Differential Analysis And Molecular Mechanism Of Amniotic Fluid Mesenchymal Stromal/Stem Cells Derived From Different Stages Of Embryo Development

Objective:Amniotic fluid derived stromal/stem cells?AF-MSCs?have good self-renewal capacity,flexible plasticity,lower immunogenicity and tumorigenicity,lower senescence rate in vitro culture,which make AF-MSCs become a better prospect seed cells in regenerative medicine than adult stem cells.However,due to the lacking uniform culture standards for MSCs in vitro culture,their various biological characteristics,which leads to lower comparability among the AF-MSCs related studies;furthermore it is difficult to identify the differences of AF-MSCs derived from different stages of pregnancy,which in turn limit its application in the future.In addition,although the heterogeneity of morphology,proliferation and differentiation on AF-MSCs are widely known,the origin and the mechanism of heterogeneity remains unclear,it urgently need some technical measures to analyze the differences.Transcription sequencing was the study of RNA that extracted from the particular cells consisting of certain stage of development or functional state.Transcription sequencing can reveal specific biological processes in order to study relevant gene function and structure from the overall level,which has been widely used in basic research,clinical diagnosis and drug development.Therefore,using the transcriptome to study the different stages of AF-MSCs can help us to better understand the differences among AF-MSCs derived from the different developmental stages.Methods:1.Isolation and culture of AF-MSCsCollected the rat amniotic fluid of E12,E15,E18 and E21 on aseptic conditions,centrifuging 1800 rpm for 15 minutes at 4?,washing with PBS 2 times,and then resuspended with DMEM/F12 containing 10%FBS,incubated on 37?,5%CO2 humidity incubator.Day 5 was the first time of changing the medium,and then the medium was changed every 2-3 days,subcultured cells until the 70%confluence.Cell passaged more than 3 times identified as the isolated successfully.2.AF-MSCs identificationAccording to the instructions,we detected the CDs surface markers by the flow cytometry,and induced the tri-lineage differentiation with the differentiation mediums.3.BrdU assay P5 AF-MSCs cultured on 6-well plates,when the cells reached 70-80%confluence,added20?L 1-mM BrdU solution per well,and maintained another 4 hours in the incubator.Detached the AF-MSCs and treated the cells by BrdU Kit,and then detected by a flow cytometry.4.CCK-8 assaySubculture P4 AF-MSCs at a density of 1.5×10⁴ per well in 96-well plates,took out a plate and added 10?L CCK-8 per well and measure the ODs on 450nm every 24 hours.5.Transwell migration experimentThe P4 AF-MSCs re-suspended with DMEM/F12 supplemented with 2%FBS and culture on the upper chamber,the lower chamber added 600?L DMEM/F12 supplemented with12%FBS,maintained in a 37?incubator for 18 hours.Used the crystal violet solution to stain for 20 minutes,and observed by the microscope and take the pictures.After taking the pictures,the crystal violet decolorization by the 10%acetic acid for 10 minutes,and detected the eluate by Tecan.The higher the ODs represented the more cells migrated to the bottom of the chamber.6.Scratch experimentP4 AF-MSCs cultured at a density of 2.5×10⁵ per well on 6-well plates.The next day,the cells reached 70%confluence,with a sterile 10?L pipette tip scratch the cells,after 12hours and 24 hours to photograph the scratch.7.Transcriptome of AF-MSCsWe randomly selected 3 samples of AF-MSCs at P4 on each pregnant stages,which were12 samples for four groups of AF-MSCs in total.The whole transcription sequencing was completed by BGI.Briefly,used the biotin labeling probe(Ribo-Zero^TM rRNA Removal Kit)to remove the ribosome rRNA in the total RNA,after purification,fragmented the RNA under the environment of certain temperature and the ion.Then use the random primers and reverse transcriptase in Stranded TruSeq?Stranded kit to synthesize the first strand cDNA,then use DNA polymerase I and RNaseH to synthesize double-stranded cDNA.In cDNA second strand synthesis process,RNA template has been removed,dTTP was replaced by dUTP.The product of the double strand cDNA followed to add adenosine and joint connection,and then be expanded and purified,cDNA library finally being built and sequencing.The Illumina HiSeq platform was used to sequencing.8.Bioinformatics AnalysisWe filter out reads that contained rRNA or excessive amount of unknown nucleobase N,eliminate the low-quality reads,adaptors and other contaminants to get clean reads.The clean reads are then mapped to the reference genome?Rnor 6.0?and then assembled with the software StringTie and Cufflinks.These transcripts followed to predict coding capacity by three softwares-CPC,txCdsPredict and CNCI and a database pfam,then used the software Bowtie2 to compare transcripts to the reference sequence and then used the software RSEM to measure the FPKM.At last,we use DEGseq method to do Differentially Expressed Genes?DEGs?screening analysis.The filtration conditions of the significant differentially expressed were defined as fold change?2.00 and Adjusted Pvalue?0.001.Further analysis was as following,the OD values of cell proliferation curve and the mRNA expressions from 4 groups transcription data combined to make the linear regression analysis,screening for proliferation-related differentially expressed genes,and using TargetScan and miRanda for microRNA target prediction,to find the microRNA interaction with the target mRNA.9.Genes transfection and detection through PCR and western blottingTransfected the plasmids or siRNAs or mimics into AF-MSCs according to the transfection reagent instruction.After observing the cell biological function?proliferation,migration?,the cells were collected to extract RNAs and proteins.The total RNAs reversed to cDNA and performed the real-time quantitative PCR.The reaction condition was as following,95?pre-denaturation for 30 seconds,95?for 5 seconds,and 58?for 45 seconds,40cycles,the dissolution curve stage was 95?for 5 seconds,60?for 30 seconds.The extracted proteins performed western blot with the 10%separation gels,5%upper gel,the period of electrophoresis was 2 hours,and the protein transferred to the PVDF membrane for 2 hours,and blocked for another 2 hours,and incubated primary antibody overnight,the next day,incubated the second antibody at room temperature for 2 hours,then detected the protein by ECL.10.Dual luciferase reporter gene experimentHEK293T cells planted on the 24-well plates overnight and made the confluence reach60%,replaced the culture medium to serum-free medium,co-transfected mimic and wild-type or mutant plasmids into 293T cells,and incubated in 37?,5%CO₂ incubator for 6hours,changed to the fresh medium contained the 10%FBS and incubated another 48hours.The luciferase reporter assays Kit was used in this experiment according to the manufacturer's introduction.Calculated the ratios of the firefly luciferase and the renilla luciferase,and normalized the ratios to miR-NC which normalized as 1.11.Statistical analysisThe counting data was written in the form of mean±standard deviation,the difference of4 groups data was analyzed by one way analysis of variance?ANOVA?,and the post-hoc analysis was the LSD?homogeneity of variance?or Tamhane T2?heterogeneity of variance?,the difference of the 2 groups were analyzed by student's t test?homogeneity of variance?or Mann-Whitney U?heterogeneity of variance?.SPSS 17.0 software was performed statistical analysis above,p<0.05 was considered a significant difference.Results:1.AF-MSCs derived from rat early pregnancy,especially the E15 have good proliferation and migration capacity.We successfully collected amniotic fluid from 125 pregnant rats,among which consist of47 derived from E12,17 from E15,44 from E18,and 17 from E21.Surprisingly,E15 stage had a 100%culture success rate,followed by E21,and the E12 had the lowest success rate.The time of the first clone emergence of E12 and E15 were 1-3 days,while 7-10 days for E18 and E21.The AF-MSCs from E12 and E15 passed over the"Hayflick Limit"continuous passaging to 60 times and maintained proliferation capacity,while E18 and E21 could not passaged up to 22 times.Both the CCK-8 assay and the BrdU assay identified the AF-MSCs derived from E12 and E15 have better proliferation capacity.The scratch experiment and Transwell migration experiment demonstrated the AF-MSCs derived from E12 and E15 have better migration capacity.2.Transcriptome of AF-MSCsA total of 12 of rat AF-MSCs samples were measured using the Illumina HiSeq platform,with an average output of 12.67 Gb per sample and an average alignment ratio of 87.26%to the reference genome.A total of 61,678 transcript expressions were detected,of which7,106 were new lncRNAs,7,210 were new mRNAs,4,966 were known lncRNAs,and42,396 were known mRNAs.The small non-coding RNA?sncRNA?sequencing were using BGISEQ-500 technology,the average number of detected sncRNAs for each sample were 1434.There was an average of 593 new microRNAs and an average of 112 known microRNAs.3.Enrichment analysisFrom the principal component analysis?PCA?and the hierarchical cluster analysis of the mRNA Fragments Per Kilobase per Million?FPKM?of four stages of AF-MSCs in the transcriptome,we also observed that E12 and E15 located in the left side of PC1 axis,and the E18 and E21 located in the right side of PC1 axis,and all the abundance profiles were group into two groups,the E12&E15 and the E18&E21 on the hierarchical clustering,which was similar to that in the cells proliferation tendency.Therefore,we grouped the E12 and E15 into the early groups,the E18 and E21 into the later groups for the further analysis.We got 604 of DE mRNAs between the early groups and later groups.These DEGs enriched in biological process such as chromosome segregation,nuclear division,organelle fission,and DNA replication.Subsequently,KEGG Pathway enrichment of DEGs was conducted.We can see that each group of DEGs clustered in cell cycle,DNA replication,purine metabolism,cellular senescence,and mismatch repair,which were important roles for cells growth and proliferation,further supported the growth difference between AF-MSCs from the early and later groups.Furthermore,we performed the linear regression on the ODs values of proliferation curve and the expression of RNAs,Abca4/miR-351-3p were selected as the gene pair related to proliferation.In order to explore the mechanism of AF-MSCs migration and proliferation,we reanalyzed the transcriptome data above.We performed the differentially expressed analysis based on E15 as the reference group,which had higher proliferation capacity and easier cultured.We observed that the E15 and E12 had similar transcript profiles,contrasted to the E15 VS E18 or E15 VS E21 who had the more differentially expressed genes.We had screened the Chrdl1/miR-532-3p and Chrdl1/miR-672-3p as the gene pairs that related to AF-MSCs proliferation and migration.4.Function of miR351-3p and its potential target gene ABCA4 in association with proliferationThrough the linear regression analysis,we found out the Abca4 was differentially expressed gene of AF-MSCs at different stages of development,which have the ability of inhibiting cell proliferation.Knock-in and knock-down of miR-351-3p,which induced decreased and increased of Abca4,and as well as the proliferation or suppressed proliferation of AF-MSCs,respectively.Dual luciferase reporter gene experiment showed that there is a microRNA binding site of miR-351-3p in the CDS region of Abca4.5.Function of miR-532-3p and its potential target gene Chrdl1 in association with proliferation and migrationSelected Chrdl1 as the differentially expressed gene of AF-MSCs at different stages of development by filtering the 8 fold change.The miRranda software used as the microRNA target prediction,and the results were that 2 microRNAs bound to the 3'UTR region of Chrdl1,where in which miR-672-3p was removed because of its expression not match the transcription sequencing data by RT-qPCR test,then miR-532-3p became the only selected differentially expressed microRNA.It was confirmed that Chrdl1 have the ability of inhibiting cell proliferation and migration by CCK-8 and transwell experiments.Knock-in and knock-down of miR-532-3p,which induced decreased and increased of Chrdl1,and the AF-MSCs show the corresponding trend of increasing and decreasing proliferation and migration.Dual luciferase reporter gene experiment showed that miR-532-3p bind to the3'UTR region of Chrdl1.Conclusion:Our study collected the amniotic fluid at different stages of embryonic development,cultured AF-MSCs with the basic mediums,and observed the differences in the attachment,growth,proliferation,differentiation and migration of cells at various stages.We suggested that the AF-MSCs derived from rat E15 gestational age are readily isolated and cultured,had a higher culture successful rate,and the AF-MSCs derived from rat first trimester?E12and E15?have better proliferation and migration capacity compared to AF-MSCs from the mid-and third trimesters?E18 and E21?.We used the whole transcriptome to compare the difference of AF-MSCs from different stages of development,we found that AF-MSCs come from the early stages?E12 and E15?or the later stages?E18 and E21?shared the common transcription data.The miR-351-3p promoted the proliferation of AF-MSCs through targeting to Abca4,on the other hand,miR-532-3p promoted the proliferation and migration of AF-MSCs through targeting to Chrdl1.