In vitro Myogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells as a Potential Treatment for Urethral Sphincter Muscle Repair
In vitro Myogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells as a Potential Treatment for Urethral Sphincter Muscle Repair
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DOI：
10.1111/j.1749-6632.2009.04610.x
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发表时间：
2009-01-01
期刊：
影响因子：
--
通讯作者：
Moehle, Robert
中科院分区：
文献类型：
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作者：
Drost, Adriana C.;Weng, Sibylle;Moehle, Robert
为探索治疗尿失禁的新策略，采用5-氮杂胞苷（5-azacytidine，AZA）诱导体外第1代人骨髓间充质干细胞（MSC）向肌源性细胞分化，共培养6代。在每次传代结束时通过流式细胞术检查干细胞表面抗原和细胞内α-肌动蛋白的表达，并与平行培养的天然MSC（未暴露于AZA）的表达进行比较。为了分析向横纹肌的分化，通过RT-PCR检测转录因子MyoD 1和肌球蛋白重链（MyHC）的表达。所有传代的天然和AZA暴露MSC的祖细胞/内皮抗原CD 34、白细胞CD 45和内皮/单核细胞CD 31均为阴性。相反，在整个培养期间，在两组MSC中均检测到MSC标志物CD 73、CD 90、CD 105和细胞内肌动蛋白。在最初增加后，MSC抗原的表达水平随时间降低，特别是在AZA暴露的MSC中。平滑肌α-肌动蛋白的表达也下降，但在整个培养期间，AZA暴露的MSC中表达更大。不同百分比的MSC培养物表达MyoD 1和MyHC mRNA。在晚期传代中，AZA暴露的MSC倾向于比天然MSC更频繁地呈阳性。在初步实验中，MSC移植到无胸腺大鼠的膀胱颈组织中是可行的;长期分析待定。我们的结论是独立的AZA暴露，MSC表达平滑肌和横纹肌抗原。用AZA治疗略微增加肌源性分化，但在将来MSC作为尿失禁治疗方式的研究中可能没有必要。
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 a-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 a-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.