Generation of gastirc insulin-secreting organoids from human stomach sample

Stomach stem cells are accessible by biopsy and propagate robustly in culture, offering an invaluable resource for autologous cell therapies. Here we describe a detailed protocol to isolate, expand, engineer and differentiate human gastric stem cells (hGSCs) into pancreatic islet-like organoids containing abundant gastric insulin-secreting (GINS) cells that resemble beta-cells in molecular hallmarks and function. Sequential activation of the inducing factors NGN3 and PDX1-MAFA led hGSCs onto a novel differentiation path, including endocrine progenitor and GINS precursor, before adopting beta-cell identity, at efficiencies close to 70%. GINS organoids acquired glucose-stimulated insulin secretion in 10 days post differentiation.


Introduction
Gut stem cells are highly proliferative and power the weekly self-renewal of the gut mucosal lining [1][2][3] .
Harvested from biopsies, human gut stem cells can be propagated in culture as organoids or primary cell lines over many generations, providing abundant tissues for potential autologous transplantation therapies [4][5][6] . Gut stem cells produce gut-speci c tissues, including hormone-secreting enteroendocrine cells (EECs). Rare insulin expressing EECs have been reported in fetal human small intestine 7 . Whether such cells secret insulin is unknown, but their presence suggests an intrinsic permissiveness for insulin production in the fetal if not postnatal intestine. Generating functional insulin-secreting cells has tremendous therapeutic value, offering treatments for insulin-dependent diabetes, including the autoimmune type 1 diabetes [8][9][10][11][12][13] . An attractive feature of using gut stem cells to make beta-cell mimics is the ease of establishing autologous organoids from biopsies, which can enable mass production and personalized therapies.
Previously, we reported that co-expression of the endocrine regulator NEUROG3 (also known as NGN3) and pancreatic beta-cell regulators PDX1 and MAFA could induce insulin-secreting cells from murine intestine and stomach 14 . Here, we provided a detailed step-by-step protocol to induce cultured hGSCs derived from human donors to differentiate into islet-like organoids at high e ciency, containing approximately 70% beta-like cells and other islet-like endocrine populations. The protocol contains 4 sections including (1) isolation of primary hGSCs from human stomach sample, (2) expansion and cryopreservation of hGSCs, (3) generation of Ngn3ER-hGSC line, and (4) generation of GINS organoids. GINS organoids generated from this protocol exhibited glucose responsiveness 10 days after induction. agreement. Virus can be packaged as previously decribed 15,16 or following the protocol on the website (https://www.thermo sher.com/us/en/home/life-science/cell-culture/cell-culture-learning-center/cellculture-resource-library/cell-culture-transfection-application-notes/improve-lentiviral-production-usinglipofectamine-3000-reagent.html). 11. Resuspend pellet in the warmed isolation medium using the coated pipet.
12. (CRITICAL) Incubate tissue at 37℃ in the water bath for 15-30 min with pipetting rigorously using the coated 10 mL pipet (when the size of tissue allows, use the coated P1000 tip instead) until clusters of crypt cells released. NOTE: duration of this step should be adjusted case by case.
13. (Optional) Let stand for 1 min and collect the tissue that sink to the bottom and repeat step 11-12 in a separate tube.
17. Resuspend pellet in hGSC medium and seed cells on 1 well of 6-well plate that coated with con uent inactivated MEF feeder cells.
19. Change medium every other day.

Expansion and cryopreservation of hGSCs
Note: hGSC colonies should be passaged when the colonies begin to make contact to each other (~70% con uency).

Detach cells by pipetting.
4. Transfer cells into a centrifuge tube that contains DMEM complete medium. 5. Centrifuge cells at 300 x g for 5 min.
7. Maintain hGSCs in hGSC medium at 37 °C in a 5-7.5% CO 2 humidi ed incubator. 8. Change medium every other day. 9. For hGSC cryopreservation, pellet from step 6 should be resuspended in freezing solution (10% DMSO in FBS) and frozen using standard mammalian cell cryopreservation protocol.
4. Spin the cell culture with lentivirus in plate at 1000 g for 30 min at 37°C. 5. Culture the infected hGSCs at 37°C in a 5-7.5% CO 2 incubator for 48 hours.