Figure 6. Bone marrow transplantation reveals a haematopoietic contribution to the adult epicardium that alters with age.

Two months after whole bone marrow (BM) transplantation of β-actin-Cre;R26R-EYFP labelled BM into lethally irradiated unlabelled hosts (Supplementary Fig. 9a), YFP+ cells are localized to the adult epicardium (a,b; as highlighted by white arrowheads) and the YFP+ BM cells contribute to epicardial clusters (c,d). The YFP+ cells persist at 6 months post transplantation and are localized to the epicardium (e–h; as highlighted by white arrowheads) and there was a significant increase in epicardial YFP+ cells over the intervening 4 months (mean %YFP+ cells ± s.e.m.; P≤0.001; n=6 hearts; i). At 2 and 6 months, podoplanin immunostaining confirmed the epicardial localization of YFP+ BM cells (j,n) and these were CD45+ (k,o; white arrowheads; white asterisk highlights a YFP+/CD45− cell), suggesting they represented the haematopoietic compartment of the whole BM inoculum, but were negative for Ly6C, thus excluding the mature myeloid lineage (l,p; white arrowheads indicate YFP+ cells in red which do not co-express Ly6C in green). A sub-population of YFP+ BM cells within the underlying myocardium expressed NG2, suggesting they had differentiated into pericytes (m,q; highlighted by white arrowheads). Two months after transplantation of flow-sorted Vav1+ cells, from Vav1-Cre;R26R-tdTomato-labelled bone marrow into lethally irradiated unlabelled hosts (Supplementary Fig. 9b), tdTomato+ cells are localized to the podoplanin+ adult epicardium (r; as highlighted by white arrowheads). The tdTomato+ BM cells were CD45+ (s; white arrowheads), confirming them as representative of the haematopoietic compartment of the whole BM inoculum. The tdTomato+ donor cells were negative for Ly6C, thus excluding the mature myeloid lineage (t; white arrowheads indicate tdTomato+ cells in red, which do not co-express Ly6C in green). A sub-population of tdTomato+ BM cells within the underlying myocardium expressed NG2, suggesting they had differentiated into pericytes (u; highlighted by white arrowheads). The number of CD45+ cells increased in the adult epicardium with age up to 24 weeks (v,w) but decreased thereafter as measured at 52 and 78 weeks (x,y). The increases between 1 and 8, and 8 and 24 weeks were significant, as was the decrease between 24 and 52 weeks (mean % CD45+ cells±s.e.m. of n=3 hearts, 6 sections analysed per heart; P≤0.001; z), suggesting an increased haematopoietic epicardial reserve with adulthood to maturity but depletion or impaired replenishment with advanced age (12 and 18 months). ep, epicardium; my, myocardium. Scale bars, 50 μm (a,b,e,f,v–y); 20 μm (c,d,j,k–o,q–s,u); 10 μm; (g,h); 10 μm (i,p,t). All statistics are calculated by Student’s t-test; *P≤0.001.