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Cardiomyocytes Derived from Human ^{Cardiopoietic}Amniotic Fluids.

ABSTRACT: Human amniotic fluid (hAF) cells share characteristics of both embryonic and adult stem cells. They proliferate rapidly and can differentiate into cells of all embryonic germ layers but do not form teratomas. Embryoid-bodies obtained from hAF have cardiac differentiation potential, but terminal differentiation to cardiomyocytes (CMs) has not yet been described. Our purpose was to promote cardiac differentiation in hAFcells. Cells were exposed to inducing factors for up to 15 days. Only the subset of hAF cells expressing the multipotency markers SSEA4, OCT4 and CD90 (^{Cardiopoietic}AF cells) responded to the differentiation process by increasing the expression of the cardiac transcription factors Nkx2.5 and GATA4, sarcomeric proteins (cTnT, α-MHC, α-SA), Connexin43 and atrial and ventricular markers. Furthermore, differentiated cells were positive for the calcium pumps CACNA1C and SERCA2a, with approximately 30% of ^{Cardiopoietic}AF-derived CM-like cells responding to caffeine or adrenergic stimulation. Some spontaneous rare beating foci were also observed. In conclusion, we demonstrated that ^{Cardiopoietic}AF cells might differentiate toward the cardiac lineage giving rise to CM-like cells characterized by several cardiac-specific molecular, structural, and functional properties.

SUBMITTER: Di Baldassarre A

PROVIDER: S-EPMC6089907 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Cardiomyocytes Derived from Human <sup>Cardiopoietic</sup>Amniotic Fluids.

Di Baldassarre Angela A D'Amico Maria A MA Izzicupo Pascal P Gaggi Giulia G Guarnieri Simone S Mariggiò Maria A MA Antonucci Ivana I Corneo Barbara B Sirabella Dario D Stuppia Liborio L Ghinassi Barbara B

Scientific reports 20180813 1

Human amniotic fluid (hAF) cells share characteristics of both embryonic and adult stem cells. They proliferate rapidly and can differentiate into cells of all embryonic germ layers but do not form teratomas. Embryoid-bodies obtained from hAF have cardiac differentiation potential, but terminal differentiation to cardiomyocytes (CMs) has not yet been described. Our purpose was to promote cardiac differentiation in hAFcells. Cells were exposed to inducing factors for up to 15 days. Only the subse ...[more]

PMID: 30104705

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Project description:: 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 β-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.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 β-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.

Project description:IntroductionHuman induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called "iCell cardiomyocytes" under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents.MethodsiCell cardiomyocytes were cultured for 8-14 days (38-44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (INa) and L-type Ca2+ channel current (ICaL) were evaluated by the whole-cell patch clamp.ResultsiCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of INa, ICaL, and rapidly-activating delayed-rectifier K+ channel current were -62.7 ± 13.7, -8.1 ± 0.7, and 3.0 ± 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K+ channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked INa in a concentration-dependent manner with IC50 of 0.87 ± 0.37 and 0.88 ± 0.16 μM, respectively; the class Ib agent mexiletine revealed weak INa block with a higher IC50 of 30.0 ± 3.0 μM. Pirmenol, pilsicainide and mexiletine blocked ICaL with IC50 of 2.00 ± 0.39, 7.7 ± 2.5 and 5.0 ± 0.1 μM, respectively.ConclusionsIn iCell cardiomyocytes, INa was blocked by the class Ia and Ic antiarrhythmic agents and ICaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity.

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Cardiomyocytes Derived from Human ^{Cardiopoietic}Amniotic Fluids.

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Cardiomyocytes Derived from Human <sup>Cardiopoietic</sup>Amniotic Fluids.

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Cardiomyocytes Derived from Human CardiopoieticAmniotic Fluids.

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Cardiomyocytes Derived from Human <sup>Cardiopoietic</sup>Amniotic Fluids.

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OmicsDI is part of the ELIXIR infrastructure

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Cardiomyocytes Derived from Human ^{Cardiopoietic}Amniotic Fluids.