Abstract
INTRODUCTION
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. DMD is the commonest form of muscular dystrophy, and is characterized by the progressive degeneration of muscle fibers and their subsequent replacement by fat or fibrotic tissue, with eventual deterioration of muscle function [1]. Although curative treatments remain to be developed, several therapies have been proposed for DMD and other degenerative muscle diseases [2–4], including cell transplantation therapy. Satellite cells are stem cells in skeletal muscle and represent an important cell source for transplantation therapy, because their depletion leads to complete impairment of muscle regeneration [5, 6]. Although primary satellite cells immediately after isolation shows high levels of stemness, culturing and passaging of satellite cells results in loss of their undifferentiated state, and gradual reductions in transplantation efficiency [7, 8].However, the culture and expansion of human skeletal muscle cells in vitro is necessary to ensure the adequate cell numbers for transplantation therapy. Although growth factors, cytokines [9], and chemicals [10] have been used in muscle cell cultures, the optimal culture conditions required to maintain the undifferentiated state, inhibit differentiation, and enhance eventual transplantation efficiency have not yet been established.
The expression of leukemia inhibitory factor (LIF) has been reported to be increased in patients with muscular dystrophy and muscle trauma [11]. Furthermore, LIF is thought to be functionally involved in muscle regeneration, because LIF knockout mice showed impaired muscle regeneration [12], and administration of LIF ameliorated the pathology of the mouse model of DMD [13, 14]. However, although its effects on the proliferation of muscle cell lines or primary muscle cells have been studied [12, 15–18], the roles of LIF in the transplantation efficiency have not been analyzed. We therefore examined the effects of LIF on the undifferentiated state and transplantation efficiency in primary, freshly isolated mouse satellite cells. The results of this study will contribute to the development of optimal culture conditions for cell transplantation therapy.
MATERIALS AND METHODS
Animals
Twelve- to 16-week-old male C57BL/6 mice were purchased from Nihon CREA. Four- to 8-week-old NOD/SCID mice were purchased from Oriental Yeast. C57BL/6-green fluorescent protein (GFP)-transgenic mice were kindly provided by Dr. Masaru Okabe (Osaka University, Osaka, Japan). C57BL/6-mdx mice were kindly provided by Dr. Toshikuni Sasaoka (National Institute for Basic Biology, Aichi, Japan). All mice were housed in the institutional animal facility. All experimental procedures were approved by the Experimental Animal Care and Use Committee at the National Center of Neurology and Psychiatry.
Satellite cells preparation and culture
Primary satellite cells were prepared from extensor digitorum longus (EDL) muscles from C57BL/6 or C57BL/6-GFP mice by dissociation with type 1 collagenase (Worthington), as described previously [19]. Briefly, isolated muscles were dissociated by incubation in 0.2% type 1 collagenase/DMEM for 80–90 min and unraveled by gentle pipetting. Single muscle fibers were then isolated and plated on Matrigel-coated plastic dishes. Isolated muscle fibers with primary satellite cells were cultured in DMEM (high-glucose, sodium pyruvate and GlutaMAX supplement, Thermo Fisher Scientific) supplemented with 20% fetal bovine serum, 1% chicken embryo extract (US Biological) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO2 with or without 5 ng/ml LIF (PROSPEC) for 5 days.
RNA isolation and quantitative reverse transcription–polymerase chain reaction analysis
Total RNA was isolated using TRIzol (Invitrogen) and single-strand cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen). The expression levels of Pax7, MyoD, Myogenin, and Tbp (encoding TATA-binding protein) were evaluated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) using SYBR Premix Ex Taq II (Takara) on a MyiQ single-color system (Bio-Rad). Primer sequences for qRT–PCR were as follows: Pax7 forward: 5′-gcccacagaacctgtcactc-3′,reverse: 5′-ggacttgaaagcttggtgctct-3′, MyoD forward:5′-tgagccttgcacacctaagcc-3′, reverse: 5′-ctccgcaagctgtggggaaa-3′, Myogenin forward: 5′-tgtgcacatctgttctagtctc-3′, reverse: 5′-gctttggaaccggatagctc-3′, Tbp forward: 5′-cagcctcagtacagcaatcaac-3′, reverse: 5’-taggggtcataggagtcattgg-3′. The expression levels of Pax7, MyoD, and Myogenin were normalized to that of Tbp.
Satellite cells transplantation
To induce muscle regeneration, 100 μl of 10 μM cardiotoxin (Sigma-Aldrich) was injected into the tibialis anterior (TA)/EDL muscles of NOD/SCID mice or mdx mice 24 hours before transplantation. Thirty microliters of cell suspension containing 3×104 C57BL/6-GFP transgenic-mouse-derived satellite cells cultured with or without 5 ng/ml LIF for 5 days were injected directly into the TA/EDL muscles. After 1, 2, 4 or 14 days, the muscles were dissected, fixed in 4% paraformaldehyde for 30 min at 4°C, immersed in 10% sucrose/PBS followed by 20% sucrose/PBS, and then frozen in isopentane cooled with liquid nitrogen.
Immunocytochemical analysis
The obtained cells were fixed with 4% parafor-maldehyde for 10 min at room temperature, washed, and treated with 0.1% Triton X-100/phosphate-buffered saline (PBS) for 10 min for permeabilization. The cells were blocked with 5% goat serum (Cedarlane) in 2% bovine serum albumin (BSA)/PBS for 15 min, and then incubated with anti-Pax7 (1:80, sc-81648, Santa Cruz), anti-MyoD (1:400, sc-32758, Santa Cruz) or anti-Myogenin antibody (1:1000, sc-12732, Santa Cruz) in 2% BSA/PBS at 4°C overnight. The cells were then incubated with Alexa Fluor 488- or Alexa Fluor 568-labeled secondary antibodies (1:1000, Thermo Fisher Scientific) in 2% BSA/PBS. After several washings, nuclei were stained with Hoechst (DOJINDO). Immunofluorescent images were evaluated by fluorescence microscopy (Olympus).
Immunohistochemical analysis
The transplanted TA/EDL muscles were cut into 8 μm transverse sections using a cryostat. The sections were fixed with cooled acetone for 10 min and air-dried. They were then blocked with 5% goat serum in 1% BSA/PBS for 15 min, or M. O. M Mouse Ig Blocking reagent (VECTOR) for 1 hour followed by 5% goat serum in 1% BSA/PBS for 15 minutes. The sections were incubated with anti-GFP antibody (1:300, AB3080, Chemicon) and anti-laminin α2 antibody (1:200, ALX-804-190, Alexis) in 1% BSA/PBS, or anti-GFP antibody and anti-Dystrophin antibody (1:100, Clone: Dy8/6C5, Leica) or anti-GFP antibody and anti-Ki-67 antibody (1:200, Clone: B56, BD Pharmingen) in M.O.M. Diluent at 4°C overnight. The sections were then incubated with Alexa Fluor 488- or Alexa Fluor 568-labeled secondary antibodies (1:1000, Thermo Fisher Scientific) in 1% BSA/PBS or M.O.M. Diluent. For TUNEL assay, ApopTag Red In Situ Apoptosis Detection Kit (Chemicon) was used. After several washings, nuclei were stained with DAPI (VECTOR). Immunofluorescent images were evaluated under a fluorescence microscope (BZ-9000; Keyence), and GFP-positive fibers or cells, GFP/Dystrophin double-positive fibers, GFP/Ki-67 double-positive cells or GFP/TUNEL double-positive cells were counted.
Statistical analysis
All values were expressed as mean±s.e.m. Statistical differences were assessed by the Student’s t-test. Probabilities less than 5%, 1% or 0.1% were considered to be statistically significant.
RESULTS
Single muscle fibers and attached primary satellite cells were cultured with or without 5 ng/ml LIF for 5 days (Fig. 1A). Similar to previous report [15], the expansion of satellite cells was promoted by LIF (Fig. 1B). We first analyzed the expression of Pax7, MyoD and Myogenin by qRT-PCR. We found that the expression of Pax7, a satellite cell marker which expression level was related to its stemness [20], was elevated by LIF (Fig. 1C). Expression of Myogenin, an essential transcription factor required for differentiation, was reduced, though the difference was not significant. These results suggest that LIF affected the maintenance of undifferentiated state in satellite cells. Expression of MyoD was slightly increased by LIF (Fig. 1C). We also found that the incidence of Pax7-negative differentiated cells were significantly decreased by LIF (Fig. 1D and E),while Myogenin-negative undifferentiated cells were increased, thus supporting a role for LIF in preventing the differentiation of satellite cells (Fig. 1Dand E).
We investigated the effects of LIF on the transplantation efficiency of satellite cells, which reflects the number of engraftable satellite cells that retained their stemness. Primary satellite cells isolated from C57BL/6-GFP mice [21] cultured with or without LIF were transplanted into cardiotoxin-injected regenerating TA/EDL muscles in NOD/SCID mice. We found that LIF-treated satellite cells formed two to three times as many GFP-positive fibers which were surrounded by laminin α2 as vehicle-treated satellite cells did (Fig. 2A and B). We also observed higher number of LIF-treated GFP-positive cells compared with that of vehicle-treated cells at 1, 2, or 4 days after transplantation. To elucidate the cause of this differences, we analyzed the proliferation or apoptosis of GFP-positive transplanted cells. We found that the majority of transplanted cells lost their proliferation ability immediately after transplantation (Fig. 2D and E). In addition, no difference in the number of GFP/Ki-67 double positive cells between vehicle-treated and LIF-treated satellite cells transplanted muscle was observed (Fig. 2DD and E). In contrast we found that the number of GFP/TUNEL double positive cells was decreased in LIF-treated satellite cells transplanted muscle at 1 day after transplantation (Fig. 2F and G). These results suggested that treatment with LIF in vitro enhanced the transplantation efficiency of satellite cells by inhibiting their apoptosis in vivo.
Finally, we transplanted C57BL/6-GFP mice-derived satellite cells in mdx mice, a mouse model of Duchenne muscular dystrophy. Similar to the observation in NOD/SCID mice, we observed two to three times many GFP/Dystrophin-double positive fibers in LIF-treated satellite cells-transplanted mdx mice compared with those in vehicle-treated satellite cells-transplanted mice (Fig. 2H and I). Overall, these results indicated that LIF maintained the undifferentiated state, inhibited the differentiation of satellite cells in vitro, and enhanced their transplantation efficiency in vivo.
DISUCUSSION
To the best of our knowledge, this study provides the first report of the effect of LIF on the transplantation efficiency of primary satellite cells. In response to several stimuli, quiescent satellite cells are activated, proliferate, and form new muscle fibers [22]. Culturing of satellite cells lead to proliferation of their descendants, with loss of their undifferentiated state and transplantation efficiency. In addition to our results, Hashimoto et al., showed the role of LIF in the increase of round stem-like cells in vitro [23], which also support that LIF might facilitate the expansion of primary muscle cells while maintaining their undifferentiated state. However, further studies are needed to confirm the effects of LIF in long-term cultures, and in human muscle stem cells. Although we showed the effects of LIF on the enhancement of transplantation efficiency, the total number of engrafted GFP-positive fibers were approximately 3% of the total muscle fibers, and the limited distribution of transplanted satellite cells from the injected site was not improved (Fig. 2H and I), suggesting that the combination of LIF with other cytokines, such as bFGF, or transplantation with other cell population that improve migration of transplanted satellite cells would be required [24]. The use of immune deficient mdx mice or immunosuppression would also enhance the survival of transplanted cells.
By analyzing the proliferation or apoptosis of transplanted cells, we found that treatment with LIF inhibited the apoptosis of transplanted cells (Fig. 2F and G), suggesting the relationship between stemness of satellite cells in vitro and their survival in vivo. However, precise mechanisms of LIF for the enhancement of transplantation efficiency remain unknown. Investigations aimed at determining the downstream targets of LIF would help to clarify the functional importance of LIF in muscle regeneration, and further its potential application in cell transplantation therapy.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
Footnotes
ACKNOWLEDGMENTS
We thank Dr. Isao Kii and Dr. Takashi Nishiyama for valuable discussions. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science, Intramural Research Grant (28-6) for Neurological and Psychiatric Disorders of NCNP, and Research Center Network Project for Realization of Regenerative Medicine from Japan Agency for Medical Research and development (AMED).