Therapeutic effect of lung mixed culture-derived epithelial cells on lung fibrosis

Abstract

Cell-based therapy is recognized as one of potential therapeutic options for lung fibrosis. However, preparing stem/ progenitor cells is complicated and not always efficient. Here, we show easily prepared cell populations having therapeutic capacity for lung inflammatory disease that are named as 'lung mixed culture-derived epithelial cells' (LMDECs). LMDECs expressed surfactant protein (SP)-C and gave rise to type I alveolar epithelial cells (AECs) in vitro and in vivo that partly satisfied type II AEC-like characteristics. An intratracheal delivery of not HEK 293 cells but LMDECs to the lung ameliorated bleomycin (BLM)-induced lung injury. A comprehensive analysis of bronchoalveolar fluid by western blot array revealed that LMDEC engraftment could improve the microenvironment in the BLM-instilled lung in association with stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 signaling axis. SDF-1 enhanced both migration activity and differentiating efficiency of LMDECs. Further classification of LMDECs by flow cytometric study showed that a major population of LMDECs (LMDEC Maj , 84% of total LMDECs) was simultaneously SP-C þ , CD44 þ , CD45 þ , and hematopoietic cell lineage þ and that LMDECs included bronchioalveolar stem cells (BASCs) showing SP-C þ Clara cell secretory protein þ stem cell antigen (Sca)1 þ as a small population (1.8% of total LMDECs). CD44 þ -sorted LMDEC Maj and Sca1 þ -sorted LMDECs equally ameliorated fibrosis induced by BLM like LMDECs did. However, infiltrated neutrophils were observed in Sca1 þ -sorted LMDEC-treated alveoli that was not typical in LMDEC Maj -or LMDEC-treated alveoli. These findings suggest that the protective effect of LMDECs against BLM-induced lung injury depends greatly on that of LMDEC Maj . Furthermore, the cells expressing both alveolar epithelial and hematopoietic cell lineage markers (SP-C þ CD45 þ ) that have characteristics corresponding to LMDEC Maj were observed in the alveoli of lung and increased approximately threefold in response to BLM instillation. Taken together, LMDECs newly classified in the present study are easily culture expanded and have a potential role in future regenerative cell therapy for pulmonary fibrosis.

Antifibrotic effects of cyclosporine A on TGF‐β1–treated lung fibroblasts and lungs from bleomycin‐treated mice: role of hypoxia‐inducible factor‐1α

“…All sections were analyzed in a blinded manner by 3 independent researchers. In some experiments, sections were subjected to double staining with anti–HIF‐1α Ab in combination with anti‐αSMA Ab, rabbit anti‐S100A4 Ab, rabbit anti–ionized calcium‐binding adaptor molecule‐1 Ab (Wako), hamster anti‐podoplanin/gp36 Ab (Abcam), or rabbit anti–prosurfactant protein‐C Ab (18), and this was followed by a reaction with appropriate fluoresceinconjugated secondary Abs.…”

Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis

“…Primary cultured alveolar epithelial cells were prepared from mice according to a method previously described with modifications [ 26 ]. The primary cultured cells were characterized by immunofluorescence study with rabbit anti-proSP-C antibody [ 27 ] and hamster anti-podoplanin/gp36 antibody (Abcam) in combination with Alexa Fluor 488-chicken anti-rabbit IgG and Alexa Fluor 594-goat anti-hamster IgG (Life Technologies, Carlsbad, CA). Then, SP-C + gp36 − cells (purity > 90 %) were used as alveolar epithelial cells for the following experiments.…”

Transcriptomic Evaluation of Pulmonary Fibrosis-Related Genes: Utilization of Transgenic Mice with Modifying p38 Signal in the Lungs

“…Male and female C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan). Using the 3.7SP-C/SV40 vector kindly provided by Dr. Jeffrey A. Whitsett (Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio), we generated the following transgenic mice: C57BL/6J-hSP-C-M2 flag-p38α dominant-negative (d.n., dual mutations in wild mouse p38α: Thr180 to Ala; Tyr182 to Phe) TG (p38-DN) mice [62] and C57BL/6J-hSP-C-3HA-tag-MKK6 constitutive-active (c.a., dual mutations in wild human MKK6: Ser207 to Asp; Tyr211 to Asp) TG (MKK6-CA) mice [63]. We confirmed that each transgene-derived product was expressed at least in surfactant protein C (SP-C)-positive AEC II in the lung by using anti-M2-Flag or anti-HA tag antibody.…”