Adult cardiac-resident MSC-like stem cells with a proepicardial origin

Abstract

Colony-forming units - fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.

Figure 1. cCFU-F Growth and Differentiation Assays

(A) Colony morphology. (B) Growth curves of bulk cCFU-F colonies and clonally isolated large and small colonies. (C and D) Marker expression and cell conversion graphs after differentiation of cCFU-F colonies into cardiomyocyes (CM), endothelial cells (Endo), and smooth muscle cells (SMC) for in vitro and in vivo differentiation assays as indicated. All immunofluorescence panels were costained with Hoechst to detect nuclei. Graphs show percent conversion to marker-positive cells (black bars) relative to undifferentiated cells (open bars). Right SMC panels in (D) show cCFU-F-derived smooth muscle contraction after treatment with carbachol. Graphs show percentage of cells contracting and percentage of contraction of cell area. For (C), error bars = SEM between independent experiments. For (D), error bars = SD between independent experiments. Marker abbreviations: CX43: CONNEXIN 43; VE-CAD: VE-CADHERIN; CAV-1: CAVEOLIN 1; vWF: VON WILLEBRAND's FACTOR; eNOS: ENDOTHELIAL NITRIC OXIDE SYNTHASE; SRF: SERUM RESPONSE FACTOR; MYOCD: MYOCARDIN; AP: ALKALINE PHOSPHATASE; Ac-LDL: acetylated low density lipoprotein; MYH11: SMOOTH MUSCLE MYOSIN HEAVY CHAIN; SMA: SMOOTH MUSCLE ACTIN (bar = 50 μm). See also Figure S1.

Figure 2. cCFU-F Trans-Germ Layer Differentiation

(A) Marker expression and cell conversion graphs for direct differentiation into hepatic, endoderm, and neuronal lineages. Graphs in (A) and (D) below show percentage conversion to marker-positive cells (black bars) relative to undifferentiated cells (open bars). (B) Descendant lineages of cCFU-F colony cells in ESC coculture teratomas. Brown staining indicates GFP expression in cCFU-F lineage descendants (arrows). (C) Immunostaining (red) and GFP fluorescence (green) for endothelial cells and smooth muscle (left panels) and cardiomyocytes in ESC coculture teratomas. (D) Percent conversion graphs for in vitro differentiation of freshly isolated S+P+ fraction. Marker abbreviations: AFP: ALPHA-FETOPROTEIN; ALB: ALBUMIN; β3-TUB: B3 TUBULIN; GFAP: GLIAL FIBRILLARY ACIDIC PROTEIN; O4: OLIGODENDROCYTE MARKER O4. Error bars = SEM between independent experiments. See also Figure S2.

Figure 3. PDGFRα Expression and cCFU-Fs in the Developing Heart

(A) Colony forming cells were derived from the SCA1⁺/CD31^–/PDGFRα⁺ fraction of adult cardiac nonmyocytes (S⁺P⁺ fraction, gate). (B and C) Whole-mount ISH (B) and immunohistochemistry (C) of PDGFRα expression at 9.5 dpc (arrows show proepicardium; pe). (D and E) Immunohistochemistry at 12.5 dpc (D) and 14.5 dpc (E) showing increasing PDGFRα expression in the epicardium (epi) and subepicardium. (F–J) Pdgfra-GFP-expressing cells at 12.5–15.5 dpc relative to indicated markers showing expression in epicardium, the interstitium, and interventricular and atrioventricular grooves (ivg and avg, respectively), as well as in endocardial cushions (ec), at 12.5 dpc. Arrowheads in (G) and (H) indicate interstitial cells, and in (J), Pdgfra-GFP⁺ cells in a perivascular position. (K and L) Smooth muscle fate of some Pdgfra-GFP⁺ cells is suggested by CALPONIN coexpression with GFP^low cells in coronary vessels and aorta at 15.5 dpc (arrows). (M) Colony formation in the 12.5 dpc heart is limited to Pdgfra-GFP⁺/PDGFRα⁺ cells (gate). Although not shown on this plot, some PDGFRα⁺/Pdgfra-GFP^– cells appear variably at fetal stages and may be nonspecific. (N) Cardiac CFU-F colony formation is found in the proepicardium (pe), and embryonic, early postnatal (free ventricular walls), and 8-week-old adult heart. Error bars = SD between independent experiments. See also Figure S3.

Figure 4. cCFU-Fs Are Lost after Irradiation and Cannot Be Rescued by Whole BM Transplantation

(A) Overview of BM transplant experimental design. All transplants were on mice 8 weeks of age. (B) Large colony formation was completely lost in BM transplanted mice. Colony formation was not rescued after 3 months or 6 months aging, or after MI. (C) Reduced BM CFU-F colonies from BM-GFP chimeric mice, which do not recover after 6 months. (D) Majority of remaining BM CFU-F colonies are GFP⁺, indicating donor origin. Error bars = SEM between independent experiments. See also Figure S4.

Figure 5. Large cCFU-Fs Are Not BM Derived

(A) Cardiac lead shielding during irradiation preserved large colony formation after BM transplantation. Large colony formation was from the non-BM-derived fraction after 3 and 6 months aging, MI (6 weeks posttransplant; cells isolated either 5 or 30 days after MI), or MI with G-CSF administration for 6 days. (B and C) Crystal-violet-stained colonies from GFP^– and GFP⁺ fractions. Error bars = SEM between independent experiments. See also Figure S5.

Figure 6. Lineage Tracing Studies Suggest an Epicardial Origin for cCFU-Fs

(A) Overview of lineage tracing strategy. (B) Expression of GFP and LACZ in colonies from control mice. (C and D) Percentage of GFP⁺/LACZ^– or GFP^–/LACZ⁺ cCFU-F colonies from (C) adult whole hearts and (D) ventricles of 17.5 dpc lineage-CRE × Z/EG mice. (E and F) Colonies derived from adult BM (E) or adult proximal aorta (F). Error bars = SEM between independent experiments. See also Figure S6.

Figure 7. Conditional Lineage Tracing with WT1^CreERT2

(A–E) LacZ staining in 11.75 dpc embryonic hearts from Wt1^CreERT2 × R26R crosses shows patchy labeling of epicardium, interstitial and interventricular groove (ivg) cells, and pericardium (p) (arrowheads). (F and G) Percentage of LACZ⁺ or LACZ^– cCFU-F large colonies from Wt1^CreERT2 × R26R progeny at 14.5 dpc (F) and adult whole hearts (G). (H) Percentage of GFP⁺/LACZ^– cCFU-F large colonies from Gata5Cre × Z/EG adult mice was not significantly different 5 or 30 days post-MI compared to sham-operated controls. Error bars = SEM between independent experiments. See also Figure S7.