Myogenic potential of whole bone marrow mesenchymal stem cells in vitro and in vivo for usage in urinary incontinence

Abstract

Urinary incontinence, defined as the complaint of any involuntary loss of urine, is a pathological condition, which affects 30% females and 15% males over 60, often following a progressive decrease of rhabdosphincter cells due to increasing age or secondary to damage to the pelvic floor musculature, connective tissue and/or nerves. Recently, stem cell therapy has been proposed as a source for cell replacement and for trophic support to the sphincter. To develop new therapeutic strategies for urinary incontinence, we studied the interaction between mesenchymal stem cells (MSCs) and muscle cells in vitro; thereafter, aiming at a clinical usage, we analyzed the supporting role of MSCs for muscle cells in vitro and in in vivo xenotransplantation. MSCs can express markers of the myogenic cell lineages and give rise, under specific cell culture conditions, to myotube-like structures. Nevertheless, we failed to obtain mixed myotubes both in vitro and in vivo. For in vivo transplantation, we tested a new protocol to collect human MSCs from whole bone marrow, to get larger numbers of cells. MSCs, when transplanted into the pelvic muscles close to the external urethral sphincter, survived for a long time in absence of immunosuppression, and migrated into the muscle among fibers, and towards neuromuscular endplates. Moreover, they showed low levels of cycling cells, and did not infiltrate blood vessels. We never observed formation of cell masses suggestive of tumorigenesis. Those which remained close to the injection site showed an immature phenotype, whereas those in the muscle had more elongated morphologies. Therefore, MSCs are safe and can be easily transplanted without risk of side effects in the pelvic muscles. Further studies are needed to elucidate their integration into muscle fibers, and to promote their muscular transdifferentiation either before or after transplantation.

Figure 1. Mouse BM-mMSCs.

(A) Desmin-positive multinucleated myotubes (nuclei labelled in blue with bisbenzimide) derived from the fusion of C2C12 myoblasts. (B) EGFP-MSCs, plated alone, display a fibroblast-like shape. (C–F) In co-culture with C2C12 cells (desmin-positive, labelled in red), MSCs (green) show long cytoplasmatic processes (arrow). (G–J) EGFP-MSCs adhere to desmin-positive myotubes (arrowhead). Scale bar = 50 µm.

Figure 2. Cytofluorimetric and morfological analysis of a representative wBM-hMSCs.

(A) Immunophenotypic analysis of wBM hMSCs showing the negativity of haematopoietic markers CD45, CD14, CD34, and the positivity of CD90, CD29, CD73, CD105, CD44. (B) Analyses of growth rate of wBM hMSCs in terms of cumulative PD. The graphic refers to the median cumulative values (1^st passage: median 2.3 - range 1.4–2.6; 2^nd: median 4.5 - range 2.9–5.9; 3^rd: median 6.4 - range 4.9–8.5).

Figure 3. Immunofluorescence and Molecular analysis of Calcium ion channel subunits in wBM-hMSC.

(A) Positive control normal Human Skeletal Muscle Myoblast (HSMM). Scale bar = 10 µm (alpha SMA) and 25 µm (SA, Myosin, Myogenin and Desmin). (B) Undifferentiated wBM hMSC analyzed at 28 days. Scale bar = 10 µm (a-SMA), 25 µm (SA, Myosin, and Desmin), 50 (Myogenin). (C) Original gels demonstrating amplification of calcium ion channel subunit transcripts in wBM hMSCs: -RT: control of reverse transcription without RT enzyme; C−: negative control (water); C+: positive control (HSMM); line 1: wBM hMSCs from first passage; line 2: wBM hMSCs from second passage; line 3: wBM hMSCs from fourth passage; β-actin, housekeeping gene.

Figure 4. Myogenic differentiation on laminin matrix and L-type Calcium ion channel subunits analysis.

(A) Phase contrast images of laminin cells: presence of some binucleated cells. Scale bar = 25 µm. (B) Immunofluorescence analysis confirmed the presence of a few binucleated structures positive for desmin, SA, myogenin in BM-hMSC cultured on laminin cells. Scale bar = 25 µm. (C) Original gels demonstrating amplification of calcium ion channel subunit transcripts in laminin cells: –RT: control of reverse transcription without RT enzyme; C−: negative control, water; C+: positive control, HSMM; line 1: control wBM-hMSCs; line 2: laminin cells cultured in DMEM-F12 supplemented with 15% FBS; line 3: laminin cells induced to myogenic differentiation with EGF for 7 days.

Figure 5. Bisbenzimide-stained BM-hMSCs (in blue) transplanted into rat bulbocavernosus muscle.

(A) 24 hours after transplantation BM-hMSCs appear undifferentiated with a typical round shape (inset in A, scale bar = 500 µm); (B) one month after engraftment many BM-hMSCs with elongated shape are recognizable among muscular fibers (inset in B, scale bar = 100 µm). (C) At 4 months, migration toward muscle fibers is confirmed by elongated appearance of cells occupying peripheral position (inset b), whereas undifferentiated cells are observed in the core of graft (inset a). Scale bar = 500 µm.

Figure 6. Integration of BM-hMSCs in the striate muscle.

(A) Bisbenzimide-labelled BM-hMSCs (in blue) appear integrated into desmin-positive striated muscle fibers (in green). (B) At 4 months, several BM-hMSCs are located in close vicinity (arrowheads) to acetylcholine receptors (α-BTX staining, in red) (C). Proliferative profile of transplanted BM-hMSCs at one and (D) four months after transplantation, as revealed by Ki67 immunohistochemistry (in purple). Scale bar = 50 µm.