Controllable Expansion of Primary Cardiomyocytes by Reversible Immortalization

Abstract

Cardiac tissue engineering will remain only a prospect unless large numbers of therapeutic cells can be provided, either from small samples of cardiac cells or from stem cell sources. In contrast to most adult cells, cardiomyocytes are terminally differentiated and cannot be expanded in culture. We explored the feasibility of enabling the in vitro expansion of primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then reverting the phenotype of the expanded cells back to cardiomyocytes state. Primary rat cardiomyocytes were transduced with SV40 large T Antigen (TAg) or Bmi-1 followed by human telomerase (hTERT) gene, the cells were expanded, and the transduced genes were removed by adenovirus vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than Bmi-1/hTERT gene, based on the rate of cells proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (α-actin, connexin 43 (Cx-43), calcium transients). After the immortalization was reversed, cells returned to their differentiated state. This strategy for controlled expansion of primary cardiomyocytes by gene transfer has potential for providing large amounts of patient's own cardiomyocytes for cell therapy, and the cardiomyocytes derived by this method could be a useful cellular model to study cardiogenesis.

Multiscale Biomimetic Topography for the Alignment of Neonatal and Embryonic Stem Cell-Derived Heart Cells

“…38 Unfortunately, neonatal mouse hearts are limited by their size and thus in the number of cells available for experiments. Isolation of neonatal ventricular CM is challenging, in part because these cells are fully differentiated and cannot be expanded in vitro.…”

“…Isolation of neonatal ventricular CM is challenging, in part because these cells are fully differentiated and cannot be expanded in vitro. 38 We hypothesized that the inclusion of noncardiac cell populations would benefit our CM cultures by inducing them to physiologically respond more like native tissue. We consistently isolated *1 million cells per neonatal mouse heart by following the general protocol described by Heidi et al 27 The heterogeneous cellular composition of the heart includes CM, endothelial cells, fibroblasts, and smooth muscle cells.…”

Controlled Expansion of Mammalian Cell Populations by Reversible Immortalization

“…Reversible immortalization of cardiomyocytes has also been reported [63]. The immortalized cardiomyocytes exhibited the morphological features of dedifferentiation (an increased expression of vimentin and reduced expressions of troponin I and Nkx2.5), along with the continued expression of cardiac markers (alpha-actin, connexin-43 and calcium transients).…”

2.1 Generation of Cell Lines and Biotechnological Applications

“…For this purpose, site-specific recombinases such as Cre or Flp recombinase were successfully employed to facilitate excision of the immortalizing gene(s) flanked with the cognate recombination target sites (e.g., loxP or FRT sites) [82][83][84][85]. However, in contrast to the previously described transcriptional and post-translational control systems, the reversion of the genetic switches is irreversible since the immortalizing gene is eliminated.…”