Heart Cells with Regenerative Potential from Pediatric Patients with End Stage Heart Failure: A Translatable Method to Enrich and Propagate

Abstract

Background. Human cardiac-derived progenitor cells (hCPCs) have shown promise in treating heart failure (HF) in adults. The purpose of this study was to describe derivation of hCPCs from pediatric patients with end-stage HF. Methods. At surgery, discarded right atrial tissues (hAA) were obtained from HF patients (n = 25; hAA-CHF). Minced tissues were suspended in complete (serum-containing) DMEM. Cells were selected for their tissue migration and expression of stem cell factor receptor (hc-kit). Characterization of hc-kitpositive cells included immunohistochemical screening with a panel of monoclonal antibodies. Results. Cells, including phase-bright cells identified as hc-kitpositive, spontaneously emigrated from hAA-CHF in suspended explant cultures (SEC) after Day 7. When cocultured with tissue, emigrated hc-kitpositive cells proliferated, first as loosely attached clones and later as multicellular clusters. At Day 21~5% of cells were hc-kitpositive. Between Days 14 and 28 hc-kitpositive cells exhibited mesodermal commitment (GATA-4positive and NKX2.5positive); then after Day 28 cardiac lineages (flk-1positive, smooth muscle actinpositive, troponin-Ipositive, and myosin light chainpositive). Conclusions. C-kitpositive hCPCs can be derived from atrial tissue of pediatric patients with end-stage HF. SEC is a novel culture method for derivation of migratory hc-kitpositive cells that favors clinical translation by reducing the need for exogenously added factors to expand hCPCs in vitro.

Ex vivo paracrine properties of cardiac tissue: Effects of chronic heart failure

“…27 The c-kit þ cells in this study unequivocally reside in heart tissue at the time of harvest and exhibit progenitor cell behavior, including acquisition of cardiomyocyte markers in vitro. 22 Cardiacderived cells expressing c-kit were defined as CSCs, and additional characterization was not performed with each analysis reported in this study. Huc-kit þ cells were selected using monoclonal antibody and magnetic nanoparticle separation techniques with minor modifications of the manufacturer's protocol (Stemcell Technologies Inc, Vancouver, BC, Canada).…”

“…22 The substantive modification is that freshly obtained atrial tissues are aseptically minced and completely suspended in pre-warmed Dulbecco's Modified Eagle's Medium with 20% fetal calf serum (DMEM/ FCS; Invitrogen, Carlsbad, CA) at 371C and 5% CO 2 . 22 Media were partially exchanged (10% v/v) with fresh media every 3 days.…”

“…22 The substantive modification is that freshly obtained atrial tissues are aseptically minced and completely suspended in pre-warmed Dulbecco's Modified Eagle's Medium with 20% fetal calf serum (DMEM/ FCS; Invitrogen, Carlsbad, CA) at 371C and 5% CO 2 . 22 Media were partially exchanged (10% v/v) with fresh media every 3 days. This results in the mobilization and emigration of cells, including a sub-population of c-kit þ CD34 -CD45 -CD3 -cells with properties of cardiac progenitor/stem cells from mouse 10 and human heart tissues.…”

“…This results in the mobilization and emigration of cells, including a sub-population of c-kit þ CD34 -CD45 -CD3 -cells with properties of cardiac progenitor/stem cells from mouse 10 and human heart tissues. 22 We have preliminarily reported that a number of protein factors can be identified during suspension explant culturing. 23 Because suspension explant culturing is a relatively non-destructive cardiac culture technique compared with enzymatic dispersion, the factors produced ex vivo would be expected to emulate the in vivo cardiac tissue responses, albeit with "ischemia" and in the hibernating, non-working state, without confounding effects from circulating factors and cells.…”

“…Suspension explant culturing, a novel method of culturing explanted cardiac tissues, can be used to selectively enrich c-kit þ cluster of differentiation (CD) 34 -CSCs based on their spontaneous emigration from suspended murine 10 and human 22 cardiac tissues. In the course of these experiments, we observed that retaining the cardiac tissue in serum-containing media was necessary and sufficient for the long-term in vitro proliferation and survival of CSCs derived from mouse heart.…”