Cardiac Fibroblast-Induced Pluripotent Stem Cell-Derived Exosomes as a Potential Therapeutic Mean for Heart Failure

Abstract

The limited regenerative capacity of the injured myocardium leads to remodeling and often heart failure. Novel therapeutic approaches are essential. Induced pluripotent stem cells (iPSC) differentiated into cardiomyocytes are a potential future therapeutics. We hypothesized that organ-specific reprogramed fibroblasts may serve an advantageous source for future cardiomyocytes. Moreover, exosomes secreted from those cells may have a beneficial effect on cardiac differentiation and/or function. We compared RNA from different sources of human iPSC using chip gene expression. Protein expression was evaluated as well as exosome micro-RNA levels and their impact on embryoid bodies (EBs) differentiation. Statistical analysis identified 51 genes that were altered (p ≤ 0.05), and confirmed in the protein level, cardiac fibroblasts-iPSCs (CF-iPSCs) vs. dermal fibroblasts-iPSCs (DF-iPSCs). Several miRs were altered especially miR22, a key regulator of cardiac hypertrophy and remodeling. Lower expression of miR22 in CF-iPSCs vs. DF-iPSCs was observed. EBs treated with these exosomes exhibited more beating EBs p = 0.05. vs. control. We identify CF-iPSC and its exosomes as a potential source for cardiac recovery induction. The decrease in miR22 level points out that our CF-iPSC-exosomes are naïve of congestive heart cell memory, making them a potential biological source for future therapy for the injured heart.

Keywords: exosome; heart failure; iPSCs.

Figure 1

Generation of induced pluripotent stem cells (iPSCs). (A,a) A representative image of skin fibroblasts isolated from skin biopsies. (b) A representative image of cardiac apex fibroblasts isolated from cardiac apex biopsies. Scale bars (a,b), 100 µm. (c) Immunostaining of live cells using TRA-1-60 marker, derived from reprogramming the skin fibroblasts. (d) Representative image of a skin fibroblast-derived iPSC colony. (e) Representative image of a cardiac apex fibroblast-derived iPSC colony. Scale bars (C,E), 200 µm. (B) Immunofluorescence detection of cellular marker expression in iPSC. iPSC demonstrated staining of TRA 1–60 (a), Nanog (c), Sox2 (e), Oct4 (g), and TRA-1-81 (i). DAPI (4′,6-diamidino-2-phenylindole) staining of the nuclei (b), (f), and (j). Merged staining images (d), (h), and (k). Scale bars (a–k), 200 µm. (C) Karyotype of CF-iPSC (a) and DF-iPSC (b). (D) Alkaline phosphatase staining of cardiac fibroblasts (CF-iPSC)- and skin (DF-iPSC)-derived iPSCs. Scale bars, 100 µm. (E) iPSC lines can spontaneously differentiate into the three germ layers. Microdissected contracting areas were stained for typical myofilament proteins. Immunostaining of iPS-derived embryoid bodies (EBs) revealed the expression of ectodermal (βll-tubulin; scale bars, 200 µm), endodermal (AFP (alpha fetoprotein); scale bars, 10 µm), and mesodermal (CD31, SMA (smooth muscle actin); scale bars, 20 µm) markers. Nuclei were stained with DAPI.

Figure 2

Immunofluorescence staining of cardiac proteins in iPSC-derived cardiomyocytes at day 28 of cells’ spontaneous differentiation. (A) Microdissected contracting areas were stained for typical myofilament proteins. Cells were labeled with antibodies specific to myosin (a), troponin (c), and α-actinin (e). DAPI staining of the nuclei; (b,f). Merged images; (d,g). Scale bars (a–g), 100 µm. (B,C) (n = 11). Cardiac differentiation; Plated EBs were monitored over 4 weeks of culture for (B) the onset of the first contracting areas (* p = 0.02). Number of contracting EBs were counted out of the total number of plated EBs ((C), * p = 0.009).

Figure 3

Microarray chip gene expression; 6367 differentially expressed probes selected by padj ≤ 0.05 and absolute log FC ≥ 0.75 were clustered to 11 clusters (data not shown); clustering of differentially expressed genes in CF-iPSCs versus DF-iPSCs. Cluster 1: Highly expressed in DF-iPSC vs CF-iPSC; cluster 2: Highly expressed in CF-iPSC vs DF-iPSC (p ≤ 0.05 and absolute log FC ≥ 0.75).

Figure 4

Western blot for iPSC protein (p16–18). (A) GSTM2 * p < 0.05. (B) ARGBP2 * p < 0.05. (C) GPR177 * p < 0.05. (D) CDH11. (E) ACTA2. (F) Blotting: CF-iPSC (n = 4), CF-iPSC (n = 5).

Figure 5

Verification of nanoparticles as exosomes. Nano sight for nanoparticles size; avg 115 ± 7, mode 111 ± 6 nm, SD 27 ± 3.

Figure 6

miR expression in iPSCs-derived exosomes. (A) miR22 expression in iPSCs exosomes, * p < 0.05. (B) miR371 expression, * p < 0.05. (C) miR372 expression, p < 0.05. (D) miR373 expression, p < 0.05. CF-iPSC (n = 5), DF-iPSC (n = 5), CF (n = 4), DF (n = 5).

Figure 7

Exosome implication on beating EBs. EBs were seeded on a 6-well plate. The number of contracting EBs was calculated as a percentage of the total number of EBs. EBs treated with exosomes were compared to control,* p = 0.05. Control (n = 5), exosome (n = 5).