Characterization of Long-Term Cultured c-kit⁺Cardiac Stem Cells Derived From Adult Rat Hearts

Abstract

Previous studies have revealed c-kit-positive (c-kit(+)) cardiac stem cells (CSCs) in the adult mammalian heart and these cells could be a suitable cell source for heart regeneration therapy. However, these cells have not been fully evaluated in terms of characterization and effect of long-term culture, which is necessary for their safe and optimal usage. Therefore, we isolated c-kit(+) CSCs from adult rat hearts to characterize these cells and investigate stability over long-term culture. We performed isolations of c-kit(+) CSCs 11 times and passaged them 40 times in a bulk culture system; we termed these cultures, bulk culture CSCs (CSC-BC). c-kit(+) CSCs expressed stemness genes and exhibited stem cell properties of single cell-derived clone formation, cardiosphere generation, and potential to differentiate into the three main cardiac lineages: cardiomyocyte, smooth muscle, and endothelial cells in vitro. Over long-term culture, some CSC-BC up-regulated GATA-4 expression, which resulted in enhanced cardiomyocyte differentiation, suggesting that the GATA-4 high c-kit(+) CSCs have potent cardiac regenerative potential. We also observed the spontaneous differentiation into cells other than cardiac lineages, such as adipocyte and skeletal myocyte. This effect of long-term culture on the c-kit(+) CSCs has not been previously reported. Interestingly, when c-kit(+) CSCs were co-cultured with adult rat cardiomyocytes, we found increased cardiomyocyte survival, and the growth factors, insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), appeared to be responsible factors. The present study suggests that c-kit(+) CSCs have great therapeutic potential yet should be further investigated and optimized as a cell source for regenerative therapies prior to transplantation.

Specific inhibition of HDAC4 in cardiac progenitor cells enhances myocardial repairs

“…The isolation of CSCs is the same as described before (31). Myocardial tissues from 3-mo-old CD-1 male mice were minced into small pieces and subjected to enzymatic dissociation, and the remaining tissue fragments were cultured as explants in explant medium [Iscove's Modified Dulbecco's IMDM with 10% fetal calf serum (FBS), 100 U/ml penicillin G, 100 g/ml streptomycin, 2 mmol/l L-glutamine, and 0.1 mmol/l 2-mercaptoethanol] at 37°C and 5% CO 2.…”

Are We There Yet? A Story About Cardiac Stem Cells

“…Sca1+ CSCs CD34-, CD45-, FLK1-, ckit+/-, GATA4+, NKX2-5+/-, MEF2C+ (Oh et al, 2003;Forte et al, 2008;Matsuura et al, 2004;Rosenblatt-Velin et al, 2005;Tateishi et al, 2007;Wang et al, 2006;Wu et al, 2006) c-kit+ CSCs CD34-, CD45-, Sca1+, GATA4+, NKX2-5+, MEF2C+ Beltrami et al, 2003;Dawn et al, 2005;Linke et al, 2005;Miyamoto et al, 2010;Tillmanns et al, 2008;Urbanek et al, 2003) Isl-1+ CSCs CD31-, Sca1-, c-kit-, GATA4+, NKX2-5+ (Laugwitz et al, 2005;Moretti et al, 2006) Side population (SP Due to the importance of heart as a vital organ, several attempts have been made to characterize CSCs using various techniques. Numerous review articles were published covering the field of CSCs (Anversa et al, 2006;Bollini et al, 2011;Di Nardo et al, 2010;Musunuru et al, 2010;Tateishi et al, 2008).…”

“…Considering the acclaimed types of CSCs, it seems that heart, once considered to be a post-mitotic organ, harbours the highest number of adult stem cells in the body. At present, it is not clear whether CSCs can be called "true" adult stem cells with a "true" multipotency (Ellison et al, 2010;Stamm et al, 2009). The concept of multipotent adult stem cells and progenitor cells (sometime classified as "transient-amplifying cells") (Bryder et al, 2006) is derived from the haematopoietic system and might not necessarily apply to CSCs.…”

Molecular Analysis of Long-Term Cultured Cardiac Stem Cells for Cardiac Regeneration

“…The results suggested that these CSCs retained multiple developmental potential to some extent. Consequently, we investigated these CSCs in detail, hoping to establish the regeneration method by using c-Kit-positive cardiac cells [1][2][3][4][5][6][7][8][9][10][11][12].…”