Supplementary MaterialsSupplementary information 41598_2020_68373_MOESM1_ESM. may therefore explain the transient incidence of post-transplant ventricular tachycardia, although further large animal model studies will be required to control post-transplant arrhythmia. test, **P?=?0.0002 by Students test. (c,d) Expression of the nodal marker SHOX2 and cardiac marker cardiac Mitoxantrone Hydrochloride troponin T (cTNT); MLC2A and MLC2V in hESC-CMs on day 20. See also SI Figures S1 and S2. Grafted hESC-CMs grow and become mature over time To evaluate the in vivo chronological characteristics of Mitoxantrone Hydrochloride hESC-CMs, we transplanted hESC-CMs into the athymic rat heart and harvested the hearts at 2 (2?weeks; n?=?5), 4 (4 weeks; n?=?5), or 12 (12 weeks; n?=?5) weeks post transplantation. These endpoints were designed based on our previous transplantation study in which post-transplant arrhythmia was frequently observed between 2 and 4?weeks whereas no sustained VT was detected at 12?weeks post transplantation9. All of the recipients sacrificed at 2, 4, and 12?weeks showed surviving grafts without apparent infiltration of inflammatory cells (Fig.?2aCc). Graft tissue exclusively consisted of cardiomyocytes as determined by the cardiac specific markers -myosin heavy chain (-MHC, Fig.?2dCf) and cTNT (Fig.?2gCi). Grafted cardiomyocytes at 12?weeks post transplantation often showed a clear sarcomere and were arranged in a more serried manner and aligned (Fig.?2gCi). Moreover, co-staining against -MHC, and the proliferation marker KI-67, demonstrated that graft cardiomyocytes retained substantial proliferative capacity up to 4?weeks following transplantation, however, the proliferative capacity was significantly decreased by 12?weeks (Fig.?2jCl,n). * The fraction of MLC2A-positive cardiomyocytes, which reflects either atrial, nodal, or immature ventricular cells, was decreased at 12 significantly?weeks post transplantation in comparison to that in 2 or 4?weeks post transplantation. On the other hand, the fraction of MLC2V-positive mature ventricular cells was increased at 12 significantly?weeks post transplantation (SI Fig. S3iCl on-line). The recipients sacrificed at 12?weeks post transplantation tended showing a more substantial graft region, although this difference didn’t reach statistical significance (Fig.?2m). Used together, the full total outcomes of histological analyses indicated how the grafted cardiomyocytes grew and became mature in vivo, consistent with earlier reviews8,13,14. Open up in another window Shape 2 Engraftment of human being Sera cell-derived cardiomyocytes (hESC-CMs) in athymic rat hearts. hESC-CMs had been injected straight into the athymic rat center and histological evaluation was performed Rabbit polyclonal to Vang-like protein 1 at 2, 4, or 12?weeks after transplantation. (aCc) HematoxylinCeosin Mitoxantrone Hydrochloride (H&E) staining of grafted hESC-CMs in the sponsor hearts. (dCf) Human being grafted cardiomyocytes determined from the cardiac marker, -myosin weighty string (-MHC, arrowheads). Remember that -MHC was specifically indicated in grafted human being cardiomyocytes however, not in sponsor rat cardiomyocytes. Dark squares indicate the region where photos below (gCl) had been extracted from. (gCl) Quadruple staining against cTNT (green), -MHC (reddish colored), KI-67 (white), and DAPI (blue) in the graft region. Grafted cardiomyocytes at 12?weeks post transplantation (we,l) showed an adult appearance characterised by development of the aligned sarcomere framework and serried deposition of cardiomyocytes, aswell as rare manifestation from the proliferative marker KI-67. (m) Percentage of grafted region divided by total left-ventricular region (n?=?5 per group). left ventricle. (n) Percentage of KI-67 positive graft cells at 2, 4, and 12?weeks post transplantation. *P?=?0.0019 vs. 2?weeks,?+?P?=?0.0019 vs. 4?weeks by ANOVA with Tukeys post hoc test. See also SI Figure S3. Transient engraftment of nodal-like cardiomyocytes in vivo We next traced the nodal-like cardiomyocytes in grafted tissue Mitoxantrone Hydrochloride by histology. As no perfectly specific antigen for nodal cardiomyocytes has yet been identified to our knowledge, we used three antibodies against HCN4, SHOX2, and TBX3 to trace nodal-like grafted cardiomyocytes. The expression of the pacemaker channel HCN4 in the graft was substantially decreased at 12?weeks post transplantation (Fig.?3aCc). Similarly, the fractions of cells expressing SHOX2 and TBX3 were significantly decreased at 12?weeks post transplantation (Fig.?3dCk). Open in a separate window Figure 3 Mitoxantrone Hydrochloride Chronological expression of nodal markers in grafted human ES cell-derived cardiomyocytes (hESC-CMs) in intact hearts. (aCc) HCN4 (red) staining in the graft area. (dCf) SHOX2 (red) and human specific Lamin A+C (green) staining. Note that SHOX2 was expressed not only in graft cells but also in host cells, such as fibroblasts. As such, we designated SHOX2+/Lamin A+C+ cells (arrow heads) as graft nodal cells. (gCi) Staining against -MHC (red) and TBX3 (green). (j) Quantitative analysis of the number of SHOX2+/Lamin A+C+ cells divided by the number of total Lamin A+C+ graft cells (n?=?5 per group). Data are mean??SEM. *P?=?0.0135 vs. 2?weeks by ANOVA with Tukeys post hoc test. (k) Quantitative analysis of the number of TBX3+?cells divided by that of -MHC+ graft cardiomyocytes (n?=?5 per group). Data are mean??SEM. *P? ?0.0001 vs. 2?weeks,?+?P?=?0.0002.