No evidence of transdifferentiation of human mesenchymal or hematopoietic stem cells into cardiomyocytes following coculture with neonatal rat cardiomyocytes

Abstract

Background: Several clinical trials have shown that stem cell based therapy may improve heart function. Both bone marrow derived mesenchymal (MSC) as well as hematopoietic stem cells (HSC) are reported to be multipotent. This study investigates whether neonatal rat cardiomyocytes (NRCM), when co-cultured, can induce transdifferentiation of either MSC or HSC into cardiomyocytes (CM). Methods and results: Ex vivo expanded human bone marrow derived MSC showed expression of CD49c, CD73, CD90, CD105 but not of CD34, CD45, CD106 and CD184. The expanded MSC kept their multipotent characteristics. In contrast, HSC were freshly isolated by flow-sorting based on their expression of CD133 CD34 . Co-cultures, using cell tracker green (5-chloromethylfluorescein diacetate) labelled MSC or HSC and cell tracker red (5-(and6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamin) labelled NRCM, were performed at different ratios (1:6, 1:4, 1:3 and 1:1). Since several reports indicate that dimethylsulfoxide (DMSO) and 5-azacytidin (5-aza) induce myocardial differentiation, 1% DMSO for 48h or 3 M 5-aza for 24h was added to the co-cultures and compared to conditions without additives. After three weeks green and red labelled cells were separated by flow-sorting and cardiac gene expression was analyzed by RT-PCR. Co-culturing MSC induced the expression of troponin T (TnT) and GATA-4. However, no expression of -actinin, myosin heavy chain (MHC) or troponin I (TnI) could be detected. Furthermore, co-culturing HSC, could only induce the expression of TnT, but no expression of -actinin, MHC, TnI or GATA-4 was observed. Transmission electron microscopy confirmed the absence of sarcomeric organization both in co-cultured MSC and HSC. Conclusions: This in vitro co-culture study obtained no convincing evidence of transdifferentiation of either MSC of HSC into CM. Despite the induction of Tnt and GATA-4, several cardiac specific genes are not expressed after 3 weeks of co-culture. No influence of DMSO or 5-aza on the transdifferentiation of MSC or HSC could be detected. Therefore, the reported functional improvement following cell based therapy for myocardial infarction may be due to other mechanisms than transdifferentiation of MSC or HSC.