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This article presents a protocol for directed differentiation and functional analysis of β-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional β-cell like cells secreting insulin in response to glucose.
Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic β-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for β-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic β-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic β-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.
Human pluripotent stem cells (hPSCs) have the unique ability to differentiate into various cell types, making them a viable alternative to human pancreatic β-cells1. These hPSCs are categorized into two types: embryonic stem cells (hESCs), derived from the blastocyst2, and induced pluripotent cells (hiPSCs), generated by reprogramming somatic cells directly3. The development of techniques to differentiate hPSCs into β-cells, has important implications for both fundamental research and clinical practice1,4. Diabetes mellitus is a ....
Prior to initiating differentiation, it is recommended to determine the required number of islet-like organoids for experimental purposes. In a 6 well plate, a single well with over 80% confluency typically consists of 2-2.3 million hPSCs. While an accurate prediction is challenging due to variations in hPSC lines and differentiation efficiency, a rough estimate is 1.5 times the number of initial wells. An effectively directed differentiation usually yields 1.6 to 2 million cells per well in six-well plates, encompassing.......
The protocol described in this paper offers a highly efficient approach for differentiating β-like cells from hPSCs10. This process utilizes a 2D culture system that is easily scalable, enabling its use in various experimental settings, such as learning differentiation, smaller projects and laboratories, and pilot tests to assess the potential of an iPSC line for differentiation.
It is essential to characterize the functional properties of differentiated β-cel.......
The successful differentiation of hPSCs into pancreatic β-cells depends on optimizing all aspects of routine culturing and passage of the selected hPSCs. This includes ensuring that the cell line has a normal karyotype, is negative for mycoplasma infection, and is free of plasmid or viral vector genomes. Furthermore, when using hiPSCs, it is important to avoid using the earliest passage which are still undergoing reprogramming, for pilot experiments. These experiments should be conducted on a small scale to identify.......
Ines Cherkaoui was supported by a Diabetes UK studentship (BDA 18/0005934) to GAR, who also thanks the Wellcome Trust for an Investigator Award (212625/Z/18/Z), UKRI MRC for a Programme grant (MR/R022259/1), Diabetes UK for Project grant (BDA16/0005485), CRCHUM for start-up funds, Innovation Canada for a John R. Evans Leader Award (CFI 42649), NIH-NIDDK (R01DK135268) for a project grant, and CIHR, JDRF for a team grant (CIHR-IRSC:0682002550; JDRF 4-SRA-2023-1182-S-N). Camille Dion and Dr Harry Leitch for their help with human hiPSCs generation and culture, the NIHR Imperial BRC (Biomedical Research Centre) Organoid facility, London.
....Name | Company | Catalog Number | Comments |
1.5 mL TubeOne Microcentrifuge Tube | Starlabs | S1615-5500 | |
6-well Cell culture plate | ThermoFisher Scientific | 165218 | |
AggreWell 400 6-well plate | STEMCELL Technologies | 34425 | |
Anti-Glucagon | Sigma-aldrich | G2654-100UL | |
Anti-Insulin | Dako | A0564 | |
Anti-NKX6.1 | Novus Biologicals | NBP1-49672SS | |
Anti-PDX1 | Abcam | ab84987 | |
Aphidicolin | Sigma-Aldrich | A4487 | |
B-27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504044 | |
Bovine Serum Albumin, fatty acid free | Sigma-Aldrich | A3803-100G | |
Calcium chloride dihydrate | Sigma-Aldrich | C3306 | |
Calcium/Magnesium free D-PBS | Thermo Fisher Scientific | 14190144 | |
Cyclopamine-KAAD | Calbiochem | 239804 | |
D-(+)-Glucose,BioXtra | Sigma-Aldrich | G7528 | |
Disodium hydrogen phosphate, anhydrous | Sigma-Aldrich | 94046-100ML- | |
DMEM plus GlutaMAX | Thermo Fisher Scientific | 10566016 | For Washing Medium 2: DMEM plus GlutaMAX 1% PS. |
DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) | Thermo Fisher Scientific | 10565-018 | |
Epredi SuperFrost Plus Adhesion slides | Thermo Fisher Scientific | 10149870 | |
Ethanol | VWR | 20821.33 | |
Fetal Bovine Serum | Thermo Fisher Scientific | 10270098 | |
Gamma-Secretase Inhibitor XX | Thermo Fisher Scientific | J64904 | |
Geltrex LDEV-Free Reduced Growth Factor Basement | Thermo Fisher Scientific | A1413302 | Geltrex 1:1 into cold DMEM/F-12 medium to provide a final dilution of 1:100. |
Goat Anti-Guinea pig, Alexa Fluor 555 | Thermo Fisher Scientific | A-21435 | |
Goat Anti-Guinea pig, Alexa Fluor 647 | Abcam | ab150187 | |
Goat anti-Mouse Secondary Antibody, Alexa Fluor 633 | Thermo Fisher Scientific | A-21052 | |
Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | A-11011 | |
Heparin | Sigma-Aldrich | H3149 | |
HEPES buffer | Sigma-Aldrich | H3375-500G | |
Hoechst 33342, Trihydrochloride | Thermo Fisher Scientific | H1399 | |
Human FGF-7 (KGF) Recombinant Protein | Thermo Fisher Scientific | PHG0094 | |
Hydrogen chloride | Sigma-Aldrich | 295426 | |
ImmEdge Hydrophobic Barrier PAP Pen | Agar Scientific | AGG4582 | |
LDN193189 | Sigma-Aldrich | SML0559-5MG | |
Magnesium chloride hexahydrate | Sigma-Aldrich | M9272-500G | |
OCT Compound 118 mL | Agar Scientific | AGR1180 | |
PBS Tablets, Phosphate Buffered Saline, Fisher BioReagents | Thermo Fisher Scientific | 7647-14-5 | |
Penicillin-Streptomycin (PS) | Thermo Fisher Scientific, | 15070-063 | |
Potassium chloride | Sigma-Aldrich | 7447-40-7 | |
Recombinant Human EGF Protein | R&D Systems | 236-EG-200 | |
Rectangular cover glasses, 22×50 mm | VWR | 631-0137 | |
RepSox (Hydrochloride) | STEMCELL Technologies | 72394 | |
RPMI 1640 Medium, GlutaMAX Supplement | Thermo Fisher Scientific | 61870036 | For Washing Medium 1: RPMI 1640 plus GlutaMAX 1% PS. |
Shandon Immu-mount | Thermo Fisher Scientific | 9990402 | |
Sodium bicarbonate | Sigma-Aldrich | S6014-500G | |
Sodium chloride | Sigma-Aldrich | S3014 | |
Sodium dihydrogen phosphate anhydrous | Sigma-Aldrich | 7558-80-7 | |
STEMdiff Endoderm | STEMCELL Technologies | 5110 | |
StemFlex Medium | Thermo Fisher Scientific | A3349401 | Thaw the StemFlex Supplement overnight at 4°C, transfer any residual liquid of the supplement bottle to StemFlex Basal Medium. |
Stemolecule All-Trans Retinoic Acid | Reprocell | 04-0021 | |
Thyroid Tormone 3 (T3) | Sigma-Aldrich | T6397 | |
Trypan Blue Solution, 0.4% | ThermoFisher Scientific | 15250061 | |
TrypL Express Enzyme (1X) | Thermo Fisher Scientific | 12604013 | |
TWEEN 20 | Sigma-Aldrich | P2287-500ML | |
Ultra-Low Adherent Plate for Suspension Culture | Thermo Fisher Scientific | 38071 | |
UltraPure DNase/RNase-Free Distilled Water | Thermo Fisher Scientific | 10977015 | |
Y-27632 (Dihydrochloride) | STEMCELL Technologies | 72302 | |
Zinc Sulfate | Sigma-Aldrich | Z4750 |
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