CD45/CD11b positive subsets of adult lung anchorage-independent cells harness epithelial stem cells in culture

Abstract

Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between the stem/progenitor cells that proliferate or differentiate and the somatic cells of lung, we utilized a novel ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3, and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conserved population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. Over 50% of the AIC population was cKit+ or double-positive for CD45+ and CD11b+ antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. AICs undergo remodeling to form a cellular lining at the air/gel interface, and TGFβ1 treatment modifies protein expression, implying direct-differentiation of this population. These data confirm the active participation of clonogenic hematopietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which embrace sustained cell viability, proliferation, and differentiation, for use in studies of compensatory pulmonary growth.

Very Late Antigen-5 Facilitates Stromal Progenitor Cell Differentiation Into Myofibroblast

“…The AIC population of cells was previously reported to compose a subset of epithelial progenitor cell types [16]. Because we had established the presence of a subset of stromal cell progenitors with the inherent capability to attach, grow, and differentiate in culture, we next sought to determine the differentiation properties of these cells in an in vivo model.…”

“…Tissue culture media and supplements, including Dulbecco's modified Eagle's medium (DMEM), bovine growth serum (BGS) or fetal bovine serum (FBS), and 1% penicillin‐streptomycin were purchased from Life Technologies (Grand Island, NY, http://www.lifetechnologies.com) or Atlanta Biologicals (Lawrenceville, GA, http://www.atlantabio.com). Organotypic isolation of lung AICs has been previously described by our laboratory [16]. In brief, lung, heart, and kidney organs were extracted and minced into ∼1 mm 2 cubes with two randomly collected fragments submerged in a DMEM + 10% BGS (upper layer) situated on a polymerized (DMEM + 20% BGS) 3.3% alginate substrate (bottom layer).…”

“…Using a method designed to model tissue regeneration ex vivo, we described an organotypic lung population of anchorage‐independent cells (AICs) that proliferated and differentiated in suspension culture [16]. This physiologically distinct AIC population, which could be isolated from multiple organisms, including humans, was immunophenotypically mixed and included lung epithelial progenitor and hematopoietic subsets decorated by CD45, c‐KIT, and/or the CD11b (ITGAM) integrin.…”

Discrimination Between Lung Homeostatic and Injury-Induced Epithelial Progenitor Subsets by Cell-Density Properties

“…As seen in Fig. 3A, although variability in protein expression existed, BrdU-incorporating CD45-positive cells comprised over 50% of the lighter fractions (FR1 and FR2), to possibly correspond with a previously reported free-floating anchorage-independent progenitor cell population [25]. Meanwhile, in FR4, proliferating cells were largely characterized by SCA-1 and CD31 and c-KIT protein presentation suggesting equilibrium of fibroblastic, endothelial, and/or (human) multilineage stem cells, respectively [10].…”

“…The absence of primer-dimers was verified postamplification by melting curve analysis. Except for actin, alpha-1 (Acta1) forward 5¢-CGCAAATGCTTCTAGGCGCACT-3¢ and reverse 5¢ ACACGTCAAAAACAGGCGCCGG-3¢, and telomerase (Tert) forward 5¢-CATGGAGAACAAGCTGTTTGC-3¢and reverse 5¢-CAGGGAAGTTCACCACTGTC-3¢, all primer sequences have been previously reported [25]. These include Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), CCAAT/ enhancer-binding protein alpha (Cebpa), secretoglobin, family 1A, member 1 (Scgb1a1), Sftpc, Aqp5, surfactant protein B (Sftpb), and CD31.…”