title: 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 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.
  
  
  
  This 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.
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.|Pluripotent Stem Cell Laboratory, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Guihua
authors.initials: G
authors.lastname: Jiang
authors.affiliation: Department of Internal Medicine, Cardiovascular Research Center, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Todd J
authors.initials: TJ
authors.lastname: Herron
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Di Bernardo
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.|Pluripotent Stem Cell Laboratory, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Kendal A
authors.initials: KA
authors.lastname: Walker
authors.affiliation: Pluripotent Stem Cell Laboratory, University of Michigan Medical School, Ann Arbor, Michigan, USA.|Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: K Sue
authors.initials: KS
authors.lastname: O'Shea
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA shaunkun@umich.edu.|Pluripotent Stem Cell Laboratory, University of Michigan Medical School, Ann Arbor, Michigan, USA.|C.S. Mott Children's Hospital and Von Voigtlander Women's Hospital, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Shaun M
authors.initials: SM
authors.lastname: Kunisaki
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D002454:Cell Differentiation; D019070:Cell Lineage; D065150:Cellular Reprogramming; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D000090062:Kruppel-Like Factor 4; D059630:Mesenchymal Stem Cells / Q000166:cytology; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D029082:Sendai virus / Q000378:metabolism; D012867:Skin / Q000166:cytology; D019076:Transgenes
keywords: Amniotic fluid; Cardiac; Fetal stem cells; Induced pluripotent stem cells; Pluripotent stem cells