Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and β-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.

Objective Abnormal pulmonary vasculature directly affects the development and progression of congenital diaphragmatic hernia (CDH)‐associated pulmonary hypertension (PH). Though overarching structural and cellular changes in CDH‐affected pulmonary arteries have been documented, the precise role of the extracellular matrix (ECM) in the pulmonary artery (PA) pathophysiology remains undefined. Here, we quantify the structural, compositional, and mechanical CDH‐induced changes in the main and distal PA ECM and investigate the efficacy of mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) as a therapy to ameliorate pathological vascular ECM changes. Methods Pregnant Sprague‐Dawley rodents were administered nitrofen to induce CDH‐affected pulmonary vasculature in the offspring. A portion of CDH‐affected pups was treated with intravenous infusion of MSC‐EVs (1 × 1010/mL) upon birth. A suite of histological, mechanical, and transmission electron microscopic analyses were utilized to characterize the PA ECM. Results The CDH model main PA presented significantly altered characteristics—including greater vessel thickness, greater lysyl oxidase (LOX) expression, and a relatively lower ultimate tensile strength of 13.6 MPa compared to control tissue (25.1 MPa), suggesting that CDH incurs ECM structural disorganization. MSC‐EV treatment demonstrated the potential to reverse CDH‐related changes, particularly through rapid inhibition of ECM remodeling enzymes (LOX and MMP‐9). Additionally, MSC‐EV treatment bolstered structural aspects of the PA ECM and mitigated pathological disorganization as exhibited by increased medial wall thickness and stiffness that, while not significantly altered, trends away from CDH‐affected tissue. Conclusions These data demonstrate notable ECM remodeling in the CDH pulmonary vasculature, along with the capacity of MSC‐EVs to attenuate pathological ECM remodeling, identifying MSC‐EVs as a potentially efficacious therapeutic for CDH‐associated pulmonary hypertension.

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