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Mesenchymal properties of iPSC-derived neural progenitors that generate undesired grafts after transplantation – Communications Biology

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Ethics

This study was conducted following the principles of the Helsinki Declaration. The use of human iPSCs was approved by the ethics committee at Keio University School of Medicine (Approval numbers: 20130146 and 20030092).

Animals

All animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals of the Central Institute for Experimental Animals (CIEA; Kanagawa, Japan). The experimental protocols were approved by the CIEA Animal Care Committee (Permit number: 11029A) and Keio University School of Medicine (Tokyo, Japan) (Permit number: 16-096-25).

Cell culture

The integration-free hiPSC lines (1210B2 and 1231A3) established from peripheral blood mononuclear cells under xeno-free and feeder-free conditions were kindly provided by the Center for iPS Cell Research and Application (CiRA: Kyoto, Japan)19,45. The hiPSC line 201B7 established from dermal fibroblasts46 was kindly provided by Dr. Shinya Yamanaka. The hiPSC line WD39 was established from dermal fibroblasts35. The feeder-free hiPSCs were maintained using human recombinant laminin fragment iMatrix-511 (Nippi) and xeno-free medium StemFit®AK03 (Ajinomoto)19,45. For hiPSC-NS/PC-induction, feeder-free hiPSCs were dissociated into single cells using TrypLE Select (Thermo Fisher Scientific) and re-aggregated to form embryoid bodies (EBs) using 96-well low cell-adhesion plates (Sumilon PrimeSurface plate M; Sumitomo Bakelite) at a density of 5000 cells/well (100 μl) in StemFit®AS200 (Ajinomoto) without FGF2 supplemented with 100 nM LDN-193189 (Stemgent) and 500 nM A83-01 (Stemgent). The medium was changed every day. On day 5, EBs were attached to tissue culture plates precoated with 0.1 μg/cm2 iMatrix-511 in StemFit®AS200. During the subsequent 7-day culture, neural rosette structures were developed. On day 12, they were manually selected with needles under a microscope and maintained in suspension culture for 3 days in StemFit®AS200. On day 15, floating neural rosette-derived spheres were plated on tissue culture plates in the same medium. On day 18, the attached neural rosettes were dissociated into single cells with TrypLE Select and plated on a PO/laminin-coated plate in StemFit®AS200. The medium was changed every other day. Cells were passaged every 3–4 days16,20. 201B7-Neurospheres were kindly provided by Dr. Yohei Okada4. 1210B2-EB-NS/PCs were kindly provided by Dr. Yonehiro Kanemura9. AF22 and AF23 were kindly provided by Dr. Austin Smith20. For neuronal and glial differentiation of hiPSC-NS/PCs, cells were plated on PO/laminin-coated 8-well chamber glass slides at a density of 2.5 × 104 cells/cm2 in Neurobasal medium (Gibco; Thermo Fisher Scientific) containing 2% B27 and 1% GlutaMAX (Gibco; Thermo Fisher Scientific)16. Osteogenic differentiation and adipogenic differentiation were performed according to the manufacturer’s instructions (Human Mesenchymal Stem Cell (hMSC) differentiation Bullet Kit, Lonza). For osteogenic differentiation, a total of 7.0 × 103 cells was transferred to a 24-well plate and cultured overnight in a culture medium. Adherent cells were cultured in an osteogenic differentiation medium (Lonza) that was changed every 3 days. After 15 days, the differentiation of these cells into osteoblasts was assessed by alizarin red S staining (Millipore)47. For adipogenic differentiation, 7.0 × 104 cells were transferred to a 24-well plate and cultured until the cells reached confluency. At 100% confluence, three cycles of induction/maintenance were performed. Each cycle consisted of feeding cells with Adipogenesis Induction Medium (Lonza) and culturing for 3 days, followed by 2 days of culture in an Adipogenic Maintenance Medium (Lonza). After 3 complete cycles, the cells were further incubated with Adipogenic Maintenance Medium for 7 days, and the medium was replaced twice a week. After 22 days, oil red O staining (Muto Pure Chemicals) was performed to examine whether cells differentiated into adipocytes.

For hiPSC-NCC-induction from hiPSCs (1210B2), feeder-free hiPSCs were incubated with 2 mg/ml collagenase IV (Thermo Fisher Scientific). Detached colonies of iPSCs were broken into pieces by mild pipetting, and the clusters consisting of ~200 cells were plated onto a 100 mm petri dish (Beckton Dickinson) in the medium for the NCC-induction30. The medium for NCC induction consisted of 1:1 neurobasal medium (Thermo Fisher Scientific) and DMEM/F-12 medium containing 1x GlutaMax (Thermo Fisher Scientific), 5 mg/ml insulin (Sigma-Aldrich), 0.5% penicillin and streptomycin, 0.5x GEM 21 NeuroPlex serum-free supplement (Gemini Bio Products, West Sacramento, CA), 0.5x N2 supplement and supplemented with 20 μg/ml human recombinant EGF (Peprotech) and 20 μg/ml FGF2. The medium was changed every other day. 7-days after the plating, migratory NCCs were observed from the attached cell clusters.

For preparation of MSCs and WBM48, human bone marrow mononuclear cells (BM-MNCs) (Lonza, 2M-125C) were stained for 30 min on ice with a monoclonal antibody [LNGFR-PE (Miltenyi Biotec; 1:40) and THY-1-APC (BD Pharmingen; 1:200)]. Propidium iodide (PI) (Sigma) was used to exclude dead cells. Flow cytometric analysis of controls determined the setting for gating and sorting of PI(-)LNGFR(+)THY-1(+) BM-MNCs (MSCs). The PI-negative cells were sorted as Whole Bone Marrow cells (WBM). The flow cytometric analysis and sorting procedures were carried out using a FACSAria cell sorter (BD Biosciences). Sorted cells were washed by MSC medium [DMEM (Nacalai Tesque) containing 20%FBS and FGF2 (5 ng/ml)] and seeded onto culture plates. Prior to reaching confluency, the cells were passaged using 0.05% Trypsin-EDTA (Gibco). For the experiments, the cells that had undergone 3 passages were utilized.

Single cell cloning

hiPSC-NS/PCs (NS/PC-B) were dissociated using TrypLE Select (Thermo Fisher Scientific). The dissociated cells were suspended in StemFit AS200 (Ajinomoto Co., Inc.). Single cell sorting was performed using an SH800 flow cytometer (Sony). The sorted cells were cultured in StemFit AS200 in a Matrigel (Corning)-coated 96-well plate (Greiner Bio-One). Each clone derived from the single sorted cell was passaged at confluency. The clones were expanded further for analyses.

Immunocytochemistry

Cells were fixed in 4% PFA/PBS for 15–20 min at room temperature and washed three times in PBS. The cells were then permeabilized and blocked with PBS containing 5% fetal bovine serum and 0.3% Triton X-100 for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies: anti-SOX1 (R&D Systems, AF3369; 1:500), anti-SOX2 (R&D Systems, MAB2018; 1:500), anti-human Nestin (Immuno-Biological Laboratories Co., 18741; 1:500 and Millipore, MAB5326; 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095; 1:200), anti-βIII-tubulin (Sigma-Aldrich, T8660; 1:500), anti-AP2α (Santa Cruz Biotechnology, sc-12726; 1:50), anti-NeuN (abcam, ab177487; 1:500), anti-MAP2ab (Sigma-Aldrich, M1406; 1:500), anti-GalC (Millipore, MAB342; 1:500), anti-GFAP (Thermo Scientific, 13-0300; 1:500) and anti-Ki67 (abcam, ab15580; 1:1000). After 3 washes with PBS, the cells were incubated for 1 h at room temperature with Alexa Fluor 488-, 555-, and 647-conjugated secondary antibodies (Thermo Fisher Scientific; 1:1000). Cell nuclei were counterstained with 1 μg/ml Hoechst 33258 (Sigma-Aldrich). Images were acquired under an Apotome fluorescence microscope (Carl Zeiss), Axio Imager Z2 (Carl Zeiss), or IN Cell Analyzer 6000 (Cytiva). For the quantitative analysis of SOX2+ and NESTIN+ cells in Fig. 1c, 9 fields were randomly selected, and >150 cells/field were analyzed. The number of positive cells was quantified and normalized to total nucleus counts by the Multi-Target Analysis module of IN Cell Analyzer Workstation (Cytiva).

Transplantation of hiPSC-NS/PCs into immunodeficient mice

Intrastriatal transplantation was performed by injecting hiPSC-NS/PCs bilaterally into the striata of 9-week-old female NOG mice (Clea Japan) (1.0 × 106 cells per site). Intraspinal transplantation was performed by injecting hiPSC-NS/PCs into the epicenter of the injured spinal cord 9 days after the moderate contusion injury (IH impactor, 60-70kdyn) of 9-week-old NOD/SCID mice (Charles River Laboratories Japan) (5.0 × 105 cells per site)9. Dissected brains were dehydrated in 100% ethanol, cleared in xylene, and embedded in paraffin. Then, 5-μm-thick serial coronal sections of the brain and sagittal sections of the spinal cord were prepared and processed for hematoxylin-eosin (H&E) staining and immunohistochemistry.

Immunohistochemistry

3,3-Diaminobenzidine staining was performed using Bond-Max automated staining system (Leica) according to the manufacturer’s instructions. Paraffin-embedded brain sections were deparaffinized and rehydrated, followed by antigen retrieval by heating for 10 min at 100 °C in BOND Epitope Retrieval Solution1 (Leica). Sections were incubated at room temperature for 30 min with primary antibodies; anti-human cytoplasm (STEM121, Takara Bio, Y40410; 1:1000), anti-Ki67 (Dako, M7240; 1:50 and Leica Biosystems, KI67; 1:100), anti-Lamin A + C (abcam, ab108595; 1:400), anti-human NESTIN (IBL, 18741; 1:100), anti-human GFAP (STEM123, Takara Bio, Y40420; 1:1000), anti-NeuN (abcam, ab177487; 1:250), anti-cleaved caspase 3 (Cell Signaling Technology, 9661; 1:250), anti-human synaptophysin (R&D Systems, AF5555; 1:250), anti-RUNX2 (abcam, ab192256; 1:500).

For fluorescent immunohistochemistry, paraffin-embedded brain sections were deparaffinized and rehydrated, followed by antigen retrieval by heating for 10 min at 121 °C in Target Retrieval Solution (Dako, S1699). Sections were then blocked with Blocking One (Nacalai Tesque) at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies: anti- nELAVL (Thermo Fisher Scientific, A21271; 1:50), anti-human nuclear antigen (HNA; Millipore, MAB4383; 1:100), anti-human cytoplasm (STEM121, Takara Bio, Y40410; 1:100), anti-SOX1 (R&D Systems, AF3369; 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095; 1:100), anti-AP2α (Santa Cruz Biotechnology, sc-12726; 1:50), anti-human Vimentin NL493-conjugated rat IgG2a, and anti-human Snail NL557-conjugated goat IgG (Human EMT 3-Color Immunocytochemistry Kit, R&D Systems, SC026). Alexa Fluor 488-, 555-, and 647-conjugated secondary antibodies were used at 1:1000. Cell nuclei were counterstained with 1 μg/ml Hoechst 33258. Images were acquired under the Apotome fluorescence microscope, Axio Imager Z2, confocal laser scanning microscope (LSM700; Carl Zeiss), or confocal image cytometer (CQ1; Yokogawa Electric Corp.). Fluorescence images were captured using a 20× primary objective. The number of positive cells, such as HNA+, SOX1+, SOX9+, AP2α+, and nELAVL+ cells, was counted in each section.

Bone staining

Von Kossa staining was performed based on Calcium Stain kit (ScyTek Laboratories, Inc., CVK-1). Tissue sections were incubate in 5% Silver Nitrate Solution for 60 min under the ultraviolet light. After the incubation, the sections were washed by distilled water three times. Then, slides are immersed in a 5% sodium thiosulfate solution for 2 min, washed under running water for 2 min, and washed twice in distilled water. The sections were further stained with a nuclear dye solution for 5 min. Finally, the sections dehydrated by Ethanol and mounted for observation. For alizarin red S staining, the staining solution was prepared from diluting alizarin red S (Nacalai Tesque) by distilled water to a final concentration of 1%. pH of the solution was adjusted to 6.3–6.4 with aqueous ammonia. Tissue sections were incubated in the alizarin red S staining solution for 10 min at room temperature.

Cell surface marker screening using Lyoplate

Cells were analyzed using the BD Lyoplate™ Human Cell Surface Marker Screening Panel BD Biosciences; 560747), following the manufacturer’s protocol with slight modifications. A single cell suspension was prepared at 0.5–1.0 × 106 cells/100 μl and incubated with 0.5 μg/20 μL of primary antibodies for 30 min at 4 °C. After 2 washes with FACS Buffer containing BD Pharmingen Stain Buffer (BD Biosciences, 554656), 5 mM EDTA, and 10 μM Y-27632 (Wako), the cells were incubated with an Alexa Fluor 647-conjugated secondary antibody at 4 °C for 30 min in the dark. After washing with FACS buffer, the cells were fixed with BD CellFIX (BD Biosciences, 341081). Flow cytometric analysis was performed on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo, version 7.6 (TreeStar).

Flow cytometry

Cells were suspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA (pH 8.0) at 3.0 × 105 cells/50 μl and stained for 30 min on ice in the dark with fluorescent dye-conjugated antibodies: PSA-NCAM (Millipore, MAB5324; 1:50), PSA-NCAM-APC (Miltenyi Biotec, 130-093-273; 1:50), CD133-APC (Miltenyi Biotec, 130-090-826; 1:50), CD15-Brilliant Violet 421 (BioLegend, 323039; 1:50), CD49α-FITC (BioLegend, 328308; 1:50), CD73-PE-Cy7, (BioLegend, 127224; 1:50), and CD105-APC (BioLegend, 323208; 1:50). In addition, 7-AAD (BD Biosciences, 559925; 1:1000) was applied for live/dead discrimination. An isotype control was used to subtract background fluorescence. Flow cytometric analysis was performed on a FACSVerse flow cytometer (BD Biosciences). Cell sorting was performed on the FACSAria cell sorter (BD Biosciences). Data were analyzed using FlowJo, version 7.6. The gating strategies are shown in Supplementary Fig. 13.

Purification of CD15 + CD73− CD105− NS/PCs from NS/PC-B for in vivo evaluation

The hiPSC-NS/PCs (NS/PC-B) were suspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA (pH 8.0) at 3.0 × 105 cells/50 μl and with fluorescent dye-conjugated antibodies for 30 min on ice in the dark: CD15-Brilliant Violet 421 (BioLegend, 323039), CD73-PE-Cy7, (BioLegend, 127224), and CD105-APC (BioLegend, 323208). 7-AAD (BD Biosciences, 559925) was also used for live/dead discrimination. An isotype control was used to subtract background fluorescence. Cell sorting was performed on FACSAria cell sorter (BD Biosciences), and the sorted cells were plated and cultured in AS200 in a Matrigel (Corning)-coated six-well plate (Greiner Bio-One). Intrastriatal transplantation into 9-week-old NOG mice (Clea Japan, In-Vivo Science Inc.) was performed as previously described9. All mice were anesthetized and euthanized by transcardial perfusion of 0.1 M PBS containing 4% PFA at 7–10 weeks after the transplantation. The dissected brains were further fixed in 4%PFA for 24 h and processed for immunohistochemical analysis.

Microarray analysis

Total RNA was extracted with an RNAeasy Kit (Qiagen). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA (200 ng) was reverse transcribed, labeled with biotin using a Target Amp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre, Illumina), and hybridized to a HumanHT-12_v4_BeadChip (Illumina) in accordance with the manufacturer’s instructions. The array was washed and stained using an Illumina gene expression kit. Raw intensity values were acquired using an iScan microarray scanner (Illumina). Raw probe intensity files were exported using Illumina GenomeStudio gene expression software (v1.9.0) and loaded into R for background correction, quantile normalization, and log (base 2) conversion with the limma package. Finally, the gene set was filtered by expression levels to remove genes that were not expressed in all samples.

Genes differentially expressed 1.2-fold between NS/PC-like scNS/PCs and NCC-like scNS/PCs were extracted and applied to GO analysis using DAVID Bioinformatics Resources (http://david.ncifcrf.gov). Box plot evaluation of gene expression was performed using BoxPlotR (http://shiny.chemgrid.org)49.

RNA-seq

Samples for RNA-seq were prepared using a TruSeq RNA Sample Prep Kit (Illumina) in accordance with the manufacturer’s protocol. The sequencing library was sequenced on a HiSeq 2500 (Illumina). Base calling and chastity filtering were performed using Real-Time Analysis Software version 1.18.61. Raw reads were mapped to the reference genome hg19 using sailfish (v0.7.6) with default settings. Count matrix data from sailfish were loaded into R software (v4.1.1), and downstream analysis was performed.

Single cell RNA-seq

hiPSC-NS/PCs were sorted into a 96-well plate by the SH800 flow cytometer and dissolved with cell lysis buffer (0.5% NP40). These solutions were mixed using a bench-top mixer at 2,500 rpm and 4 °C for 15 s and then at 3000 × g and 4 °C for 10 s. Immediately after the second centrifugation, 0.8 μl of priming buffer (1.5× PCR buffer with MgCl2; TaKaRa Bio), 41.67 pmol/l of the RT primer (TATAGAATTCGCGGCCGCTCGCGATAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTTTTT), 4 U/μl of RNase inhibitor (Promega Corp), and 50 μmol/l dNTPs were added to each well, and. the solutions were mixed at 2500 rpm and 4 °C for 15 s. The denaturation and priming were conducted using a thermal cycler (C1000 and S1000; BioRad Laboratories) at 70 °C for 90 s and 35 °C for 15 s. The 96-well plate was then placed into an aluminum PCR rack at 0 °C. Afterward, 0.8 μl of RT buffer (1× PCR buffer, 25 U/μl reverse transcriptase (SuperScript III; Life Technologies), and 12.5 mmol/l DTT) was added to each well, and the reverse transcription was performed at 35 °C for 5 min and 45 °C for 20 min. The reactions were heat-inactivated at 70 °C for 10 min, and the 96-well plate was again placed into an aluminum PCR rack at 0 °C. After centrifugation at 3000 × g and 4 °C for 10 s, 1 μl of the exonuclease solution (1× Exonuclease buffer and 1.5 U/μl exonuclease I; both TaKaRa Bio) was added to each well. The primer digestion was performed at 37 °C for 30 min, and the reactions were heat-inactivated at 80 °C for 10 min. The reaction plate was placed into an aluminum PCR rack at 0 °C. After centrifugation at 3000 × g and 4 °C for 30 s, 2.5 μl of poly-A-tailing buffer (1× PCR buffer, 3 mmol/l dATP, 33.6 U/μl terminal transferase (Roche Applied Science), and 0.048 U/μl RNase H (Invitrogen) was added to each tube in the aluminum PCR rack at 0 °C. The reaction plate was mixed at 2500 rpm and 4 °C for 15 seconds. Immediately after centrifugation at 3000  × g and 0 °C for 10 s, the reaction plate was placed into a thermal cycler block, which was pre-chilled to 0 °C. Subsequently, the poly-A-tailing reaction was performed at 37 °C for 50 s and heat-inactivated at 65 °C for 10 min. The reaction plate was then placed into an aluminum PCR rack at 0 °C. After centrifugation at 3000 × g and 4 °C for 30 seconds, the reaction plate was placed into an aluminum PCR rack at 0 °C. We then added 23 μl of the second strand buffer (1.09× MightyAmp Buffer v2 (TaKaRa), 70 pmol/l tagging primer (TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT), and 0.054 U/μl MightyAmp DNA polymerase (TaKaRa)) to each well. The reaction plate was mixed at 2500 rpm and 4 °C for 15 s. After centrifugation at 3000 × g and 4 °C for 10 s, the second-strand synthesis was performed at 98 °C for 130 s, 40 °C for 1 min, and 68 °C for 5 min. Subsequently, the reaction plate was then immediately transferred to an aluminum PCR rack that had also chilled to 0 °C, and 25 μl of PCR buffer (1× MightyAmp Buffer version 2 and 1.9 μmol/l suppression PCR primer (NH2)-GTATAGAATTCGCGGCCGCTCGCGAT) was added. After centrifugation at 3000 × g and 4 °C for 10 s, the PCR enrichment was performed using the following conditions per cycle for a total of 21 PCR cycles: 98 °C for 10 s, 65 °C for 15 s, and 68 °C for 5 min. After the PCR step, the reaction plate was incubated at 68 °C for 5 min. The reaction plate was then placed into an aluminum PCR rack at 25 °C. The amplified cDNA was purified using a PCR purification bead system (Agencourt AMPure XP; Beckman Coulter Inc). Amplified cDNA was processed for library preparation using a Nextera XT Library Prep Kit (Illumina). The DNA sequencing library was analyzed with the massively parallel sequencer HiSeq 2500. Raw reads were trimmed by read quality and read length using Trimmomatic software (v0.33). Trimmed reads were aligned to the reference genome hg19 using sailfish (v0.7.6) with default settings. Samples were filtered by the following parameters and used for analysis: read number > 1 million, aligned rate > 70%, and detected gene number > 5000.

Correlation analysis of differentially expressed genes

The correlation of differentially expressed genes in individual cells with publicly available datasets for representative tissues or cells was evaluated using ExAtlas (https://lgsun.irp.nia.nih.gov/exatlas/)27. The datasets for somatic tissues were preloaded in ExAtlas. Other datasets used for comparison were as follows: iPSC-derived NS/PCs (GSM1553289, GSM1553290, GSM1553291, and GSM2030405, GSM2040306), iPSC-derived NCCs (GSM1470883, GSM1470884, and GSM1470885), iPSC-derived NCMSCs (GSM1470886, GSM1470887, and GSM1470888), and PSA-NCAM+ or PSA-NCAM− embryonic stem cell-derived NS/PCs (GSE67383).

Reanalysis of RNA-seq data

RNA-seq data for human neuroepithelial stem cells from the neocortex and spinal cord were obtained from the Gene Expression Omnibus (GEO) database (GSE107514). SRA raw data were downloaded and converted to fastq data using SRA Toolkit. Raw reads were mapped to the reference genome hg38 using kallisto (v0.46.2) with default settings. Count matrix data from kallisto were loaded into R software (v4.1.1) and downstream analysis was performed.

Statistics and reproducibility

All data are shown as the mean ± SD. Statistical significance was determined by Student’s t-test. There were no statistical tests to determine the sample size. Sample sizes were determined based on previous reports with similar experiments. The sample size in each experiment is described in the part of figure legend. The degree of statistical significance is represented by Asterisks, *P < 0.05; **P < 0.01; ***P < 0.001. In vitro experiments, except for single-cell cloning, were performed at least twice with similar outcomes.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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