3D reconstitution of nerve-blood vessel networks using skeletal muscle-derived multipotent stem cell sheet pellets.

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.

Caution in evaluation of removal of virus by filtration: Misinterpretation due to detection of viral genome fragments by PCR.

The testing of biological products at different stages of the manufacturing process currently involves quantitative polymerase chain reaction (Q-PCR)-based assays. Q-PCR techniques are able to detect not only the viral genome in viral particles but also fragments of degraded genome in samples. The ability of 15 and 19-nm filters to remove viruses was examined by conducting infectivity assays and Q-PCR assays using parvovirus B19 (B19), one of the smallest non-enveloped viruses. Although the filtered samples showed no infectivity, viral DNA was detected by Q-PCR. Interestingly, approximately 90% of the total viral genome in 15-nm filtrates had a detectable size of less than 0.5kb by the Q-PCR and as a consequence reduction factors were underestimated using Q-PCR. The reduction factors using Q-PCR might be underestimated due to the presence of a large amount of free B19 DNA which shows no infectivity in the tested filtrates. Therefore, the results of Q-PCR should be interpreted with caution. The careful design of primers is needed to eliminate amplification from fragments of viral DNA by Q-PCR.

Origin and hierarchy of basal lamina-forming and -non-forming myogenic cells in mouse skeletal muscle in relation to adhesive capacity and Pax7 expression in vitro.

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.

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.

Clonal differentiation of skeletal muscle-derived CD34(-)/45(-) stem cells into cardiomyocytes in vivo.

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.

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.

Synchronized reconstitution of muscle fibers, peripheral nerves and blood vessels by murine skeletal muscle-derived CD34(-)/45 (-) cells.

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34(-)/CD45(-) (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2-8 x 10(4)); however, the number increased 10-20 fold (2-16 x 10(5)) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.

Clonal multipotency of skeletal muscle-derived stem cells between mesodermal and ectodermal lineage.

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 x 10(5) 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 (alpha-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.