Fig 1.
Fluorescence activated cell sorting (FACS) analysis of human Sk-SCs before and after expansion culture, and sorting of human Sk-34 and Sk-DN cells.
(A) Characteristics of human Sk-SCs just after thawing, in a putative freshly isolated state. First, CD45+ cells were discarded as hematopoietic cells, and the remaining cells were analyzed further. Based on the results in (A), possible markers for cell sorting were CD29, CD34, CD73, and p75 (CD271). (B) We then selected CD29 and CD34, and sorted three types of cell: Sk-34/29- (CD34+/45-/29-), Sk-34/29+ (CD34+/45-/29+), and Sk-DN/29+ (CD34-/45-/29+). We also confirmed the concentration of p75+ cells (see panels P6, P7, and P8). (C) Characteristics of the three types of cell after 2 weeks of expansion culture. Basically, three types of cell showed similar characteristics, while a lower distribution of p75+ cells was seen in Sk-34/29- cells than in the initial state (see P6 in B). Note that the patterns of Sk-34/29+ and Sk-DN/29+ are similar, but that they showed quite different behaviors after in vivo transplantation (see Fig 1). Therefore, in our cells, FACS characteristics after expansion culture did not reflect in vivo differentiation capacity after transplantation. The validity of using CD29 in the detection of human cells (see Fig 2) is also evident (C), because of their whole positivity. Standard cell sorting was performed on all samples, and detailed FACS analysis was performed using 3 males and 1 female (ages: 17, 27, 60, and 79; muscles: soleus, gastrocnemius, and tibialis anterior).
Fig 2.
Schematic drawing of the nerve gap bridging method and extraction of histological sections.
A cellular conduit was prepared using a mouse esophagus dehydrated in 70% ethanol for 3 days. Cells were injected with a fine glass pipet after suturing.
Fig 3.
Differentiation potential of Sk-34/29+, Sk-34/29-, and Sk-DN/29+ cells in a severely crushed nerve niche.
Results were obtained from a nude rat experiment, after transplantation of a male patient sample (age 62, tibialis anterior). (A) Cross-sectional profiles of crushed nerves 6 weeks after surgery stained with HNA (human cell nuclei, pink) and N200 (axon, green). (B) Comparison of the number of axons and myelin in the whole nerve cross-sections of four experimental groups. Dotted square shows the average range of normal non-operated control values. Complete recovery of both axon and myelin was observed in the media group, suggesting that this crush injury model is a possible self-recovery model. *P < 0.05. (C) Triple staining with HNA (purple), N200 (red), and p75 (immature Schwann cells, green) in Sk-34/29+ cell-transplanted nerves, showing donor human cell-derived Schwann cells (arrows). Blue = nuclear staining with DAPI. (D) Immunoelectron microscopic detection of engrafted Sk-34/29- cells. Note that arrows shows HNA- nuclei, and HNA+ nuclei show darker staining because of their DAB reaction products (higher electron density of heavy metal-binding; compare nuclei with arrows and arrowheads). Human Sk-34/29- cells differentiated into Schwann cells (S), perineurial cells (P), endoneurial cells (E), endothelial cells (EC), and pericytes (PC). Note that opposite pattern of immunostaining and immunoelectron microscopy of Sk-34/29- and Sk-34/29+ cells were presented in the S1 Fig, and confirmed that the same differentiation capacity of both cells. Bars in A = 200 μm, B = 50 μm, and C = 10 μm.
Fig 4.
Relative expression of mRNAs specific for peripheral nerve, vascular, and skeletal muscle lineages before and after transplantation of Sk-34 and Sk-DN/29+ cells.
Results were obtained from a nude rat experiment. (A) Expression of specific mRNAs of expanded Sk-34 and Sk-DN/29+ cells just before transplantation. Numbers (1–36) and colors (black, pink, green, red) in bars correspond to factor names and numbers (1–36). (B) Sorting of cells from nerves treated with bridging conduits using human cell-specific CD29 antibody 6 weeks after transplantation. Non-reactivity of human CD29 to the rat cells were preliminary confirmed. There were no human cells present in Sk-DN/29+ cell-transplanted nerves, but numerous cells were seen in the Sk-34 transplanted nerves. (C) Expression of specific mRNAs in sorted (engrafted) Sk-34 cells. Disappearance of skeletal muscle-related markers is clear (No.1–9). Expression was evaluated based on a housekeeping control (β-actin) provisionally as follows: 0 = none, 1 = apparently low, 2 = intermediate, 3 = apparently strong (relative to β-actin = 2), and averaged across five samples (4 males and 1 female; age: 16, 17, 32, 50, and 62; muscles: gastrocnemius and tibialis anterior).
Fig 5.
Cross-sectional profiles of bridging conduits transplanted with Sk-34, Sk-DN/29+, media, and mixed cells (Sk-34+Sk-DN/29+) 8 weeks after surgery.
Results were obtained from a nude mice experiment. Upper images = section-1, lower images = section-3. A large numbers of HNA+ cells (red reactions) were observed limitedly in the Sk-34- and mixed-transplanted nerve (first row in both sections), with reconstitution of axons and myelin that was favorable when compared to the other two groups (green reactions in second and third row). However, there are no HNA+ cells in the Sk-DN/29+ group, but showed better axon and myelin recovery than the media group (green reactions in second and third row in columns 2 and 3). Bars = 100 μm. Data were obtained from 2 male patients (ages: 35 and 60; muscles: gastrocnemius and tibialis anterior).
Fig 6.
Quantitative comparison of the number of axons and myelin formation in sections-1 and -3 at 2 and 8 weeks after transplantation.
Results were obtained from a nude mice experiment. Reconstitution in section-1 (proximal portion) clearly came before that in section-3 (distal portion). This was also the case for axonal regeneration and myelin formation. Note that the Sk-34 and mixed groups showed more axonal regeneration than controls in section-1 at 2 weeks, but they were both within the mean range at 8 weeks. The dotted line shows normal control values ± SE. Thus, Sk-34 group in section-1 shows 100% recoveries. *P < 0.05 (vs. media), # P < 0.05 (vs. Sk-DN/29+).
Fig 7.
Quantitation of blood vessels formed in the conduit at 2 weeks after transplantation and the protein expression of angiogenetic cytokines.
(A) Detection of newly formed blood vessels in the conduit using CD31 immunostaining in the Sk-34 (left), Sk-DN/29+ (center), and media (right) groups. Upper panels show Section-1 and lower panels show Section-3. Bars = 100 μm. (B) Comparison of the number of blood vessels among three groups. *P < 0.05. (C) Measurement of the protein levels of angiogenesis related cytokines. Ang: Angiogenin, IGFBP-3: Insulin-like growth factor binding protein-3, IL-8: Interleukin-8, MCP-1: Monocyte chemotactic protein-1, MMP-9: Matrix metalloprotease-9, PlGF: Placenta growth factor, and VEGF: Vascular endothelial growth factor. Levels are expressed as pixel intensity. Data were obtained from 2 male patients (ages: 35 and 60; muscles: gastrocnemius and tibialis anterior).
Fig 8.
Profiles of engrafted human Sk-34 and Sk-DN/29+ cells in the bridging conduit.
Results were obtained from a nude mice experiment. A–E = 8 weeks, F = 7 days after transplantation. Pink coloring indicates HNA+ nuclei of human cells. (A, B) Relationship between engrafted Sk-34 cells and myelin (MBP, green staining) formation in cross- and longitudinal-sections. (C) Engrafted human Sk-34 cell-derived, putative newly formed Schwann cells (arrows) stained with p75 (green). (D) Human Sk-34 cell-derived perineurial cells and/or perineurium stained with GLUT-1 (green, arrows). (E) Human Sk-34 cell-derived endothelial cells (CD31+; green, arrows). (F) Human Sk-DN/29+ cell-derived myotubes (skeletal muscle actin+; green, arrows) detected at 7 days. Blue = nuclear staining with DAPI. Bars = 50 μm. Photographs were obtained from 2 male patients (ages: 27 and 55; muscles: gastrocnemius and tibialis anterior). In addition, disappearance of Sk-DN/29+ cells in the bridging conduit during the 3 weeks after transplantation is confirmed in the S2A Fig. Response of Sk-actin in the Sk-34 cell transplanted nerve conduit is also showed in the S2B Fig. Confirmation of p75 (C) and CD31 (E) staining were further performed by confocal microscopy (S3 Fig). Furthermore, in the present immunohistochemistry, detection of Schwann cells and perineurium were performed by p75 and GULT-1. However, anti-p75 also react to endoneurium (endoneurial cells) and perineurium (perineurial cells), and there were confirmed by IEM (S4 Fig). The standard for these immunohistochemistry (N200, MBP, p75, and GLUT-1) as presented in Figs 3, 5 and 8 is shown in S5 Fig as for the staining for the normal control nerve.
Fig 9.
Recovery of downstream muscle mass and tension output.
Results were obtained from a nude mice experiments, after transplantation of 7 patient samples (5 males and 2 female; ages: 16, 17, 27, 35, 55, 62, and 79; muscles: soleus, gastrocnemius, and tibialis anterior). (A) Changes in lower hindlimb muscle mass during the 12-week recovery phase. Values represent the total muscle mass of the gastrocnemius, plantaris, soleus, extensor digitorum longus, and anterior tibialis muscles. (B) Changes in the tetanic tension output of plantar flexor muscles (gastrocnemius, plantaris, and soleus) during the 12-week recovery phase. Mean values in each group at each stage are summarized in S2 Table. *P < 0.05.
Fig 10.
Characteristics of the tibial nerve and its branches associated with muscle-spindles in the plantaris muscle.
Results were obtained from a nude mice experiment. Photographs (Sk-34) were obtained from the data of a female patient (age: 17; muscle: tibialis anterior). (A–C) Tibial nerve along with the plantaris muscle. (D–F) Tibial nerve branches (arrows) and muscle spindles (arrowheads) in the plantaris. (G-I) Portion of the gastrocnemius muscle stained with ATPase (acid) taken at the same magnification. Left column (A, D, G) = Sk-34, mid column (B, E, H) = media, and right column (C, F, I) = control. Panels A–F show staining with N200 (red) + laminin (green) at the same magnification, and blue = nuclear staining with DAPI. Bars in C, F = 50 μm, and I = 200 μm. (J) The number of axons in the tibial nerve was counted. A 50% recovery was observed in the Sk-34 group, whereas media group showed only less than 10% recovery. *P < 0.05.