Human Cardiomyocytes Prior to Birth by Integration-Free Reprogramming of Amniotic Fluid Cells

Abstract

The establishment of an abundant source of autologous cardiac progenitor cells would represent a major advance toward eventual clinical translation of regenerative medicine strategies in children with prenatally diagnosed congenital heart disease. In support of this concept, we sought to examine whether functional, transgene-free human cardiomyocytes (CMs) with potential for patient-specific and autologous applications could be reliably generated following routine amniocentesis. Under institutional review board approval, amniotic fluid specimens (8-10 ml) at 20 weeks gestation were expanded and reprogrammed toward pluripotency using nonintegrating Sendai virus (SeV) expressing OCT4, SOX2, cMYC, and KLF4. Following exposure of these induced pluripotent stem cells to cardiogenic differentiation conditions, spontaneously beating amniotic fluid-derived cardiomyocytes (AF-CMs) were successfully generated with high efficiency. After 6 weeks, quantitative gene expression revealed a mixed population of differentiated atrial, ventricular, and nodal AF-CMs, as demonstrated by upregulation of multiple cardiac markers, including MYH6, MYL7, TNNT2, TTN, and HCN4, which were comparable to levels expressed by neonatal dermal fibroblast-derived CM controls. AF-CMs had a normal karyotype and demonstrated loss of NANOG, OCT4, and the SeV transgene. Functional characterization of SIRPA + AF-CMs showed a higher spontaneous beat frequency in comparison with dermal fibroblast controls but revealed normal calcium transients and appropriate chronotropic responses after b-adrenergic agonist stimulation. Taken together, these data suggest that somatic cells present within human amniotic fluid can be used to generate a highly scalable source of functional, transgene-free, autologous CMs before a child is born. This approach may be ideally suited for patients with prenatally diagnosed cardiac anomalies. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:1595-1606 SIGNIFICANCEThis study presents transgene-free human amniotic fluid-derived cardiomyocytes (AF-CMs) for potential therapy in tissue engineering and regenerative medicine applications. Using 8-10 ml of amniotic fluid harvested at 20 weeks gestation from normal pregnancies, a mixed population of atrial, ventricular, and nodal AF-CMs were reliably generated after Sendai virus reprogramming toward pluripotency. Functional characterization of purified populations of beating AF-CMs revealed normal calcium transients and appropriate chronotropic responses after b-adrenergic agonist stimulation in comparison with dermal fibroblast controls. Because AF-CMs can be generated in fewer than 16 weeks, this approach may be ideally suited for eventual clinical translation at birth in children with prenatally diagnosed cardiac anomalies.

Cardiomyocytes Derived from Human CardiopoieticAmniotic Fluids

“…Recently, Jiang et al . 45 showed that hAFs can be reprogrammed and subsequently used to generate functional CMs with robust and highly organized myofilament structures. In this study, we demonstrated that if the hAFs present the cardiopoietic phenotype (high expression of OCT4, SSEA4, CD90), it is possible to obtain CM-like cells that recapitulate the iPS- or ES-cell-derived CM phenotype with a time- and cost-saving method that avoids cell reprogramming and all the risks related to iPS cell utilization 11 .…”

Cardiac Restoration Stemming From the Placenta Tree: Insights From Fetal and Perinatal Cell Biology

“…Hence, the quest is now on defining the most suitable cell to efficiently induce iPS from and since amniotic fluid stem cells are immature fetal progenitors endowed with some degree of instrinsic pluripotency and active expression of embryonic genes including OCT4, NANOG, and SOX2 (Loukogeorgakis and De Coppi, 2017 ), they represent an ideal candidate. Recent studies have reported that murine and human AFSC can be reprogrammed into iPS more easily than adult stem cells by applying either transgene-free approaches, like chemical defined conditions via stimulation with the histone deacetylase inhibitor valproic acid (Moschidou et al, 1953 ), as well as non-integrating methods by episomal plasmid, transposon system, sendai virus or mRNA delivery by lipofection (Jiang et al, 2016 ; Slamecka et al, 2016 ; Bertin et al, 2017 ; Velasquez-Mao et al, 2017 ); notably the obtained AFSC-iPS have been proven capable of functional cardiac differentiation, thus providing important impact for future cardiac regenerative therapy and specific relevance for the treatment of neonatal cardiac congenital disease (Jiang et al, 2016 ; Velasquez-Mao et al, 2017 ). Indeed, hAFSC can be effortlessly harvested during prenatal diagnosis, treated by gene therapy and iPS technology to derived healthy myocardial and cardiovascular cells to be then processed by tissue engineering approaches so to provide cardiac grafts developed in parallel with gestation and promptly implanted in utero or at birth.…”

Decellularized Extracellular Matrices and Cardiac Differentiation: Study on Human Amniotic Fluid-Stem Cells

“…Finally, hiPSC-derived CMs differentiated using both dECMs showed percentages of spontaneous beating foci comparable to the same cells differentiated using Matrigel ® , while the percentage of the contracting area in Cardiopoietic AF-derived CMs was surprisingly significantly higher in both the Cor TM PATCH and Matrix Patch TM than in the control cells. Even if this percentage never exceeded 30% of the total area, it was still a promising result, since very few groups were able to obtain beating CMs from amniotic stem cells without prior reprogramming [ 2 , 44 , 45 ]. This was possibly because, as expected [ 5 , 46 , 47 ] CMs with different levels of maturity were obtained, and a scaffold with a rigid elasticity niche may strongly promote beating induction, in line with what was reported by Hirata and colleagues [ 48 ].…”