A Preliminary Study On The Characteristics Of Tracheal And Lung Epithelial Stem Cells And Their Influencing Factors

Backgrounds and ObjectiveTissue and organ damage and failure have been the greatest threats to human health.Exploring new ways to repair or replace irreversible injury due to illness or the genetic defect is the most concerned direction of life sciences.Stem cell who has the ability of self-renewal and multi-lineage differentiation undoubtedly become the most competent candidates to repair damaged tissue or organ and recover its function in long-term.At present,the stem cell researches and clinical applications are focusing on the hematopoietic disease,diabetes,neurological disorders,primary immunodeficiency diseases,bone and cartilage diseases,cancer,and other fields.In particular,hematopoietic stem cell transplantation,based on its advanced development on research and clinical applications,has become a reliable choice for treating hematologic malignancies and solid tumors.In recent years,mesenchymal stem cells have been preliminary applied to treat certain degenerative diseases such as Parkinson's disease,Alzheimer disease,muscular dystrophy and other degenerative diseases bringing great hope to the patients due to their high self-renewal and differentiation capacities and easy-to-obtaining.However,due to a lot of reasons such as the complexity of the lung tissue itself on its structure and function,a very slow turnover time of epithelium cells,technical limitations for isolating and purifying lung stems cells,the research and the application of lung epithelial stem cell is far lagging behind other organs.A great deal of explorations is needed to this unknown field.For example,characteristics of human and mouse trachea and lung epithelial stem cells have not been entirely clear yet.How the mechanisms of stem cell microenvironment maintain the undifferentiated state of stem cells?What is the regulatory mechanism of the trachea and lung stem cells differentiation?How to get high number of undifferentiated cells with great purity?And which factors play regulatory roles in the microenvironment inducing the stem cells differentiation and how they work?Trachea and lung stem cells can form various types of differentiated cells and simple tissues,but it is far from clear whether they have abilities to form complex organs.In addition,refractory lung diseases such as chronic obstructive pulmonary emphysema,cystic fibrosis,adult respiratory distress syndrome and congenital pulmonary insufficiency are likely due to the presence of lung stem cell proliferation and differentiation defects,so that the damages caused by above diseases can not be repaired.In future,researches on the characteristics of stem cells in disease models may help to find an entry point for further studies.Current researches on lung epithelial stem cells of these disease models are remaining many gaps to be further defined and explored.Our study is to establish a simple and easily reproducible assay to study lung stem cells behavior and niche interactions in vitro.To study stem cells behavior based on its capacity to evaluate self-renewal,differentiation and the effect of co-culturing with fibroblasts and endothelial cells and/or treating with several biologically relevant substances as possible contributors to their niche.MethodsThe use of the extracellular matrix substitute Matrigel and Transwell insert in a 3D in vitro culture system to mimic the microenvironment the trachea and lung epithelial stem cells reside in vivo characterize their proliferation and differentiation potential by observing their colony's size,number,morphology,type selection,colony forming efficiency and lineage differentiation by immunostaining a series of cells makers including basal cells,ciliated cells,club cells,alveolar type ? cells and alveolar type ? cells.After identifying the morphological characteristics of the different colony types growing in this assay and their differentiation spectrum at baseline culture conditions,we continued to explore the effect manipulating several potential niche components including FGF-9,FGF10 or FGF2,Rho-associated protein kinase inhibitor(ROCK-I)Y-27632,transforming growth factor-beta superfamily type I activin receptor-like kinase-5 inhibitor(Alk5-I)SB-431542 or Leukemia Inhibitory Factor(LIF)or FGF Receptor(FGFR)inhibitor PD 173074 on lung stem cells' activation,proliferation and differentiation.Results1.We established a fit assay to study lung stem cells behavior and niche interactions in vitro Our work revealed that lung epithelial stem cells require co-culture with fibroblasts to be able to proliferate and form clonal spheres in vitro and identified the optimum ratio for co-culture with lung epithelial cells and fibroblasts at 1:1.2.Under these culture conditions,lung epithelial cells yielded three morphologically distinct colony types.Type A is big,rounded shape with big lumen and thin wall,mainly expressed markers of airway secretory cells;Type B is big,irregular shape(locular or budding)with big or small lumens but thick wall.Type B colonies had differentiation spectrum similar to Type A,but percentages of cellular subtypes were different.Type B colonies mainly expressed alveolar cell markers.Type C is small,rounded or oval shape with no or small lumen.They originated from alveolar type ? progenitor/stem cells and they have limited differentiation potential only expressed maker of alveolar cells.The presence of basal-like cells in Type A&B colonies is most probably through de-differentiation of an airway secretory stem/progenitor cells.Fibroblasts played its role by direct cell-cell contact and wrapped tightly around the 3 different colony types and cells in close proximity to the fibroblasts are proliferating while cells towards the center of the colony were not.Trachea epithelial basal cells mainly formed type A colonies and mainly expresses basal cell maker in all basally-positioned cells of colonies.They are not dependent on fibroblasts co-culture for their growth.3.Treated with several potential niche components,the proliferation and differentiation characteristics were changed.(1)Co-culture wells treated with FGFs significantly increased the number and size of all colony types.When added with FGFR blocker to FGFs-treated co-culture wells,it abolished the effect induced by FGF10 and FGF2 but not FGF9 on colonies' size and number.FGF treatment caused near-complete disappearance of the basal cell marker and an increase in the alveolar cell marker expression.Notably,neither FGFs nor FGFR blocker treatments influenced the tight wrapping of fibroblasts around the epithelial colonies.Trachea basal cells treated with FGFs,not only increased number of forming spheres but also their size and differentiation towards secretory lineages.FGFR blocker reduced both sphere number and size of untreated wells,and abolished the FGFs-inducing effect on sphere numbers.(2)Lung epithelial cells treated with LIF,ALK5-I or ROCK-I co-cultured with fibroblasts significantly increased the number and size of spheres compared to the co-culture alone.Interestingly,each of these factors caused variable effects on colony types and differentiation spectrum.None of the treatments abolished the tight wrapping of fibroblasts around the epithelial colonies.Treating trachea basal cells with LIF,ALK5-I or ROCK-I significantly increased the number and size of colonies inducing differentiation towards the secretory or ciliated lineage.(3)Both liver and lung endothelial cells could support the growth of colonies from lung epithelium but showed limited differentiation spectrum compared to fibroblast co-culture.The tight wrapping seen with fibroblasts was not detected.(4)Notably,neither fibroblasts nor endothelial cells wrapped around the spheres co-culturing trachea epithelial basal cells.Co-culture with lung endothelial cells resulted in higher colony forming efficiency than with liver endothelial cells but both promoted limited differentiation.ConclusionsLung epithelial(but not tracheal basal)stem cells require co-culture with fibroblasts to undergo clonal proliferation and differentiation and fibroblasts played its role by direct cell-cell contact and wrapped tightly around the colony.Treating lung epithelial or trachea basal stem cells with FGF2,FGF9,FGF10,LIF as well as ALK5 and ROCK inhibitors increased their colony formation efficiency and resulted in variable effects on colonies number,size and differentiation spectrum.FGFR blocker could abolish the inducing effect of FGFs.Endothelial cells could support the growth of colonies of both lung epithelial and trachea epithelial basal stem cells but showed limited differentiation spectrum.Backgrounds and ObjectiveThe role of Aldehyde dehydrogenase 2(ALDH2)in the occurrence and development of diseases has been concerned by researchers.Given the polymorphisms of human ALDH2 in Asian populations,most attention have been paid on correlation between the populations carrying different ALDH2 genotypes and certain diseases.There is plenty of evidence given by the animal experiments or the epidemiological studies to elucidate that many diseases are closely associated with excessively high organ or tissue aldehydes load.ALDH2 can reduce the aldehydes load,thereby reduce the damage of tissue and organs caused by aldehydes,so that to improve and restore the pathological state.Roles of ALDH2 in heart disease,diabetes,neurodegenerative diseases,alcohol-induced pathological damages,upper gastrointestinal cancer,fanconi anemia,osteoporosis,radiation dermatitis and other diseases have been reported.In recent years,the important role of ALDH2 on the occurrence,development and pathological damage of lung diseases has been concerned,but the roles and the possible mechanisms of ALDH2 on the trachea and lung epithelial stem cell characteristics including proliferation,differentiation and repair after damage have not been reported yet.To this end,we used mouse and human trachea and lung epithelial stem cells with different ALDH2 genotypes as samples to explore the roles and mechanisms of ALDH2 polymorphisms on the trachea and lung epithelial stem cell proliferation,differentiation and repair,providing basis for discovering more impact factors to the characteristics of stem cells so that to reveal the mechanisms of ALDH2 effecting on respiratory diseases.MethodsComparison of epithelial thickness,nuclear density and percentage of basal cells between groups by using ALDH2 wild-type,ALDH2homozygous knockout,ALDH2heterozygous knockout and ALDH2transgenic mice;Acute injury was induced by intra-trachea instillation of 10ul 2%polidocanol and trachea epithelium was dynamically evaluated by H&E staining and immune-staining of paraffin slides at 12h,48h and 7 days after injury to explore whether there is significant difference of epithelium repairing among different ALDH2 genotyping groups;Comparing colony forming efficiency in vitro by isolating and culturing mouse trachea epithelial stem cells from different ALDH2 genotyping groups;Comparing stem cell proliferation potential by isolating and culturing human trachea and lung epithelium stem cells from different ALDH2 genotyping groups via sphere forming efficiency.Compare stem cell differentiation potential by H&E staining and immuno-staining of paraffin slides between different huamn ALDH2 genotyping groups.ResultsA.Effect of ALDH2 on mouse trachea epithelium pathology and trachea epithelium stem cells proliferation capacity in vitro1.Effect of ALDH2 on mouse trachea epithelium pathology and injury repair(1)In naive state,trachea epithelium thickness was significantly thinner in ALDH2*2 mice and ALDH2-/-mice(p<0.05)while not significantly different in ALDH2+/-mice(p>0.05)compared to ALDH2 wild type mice.Cell nucleus density and basal cells proportion were significantly lower in all ALDH2 absence or disturbance groups compared to ALDH2 wild type group(p<0.05).(2)7 days after acute injury,trachea epithelium was repaired in each ALDH2 genotyping group with different ALDH2 activity.The epithelium thickness and nucleus density were higher than non-injured state of each genotyping group.However,there was no significant difference of trachea epithelium thickness,cell nucleus density and basal cells proportion in each ALDH2 absence or disturbance group compared to ALDH2 wild type mice,neither did differentiation to ciliated cells and secretory cells at the this time point2.Effect of ALDH2 on mouse trachea epithelial stem cells colony forming capacity(1)In naive state,the trachea epithelium stem cells colony forming efficiency is highest in ALDH2 wild type mice at 0.45%,followed by decreased colony forming efficiency of ALDH2-/-and ALDH2*2 at 0.35%and 0.29%,respectively,the lowest is of ALDH2+/-at 0.19%.However,there is no significant difference of colony forming efficiency between ALDH2 wild type mice and any ALDH2 absence or disturbance group(p>0.05);(2)Treatment with ALDH2 agonist Alda-1 and H2O2 which could induce ROS.Treated with Alda-1,trachea epithelium stem cells colony forming efficiency increased in both ALDH2 wild type mice and ALDH2+/-mice by 56%and 23.8%,respectively;Treated with H2O2,trachea epithelium stem cells colony forming efficiency decreased in ALDH2 wild type mice while increased in ALDH2+/-mice;Co-treated with Alda-1 and H2O2,trachea epithelium stem cells colony forming efficiency increased in both ALDH2 wild type mice and ALDH2+/-mice.B.Effect of ALDH2 on human bronchial epithelium stem cells and lung epithelium stem cells proliferation and differentiation capacity in vitro.1.Effect of ALDH2 on human bronchial epithelium stem cells and lung epithelium stem cells colony forming capacity(1)In naive state,human bronchial epithelial stem cells can form colonies without fibroblasts support while lung epithelium stem cells must rely on fibroblasts to form colonies;The colony forming efficiency of ALDH2 wild type primary bronchial epithelium stem cells was 3.13%and that of lung epithelium stem cells was 0.3%,significantly lower than the former(p<0.05).The colony forming efficiency of ALDH2 mutant primary bronchial epithelium stem cells was 1.58%and that of lung epithelium stem cells was 0.12%,also significantly lower than the former(p<0.05).There is no significant difference of bronchial epithelium stem cells colony forming efficiency between ALDH2 wild type and mutant groups(p>0.05).Likewise,there is no significant difference of lung epithelium stem cells colony forming efficiency between ALDH2 wild type and mutant groups(p>0.05).(2)Treated with ALDH2 agonist Alda-1 and H2O2 which could induce ROS.Treated with Alda-1,bronchial epithelium stem cells colony forming efficiency in ALDH2 wild type group increased by 41%compared to non-treatment control;Treated with H2O2,bronchial epithelium stem cells colony forming efficiency decreased by 27%and co-treated with Alda-1 and H2O2,bronchial epithelium stem cells colony forming efficiency increased by 48.4%compared to only H2O2 treated ones.Treated with Alda-1,lung epithelium stem cells colony forming efficiency in ALDH2 wild type group increased by 36%compared to non-treatment control;Treated with H2O2,lung epithelium stem cells colony forming efficiency decreased by 82%and co-treated with Alda-1 and H2O2,lung epithelium stem cells colony forming efficiency increased by 11.7%compared to only H2O2 treated ones Treated with Alda-1,bronchial epithelium stem cells colony forming efficiency in ALDH2 mutant group increased by 54%compared to non-treatment control;Treated with H2O2,bronchial epithelium stem cells colony forming efficiency increased by 26%and co-treated with Alda-1 and H2O2,bronchial epithelium stem cells colony forming efficiency increased by 51%compared to only H2O2 treated ones.2.Effect of ALDH2 on human bronchial epithelium stem cells and lung epithelium stem cells colony differentiation capacityBronchial epithelial stem cells formed colonies with big size and large lumen,single or multiple,mainly expressed basal cell markers,also with the expression of secreted products in the lumen.Fibroblasts appeared and wrapped around the colonies;Lung epithelial stem cells colonies were also with large size,big or smaller lumen,mainly expressed alveolar epithelial cell marker.Fibroblasts wrapped colonies.There is no significant difference of colony differentiation characteristics between ALDH2 wild type group and ALDH2 mutant group;Treated with Alda-1 and/or H2O2,there was no significant difference of colony differentiation characteristics between treated group and non-treated group.Conclusions1.Effect of ALDH2 on mouse trachea epithelium stem cells(1)ALDH2 absence or disturbance contributed to abnormal trachea structure while no effect on sphere forming efficiency of trachea epithelium stem cells in vitro.(2)After acute injury,trachea epithelium was repaired in each ALDH2 genotyping group with different ALDH2 activity indicating that ALDH2 has little effect on trachea epithelium repair.(3)Enhancing ALDH2 activity,trachea epithelium stem cells colony forming efficiency increased in both normally-functional ALDH2 mice and ALDH2 disturbance mice;ROS induced different effect on trachea epithelium stem cells colony forming efficiency with the mechanism to be explored.2.Effect of ALDH2 on human bronchial epithelium stem cells and lung epithelium stem cells.(1)Human bronchial epithelium stem cells can form colonies without fibroblasts support while lung epithelium stem cells must rely on fibroblasts to form colonies;Bronchial epithelium stem cells mainly expressed basal cell marker and epithelial-mesenchymal transition was found for the evidence that fibroblasts appeared and wrapped around the colonies;lung epithelial stem cells mainly expressed alveolar epithelial cell marker.(2)ALDH2 mutation have little effect on colony forming efficiency and colony differentiation characteristics of human bronchial epithelium stem cells and lung epithelium stem cells.(3)Enhancing ALDH2 activity,human bronchiole epithelium stem cells colony forming efficiency increased in both normally-functional ALDH2 and ALDH2 disturbance individuals;ROS induced different effect on bronchiole epithelium stem cells colony forming efficiency of human individuals with different ALDH2 activity and potential mechanism need to be explored.