Restoration of cardiac functionality after myocardial infarction represents a major clinical challenge1. Recently, we found that transient transfection with a microRNA combination (miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocyte (iCMs) in vitro2. However, achieving efficient direct reprogramming still remains a challenge. Direct reprogramming of human fibroblasts could be enhanced by culturing miRcombo-transfected cells in a three-dimensional (3D) environment with biomimetic biophysical and biochemical cues3,4. Herein, the ability of cardiac-like extracellular matrix (called Biomatrix, BM) to enhance miRcombo-mediated direct cell reprogramming was studied. Then, miRcombo-transfected cells were cultured in a biomimetic microenvironment consisting of a 3D fibrin hydrogel containing BM.
BM was produced by in vitro culture of AHCFs for 21 days, followed by decellularization. AHCFs were transfected with miRcombo and then cultured for 2 weeks on the surface of: uncoated and BM-coated polystyrene (PS) dishes, on the top of fibrin hydrogels (2D hydrogel), or embedded into 3D fibrin hydrogels without (3D hydrogel) or with BM (3D BM hydrogels). The expression of cardiac markers and cell maturation was analysed by ddPCR, immunofluorescence and calcium transient analysis.
Culture of miRcombo-transfected cells on BM-coated vs. uncoated PS dishes enhanced direct reprogramming efficiency, enhancing TNNT2, ACTC1 and CACNA1C expression and increasing the percentage of cardiac troponin T (cTnT)-positive cells at 15 days culture time. Culture in 3D hydrogel after miRcombo transfection significantly improved direct reprogramming efficiency respect to uncoated PS and 2D hydrogel, increasing TNNT2, SCN5A and MYL7 expression. Finally, miRcombo-transfected cells were cultured in 3D BM hydrogel providing cardiac tissue-mimetic biophysical and biochemical cues. The expressions of cardiomyocyte genes and cTnT were significantly enhanced in cells cultured for 15 days in 3D BM compared to 3D hydrogels. Calcium transient was enhanced in 3D BM compared to 3D hydrogels in miRcombo cells, showing higher slope of calcium upstroke.
Overall results demonstrated that a biomimetic 3D culture microenvironment can enhance the direct reprogramming efficiency of miRcombo-transfected human adult cardiac fibroblasts into iCMs. Future investigations will elucidate which molecular barriers to direct reprogramming can be overcome by the use of 3D biomimetic cell culture substrates, paving the way to the research for more efficient strategies for direct cardiac reprogramming.
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2. Paoletti, C. et al. Front. Bioeng. Biotechnol. (2020).
3. Li, Y. et al. Sci. Rep. 6, (2016).
4. Paoletti, C. & Chiono. Front. Cardiovasc. Med. 8, (2021).