Key Resources Table.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| rat anti-insulin (pro-)/C-peptide | DSHB | GN-ID4 |
| mouse anti-glucagon | Santa Cruz | sc-514592 |
| guinea pig anti-insulin | Dako | A0564 |
| mouse anti-Nkx6.1 | DSHB | F55A12 |
| rabbit anti phospho-S6 (Ser240/244 | Cell Signaling | 5364 |
| rabbit anti Mafa | Cell signaling; | 79737 |
| goat anti Pdx1 | R&D Systems | AF2419 |
| rabbit anti phospho-AMPKα | Cell signaling | 2535 |
| Biological Samples | ||
| Cadaveric human islets | Prodo labs | N/A |
| Fetal human pancreas | University of Washington | N/A |
| Chemicals, Peptides, and Recombinant Proteins | ||
| mTeSR1 medium | StemCell Technologies | 85850 |
| nutrient-free RPMI-1640 medium | mybiosource | MBS652918 |
| RPMI-1640 medium with no glucose | Life Technologies | 11879–020 |
| RPMI-1640 medium | Life Technologies | 11875–093 |
| CMRL 1066 Supplemented | Mediatech | 99–603-CV |
| FBS (HyClone) | VWR | 16777 |
| insulin | Sigma | 19278 |
| S961 | Novonordisk | N/A |
| exendin-4 | Sigma | E7144 |
| forskolin | Santa Cruz | SC3562 |
| Torin-1 | Thermo Fisher | 424710 |
| leucine | Sigma | N/A |
| diazoxide | Sigma | D9035 |
| perm/fix solution | BD | 554714 |
| Amino acids | Sigma | N/A |
| Critical Commercial Assays | ||
| 500ml spinner flasks | Corning | |
| flow cytometry tubes | BD Falcon | 352235 |
| RLT Lysis Buffer | QIAGEN | 79216 |
| Human Ultrasensitive Insulin ELISA | ALPCO | 80-INSHUU-E1.1 |
| TrypLE Express | GIBCO | 12604013 |
| Deposited Data | ||
| mRNA sequencing of stem cell-derived beta cells before and after transplantation into mice | GEO NCBI | GSE147346 |
| mRNA sequencing of stem cell-derived beta cells cultured in different AA concentrations | GEO NCBI | GSE147347 |
| single-cell mRNA sequencing of human embryonic pancreas | GEO NCBI | GSE147349 |
| Experimental Models: Cell Lines | ||
| HUES8 human embryonic stem cells | NIH | #0021 |
| Experimental Models: Organisms/Strains | ||
| C57BL/6 | Jackson Laboratory | N/A |
| SCID-Beige mice | Jackson Laboratory | N/A |
| Prkaa1tm1.1Sjm/J | Jackson Laboratory | 014141 |
| Prkaa2tm1.1Sjm/J | Jackson Laboratory | 014142 |
| Tsc1tm1Djk/J | Jackson Laboratory | 005680 |
| B6.Cg-Tg(Ins1-cre/ERT)1Lphi/J | Jackson Laboratory | 024709 |
| Oligonucleotides | ||
| Custom Taqman probes for TSC1, TSC2, LKB1, PRKAA1, PRKAA2, SESN1, SESN2, UBC and GAPDH | Applied Biosystems | |
| Cre forward: GAACCTGATGGACATGTTCAGG Cre reverse: AGTGCGTTCGAACGCTAGAGCCTGT |
||
| Prkaa1 forward: CCC ACC ATC ACT CCA TCT CT Prkaa1 reverse: AGC CTG CTT GGC ACA CTT AT |
||
| Prkaa2 forward: GCA GGC GAA TTT CTG AGT TC Prkaa2 reverse: TCC CCT TGA ACA AGC ATA CC |
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| TSC1 forward: GTC ACG ACC GTA GGA GAA GC TSC1 reverse: GAA TCA ACC CCA CAG AGC AT |
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| Recombinant DNA | ||
| Software and Algorithms | ||
| Biorender | https://app.biorender.com/ | |
| Prism | Graphpad | https://www.graphpad.com/scientific-software/prism/ |
| ImageJ | Schneider et al., 2012 | https://imagej.nih.gov/ij/ |
| FlowJo | FlowJo | https://www.flowjo.com |
| Other | ||