Very small embryonic-like stem cells from the murine bone marrow differentiate into epithelial cells of the lung

Abstract

The view that adult stem cells are lineage restricted has been challenged by numerous reports of bone marrow (BM)-derived cells giving rise to epithelial cells. Previously, we demonstrated that nonhematopoietic BM cells are the primary source of BM-derived lung epithelial cells. Here, we tested the hypothesis that very small embryonic like cells (VSELs) are responsible for this engraftment. We directly compared the level of BM-derived epithelial cells after transplantation of VSELs, hematopoietic stem/progenitor cells, or other nonhematopoietic cells. VSELs clearly had the highest rate of forming epithelial cells in the lung. By transplanting VSELs from donor mice expressing H2B-GFP under a type 2 pneumocyte-specific promoter, we demonstrate that this engraftment occurs by differentiation and not fusion. This is the first report of VSELs differentiating into an endodermal lineage in vivo, thereby potentially crossing germ layer lineages. Our data suggest that Oct4+ VSELs in the adult BM exhibit broad differentiation potential.

Keywords: Adult stem cells; Bone marrow; Differentiation; Fluorescent protein reporter genes; Plasticity; Respiratory tract; Stem cell transplantation; Tissue regeneration.

Figure 1

Sorting strategy for isolation of VSELs and non-hematopoietic non-VSEL-cells, experimental approach and expression of Oct4 and Nanog in VSELs isolated from BM. A: FSC-SSC gate was set around events ranging from 2–10µm using flow cytometry size beads. B–D: VSELs were sorted from this gate (R1) as lineage-negative and Sca-1 positive cells (R2), that are also CD45-negative (R3), while HSPC are CD45-positive (R4). E and F: Sorting of non-VSEL cells. Excluding the cells between 2 and 6µm, Lin− CD45− negative cells were sorted (Lin− CD45− Non-VSELs). G: Immunofluorescent staining of freshly isolated VSELs or HSPC for Oct4 (green) and Nanog (red). Murine embryonic stem cells (ESC) served as positive control. Images were acquired with a 63× objective. H: Experimental approach: VSELs, Lin negative, CD45-negative CD45− Non-VSELs or HSPC were sorted from WT BM and transplanted into SPC-KO recipients, and 2 months post transplant, lung tissue was harvested and analyzed.

Figure 2

VSELs give rise to T2 cells: Detection by ImageStream. Isolated cells from lung tissue harvested from transplant recipients after 2 months were analyzed for SPC (yellow) and cytokeratin (orange). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Cells were analyzed using an ImageStream imaging flow cytometer with 40× magnification. Examples of 2 cells from each group are shown. A: T2 cells from a WT control express SPC in intracellular vesicles as well as cytoplasmic cytokeratin. B and D: Lung cells from SPC-KO mice transplanted with non-VSELs or HSPC express cytokeratin but not SPC. C: SPC-positive T2 cells derived from VSELs express SPC in intracellular vesicles and cytokeratin. E: Percentages of T2 cells derived from donor BM detected by ImageStream calculated based on the number of T2 cells among CD45-negative, round cells in corresponding WT samples, which was set to 100%. See also supplemental figures S1 and S3.

Figure 3

SPC-positive T2 cells derived from VSELs detected on tissue sections. Lung tissue was stained for SPC (red) and TTF1 (blue). VSEL derived T2 cells are marked with arrows. A: T2 cells from WT controls express SPC in intracellular compartments and co-express nuclear TTF1 B: T2 cells in SPC-KO lungs transplanted with non-VSELs express nuclear TTF1, but not SPC. C and D: Lungs from SPC-KO mice that received VSELs contain donor derived T2 cells located at the alveolar junction (arrows). Confocal microscopic images were taken with a 63× lens. The left-most panels show images taken at low power, merged with brightfield images to show localization of T2 cells at the alveolar junction. Middle panel: High power. Right panel: Images were taken in 0.4 µm steps from the top to the bottom of each cell. Optical cross sections through one focal plane show SPC expression in cytoplasmic vesicles surrounding the nucleus, excluding cell overlay. Cross-hairs indicate planes of optical x and y sections projected on bottom and right side.

Figure 4

VSELs differentiate into T2 cells, activating the SPC-promoter. A: VSELs and Non-VSELs were isolated from mice expressing the nuclear H2B-GFP under the SPC-promoter and transplanted into SPC-KO recipients expressing red fluorescent protein (DsRed) under the ubiquitous beta-actin promoter. B: If VSELs give rise to T2 cells by differentiation without fusion, the resulting cell would express SPC and GFP but not DsRed. C–G: Isolated lung cells were analyzed by Imagestream for SPC (red), GFP (green), DsRed (orange) and CD45 (not shown). Nuclei were stained with DAPI. C: T2 cells from SPC-H2B-GFP WT (SPC+) controls express SPC and nuclear GFP but not DsRed. D: T2 cells from DsRed+SPC-KO mice express DsRed but not SPC or GFP. E: VSEL derived T2 cells express SPC and GFP, but not DsRed. F: Rare T2 cells derived from non-VSELs express SPC and GFP but not DsRed. Images were acquired with a 63× objective G: In DsRed+SPC=KO mice, 40–60% of T2 cells are positive for DsRed. Of 41 VSEL derived SPC-positive T2 cells, 32 were positive for SPC-H2B-GFP and none expressed DsRed.

Figure 5

FACS-sorting of GFP-positive VSEL-derived T2 cells and expression of SPC-mRNA. Donor derived, GFP-positive type 2 pneumocytes were sorted from SPC-KO mice transplanted with VSELs from SPC-H2B-GFP donors and analyzed for expression of WT SPC-mRNA A: Expression of Epcam and CD49f, gated on CD45−CD31−Lin− and DAPI- (viable) cells. B: Epcam+ CD49f+ double positive cells containing all SPC-positive cells were assessed for expression of GFP in a SPC-H2B-GFP mouse. GFP-positive cells were sorted. C: Epcam+ CD49f+ double positive cells in SPC-KO lungs contain 0.7% autofluorescent cells in the GFP+ channel. GFP− negative cells and autofluorescent cells were sorted. D: In SPC-KO recipients of SPC-H2B-GFP VSELs, GFP-positive cells were sorted. E: Expression of WT SPC-mRNA in GFP-positive T2 cells isolated from SPC-KO recipients. Fold increase of expression is shown compared to T2 cells from SPC-KO controls, which was set to 1.