Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies

Abstract

The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.

578 Human Blood and Cardiac Stem Cells Synergize to Enhance Cardiac Repair When Co-Transplanted Into Ischemic Myocardium

“…5,14 This product has been shown to provide equivalent repair to established cell products with hints of superiority. 10,15 The current work extends previous studies by providing the first unambiguous head-to-head comparison of autologous CACs and CSCs from clinical patients. Although earlier work from our laboratory demonstrated that expanded populations of CSCs from human cardiac samples secrete significant quantities of vascular endothelial growth factor, insulin-like growth factor-1, and hepatocyte growth factor, we used a wide-ranging cytokine protein array to show that CSCs also secrete significant amounts of angiopoietin-1, angiogenin, and inter-leukin-6.…”

“…CSCs were cultured using standard culture techniques described previously. 10 CACs were isolated from peripheral blood samples using standard techniques. 2,11 Normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells were cultured according to the manufacturer's directions.…”

The effect of encapsulation of cardiac stem cells within matrix-enriched hydrogel capsules on cell survival, post-ischemic cell retention and cardiac function

“…2A) [15]. Briefly, tissue sections were minced and digested prior to being placed in cardiac explants media (CEM; Iscove's Modified Dulbecco's Medium (Invitrogen), 20% Fetal Bovine Serum (FBS; Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 2 mmol/l L-glutamine (Invitrogen) and 0.1 mmol/l 2-mercaptoethanol (Invitrogen).…”

Cellular and Molecular Characterization of Human Cardiac Stem Cells Reveals Key Features Essential for Their Function and Safety

“…Several studies have been carried out to isolate and characterize cardiac precursor cells (mainly c-Kit + , Sca-1 + , and cardiospheres) from normal and pathologic fetal and adult heart biopsies [12][13][14][15]. Different protocols have been developed to improve CSC isolation methods, resulting in the generation of a specific population of CSCs [16][17][18][19]. In addition to cell sorting and culture of explants, clonogenic expansion of cells has been described as another CSCs isolation method [20].…”