Differentiation of human adipose-derived stem cells into beating cardiomyocytes

Abstract

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.

Fig 1

Real-time RT-PCR analysis of cardiac actin and TropT mRNA in hASCs 1, 2, 3 weeks after 5-aza treatment or culture in MCM. (A) Cardiac actin, (B) TropT. GAPDH was used as internal control. Bar graphs indicate fold change of mRNA compared with untreated ASCs (dotted line). Results are shown as mean ± S.E.M. (n= 3). *P < 0.05 versus untreated ASCs as control.

Fig 2

Immunocytochemistry for ASCs (A, D), 5-aza treated ASCs (B, E), ASCs cultured in MCM (C, F) after 3 weeks. Images show expression of Nkx2.5 (green) and α-actin (red) (A–C); Nkx2.5 (green) and TropI (red) (D–F). Scale bar = 100 μm.

Fig 3

Real-time RT-PCR analysis of cardiac actin mRNA in hASCs after trichostatin A treatment for 24 hrs, and cultured in control medium or continuously for 1, 2 and 3 weeks (A). ASCs also treated by TSA continuously for 1 week were cultured in control medium or CCM for 2 weeks (B). GAPDH was used as internal control. Bar graphs indicate fold change of mRNA compared with ASCs (dotted line). Results are shown as mean ± S.E.M. (n= 3). *P < 0.05 versus other groups

Fig 4

Immunocytochemistry for 1 week TSA treated ASCs after culturing in CCM for 2 weeks. Cells were positively stained by cMHC (A), α-actin (B), cTropI (C) and Cx43 (D). Primary antibodies were conjugated FITC. Scale bar = 100 μm.

Fig 5

Cardiac actin mRNA level of ASCs in co-culture at day 3, 5, 7, 14, 21 by real-time RT-PCR. GAPDH was used as internal control. Stem cells sorted from co-cultures by FACS at day 14 retained expression levels, confirming human probe specificity. Bar graphs indicated fold change of mRNA compared with ASCs (dotted line). Results are shown as mean ± S.E.M. (n= 3). *P < 0.05 versus untreated ASCs as control.

Fig 6

Immunocytochemistry of ASCs after sorting from co-culture with cardiomyocytes by CM-DiI labelling. Cells were stained by GATA4 (A), Nkx2.5 (B), α-actin (C), TropI (D), cMHC (E) and MyoD (F) conjugated by FITC (ASCs were pre-labelled by CM-DiI which shown in red). MyoD was used as negative control. Scale bar = 100 μm.

Fig 7

Gap junction between ASC and cardiomyocyte. ASCs were labelled by CM-DiI (Red, not able to transfer to adjacent cells through gap junction) and rat cardiomyocytes were labelled by calcein AM (Green, able to transfer to adjacent cells through gap junction). Red cells are hASCs under Texas Red filter (A, D) and Green cells were rat cardiomyocytes or hASC with calcein AM transferred from cardiomyocytes under FITC filter (B, E) and two pictures were merged (C, F). Gap junction between two cell types by direct contact allowed calcein AM transfer from rat cardiomyocyte to hASC. White arrow showed hASCs with calcein AM transferred from beating rat cardiomyocytes in merged images. Scale bar = 100 μm.