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Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45− (Sk-34) and CD34−/45−/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice.

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined by in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was totally stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs were closer to those in humans than to those in mice.

The effects of total body irradiation (TBI) to the capacity of skeletal muscle hypertrophy were quantified using the compensatory muscle hypertrophy model. We additionally assessed the responses of stem and/or progenitor cells in the muscles. A single TBI of 9.0, 5.0 and 2.5 Gy was delivered to C57BL/6 mice. Bone marrow stromal cells were obtained from GFP-Tg mice, and were injected into the tail vein of the recipient mice (1 × 106 cells/mouse), for bone marrow transplantation (BMT). Five weeks after TBI, the mean GFP-chimerism in the blood was 96 ± 0.8% in the 9 Gy, 83 ± 3.9% in the 5 Gy, and 8.4 ± 3.4% in the 2.5 Gy groups. This implied that the impact of 2.5 Gy is quite low and unavailable as the BMT treatment. Six weeks after the TBI/BMT procedure, muscle hypertrophy was induced in the right plantaris muscle by surgical ablation (SA) of the synergist muscles (gastrocnemius and soleus), and the contralateral left side was preserved as a control. The muscle hypertrophy capacity significantly decreased by 95% in the 9 Gy, 48% in the 5 Gy, and 36% in the 2.5 Gy groups. Furthermore, stem/progenitor cells in the muscle were enzymatically isolated and fractionated into non-sorted bulk cells, CD45-/34-/29+ (Sk-DN), and CD45-/34+ (Sk-34) cells, and myogenic capacity was confirmed by the presence of Pax7+ and MyoD+ cells in culture. Myogenic capacity also declined significantly in the Bulk and Sk-DN cell groups in all three TBI conditions, possibly implying that skeletal muscles are more susceptible to TBI than bone marrow. However, interstitial Sk-34 cells were insusceptible to TBI, retaining their myogenic/proliferative capacity.

The effects of liposome-encapsulated hemoglobin with high O2 affinity (h-LEH), an artificial O2 carrier in skeletal muscle, were studied by in situ fatigue resistance test in fast-type plantaris (PLT) and slow-type soleus (SOL) muscles with or without ischemia in the rat. The distal tendons of PLT and SOL muscles were isolated in situ and individually attached to the force transducers to record the developed tension in response to stimuli (80 Hz tetanus train, 1.5 minutes) to the ipsilateral sciatic nerve. The fatigue resistance test (five sets separated by 2-minute rests) was evaluated in terms of tension attenuation (fatigue) from the initial to the last tension (A) during each set, attenuation of the initial (B) or last tension (C) in each set, as compared to the first set in the presence or absence of ischemia or h-LEH (10 mL/kg). While ischemia significantly enhanced fatigue only in PLT, h-LEH showed no effect regardless of the perfusion pattern (normal/ischemia) or muscle-type (PLT/SOL) during each set (A). In parameter (B), set-by-set fatigue development was observed in PLT, whereas h-LEH-SOL showed a trend of advanced fatigue resistance. Such trends became clear in the parameter C (last tension), because h-LEH-SOL exerted, rather than decreased, the tension enhancement regardless of the presence or absence of ischemia, whereas there were no h-LEH effects in PLT. In addition, faster recovery of the nicotinamide adenine dinucleotide content in the muscle after 10 minutes of all fatigue tests was observed in h-LEH-SOL, while saline-SOL still showed a significantly higher value than that of control. These results suggested that additional O2 supply by h-LEH may accelerate the tricarboxylic acid cycle/electron transport chain in slow-type aerobic SOL muscle containing abundant mitochondria and contribute to the faster removal of muscle fatigue substances such as lactate.

Severe peripheral nerve injury, which does not promise natural healing, inevitably requires clinical treatment. Here, we demonstrated the facilitation effect of peripheral nerve regeneration using a cytokine cocktail secreted by skeletal muscle-derived stem cells (Sk-MSCs). Mouse sciatic nerve was transected with a 6 mm gap and bridged collagen tube, and the culture supernatant of Sk-MSCs with 20% adult mouse serum (AMS)/Iscove’s modified Dulbecco’s medium (IMDM) was administered into the tube immediately after the operation, followed by an injection once a week for six weeks through the skin to the surrounding tube of the cytokine (CT) group. Similarly, 20% AMS/IMDM without cytokines was administered to the non-cytokine control (NT) group. Tension recovery in the plantar flexor muscles via electrical stimulation at the upper portion of the damaged nerve site, as well as the numerical recovery of axons and myelinated fibers at the damaged site, were evaluated as an index of nerve regeneration. Specific cytokines secreted by Sk-MSCs were compared with damaged sciatic nerve-derived cytokines. Six weeks after operation, significantly higher tension output and numerical recovery of the axon and myelinated fibers were consistently observed in the CT group, showing that the present cytokine cocktail may be a useful nerve regeneration acceleration agent. We also determined 17 candidate factors, which are likely included in the cocktail.

Skeletal muscle-derived stem cells (Sk-MDSCs) are considered promising sources of adult stem cell therapy. Skeletal muscle comprises approximately 40-50% of the total body mass with marked potential for postnatal adaptive response, such as muscle hypertrophy, hyperplasia, atrophy, and regenerative capacity. This strongly suggests that skeletal muscle contains various stem/progenitor cells related to muscle-nerve-vascular tissues, which would support the above postnatal events even in adulthood.The focus of this review is the therapeutic potential of the Sk-MDSCs as an adult stem cell autograft. For this purpose, the validity of cell isolation and purification, tissue reconstitution capacityAlthough the clinical application of Sk-MDSCs began as a therapy for the systemic disease of Duchenne muscular dystrophy, here, through the unique local injection method, therapy for severely damaged peripheral nerves, particularly the long-gap nerve transection, has been introduced. The beneficial aspects of the use of Sk-MDSCs as the source of local tissue transplantation therapy have also been discussed.

Artificial oxygen (O2 ) carriers were reported to be protective in ischemia/reperfusion (I/R) in various organs including the heart. In the current study, 20 rats underwent ligation (MI) of the left anterior descending artery, were treated with 10 mL/kg of PEGylated carboxyhemoglobin bovine (SANGUINATE, S+, n = 10) or saline (S-, n = 10) 10 minutes after MI and daily thereafter for 3 days, and were followed by weekly echocardiography for 4 weeks, when they had left ventricular pressure volume relationship (PVR) analyses followed by necropsy. Echocardiography showed an increase in end-systolic dimension rather than end-diastolic dimension, preserved fractional shortening (36 vs. 26%, P < .01), and milder mitral regurgitation in S+ compared with S- rats. PVR revealed a milder increase in end-systolic volume, larger stroke volume (101 vs. 74 μL, P < .005) and cardiac output (33.4 vs. 23.8 mL/min, P = .004) in S+ rats in actual determination and under a wide range of standardized loading conditions 4 weeks after MI. Excised heart showed significantly limited area of MI (8.9 vs. 13.3%, P = .028). The results suggest that SANGUINATE in short-term repeated doses may accelerate weight recovery, preserving the myocardium, mitral competence, and cardiac function after MI. The mechanism of action and optimal treatment for MI remain to be studied.

Hybrid transplantation of skeletal muscle-derived multipotent stem cells (Sk-MSCs) and bioabsorbable polyglyconate (PGA) felt was studied as a novel regeneration therapy for the transected recurrent laryngeal nerve (RLN). Sk-MSCs were isolated from green fluorescence protein transgenic mice and then expanded and transplanted with PGA felt for the hybrid transplantation (HY group) into the RLN transected mouse model. Transplantation of culture medium (M group) and PGA + medium (PGA group) were examined as controls. After eight weeks, trans-oral video laryngoscopy demonstrated 80% recovery of spontaneous vocal-fold movement during breathing in the HY group, whereas the M and PGA groups showed wholly no recoveries. The Sk-MSCs showed active engraftment confined to the damaged RLN portion, representing favorable prevention of cell diffusion on PGA, with an enhanced expression of nerve growth factor mRNAs. Axonal re-connection in the HY group was confirmed by histological serial sections. Immunohistochemical analysis revealed the differentiation of Sk-MSCs into Schwann cells and perineurial/endoneurial cells and axonal growth supportive of perineurium/endoneurium. The number of axons recovered was over 86%. These results showed that the stem cell and cytokine delivery system using hybrid transplantation of Sk-MSCs/PGA-felt is a potentially practical and useful approach for the recovery of transected RLN.

Artificial oxygen (O2) carriers were reported to be protective in ischemia/reperfusion (I/R) in various organs including the heart. In the current study, 20 rats underwent ligation (MI) of the left anterior descending artery, were treated with 10 mL/kg of PEGylated carboxyhemoglobin bovine (SANGUINATE, S+, n = 10) or saline (S−, n = 10) 10 minutes after MI and daily thereafter for 3 days, and were followed by weekly echocardiography for 4 weeks, when they had left ventricular pressure volume relationship (PVR) analyses followed by necropsy. Echocardiography showed an increase in end‐systolic dimension rather than end‐diastolic dimension, preserved fractional shortening (36 vs. 26%, P

Postoperative damage of the urethral rhabdosphincter and nerve-vascular networks is a major complication of radical prostatectomy and generally causes incontinence and/or erectile dysfunction. The human skeletal muscle-derived stem cells, which have a synchronized reconstitution capacity of muscle-nerve-blood vessel units, were applied to this damage.Cells were enzymatically extracted from the human skeletal muscle, sorted using flow cytometry as CD34/45 (Sk-34) and CD29/34/45 (Sk-DN/29) fractions, and separately cultured/expanded in appropriate conditions within 2 weeks. Urethral damage was induced by manually removing one third of the wall of the muscle layer in nude rats. A mixture of expanded Sk-34 and Sk-DN/29 cells was applied on the damaged portion for the cell transplantation (CT) group. The same amount of media was used for the non-CT (NT) group. Urethral pressure profile was evaluated via electrical stimulation to assess functional recovery. Cell engraftments and differentiations were detected using immunohistochemistry and immunoelectron microscopy. Expression of angiogenic cytokines was also analyzed using reverse transcriptase-polymerase chain reaction and protein array.At 6 weeks after transplantation, the CT group showed a significantly higher functional recovery than the NT group (70.2% and 39.1%, respectively; P0.05). Histological analysis revealed that the transplanted human cells differentiated into skeletal muscle fibers, nerve-related Schwann cells, perineuriums, and vascular pericytes. Active paracrine angiogenic cytokines in the mixed cells were also detected with enhanced vascular formation in vivo.The transplantation of Sk-34 and Sk-DN/29 cells is potentially useful for the reconstitution of postoperative damage of the urethral rhabdosphincter and nerve-vascular networks.

As a novel approach to distinguish skeletal myogenic cell populations, basal lamina (BL) formation of myogenic cells was examined in the mouse compensatory enlarged plantaris muscles in vivo and in fiber-bundle cultures in vitro. MyoD(+) myogenic cells located inside the regenerative muscle fiber BL were laminin(-) but interstitial MyoD(+) cells were laminin(+). This was also confirmed by electron microscopy as structural BL formation. Similar trends were observed in the fiber-bundle cultures including satellite cells and interstitial myogenic cells and laminin(+) myogenic cells predominantly showed non-adhesive (non-Ad) behavior with Pax7(-), whereas laminin(-) cells were adhesive (Ad) with Pax7(+). Moreover, non-Ad/laminin(+) and Ad/laminin(-) myotubes were also observed and the former type showed spontaneous contractions, while the latter type did not. The origin and hierarchy of Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells were also examined using skeletal muscle interstitium-derived CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) multipotent stem cells, which were composed of non-committed myogenic cells with a few (1%) Pax7(+) cells in the Sk-DN cells at fresh isolation. Both cell types were separated by Ad/non-Ad capacity in repetitive culture. As expected, both Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells consistently appeared in the Ad and non-Ad cell culture. However, Ad/Pax7(+)/laminin(-) cells were repeatedly detected in the non-Ad cell culture, while the opposite phenomenon did not occur. This indicates that the source of non-Ad/ Pax7(-)/laminin(+) myogenic cells was present in the Sk-34 and Sk-DN stem cells and they were able to produce Ad/ Pax7(+)/ laminin(-) myogenic cells during myogenesis as primary myoblasts and situated hierarchically upstream of the latter cells.

The therapeutic effects of voluntary exercise on the recovery of long-gap nerve injury following the bridging of an acellular conduit filled with human skeletal muscle-derived stem cells (Sk-SCs) have been described. Human Sk-SCs were sorted as CD34+/45− (Sk-34) cells, then cultured/expanded under optimal conditions for 2 weeks. Surgery to generate a long-gap sciatic nerve injury was performed in athymic nude mice, after which the mice were divided into exercise (E) and non-exercise (NE) groups. The mice were housed in standard individual cages, and voluntary exercise wheels were introduced to the cages of the E group one week after surgery. After 8 weeks, the human Sk-34 cells were actively engrafted, and showed differentiation into Schwann cells and perineurial cells, in both groups. The recovery in the number of axons and myelin in the conduit and downstream tibial nerve branches, and the lower hindlimb muscle mass and their tension output, was consistently higher by 15–25% in the E group. Moreover, a significantly higher innervation ratio of muscle spindles, reduced pathological muscle fiber area, and acceleration of blood vessel formation in the conduit were each observed in the E group. These results showed that the combined therapy of tube-bridging, Sk-34 cell transplantation, and voluntary exercise is a potentially practical approach for recovery following long-gap nerve injury.

The differentiation and/or therapeutic potential of skeletal muscle-derived stem cells for cardiac infarction have been studied extensively for use in cellular cardiomyoplasty, as injured cardiomyocytes exhibit limited regenerative capacity. We previously reported cardio-myogenic differentiation of skeletal muscle-derived CD34+/45(-) (Sk-34) stem cells after therapeutic transplantation. However, the clonal differentiation potential of these cells remains unknown. Here, we show that skeletal muscle-derived CD34(-)/45(-) (Sk-DN) stem cells, which are situated upstream of Sk-34 cells in the same lineage, exhibit clonal differentiation into cardiomyocytes after single cell-derived single-sphere implantation into myocardium. Sk-DN cells were enzymatically isolated from green fluorescent protein (GFP) transgenic mice and purified by flow cytometry, and were then clonally cultured in collagen-based medium with bFGF and EGF after clonal cell sorting. Single cell-derived single-sphere colonies of Sk-DN cells were directly implanted into the wild-type mouse myocardium. At 4 weeks after implantation, donor cells exhibited typical cardiomyocyte structure with the formation of gap-junctions between donor and recipient cells. Expression of specific mRNAs for cardiomyocytes, such as cardiac actin and GATA-4, Nkx2-5, Isl-1, Mef2, and Hand2, were also seen in clonal cell cultures of Sk-DN cells. Cell fusion-independent differentiation was also confirmed by bulk cell transplantation using Cre- and loxP (enhanced GFP)-mice. We conclude that Sk-DN cells can give rise to cardiac muscle cells clonally, and that skeletal muscle includes a practical cell source for cellular cardiomyoplasty.

The hierarchical relationship of skeletal muscle-derived multipotent stem cells sorted as CD34(+)/CD45(-) (Sk-34) and CD34(-)/CD45(-) (Sk-DN) cells, which have synchronized reconstitution capacities for blood vessels, peripheral nerves, and muscle fibers, was examined. Expression of Sca-1 and CD34 (typical state of freshly isolated Sk-34 cells) in Sk-DN cells was examined using in vitro culture and in vivo cell implantation. Sk-DN cells sequentially expressed Sca-1 and CD34 during cell culture showing self-maintenance and/or self-renewal-like behavior, and are thus considered hierarchically upstream of Sk-34 cells in the same lineage. Sk-34 and Sk-DN cells were further divided into small and large cell fractions by cell sorting. Immunocytochemistry using anti-Pax7 was performed at the time of isolation (before culture) and revealed that only 1% of cells in the large Sk-DN cell fraction were positive for Pax7, while Sk-34 cells and 99% of Sk-DN cells were negative for Pax7. Therefore, putative satellite cells were possibly present in the large Sk-DN cell fraction. However, serial analysis of Pax7 expression by RT-PCR and immunocytochemistry for single and 2 to >40 clonally proliferated Sk-34 and Sk-DN cells revealed that both cell types expressed Pax7 after several asymmetric cellular divisions during clonal-cell culture. In addition, production of satellite cells was seen after muscle fiber formation following Sk-34 or Sk-DN cell transplantation into damaged muscle, and even in the nonmuscle tissue environment (beneath the renal capsule). Thus, Sk-DN cells are situated upstream of Sk-34 cells and both cells can produce Pax7+ cells (putative satellite cells) after cellular division.

The differentiation potential of skeletal muscle-derived stem cells (MDSCs) after in vitro culture and in vivo transplantation has been extensively studied. However, the clonal multipotency of MDSCs has yet to be fully determined. Here, we show that single skeletal muscle-derived CD34−/CD45− (skeletal muscle-derived double negative [Sk-DN]) cells exhibit clonal multipotency that can give rise to myogenic, vasculogenic, and neural cell lineages after in vivo single cell-derived single sphere implantation and in vitro clonal single cell culture. Muscles from green fluorescent protein (GFP) transgenic mice were enzymatically dissociated and sorted based on CD34 and CD45. Sk-DN cells were clone-sorted into a 96-well plate and were cultured in collagen-based medium with basic fibroblast growth factor and epidermal growth factor for 14 days. Individual colony-forming units (CFUs) were then transplanted directly into severely damaged muscle together with 1 × 105 competitive carrier Sk-DN cells obtained from wild-type mice muscle expanded for 5 days under the same culture conditions using 35-mm culture dishes. Four weeks after transplantation, implanted GFP+ cells demonstrated differentiation into endothelial, vascular smooth muscle, skeletal muscle, and neural cell (Schwann cell) lineages. This multipotency was also confirmed by expression of mRNA markers for myogenic (MyoD, myf5), neural (Musashi-1, Nestin, neural cell adhesion molecule-1, peripheral myelin protein-22, Nucleostemin), and vascular (α-smooth muscle actin, smoothelin, vascular endothelial-cadherin, tyrosine kinase-endothelial) stem cells by clonal (single-cell derived) single-sphere reverse transcription-polymerase chain reaction. Approximately 70% of clonal CFUs exhibited expression of all three cell lineages. These findings support the notion that Sk-DN cells are a useful tool for damaged muscle-related tissue reconstitution by synchronized vasculogenesis, myogenesis, and neurogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2-8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

Skeletal muscle makes up 40-50% of body mass, and is thus considered to be a good adult stem cell source for autologous therapy. Although, several stem/progenitor cells have been fractionated from mouse skeletal muscle showing a high potential for therapeutic use, it is unclear whether this is the case in human. Differentiation and therapeutic potential of human skeletal muscle-derived cells (Sk-Cs) was examined. Samples (5-10 g) were obtained from the abdominal and leg muscles of 36 patients (age, 17-79 years) undergoing prostate cancer treatment or leg amputation surgery. All patients gave informed consent. Sk-Cs were isolated using conditioned collagenase solution, and were then sorted as CD34(-)/CD45(-)/CD29(+) (Sk-DN/29(+)) and CD34(+)/CD45(-) (Sk-34) cells, in a similar manner as for the previous mouse Sk-Cs. Both cell fractions were appropriately expanded using conditioned culture medium for about 2 weeks. Differentiation potentials were then examined during cell culture and in vivo transplantation into the severely damaged muscles of athymic nude mice and rats. Interestingly, these two cell fractions could be divided into highly myogenic (Sk-DN/29(+)) and multipotent stem cell (Sk-34) fractions, in contrast to mouse Sk-Cs, which showed comparable capacities in both cells. At 6 weeks after the separate transplantation of both cell fractions, the former showed an active contribution to muscle fiber regeneration, but the latter showed vigorous engraftment to the interstitium associated with differentiation into Schwann cells, perineurial/endoneurial cells, and vascular endothelial cells and pericytes, which corresponded to previous observations with mouse SK-Cs. Importantly, mixed cultures of both cells resulted the reduction of tissue reconstitution capacities in vivo, whereas co-transplantation after separate expansion showed favorable results. Therefore, human Sk-Cs are potentially applicable to therapeutic autografts and show multiple differentiation potential in vivo.

Objective—Tissue regeneration requires both growth factor and extracellular matrix such as collagen, serving as a scaffold for cell growth. We established FNCBD-VEGF121, consisting of the fibronectin collagen-binding domain (FNCBD) and vascular endothelial growth factor (VEGF) 121, and investigated its properties.Methods and Results—FNCBD-VEGF121 specifically bound to gelatin and type I, II, III, IV, and V collagen. This collagen-bound FNCBD-VEGF121 captured soluble VEGF receptor 2 (VEGFR-2)/Fc chimeric protein. Cell growth-promoting activity of FNCBD-VEGF121 was almost identical to that of VEGF121. The VEGF fusion protein significantly enhanced the expression of VEGFR-2 (71.6±0.8%) on endothelial progenitor cells (EPCs) derived from umbilical cord blood. Expectably, the collagen-bound VEGF fusion protein not only promoted the growth of endothelial cells (ECs) but also induced the expression of VEGFR-2 (63.7±0.8%) on non-adherent cells expanded from bone marrow CD34+cells. Moreover, the VEGF fusion protein enhanced sprout formation of ECs in a matrigel model. In vivo experiments revealed that FNCBD-VEGF121 had local effects but not systemic effect on EPC mobilization.Conclusions—These results suggest that FNCBD-VEGF121 stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates both ECs and EPCs in situ, supplying a vascular regeneration niche.

Endometrium is a highly regenerative adult tissue that undergoes repeated degeneration and regeneration following menarche. Therefore, it is believed that endometrium contains stem and/or progenitor cells in order to compensate for the regeneration of tissue components. We report here that stem-like cells having vasculogenic potential are present in the uterus. Enzymatically extracted cells from murine uteri were characterized and fractionated into four subpopulations by flowcytometry; CD34(+)/45(-) (Ut-34), CD34(-)/45(-) (Ut-DN) and the remaining CD45(+) cell fractions (CD34(+)/45(+) and CD34(-)/45(+) cells). The Ut-34 and Ut-DN fractions were mostly negative for putative endothelial cell (EC) markers, such as CD31, Flk-1, c-kit and VE-cadherin, although the Ut-DN fraction contained 2.8% CD31(+) cells. Ut-DN cells were further divided into CD31(+) and CD31(-) fractions. Three cell populations were obtained from green fluorescence protein (GFP) transgenic mice and were transplanted into injured wild-type mouse skeletal muscle. At 4 weeks after cell transplantation, donor-derived vascular smooth muscle and ECs were observed in the injured recipient muscle. A similar trend was observed in the Ut-34 group, but differentiation into vascular smooth muscle was predominant. In contrast, the Ut-DN/31(+) cell-transplanted group showed preferential differentiation into vascular ECs, thus suggesting that they were relatively committed preexisting ECs. These characteristics were also seen in vitro, in clonal cell cultures. Interestingly, donor derived Ut-DN/31(+), Ut-DN/31(-) and Ut-34 cells could not be identified after bone marrow (BM) transplantation, thus confirming that they are not derived from BM. It therefore appeared that tissue-specific vasculogenic cells are present in the murine uterus and that they exhibit vascular formation, even in different tissue microenvironments.

Background—Recent studies have shown that skeletal muscle–derived stem cells (MDSCs) can give rise to several cell lineages after transplantation. However, the potential therapeutic uses of MDSCs, the functional significance of the transplanted tissue, and vasculogenesis, myogenesis, and reconstitution of other tissues have yet to be investigated in detail. In addition, the relationship between MDSCs and mesenchymal bone marrow cells is of interest.Methods and Results—We developed a severe-damage model of mouse tibialis anterior muscle with a large deficit of nerve fibers, muscle fibers, and blood vessels. We investigated the potential therapeutic use of freshly isolated CD34+/45−(Sk-34) cells. Results showed that, after transplantation, implanted cells give rise to myogenic, vascular (pericytes, vascular smooth muscle cells, and endothelial cells), and neural (Schwann) cells, as well as contributing to the synchronized reconstitution of blood vessels, muscle fibers, and peripheral nerves, with significant recovery of both mass and contractile function after transplantation. Investigation of Sk-34 cell transplantation to the renal capsule (nonmuscle tissue) and fluorescence in situ hybridization analysis for the transplanted muscle detecting the Y chromosome revealed the intrinsic plasticity of the Sk-34 cell population. In addition, there were no donor-derived Sk-34 cells in the muscle of lethally irradiated bone marrow–transplanted animals, indicating that the Sk-34 cells were not derived from bone marrow.Conclusions—These findings indicate that freshly isolated skeletal muscle–derived Sk-34 cells are potentially useful for reconstitution therapy of the vascular, muscular, and peripheral nervous systems. These results provide new insights into somatic stem and/or progenitor cells with regard to vasculogenesis, myogenesis, and neurogenesis.

TAMAKI, T., T. SHIRAISHI, H. TAKEDA, T. MATSUMIYA, R. R. ROY, and V. R. EDGERTON. Nandrolone Decanoate Enhances Hypothalamic Biogenic Amines in Rats. Med. Sci. Sports Exerc., Vol. 35, No. 1, pp. 32-38, 2003. Purpose: To identify possible mechanisms for an anabolic-androgenic steroid induced increase in aggressive behavior and work capacity, the levels of some biogenic amines considered to be closely related to a systemic hyper-adrenergic state were measured in selected regions of the brain. Methods: Wistar male rats were divided randomly into five groups: nontreated (control), oil-vehicle-treated (vehicle) or one of three (therapeutic dose and 10- or 100-fold higher dose) anabolic-androgenic steroid-treated (steroid-1, -2, -3) groups. Rats in the steroid and vehicle groups were given a single dose of nandrolone decanoate or oil vehicle, respectively, one week before tissue sampling. The levels of norepinephrine (NE) and its metabolite, 4-hydroxy-3-methoxyphenylglycol (MHPG), serotonin (5-HT) and its metabolite, 5-hydroxy- indole-3-acetic acid (5-HIAA) were measured in the cerebral cortex, hypothalamus and cerebellum by high-performance liquid chromatography. Immunostaining for c-fos was performed as a confirmation of increased neural activity. Results: The levels of NE and MHPG were increased by ~2- and ~7-fold in the hypothalamus of the steroid-2 compared with the control and vehicle groups. The levels of 5-HT and 5-HIAA were ~40 and ~50% higher in the steroid-2 compared with the control and vehicle groups. A significantly higher number of c-fos expressing neurons were observed in the periventricular region of the steroid-2 than the control and vehicle groups, indicating enhanced neuronal activity after nandrolone decanoate treatment. Conclusions: The present results, combined with previously reported findings of physical performance enhancement after anabolic-androgenic steroid treatment, are consistent with the interpretation that elevated levels of adrenergic and serotonergic amines in the hypothalamus could contribute to aggressive behaviors as well as improved physical performance. Key Words: NANDROLONE-DECANOATE, HYPOTHALAMUS, NOREPINEPHRINE, SEROTONIN, C-FOS

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45− fraction. Cells in this fraction were ∼94% positive for Sca-1, and mostly negative (

Qualitative changes in the peripheral motor system were examined using young, adult, middle-aged, and old-aged rats in order to assess before and after the appearance of sarcopenia symptoms. Significant loss of muscle mass and strength, and slow-type fiber grouping with a loss of innervated nerve fibers were used as typical markers of sarcopenia. Dynamic twitch and tetanus tension and evoked electromyogram (EEMG) were measured via electrical stimulation through the sciatic nerve under anesthesia using our force-distance transducer system before and after sciatectomy. Digital and analog data sampling was performed and shortening and relaxing velocity of serial twitches was calculated with tension force. Muscle tenderness in passive stretching was also measured as stretch absorption ability, associated with histological quantitation of muscle connective tissues. The results indicated the validity of the present model, in which old-aged rats clearly showed the typical signs of sarcopenia, specifically in the fast-type plantaris muscles, while the slow-type soleus showed relatively mild syndromes. These observations suggest the following qualitative alterations as the pathophysiological mechanism of sarcopenia: (1) reduction of shortening and relaxing velocity of twitch; (2) decline of muscle tenderness following an increase in the connective tissue component; (3) impaired recruitment of motor units (MUs) (sudden depression of tetanic force and EEMG) in higher stimulation frequencies over 50–60 Hz; and (4) easy fatigability in the neuromuscular junctions. These findings are likely to be closely related to significant losses in fast-type MUs, muscle strength and contraction velocity, which could be a causative factor in falls in the elderly. Importantly, some of these symptoms began in middle-aged rats that showed no other signs of sarcopenia. Thus, prevention should be started in middle age that could be retained relatively higher movement ability.

The purpose of the present study was to compare the myogenic response of hindlimb muscles in young (14–20 wk of age) and old (>120 wk of age) rats with a single exhaustive bout of heavy resistance weight lifting. [3H]thymidine and [14C]leucine labeling were monitored for up to 2 wk after the exercise bout to estimate serial changes in mitotic activity and the level of amino acid uptake and myosin synthesis. Histological, histochemical, and immunohistochemical [anti-5-bromo-2′-deoxyuridine and myogenic determination genes (MyoD)] analyses of whole muscles and analysis of muscle-specific gene expression (MyoD) using Western blotting and RT-PCR were performed. Old rats showed significant muscle atrophy and a lower exercise capacity than young rats. Exercise-induced muscle damage, as assessed in histological sections, and increases in serum creatine kinase activity were evident in both young and old exercised groups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in [14C]leucine uptake during the 14 days postexercise (peaks at 1–4 and 10 days) in young rats: only the first peak was observed in old rats. There was a lower uptake of [14C]leucine in the myosin fraction and an impaired expression of MyoD at the protein (immunohistochemistry and Western blotting) and mRNA (RT-PCR) levels in old rats throughout the postexercise period. These results demonstrate a reduced reparative capability of muscle in response to a single bout of exercise in old compared with young rats.

Aim: To cover the large tissue deficits associated with significant loss of function following surgery, a 3D gel-patch-like nerve–vascular reconstitution system was developed using the skeletal muscle-derived multipotent stem cell (Sk-MSC) sheet pellet. Materials & methods: The Sk-MSC sheet pellet was prepared from GFP transgenic mice by the collagenase extraction and 7 days expansion cell culture, and transplanted into a severe muscle damage model with large disruptions to muscle fibers, blood vessels and peripheral nerves. Results: At 4 weeks after transplantation, engrafted cells contributed to nerve–vascular regeneration associated with cellular differentiation into Schwann cells, perineurial/endoneurial cells, vascular endothelial cells and pericytes. However, skeletal myogenic differentiation was scarcely observed. Paracrine effects regarding donor cells/tissues could also be expected, because of the active expression of neurogenic and vasculogenic factor mRNAs in the sheet pellet. Conclusion: These results indicate that the vigorous skeletal myogenic potential of Sk-MSCs was clearly reduced in the sheet pellet preparation and this method may be a useful adjuvant for nerve–vascular regeneration in various tissue engineering applications.

Losses in vital functions of the somatic motor and sensory nervous system are induced by severe long-gap peripheral nerve transection injury. In such cases, autologous nerve grafts are the gold standard treatment, despite the unavoidable sacrifice of other healthy functions, whereas the prognosis is not always favorable. Here, we use human skeletal muscle-derived stem cells (Sk-SCs) to reconstitute the function after long nerve-gap injury. Muscles samples were obtained from the amputated legs from 9 patients following unforeseen accidents. The Sk-SCs were isolated using conditioned collagenase solution, and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN/29+) cells. Cells were separately cultured/expanded under optimal conditions for 2 weeks, then injected into the athymic nude mice sciatic nerve long-gap model (7-mm) bridging an acellular conduit. After 8-12 weeks, active cell engraftment was observed only in the Sk-34 cell transplanted group, showing preferential differentiation into Schwann cells and perineurial/endoneurial cells, as well as formation of the myelin sheath and perineurium/endoneurium surrounding regenerated axons, resulted in 87% of numerical recovery. Differentiation into vascular cell lineage (pericyte and endothelial cells) were also observed. A significant tetanic tension recovery (over 90%) of downstream muscles following electrical stimulation of the sciatic nerve (at upper portion of the gap) was also achieved. In contrast, Sk-DN/29+ cells were completely eliminated during the first 4 weeks, but relatively higher numerical (83% vs. 41% in axon) and functional (80% vs. 60% in tetanus) recovery than control were observed. Noteworthy, significant increase in the formation of vascular networks in the conduit during the early stage (first 2 weeks) of recovery was observed in both groups with the expression of key factors (mRNA and protein levels), suggesting the paracrine effects to angiogenesis. These results suggested that the human Sk-SCs may be a practical source for autologous stem cell therapy following severe peripheral nerve injury.

Background.Significant and/or complete rupture in the musculotendinous junction (MTJ) is a challenging lesion to treat because of the lack of reliable suture methods. Skeletal muscle-derived multipotent stem cell (Sk-MSC) sheet-pellets, which are able to reconstitute peripheral nerve and muscular/vascular tissues with robust connective tissue networks, have been applied as a “bio-bond”.Methods.Sk-MSC sheet-pellets, derived from GFP transgenic-mice after 7 days of expansion culture, were detached with EDTA to maintain cell–cell connections. A completely ruptured MTJ model was prepared in the right tibialis anterior (TA) of the recipient mice, and was covered with sheet-pellets. The left side was preserved as a contralateral control. The control group received the same amount of the cell-free medium. The sheet-pellet transplantation (SP) group was further divided into two groups; as the short term (4–8 weeks) and long term (14–18 weeks) recovery group. At each time point after transplantation, tetanic tension output was measured through the electrical stimulation of the sciatic nerve. The behavior of engrafted GFP+tissues and cells was analyzed by fluorescence immunohistochemistry.Results.The SP short term recovery group showed average 64% recovery of muscle mass, and 36% recovery of tetanic tension output relative to the contralateral side. Then, the SP long term recovery group showed increased recovery of average muscle mass (77%) and tetanic tension output (49%). However, the control group showed no recovery of continuity between muscle and tendon, and demonstrated increased muscle atrophy, with coalescence to the tibia during 4–8 weeks after operation. Histological evidence also supported the above functional recovery of SP group. Engrafted Sk-MSCs primarily formed the connective tissues and muscle fibers, including nerve-vascular networks, and bridged the ruptured tendon–muscle fiber units, with differentiation into skeletal muscle cells, Schwann cells, vascular smooth muscle, and endothelial cells.Discussion.This bridging capacity between tendon and muscle fibers of the Sk-MSC sheet-pellet, as a “bio-bond,” represents a possible treatment for various MTJ ruptures following surgery.

Change in body weight and lower hindlimb length as physical properties, muscle weight and length as whole-muscle properties, and fiber numbers, diameters, and numbers of proliferating cells as intramuscular properties, were measured in 2- to 60-week-old Wistar male rats. Results showed that the rapid increase in muscle weight (muscle mass) in the postnatal "growing phase" depended on the longitudinal and transverse growth. The increase in muscle length is due to longitudinal growth of muscle fiber, which may be derived from an increase in the number of sarcomeres. The increase in muscle girth depends on the increase of individual fiber diameter (muscle fiber hypertrophy) and fiber numbers (hyperplasia). The remaining increase in muscle mass in the "steady phase" (after the 10th week) is caused entirely by transverse growth, depending mainly on the muscle fiber hypertrophy (but may include increase of connective tissues). In conclusion, to study the effects of exercise and/or various overloads on muscle hypertrophy or hyperplasia, especially in the case of the plantaris muscle in Wistar rats, they should be 10-30 weeks old, and over 300 g in body weight, because in this period their characteristics remain most constant.

Background: To examine whether the complex branched fibers observed in mdx mutant mice were formed in normal skeletal muscles, long-term repetitive muscle trauma was applied to muscle of normal Wistar male rats. Methods: Three kinds of artificial muscle trauma—crush injury, bupivacaine hydrochloride treatment, and forced stretching of contracting muscle (eccentric contraction)—were performed once a week for 10 weeks to achieve a state of repetitive degeneration and regeneration in the muscles. Two weeks after the final treatment, numerical, histochemical, and three-dimensional analyses by scanning electron microscopy were performed. Results: Mean numbers of total branched fibers of the three groups were increased compared with normal control values, especially in the bupivacaine treatment group (three- to fivefold greater than in the other two groups). Aggregations of fibers of the same type which usually appear in mdx mice were observed in various parts of histological sections of the bupivacaine treatment group and only in a part of the crush injury group. No aggregations were observed in sections of the forced stretching group. In the three-dimensional analysis, complex branched fibers appearing as an “anastomosing syncytial reticulum” were observed only in the bupivacaine treatment group. Conclusions: These findings suggest that the formation of an anastomosing syncytial reticulum is one of the adaptation mechanisms of normal skeletal muscle rather than a specific event in mdx mutant mice, and long-term repeated trauma of the same fiber is necessary for this formation. Adaptive changes in the muscles with the three different types of muscle trauma are discussed. © 1994 Wiley-Liss, Inc.

Skeletal muscle-derived myoblasts were initially considered to be a potential source for cardiomyoplasty. These cells were expected to be able to trans-differentiate into cardiomyocytes after transplantation in the cardiac muscle environment. However, several studies have shown differentiation into multinucleated myotubes, and no differentiation into cardiomyocytes has been observed. Complete differentiation of murine skeletal muscle interstitium-derived multipotent stem cells (SKMI-DMSCs) into cardiomyocytes was recently demonstrated following engraftment into the infarcted heart muscle (left ventricle). Engrafted SKMI-DMSCs showed typical formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells in vivo. When SKMI-DMSCs were co-cultured with embryonic cardiomyocytes on a glass slide, cells typically formed sphere-like cell aggregations together in vitro. These mixed cell aggregations showed spontaneous synchronous contractions with the formation of gap-junctions between aggregated cells. Cardiomyocyte differentiation was also confirmed by the expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2. Vigorous expression of these factors in the SKMI-DMSCs was seen after co-culture, and lower expression of these factors was also observed in their clonal cell culture. These results indicate that SKMI-DMSCs are a potential source for practical cellular cardiomyoplasty, and that they should be isolated and transplanted in a more primitive state for milieu-dependent differentiation of stem cells.

INTRODUCTION AND OBJECTIVES: Reconstruction of contracted bladder by illeocystoplasty using a segment of bowel has got a wide acceptance in the urological field. In this case, however, nervous control is disrupted due to the complete section of bladder-wall and replaced bowel. Thus, conservation of bladder functions such as wall contractions and sensations is difficult, whereas capacity growth is possible. Here, we tried to dilate the bladder wall associate with vascular and nerve reconstitutions using transplantation of stem cell sheet-pellets, which is formed by expanded skeletal muscle-derived multipotent stem cells (MDSCs) that have a synchronized reconstitution capacity of vascular, muscular and peripheral nervous systems. METHODS: MDSCs were obtained from green fluorescent protein (GFP) transgenic mice muscles. Muscles were enzymatically dissected into several fiber-bundles, and totally cultured for 5 days. Then, cells were suspended by EDTA, and removed fibers and other debris. Remaining cells were cultured for 4 days to obtaining confluent cellular sheets. Sheets were softly suspended by EDTA, and corrected by centrifuge (MDSCs sheet-pellets). Wild-type C57/BL mice were used for recipients. Surface of the bladder-wall was sectioned from smooth muscle layers along midline legion maintaining mucosal layer, and tag ends were spread out for a transverse plane. The MDSCs sheet-pellets were pasted on the open up thin-walled bladder. Cellular engraftment and differentiation were determined by fluorescence immunohistochemistry 4 wks after transplantation. RESULTS: GFP transplanted cells were actively engrafted on the bladder-wall and contributed to recover the thickness of the wall (Fig. 1). In addition, transplanted GFP cells differentiated into vascular cells (pericytes, endothelial cells, and adventitial cells) and nervous cells (Schwann cells and perineurial cells), and contribute to the reconstitution of blood vessels and peripheral nerves (arrows in Fig. 1). CONCLUSIONS: Present Bladder augmentation method using one-half wall section cystoplasty and MDSCs sheet-pellets transplantation was potentially useful for the reconstruction of contracted bladder associate with native bladder functions.

Background We herein describe a surgical technique for the repair of complete tear of the pectoralis major (PM) tendon using endobuttons to strengthen initial fixation. Methods Five male patients (3 judo players, 1 martial arts player, and 1 body builder) were treated within 2 weeks of sustaining complete tear of the PM tendon. Average age at surgery and follow-up period were 28.4 years (range, 23-33) and 28.8 months (range, 24-36). A rectangular bone trough (about 1 × 4 cm) was created on the humerus at the insertion of the distal PM tendon. The tendon stump was introduced into this trough, and fixed to the reverse side of the humeral cortex using endobuttons and non-absorbable suture. Clinical assessment of re-tear was examined by MRI. Shoulder range of motion (ROM), outcome of treatment, and isometric power were measured at final follow-up. Results There were no clinical re-tears, and MRI findings also showed continuity of the PM tendon in all cases at final follow-up. Average ROM did not differ significantly between the affected and unaffected shoulders. The clinical outcomes at final follow-up were excellent (4/5 cases) or good (1/5). In addition, postoperative isometric power in horizontal flexion of the affected shoulder showed complete recovery when compared with the unaffected side. Conclusions Satisfactory outcomes could be obtained when surgery using the endobutton technique was performed within 2 weeks after complete tear of the PM tendon. Therefore, our new technique appears promising as a useful method to treat complete tear of the PM tendon.

Three-dimensional cytoarchitecture and types and features of muscle fibers were examined in soleus muscles from mdx mutant mice at different stages of development. In the 2-week-old mice, no abnormal muscle fibers were observed light microscopically, whereas in the 4-week-old animals, disrupted fibers were frequent in light microscopy and scanning electron microscopy. Muscle fibers fused with several short fiber branches appeared at the sixth week after birth and increased in number until the tenth week. In the 1-year-old mice, approximately ten or more muscle fibers were seen fused together. They had many complex branches forming an “anastomosing syncytial reticulum.” Muscle fibers with irregular diameters and aggregations of the same type fibers were also observed. Our results demonstrated that these complex branched fibers might be formed by long term repetition of the degeneration and regeneration cycle during the development of soleus muscles, indicating that the characteristic features of muscle fibers with irregular diameters and aggregations of the same type fibers are certainly dependent on the existence of the complex branched fibers. © 1993 Wiley-Liss, Inc.

Stem cells other than satellite cells that can give rise to primary myoblasts, which are able to form additional new fibers postnatally, are present in the interstitial spaces of skeletal muscle. These cells are sorted into CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) cell fractions, and they are wholly (>99%) negative for Pax7 at initial isolation. Colony-forming units of these cells typically include non-adherent type myogenic cells, while satellite cells are known to be adherent in cell culture. In addition, both Pax7(-) and Pax7(+) cells are produced, depending on asymmetric cell division. A large number of myotubes are also formed in each colony, thus suggesting that putative Pax7(+) satellite cells also present in each colony. Interestingly, interstitial myogenic cells show basal lamina formation at early stages of myogenesis in response to various types of stimulation in compensatory enlarged muscle, a property that satellite cells do not possess in the parent fiber basal lamina cylinder. Basal lamina formation and production of satellite cells are essential before muscle fiber establishment in vivo. It is therefore likely that myogenic cells in skeletal muscle can be divided into two populations: 1) basal lamina-producing myogenic cells; and 2) basal lamina-non-producing myogenic cells. The latter population may be Pax7(+) satellite cells showing adherent capacity in cell culture, while the lamina-producing myogenic population derived from interstitial multipotent stem cells, which is predominant among Sk-34 and Sk-DN cells, plays a role in primary myoblast generation and shows non-adherent behavior in culture. Therefore, the physiological role of interstitial myogenic cells is as a source for new postnatal muscle fiber formation, and multinucleated muscle fibers (cells) are potentially formed clonally.

Cellular responses in the compensatory hypertrophied (plantaris) muscle induced by surgical ablation of synergistic muscles (soleus and gastrocnemius) were determined during 10-week anabolic androgenic steroid (AAS) treatment. Adult Wistar male rats were divided randomly into the Control and Steroid groups, and contralateral surgery was performed. Nandrolone decanoate was administered to the Steroid group. [3H]thymidine and [14C]leucine labeling were used to determine the serial changes in cellular mitotic activity and amino acid uptake. Myogenic cells and cellular responses in blood vessels and nerve fibers were analyzed by immunohistochemistry. Significantly lower cellular mitotic activity associated with lower volume of muscle fiber necrosis was observed in the Steroid group during the first week. However, amino acid uptake and final muscle wet weight gain did not differ between the groups. Marked activation/proliferation of muscular, vascular, and peripheral nerve-related cells was seen with the inflammatory responses in both groups. However, this activation was dependent on the volume of muscle fiber damage and was not preferentially accelerated by AAS loading. These results indicated that AAS loading significantly diminished muscle fiber damages, but they did not accelerate final muscle wet weight gain and activation of myogenic, vascular, and peripheral nerve related cells in the compensatory enlarged muscles.

Tissue inflammation and multiple cellular responses in the compensatory enlarged plantaris (OP Plt) muscle induced by surgical ablation of synergistic muscles (soleus and gastrocnemius) were followed over 10 weeks after surgery. Contralateral surgery was performed in adult Wistar male rats. Cellular responses in muscle fibers, blood vessels and nerve fibers were analyzed by immunohistochemistry and electron microscopy. Severe muscle fiber damage and disappearance of capillaries associated with apparent tissue edema were observed in the peripheral portion of OP Plt muscles during the first week, whereas central portions were relatively preserved. Marked cell activation/proliferation was also mainly observed in peripheral portions. Similarly, activated myogenic cells were seen not only inside but also outside of muscle fibers. The former were likely satellite cells and the latter may be interstitial myogenic cells. One week after surgery, small muscle fibers, small arteries and capillaries and several branched-muscle fibers were evident in the periphery, thus indicating new muscle fiber and blood vessel formation. Proliferating cells were also detected in the nerve bundles in the Schwann cell position. These results indicate that the compensatory stimulated/enlarged muscle is a suitable model for analyzing multiple physiological cellular responses in muscle-nerve-blood vessel units under continuous stretch stimulation.

Background. Postoperative damage of the urethral rhabdosphincter (URS) and neurovascular bundle (NVB) is a major operative complication ofradical prostatectomy. It is generally recognized to be caused by unavoidable surgical damage to the muscle-nerve-blood vessel units around the urethra. We attempted to treat this damage using skeletal muscle-derived stem cells, which are able to reconstitute muscle-nerve-blood vessel units. Methods. Cells were enzymatically extracted and sorted by flow cytometry as CD34 + /45 - (Sk-34) and CD34 - /45 - (Sk-DN) cells from green fluorescent protein transgenic mice and rats. URS-NVB damage was induced by manually removing one-third of the total URS and unilateral invasion of NVB in wild-type Sprague-Dawley and node rats. Freshly isolated Sk-34, Sk-34+Sk-DN cells, and cultured Sk-DN cells were directly transplanted into the damaged portion. Results. At 4 and 12 weeks after transplantation, urethral pressure profile by electrical stimulation through the sacral surface (L6-S1) was evaluated as functional recovery. The recovery ratio in the control and transplanted groups was 37.6% and 72.9%, at 4 weeks, and 41.6% and 78.4% at 12 weeks, respectively (P