Inhibition of stearoyl-coA desaturase selectively eliminates tumorigenic Nanog-positive cells: improving the safety of iPS cell transplantation to myocardium

Abstract

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, α-MHC, or MLC-2v, markers of cardiac differentiation (P>0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.

Keywords: Nanog; PluriSin#1; iPS; myocardial infarction; tumorigenicity.

Figure 1. Characterization of CF-derived iPS cells. (A) Mouse iPS cells cultured on feeder free matrigel coated dish; (B) Immunostaining of iPS cells for the classic ES cell marker Nanog (red). Nuclei were stained with DAPI (blue); (C) Image of an explanted teratoma; (D–F) Hematoxylin and eosin staining of teratoma sections 3 wk after iPS cell transplantation.

Figure 2. Cardiomyocyte differentiation of mouse iPS cells. (A) Schematic diagram of protocol used for cardiac differentiation of mouse iPS cells; (B) Appearance of iPS-derived EB in suspension culture; (C) Immunostaining for cardiac troponin I (cTnI) in iPS-derived cells (IPSD); nuclei were counterstained with DAPI.

Figure 3. Effects of PluriSin#1 on Nanog-positive iPSD. (A1 and A2) Mouse iPSD were incubated with DMSO or PluriSin#1 for 4 d; note the appearance of cell death in the central region of iPSD treated with PluriSin#1; (B1 and B2) DMSO- and PluriSin#1-treated iPSD were assayed for apoptosis using TUNEL staining; note extensive TUNEL positivity in the center of PluriSin#1-treated iPSD; (C) Real-time RT-PCR analysis demonstrating the effect of PluriSin#1 on Nanog mRNA expression; (D) Protein was isolated from DMSO or PluriSin#1 treated IPSD and then used for immunoblotting; (E1 and E2) Immunostaining for Nanog demonstrating elimination of Nanog-positive iPSD cells by PluriSin#1. Nuclei were counterstained with DAPI; (F and G) Mouse iPSD were incubated with DMSO or PluriSin#1 for 1 d, a combined TUNEL and Nanog immunofluorescent staining demonstrating apoptosis of Nanog-positive iPSD cells by PluriSin#1. Nuclei were counterstained with DAPI.

Figure 4. PluriSin#1 treatment inhibits tumorigenic potential of iPSD in vivo. (A1 and A2) DMSO- or PluriSin#1-treated iPSD cells were injected intramyocardially into the border zone of infarcted hearts of C57BL/6 mice. Mice were anesthetized and sacrificed on day 14 after injection. Hearts were harvested and photographed (tumor in A1 is outlined by broken line); (B1 and B2) H&E staining of heart sections showing tumor in hearts transplanted with DMSO-treated iPSD, tumors are indicated by arrows; (C and D) Immunostaining for Nanog (red) in hearts transplanted with DMSO- or PluriSin#1-treated iPSD (green). Note the diffuse GFP expression and co-localization with Nanog in tumors of hearts injected with DMSO-treated iPSD, which is prevented by treatment with PluriSin#1.

Figure 5. Effects of PluriSin#1 on cardiac differentiation and survival of iPSD in vitro and in ischemic myocardium in vivo. (A–C) Real-time RT-PCR detection of cTnI, α-MHC and MLc-2v in DMSO- and PluriSin#1-treated iPSD. Four biological replicates were analyzed for each sample. The relative gene expression values represent the level of gene expression for PluriSin#1-treated samples compared with DMSO control; (D1–4) Apoptotic cardiomyocytes expressed as cTnI positive (green) and TUNEL positive (red) cells; (E and F) Engrafted iPSD (green) cells in ischemic myocardium 2 wk after transplantation. CTnI-positive (red) iPSD indicate iPS-derived cardiomyocytes. Nuclei were stained with DAPI (blue).