Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts

Abstract

The origin and turnover of connective tissue cells in adult human organs, including the lung, are not well understood. Here, studies of cells derived from human lung allografts demonstrate the presence of a multipotent mesenchymal cell population, which is locally resident in the human adult lung and has extended life span in vivo. Examination of plastic-adherent cell populations in bronchoalveolar lavage samples obtained from 76 human lung transplant recipients revealed clonal proliferation of fibroblast-like cells in 62% (106 of 172) of samples. Immunophenotyping of these isolated cells demonstrated expression of vimentin and prolyl-4-hydroxylase, indicating a mesenchymal phenotype. Multiparametric flow cytometric analyses revealed expression of cell-surface proteins, CD73, CD90, and CD105, commonly found on mesenchymal stem cells (MSCs). Hematopoietic lineage markers CD14, CD34, and CD45 were absent. Multipotency of these cells was demonstrated by their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. Cytogenetic analysis of cells from 7 sex-mismatched lung transplant recipients harvested up to 11 years after transplant revealed that 97.2% +/- 2.1% expressed the sex genotype of the donor. The presence of MSCs of donor sex identity in lung allografts even years after transplantation provides what we believe to be the first evidence for connective tissue cell progenitors that reside locally within a postnatal, nonhematopoietic organ.

Figure 1. Isolation and characterization of mesenchymal cells from BAL of lung transplants.

(A) Mononuclear cells obtained from BAL of lung allografts were plated and maintained in culture. Giemsa staining at day 10 identified the CFU-Fs (left). The top right panel shows an individual CFU-F (original magnification, ×10). On subsequent trypsinization and culturing, a homogeneous population of mesenchymal cells was obtained by passage 2 (bottom right). (B) Immunofluorescence staining of mesenchymal cells at passage 3 demonstrated expression of vimentin (an intermediate filament protein), prolyl-4-hydroxylase (a collagen cross-linking enzyme), and Ki67 (a marker of proliferation). Merged images (right) demonstrated that the majority of vimentin- and prolyl-4-hydroxylase–expressing mesenchymal cells were actively proliferating.

Figure 2. Immunophenotyping of mesenchymal cells isolated from BAL fluid of lung transplant recipients by flow cytometric analysis.

Lung mesenchymal cells were isolated at passage 2 and immunostained for cell-surface markers with specific mAbs. These cells were predominantly positive for CD73, CD90, and CD105 (upper panels) and uniformly negative for the hematopoietic lineage markers CD14, CD34, and CD45 (bottom panels). All histograms show specific mAbs in purple and control isotype-specific IgGs in green. The percentage of positive cells relative to total cells analyzed (mean ± SD) is shown above each histogram.

Figure 3. Mesenchymal cells isolated from BAL fluid of lung transplant recipients differentiate to multiple connective tissue lineages.

(A) Cultured mesenchymal cells from 15 BAL samples and 10 individual CFU-Fs were investigated for in vitro multilineage differentiation capacity. Osteocytic differentiation was indicated by calcium deposition as demonstrated by alizarin red staining (red) in treated cells (bottom left). This was absent in control untreated cells (top left). Accumulation of lipid droplets (indicating adipocytic differentiation) was demonstrated by staining with oil red O in treated cells (bottom center; untreated control is shown in top center). Immunofluorescence staining with anti-human aggrecan polyclonal antibody demonstrates the presence of the extracellular matrix proteoglycan aggrecan, indicative of chondrocytic differentiation in treated cells (bottom right panel; aggrecan-FITC: green; DAPI: blue). A contiguous section stained with control IgG antibody is shown (top right). (B) RT-PCR was performed to analyze the expression of mRNAs specifically related to adipogenic, chondrogenic, and osteogenic activity under inductive culture conditions. Ten cell lines generated from individual CFU-Fs were cultured in either control or differentiation-inducing conditions. Expression of PPARγ and aP2 (indicative of adipogenic activity), type II collagen and type IX collagen (Coll. type II and IX; indicative of chondrogenic activity), and osteopontin (indicative of osteogenic activity) was upregulated in treated cells. Lanes demonstrate RNA isolated from untreated cells, RNA isolated from cells undergoing differentiation, and RNase-Free Distilled Water (UltraPure; Invitrogen), respectively. B2M, β₂-microglobulin. (C) Real-time PCR provided a quantitative analysis of increased relative expression of mRNAs specifically related to adipogenic, chondrogenic, and osteogenic activity under inductive culture conditions.

Figure 4. MSCs isolated from BAL fluid of transplanted lungs are of donor sex genotype.

FISH of mesenchymal cells isolated from BAL fluid of sex-mismatched lung transplant recipients was performed using CEP X Spectrum Orange and Y Spectrum Green DNA probes to distinguish cells of donor versus recipient origin. The top row shows the male sex chromosome status of mesenchymal cells obtained from BAL of a male donor lung transplanted into a female host. Red signal indicates X chromosome, and green signal indicates Y chromosome. Cells were scored in interphase (left). A cell in metaphase demonstrating specificity of the DNA probes is shown on the right. Bottom panels demonstrate the female sex chromosome status of mesenchymal cells obtained from BAL of a female donor lung transplanted into a male recipient (left: interphase cells; right: representative cell in metaphase).