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. 2014 Mar 10;9(3):e91257.
doi: 10.1371/journal.pone.0091257. eCollection 2014.

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

Tetsuro Tamaki  1 , Maki Hirata  2 , Shuichi Soeda  3 , Nobuyuki Nakajima  3 , Kosuke Saito  4 , Kenei Nakazato  5 , Yoshinori Okada  6 , Hiroyuki Hashimoto  7 , Yoshiyasu Uchiyama  7 , Joji Mochida  8
Affiliations

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

Tetsuro Tamaki et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exist.

Figures

Figure 1
Figure 1. Evaluation of nerve crush injury model, and macroscopic observations at 4 weeks after transplantation.
(A) Determination of crush distance and (B) nerve crush by forceps and (C) features immediately after crush damage under stereomicroscope. Nerve length was strictly determined by forceps fixed at 7-mm distance (A), and crush damage was added using other forceps from the vertical direction to the nerve (B). Translucent bands, which are evidence of nerve crush damage, were clearly evident immediately after surgery (C). (D and E) To confirm the details of the present nerve damage model, 10-minute post-damage nerve was prepared as a resin-section, and was stained with toluidine-blue. Several complete disruptions of nerve fiber bundles could be seen in the longitudinal section (arrows in D), corresponding to the translucent bands in (C). However, continuous epineurium (an envelope of entire nerve) was maintained (arrows in E). (F–H) GFP+ tissues were also detectable under the stereomicroscope at 4 weeks after transplantation. Stronger and widespread emission was observed with Sk-MSC transplantation. Scale bars represent 1 mm (C, F–H), 500 µm (D) and 200 µm (E).
Figure 2
Figure 2. Cellular engraftment and comparison of regenerated axons, myelin and blood vessels in damaged portion of Sk-MSC-7d-, BMSC-7d- and SNDC-D-transplanted nerves at 4 weeks after injection.
Comparisons were performed among 7-day cultured Sk-MSCs (Sk-MSC-7d) and BMSCs (BMSC-7d), freshly isolated SNDCs from damaged sciatic nerve (SNDC-D), and medium control (MC) groups based on the results shown in Fig. 1. (F–H). (A–C) Typical engraftment on whole cross-sections of each transplantation group (except for MC group). (D–F) Typical staining of regenerated axons as N200+, and myelin as MBP+ (G–I) and blood vessel formation as CD31+ regions (J–L). (M–P) Comparison of the above factors (Sk = Sk-MSC-7d, BM = BMSC-7d, SN = SNDC-D and MC = medium control). (M) Percentage of mean GFP+ area/total area on whole cross-sections, as compared to relative engraftment ratio. (N) Mean number of axons. (O) Mean number of myelin signals. (P) Mean number of blood vessels. Dotted lines in (N, O and P) indicate the mean number of axons, myelin signals and blood vessels in the corresponding portion of normal sciatic nerve (4625±470, 3179±760 and 27±4, respectively). Significantly greater cellular engraftment ratio and blood vessel formation was evident in the Sk-MSC group. N-200; Neurofilament 200, MBP; Myelin basic protein. *P≤0.05; all scale bars represent 200 µm.
Figure 3
Figure 3. Cellular differentiation of Sk-MSCs in damaged nerve niche at 4 weeks after injection.
(A) Tissues having strong GFP emission actively encircled single or multiple axons stained by N-200, thus suggesting formation of perineurium and endoneurium in the Sk-MSC-7d group. This was a common trend in the Sk-MSC-transplanted groups throughout the experiment. (B) GFP+ circles (probably perineurium/endoneurium) also enclosed single/multiple myelin sheaths stained by MBP. (D) Some showed double positive staining for BMP and GFP, thus suggesting donor cell-derived myelin formation (arrows in D). (C) Several claudin+ reactions were located on donor-derived GFP+ perineurium (arrows in C), demonstrating the formation of tight-junctions. (E) Incorporation of GFP+/CD31+ cells into blood vessels inside the nerve bundle (arrows in E). Incorporation was seen frequently, but not in all cases. (F) Similar contributions were observed in the large conduit blood vessels located outside of nerve bundles (arrows in F), and GFP+ donor cell contributions extended from the tunica media to the adventitia in this case. All scale bars represent 50 µm.
Figure 4
Figure 4. Cellular differentiation of SNDC and BMSC in damaged nerve niche at 4 weeks after injection.
(A–C) Typical differentiation of transplanted damaged sciatic nerve-derived freshly isolated cells (SNDC-D). SNDC-D showed differentiation into perineurial/endoneurial cells that encircled the axon and myelin (A), myelin-forming Schwann cells showed double-positive staining for MBP and GFP (B), and endothelial cells (CD31+/GFP+) were incorporated into blood vessels (C). These differentiation potentials correspond to Sk-MSC-7d (see Fig. 3), whereas the relative engraftment capacity was significantly lower in SMDC-D (see Fig. 2M). (D–H) Typical behavior of transplanted BMSC-7d. Definitive perineurium/endoneurium formation closely encircling axons and myelin, which were observed in both Sk-MSCs and SNDCs, was not seen in the BMSC-7d group; instead, axons with very small diameter and weak reactions for MBP were prominent (D, E). In addition, hollow cavity-like structures around engrafted GFP+ tissues (cells) were frequently observed (arrows in D, E). This trend was consistently observed throughout BMSC-7d transplantation. Similarly, GFP+ cells (tissues) were not incorporated into CD31+ blood vessels (arrows in F). Interestingly, some osteogenesis was observed with BMSC transplantation (G, H), but only in one case (1/13). A large dark area that superficially resembled a large hollow cavity under fluorescence immunohistochemistry (arrows in G) was positive for von Kossa staining (H), representing calcified bone formation in the damaged peripheral nerve niche. Scale bars represent 50 µm (A–F), 200 µm (G).
Figure 5
Figure 5. Detailed analysis of engrafted cell differentiation into peripheral nerve tissues in damaged sciatic nerve by immunoelectron microscopy at 4 week.
Three mice/group were used in this analysis. Anti-GFP antibody was used and positive reactions are represented as black dots. (A, B) Confirmation of Sk-MSC-7d differentiation into Schwann cells and formation of perineurium. (E–F) Similar confirmation of SNDC-D differentiation into Schwann cells and formation of perineurium. (C–D) Localization of BMSC-7d-derived fibroblast-like structure cells between Schwann cells and perineurium. Note that there were no other specific characteristics observed in BMSC-7d transplantation. S = Schwann cell. Fb = Fibroblast. Scale bars represent 2 µm.
Figure 6
Figure 6. Expression of specific mRNAs for peripheral nerves, vascular and skeletal muscle lineages in three types of cells before transplantation, and in re-isolated Sk-MSC-3d and -7d cells after transplantation into damaged sciatic nerve.
Thirty-six primers were used in this analysis. Blue bars represent differentiation markers for peripheral nerve cells. Pink bars represent nerve growth and neurotrophic factors. Red bars are differentiation markers for skeletal muscle, and green bars are common factors. Full names, details and roles of each primer used in this analysis are summarized in Table S1. (A–C) Upper panels show expression patterns in the three types of cells before and after expansion culture (before transplantation); Sk-MSCs (A), BMSCs (B) and SNDCs (C). Freshly isolated Sk-MSCs (Fr) expressed all markers in the above 4 categories, and this expression was consistently enhanced during expansion cell culture (-3d and -7d in A), except for GFAP (mature Schwann cell marker naturally expressed in intact nerve, blue bar No. 31). In freshly isolated BMSCs (Fr), relatively strong expression of vascular-related growth factors (black) was observed, but the other three categories showed weak expression (B). After 7 days of expansion culture (-7d), expression of nerve, vascular and common factors were enhanced, but no enhancement of skeletal muscle markers was seen (black bars in B). There were no assertive characteristics in freshly isolated SNDCs from intact sciatic nerve (Fr-nonD in C), but uniform and strong expression of factors other than skeletal muscle lineage were observed in freshly isolated SNDCs from 4 days after damage (Fr-D in C). (D–E) Lower panels show expression in Sk-MSC-3d and -7d after transplantation into damaged sciatic nerve niche. Engrafted Sk-MSC-3d and -7d were enzymatically re-isolated from regenerating sciatic nerve at 7, 12, 17 days and 4 weeks after transplantation, and were sorted as GFP+ cells and subjected to mRNA analysis (D, E). Strong expression of skeletal myogenic mRNAs, which was observed in -3d and -7d cultured Sk-MSC preparations (red bars No. 1–9 in A) were gradually diminished with time after transplantation (compare A to D, E), but these decreases were faster in 7d-cul than in 3d-cul (compare at 12 and 17 days after transplantation in D and E), thus suggesting that myogenic potential was reduced after longer culture periods. However, expression of the remaining 3 categories (No. 10–36) was consistently/continuously observed in both preparations (D, E). Expression patterns of Sk-MSC-7d after 17 days closely resemble to those in SNDC from damaged nerve (Fr-D in c vs. 17 days in E).
Figure 7
Figure 7. In vivo differentiation potential of re-isolated Sk-MSC-3d after “2nd transplantation” into damaged skeletal muscle and sciatic nerve.
To confirm the differentiation potential of once engrafted Sk-MSC-3d, GFP+ cells were re-isolated from transplanted crushed nerves (using same method as described in previous RT-PCR analysis) from 1st transplanted mice (n = 10), and re-transplanted into both the damaged skeletal muscle and sciatic nerve models (2nd transplantation). Re-isolation and 2nd transplantation were performed at 7, 12, 17 days and 4 weeks after 1st transplantation (n = 3 in each stages in both the damaged skeletal muscle and sciatic nerve), and final sampling was performed at 4 weeks after 2nd transplantation. The left column shows transplantation into damaged skeletal muscle, and the right column shows transplantation into damaged sciatic nerve. (A) Some GFP+ muscle fibers were detected in limited areas (arrows in A). (B, C) Incorporation of GFP+ cells to the vascular (arrows in B) and peripheral nerve (arrows in C) tissues were also evident. There were no GFP+ muscle fibers detected thereafter, with a lower rate of cellular engraftment (at 12 and 17 days, data not shown). (D) Few GFP+ cells related to nerves were seen in the case of re-isolated-4w (arrows in D). (E) There was a large number of GFP+ muscle fibers near the N-200+ nerve bundles when Sk-MSC-3d were directly transplanted into damaged muscle (as 1st transplantation), thus indicating vigorous skeletal myogenic potential. These data indicate that diminished engraftment capacity of Sk-MSC-3d is associated with reduced myogenic potential in the 2nd transplantation. (F) When original SNDC-D was directly transplanted into damaged skeletal muscle niche, they were unable to differentiate into muscle fibers, and their engraftment capacity was quite low. (G–I) 2nd transplantation of re-isolated Sk-MSC-3d cells at -7d (G), -12d (H) and -4 w (I) into damaged sciatic nerve. Number of engrafted cells increased gradually with time after 2nd transplantation. (J–L) Similar trends in cell differentiation which was observed in 1st transplantation were seen for 2nd transplantation (refer to Figs. 2 and 3). Scale bars represent 50 µm (A–F, and J–L), 20 µm (inset of F) and 200 µm (G–I).
Figure 8
Figure 8. Disappearance of skeletal myogenic cells in damaged sciatic nerve.
Data are presented as (A) 7 days, (B–E) 17 days and (F) 4 weeks after Sk-MSC-3d transplantation. (A) SkMA+ cells were observed in the damaged sciatic nerve at 7 days after transplantation and were typically aggregated. These cells typically showed strong SkMA+ reactions at this time point. However, the number of SkMA+ cells was markedly lower at 17 days after transplantation (B), and reactions for anti-SkMA and emission of GFP were both weaker (arrows in C). (D, E) Immunoelectron microscopy of SkMA+ cells. The same antibody, which was used for immunohistochemistry, was used and reactions against DAB were represented as black dots. Typical immature features of myogenic cells, such as central nuclei, fewer myofibrils and low nuclear-cytoplasmic ratio, were evident at 17 days after transplantation. This may be representative of the degenerative phase of myotubes, as more intense reactions of SkMA were detectable at 7 days, and there were no SkMA+ cells at 4 weeks after transplantation. (F) In contrast, freshly isolated non-cultured Sk-MSCs showed vigorous myogenic potential, and these fibers did not disappear, even at 4 weeks after transplantation. These results suggest that the strong myogenic potential in the freshly isolated Sk-MSCs was not affected by the crushed nerve niche, and muscle fiber formation could be established. We also performed the same analysis for Sk-MSC-7d, and compared the residual ratio of SkMA+ cells at 17 days after transplantation among three groups. For Sk-MSC-3d, SkMA+ cells remained in 50% of cases (9/18), while -7d showed 33% (7/21), but all SkMA+ cells disappeared at 4 weeks after transplantation, thus, residual ratio was 0%. However, freshly isolated Sk-MSCs showed a 100% residual ratio (8/8), and this was maintained even after 4 weeks (not disappeared, thus, 100% residual ratio). SkMA = skeletal muscle actin. Scale bars represent 200 µm (A, B), 10 µm (C), 2 µm (D, E) and 50 µm (F).
Figure 9
Figure 9. Characterization of Sk-MSC-3d and SNDC-D before transplantation by FACS, and features of p75+ cells in Sk-MCS-3d culture.
(A) Characteristics of Sk-MSC-3d before transplantation. (B–D) Double-staining of MyoD (pink)- and p75 (yellow)-positive cells in Sk-MSC-3d. Typical bipolar features of p75+ cells (immature Schwann cell marker) in the Sk-MSC-3d culture were evident independently of MyoD+ cells, and there were no MyoD+/p75+ cells. Nuclei are stained with DAPI. Scale bars represent 50 µm. (E) Characteristics of freshly isolated SNDC-D.
Figure 10
Figure 10. Immunohistochemical analysis in the bridging conduit with Sk-MCS-7d at 4 weeks after surgery.
(A) Macroscopic observation of bridging conduit with transplantation. Yellow arrows show both ends of the conduit. Engrafted GFP+ donor cells/tissues stretch back to the proximal portion of the recipient nerve. (B–D) Cross-sections obtained from solid line in panel (A). (E–H) Longitudinal sections obtained from dotted square in panel (A). White arrows in (E–H) show the distal donor-recipient junction. A number of N200+ axons and MBP+ myelin was present, and was mostly encircled by GFP+ perineurium (B, C). A large number of blood vessels were also evident (D). These properties were exerted in the distal portion of the conduit, as represented in the longitudinal sections (E–H). Regenerated/extended axons in the conduit already reached the distal donor-recipient junction (arrows in F), thus, axonal continuity was achieved at the 4 week time point. Myelin formation in the junctional portion was likely to be delayed when compared with the axon (arrows in G), but relatively clear reactions could be seen in the downstream recipient nerve portion. Uniform vascular formation through the conduit was also evident (H). Scale bars represent 1 mm (A) and 200 µm (B–H).
Figure 11
Figure 11. Immunohistochemical analysis in the bridging conduit with Sk-MCS-7d at 8 weeks after surgery.
(A) Macroscopic observation of bridging conduit with transplantation. Yellow arrows show both ends of the conduit, and GFP+ cells/tissues extended beyond the conduit portion at both the proximal and distal ends. Solid line and dotted squares, which are specified in panel (A) correspond to the panels hereafter. (B, C) Longitudinal sections showed spreading of GFP+ cells/tissue beyond the conduit portion and introduction into recipient nerve area. (D–F) Whole cross-sections from the central portion of the conduit. (G–I) High magnification images of staining for N200 (G), MBP (H) and CD31 (I). GFP+ perineurium/endoneurium encircled the axons and myelin (G and H), and this is associated with blood vessel formation (F and I), thus showing sufficient recovery of the transected long nerve gap. These properties of engrafted GFP+ cells/tissues were similar to those in the former injection experiments into the crash nerve (refer to Fig. 2), showing that Sk-MSCs exert the same differentiation/reconstitution potential for neural/vascular lineages, even in the nerveless bridging conduit. Scale bars represent 1 mm (A), 200 µm (B–F) and 50 µm (G–I).
Figure 12
Figure 12. Immunohistochemical analysis in the bridging conduit with healthy nerve graft at 8 weeks after surgery.
(A) Macroscopic observation. Yellow arrows show both ends of the conduit. Solid line and dotted squares, which are specified in panel (A), correspond to the panels hereafter. Some GFP+ cells/tissues were observed around the central portion of the conduit (A). (B, C, F–K) Cross-sections obtained from the solid line in (A). (D, E) Longitudinal section obtained from dotted square in (A). Interestingly, formation of perineurium/endoneurium was scarcely seen (B, C, I, and J), but a close relationship between GFP circles and N200+ reactions (I) and/or double labeling of GFP+/MBP+ reactions (yellow circle reactions) were frequently observed (J). This indicates that the main contribution of nerve grafts was limited by the supply of Schwann cells. There was no relationship between GFP+ cells and CD31+ reactions can be seen in panel (K); thus, donor-derived endothelial cells were also unavailable, in contrast to the former injection experiments into the crush nerve. Scale bars represent 1 mm (A), 200 µm (B–H) and 50 µm (I–K).
Figure 13
Figure 13. Quantitative data for reconstruction of axon, myelin and blood vessels in bridging conduit and narrow corduroy walking scores at 8 weeks after surgery.
Sk-MSC transplantation shows significant and/or favorable quantitative and functional recoveries in all four factors. Dotted lines indicate the mean number of axons, myelin signals and blood vessels in the corresponding portion of normal sciatic nerve (4625±470, 3179±760 and 27±4, respectively). Sk = Sk-MSC-7d, NG = nerve graft and MC = medium control. *P<0.05.
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This work was supported by the grant 2013 Tokai University School of Medicine, Project Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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