Menstrual blood-derived cells confer human dystrophin expression in the murine model of Duchenne muscular dystrophy via cell fusion and myogenic transdifferentiation

Abstract

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood-derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood-derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood-derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

Figure 1.

Surface marker expression of endometrium-derived cells. (A and B) Morphology of menstrual blood–derived cells, regarded as being PD 1 or 2. Scale bars, 200 μm (A), 100 μm (B). (C and D) Flow cytometric analysis of cell surface markers of EM-E6/E7/hTERT-2 cells (C) and menstrual blood–derived cells (D). (E) Further phenotypic analysis in EM-E6/E7/hTERT-2 cells and menstrual blood–derived cells are summarized. Peak intensity was estimated in comparison with isotype controls. +++, strongly positive (>100 times the isotype control); ++, moderately positive (<100 times but more than 10 times the isotype control); +, weakly positive (<10 times but more than twice the isotype control); −, negative (less than twice the isotype control).

Figure 2.

Implantation of endometrium-derived cells-derived cells into the muscle of NOG mice. EM-E6/E7/hTERT-2 cells (A–F) or menstrual blood–derived cells (G–J) cultured in absence of any stimuli were directly injected into the right thigh muscle of NOG mice. Immunohistochemical analysis was performed using antibody that reacts to human vimentin but not to murine vimentin. (A, C, E, G, I, and K) hematoxylin and eosin stain. (B, D, F, H, J, and L) immunohistochemistry. Note that vimentin-positive EM-E6/E7/hTERT-2 cells and menstrual blood–derived cells with a spindle morphology (C–J, arrowheads) extensively migrated into muscular bundles at 3 wk after injection, and some of the injected cells exhibited round structure (D, F, and J, arrows). Isotype mouse IgG1 served as a negative control (L). Scale bars, 100 μm (A, B, K, and L), 50 μm (C–F, I, and J), 90 μm (G and H).

Figure 3.

Expression of myogenic-specific genes during myogenic differentiation of EM-E6/E7/hTERT-2 cells. (A–E) Immunocytochemical analysis of EM-E6/E7/hTERT-2 cells using an antibody to desmin. (A) Omission of only the primary antibody to desmin serves as a negative control. (C) Higher magnification of inset in B. (F) Myogenic differentiation of EM-E6/E7/hTERT-2 cells with exposure to different concentrations (B, 5 μM; C, 5 μM; D, 10 μM; E, 100 μM) of 5-azacytidine. To estimate myogenic differentiation, the number of all the desmin-positive cells was counted for each dish (n = 3). Data were analyzed for statistical significance using ANOVA. EM-E6/E7/hTERT-2 cells were cultured in the DMEM supplemented with 2% HS, and serum-free ITS. (G and K) RT-PCR analysis with PCR primers allows amplification of the human MyoD, Myf5, desmin, myogenin, myosin heavy chain type IIx/d (MyHC-IIx/d), and dystrophin cDNA (from top to bottom). RNAs were isolated from EM-E6/E7/hTERT-2 cells at the indicated day after treatment with 5-azacytidine. RNAs from human muscle and H₂O served as positive (P) and negative (N) controls, respectively. Only the 18S PCR primer reacted with the human and murine cDNA. (H) Time course of MyoD, desmin, myogenin, MyHC-IIx/d, and dystrophin expression in the cells incubated with 2% HS for up to 21 d after 5-azacytidine treatment. Relative mRNA levels were determined using Multi Gauge Ver 2.0 (Fuji Film). The signal intensities of MyoD, desmin, and dystrophin mRNA at day 0, myogenin mRNA at day 3, and MyHC-II/d mRNA at day 21 were regarded as equal to 100%. (I and J) The cells were exposed to 5 μM 5-azacytidine for 24 h and then subsequently cultured in DMEM supplemented with 2% HS for 21 d. α-Sarcomeric actin (I) and skeletal myosin heavy chain (J) was detected by immunocytochemical analysis. Scale bars, 2 mm (A and B), 300 μm (C–E), 900 μm (Ia and Ja), 425 μm (Ib and Jb).

Figure 4.

Expression of myogenic-specific genes in differentiated menstrual blood–derived cells. Menstrual blood–derived cells were cultured in DMEM supplemented with 20% FBS, 2% HS, or serum-free ITS medium. (A) RT-PCR analysis with PCR primers that allows amplification of the human MyoD, Myf5, desmin, myogenin, MyHC-IIx/d, and dystrophin cDNA (from top to bottom). RNAs were isolated from menstrual blood–derived cells in DMEM supplemented with 20% FBS at the indicated day after treatment with 5 μM 5-azacytidine for 24 h. RNAs from human muscle and H₂O served as positive (P) and negative (N) controls. Only the 18S PCR primer reacted with the human and murine cDNA. (B) Immunocytochemical analysis using an antibody to desmin (a–f) was performed on the menstrual blood–derived cells at 2 wk after exposure to 5 μM of 5-azacytidine for 24 h. The desmin-positive cells are shown at higher magnification (d–f). Merge of a and b is shown in c, and merge of d and e is shown in f. The images were obtained with a laser scanning confocal microscope. Scale bars, 200 μm (a–c) and 75 μm (d—f). (C and D) RT-PCR analysis of menstrual blood–derived cells on DMEM supplemented with 2% HS (C) or serum-free ITS medium (D) at the indicated day after exposure to 5 μM 5-azacytidine for 24 h. (E–G) Western blot analysis was performed on the cells cultured in myogenic medium indicated for 21 d. The blot was stained with desmin (E), myogenin (F), and dystrophin (G) antibodies followed by an HRP-conjugated secondary antibody.

Figure 5.

Conferral of dystrophin to mdx myocytes by human endometrial cells. (A and B) Immunohistochemistry analysis using an antibody against human dystrophin molecule (green), human nuclei (HuNucl, red), and DAPI staining (blue) on thigh muscle sections of mdx-scid mice after direct injection of EM-E6/E7/hTERT-2 cells (A) or menstrual blood–derived cells (B) without any treatment or induction. (C) EGFP-labeled EM-E6/E7/hTERT-2 cells without any treatment or induction were directly injected into the thigh muscle of mdx-scid mice. Immunohistochemistry revealed the incorporation of implanted cells into newly formed EGFP-positive myofibers, which expressed human dystrophin 3 wk after implantation. (A and B) As a methodological control, the primary antibody to dystrophin was omitted (e and f). (D) Immunohistochemistry analysis using an antibody against human dystrophin molecule (green, arrowheads), human nuclei (HuNucl, red, arrow), and DAPI staining (blue) on thigh muscle sections of mdx-scid mice after direct injection of human EM-E6/E7/hTERT-2 cells without any treatment or induction. (A and B) Merge of a–c is shown in d, and merge of e–g is shown in h. (C and D) Merge of a–c is shown in d. Scale bars, 50 μm (A and B), 20 μm (C and D). (E) Quantitative analysis of human dystrophin-positive myotubes. Menstrual blood–derived cells or EM-E6/E7/hTERT-2 cells without any treatment or induction were directly injected into thigh muscle of mdx-scid mice. The percentage of human dystrophin–positive-myofiber areas was calculated 3 wk after implantation of the EM-E6/E7/hTERT-2 cells or menstrual blood–derived cells. Injection of PBS without cells into mdx-scid myofibers was used as a control.

Figure 6.

Detection of human endometrial cell contribution to myotubes in an in vitro myogenesis model. EGFP-labeled EM-E6/E7/hTERT-2 cells (A) or EM-E6/E7/hTERT-2 cells (B) or menstrual blood–derived cells (C and D) were cocultured with C2C12 myoblasts for 2 d under conditions that favored proliferation. The cultures were then changed to differentiation media for 7 d to induce myogenic fusion. (A) Myotubes were revealed by EGFP (green); human nuclei were detected by antibody specific to human nuclei (HuNucl, red, arrows). (B–D) Myotubes were revealed by specific human dystrophin mAb NCL-DYS3 (B and C, red) or anti-myosin heavy chain mAb MF-20 (D, red). (D) Human nuclei were detected by antibody specific to human nuclei (HuNucl, green, arrows). Total cell nuclei in the culture were stained with DAPI (blue, arrowheads). (B–D) Merge of a–c are shown in d. The cultures were then changed to differentiation media for 7 d to induce myogenic fusion. Scale bars, 100 μm (A–D).