Hyperthermia differentially affects specific human stem cells and their differentiated derivatives

Materials and Methods Cell culture hESCs (Line H9, WA09, WiCell Research Institute, Inc.) were cultured on feeder cells of Mitomycin C (MMC) (Selleck)-inactivated mouse embryonic fibroblast (MEF) in DMEM/F12 medium (Gibco) containing 20% KnockOut Serum Replacement (Gibco), 0.1 mM non-essential amino acids (NEAA, Gibco), 2 mM GlutaMAX (Gibco), 1% penicillin/streptomycin (Gibco), 55 μM β-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/mL FGF2 (Joint Protein Central, JPC) or on Matrigel (Corning) in mTeSR medium (STEMCELL Technologies). hMSCs were cultured in α-MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 0.1 mM NEAA (Gibco), 1% penicillin/streptomycin (Gibco), and 1 ng/mL FGF2 (JPC). hVSMCs were cultured in N2B27 medium supplemented with 50% Neurobasal (Gibco), 50% DMEM/F12 medium (Gibco), 1× N2 (Gibco), 1× B27 (Gibco), 10 ng/mL PDGF-AB (Peprotech), 55 μM β-mercaptoethanol (Gibco) and 1% penicillin/streptomycin (Gibco). hVECs were maintained in EGM-2 (Lonza) medium supplemented with 50 ng/mL VEGF165 (HumanZyme), 20 ng/mL FGF2 (JPC) and 10 nM SB431542 (Selleck). hCardiomyocytes (hCMs) were cultured in RPMI1640 medium (Gibco) supplemented with S12 (plus insulin). The S12 medium was an albumin-free and chemically defined supplement for cardiac differentiation including fatty acid, protein, chemicals, and antioxidants (Jiang et al., 2012). hNSCs were grown in neural stem cell maintenance medium (NSMM) containing 50% Advanced DMEM/F12 (Gibco), 50% Neurobasal (Gibco), 2 mM GlutaMAX, 0.1 mM NEAA (Gibco), 1% penicillin/streptomycin (Gibco), 1× N2 (Gibco), 1× B27 (Gibco), 10 ng/mL human leukemia inhibitory factor (hLIF, Millipore), 2 μM SB431542 (Selleck) and 3 μM CHIR99021 (Selleck). hNeurons were maintained in Advanced DMEM/F12 medium supplemented with 1× N2, 1× B27, 200 μM Ascorbic acid (Sigma), 400 μM dbcAMP (Sigma), 10 ng/mL GDNF (Peprotech) and 10 ng/mL of BDNF (Peprotech), 1% penicillin/streptomycin (Gibco).

transferred to 39 ºC and cultured for additional 48 h, while the other set was maintained at 37 ºC.
Generation of hVSMCs from hESCs hVSMC induction was performed according to a previous study (Ling et al., 2019). Briefly, H9-hESCs were split into single cells with TrypLE (Thermo Fisher Scientific) and about 3 × 10 5 cells for each well were cultured onto Matrigel-coated 6-well plates in mTeSR medium supplemented with 10 μM Y-27632 (Selleck) at the first day. Cultures were then maintained in N2B27 medium supplemented with 25 ng/mL BMP4 (R&D), 8 μM CHIR99021 (Selleck). After three days, cells were then cultured in N2B27 medium with 10 ng/mL PDGF-AB (Peprotech) and 2 ng/mL Activin A (HumanZyme) for another two days and the medium was changed every day. The cells were then stained with anti-human CD140b-PE (BD biosciences, 558821) antibody and sorted by flow cytometry (BD, Aria II) (Ling et al., 2019;Wang et al., 2018). For all febrile temperature experiments, hVSMCs at passage 2 or 3 were first plated in two sets at 37 ºC for 24 h. Afterward, one set of cells was transferred to 39 ºC and cultured for another 48 h, and the other set was maintained at 37 ºC.

Generation of hCMs from hESCs
Differentiation of hESCs into hCMs was performed as previously described (Jiang et al., 2012;Lee et al., 2017). hESCs were split into small clusters with 0.5 mM EDTA (Sigma, EDS-500G) and plated onto Matrigel-coated 24-well plates in mTESR medium. Differentiation of hESCs into hCMs was carried out when the density of hESCs reached a confluency of 80% to 90%. Culture medium was switched to differentiation medium constituting with RPMI1640 and S12 (without insulin). 3 μM CHIR99021 was added at day 0. After 24 h, the medium was changed back to differentiation medium. 5 μM IWR-1 was added at days 3-5. From day 5 to day 11, the differentiation medium was changed to RPMI1640 supplemented with S12 (plus insulin). To purify the hCMs, the medium was changed to RPMI1640 (without glucose) supplemented with S12 (plus insulin) and lactate every two days for three times starting from day 12. For all febrile temperature experiments, one set of hCMs (about at 21 days) was transferred to 39 ºC and cultured for additional 48 h, and another set was maintained at 37 ºC.

Neuronal differentiation from hNSCs
Neuronal differentiation from hNSCs was as previously described (Zhang et al., 2019). Firstly, hNSCs were plated onto Matrigel-coated 24-well plates at a density of 3×10 4 cells per well, and maintained in NSMM for 1-3 days. The cultures were then maintained in differentiation medium with Advanced DMEM/F12, 1× N2, 1× B27, 200 μM Ascorbic acid (Sigma), 400 μM dbcAMP (Sigma), 10 ng/mL GDNF (Peprotech) and 10 ng/mL of BDNF (Peprotech), 1% penicillin/streptomycin for two days before 20 µg/mL laminin (Sigma) was added to further facilitate differentiation. About 14 days later, differentiated hNeurons were immunostained with an antibody against the neuron marker MAP2. For febrile temperature experiments, one set of hNeurons (about at 21 days) was transferred to 39 ºC and cultured for another 48 h and the other set was maintained at 37 ºC.

Apoptosis analysis
Cell apoptosis assays were performed using Annexin V-EGFP Apoptosis Detection Kit (Vigrous Biotechnology) according to the manufacturer's instructions. Cells were cultured at 37 ºC or 39 ℃ in 5% CO2 for 48 h, respectively. Then the cells were stained with Annexin V-EGFP Apoptosis Detection Kit and analyzed by flow cytometry (BD LSRForteas). The percentage of apoptotic cells were analyzed by FlowJo_V10 software.

Immunofluorescence microscopy
Cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed with PBS, permeabilized with 0.4% Triton X-100 in PBS at room temperature for another 30 min and then blocked with 10% donkey serum (Jackson ImmunoResearch Labs) in PBS at room temperature for 1 h. The cells were then incubated with primary antibodies in blocking solution at 4 °C overnight followed by incubation with corresponding fluorescent secondary antibodies and Hoechst 33342 (Thermo Fisher Scientific) at room temperature for 1 h. Images were captured using a confocal microscope (Leica SP5). The primary antibodies used were as follows (company,

Cell cycle analysis
Cells were collected and fixed in 70% ice-cold ethanol at -20 °C overnight. Next day, the cells were stained with 0.2 mg/mL RNase and 0.02 mg/ml propidium iodide (PI) at 37 ℃ for 30 min. Samples were then analyzed using BD LSRFortesa, and cell-cycle phase distributions were analyzed by ModFit software.

Western blotting
Cells were lysed in lysis buffer (Millipore) with protease inhibitor cocktail (Roche) for 30 min. Then cell lysates were centrifuged at 17,000 × g for 30 min. A following BCA kit was used for protein quantification. 20 μg of cell lysate was loaded onto SDS-PAGE gels for electrophoresis and electrotransferred to PVDF membranes (Millipore). After blocking with 5% (w/v) nonfat powdered milk (BBI Life Sciences), the membrane was incubated with primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies followed at room temperature for 1 h. Finally, imaging was performed with the ChemiDoc XRS system (Bio-Rad). The primary antibodies used for western blotting in this study were anti-HSP90AA1 (Abcam, ab79849), anti-GAPDH (Sigma, G8795), and anti-CDC20 (Santa Cruz, 13162).

RNA-seq library construction and sequencing
Briefly, total RNAs were extracted using TRIzol Reagent, and genomic DNA was removed using a DNA-free Kit. Library preparation was conducted using a NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina (New England Biolabs). Quality control and sequencing on Illumina HiSeq X Ten platforms were performed by Novogene Bioinformatics Technology Co., Ltd.

RNA-seq data processing
RNA-seq data processing was performed as previously described (Bi et al., 2020). We first inspected RNA-seq read quality using FASTQC (v0.11.6), low-quality reads and adaptors were then trimmed using TrimGalore (v0.4.4_dev). The remaining clean reads were mapped to the UCSC human hg19 genome using HISAT2 software (v2.1.0). Reads on each annotated gene were counted using HTSeq (v0.6.1). Differentially expressed genes (DEGs) were calculated using DESeq2 with a cutoff adjust P-value less than 0.05 and |Log2 (fold change)| more than 0.5. Gene Ontology (GO) term enrichment analysis was performed using Metascape with P-value less than 0.05 (http://metascape.org/gp/index.html). The principal component analysis (PCA) and Euclidian distance were performed using R based on Log2 (FPKM + 1). Gene lists of "cell cycle", "DNA repair" and "positive regulation of cell death" were obtained from Gene Ontology Resource. Core regulatory transcription factors were predicted based on the DEGs under febrile temperature (39 ℃) in seven cell types. Transcription factorbinding motifs were identified by using R/Bioconductor package RcisTarget (v1.10.0). The DEGs and GO terms are listed in Supplementary Table S1 and S2.

Statistical analysis
Data are presented as the mean ± SEM. GraphPad Prism software was used to perform a two-tailed Student's t-test. Statistical significance is presented as * P < 0.05, ** P < 0.01 and *** P < 0.001.

Data availability
The sequencing data have been deposited in the Genome Sequence Archive (GSA) in the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation) of the Chinese Academy of Sciences, under accession number HRA000922 that are publicly accessible at http://bigd.big.ac.cn/gsa-human. The data can also be accessed via an interactive user-friendly webtool at Aging Atlas (https://bigd.big.ac.cn/aging/index) (Aging Atlas, 2021).    Table S1. Hyperthermia-associated differentially expressed genes (DEGs) and the corresponding Gene Ontology (GO) terms across seven cell types. Table S2. DEGs upon knockdown or activation of CDC20 in hMSCs and the shared GO terms between different conditions. Figure S1