Identification and gene expression profiling of human gonadotrophic pituitary adenoma stem cells

Patients and samples

Fourteen human pituitary adenoma samples obtained during neurosurgery in our department were used for hPASC culture and sequencing. The detailed clinical information of each patient is shown in Additional file 1: Table S1.

Clinically, invasive pituitary adenoma can be defined using the Knosp grade on magnetic resonance imaging [13]. If the edge of the tumor is extended beyond the lateral tangent of the intra- and supra-cavernous internal carotid artery, it is defined invasive and the grade will be 3 or 4. Wherease, non-invasive tumors were located within the tangent of the internal carotid artery and categorized as grades 0, 1 and 2 [13]. Seven non-invasive and nine invasive gonadotrophic pituitary adenoma samples were used for the verification of candidate gene expression. The clinical information is summarized in Additional file 1: Table S2. All the samples were pathologically diagnosed as gonadotrophic pituitary adenoma by two pathologists. This study was approved by the Ethics Committee of Beijing Tiantan Hospital, and written informed consent was available for all patients.

Culture and differentiation of hPASCs

During surgery, the pituitary adenoma specimen was resected and immediately divided into two portions. One portion was “snap-frozen” and stored in liquid nitrogen for pathological immunostaining and sequencing analysis, and the other portion was transferred to the laboratory for hPASC culture. The tumor specimen was thoroughly washed with 1X phosphate buffer saline (PBS) and minced with a sharp scalpel into small pieces under sterile conditions. The cells were collected in 10 ml Dulbecco’s modified Eagle’s Medium /F-12 (DMEM/F-12) and centrifuged at 1000 rpm for 2 min. The cell pellet was dissolved in 2 ml 1X Accutase enzyme (Stemcell Technology, USA) and incubated at 37 °C for 5 min. The dissociated cells were centrifuged at 1,000 rpm for 3 min, and the cell pellet was incubated with red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) for 5 min to eradicate contamination from red blood cells. After washing, the cells were dissolved and cultured in the stem cell medium composed of DMEM/F-12 supplemented with 2 mM L-glutamine, 1% penicillin–streptomycin, 1X B27 (50X, Life Technologies, USA), 20 ng/ml basic fibroblast growth factor (bFGF, Peprotech) and 20 ng/ml epidermal growth factor (EGF, Peprotech) at 37 °C with 5% CO2 in an incubator. The stem cell medium was refreshed every 5 days.

For differentiation, the cultured hPASCs were seeded in a 24-well plate, and the culture medium was replaced with the differentiation medium composed of DMEM/F-12 supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin–streptomycin. The differentiation medium was refreshed every 5 days.

Immunohistochemistry

The hPASCs were cultured in a poly-D-lysine-coated 6-well plate for attachment, then washed with 1X PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were incubated in 0.3% Triton™ X-100 (Sigma-Aldrich, USA) for 15 min and washed with 1X PBS three times. The cells were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibody at 4 °C overnight. The cells were then washed with 1X PBS and incubated with secondary antibody conjugated to Alexa-Fluor 488 or Alexa-Fluor 647 (abcam, USA) at 1:1000 dilution for 1 h at room temperature. The images were captured using a Zeiss microscopic imaging system with fluorescence emission system at different magnifications. For tissue staining, the samples were sectioned at a thickness of 10 μm per slice and subjected to the above procedures.

All antibodies were purchased from Abcam unless otherwise stated. The antibodies used in the experiments were as follows: rabbit anti-Sox2 (1:800), rabbit anti-Oct4 (1:500), rabbit anti-Nestin (1:200), rabbit anti-CD133 (1:500), rabbit anti-ANXA2(1:1000), and mouse anti-FSHβ (1:800, Santa Cruz Biotechnology).

Cell count

The cells in each culture well were washed carefully with 1X PBS before harvesting. Then, the cells were collected in a 1.5-ml tube and centrifuged at 1000 rpm for 20 s. The cell pellet was resuspended in 1-ml DMEM. For counting, 10-μl of cell suspension was added to a hemacytometer, and the number of cells in the central gridded area was manually counted under the microscope. For each sample, the total number was calculated with four separate wells, each performed in duplicate.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was performed to quantify the level of FSH in the culture medium according to manufacturer’s protocol (Elabscience, USA). Briefly, 100 μl standard or culture medium sample was added to each well and incubated for 90 min at 37 °C. The liquid was then replaced with 100 μl biotinylated detection Ab for 1 h at 37 °C. After washing, 100 μl HRP conjugate was added to the well and incubated for 30 min at 37 °C. Each well was washed thoroughly before 90 μl substrate reagent was added and incubated for 15 min at 37 °C. The reaction was stopped by adding 50 μl stop solution, and the mixture was evaluated at 450 nm using a spectrophotometer (Bio-Tek, USA).

RNA extraction

Total RNA from the hPASCs or tumor specimen was extracted by TRIzol reagent (Invitrogen, USA) according to manufacturer’s instructions. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, USA). Total RNA was stored at − 80 °C before use.

Preparation of cDNA library and RNA-sequencing

RNA sequencing was performed by the commercial provider Novogene Co. Ltd. Briefly, first-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. After the preferential selection of 250–300-bp cDNA fragments, polymerase chain reaction (PCR) was performed using Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer. PCR products were purified (AMPure XP system), and the quality of cDNA library was assessed on the Agilent Bioanalyzer 2100 system.

Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform.

Abundance of differentially expressed genes (DEG)

After read mapping, the counts for differentially expressed genes from each sequenced library were adjusted and analyzed using the “edgeR” package from R software (3.22.5). The P-values were adjusted by the Benjamini & Hochberg method. An adjusted P-value of 0.05 and fold change of Log2FC were set as the threshold for significant differential expression.

Enrichment analysis of differentially expressed genes

Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented using the cluster Profiler R package. GO terms with corrected p < 0.05 were considered significantly enriched by differentially expressed genes.

The Cluster Profiler R package was used to test the statistical enrichment of differential expression genes in the pathways.

Protein–protein interaction (PPI) network

GSE26966 dataset from GEO (Gene Expression Omnibus) contains the expression profiles of 14 gonadotrophic pituitary adenomas in comparison to 3 normal pituitary tissues in the published research [14]. The differentially expressed genes of hPASCs were selected according to the following criteria: Log2FC > 1.5 or <  − 1.5, and adjusted p < 0.05. The genes presented in both datasets were illustrated using a Venn diagram, which were further subjected to PPI analysis using the STRING database (www.string-db.org). The network was outlined by Cytoscape software (3.9.1).

Real-time PCR

First-strand cDNA was synthesized by combining total RNA (2 μg) with a reverse transcription mixture (RevertAid™ First Strand cDNA Synthesis Kit, Thermo-fisher, USA) in a total reaction volume of 20 μl, according to the manufacturer’s instructions. The mixture was incubated at 42 °C for 60 min and 70 °C for 10 min and then returned to ice. One microliter of the mixture was extracted for Real-time PCR quantification together with 25-μl SYBR Green PCR Master Mix (TaKaRa, Japan) in a TaKaRa 7500 Real-time system (TaKaRa, Japan). β-Actin served as the internal control. The relative expression level was quantified using the 2−∆∆ct method. The primers used for qPCR are mentioned in Additional file 1: Table S3.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., USA). Data are shown as the mean ± standard error of the mean (SEM) or the mean ± standard deviation (SD). A t-test was used to compare the statistical differences. A P-value < 0.05 was considered to indicate a significant difference.

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