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Series GSE164092 Query DataSets for GSE164092
Status Public on Feb 04, 2021
Title Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation
Organisms Homo sapiens; Oryctolagus cuniculus
Experiment type Expression profiling by high throughput sequencing
Summary Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here we conduct single-cell transcriptomic profiling of a hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, in which cells rapidly acquired a rostral embryo patterning signature, before converging towards the RPE lineage. At intermediate steps, we identified and examined the potency of a NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.
Overall design hESC lines HS980 and KARO1 were previously derived and cultured under xeno-free and defined conditions (Rodin et al. 2014). Donors gave their informed consent for the derivation and subsequent use of hESC lines (Swedish Ethical Review Authority: 2011/745:31/3). The hESC line E1C3 (NN GMP0050E1C3) was provided as a research cell bank of the clinical GMP cell line by NovoNordisk (UCSF IRB:1518222, for RPE differentiation Projekt-ID: H-18016740, Anmeldelsesnr.: 73105). All hESCs maintain a normal karyotype and trilineage differentiation potential, and they are routinely tested for mycoplasma. Cells were maintained by clonal propagation on hrLN-521-coated plates and on iMatrix-511 for the E1C3 line (0.25 μg/cm2, Nippi, T303) in NutriStem hPSC XF medium (Biological Industries) and a 5% CO2/5% O2 incubator.

All cells were transported at 4°C to the Eukaryotic Single Cell Genomics Facility (ESCG, SciLifeLab, Stockholm, Sweden) where a 3’ cDNA library was prepared for single cell RNA sequencing (scRNA-seq) using the 10X Genomics platform and NovaSeq 6000 software. Cell Ranger 2.1.1 was used to convert Illumina base call files to FASTQ format. Cell Ranger 3.1.0 was used to map FASTQ sequencing reads to the human GRCh38 reference transcriptome with the STAR aligner and to generate feature-barcode count matrices.

A step-by-step protocol describing the differentiation procedure has been reported previously (doi 10.1038/s41467-020-15326-5 and doi 10.21203/rs.3.pex-635/v1). hESCs were plated at a cell density of 2.4x104 cells/cm2 on hrLN-521 (20 μg/mL) using NutriStem hPSC XF medium. A Rho-kinase inhibitor (Millipore, Y-27632) at a concentration of 10 μM was added during the first 24h, while cells were kept at 37°C, 5% CO2/5% O2. After 24h, hPSC medium was replaced with differentiation medium NutriStem hPSC XF without bFGF and TGFβ and cells were placed at 37°C, 5% CO2/21%O2. From day 6 after plating, 100 ng/mL of Activin A was added to the media. Cells were fed three times a week and kept for 30 days. Monolayers were then trypsinized using TrypLE Select for 10 min at 37°C, 5% CO2. The enzyme was carefully removed and the cells were collected in fresh pre-warmed NutriStem hPSC XF medium without bFGF and TGFβ by gentle pipetting to obtain a single cell suspension. Cells were centrifuged at 300 g for 4 min, the pellet was resuspended, passed through a cell strainer and seeded on laminin coated dishes at different cell densities ranging from 1.4x106 to 1.4x104 cells/cm2. Replated cells were fed three times a week during the subsequent 30 days with NutriStem hPSC XF medium without bFGF and TGFβ. Single cell RNA-sequencing was performed on undifferentiated hESCs and at days 7, 14, 30, 38, 45, and 60 of this protocol.

hESC-RPE monolayers were washed with DPBS, incubated with TrypLE and dissociated to single cell suspension as described above. Cells were counted in a Neubauer hemocytometer chamber using 0.4% trypan blue, centrifuged at 300g for 4 min, and the cell pellet was resuspended in freshly filter-sterilized DPBS to a final concentration of 1000 cells/μL. The cell suspension was then aseptically aliquoted into 600 μL units and kept on ice until surgery.

After approval by the Northern Stockholm Animal Experimental Ethics Committee (DNR N56/15), two female New Zealand white albino rabbits aged 5 months and weighing 3.5 to 4.0 kg were used in this study. All experiments were conducted in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research.

Animals were put under general anesthesia by intramuscular administration of 35 mg/kg ketamine and 5 mg/kg xylazine, and the pupils were dilated with a mix of 0.75% cyclopentolate / 2.5% phenylephrine. Microsurgeries were performed on both eyes using a 2-port 25G transvitreal pars plana technique. 25G trocars were inserted 1 mm from the limbus and an infusion cannula was connected to the lower temporal trocar. The cell suspension was drawn into a 1 mL syringe connected to an extension tube and a 38G polytip cannula. Without infusion or prior vitrectomy the cannula was inserted through the upper temporal trocar. After proper tip positioning, ascertained by a focal whitening of the retina, 50 μL of hESC-RPE cell suspension (equivalent to 50,000 cells) was injected slowly subretinally, approximately 6 mm below the inferior margin of the optic nerve head, forming a uniform bleb that was clearly visible under the operating microscope

Human post-mortem research-consented donor eyes were obtained from the cornea bank at St. Erik Eye Hospital, Stockholm, Sweden. The use of human tissue was in accordance with the tenets of the Declaration of Helsinki and was approved by the Swedish legislative and ethical committee (#2019-02032) for the use of human donor material for research. Donors did not present any clinical diagnosis of ocular disease, and samples were anonymized and processed under the general data protection regulation. Two human eyes from the same donor were used (45-year-old male, 32 hours post-mortem). The lens was dissected out and the rest of the retina, except the sclera, was chopped in several small pieces mixed together in 500 uL of digestion buffer (described below). Two embryonic eyes from the same embryo were used from a 7.5 post-conception week embryo (Table S1). The optic cups were dissected out and chopped in several small pieces to facilitate dissociation in 500 uL of digestion buffer (described below). Donors (deceased, family for adult human eyes, or couples for the embryos) gave their informed consent for the donation and subsequent use for research purposes. The embryonic eyes were acquired from a clinical routine abortion after informed consent by the pregnant woman, in accordance with permissions from the regional ethical review board and the Swedish National Board of Health and Welfare (“Socialstyrelsen” #8.1-11692/2019) and the Swedish Ethical Review Authority (“EPN” #2007/1477-31/3).

Two rabbit eyes (from different animals) with 30-day integrated hESC-RPE were enucleated and pigmented areas including neuroretina, choroid and RPE layer was dissected out, trimmed and mixed together in 500 uL of digestion buffer. Eppendorfs containing the samples were rotated and incubated at 37°C on a thermocycler at 300g for 25 min until samples were homogenized. Samples were pipetted every 5 min to digest the tissue sample into single cells. Digestion was stopped by adding equivalent volume of 10% embryonic bovine serum in PBS, the samples were filtered using a 30µm MACS Smart Strainer followed by Dead Cell Removal kit to remove dead cells and debris. At this stage, one of the rabbit eye cell samples was stained with mouse anti-human HLA-ABC-FITC, and anti-human HLA-ABC-positive cells were FACS-sorted as specified above, collected and resuspended to 1000 cells/uL in 1% BSA in PBS for further scRNA-sequencing. Otherwise, cells were finally resuspended to 1000 cells/uL in 1% BSA in PBS prior to scRNA-seq.
Contributor(s) Petrus-Reurer S, Lederer AR, Baqué-Vidal L, Douagie I, Pannagel B, Khven I, Aronsson M, Bartuma H, Wagner M, Wrona A, Efstathopoulos P, Jaberi E, Willenbrock H, Shimizu Y, Villaescusa JC, André H, Sundström E, Bhaduri A, Kriegstein A, Kvanta A, La Manno G, Lanner F
Citation missing Has this study been published? Please login to update or notify GEO.
Submission date Dec 31, 2020
Last update date Dec 26, 2021
Contact name Gioele La Manno
Organization name EPFL
Department BMI
Street address Station 19
City Lausanne
State/province Vaud
ZIP/Postal code 1015
Country Switzerland
Platforms (2)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
GPL29549 Illumina NovaSeq 6000 (Homo sapiens; Oryctolagus cuniculus)
Samples (24)
GSM4996489 In vitro hESC-RPE differentiation day 7
GSM4996490 In vitro hESC-RPE differentiation day 14
GSM4996491 In vitro hESC-RPE differentiation day 30
BioProject PRJNA688888
SRA SRP299892

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Supplementary file Size Download File type/resource
GSE164092_E1C3_D60_Rep2.h5ad.gz 34.8 Mb (ftp)(http) H5AD
GSE164092_E1C3_D60_Rep3.h5ad.gz 55.5 Mb (ftp)(http) H5AD
GSE164092_E1C3_D60_Rep4.h5ad.gz 64.9 Mb (ftp)(http) H5AD
GSE164092_RAW.tar 1.0 Gb (http)(custom) TAR (of LOOM, TAR)
GSE164092_all_processed_data_annotated.tar.gz 2.2 Gb (ftp)(http) TAR
GSE164092_pyscenic_metadata.tar.gz 95.6 Mb (ftp)(http) TAR
GSE164092_refdata-custom-cellranger-GRCh38-OryCun.tar.gz 1.6 Gb (ftp)(http) TAR
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Raw data are available in SRA
Processed data provided as supplementary file

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