Numbers of EPC colony-forming units (CFUs) from healthy control patients, ICU control patients, and patients with ALI. Both ICU control patients and patients with ALI have significantly more EPC CFUs than do healthy control patients. ^*p=0.95 versus patients with ALI, ^**p<0.05 versus patients with ALI. Boxes represent the median, 25th,and 75th percentiles. The 10th and 90th percentiles are signified by floating red bars.

Kaplan-Meier curve of patients with ALI stratified by EPC CFU count of greater than or equal to 35 (red line) or less than 35 (green line). Patients with ALI with an EPC CFU count of greater than 35 had a survival benefit, with approximately 70% of these patients alive at 28 days, compared with 35% in those patients with EPC CFUs less than 35. p<0.03 for comparison between the two groups.

Reduction of circulating progenitor cells in patients with severe lung disease. (A): Comparisons of the levels of all progenitor cell subtypes in control subjects, in all patients with hypoxemia due to pulmonary disease, in patients with RLD, and in patients with COPD. Analysis of variance (ANOVA) p<.05 for all cell types. (B): Percentages of progenitor cells positive for Annexin V binding in the same four groups. ANOVA p<.05 for CD34 KDR and CD133 KDR cells. ^*p<.05 for t test comparing patients with controls. ^‡p<.05 for t test comparing COPD with RLD patients. Abbreviations: COPD,chronic obstructive pulmonary disease; CTRL, control subjects; RLD, restrictive lung disease.

SEM of generated airway epithelium (Aa) showing ciliated cells (white arrowheads) and cells exhibiting a nonciliated bulging apical membrane, characteristic of Clara cells (white arrows). Ultrastructural features of in vitro ES-derived bioengineered airway epithelium observed by TEM (Ab, Ac, Ad, Ae, and Af). This epithelium exhibits active ciliogenesis demonstrated by centriole migration (black arrows) toward the apical plasma membrane (b) leading to the formation of mature cilia composed of nine tubule-pairs at the periphery and a tubule-doublet in the center of the cilia (c). The bioengineered epithelium exhibits tight junctions (black arrow) at the apical part of lateral plasma membranes (d), desmosomes (black arrow) at the lateral plasma membranes (e) and hemidesmosomes (black arrows) connecting the basal plasma membranes to the underlying basement membrane (BM) (f). Bars 10 µm (a); 150 nm (b), 30 nm (c), 75 nm (d), 295 nm (e), and 50 nm (f). (B) Functionality of the tight junctions analyzed using the lanthanum nitrate diffusion technique and visualized by TEM. Ultrathin sections show that lanthanum nitrate remains located at the apical surface of the ES-derived bioengineered airway epithelium (black arrows) and does not penetrate through the tight junctions (black arrowheads) between electron-dense secretory granule (SG)-containing Clara cells. Bar 50 nm. (C) Electrophoresis of RT-PCR products of CC10 mRNA extracted from control mouse trachea (lane 1), undifferentiated ES cells (lane 2) and ES-derived airway epithelium (lane 3) shows the expected 198-bp transcript in bioengineered airway epithelium. 28S housekeeping gene generates the expected 212-bp transcript. (D) Western blot analysis of the CC10 protein in the secretions covering the air-liquid interface cultures reveal a 5-kD protein corresponding to the CC10 protein (lane 3) also observed in native mouse lung (lane 1) and tracheal (lane 2) tissues

CFTR-corrected CF-patient MSCs retained their multipotency and responded to cAMP stimulation by secreting chloride to the apical side. (a) Schematic for CF-patient MSC isolation, expansion, gene correction, and positive drug selection. (b) RT-PCR to verify the successful CFTR gene transfer. RT-PCR was performed to amplify WT CFTR transcripts but not ΔF508 mutant transcripts. The gene-corrected CF MSCs and positive control Calu-3 cells have WT CFTR transcription, whereas non-gene-corrected CF MSCs and the no-RT control show negative amplification. In the TBP RT-PCR control, all of the samples except the no-RT control show positive PCR products. (c) Phase-contrast microscopic view of the CFTR gene-corrected CF-patient MSCs. (d) Photomicrograph of a representative stem cell colony plate. Purple-stained foci are the MSC colonies. (e) Osteogenesis of the CFTR gene-corrected CF-patient MSCs. After differentiation in an osteogenic medium, cells had mineral deposits visualized in red by Alizarin red staining. (f) Adipogenesis of the CFTR gene-corrected CF-patient MSCs. After differentiation in an adipogenic medium, cells had lipid droplet accumulation stained in red with oil red O. (g and h) CFTR gene-corrected MSCs from CF patients contributed to the apical cAMP-stimulated Cl- secretion. CFTR gene-corrected CF-patient MSCs or non-gene-corrected CF-patient MSCs were mixed with ΔF508 CF AECs at different ratios as indicated. After 1 month in culture at the air-liquid interface, chloride efflux assays were performed as described in Materials and Methods. A two-way ANOVA test revealed that cocultures with the CFTR gene-corrected CF-patient MSCs (F) had a greater chloride secretion in response to the IBMX and forskolin stimulation than the cocultures with non-gene-corrected CF-patient MSCs (OE) (n 4; P≤0.05).

Donor BMDMSC localizing to injured lung assume lung cell phenotypes. Sections were analyzed in double-stained IIFA with anti-GFP (green) and antibodies to specific cell type markers (red); co-localization in each case appears yellow (arrows point to double positive cells). (a-d) Anti-vimentin (fibroblast). (a) Normal control; (b) 14 d after bleomycin; (c) 14 d after busulfan followed by BMDMSC (no lung injury); (d) 14 d after bleomycin followed by BMDMSC in a busulfan myelosuppressed animal. (e, f) Anti-aquaporin (type I alveolar epithelium). (e) Fourteen days after busulfan followed by BMDMSC (no lung injury); (f) 14 d after bleomycin followed by BMDMSC in busulfan myelosuppressed animal. (g, h) Anti-pro-surfactant protein C (type II alveolar epithelium). (g) Fourteen days after busulfan followed by BMDMSC (no lung injury); (h) 14 d after bleomycin followed by BMDMSC in busulfan myelosuppressed animal. (i, j) Anti-smooth muscle actin (SMA-1, myofibroblasts). (i) Fourteen days after busulfan followed by BMDMSC (no lung injury); (j) 14 d after bleomycin followed by BMDMSC in busulfan myelosuppressed animal. (k) Percentage of GFP-positive cells that express lung cell phenotype markers in myelosupressed mice treated with bleomycin and infused with BMDMSC. All microphotographs were taken at 40 magnification.