polyadenylation sequence could not completely compensate for this effect. We observed three patterns of transgene expression in cells transduced with these tet-on retroviral vectors: (1) high levels of both basal and inducible expression, (2) low levels of both basal and inducible expression, and (3) low levels of basal and high levels of inducible expression. After using the optimal vector to transduce muscle-derived stem cells, we were able to regulate the strong in vitro expression of transgenes-including enhanced green fluorescent protein and bone morphogenetic protein 4-via the addition or withdrawal of doxycycline (Dox). Implantation of the transduced cells and subsequent Dox-dependent induction of gene expression resulted in bone formation in vivo. Thus, we have developed an optimal SI retroviral vector that maintains a high titer, efficiently transduces muscle-derived stem cells, and enables both high levels of inducible gene expression in vitro and robust regulated bone formation in vivo.