Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells

Abstract

Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.

Figure 1. Vav-Cre-ROSA-YFP lineage tracing and experimental approach

(A): Expression of Cre recombinase under the hematopoietic vav-promoter leads to excision of the Stop-codon with subsequent expression of YFP in vav-Cre/ROSA26R-YFP (vav-YFP) transgenic mice. (B): Fluorescence-activated cell sorting (FACS) analysis of bone marrow from vav-YFP mice. (C): FACS-sorting of vav-YFP-negative and vav-YFP-positive BM cell populations and purity after sorting. (D): Experimental approach. Hematopoietic (YFP positive) or nonhematopoietic (YFP negative) BM cells were sorted by flow cytometry from vav-YFP transgenic mice and transplanted into irradiated SPC-KO recipient mice. Lung tissue was harvested for analysis 2-6 months after transplant. Abbreviations: BM, bone marrow; WBM, whole bone marrow.

Figure 2. Analysis of lung tissue sections for donor-derived, SPC-positive cells

(A)–(F): Confocal microscopic images of lung tissue sections stained for SPC, YFP, and TTF1. The left-most panels show images taken at low zoom, merged with bright-field images to show localization of type 2 cells within lung architecture. Donor-derived type 2 cells are marked with arrows. Middle panel: confocal images, ×630. 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. (A): Type 2 pneumocytes from vav-YFP control lungs coexpress vesicular SPC and nuclear TTF1. YFP is expressed in blood cells but not epithelial cells. (B): Type 2 pneumocytes in SPC-KO lungs express nuclear TTF1 but not SPC. (C) and (D): Lungs from two different SPC-KO mice that received nonhematopoietic BM cells contain donor-derived type 2 pneumocytes (vesicular cytoplasmic SPC and nuclear TTF1) located at the alveolar junction (arrows). (E): Lungs from SPC-KO mice transplanted with vav-YFP positive, hematopoietic BM cells contain vav-YFP positive blood cells. (F): Lung from SPC-KO mouse transplanted with wild-type HSPC. (G): The percentage of donor-derived, SPC-positive type 2 pneumocytes on tissue sections was calculated based on the percentage of type 2 pneumocytes among nucleated cells in corresponding wild-type samples, which was set to 100%. Abbreviations: BM, bone marrow; HSPC, hematopoietic stem and progenitor cell; TTF1, thyroid transcription factor 1; WBM, whole bone marrow; and Wt, wild type.

Figure 3. Detection of bone marrow-derived lung epithelial cells by ImageStream and confocal microscopy on single cells

(A)–(E): Lung tissues from control (Wt and SPC-KO) mice or SPC-KO mice transplanted with the indicated donor BM population were digested with dispase, fixed, and analyzed for SPC (yellow), cytokeratin (orange), and YFP (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Cells were analyzed using an ImageStream imaging flow cytometer with ×40 magnification (Amnis ImageStreamX). Examples of two cells from each group are shown. (A): Type 2 pneumocytes from a WT control mouse. (B): Lung cells from SPC-KO control mice. (C)–(E): Donor-derived, type 2 pneumocytes expressing SPC and cytokeratin were detected in SPC-KO mice transplanted with nonhematopoietic BM cells but not in mice receiving hematopoietic BM cells or purified HSPC. (F): The percentage of donor-derived, SPC-positive type 2 pneumocytes detected by ImageStream was calculated based on the number of type 2 pneumocytes among CD45-negative, round cells in corresponding wild-type samples, which was set to 100%. (G)–(J): Confocal microscopic analysis of sorted lung epithelial cells. Sorted cells were adhered to slides, fixed, and stained for SPC and cytokeratin. Nuclei were stained with DAPI. All cells shown were also CD45 negative (not shown). SPC-positive cells were detected in SPC-KO mice transplanted with nonhematopoietic BM cells but not in mice receiving hematopoietic BM cells. Scale bar = 7.5 μm is the same for all images. Abbreviations: BM, bone marrow; HSPC, hematopoietic stem and progenitor cell; WBM, whole bone marrow; and Wt, wild type.

Figure 4. Detection of SPC-mRNA in SPC-KO recipients by nested PCR

(A): Wild-type SPC-mRNA was detected by nested RT-PCR with primers designed to amplify the wild-type SPC-allele, with no PCR product generated in SPC-KO mice receiving bone marrow from SPC-KO donors. (B): SPC-mRNA was not expressed in mice receiving HSPC (lanes 1-3), and in only two out of nine mice receiving hematopoietic BM cells (lanes 4-12) but in seven out of nine lungs from SPC-KO mice receiving nonhematopoietic BM cells (lanes 13-21). (C): Total number of mice analyzed, number of SPC-positive cells detected, and number of cells analyzed with each indicated method. Total number of mice with SPC-positive cells detected (numerator) and total number of mice analyzed (denominator). Total number of SPC-positive cells detected (numerator) and total number of cells analyzed with each method (denominator). Denominators: ImageStream, CD45– intact cells; tissue sections, nucleated cells; sorted, CD45-CD31-CD11b– nucleated cells. Abbreviations: BM, bone marrow; DAPI, 4′,6-diamidino-2-phenylindole; HSPC, hematopoietic stem and progenitor cell; MDLE, marrow derived lung epithelial cells; ND, not done; PCR, polymerase chain reaction; WBM, whole bone marrow; and Wt, wild type.