Additional HAND2 or microRNA-1 could facilitate the progress of human iCM-reprogramming by GMTEMMZ 7 factors (7Fs).

<p>A) A t-SNE embedding was utilized to visualize the overall reprogramming degree in individual iCMs reprogrammed by 7Fs plus one extra factor, including microRNA-1 (n = 39), <i>HAND2</i> (n = 61), <i>RXRG</i> (n = 18), <i>SMYD1</i> (n = 74), and <i>TBX20</i> (n = 74). Two dash lines were inserted to assort iCMs into three populations: fibroblast-like, intermediate-, and CM-like reprogrammed iCMs. The data of H9CMs, H9Fs, 4 weeks-reprogrammed 7Fs-iCMs (4W-7Fs-iCMs) and 12W-7Fs-iCMs were included as control groups. B) The quantification of three subpopulations in panel A showed that additional HAND2 or microRNA-1 significantly decreased the subpopulation of fibroblast-like reprogrammed iCMs, while the CM-like subpopulation was significantly enhanced only in 12W-7Fs-iCMs. *p<0.05 vs. 4W-7Fs-iCMs. C) The expression of HAND2 in individual H9Fs and iCMs reprogrammed by 7Fs or 7Fs+HAND2. 7Fs+HAND2-reprogrammed iCMs were classified into three sub-populations with low-, medium- and high-expression of HAND2. D) Comparison of cardiac (upper panel) and fibroblast-enriched (lower panel) genes among low-HAND2 (n = 5), medium-HAND2 (n = 14) and high-HAND2 (n = 42) subpopulations of 7Fs+HAND2-reprogrammed iCMs. *p<0.05, ** p<0.01, *** p<0.001.</p

Single cell qPCR reveals that additional <i>HAND2</i> and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes

<div><p>The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan^® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional <i>HAND2</i> or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more <i>HAND2</i> expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, <i>HAND2</i> and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.</p></div

Additional reprogramming factors didn’t increase the yield of αMHC-mCherry⁺ iCMs.

<p>A) Heatmap of gene expression profiles for a panel of transcription factors that were expressed at a significantly higher level in pooled CMs (H9CMs and fetal human CMs [fHCMs]) than that in H9Fs and HDFs. B) Fold changes (normalized to H9Fs) of cardiac transcription factors activated in pooled human iCMs reprogrammed by 7 factors (7Fs) of GMTEMMZ for 4 weeks (H9FiCMs-4W) and 12 weeks (H9FiCMs-12W). C-D) The effect of adding one extra transcription factor (C) or microRNA-1 (D) on the induction of αMHC-mCherry⁺ (upper panel, n = 6) or cardiac troponin T (cTnT⁺, lower panel, n = 4) iCMs reprogrammed by 7Fs. *p<0.05, **p<0.01 compared to the control group of 7Fs+GFP.</p

Validation of 96 TaqMan^® primers/probes for single-cell qPCR in individual H9CMs (n = 56), H9Fs (n = 45), and HDFs (n = 41).

<p>Pooled samples of H9CMs and human fibroblasts (P-CMs and P-HFs) were used as positive controls. A) Heatmap of gene expression profiles in our microarray assay of H9Fs and H9CMs. B) A panel of 46 primers/probes had sufficient efficiency and sensitivity for single-cell qPCR. C) A principal component analysis (PCA) displayed a global view of the expression profile of those 46 genes in individual H9CMs, H9Fs, and HDFs.</p

The expression profile of the identified 46 genes is able to estimate the quality of human iCM reprogramming.

<p>A) A hierarchical clustering analysis showed that most human iCMs reprogrammed from H9Fs by 7 factors of GMTEMMZ for 12 weeks (H9FiCM-12W, n = 39) were clustered close to H9CMs, while many iCMs reprogrammed from H9Fs (H9FiCM-4W, n = 36) and HDFs (HDFiCM-4W, n = 37) for 4 weeks were clustered close to fibroblasts. B) Principal component (PC) projections of individual cells. The dash-line circle surrounds a population of iCMs that had a good quality of reprogramming; the arrow indicates another population of iCMs that were poorly reprogrammed.</p

Harvesting individual reprogrammed αMHC-mCherry⁺ H9F iCMs for single-cell qPCR.

<p>A) Many αMHC-mCherry⁺ iCMs were observed in reprogrammed H9Fs 4 weeks after retroviral infection of GMTEMMZ. B) αMHC-mCherry⁺ iCMs were quantified and purified by FACS sorter. C) Representative single αMHC-mCherry⁺ iCM captured by C1 Single-Cell Auto Prep System. A positive staining of calcein indicates cell viability. Scale bars, 20 μm.</p

Additional file 1: of Intratumoral and peritumoral radiomics for the pretreatment prediction of pathological complete response to neoadjuvant chemotherapy based on breast DCE-MRI

Supplementary methods. (DOCX 12.4 kb