In vitro Myogenic Differentiation of Human Bone Marrow–Derived Mesenchymal Stem Cells as a Potential Treatment for Urethral Sphincter Muscle Repair

Abstract

To explore a new treatment strategy for urinary incontinence, human bone marrow mesenchymal stem cells (MSC) of the first in vitro passage were exposed to 5-azacytidine (AZA) to induce myogenic differentiation, and cultured for a total of six passages. Expression of stem cell surface antigens and intracellular alpha-actin was examined by flow cytometry at the end of each passage and compared to that of native MSC (not exposed to AZA) cultured in parallel. To analyze differentiation into striated muscle, expression of the transcription factor MyoD1 and myosin heavy chain (MyHC) was examined by RT-PCR. Both native and AZA-exposed MSC of all passages were negative for the progenitor/endothelial antigen CD34, leukocytic CD45, and endothelial/monocytic CD31. In contrast, the MSC markers CD73, CD90, CD105, and intracellular actin were detected in both groups of MSC throughout the culture period. After an initial increase, the expression level of MSC antigens decreased over time particularly in AZA-exposed MSC. Expression of smooth muscle alpha-actin also declined, but was greater in AZA-exposed MSC throughout the culture period. Varying percentages of MSC cultures expressed MyoD1 and MyHC mRNA. In late passages, AZA-exposed MSC tended to be more frequently positive than native MSC. In pilot experiments, transplantation of MSC into the bladder neck tissue of athymic rats was feasible; long-term analyses are pending. We conclude that independent of AZA exposure, MSC express smooth and striated muscle antigens. Treatment with AZA slightly increases myogenic differentiation, but may not be necessary in future studies of MSC as a treatment modality for urinary incontinence.

Implantation of Autologous Bone-Marrow-Derived Cells Reconstructs Functional Urethral Sphincters in Rabbits

“…Bone-marrow-derived cells are especially suited for this purpose because they can differentiate into striated or smooth muscle cells, both in vitro and in vivo. 31 Previously, we showed that bone-marrow-derived cells of wild-type mice, when implanted into freeze-injured urinary bladders of nude mice where most of the smooth muscle was lost, differentiate into smooth muscle cells. 36 Further, the bonemarrow-derived cells, when situated in areas of damage, produce cytokines and growth factors based on available signals in the surrounding tissues.…”

“…3,30 Our strategy to accomplish the regeneration of functional urethral sphincters is the utilization of autologous bone-marrow-derived cells. These cells are capable of differentiating both in vitro and in vivo along multiple pathways that include striated and smooth muscle 31 as well as bone, cartilage, adipose, neural cells, tendon, and connective tissue. [32][33][34] Further, in the translation to clinical therapy, autologous cells are superior to allogenic cells because the autologous cells are not burdened with immunological rejection or ethics problems.…”

The Impact of Autologous Bone Marrow Mesenchymal Stem Cells (MSCs) in the Treatment of Stress Urinary Incontinence: Short term Follow-up

“…Mesenchymal stem cells have many advantages in this setting; namely, easy isolation, high proliferative potential and efficient plasticity in vivo. They can be derived from adipose tissue, synovial membrane or bone marrow [22]. A number of workers used adipose-derived MSCs in-vitro for myogenic differentiation [23] and in experimental animal models [24].…”

Stem Cell Therapy for Male Urinary Incontinence

“…These cells can be isolated from many different sources such as bone marrow, muscle, adipose tissue, liver tissue, amniotic fluid, placenta, umbilical cord and dental pulp [16]. Table 1 summarizes the main animal studies which have been conducted in the last years using adult stem cells [17,18,19,20,21,22,23,24,25,26]. …”