Transcriptome analysis of umbilical cord mesenchymal stem cells revealed fetal programming due to chorioamnionitis

Subjects and samples

The present study was approved by the ethical board of Tokyo Medical and Dental University Graduate School of Medicine (M2017-28) and conducted in accordance with the approved guidelines. Written informed consent was obtained from the parents of each neonate. Human umbilical cords were collected from nine very low birth weight infants delivered by cesarean sections at 25–30 weeks of gestation. Clinical data were prospectively collected from the medical records of the neonates and their mothers.

We divided the nine neonates into CAM (n = 4) and non-CAM groups (n = 5). The neonates who exhibited “Triple I,” namely intrauterine inflammation, or infection, or both, were categorized into the CAM group. More specifically, besides pathological findings, maternal fever (> 38.0C), leukocytosis (> 15,000), fetal tachycardia (> 160/min), and definite purulent fluid from the cervical os, were considered as symptoms of CAM8. Further, we confirmed CAM by histological analysis of the placenta. On the other hand, with or without identifying histological CAM in placentas, asymptomatic cases were categorized into non- CAM group.

The criterion for chronic lung disease was the requirement of oxygen support at 36 weeks’ corrected postnatal gestational age9. Neuromotor development was evaluated according to the Kyoto Scale of Psychological Development 2001, a developmental test that has been widely used by Japanese clinicians working with infants, toddlers, and children. We classified the subjects into three groups based on the TDQ score (normal:> 85, border: 70 ~ 85 and retardation: <70) (Shinpan K Shiki Hattatsu Kensahou 2001 Nenban)10.

Preparation of UCMSCs

Umbilical cord-derived mesenchymal stem cells (UCMSCs) were established according to an improved explant method previously reported11. Briefly, a small fragment of the umbilical cord was cultured at 37 ° C (5% CO2 and 95% air) in MEM-α (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS and 2% penicillin – streptomycin (Thermo Fisher Scientific). The outgrowth monolayer cells (Passage1: P1) were collected by disassociating with TrypLE ™ Express enzyme (Thermo Fisher Scientific). The collected cells were seeded into the new dishes and frozen stock was collected after reaching confluence (P2). In the present study, we used the cells from the freeze stock (P3).

RNA extraction

Total RNA from UCMSCs was extracted and purified using the RNeasy Micro Kit (#74106, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was measured using a Nanodrop ND-8000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).

Cell surface marker analysis

UCMSCs were dissociated with TrypLE ™ Express enzyme (Thermo Fisher Scientific), washed with PBS and suspended. The cells were incubated with phycoeryhrin- (PE-) or Fluoresceinisothiocyanate isomer-I (FITC-) conjugated mouse primary antibodies against CD14, CD19, CD34, CD45, CD73, CD90, CD105, or HLA-DR (BD Bioscience, Franklin Lakes, NJ) for 10 min at room temperature and washed with PBS. Flow cytometry and analysis was performed using BD LSRFortessa ™, FACSDiva software and FlowJo ™ software (BD Bioscience).

RNA seq

Library preparation and sequencing

The sequencing libraries from total RNA of USMSC were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (#E7760, New England Biolabs, MA, USA) with NEBNext Poly (A) mRNA Magnetic Isolation Module according to the manufacturer’s protocols. The quality of the libraries was assessed using an Agilent 2200 TapeStation High Sensitivity D1000 (Agilent Technologies, Inc., Santa Clara, CA, USA). The pooled libraries of the samples were sequenced using the Illumina NextSeq 500 (Illumina, Inc., San Diego, CA, USA) in 76-base-pair (bp) single-end reads.

Alignment to the whole transcriptome

Sequencing adapters, low quality reads, and bases were trimmed using the Trimmomatic-0.38 tool12. The sequence reads were aligned to the human reference genome (hg19) using STAR 2.713. For the whole transcriptome alignment with the STAR, files of the gene model annotations and known transcripts were downloaded from the Illumina’s iGenomes website (http://support.illumina.com/sequencing/sequencing_software/igenome.html).

Quantifying the gene expression levels and detection of differentially expressed genes

The aligned reads were subjected to downstream analyzes using StrandNGS 3.2 software (Agilent Technologies). The read counts allocated for each gene and transcript RefSeq Genes (2015.10.05) were quantified using the trimmed mean of M-value (TMM) method14. To investigate gene expression differences, we selected genes through moderated t-test (Benjamini – Hochberg multiple test correction FDR-q-value <0.05) and up- or downregulated them by setting a threshold of twofold. To summarize the biological aspects of the selected genes, we employed a volcano plot, Gene Ontology (GO) terms, and pathway analysis.

For the volcano plot, the genes of each category were selected using the following procedures. The representative genes involved in the contractile apparatus and extracellular matrix were selected based on previous reports15,16,17,18. We selected cell cycle genes that annotated GO: 0045787 positive regulation of cell cycle, fold change <0.5, and p <0.05, or that annotated GO: 0045786 negative regulation of cell cycle, fold change> 2, and p <0.05. We used R software version 4.1.1 (R-Tools Technology Inc., ON, Canada) for statistical analysis.

Pathway statistical analysis was performed on a pathway collection of the WikiPathways19 database using PathVisio tool20 to determine pathways containing the most changed expression, taking into consideration the number of genes in the pathway that were measured in the experiment and the number of genes that were differentially expressed.

Quantitative real-time PCR

cDNA was synthesized from 800 ng of total RNA from UCMSCs by using a High Capacity cDNA Reverse Transcription Kit (#4368814, Thermo). Real-time PCR analysis was performed with a Roche Lightcycler 480II real-time PCR system (Roche Diagnostics, Mannheim, Germany) using FastStart Universal SYBR Green master mix (#4913914001, Roche) with 0.5 μM sense and antisense primers and cDNA (corresponding to 25 of total RNA) according to the manufacturer’s instructions. The relative expression of each transcript was calculated based on the calibration curve method using GAPDH as an endogenous reference for normalization. The primer sets are listed in Table S2. Biologically independent (n = 4 or 5) experiments were performed, and all sample measurements were repeated at least three times.

Cell proliferation assay

UCMSCs were seeded at the density of 1 × 106 cells per 10 cm cell culture dish. The cells were passaged three times every 2 days and the number of the cells at each passage was counted. Biologically independent (n = 4 or 5) experiments were performed, and all sample measurements were repeated at least twice.

MTS assay

At the density of 1 × 104 cells per 96 well plate, we seeded UCMCSs, and the cells were incubated at 37 ° C (5% CO2 and 95% air) for 24 h. Cell proliferation was measured by the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (#G3582, Promega, Madison, WI, USA) according to the manufacturer’s instruction. Briefly, 20 μl of MTS reagent (a tetrazolium compound and an electron coupling reagent) was added into each well and incubated at 37 ° C (5% CO2 and 95% air) for 4 h. The absorbance at 450 nm was measured using an iMark ™ Microplate Reader (#168-1130JA, BIO-RAD Laboratories, Inc, Hercules, CA). Biologically three independent experiments were performed, and all samples were measured with five replicates. We calculated the average and SE of each sample.

Cell cycle analysis

UCMSCs were seeded at 6 × 105 cells/per 10 cm dish and incubated at 37 ° C (5% CO2 and 95% air) for 24 h. At 60–70% confluence, cells were performed cell cycle analysis. Briefly, cells were washed and fixed in 70% ethanol for 2 h at – 20 ° C. Fixed cells were washed and incubated in 0.25 mg/ml RNase A (#12091039, Thermo Fisher Science) for 30 min at 37 ° C. Subsequently, cells were stained with 50 μg/ml propidium iodide (PI) (#25535-16-4, BioVison, Inc, Milpitas, CA) for 30 min at 4 ° C in the dark. Cell cycles were assessed by flow cytometry and analysis was performed using BD LSRFortessa ™, FACSDiva software and FlowJo ™ software (BD Bioscience), and counted the number of the cells at each cell cycle phase.

Statistical analysis

Real-time PCR was analyzed using the Mann – Whitney U test. Student’s t test was used for cell proliferation and cell cycle analyzes. Clinical data and experimental data were compared using Fisher’s exact test, Mann – Whitney U test, or Student’s t test, as required. Cell cycle analysis.

For statistical analysis, we used JMP Pro version 15.1.0 (SAS Institute Inc, NC, USA). Statistical significance was set at p<0.05. Significant differences were expressed as '*' for Pvalues, 0.05, ‘**’ for Pvalues, 0.01 and ‘***’ for P-values, 0.001 respectively.

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