Isolation and Characterization of Murine Multipotent Lung Stem Cells

Abstract

The capacity of the lung to repair itself after injury is well known, but the cell types involved in lung regeneration remain undefined. The aim of this study was to isolate and characterize resident progenitor/stem cells from adult mouse lung. We report the isolation and characterization of resident stem cells that have a Sca1+/CD45(-)/CD31(-) phenotype. Their immunophenotype and differentiative potentiality were distinct from that of other previously described lung stem cells. These cells underwent extensive self-renewal in culture and could differentiate into endothelial and lung epithelial (alveolar type I, II, and Clara) cells in vitro. They have exhibited some mesenchymal but no neural differentiation ability. Transfer of these cells into mouse models of lung injury significantly improved survival and minimized lung destruction. These cells may provide useful tools for the study of lung stem cells and the assessment of new therapeutic approaches for lung diseases.

Lung-Derived Mesenchymal Stromal Cell Post-Transplantation Survival, Persistence, Paracrine Expression, and Repair of Elastase-Injured Lung

“…Myofibroblast differentiation was assessed by exposing serum-starved (6 h) L-MSCs with human recombinant transforming growth factor (TGFb1) (10 ng/mL, R&D Systems) for 48 h. Outcome was evaluated by qPCR of myofibroblastic (Acta2, Fn, col1A1, Tnc, and matrix metalloproteinase 9 [MMP9]), senescence (p21/Cdknb1, p53), survival (Birc5, midkine), and fibroblast activation (Fapa, Dpp4, and S100A4) genes using commercial primers (Supplementary Table S2); in addition, cultures were immunostained for alpha-sma (n = 3 samples/ experiment). Differentiation to smooth muscle cells was evaluated 10 days after exposure to 5 Aza-Cytidine (5 mM, Sigma, 24 h) by morphology and qPCR (Des/Desmin, MyoD1, Acta2/a-sma) [25]. Differentiation to lung epithelial cells was assessed by incubation of L-MSCs in small airway growth media (SAGM with SingleQuots) containing rhEGF (0.5 ng/mL) for 8 days, and measurement of Aqp5, Ttf-1, proSPC (Sftpc), and CCSP (Scgb1A1) mRNA expression.…”

“…For instance, BM or adipose-derived MSCs significantly reduced injuries caused by elastase [4][5][6][7], lipopolysaccharide [8,9], sepsis [10,11], hyperoxia [12][13][14][15][16][17], and bleomycin [18][19][20][21][22]. In general, the reparative effects of MSCs were compared with control treatments such as irradiated BMMSCs, lung fibroblasts, dermal fibroblasts, or embryonic fibroblast cell lines, rather than MSCs of lung origin, which have only recently been described in mice [23][24][25][26][27], humans [28][29][30], and sheep [31,32]. Whether lung-derived MSCs (L-MSCs) are close or distant relatives of BM-MSCs with respect to in vivo identity, cellular physiology, and therapeutic potential is unclear as is the significance of their stemness in vitro [33].…”

“…A recent study in mice showed that bleomycin injury markedly reduced the abundance of L-MSCs in the lung, and their resupply prevented bleomycin induced fibrosis [34]. In another study in mice, L-MSCs (called multipotent lung stem cells) isolated by direct sorting (Sca-1 pos , CD31 neg , CD45 neg ) were shown to protect against elastase injury through paracrine signals [25]. Two recent studies from our laboratory employing autologous L-MSCs in an ovine model of emphysema [32,35] demonstrate significant improvements in tissue mass, perfusion, and diffusion capacity when cell were delivered intrabronchially within a biological scaffold designed to improve adherence and retention of transplanted cells.…”

Isolation Of Alveolar Epithelial Type II Progenitor Cells From The Adult Human Lungs

“…12,13 We seeded nonhematopoietic lung cells on feeders. In all specimens, spindleshaped cells gave rise to colonies 6-10 days after the start of culture (Figure 1a).…”

“…[7][8][9][10][11] Recent studies on rodent models have identified stem/ progenitor populations for alveolar epithelial cells, and have revealed that the stem/progenitor populations have important roles in lung repair and carcinogenesis. [12][13][14][15][16][17][18] In distal lungs of adult human, to our knowledge, only two stem cell populations were isolated. 19,20 The first report showed that lung resident mesenchymal stem cells (MSCs) isolated from bronchoalveolar lavage fluid collected from lung transplant patients have the self-renewal ability and the differentiation capability into a mesodermal lineage.…”