A role for natural killer cells in the rapid death of cultured donor myoblasts after transplantation

BACKGROUND: The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts. METHODS: Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice. RESULTS: A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells. CONCLUSIONS: These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.     

Why do cultured transplanted myoblasts die in vivo? DNA quantification shows enhanced survival of donor male myoblasts in host mice depleted of CD4+ and CD8+ cells or Nk1.1+ cells

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.     

不同代次成人正常髓核细胞的形态及生长动力学比较

目的比较不同代次成人正常髓核细胞体外培养的形态及生长动力学差异。方法从成人正常髓核组织分离培养髓核细胞，传代后对细胞形态、生长曲线、噻唑蓝（methyl thiazolyl tetrazolium，MTT）摄取等进行比较研究。结果体外培养条件下，传3代之前的细胞形态正常，胞浆丰富；传4代起，部分细胞的形态逐渐向长梭形演化。生长曲线提示传3代之前的髓核细胞持续增殖能力强。MTT测定吸光度值，传1、3代时差异无统计学意义（P〉0．05），传5、7代与传1代相比，差异具有统计学意义（P〈0．05）。结论体外培养条件下，前3代成人正常髓核细胞形态良好，活性高，增殖能力强，适合作为组织工程椎间盘的种子细胞。     

Innate inflammatory cells are not responsible for early death of donor myoblasts after myoblast transfer therapy

BACKGROUND: Myoblast transfer therapy (MTT) is a cell-based gene therapy representing a potential treatment for Duchenne muscular dystrophy. The rapid disappearance of donor myoblasts from transplanted muscles after MTT is one of the most controversial and significant obstacles facing research in this area. Dystrophin-deficient muscles show constitutively high levels of inflammation, thus necessitating an examination of whether inflammatory cells, specifically natural killer (NK) cells, neutrophils, and macrophages, within dystrophic muscle are responsible for poor graft survival. METHODS: Female mdx mice were treated with RB6-8C5 monoclonal antibody, PK136 monoclonal antibody, or clodronate liposomes to systemically deplete neutrophils, NK cells, and macrophages, respectively. After each depletion regimen, the mice and age-matched controls received 5.0 x 10 male myoblasts injected longitudinally into each tibialis anterior muscle. Donor myoblast survival was assessed by Y-chromosome specific quantitative real-time polymerase chain reaction analysis. RESULTS.: The systemic depletion of host neutrophils and NK cells resulted in a transient improvement in donor myoblast survival at 72 hr and 7 days post-MTT, respectively. Systemic depletion of macrophages had no significant beneficial effect on myoblast survival. Overall, the number of detectable male donor myoblasts was similar at time 0 and 1 hr post-MTT; however, there was significant loss by 24 hr (approximately 50%-70%) followed by a continual decline in donor cell numbers. CONCLUSIONS: Neutrophils and macrophages do not seem to play a major role in the rapid death of donor myoblasts after transplantation into dystrophic muscle. NK cells similarly seem to have no significant effect, contrary to earlier findings reported by our group.     

Fibrin gel improves the survival of transplanted myoblasts

Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy in children and young adults. Currently, there is no cure for the disease. The transplantation of healthy myoblasts is an experimental therapeutic strategy, since it could restore the expression of dystrophin in DMD muscles. Nevertheless, this cellular therapy is limited by immune reaction, low migration of the implanted cells, and high early cell death that could be at least partially due to anoikis. To avoid the lack of attachment of the cells to an extracellular matrix after the transplantation, which is the cause of anoikis, we tested the use of a fibrin gel for myoblast transplantation. In vitro, three concentrations of fibrinogen were compared (3, 20, and 50 mg/ml) to form a fibrin gel. A stiffer fibrin gel leads to less degradability and less proliferation of the cells. A concentration of 3 mg/ml fibrin gel enhanced the differentiation of the myoblasts earlier as a culture in monolayer. Human myoblasts were also transplanted in muscles of Rag/mdx mice in a fibrin gel or in a saline solution (control). The use of 3 mg/ml fibrin gel for cell transplantation increased not only the survival of the cells as measured after 5 days but also the number of fibers expressing dystrophin after 21 days, compared to the control. Moreover, the fibrin gel was also compared to a prosurvival cocktail. The survival of the myoblasts at 5 days was increased in both conditions compared to the control but the efficacy of the prosurvival cocktail was not significantly higher than the fibrin gel.     

Autotransplantation in mdx mice of mdx myoblasts genetically corrected by an HSV-1 amplicon vector

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, characterized by a lack of dystrophin. To eliminate the need for immunosuppressive drugs, transplantation of genetically modified autologous myoblasts has been proposed as a possible therapy for this myopathy. An HSV-1 amplicon vector (HSVDGN), containing a 17.3-kb full-length MCK-driven mouse dystrophin cDNA, an eGFP gene, and a neomycin resistance gene driven by CMV or SV40 promoters, respectively, was constructed and used to transduce mdx primary myoblasts. The presence of the eGFP and neomycin resistance genes facilitated the evaluation of the initial transduction efficiency and the permanent transduction frequency. At low multiplicities of infection (MOI 1-5), the majority of myoblasts (60-90%) expressed GFP. The GFP-positive mdx myoblasts were sorted by FACS and selected with neomycin (300 microg/ml) for 2 weeks. Up to 2% of initially infected mdx myoblasts stably expressed the three transgenes without further selection at that time. These altered cells were grafted into the tibialis anterior muscles of 18 mdx mice. Some of the mice were immunosuppressed with FK506 due to the anticipation that eGFP and the product of neomycin resistance gene might be immunogenic. One month after transplantation, numerous muscle fibers expressing mouse dystrophin were detected by immunohistochemistry, in both immunosuppressed (10-50%) and nonimmunosuppressed (5-25%) mdx mice. Our results demonstrated the capability of permanently expressing a full-length dystrophin in dystrophic myoblasts with HSV-1 amplicon vector and raised the possibility of an eventual treatment of DMD based on the transplantation of genetically modified autologous myoblasts.     

Dynamics of the early immune cellular reactions after myogenic cell transplantation

The role of immune cells in the early donor cell death/survival following myoblast transplantation is confusing, one of the reasons being the lack of data about the immune reactions following cell transplantation. We used outbred mice as hosts for transplantation of primary cultured muscle cells and T-antigen-immortalized myoblasts. The host muscles were analyzed 1 h to 7 days after cell injection. No net loss of the donor primary cultured cell population was observed in this period. The immune cellular reaction in this case was: 1) a brief (<48 h) neutrophil invasion; 2) macrophage infiltration from days 1 to 7; 3) a specific response involving CTL and few NK cells (days 6 and 7), preceded by a low CD4+ cell infiltration starting at day 3. In contrast, donor-immortalized myoblasts completely disappeared during the 7-day follow-up. In this case, an intense infiltration of CTL and macrophages, with moderate CD4+ infiltration and lower amounts of NK cells, was observed starting at day 2. The nonspecific immune response at days 0 and 1 was similar for both types of donor cells. The present observations set a basis to interpret the role of immune cells on the early death/survival of donor cells following myoblast transplantation.     

Human myoblast engraftment is improved in laminin-enriched microenvironment

BACKGROUND: One major challenge in developing cell therapy for muscle diseases is to define the best condition for the recipient's muscle to niche donor cells. We have examined the efficiency of human myoblast transplantation in an immunodeficient animal model, after local irradiation, as well as the potential impact of laminin on myoblast behavior. METHODS: Human myoblasts were injected into preirradiated tibialis anterior muscles from immunodeficient mice. The donor cell engraftment, proliferation, and laminin content within the transplanted muscles were evaluated by immunocytochemistry. Additionally, the effect of laminin upon myoblast proliferation, migration, and survival was ascertained in vitro. RESULTS: Engraftment of human myoblasts into the skeletal muscle of immunodeficient Rag2-/gammac-/C5- mice presubjected to local irradiation provided the best niche for myoblast engraftment, as demonstrated by the number of viable and proliferating donor cells found in the host muscle. Local irradiation significantly enhanced laminin deposition within the recipient's muscle and donor cells were preferentially located in laminin-enriched areas. The same batch of myoblasts used for in vivo injections also responded to laminin in vitro with increased proliferation and cell survival, as well as an improved migratory response. CONCLUSIONS: We show that local irradiation enhances the laminin content in the host muscle microenvironment and provides a better engraftment of human myoblasts. In addition, laminin increases myoblast proliferation, survival, and migration in vitro. These data provide combined in vivo and in vitro evidence that laminin status should be taken into account when designing experimental and clinical cell therapy strategies for muscle disease.     

Improved success of myoblast transplantation in mdx mice by blocking the myostatin signal

BACKGROUND.: Duchenne muscular dystrophy (DMD) is caused by a dystrophin gene mutation. Transplantation of normal myoblasts results in long-term restoration of dystrophin. However, the success of this approach is compromised by the limited time of regeneration following muscle damage. Myostatin is known to be responsible for limiting skeletal muscle regeneration. Our purpose is to verify whether blocking the myostatin signal in mdx host mice or in normal myoblasts transplanted in mdx host mice would increase the extent of muscle repair and thus allow the formation of more dystrophin-positive fibers. METHODS.: Transgenic mdx mice carrying a dominant negative form of myostatin receptor (dnActRIIB) were used to test the fiber resistance to damage and to act as a host for normal myoblast transplantation. Myoblasts obtained from nondystrophic transgenic mice carrying the dominant negative myostatin receptor were also transplanted in nontransgenic mdx mice. RESULTS.: Transgenic mdx mice carrying the dnActRIIB gene have bigger muscles than mdx mice with the normal gene of ActRIIB. Their fiber resistance to exercise-induced damage was also greatly improved. Moreover, the success of normal myoblast transplantation was significantly enhanced in mdx/dnActRIIB mice. Finally, nondystrophic dnActRIIB myoblasts formed more abundant and bigger dystrophin positive fibers when transplanted in mdx mice. CONCLUSIONS.: Blocking the myostatin signal in mdx mice allowed the size of muscle fibers to increase, the fiber resistance to damage induced by exercise to increase, and the success of normal myoblast transplantation to improve. The transplantation in mdx mice of dnActRIIB myoblasts formed more abundant and larger dystrophin positive fibers.     

Heat shock treatment increases engraftment of transplanted human myoblasts into immunodeficient mice

Myoblast transfer therapy (MTT) is a strategy that has been proposed to treat some striated muscle pathologies. However, the first therapeutic trials using this technique were unsuccessful due to the limited migration and early cell death of the injected myoblasts. Various strategies have been considered to increase myoblast survival in the host muscle after MTT. Overexpression of heat shock proteins (HSPs) in mouse myoblasts has been shown to improve cell resistance against apoptosis in vitro and in vivo. Our objective was to determine whether heat shock (HS) treatment increased the survival of human myoblasts leading to better participation of the injected cells in muscle regeneration. For this study, HS-treated human myoblasts were injected into the tibialis anterior (TA) muscles of immunodeficient RAG-/- gammaC-/- mice. TA muscles were excised at 24 hour and at 1 month after injection. Our results showed that HS treatment increased the expression of the hsp70 protein and protected the cells from apoptosis in vitro. HS treatment dramatically increased the number of human fibers present at 1 month after injection when compared with nontreated cells. Interestingly, HS treatment decreased apoptosis at 24 hour after human myoblast injection, but no differences were observed concerning proliferation, suggesting that the increased fiber formation among the HS-treated group was probably due to decreased cell death. These data suggested that HS treatment might be used in the clinical context to improve the success of MTT.     

A Synthetic Mechano Growth Factor E Peptide Enhances Myogenic Precursor Cell Transplantation Success

Myogenic precursor cell (MPC) transplantation is a good strategy to introduce dystrophin expression in muscles of Duchenne muscular dystrophy (DMD) patients. Insulin-like growth factor (IGF-1) promotes MPC activities, such as survival, proliferation, migration and differentiation, which could enhance the success of their transplantation. Alternative splicing of the IGF-1 mRNA produces different muscle isoforms. The mechano growth factor (MGF) is an isoform, especially expressed after a mechanical stress. A 24 amino acids peptide corresponding to the C-terminal part of the MGF E domain (MGF-Ct24E peptide) was synthesized. This peptide had been shown to enhance the proliferation and delay the terminal differentiation of C(2)C(12) myoblasts. The present study showed that the MGF-Ct24E peptide improved human MPC transplantation by modulating their proliferation and differentiation. Indeed, intramuscular or systemic delivery of this synthetic peptide significantly promoted engraftment of human MPCs in mice. In vitro experiments demonstrated that the MGF-Ct24E peptide enhanced MPC proliferation by a different mechanism than the binding to the IGF-1 receptor. Moreover, MGF-Ct24E peptide delayed human MPC differentiation while having no outcome on survival. Those combined effects are probably responsible for the enhanced transplantation success. Thus, the MGF-Ct24E peptide is an interesting agent to increase MPC transplantation success in DMD patients.     

Paracrine anti‐fibrotic effects of neonatal cells and living cell constructs on young and senescent human dermal fibroblasts

Senescent cells observed in the area of chronic wounds have been proposed to affect wound healing. Therapeutic approaches against chronic wounds include, among others, the local application of living cell constructs (LCCs), containing fibroblasts and/or keratinocytes. Accordingly, the aim of the present work was to examine the effects of factors secreted by early passage neonatal fibroblasts and LCCs—in the form of a conditioned medium (CM)—on senescent adult dermal fibroblasts regarding functions related to the healing process, i.e., cell proliferation, alpha‐smooth muscle actin and metalloproteinase expression, and collagen synthesis. Target cells were fibroblasts senescent either due to subsequent divisions (replicative senescence) or due to an exogenous stress (stress‐induced premature senescence). No effect on the proliferation of senescent fibroblasts was observed, as expected. All CMs were found to inhibit overall collagen synthesis both in early passage and in senescent fibroblasts. The LCC‐derived CM was found to be more potent than fibroblast‐derived CMs and, furthermore, to inhibit alpha‐smooth muscle actin expression. In conclusion, these results may indicate anti‐contractile and anti‐fibrotic activities of factor(s) secreted by neonatal skin fibroblasts, and more intensely by LCCs on adult donor‐derived fibroblasts. These activities seem to persist during senescence of the target cells.     

Thymic myoid cells as a source of cells for myoblast transfer

Transplantation of disaggregated myoblasts from normal donor to the muscles of a diseased host, or reimplantation of genetically modified host myoblasts, has been suggested as a possible route to therapy for inherited myopathies such as Duchenne muscular dystrophy, or to supply missing proteins that are required systemically in diseases such as hemophilia. With two exceptions, studies of myoblast transfer in the mouse have involved transplantation of donor myoblasts isolated from adult or neonatal skeletal muscle satellite cells. In this study we present evidence that thymic myoid cells are capable of participating in the regeneration of postnatal skeletal muscle, resulting in the expression of donor-derived proteins such as dystrophin and retrovirally encoded proteins such as beta-galactosidase within host muscles. This leads us to conclude that thymic myoid cells may provide an alternative to myoblasts derived from skeletal muscle as a source of myogenic cells for myoblast transfer.     

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation

BACKGROUND: Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS: Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS: Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS: In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.     

Novel Duchenne Muscular Dystrophy Treatment Through Myoblast Transplantation Tolerance with Anti-CD45RB, Anti-CD154 and Mixed Chimerism

Duchenne muscular dystrophy (DMD) is a fatal disease caused by a defect in the skeletal muscle protein, dystrophin. One potential therapy for DMD involves transplantation of myoblasts from normal individuals. Unfortunately, myoblast allografts are particularly immunogenic and transplant tolerance in dystrophic (mdx/mdx) mice has not yet been achieved despite using strategies successful in other allograft models. Here, we attempted to induce 'central tolerance' using either haplo- or fully allogeneic bone marrow after conditioning with low-dose (3 Gy) whole body irradiation and anti-CD154 or anti-CD45RB mAbs. With one exception, these mice lacked persistent chimerism, long-term survival of myoblast allografts, or tolerance. In contrast, the addition of anti-CD45RB to anti-CD154 uniformly resulted in long-lived high-level mixed chimerism, long-term (>100 days) engraftment of allogeneic myoblasts and deletion of donor-reactive cells. Moreover, all recipients exhibited tolerance to second myoblast allografts or donor-specific tolerance to skin transplants performed >80 days after the initial graft. Thus, we now report that anti-CD45RB synergizes with anti-CD40L to promote stable mixed chimerism and robust tolerance to myoblast allografts for the first time. This novel protocol may be applicable to future clinical trials in myoblast transplantation for treatment of DMD and for transplantation of other immunogenic allografts.     

Alterations in hormone-sensitive adenylate cyclase of cloned rat osteosarcoma cells during long-term culture

The hormone-sensitive adenylate cyclase system of a cloned bone cell line (UMR-106) derived from a rat osteosarcoma was compared in preparations from cells of early passages (less than 50) and cells maintained in continuous culture for over two years (late passages). Late passage cells showed greater calcitonin (CT)-stimulated adenylate cyclase activity than did early passages, whereas stimulation by PTH and the beta-adrenergic agonist isoproterenol decreased in late passages. Hormone concentrations giving half-maximal stimulation were the same in early and late passages. Stimulation by agents (GTP and fluoride) which act at the stimulatory guanine nucleotide regulatory component (Ns) of adenylate cyclase was equivalent in early and late passages. Forskolin stimulation, which assessed catalytic component (and possibly Ns) activity, was reduced in late passages. These results are consistent with acquisition by cultured UMR-106 cells of CT receptors linked to adenylate cyclase and loss of PTH and beta-adrenergic receptors. Alteration of catalytic component (and/or Ns) function may also occur after long-term culture. Since late passage cells appear dedifferentiated by chromosomal analysis and since cAMP may regulate differentiation, altered hormone-sensitive adenylate cyclase may be a marker for and a potential modulator of differentiation occurring in UMR-106 cells over long periods.     

连续传代人胚骨骼肌成肌细胞生物学特性研究

目的 探讨成肌细胞连续传代能力,选择适宜肌组织工程研究的成肌细胞。方法 常规传代培养人胚骨骼肌细胞,以生长曲线、融合率分别观察细胞增列、分化能力,探讨成纤维细胞沾染对传代细胞的影响。结果 第６代以内细胞成纤维细胞沾染轻,主要表现出成肌细胞的生长特性,增殖较旺盛,分化能力高。第８代￣第１６代细胞成纤维细胞沾染重,表现出成纤维细胞的生长特性,增殖速度明显加快但分化能力低。第２０代细胞退变明显,细胞增殖     

Growth characteristics of human malignant tumours and cell lines in serial passages in nude mice

Two osteosarcoma cell lines, one fibrosarcoma and one undifferentiated mesenchymal tumour obtained at operation, all of human origin, were transplanted in serial passages in athymic nude mice. The growth rate was analysed and the morphologic appearance compared both with the original tumours and after each passage. All tumours retained their morphologic characteristics during the transplantation and passaging in the nude mice with only minor alterations in structure. The growth rates of the tumours showed great variability even in the same passage of each tumour, although the growth rates became somewhat more regular and predictable after some passages.     

A potential alternative strategy for myoblast transfer therapy: The use of sliced muscle grafts

Excellent long-term survival (up to 1 yr) of donor skeletal muscle cells was demonstrated using a mouse Y-chromosome specific probe, following the transplantation of grafts of whole muscles from male “normal” C57B1/ 10Sn mice into dystrophic muscles of female host mice. After the transplantation of equivalent sliced muscle grafts there was extensive movement of the male donor cells and fusion with host myofibres. This contrasts with the extremely poor survival of isolated myoblasts after injection into the same mouse model for Duchenne muscular dystrophy. The use of sliced muscle grafts may therefore represent a potential alternative approach to myoblast transfer therapy.     

Prolongation of adult skin allograft survival by cotransplantation of neonatal skin in antilymphocyte serum and donor bone marrow cell-treated mice

Adult male B6AF1 (H-2KIb/kDb/d) mice were given skin allografts from adult male C3H (H-2k) mice, with and without contralateral cotransplants. The cotransplants were of either adult or neonatal (less than 24 hr) C3H skin. In recipient mice treated peritransplantation with antilymphocyte serum and posttransplantation with an i.v. injection of donor-strain bone marrow cells, the presence of a neonatal cotransplant resulted in significantly longer survival of the adult grafts. Median survival time (MST) for adult grafts, for the group that received a neonatal cotransplant was greater than 100 days, in comparison to MSTs of 59 days and 55 days for the groups that received only single adult grafts or the adult graft with an adult cotransplant. These results suggest an active immunomodulatory contribution of neonatal tissue, and we term this phenomenon the "cotransplant effect." This prolongation of survival of the adult grafts by the neonatal cotransplants was statistically significant in animals treated with ALS and BMC, but not in recipients that were treated with ALS only (MST = 34 days, in comparison with 29 days for groups that received either a single adult graft or an adult graft with an adult cotransplant). The graft-prolonging effects of an infusion of donor BMC thus appear to potentiate the expression of the cotransplant effect. An understanding of this effect may lead to improved methods of controlling the allograft response to adult tissues in the clinical setting.