Abstract 16537: Novel Pathological Detection System of Induced Pluripotent Stem Cell-Derived Cardiomyocytes Using T-Cell Receptor Gene Locus for Cell Transplantation Therapy
Background: Recently, patient’s own induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) are expected to be cell sources for transplantation therapy for severe heart failure. In the assessment of the benefits and the adverse effects of transplantation therapy using iPSC-CMs, pathological assessment of transplanted cardiomyocytes is essential. However, engrafted autologous iPSC-CMs are indistinguishable from the recipient’s tissue. Moreover, additional gene insertion to iPSCs for pathological detection possibly causes tumorigenic transformation and therefore is unsuitable for clinical application. To address this issue, we developed a novel pathological detection method of genomic rearrangement in cardiomyocytes, which were differentiated from T cell-derived iPSCs (TiPSCs).
Methods and Results: Transgene-free TiPSCs were generated from peripheral blood of a healthy volunteer in feeder-free conditions with temperature-sensitive mutated Sendai virus which expressed OCT4, SOX2, KLF4, and c-MYC. TiPSCs showed typical iPSCs characteristics in gene expression and differentiation ability. TiPSCs were differentiated into cardiomyocytes by floating culture. RT-PCR analysis and immunohistochemistry showed that TiPSC-derived cardiomyocytes (TiPSC-CMs) had functional cardiomyocyte properties. V, D, and J segment usages in T-cell receptor beta (TCRB) gene locus were sequenced and identified by comparison to the database of the international ImMunoGeneTics information system. TiPSCs and TiPSC-CMs showed identical rearrangement patterns in TCRB gene locus. With DNA fluorescence in situ hybridization (FISH) probes which were designed according to genomic deficit area in TCRB gene locus and chromosome 7 centromere, two color FISH was performed. TiPSCs and TiPSC-CMs showed genomic deficits of the rearrangement area in TCRB gene locus.
Conclusions: We successfully detected genomic deficits of TCRB rearrangement in TiPSCs and TiPSC-CMs with the FISH method. This method enables pathological distinction of autogenous iPSC-CMs from recipient’s tissue without additional gene insertion, and plays a pivotal role in the verification of successful engraftment of donor iPSC-CMs to follow up clinical studies.
- © 2013 by American Heart Association, Inc.