Induced differentiation of adipose-derived stromal cells into myoblasts

Abstract

This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.

Keywords

adipose-derived stromal cells / 5-azacytidine / myoblasts / stress urinary incontinence / differentiation

References

[1] AbramsP., CardozoL., FallM., et al.. The standardisation of terminology in lower urinary tract function: Report from the standardisation subcommittee of the International Continence Society. Urology, 2003, 61(1): 37-49

[2] KayigilO., Iftekhar AhmedS., MetinA.. The coexistence of intrinsic sphincter deficiency with type II stress incontinence. J Urol, 1999, 162(4): 365-1366

[3] StrasserH., BerjukowS., MarksteinerR., et al.. Stem cell therapy for urinary stress incontinence. Exp Gerontol, 2004, 39(9): 1259-1265

[4] YamamotoN., AkamatsuH., HasegawaS., et al.. Isolation of multipotent stem cells from mouse adipose tissue. J Dermatol Sci, 2007, 48(1): 43-52

[5] CorcosJ., BeaulieuS., DonovanJ., et al.. Quality of life assessment in men and women with urinary incontinence. J Urol, 2002, 168(3): 896-905

[6] NortonP., BrubakerL.. Urinary incontinence in women. Lancet, 2006, 367(9504): 57-67

[7] MinassianV.A., DrutzH.P., BadrA.L.. Urinary incontinence as a worldwide problem. Int J Gynecol Obstet., 2003, 82(3): 327-338

[8] PetrosP.E., UlmstenU.I.. An integral theory and its method for the diagnosis and management of female urinary incontinence. Scand J Urol Nephrol, 1993, 153(Suppl): 1-13

[9] DelanceyJ.O.L.. Structural support of the urethra as it relates to stress urinary incontinence: the hammock hypothesis. AmJ Obstet Gynecol, 1994, 170(6): 1713-1737

[10] SmaldoneM.C., ChenM.L., ChancellorM.B.. Stem cell therapy for urethral sphincter regeneration. Minerva Urol Nefrol, 2009, 61(1): 27-40

[11] MitterbergerM., PinggeraG.M., MarksteinerR., et al.. Adult stem cell therapy of female stress urinary incontinence. Eur Urol, 2008, 53(1): 169-175

[12] MitterbergerM., PinggeraG.M., MarksteinerR., et al.. Functional and histological changes after myoblast injections in the porcine rhabdosphincter. Eur Urol, 2007, 52(6): 1736-1743

[13] StrasserH., MarksteinerR., MargreiterE., et al.. Autologous myoblasts and fibroblasts versus collagen for treatment of stress urinary incontinence in women: a randomised controlled trial. Lancet, 2007, 369(9580): 2179-2186

[14] HalvorsenY.D., FranklinD., BondA.L., et al.. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng, 2001, 7(6): 729-741

[15] ZukP.A., ZhuM., AshjianP., et al.. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002, 13(12): 4279-4295

[16] StoekU.A., VaeantiJ.P.. Tissue engineering: Current state and prospects. Annu RevMed, 2001, 52(1): 443-454

[17] GronthosS., FranklinD.M., LeddyH.A., et al.. Surface protein characteritation of human adipose tissue-derived stromal cells. Cell Physiol, 2001, 189(1): 54-63

[18] AfizahH., YangZ., HuiJ.H., et al.. A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors. Tissue Eng, 2007, 13(4): 659-666

[19] FuQ., SongX.F., LiaoG.L., et al.. Myoblasts differentiated from adipose-derived stem cells to treat stress urinary incontinence. Urology, 2010, 75(3): 718-723

[20] OgawaR., MizunoH., WatanabeA., et al.. Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice. Biochem Biophys Res Commun, 2004, 313(4): 871-877

[21] LiuT.M., MartinaM., HutmacherD.W., et al.. Identification of common pathways mediating differentiation of bone marrow and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells, 2007, 25(3): 750-760

[22] BaeouF., AndalousiR.B., DaussinP., et al.. Transplantation of adipose tissue-derived stromal cells increases mass and functional capacity of damaged skeletal muscle. Cell Transplant, 2004, 13(2): 103-l11

[23] Planat-BénardV., MenardC., AndréM., et al.. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res, 2004, 94(2): 223-229

[24] LongR.M., GiriS.K., FloodH.D.. Current concepts in female stress urinary incontinence. Surgeon, 2008, 6(6): 366-372

[25] RodriguezL.V., AlfonsoZ., ZhangR., et al.. Clonogenic multipotent stem ceils in human adipose tissue diferentiate into functional smooth muscle cells. Proc NatI Acad Sci USA, 2006, 103(32): l2167-12172

[26] JackG.S., AlmeidaF.G., ZhangR., et al.. Processed lipoaspirate cells for tissue engineering of the lower urinary tract: Implications for the treatment of stress urinary incontinence and bladder reconstruction. J Urol, 2005, 174(5): 2041-2045

[27] YamamotoT., GotohM., HattoriR., et al.. Periurethral injection of autologous adipose-derived stem cells for the treatment of stress urinary incontinence in patients undergoing radical prostatectomy: Report of two initial cases. Int J Urol, 2010, 17(1): 75-82

[28] LinG., WangG., BanieL., et al.. Treatment of stress urinary incontinence with adipose tissue-derived stem cells. Cytotherapy, 2010, 12(1): 88-95