Abstract 20576: Reporterless Reprogrammed Cardiac Progenitor Cells Attach, Migrate, and Repopulate Decellularized Whole Mouse Hearts
Cardiac progenitor cells hold great promise for cardiac repair due to their expandability and multipotency. Recently, we have reprogrammed fibroblasts (fibs) from transgenic mice with an Nkx2.5-YFP reporter and reverse tetracycline-controlled transactivator (rtTA) into induced cardiac progenitor cells (iCPCs). The purpose of this study was to advance the clinical applicability of iCPCs by generating a reporterless reprogramming protocol, assessing neoplastic risk, and determining iCPC capacity for tissue engineering. Wild type, mouse lung fibs were reprogrammed using a lentiviral infection of rtTA construct for 48hrs followed 2d later by doxycycline inducible defined factors (Mesp1, Tbx5, Gata4, Nkx2.5, Baf60c). Doxycycline was added with activation of Wnt (BIO) and JAK/STAT (LIF) signaling 2d later. iCPC colonies were identified by day 20 as proliferative aggregation of cells with high nuclear-to-cytoplasmic ratio. Reporterless iCPCs were readily expandable (>10 psgs) despite removal of doxycycline and ICC was positive for NKX2.5, GATA4, and IRX4. The iCPCs differentiated into cardiomyocytes (α-MHC, actin, actinin, MLC-2V), smooth muscle (SM-MHC), and endothelium (CD31). To test the neoplastic potential of iCPCs, 106 mESCs or iCPCs suspended in matrigel were injected subcutaneously in 12 NOD-SCID mice. After 6 wks, no teratoma formed on the iCPCs side (0/12) unlike ESC teratomas (11/12). To test the ability of iCPCs to engraft and differentiate on cardiac extracellular matrix, mouse hearts were decellularized and 107 iCPCs constitutively expressing GFP were injected retrograde via an aortic cannula. The hearts were retrograde perfused with 10% serum media for 1 wk, followed by 1 wk of transient Wnt inhibition(IWP4) in addition BMP4, FGF2, and VEGF, then switched to 1% serum media for a wk. Using sequential weekly live fluorescent imaging, iCPCs were demonstrated repopulating whole heart constructs (4/4) . IHC was notable for tri-linage differentiation (CMs, SCs, and ECs). Whole heart optical mapping demonstrated field-stimulated calcium transients. To conclude iCPC can be derived without a reporter cell line and readily repopulate cardiac extracellular matrices providing a promising new tool for tissue engineering applications.
- Cellular Engineering
- Progenitor cell
- Regenerative medicine stem cells
- Stem cell biology
- Stem/progenitor cells
Author Disclosures: R.A. Alexanian: None. P.A. Lalit: None. D. Lang: None. M.R. Lea: None. R. Vaidyanathan: None. Y.S. Markandeya: None. A.J. Zhai: None. E.G. Schmuck: None. L.L. Eckhardt: None. A. Glukhov: None. T.J. Kamp: None.
- © 2016 by American Heart Association, Inc.