Reprogramming of human somatic cells into cardiomyocytes
Jayakumar Thangavel, Buddhadeb Dawn, Johnson Rajasingh
Cardiovascular Research Institute, Division of Cardiovascular Diseases, University of Kansas Medical Center, Kansas City, KS 66160
Background and Objectives: Stem cell therapy for ischemic heart disease holds tremendous promise. One of the critical prerequisites for future cell-based regenerative therapy is a source of homogenous population of cells on specific demand. Thus, the development of a safe, efficient, and inexpensive reprogramming system is of high clinical importance. Recently, we employed a temporary, non-integrating chemical reprogramming approach to transiently activate major pluripotency genes Oct4, Nanog and Sox2 in primary mouse bone marrow progenitor cells. These factors, in turn, briefly reactivated endogenous reprogramming factors, which were maintained just long enough to allow the primary cells to be transformed into cardiomyocytes under specific culture conditions. However, the feasibility of this approach in human cells remains unknown. Thus, we hypothesized that pluripotent genes that are silenced in human keratinocytes (HKC) will be activated in response to a brief treatment with epigenetic modifying agents 5-Aza 2-deoxycytidine (Aza, a DNA methyl transferase inhibitor) and Trichostatin A (TSA, a histone deacetylation inhibitor), and thereby create cells poised to be directly transformed into cardiomyocytes under specific culture conditions.
Methods and Results: We used qRT-PCR, Western blot and immunofluorescence analyses in a series of experiments with HKC that had been treated with Aza and TSA, and made the following observations: 1) Various doses of Aza and TSA treatment induced higher levels of pluripotency gene (Oct4, Nanog and Sox2) transcript and protein expression, and downregulated the expression of abundantly present ICAM-1 and Filaggrin genes. 2) The aceH3K9 (acetylated histone H3 lysine 9) activity was higher and HDAC1 activity was lower in treated HKCs compared with control cells. 3) Under specific culture conditions, the Aza+TSA-treated HKCs differentiated into endothelial and cardiomyocytic cells.
Conclusions: The epigenetic modifying agents Aza and TSA can reprogram the HKCs into cells with a multipotent phenotype, and the de-differentiated HKCs can subsequently differentiate into multiple cell lineages, including cardiomyocytes that can be used as potential source of cells for cardiac repair.