In vivo tracking transplanted cardiomyocytes derived from human induced pluripotent stem cells using nuclear medicine imaging

Abstract

Introduction: Transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a promising treatment for heart failure. Information on long-term cell engraftment after transplantation is clinically important. However, clinically applicable evaluation methods have not yet been established.

Methods: In this study, to noninvasively assess transplanted cell engraftment, human SLC5A5, which encodes a sodium/iodide symporter (NIS) that transports radioactive tracers such as ¹²⁵I, ¹⁸F-tetrafluoroborate (TFB), and ^99mTc-pertechnetate (^99mTcO₄^-), was transduced into human induced pluripotent stem cells (iPSCs), and nuclear medicine imaging was used to track engrafted human iPSC-CMs.

Results: To evaluate the pluripotency of NIS-expressing human iPSCs, they were subcutaneously transplanted into immunodeficient rats. Teratomas were detected by ^99mTcO₄^- single photon emission computed tomography (SPECT/CT) imaging. NIS expression and the uptake ability of ¹²⁵I were maintained in purified human iPSC-CMs. NIS-expressing human iPSC-CMs transplanted into immunodeficient rats could be detected over time using ^99mTcO₄^- SPECT/CT imaging. Unexpectedly, NIS expression affected cell proliferation of human iPSCs and iPSC-derived cells.

Discussion: Such functionally designed iPSC-CMs have potential clinical applications as a noninvasive method of grafted cell evaluation, but further studies are needed to determine the effects of NIS transduction on cellular characteristics and functions.

Keywords: cell-based therapy; human induced pluripotent stem cell-derived cardiomyocytes; in vivo imaging; single photon emission computed tomography; sodium/iodide symporter.

Figure 1

Generation of SLC5A5 transgenic human induced pluripotent stem cell (iPSC) lines. (A) Transduction of human SLC5A5 gene to AAVS1 locus in human induced pluripotent stem cells (iPSCs) using TALEN. (B) The difference in size of polymerase chain reaction (PCR) product depends on the presence or absence of the transgene. (C) Determination of the presence or absence of gene transfer to the AAVS1 locus using PCR of DNA extracted from each iPSC line. (D) EGFP expression in each human iPSC line. Scale bars are 200 µm.

Figure 2

NIS-expressing human induced pluripotent stem cells (iPSCs). (A) Immunostaining of immature markers and NIS in human iPSCs. Scale bars are 100 µm. (B) Uptake of ¹²⁵I in vitro. N = 3 in each, *p < 0.001 vs. NIS (+)/¹⁸F-tetrafluoroborate (TFB) and ¹²⁵I (ANOVA/Bonferroni). (C) Uptake of ¹⁸F- TFB in vitro. NaClO₄ is an inhibitor of NIS. N = 3 in each, *p < 0.001 vs. NIS (+)/¹⁸F-TFB and ¹²⁵I (ANOVA/Bonferroni).

Figure 3

Teratoma formation assay of NIS-expressing human induced pluripotent stem cell (iPSC) lines and NIS non-expressing human iPSC lines. (A) Three cell lines per group were injected subcutaneously into the back in immunodeficient mice. Mice injected with NIS non-expressing human iPSC lines showed obvious mass formation after 6 weeks (surrounded by a black dashed line). In contrast, mice injected with NIS-expressing human iPSC lines showed no visible mass formation from the external surface. (B) Teratoma formation was confirmed histologically in NSG mice injected with NIS-expressing and NIS non-expressing human iPSC lines. Scale bars are 200 µm. (C) NIS-expressing human iPSCs were injected into the right side of the back and NIS non-expressing human iPSCs into the left side of the back in immunodeficient rats. Six weeks later, ^99mTcO₄⁻ SPECT/CT was performed to detect NIS-expressing human iPSC-derived cells. NIS-expressing human iPSC-derived cells formed a smaller mass than NIS non-expressing iPSC-derived cells (surrounded by a white dashed line). (D) NIS-expressing human cells were confirmed histologically in immunodeficient rats. Scale bars are 50 µm.

Figure 4

NIS-expressing human induced pluripotent stem cell (iPSC) lines showed slower proliferation in differentiation medium than NIS non-expressing human iPSC lines did. Sodium iodide (NaI) rescued poor proliferation of NIS-expressing cells. (A) NIS-expressing human iPSCs grew as well as NIS non-expressing human iPSCs in Essential 8 Flex Medium (maintenance medium for human pluripotent stem cells). Cells were cultured in Essential 8 Flex Medium for 4 days with or without NaI, and nuclei (blue) and F-actin (red) were stained. Scale bars are 1 mm. (B) Cell area/surface area was measured (N = 3 in each). *p < 0.001 (ANOVA/Bonferroni). (C) Cells were cultured with RPMI/B27 medium (basal medium for differentiation) for 4 days with or without NaI, and nuclei (blue) and F-actin (red) were stained. Scale bars are 1 mm. (D) Cell area/surface area was measured (N = 3 in each, Student-t test). (E) Cell counts were evaluated by MTT assay (N = 6 in each). *p < 0.01, **p < 0.001 vs. NIS (+) w/o NaI (ANOVA/Bonferroni).

Figure 5

Induction of NIS-expressing human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). (A) Immunostaining of NIS in NIS-expressing and NIS non-expressing human iPSC-CMs at differentiation day 31. Scale bars are 400 µm. (B) Immunostaining of NKX2-5, a marker of working cardiomyocytes, and cardiac troponin T (cTnT) in NIS-expressing and NIS non-expressing human iPSC-CMs at differentiation day 31. Scale bars are 200 µm. (C) Immunostaining of α-actinin and Connexin 43 in NIS-expressing and NIS non-expressing human iPSC-CMs. Scale bars are 100 µm. (D) Immunostaining of α-actinin and MLC2v, a marker of ventricular cardiomyocytes, on differentiation day 50 in NIS-expressing and NIS non-expressing human iPSC-CMs. Scale bars are 100 µm. (E) Uptake of ¹²⁵I of human iPSC-CMs at differentiation day 35 in vitro. NaClO₄ is an inhibitor of NIS. Data are shown as mean ± standard deviation (n = 5 in each). *p < 0.001 vs. NIS (+) w/o 200 µM NaClO₄ (ANOVA/Bonferroni). (F) NIS-expressing human iPSC-CMs were cultured for 10 days from differentiation day 21, and cell viability was evaluated using CCK-8 assay. Data are shown as mean ± standard deviation (n = 6 in each, ANOVA/Bonferroni).

Figure 6

Detection of transplanted human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). (A) NIS-expressing human iPSC-CMs were injected into the right inguinal subcutaneous adipose tissue and NIS non-expressing human iPSC-CMs into the left inguinal subcutaneous adipose tissue in an immunodeficient rat. One week later, ^99mTcO₄⁻ SPECT/CT was performed to detect NIS-expressing human iPSC-CMs (surrounded by a white dashed line). (B) Engraftment of both NIS-expressing human iPSC-CMs and NIS non-expressing human iPSC-CMs were histologically confirmed by immunostaining of human Ku80, NIS and cardiac troponin T (cTnT). Scale bars are 200 µm.

Figure 7

Long-term tracking transplanted human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). (A) Six weeks after injection of NIS-expressing human iPSC-CMs into the right inguinal subcutaneous adipose tissue of an immunodeficient rat, ^99mTcO₄⁻ SPECT/CT was performed to detect the injected cells (surrounded by a white dashed line). (B) ^99mTcO₄⁻ SPECT/CT image of the same rat shown in (A) 10 weeks after cell injection. Tracer uptake was observed in the right inguinal subcutaneous adipose tissue (surrounded by a white dashed line). (C) Sixteen weeks after injection of NIS-expressing iPSC-CMs into the cryoinjured heart of an immunodeficient rat, ^99mTcO₄⁻ SPECT/CT was performed to detect the injected cells (surrounded by a white dashed line). (D) Quantification of ^99mTc signals from transplanted cells over time. (E) Engraftment of NIS-expressing human iPSC-CMs was histologically confirmed by immunostaining of human Ku80, NIS, cardiac troponin T (cTnT), and MLC2v. Scale bars are 100 µm. (F) Engraftment of NIS-expressing human iPSC-CMs was histologically confirmed by immunostaining of human Ku80, NIS and cTnT. Scale bars are 100 µm.