Skeletal Muscle-derived Multipotent Stem Cells Research Articles

Regeneration of Transected Recurrent Laryngeal Nerve Using Hybrid-Transplantation of Skeletal Muscle-Derived Stem Cells and Bioabsorbable Scaffold.

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.

Reconstitution of the complete rupture in musculotendinous junction using skeletal muscle-derived multipotent stem cell sheet-pellets as a “bio-bond”

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.

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

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.

Three-Dimensional Reconstitution of Nerve Blood Vessel Units on Damaged Trachea and Bronchial Stump Using Hybrid-Transplantation of Skeletal muscle-derived stem cells and Bioabsorbable Polyglyconate Felt

A hybrid transplantation system using skeletal muscle-derived multipotent stem cells (Sk-MSCs) and bioabsorbable polyglyconate (PGA) felt was applied to coverage of the tracheal large-hole and the bronchial stump model, for the purpose of nerve-blood vessel supplementation to prevent tissue necrosis. Sk-MSCs were obtained from green-fluorescent transgenic (GFP-Tg) mouse muscles after 5 days of expansion culture. Experimental tracheal large-hole and bronchial stump following pneumonectomy were prepared using C57BL6N mice, and damaged portions were covered with PGA felt, which was then impregnated with expanded Sk-MSCs. At 2-10 weeks after transplantation, fluorescent-stereoscopy and immunohistochemical analysis were performed in order to confirm the in vivo behavior of engrafted GFP+ cells. Acceleration of nerveblood vessel formation capacity was quantified and compared between the hybrid and solo PGA felt transplantation group. RTPCR analysis of nerve-blood vessel growth, tropic, and trophic factors for re-isolated engrafted GFP+ tissue was also performed in order to confirm paracrine effects after transplantation. Acceleration of blood vessel formation was observed in both the hybrid and solo PGA transplantation groups; however, nerve formation capacity was significantly (10-fold) higher in the hybrid group. Co-transplanted GFP+ cells were stably held in the PGA area, thus confirming the prevention of unnecessary cell diffusion. Donor-derived GFP+ cells actively differentiated into vascular smooth muscle and endothelial cells, and Schwann cells and perineurium, and supported nerveblood vessel network formation. In addition, re-isolated engrafted GFP+ cells continued to express various essential nerve-blood vessel factors, confirming the prolonged biological adjuvant effects of this hybrid system. These effects were consistently observed in both the tracheal and bronchial stump damaged model. Therefore, use of the present Sk-MSC and PGA felt hybrid transplantation system is a possible treatment for the prevention of postpneumonectomy bronchial stump fistula, as an adjuvant for attributive nerve-blood vessel supplementation.

Preferential and comprehensive reconstitution of severely damaged sciatic nerve using murine skeletal muscle-derived multipotent stem cells.

Loss of vital functions in the somatic motor and sensory nervous systems can be induced by severe peripheral nerve transection with a long gap following trauma. In such cases, autologous nerve grafts have been used as the gold standard, with the expectation of activation and proliferation of graft-concomitant Schwann cells associated with their paracrine effects. However, there are a limited number of suitable sites available for harvesting of nerve autografts due to the unavoidable sacrifice of other healthy functions. To overcome this problem, the potential of skeletal muscle-derived multipotent stem cells (Sk-MSCs) was examined as a novel alternative cell source for peripheral nerve regeneration. Cultured/expanded Sk-MSCs were injected into severely crushed sciatic nerve corresponding to serious neurotmesis. After 4 weeks, engrafted Sk-MSCs preferentially differentiated into not only Schwann cells, but also perineurial/endoneurial cells, and formed myelin sheath and perineurium/endoneurium, encircling the regenerated axons. Increased vascular formation was also observed, leading to a favorable blood supply and waste product excretion. In addition, engrafted cells expressed key neurotrophic and nerve/vascular growth factor mRNAs; thus, endocrine/paracrine effects for the donor/recipient cells were also expected. Interestingly, skeletal myogenic capacity of expanded Sk-MSCs was clearly diminished in peripheral nerve niche. The same differentiation and tissue reconstitution capacity of Sk-MSCs was sufficiently exerted in the long nerve gap bridging the acellular conduit, which facilitated nerve regeneration/reconnection. These effects represent favorable functional recovery in Sk-MSC-treated mice, as demonstrated by good corduroy walking. We also demonstrated that these differentiation characteristics of the Sk-MSCs were comparable to native peripheral nerve-derived cells, whereas the therapeutic capacities were largely superior in Sk-MSCs. Therefore, Sk-MSCs can be a novel/suitable alternative cell source for healthy nerve autografts.

Sciatic nerve injury repair: a visualized analysis of research fronts and development trends.

A total of 3,446 publications regarding sciatic nerve injury repair and protection indexed by Web of Science during 2000–2004 were used for a detailed analysis of temporal-spatial distribution characteristics. Reference co-citation networks of the 100 top-cited publications as per the number of total citations were created using the Web of Science database and the information visualization tool, CiteSpaceIII. The key words that showed high frequency in these publications were included for analyzing the research fronts and development trends for sciatic nerve injury repair and protection. Through word frequency trend analysis, studies on bone marrow mesenchymal stem cells, adipose-derived stem cells, and skeletal muscle-derived multipotent stem cells combined with tissue-engineered scaffold material will become the forefronts in the field of sciatic nerve injury repair and protection in the near future.

Bridging long gap peripheral nerve injury using skeletal muscle-derived multipotent stem cells.

Long gap peripheral nerve injuries usually reulting in life-changing problems for patients. Skeletal muscle derived-multipotent stem cells (Sk-MSCs) can differentiate into Schwann and perineurial/endoneurial cells, vascular relating pericytes, and endothelial and smooth muscle cells in the damaged peripheral nerve niche. Application of the Sk-MSCs in the bridging conduit for repairing long nerve gap injury resulted favorable axonal regeneration, which showing superior effects than gold standard therapy--healthy nerve autograft. This means that it does not need to sacrifice of healthy nerves or loss of related functions for repairing peripheral nerve injury.

3D Reconstitution of Nerve–Blood Vessel Networks Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet Pellets

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. 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. 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. 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.

Functional Nerve-Vascular Reconstitution of the Bladder-Wall; Application of Patch Transplantation of Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellets

A three-dimensional gel-patch-like nerve-vascular reconstitution system using the Skeletal Muscle-Derived Multipotent Stem Cell (Sk-MSC) sheet-pellet was applied to the reconstitution of the severely damaged bladder wall as a non-skeletal muscle tissue, but has high demand for function. The Sk-MSC sheet-pellet was prepared by the mild detachment of expanded/confluent cells in culture with EDTA, then, collected in a tube and centrifuged. The sheetpellet was pasted on the open thin-walled region of the damaged bladder wall made by myotomy (remove one-third of serosal smooth muscle layer associate with large disruptions of nerve-blood vessel networks retaining the mucosal layer). At 4 wk after transplantation, significant prevention of the reduction in the passive wall-tension, and the positive wall-contraction via electrical stimulation was observed in the transplanted group. Supporting these functional results, immunohistochemical and immunoelectron microscopic analysis revealed that the engrafted cells actively contributed to the reconstitution of blood vessels and peripheral nerves with differentiation into pericytes, endothelial cells, and Schwann cells. However, skeletal and smooth muscle formation was not observed. Thus, this method is potentially useful for the reconstitution of nerve-vascular networks in the bladder-wall to be retaining function such as passive tension and contractile function.

180 DEVELOPMENT OF BLADDER AUGMENTATION METHOD USING SKELETAL MUSCLE-DERIVED MULTIPOTENT STEM CELL SHEET-PELLETS TRANSPLANTATION

You have accessJournal of UrologyStem Cell Research1 Apr 2011180 DEVELOPMENT OF BLADDER AUGMENTATION METHOD USING SKELETAL MUSCLE-DERIVED MULTIPOTENT STEM CELL SHEET-PELLETS TRANSPLANTATION Shuichi Soeda, Tetsuro Tamaki, Akio Hoshi, Maki Masuda, Masahiro Nitta, Yukio Usui, Akira Akatsuka, and Toshiro Terachi Shuichi SoedaShuichi Soeda Isehara, Japan More articles by this author , Tetsuro TamakiTetsuro Tamaki Isehara, Japan More articles by this author , Akio HoshiAkio Hoshi Isehara, Japan More articles by this author , Maki MasudaMaki Masuda Isehara, Japan More articles by this author , Masahiro NittaMasahiro Nitta Isehara, Japan More articles by this author , Yukio UsuiYukio Usui Isehara, Japan More articles by this author , Akira AkatsukaAkira Akatsuka Isehara, Japan More articles by this author , and Toshiro TerachiToshiro Terachi Isehara, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.249AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail 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. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e75 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shuichi Soeda Isehara, Japan More articles by this author Tetsuro Tamaki Isehara, Japan More articles by this author Akio Hoshi Isehara, Japan More articles by this author Maki Masuda Isehara, Japan More articles by this author Masahiro Nitta Isehara, Japan More articles by this author Yukio Usui Isehara, Japan More articles by this author Akira Akatsuka Isehara, Japan More articles by this author Toshiro Terachi Isehara, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Reconstitution of Experimental Neurogenic Bladder Dysfunction Using Skeletal Muscle-Derived Multipotent Stem Cells

BACKGROUND.: Postoperative neurogenic bladder dysfunction is a major complication of radical hysterectomy for cervical cancer and is mainly caused by unavoidable damage to the bladder branch of the pelvic plexus (BBPP) associated with colateral blood vessels. Thus, we attempted to reconstitute disrupted BBPP and blood vessels using skeletal muscle-derived multipotent stem cells that show synchronized reconstitution capacity of vascular, muscular, and peripheral nervous systems. METHODS.: Under pentobarbital anesthesia, intravesical pressure by electrical stimulation of BBPP was measured as bladder function. The distal portion of BBPP with blood vessels was then cut unilaterally (experimental neurogenic bladder model). Measurements were performed before, immediately after, and at 4 weeks after transplantation as functional recovery. Stem cells were obtained from the right soleus and gastrocnemius muscles after enzymatic digestion and cell sorting as CD34/45 (Sk-34) and CD34/45 (Sk-DN). Suspended cells were autografted around the damaged region, whereas medium alone and CD45 cells were transplanted as control groups. To determine the morphological contribution of the transplanted cells, stem cells obtained from green fluorescent protein transgenic mouse muscles were transplanted into a nude rat model and were examined by immunohistochemistry and immunoelectron microscopy. RESULTS.: At 4 weeks after surgery, the transplantation group showed significantly higher functional recovery ( approximately 80%) than the two controls ( approximately 28% and 24%). The transplanted cells showed an incorporation into the damaged peripheral nerves and blood vessels after differentiation into Schwann cells, perineurial cells, vascular smooth muscle cells, pericytes, and fibroblasts around the bladder. CONCLUSION.: Transplantation of multipotent Sk-34 and Sk-DN cells is potentially useful for the reconstitution of damaged BBPP.

Skeletal Muscle–Derived CD34+/45-and CD34-/45-Stem Cells Are Situated Hierarchically Upstream of Pax7+Cells

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.

Reconstruction of Radical Prostatectomy-Induced Urethral Damage Using Skeletal Muscle-Derived Multipotent Stem Cells

Postoperative damage of the urethral rhabdosphincter (URS) and neurovascular bundle (NVB) is a major operative complication of radical 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. 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. 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<0.05). Immunohistochemical and immunoelectron microscopic analysis revealed that transplanted cells differentiated into numerous skeletal muscle fibers having neuromuscular junctions (innervation) and nerve bundle-related Schwann cells and perineurium, and blood vessel-related endothelial cells and pericyte around the urethra. Thus, we conclude that transplantation of skeletal muscle-derived multipotent Sk-34 and Sk-DN cells is potentially useful for the reconstitution of postoperative damage of URS and NVB after radical prostatectomy.