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| Status |
Public on Jun 14, 2022 |
| Title |
organoid_d12_rat_transplant-2wk_scRNAseq_1 |
| Sample type |
SRA |
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| Source name |
IPSC
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| Organisms |
Homo sapiens; Rattus norvegicus |
| Characteristics |
type of organoid: in vivo organoid
day of maturation: day 12 - 2wk transplant
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| Treatment protocol |
In vitro organoids from growth protocol were grown in parallel and compared to in vivo transplanted organoids. Organoid transplantation protocol: Prior to initiating transplantation into rats for preclinical studies with stem cell derived organoids, persuasive evidence of promise in in vitro experimental models (Figure 2), were performed in accordance with International Society for Stem Cell Research (ISSCR) guidelines. Organoids differentiated as described above were transplanted into 3-4 weeks old athymic male nude rats (Crl:NIH-Foxn1rnu, Charles River Labs, Wilmington, MA) weighing approximately 100g each, acclimated for at least three days in conventional cage setting with rodent diet 5053 and ad libitum water, at_rat_Research test facility (Worcester, MA) in accordance with an approved IACUC study protocol. 45 rats divided randomly and equally into three experimental groups (Day 10, 12 and 14 differentiated) were anesthetized with ketamine/dexdomitor and an antibiotic (Cephazolin) as well as an analgesic (Buprenorphine-SR) was administered to facilitate post-surgical recovery. The kidneys were exposed by an incision in the abdominal wall. After creating an incision in the kidney capsule, a small niche was created under the kidney capsule to host the organoids. Organoids grown for 10, 12 or 14 days in vitro were aspirated into the tip of an 18-24G catheter (Instech, Plymouth, PA) and placed into the niche, the abdominal wall and skin were closed, and the animals were allowed to recover and followed up with routine cage side observations for general health. At 2 or 4 weeks after transplantation, 6-8 rats per experimental group were euthanized under CO2 as per IACUC approved study protocol, kidneys exposed by an incision in the abdominal wall and organoids (n≥3) were harvested for scRNA-Seq and NanoString mRNA analysis as well as analysis of graft tissue vascularization and functional connectivity in the PK/PD studies. Organoid differentiation was also evaluated at the protein level by flow cytometry analysis of two podocyte surface proteins, nephrin30 and podocalyxin31. Cells from dissociated organoids were washed with DPBS, blocked in 1% mouse serum for 30 min at 4°C, incubated with anti-nephrin primary antibody (R&D Systems, AF4269) and APC-conjugated anti-podocalyxin antibodies (R&D Systems, FAB1658A) diluted in sorting buffer (HBSS containing 1% BSA and 0.035% NaHCO3). The cells were further incubated for 10 min in Alexa Fluor 488-conjugated donkey anti-sheep LgG (H+L) antibody (ThermoFisher Scientific, A-11015), washed and resuspended in sorting buffer. Unstained cells were utilized to establish background fluorescence. Data acquisition and analysis were performed on a SONY SH800 flow cytometer. Mean fluorescence in live cells was determined for each sample and is presented after subtraction of background fluorescence.
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| Growth protocol |
High-throughput in vitro kidney organoid generation: For the development of a reproducible scalable protocol, we introduced several key modifications to published protocols5, 7, 13, 14. We started by dispersing iPSCs reaching 80 to 85% confluency into single cell suspensions using Accutase and seeding 275,000 cells per flask in hESC-qualified, matrigel-coated flasks with TeSR1 media plus Y-27632 (Tocris 1254, Bristol, United Kingdom). On the next day, the medium was changed to STEMdiff APEL2 (StemCell Technologies) supplemented with 10 M CHIR99021 (Tocris 4423). After 48 h, the medium was changed to STEMdiff APEL2 supplemented with 8 M CHIR99021. Subsequently, medium was switched to STEMdiff APEL2 with 200 ng/mL FGF9 (R&D Systems, Minneapolis, MN) and 1 g/mL heparin (StemCell Technologies) and changed every other day. On day 7, cells were passaged using Accutase and pelleted by centrifugation at 500,000 cells per pellet. The pellets were placed on transwell plates and STEMdiff APEL2 supplemented with 5 M CHIR99021 was added for 1 h before changing to STEMdiff APEL2 with 200 ng/mL FGF9 and 1 g/mL heparin. Medium was changed every other day for the next 4 days using STEMdiff APEL2 containing 200 ng/mL FGF9 and 1 g/mL heparin. On day 13, the medium was switched to STEMdiff APEL2 with 1 g/mL heparin. Thereafter, organoids were grown in STEMdiff APEL2 and medium was changed every other day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Organoids were rinsed with DPBS, collected into 1.5 ml conical tube containing 500 ml TrypLE Select and incubated for 5 min at 37°C, passed 3 to 4 times through a 27G needle, and incubated for 5 min at 37°C after adding additional 500 ml of TrypLE Select. Organoids were dissociated into single cell suspensions by passing 3 to 4 times through a 30G needle, followed by centrifugation at 600xg for 5 min. Supernatants were discarded, and cell pellets were resuspended in DPBS and filtered through a 40 mm cell strainer. Cell numbers were quantified on a Cellometer cell counter and adjusted to 1000 cells/ml in DPBS.
Single cell cDNA library preparation was performed following manufacturer's protocols (https://www.10xgenomics.com). Single cell suspension were loaded on a Chromium controller to generate single cell GEMS (Gel Beads-In-Emulsions). The scRNA-Seq library was prepared with chromium single cell 3’ reagent kit v3 (10x Genomics)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Demultiplexing, alignment, and estimation of cell-containing partitions and associated UMIs using Cell Ranger Version 3.0.2
Genome_build: GRCh38
Supplementary_files_format_and_content: For each sample countmatrixes (.mtx) and table files identifying cell barcodes and genes (.tsv)
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| Submission date |
Jan 19, 2021 |
| Last update date |
Jun 14, 2022 |
| Contact name |
Eva Fast |
| E-mail(s) |
eva.fast@pfizer.com, evaisfast@gmail.com
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| Organization name |
Pfizer
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| Street address |
1 Portland st
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL27025 |
| Series (1) |
| GSE165104 |
Transplanted human organoids empower PK/PD assessment of drug candidate for the clinic |
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| Relations |
| BioSample |
SAMN17383583 |
| SRA |
SRX9896117 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5026236_L417_109_barcodes.tsv.gz |
75.5 Kb |
(ftp)(http) |
TSV |
| GSM5026236_L417_109_features.tsv.gz |
311.3 Kb |
(ftp)(http) |
TSV |
| GSM5026236_L417_109_matrix.mtx.gz |
72.9 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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