Characterization of lung stem cell niches in a mouse model of bleomycin-induced fibrosis

被引:16
作者
Banerjee, Ena Ray [1 ,2 ]
Henderson, William Reed, Jr. [1 ]
机构
[1] Univ Washington, Dept Med, Div Allergy & Infect Dis, Ctr Allergy & Inflammat, Seattle, WA 98195 USA
[2] Univ Calcutta, Dept Zool, Kolkata 700019, W Bengal, India
关键词
SIDE POPULATION CELLS; ORIGIN;
D O I
10.1186/scrt112
中图分类号
Q813 [细胞工程];
学科分类号
摘要
Introduction: In lung fibrosis, alveolar epithelium degenerates progressively. The goal of regenerative medicine is to aid repair and regeneration of the lost tissues in parenchyma and airways for which mobilization of tissue resident endogenous or bone marrow-derived exogenous stem cells niches is a critical step. We used a lung injury model in mice to identify and characterize functional lung stem cells to clarify how stem cell niches counteract this degenerative process. Methods: Short term assay (STA) - Bleomycin-induced lung inflammation and fibrosis were assessed in a model of idiopathic pulmonary fibrosis in wild-type (WT), gp91phox-/- (NOX-/-), and gp91phoxMMP-12 double knockout (DKO) mice on C57Bl/6 background and Hoechst 33322 dye effluxing side population (SP) cells characterized. Long term assay (LTA) - In a bleomycin induced lung fibrosis model in C57Bl6 mice, the number of mature cells were quantified over 7, 14, and 21 days in bone marrow (BM), peripheral blood (PB), lung parenchyma (LP) and brochoalveolar lavage (BAL) fluid by FACS. BrdU pulse chase experiment (10 weeks) was used to identify label retaining cells (LRC). BrdU(+) and BrdU-cells were characterized by hematopoietic (CD45(+)), pluripotency (TTF1(+), Oct3/4(+), SSEA-3(+), SSEA-4(+), Sca1(+), Lin(-), CD34(+), CD31(+)), and lung lineage-specific (SPC+, AQP-5(+), CC-10(+)) markers. Clonogenic potential of LRCs were measured by CFU-c assays. Results: STA- In lung, cellularity increased by 5-fold in WT and 6-fold in NOX-/- by d7. Lung epithelial markers were very low in expression in all SP flow sorted from lung of all three genotypes cultured ex vivo. (p < 0.01). Post-bleomycin, the SP in NOX-/- lung increased by 3.6-fold over WT where it increased by 20-fold over controls. Type I and II alveolar epithelial cells progressively diminished in all three genotypes by d21 post-bleomycin. D7 post-bleomycin, CD45+ cells in BALf in NOX-/- was 1.7-fold > WT, 57% of which were Mf that decreased by 67% in WT and 83% in NOX-/- by d21. LTA-Cellularity as a factor of time remained unchanged in BM, PB, LP and BAL fluid. BrdU(+) (LRC) were the putative stem cells. BrdU(+)CD45(+) cells increased by 0.7-fold and SPC(+)CC10(+) bronchoalveolar stem cells (BASC), decreased by similar to 40-fold post-bleomycin. BrdU(+)VEGF(+) cells decreased by 1.8-fold while BrdU(-)VEGF(+) cells increased 4.6-fold. Most BrdU(-) cells were CD45(-). BrdU(-) BASCs remained unchanged post-bleomycin. CFU-c of the flow-sorted BrdU(+) cells remained similar in control and bleomycin-treated lungs. Conclusion: STA- Inflammation is a pre-requisite for fibrosis; SP cells, being the putative stem cells in the lungs, were increased (either by self renewal or by recruitment from the exogenous bone marrow pool) post-bleomycin in NOX-/-but not in DKO indicating the necessity of cross-talk between gp91phox and MMP-12 in this process; ex vivo cultured SP progressively lose pluripotent markers, notably BASC (SPC+CC10+) -significance is unknown. LTA-The increase in the hematopoietic progenitor pool in lung indicated that exogenous progenitors from circulation contribute to lung regeneration. Most non-stem cells were non-hematopoietic in origin indicating that despite tissue turnover, BASCs are drastically depleted possibly necessitating recruitment of progenitors from the hematopoietic pool. Loss of VEGF(+) LRC may indicate a signal for progenitor mobilization from niches. BrdU(-) BASC
引用
收藏
页数:21
相关论文
共 33 条
[1]   Lung cells transplanted to irradiated recipients generate lymphohematopoietic progeny [J].
Abe, S ;
Lauby, G ;
Boyer, C ;
Manouilova, L ;
Rennard, SI ;
Sharp, JG .
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, 2004, 30 (04) :491-499
[2]   Transplanted BM and BM side population cells contribute progeny to the lung and liver in irradiated mice [J].
Abe, S ;
Lauby, G ;
Boyer, C ;
Rennard, SI ;
Sharp, JG .
CYTOTHERAPY, 2003, 5 (06) :523-533
[3]   Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFGb1/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells [J].
Alipio, Zaida A. ;
Jones, Nathan ;
Liao, Wenbin ;
Yang, Jianchang ;
Kulkarni, Shilpa ;
Kumar, K. Sree ;
Hauer-Jensen, Martin ;
Ward, David C. ;
Ma, Yupo ;
Fink, Louis M. .
DIFFERENTIATION, 2011, 82 (02) :89-98
[4]   Fibrocytes and the tissue niche in lung repair [J].
Andersson-Sjoland, Annika ;
Nihlberg, Kristian ;
Eriksson, Leif ;
Bjermer, Leif ;
Westergren-Thorsson, Gunilla .
RESPIRATORY RESEARCH, 2011, 12
[5]  
[Anonymous], J ADV LAB RES BIOL
[6]   Bleomycin induces cellular senescence in alveolar epithelial cells [J].
Aoshiba, K ;
Tsuji, T ;
Nagai, A .
EUROPEAN RESPIRATORY JOURNAL, 2003, 22 (03) :436-443
[7]   Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation [J].
Asakura, A ;
Rudnicki, MA .
EXPERIMENTAL HEMATOLOGY, 2002, 30 (11) :1339-1345
[8]   Stem cells in adult skeletal muscle [J].
Asakura, A .
TRENDS IN CARDIOVASCULAR MEDICINE, 2003, 13 (03) :123-128
[9]  
Badri L, 2010, AM J RESP CRIT CARE, V181, pA5278
[10]   Human Embryonic Stem Cells Differentiated to Lung Lineage-Specific Cells Ameliorate Pulmonary Fibrosis in a Xenograft Transplant Mouse Model [J].
Banerjee, Ena Ray ;
Laflamme, Michael A. ;
Papayannopoulou, Thalia ;
Kahn, Michael ;
Murry, Charles E. ;
Henderson, William R., Jr. .
PLOS ONE, 2012, 7 (03)