Intramyocardial transplantation of undifferentiated rat induced pluripotent stem cells causes tumorigenesis in the heart

Abstract

Background: Induced pluripotent stem cells (iPSCs) are a novel candidate for use in cardiac stem cell therapy. However, their intrinsic tumorigenicity requires further investigation prior to use in a clinical setting. In this study we investigated whether undifferentiated iPSCs are tumorigenic after intramyocardial transplantation into immunocompetent allogeneic recipients.

Methodology/principal findings: We transplanted 2 × 10(4), 2 × 10(5), or 2 × 10(6) cells from the established rat iPSC line M13 intramyocardially into intact or infarcted hearts of immunocompetent allogeneic rats. Transplant duration was 2, 4, or 6 weeks. Histological examination with hematoxylin-eosin staining confirmed that undifferentiated rat iPSCs could generate heterogeneous tumors in both intracardiac and extracardiac sites. Furthermore, tumor incidence was independent of cell dose, transplant duration, and the presence or absence of myocardial infarction.

Conclusions/significance: Our study demonstrates that allogeneic iPSC transplantation in the heart will likely result in in situ tumorigenesis, and that cells leaked from the beating heart are a potential source of tumor spread, underscoring the importance of evaluating the safety of future iPSC therapy for cardiac disease.

Figure 1. Verification of M13 rat iPSC characteristics.

A, Mouse embryonic stem cell-like colonies of M13 cells on a feeder layer. B, Embryoid bodies on day 6 of a floating culture of M13 cells. C, Alkaline phosphatase staining of M13 colonies. D, M13 cells maintain a normal diploid karyotype of male rat origen (42XY) at passage 31. Scale bars: 100 µm.

Figure 2. Transplantation of undifferentiated rat iPSCs causes tumorigenesis.

A and B, Gross pathological evaluation revealed visible tumors (white arrows) extending from the left ventricular wall of the heart harvested 2 (A) or 6 (B) weeks after transplantation. The ligature site is indicated (black arrow). C-I, Representative series of hematoxylin-eosin-stained sections from harvested tumors. C, Heart section from a myocardial infracted (MI) rat sacrificed 2 weeks after cell transplantation showed an undifferentiated cell graft, magnified in D, and osteogenic structures (mesoderm), magnified in E, in addition to normal cardiac tissue (*). F, A clear-edged cartilaginous structure (mesoderm) within the cardiac tissue from an intact rat sacrificed 4 weeks after cell transplantation. G, High magnification of the inset in F. H, Neural rosettes (ectoderm) in a thoracic cavity tumor of an MI rat sacrificed 4 weeks after cell transplantation. A boxed rosette is magnified in I. Scale bars: 100 µm.

Figure 3. Tumor incidence in rats after transplantation of undifferentiated rat iPSCs and differentiation of the tumors.

A-C, Tumor incidence in MI rats receiving different cell doses and/or subjected to various transplant durations. Cell dosage had no significant influence on tumor incidence, regardless of transplant duration (A and B). Likewise, transplant duration had no significant influence on tumor incidence, regardless of cell dose (A and C). D, There was no significant difference in tumor incidence between MI and intact rats. E, Intracardiac tumor incidence was significantly higher than that of extracardiac tissues (P = 0.001). F, Infarction had no significant influence on intracardiac/extracardiac tumor incidence. G, The incidence of mesodermal differentiation was significantly higher than endodermal or ectodermal differentiation of tumors (P<0.001). H, Intracardiac tumors had a lower incidence of ectodermal differentiation than extracardiac tumors (P = 0.041), but no significant difference in the incidence of mesodermal differentiation was found between intracardiac and extracardiac tumors. I, Infarction had no significant influence on tumor differentiation.