Reprogramming of human fibroblasts toward a cardiac fate

Abstract

Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4-11 wk, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of nonmyocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach.

Fig. 1.

Screening for additional factors able to activate cardiac gene expression. (A) Representative flow cytometry plot for analyses of cTnT⁺ cells 2 wk after infection of HFFs with retroviruses expressing indicated combinations of factors. Cells infected with empty vector retrovirus were used as a control. The numbers in each plot indicate the percentage of cTnT⁺ cells. (B) Summary of flow cytometry analyses. Percentage of cTnT⁺ cells following infection of HFFs with empty vector retrovirus (control) or retroviruses expressing GHMT with an indicated individual factor as shown. Data from five independent experiments are presented as mean ± SD. Dotted line indicates the percentage of cTnT⁺ cells induced by retroviruses expressing GHMT alone. (C) Representative flow cytometry plot for subtractive analyses to determine the requirement of each individual factor in 5F (GHMMyT). Two weeks after infection of HFFs with retroviruses expressing indicated combinations of factors, cTnT⁺ (Upper) and tropomyosin⁺ (Lower) cells were quantified by flow cytometry. Cells infected with empty vector retrovirus were used as a control. The numbers in each plot indicate the percentage of cTnT⁺ or tropomyosin⁺ cells. (D) Summary of flow cytometry analyses. Percentage of cTnT⁺ (Left) or tropomyosin⁺ (Right) cells following infection of HFFs with empty vector retrovirus (control), or retroviruses expressing 5F (GHMMyT) or 5F minus an indicated individual factor as shown. Data from three independent experiments are presented as mean ± SD. Dotted line indicates the percentage of cTnT⁺ or tropomyosin⁺ cells induced by retroviruses expressing 5F.

Fig. 2.

Determining the optimal combination of factors to activate cardiac gene expression. (A) Summary of flow cytometry analyses of cTnT⁺ cells 2 wk after infection of HFFs with retroviruses expressing indicated combinations of factors. Percentage of cTnT⁺ cells following infection of HFFs with empty vector retrovirus, or retroviruses expressing 5F (GHMMyT) or 5F plus an indicated individual factor as shown. Data from three independent experiments are presented as mean ± SD. Dotted line indicates the percentage of cTnT⁺ cells induced by retroviruses expressing 5F. (B) Effect of miR-1 and miR-133 on activation of cardiac markers. Representative flow cytometry plot for analyses of cTnT⁺ cells 2 wk after infection of HFFs with retroviruses expressing indicated combinations of factors. Cells infected with empty vector retrovirus were used as a control. The numbers in each plot indicate the percentage of cTnT⁺ cells. (C) Summary of flow cytometry analyses. Percentage of cTnT⁺ cells following infection of HFFs with empty vector retrovirus, or retroviruses expressing 5F (GHMMyT) or 5F plus miR-1, miR-133, or both miR-1 and miR-133 as shown. Data from nine independent experiments are presented as mean ± SD. *P = 0.0062 (D) Summary of flow cytometry analyses to determine the necessity of individual 7F (GHMMyT, miR-1, miR-133). Percentage of cTnT⁺ (Left) or tropomyosin⁺ (Right) cells following infection of HFFs with empty vector retrovirus (control), or retroviruses expressing 7F (GHMMyT miR-1 miR-133) or 7F minus an indicated individual factor as shown. Data from three or four independent experiments are presented as mean ± SD. *P = 0.041. (E) Inefficient cardiac gene activation in the absence of miR-133. Representative flow cytometry plot for analyses of cTnT⁺ cells 1 wk after infection of retroviruses expressing GHMT into tail-tip fibroblasts isolated from either wild-type (WT) or miR-133 knockout (miR-133^−/−) mice. Tail-tip fibroblasts isolated from wild type mice infected with empty vector were used as a control.

Fig. 3.

Induction of cardiac markers in AHCFs. (A) Representative flow cytometry plot for analyses of cTnT⁺ cells 2 wk after infection of AHCFs with retroviruses expressing indicated combinations of factors. Cells infected with empty vector retrovirus were used as a control. The numbers in each plot indicate the percentage of cTnT⁺. (B) Summary of flow cytometry analyses in AHCFs. Percentage of cTnT⁺ cells following infection of AHCFs with empty vector retrovirus (control) or retroviruses expressing indicated combinations of factors as shown. Data from three independent experiments are presented as mean ± SD.

Fig. 4.

Immunostaining of cardiac markers in 5F-, 6F-, or 7F-transduced human fibroblasts. Immunofluorescence staining for α-actinin (red) or cTnT (green) was performed 5 wk after transduction of (A) HFFs, (D) AHCFs, or (E) AHDFs with 5F, 6F, or 7F. (Scale bar, 100 μm.) (B) Quantification of α-actinin⁺ cells. (C) Quantification of cTnT⁺ cells. Percentage of α-actinin⁺ (B) or cTnT⁺ (C) cells after infection of HFFs with 5F, 7F, or 7F or 6F minus an indicated individual factor as shown. Data from two or three independent experiments are presented as mean ± SD.

Fig. 5.

Gene expression profile in human fibroblasts transduced with 5F. Gene expression profile was analyzed by microarray or quantitative PCR (qPCR) in HFFs or AHCFs 4 wk after transduction with 5F. (A) Heat map of microarray data illustrating differentially expressed 2,436 genes in HFFs, 5F-transduced HFFs, and adult human heart. Red indicates up-regulated genes; green indicates down-regulated genes. (B) Heat map of selected genes. Genes that encode cardiac contractile proteins, cardiac peptides, calcium handling genes, cardiac transcription factors, and genes involved in cardiac metabolism were up-regulated. In contrast, genes encoding nonmyocyte markers were down-regulated. (C) Gene expression analyses by qPCR in HFF and AHCFs transduced with 5F. ACTC1, TNNT2, MYL7, MYH6, and TPM1 are sarcomere genes; ATP2A2, GJA1, and GJA5 are cardiac channel genes; NPPA and NPPB are cardiac peptide genes; COL1A2, COL3A1, and S100A4 are nonmyocyte genes. Expression of cardiac and nonmyocyte genes was quantified by qPCR. UD, undetectable.

Fig. 6.

Measurement of calcium transient in hiCLMs. Calcium transient of a single cell was traced upon KCL stimulation in hiCLMs derived from (A) HFFs and (B) AHCFs. HFFs or AHCFs were used as a negative control accordingly. The graph bar represents the percentage of cells displaying calcium transients. The total number of cells recorded for calcium transients was 50 for HFFs and 19 for AHCFs.