Human iPSC Line SCTi003-A Differentiation Into Kidney Organoids
发布: 2026年11月05日第16卷第21期 DOI: 10.21769/BioProtoc.5860 浏览次数: 43
评审: Jessica DavisNeha SaxenaAnonymous reviewer(s)
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Abstract
Kidney organoids derived from human induced pluripotent stem cells (hiPSCs) have emerged as valuable tools for studying human kidney development, disease mechanisms, and therapeutic responses. However, widespread application of these models has been limited by variability in differentiation efficiency, organoid composition, reproducibility, and scalability across existing protocols. Robust and standardised methods are therefore needed to improve experimental consistency and facilitate wider adoption of kidney organoid systems in basic and translational research. Here, we describe an optimised and reproducible protocol for differentiating a commercially available hiPSC SCTi003-A line into kidney organoids (KiO). The protocol builds upon earlier kidney differentiation frameworks and involves directed induction of the posterior primitive streak, intermediate mesoderm specification, and subsequent three-dimensional organoid assembly. Through systematic optimisation of differentiation media, CHIR99021 concentration and timing, growth factor supplementation, and culture format, we established a standardised workflow that yields reproducible nephron and collecting duct structures. The protocol is compatible with air–liquid interface (ALI) culture and scalable suspension-based “mini-organoid” (mKiO) and monolayer culture formats that support high-content and high-throughput applications. This validated protocol provides a reliable and accessible platform for generating human KiO, addressing key limitations of reproducibility, scalability, and assay compatibility in existing approaches, and supporting future studies of kidney development, inherited kidney diseases, and preclinical drug testing.
Key features
• An optimised kidney organoid (KiO) differentiation workflow for the commercially available normal control hiPSC line (SCTi003-A, STEMCELL Technologies), minimising line-specific re-optimisation.
• Defined control of anterior (AIM) and posterior (PIM) intermediate mesoderm specification through CHIR99021 duration and pulse timing.
• Scalable for air–liquid interface (ALI) culture, suspension-based “mini-organoid” (mKiO), and monolayer culture formats.
• Compatibility with cost-effective, high-throughput drug testing using 96-well plate formats, high content imaging, and automated image segregation.
Keywords: Kidney organoids
SCTi003-A hiPSC line
Induced pluripotent stem cells
Disease models
Background
Kidney organoids (KiO) derived from induced pluripotent stem cells (hiPSCs) have emerged as powerful in vitro systems for modelling renal development, disease mechanisms, and therapeutic responses. These three-dimensional structures recapitulate key aspects of human nephrogenesis, including segmentation of nephrons into podocyte, proximal tubule, distal tubule, and collecting duct-like domains, providing a physiologically relevant alternative to conventional two-dimensional culture systems [1,2]. KiO are particularly valuable for modelling early onset genetic forms of renal disorders that are poorly recapitulated in animal models due to species-specific differences in kidney development, signalling pathways, and drug responses.
Several protocols for differentiating hiPSCs into KiO have been established, most notably those described by Takasato et al. [2,3], which use the activation of Wnt signalling by the agonist CHIR99021 to induce posterior primitive streak and intermediate mesoderm, followed by nephron patterning and three-dimensional self-organisation [1–5]. Anterior–posterior patterning of the intermediate mesoderm is regulated by the length of CHIR99021 treatment. Exposure for up to two days biases differentiation toward anterior intermediate mesoderm (AIM), while treatment for five days or longer promotes posterior intermediate mesoderm (PIM) identity. Treatment durations of 3–4 days produce a mixed anterior/posterior intermediate mesoderm phenotype [2,6]. A transient pulse of CHIR99021 is subsequently used to stimulate Wnt signalling and drive nephrogenesis, promoting the mesenchymal-to-epithelial transition and enhancing the formation of nephron structures within the developing organoids [7]. More recent refinements by Uchimura et al. [8] include the parallel induction of anterior (AIM) and posterior (PIM) intermediate mesoderm populations to better recapitulate ureteric epithelium and nephron lineages [8]. While these methods have advanced the field, challenges remain in achieving reproducible differentiation across different hiPSC lines, minimising batch-to-batch variability, and adapting protocols for scalable and cost-effective applications such as drug screening.
Here, we present an optimised and standardised protocol for differentiating a commercially available normal control hiPSC line into KiO. We describe the key parameters that influence differentiation efficiency and reproducibility, including basal medium composition, CHIR99021 concentration and exposure duration, growth factor supplementation, ALI culture conditions, cell seeding density, and total differentiation time. We identify advanced RPMI as a robust and cost-effective alternative to the previously described APEL2 medium for differentiation and define CHIR99021 timing parameters that reliably support both AIM and PIM specification.
This protocol supports KiO generation in three complementary formats: ALI cultures that facilitate structural maturation and tissue organisation, suspension-based “mini KiO” that are compatible with 96-well plate formats for high-throughput applications [9], and monolayer culture, which allows spontaneous 3D differentiation and could be applied in fluidic devices. The protocol is suitable for nephrotoxicity assessment, genetic disease modelling, and developmental studies. Its modular design allows adaptation to additional hiPSC lines, whilst the use of widely accessible reagents and defined parameters enables standardisation and reproducibility across laboratories.
Materials and reagents
Biological materials
1. Healthy control human iPSC line, female, SCTi003-A (STEMCELL Technologies, catalog number: 200-0511); this line was derived from peripheral blood mononuclear cells (PBMCs) of a 48-year-old Caucasian healthy female (https://www.stemcell.com/products/healthy-control-human-ipsc-line-female-scti003-a.html)
Reagents
1. Advanced RPMI 1640 Medium (Gibco, catalog number: 12633020)
2. Antibiotic-antimycotic (100×) (Gibco, catalog number: 15240-096)
3. Bovine albumin fraction V (7.5% solution) (Gibco, catalog number: 15260-037)
4. CHIR99021 ≥98% (HPLC-grade purity) (6 mM) (Sigma-Aldrich, catalog number: SML1046)
5. CryoStor® CS10 (STEMCELL Technologies, catalog number: 07930)
6. DMEM/F-12, HEPES (Gibco, catalog number: 31330-038)
7. Dimethyl sulfoxide (DMSO) sterile-filtered, ≥99.7% (Sigma-Aldrich, catalog number: D2650)
8. Dulbecco’s phosphate-buffered saline (DPBS) (Sigma-Aldrich, catalog number: D8537)
9. Ethanol (Supelco, catalog number: 1.00983.2511)
10. GlutaMAXTM supplement (100×) (Gibco, catalog number: 35050061)
11. Heparin solution 0.2% (2 mg/mL) (STEMCELL Technologies, catalog number: 07980)
12. Human FGF-9 (fibroblast growth factor 9) recombinant protein (200 μg/mL) (PeproTech, catalog number: 100-23)
13. Lipidure-CM5206 powder (Amsbio, catalog number: AMS.52000032GB)
14. Matrigel hESC (human embryonic stem cells)-qualified matrix (Corning, catalog number: 354277)
15. mTeSRTM Plus (STEMCELL Technologies, catalog number: 100-0276)
16. Recombinant human/murine/rat activin A (10 μg/mL) (PeproTech, catalog number: 120-14)
17. ROCK inhibitor (Y-27632) (20 mM) (CHEMDEA, catalog number: CD0141)
18. StemProTM AccutaseTM cell dissociation reagent (Gibco, catalog number: A1110501)
19. Trypan Blue 0.4% (NanoEntek, catalog number: EBT-001)
Solutions
1. 6 mM CHIR99021 (see Recipes)
2. 20 mM Y-27632 (see Recipes)
3. 10 μg/mL activin A (see Recipes)
4. 200 μg/mL FGF9 (see Recipes)
5. Advanced RPMI 1640 Medium (ARPMI) with GlutaMAX (ARPMIG) (see Recipes)
6. ARPMIG with 6 μM CHIR99021 (see Recipes)
7. ARPMIG with 200 ng/mL FGF9, 1 μg/mL heparin, 10 ng/mL activin A (see Recipes)
8. ARPMIG with antibiotic/antimycotic (ARPMIG-ABAM) (see Recipes)
9. ARPMIG-ABAM with 5 μM CHIR99021 (see Recipes)
10. ARPMIG-ABAM with 200 ng/mL FGF9, 1 μg/mL heparin (see Recipes)
11. 0.5% w/v Lipidure (see Recipes)
Recipes
1. 6 mM CHIR99021
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| CHIR99021 | 6 mM | 5 mg |
| DMSO | n/a | 1.791 mL |
Aliquot and store at -20 °C. Stock solutions are stable for up to 6 months at -20 °C. Avoid repeated freeze/thaw cycles and protect from light.
2. 20 mM Y-27632
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Y-27632 | 20 mM | 5 mg |
| dH2O | n/a | 740 μL |
Aliquot and store at -20 °C. Avoid repeated freeze/thaw cycles and protect from light.
3. 10 μg/mL activin A
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Activin A | 10 μg/mL | 10 μg |
| dH2O | n/a | 10 μL |
DPBS BSA 7.5% | n/a 0.1% | 976.8 μL 13.2 μL |
Working aliquots stored with a carrier protein are stable for at least 7 months at -20 to -80 °C. Avoid repeated freeze/thaw cycles. It can be stored at 4 °C for 2–7 days, or up to 1 month under sterile conditions after reconstitution.
4. 200 μg/mL FGF9
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| FGF9 | 200 μg/mL | 250 μg |
| dH2O | n/a | 250 μL |
| DPBS | n/a | 986.7 μL |
| BSA 7.5% | 0.1% | 13.3 μL |
Store for up to 1 week at 4 °C or up to 12 months at -20 to -80 °C.
5. ARPMIG
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| ARPMI | n/a | 495 mL |
| GlutaMAX 100× | 1× | 5 mL |
Store at 4 °C.
6. ARPMIG with 6 μM CHIR99021 (medium for days 0–4)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| ARPMIG | n/a | 2 mL |
| 6 mM CHIR99021 | 6 μM | 2 μL |
Prepare fresh on the day.
7. ARPMIG with 200 ng/mL FGF9, 1 μg/mL heparin, 10 ng/mL activin A (medium for days 4–7)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| ARPMIG | n/a | 4 mL |
| 200 μg/mL FGF9 | 200 ng/mL | 4 μL |
| 2 mg/mL heparin | 1 μg/mL | 2 μL |
| 10 μg/mL activin A | 10 ng/mL | 4 μL |
Prepare fresh on the day.
8. ARPMIG-ABAM
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| ARPMI | n/a | 490 mL |
| GlutaMAX 100× | 1× | 5 mL |
| Antibiotic/Antimycotic 100× | 1× | 5 mL |
Store at 4°C.
9. ARPMIG-ABAM with 5 μM CHIR99021 (pulse medium day 7)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| ARPMIG-ABAM | n/a | 1.2 mL |
| 6 mM CHIR99021 | 5 μM | 1 μL |
Prepare fresh on the day.
10. ARPMIG-ABAM with 200 ng/mL FGF9, 1 μg/mL heparin (medium for days 7–12)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| ARPMIG-ABAM | n/a | 1.2 mL |
| 200 μg/mL FGF9 | 200 ng/mL | 1.2 μL |
| 2 mg/mL heparin | 1 μg/mL | 0.6 μL |
Prepare fresh on the day.
11. 0.5% w/v Lipidure
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Lipidure | 5 mg/mL | 1 g |
| Ethanol | 100% | 200 mL |
Store at room temperature (RT) in the dark for up to 3 months.
Laboratory supplies
1. 15 mL polypropylene centrifuge tubes (Corning, catalog number: BC030)
2. 50 mL polypropylene centrifuge tubes (Corning, catalog number: BC034)
3. 6-well plate (TPP, catalog number: 92406T)
4. 96-well plate (TPP, catalog number: 92097T)
5. Corning® Transwell® 6-well plates (Corning, catalog number: CLS3450)
6. C-slide disposable cell counting chamber slide (NanoEntek, catalog number: CS-050)
7. 1.5 mL Eppendorf tubes (Sarstedt, catalog number: 72.692)
8. Pipetboy (Integra, catalog number: 155017)
9. Pipette tips RT UNV (METTLER TOLEDO, catalog numbers: 30389172, 30389186, 30389165)
10. Thermo ScientificTM ARTTM wide bore filtered pipette tips P1000 (Thermo Fisher Scientific, catalog number: 2079G)
11. FinnpipetteTM F1 variable volume pipettes P2, P10, P200, P1000 (Thermo Fisher Scientific, catalog numbers: 4641010N, 4641030N, 4641080N, 4641100N)
12. FinnpipetteTM F1 multichannel pipette (Thermo Fisher Scientific, catalog number: 4661030N)
13. ThincertTM cell culture insert for 6-well plates, translucent (Greiner Bio-One, catalog number: 657640)
14. ThincertTM cell culture insert for 6-well plates, transparent (Greiner Bio-One, catalog number: 657641)
15. FisherbrandTM disposable pipette basins (Thermo Fisher Scientific, catalog number: 11988485)
Equipment
1. CountessTM3 (Thermo Fisher Scientific, catalog number: AMQAX2000)
2. EVOS FL Colour Imaging System (Life Technologies, catalog number: AMF4300)
3. Confocal laser scanning microscope or equivalent (Nikon A1R or Zeiss CellDiscoverer 7)
4. Humidified CO2 incubator (Sanyo, catalog number: MCO-20AIC)
5. CellGard Class II, Type A2 vertical laminar flow biological safety cabinet (NuAire, catalog number: NU-480-600E)
6. Sigma 1-14 Compact Microcentrifuge (Sigma, catalog number: 10014)
7. MultifugeTM X3 (ventilated) centrifuge (Thermo Fisher Scientific, catalog number: 75004500)
Software and datasets
1. Fiji (ImageJ)
2. Image-Pro (11.1.2, 2025) with the AI Deep Learning module
Procedure
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文章信息
稿件历史记录
提交日期: Jul 20, 2026
接收日期: Sep 23, 2026
在线发布日期: Oct 12, 2026
出版日期: Nov 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
如何引用
Szymanska, K., Karwatka, M., Parpas, G., Harris, E. L., Barlow, A. L. and Johnson, C. A. (2026). Human iPSC Line SCTi003-A Differentiation Into Kidney Organoids. Bio-protocol 16(21): e5860. DOI: 10.21769/BioProtoc.5860.
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