Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart

Abstract

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit(+) CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit(+) CSCs.

Fig 1

Schematic representation of the experimental design.

Fig 2

Kit/GFP⁺ CS and CS-forming-cells. (A) Kit/GFP⁺ CS-forming cells were collected from the explant of infarcted heart 13 days after the beginning of the culture, and plated onto poly-D-lysine for 1 day to allow CS formation. Note the initial appearance of slightly fluorescent cells. (B) A CS at a more advanced stage of development shows transgenic Kit/GFP expressing cells at the centre of the sphere. (C) PCR analysis of Kit/GFP in genomic DNA extracted from single CSs. 351–441 indicate different transplanted-infarcted mice; LD, sizemarker; C1a and C1b represent internal positive controls (genomic DNA from 10⁷ and 10⁶Kit/GFP BM cells); WT, genomic DNA from 10⁷ wild-type BM cells; PCR-, negative control on PCR mix. Product of amplification from sample 438 was confirmed by in vitro automatic sequencing (not shown).

Fig 3

CS and CSDCs phenotypes. Confocal analysis of a CS, derived from the infarcted heart of a lethally irradiated mouse transplanted with marrow cells of Kit/GFP transgenic mice (A) Merged image showing co-expression in some cells (arrows) of donor cell-derived GFP (green) and Nkx2.5 (an immature cardiac cell-specific transcription factor) (red); nuclei are stained blue by Hoechst dye. The figure is an average of 13 z-axis confocal sections. Single channel fluorescence intensity of some cells within the sphere is represented by the plots, depicting the fluorescence of cells traced by the white line. Co-localization is evident for the cells indicated as cell 1 and 2 whereas the third nucleus is expressing neither GFP nor Nkx2.5. (B) Single cell derived from CS dissociation (from a different CS), demonstrating nuclear co-localization of GFP and Nkx2.5. (C) RT-PCR of GFP, Nkx2.5 and cardiac actin from cells expanded on fibronectin from a single clonogenic CS, and later analysed as a monolayer on fibronectin (left) or as CSs (on polylisine).