Differentiation of human adipose-derived stem cells into beating cardiomyocytes.
Human adipose-derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5-azacytidine (5-aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co-culture with neonatal rat cardiomyocytes. 5-aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5- to 1.9-fold) and the number of cells co-expressing nkx2.5/sarcomeric alpha-actin (27.2% versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11-fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA-treated cells also stained positively for cardiac myosin heavy chain, alpha-actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non-contact co-culture showed no cardiac differentiation; however, ASCs co-cultured in direct contact co-culture exhibited a time-dependent increase in cardiac actin mRNA expression (up to 33-fold) between days 3 and 14. Immunocytochemistry revealed co-expression of GATA4 and Nkx2.5, alpha-actin, TropI and cardiac myosin heavy chain in CM-DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca(2+) transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell-to-cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co-culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.
Choi
Yu Suk
YS
O'Brien Institute, Melbourne, Australia.
Dusting
Gregory J
GJ
Stubbs
Samantha
S
Arunothayaraj
Sandeep
S
Han
Xiao Lian
XL
Collas
Philippe
P
Morrison
Wayne A
WA
Dilley
Rodney J
RJ
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D000818:Animals; D000831:Animals, Newborn; D001374:Azacitidine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D021621:Imaging, Three-Dimensional; D008875:Middle Aged; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020107:Troponin T / Q000235:genetics / Q000378:metabolism
D000199:Actins; D003470:Culture Media; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D020107:Troponin T; C012589:trichostatin A; D001374:Azacitidine; D002118:Calcium