Jun 16, 2026
Single-cell RNA sequencing of nutrient-stressed SC-islets
- Xianming Wang1,
- Nan Hu1,
- Berk Turhan1,
- Kushal Dey1,
- Danwei Huangfu1
- 1Sloan Kettering Institute
Protocol Citation: Xianming Wang, Nan Hu, Berk Turhan, Kushal Dey, Danwei Huangfu 2026. Single-cell RNA sequencing of nutrient-stressed SC-islets. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl83m29v2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 16, 2026
Last Modified: June 16, 2026
Protocol Integer ID: 319276
Keywords: scRNA-seq, hiPSCs, 10x Genomics, XM001, SC-islet, Stress treatment, glucose, cell rna, derived islet, stressed stem cell, stem cell
Funders Acknowledgements:
NHGRI
Grant ID: U01 HG012051
Abstract
single-cell RNA sequencing of nutrient-stressed stem cell-derived islets
Cell culture system and maintenance
XM001 hiPSCs were maintained in Essential 8 (E8) medium (Thermo Fisher Scientific, A1517001) on vitronectin (Thermo Fisher Scientific, A14700) pre-coated plates at 37°C with 5% CO2.
For regular maintenance, cells were passaged every 3-4 days using Accutase (STEMCELL Technologies, 07920) for dissociation.
The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (10 μM; Selleck Chemicals, S1049) was added to the E8 medium for the first 24 h after passaging or thawing of hiPSCs.
Cells were regularly tested and confirmed to be mycoplasma free by the Memorial Sloan Kettering Cancer Center (MSKCC) Antibody & Bioresource Core Facility.
Stem cell-derived islet differentiation
hiPSCs were seeded at a density of 0.75 ×105 cells/cm2 on Matrigel (Corning, 354253) pre-coated plates in E8 medium with 10 μM Y-27632.
After 48 hours, cells were washed with DPBS once and differentiated following previously described protocols with some modifications.
During stage 1 to stage 6's day 7, cells were cultured in 6-well plates. On stage 6's day 7, cells were dissociated with Accutase. 5 ×106 cells were resuspended in 5 ml ESFM medium and transferred into a single well of ultra-low attachment 6-well plates (Corning, 3471). The plate was incubated on an orbital shaker at 100 rpm.
Stage 1 (3 d):
S1/2 base medium: 500 ml MCDB 131 (Life Technologies, 10372019) supplemented with 2.07 ml 45% glucose (MilliporeSigma, G7528), 0.75 g sodium bicarbonate (MilliporeSigma, S5761), 2.59 g BSA (Proliant, 68700), 5.1 ml GlutaMAX (Invitrogen, 35050079).
S1/2 medium supplemented with 100 ng/ml Activin A (Bon Opus Biosciences, C687- 1MG) and 3 μM CHIR99021 (Stemgent, 04-0004-10) for day 1.
S1/2 medium supplemented with 100 ng/ml Activin A and 0.3 uM CHIR99021 for day 2.
S1/2 medium supplemented with 100 ng/ml Activin A for day 3.
Stage 2 (2 d):
S1/2 medium supplemented with 50 ng/ml KGF (PeproTech, AF-100-19) and 0.25 mM vitamin C (VitC) (Sigma-Aldrich, A4544).
Stage 3 (2 d):
S3/4 medium supplemented with 50 ng/ml KGF, 0.25 mM VitC, 0.25 uM SANT-1 (Sigma, S4572), 2 μM retinoic acid (MilliporeSigma, R2625), 200 nM LDN (Stemgent, 04-0019) and 500 nM TPPB (TOCRIS, 5343).
S3/4 base medium: 500 ml MCDB 131 supplemented with 0.52 ml 45% glucose, 0.877 g sodium bicarbonate, 10 g BSA, 2.5 ml ITS-X (Life Technologies, 51500056), 5.2 ml GlutaMAX.
Stage 4 (4 d):
S3/4 medium supplemented with 50 ng/ ml KGF, 0.25 mM VitC, 0.25 μM SANT-1, 0.1 μM retinoic acid, 200 nM LDN and 500 nM TPPB.
Stage 5 (7 d):
S5 medium supplemented with 10 µM ALK5i II (Cayman Chemical Company, 14794-5), 0.25 mM VitC, 0.25 μM SANT-1, 0.1 μM retinoic acid, 1 µM T3 (Sigma-Aldrich, T6397), 20 ng/ml Betacellulin (R&D, 261-CE-050/CF), 10 μg/ml Heparin (Sigma-Aldrich, H3149) and 1 µM γ-Secretase Inhibitor XXI (EMD Millipore, 565790).
S5 base medium: 500 ml MCDB 131 supplemented with 4 ml 45% glucose, 0.877 g sodium bicarbonate, 10 g BSA, 2.5 ml ITS-X, 5.2 ml GlutaMAX.
Stage 6 (14 d): ESFM medium.
ESFM medium: 500 ml MCDB 131 supplemented with 0.52 ml 45% glucose, 10.5 g BSA, 5.2 ml GlutaMAX, 5.2 ml NEAA (Invitrogen, 11140050), 1 μM ZnSO4 (MilliporeSigma, 108883), 523 μl Trace Elements A (Corning, 25-021-CI), 523 μl Trace Elements B (Corning, 25-022-CI), 10 ug /ml Heparin.
Nutrient stress treatment of SC-islet cells
A single differentiation of XM001 was performed; at stage 6 day 14 the resulting SC-islet pool was divided into six aliquots, each assigned to one nutrient-stress condition and treated independently for 72 h.
The four primary nutrient stress conditions were (i) 8 mM glucose + BSA vehicle control (Cayman Chemical, 29556), (ii) 8 mM glucose + 1 mM BSA-conjugated palmitate (Cayman Chemical, 29558), (iii) 33 mM glucose + BSA vehicle control, and (iv) 33 mM glucose + 1 mM BSA-conjugated palmitate.
Additionally, we included two secondary stress experiments comprising 8 mM glucose (no BSA) and 33 mM glucose (no BSA). After 72 h of treatment, SC-islets were dissociated into single cells, collected, and cryopreserved in Bambanker cell freezing medium (Fujifilm, 302-14681).
Single-cell RNA library preparation, sequencing and analysis
On the day of scRNA-seq library preparation
Frozen cells were thawed at 37°C, resuspended in PBS containing 2% BSA and then incubated with Live/Dead Fixable Violet Dead cell stain (Invitrogen, L34955) in FACS buffer for 15 minutes at room temperature.
Live cells were sorted by FACS and loaded onto the Chromium Controller with a targeted collection of 10,000 cells per reaction (10x Genomics Chromium Single Cell 3′ Reagent Kit v3.1 User Guide).
cDNA and sequencing libraries were prepared according to the manufacturer’s instructions and sequenced on theNovaSeq 6000 platform.
Data Processing
Transcriptome libraries were processed with Cell Ranger v8.0.0 using the GRCh38-2024-A reference and default parameters, generating gene-by-cell count matrices.
Downstream analyses were performed in Seurat v.5.0.3.
We applied a two-stage quality control strategy designed to balance broad cluster representation with precise cell quantification for downstream differential expression.
In the first stage, we applied relaxed filters (removing only cells with fewer than 200 detected genes or more than 50% mitochondrial transcripts) and performed Louvain clustering at resolution 0.6 on the resulting cells (N = 42,053 cells; 389,403,101 UMIs). Clusters were annotated based on marker gene profiles, identifying 8 transcriptionally distinct cell populations.
In the second stage, we applied stringent QC filters to the annotated dataset, retaining cells with more than 1,000 and fewer than 7,500 detected genes (nFeature_RNA) and less than 25% mitochondrial transcripts, while preserving the original cell-type annotations (N = 38,350 cells; 382,245,713 UMIs retained).
This two-stage ordering reflects the biological principle that cell types differ in their native distributions of gene detection and mitochondrial expression, and is supported by previous research.
Applying stringent filters only after cluster annotation ensures that downstream differential expression operates on high-quality cells while clustering captures the full cell-type landscape.