TY - JOUR
T1 - Human cardiomyocytes prior to birth by integration-free reprogramming of amniotic fluid cells
AU - Jiang, Guihua
AU - Herron, Todd J.
AU - Di Bernardo, Julie
AU - Walker, Kendal A.
AU - O’Shea, K. Sue
AU - Kunisaki, Shaun M.
N1 - Publisher Copyright:
© AlphaMed Press 2016.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - 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.
AB - 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.
KW - Amniotic fluid
KW - Cardiac
KW - Fetal stem cells
KW - Induced pluripotent stem cells
KW - Pluripotent stem cells
UR - http://www.scopus.com/inward/record.url?scp=84995872843&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84995872843&partnerID=8YFLogxK
U2 - 10.5966/sctm.2016-0016
DO - 10.5966/sctm.2016-0016
M3 - Review article
C2 - 27465073
AN - SCOPUS:84995872843
SN - 2157-6564
VL - 5
SP - 1595
EP - 1606
JO - Stem Cells Translational Medicine
JF - Stem Cells Translational Medicine
IS - 12
ER -