NP Cell line vs control cell
NP Cell line vs control cell
Cells and Regents hADSCs were obtained from Cyagen Biosciences (HUXMD-01001; Guangzhou, China). hADSCs were culture in medium from Cyagen Biosciences (HUXMD-90011; Guangzhou, China) in a humidified incubator at 37 °C with 5% CO2. The culture medium was replaced every 3 days. hADSCs at passages 2–4 was used for subsequent experiments.  hADSC Differentiation Culture medium  The NP differentiation medium was composed of 1× ITS, 0.1 μM dexamethasone, 1 mM sodium pyruvate, 0.35 mM proline, 0.17 mM ascorbic acid–2-phosphate, 1.25 mg/ml BSA, 10 ng/ml TGF-β1 and 100 ng/ml GDF5；1× Anti, 10ng/ml BMP2[26].  Cell pellet culture For preparation of 3D cell culture, 3 × 105 cells were centrifuged at 1500 rpm for 5 min in 15 ml polypropylene conical tubes and incubated at 37°C overnight. Pellets were formed after 24 h culture. Then pellets were cultured with different differentiation medium. The NP group was cultured with NPM differentiation medium. The control group was cultured with hADSCs culture medium. Medium was changed every three days. Pellets at 7, 14 and 21 days were selected for light microscopic analysis. After 7, 14 and 21 days culture, pellets were fixed with 4% paraformaldehyde for 24 h and then dehydrated with 30% sucrose water.   Firstly, Cutadapt [1] was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Bowtie2 [2] and Tophat2 [3] to map reads to the genome of human. The mapped reads of each sample were assembled using StringTie [4]. Then, all transcriptomes from human Samples were merged to reconstruct a comprehensive transcriptome using perl scripts. After the final transcriptome was generated, StringTie [4] and Ballgown [5] was used to estimate the expression levels of all transcripts Firstly, Cutadapt [1] was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Bowtie2 [2] and Tophat2 [3] to map reads to the genome of human. The mapped reads of each sample were assembled using StringTie [4]. Then, all transcriptomes from human Samples were merged to reconstruct a comprehensive transcriptome using perl scripts. After the final transcriptome was generated, StringTie [4] and Ballgown [5] was used to estimate the expression levels of all transcripts