KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
|
| ||
| Antibodies | ||
|
| ||
| Mouse monoclonal anti-CXCR4, PE-conjugated (clone 12G5) | StemCell Technologies | 60089PE |
| Mouse monoclonal anti-c-Kit, APC-conjugated (clone 104D2) | ThermoFisher Scientific | CD11705; RRID: AB_1463361 |
| Mouse monoclonal anti-AAT (clone B9) | Santa Cruz | sc-59438; RRID: AB_781352 |
| Rabbit monoclonal anti-AFP (clone EPR9309) | Abcam | ab169552; RRID: AB_2756827 |
| Rabbit monoclonal anti-HNF4A (clone EPR16885-99) | Abcam | ab201460 |
| Mouse monoclonal anti-AAT polymers (clone 2C1) | Hycult Biotech | HM2289 |
| Mouse monoclonal anti-CD106 (VCAM1), PE-conjugated (clone STA) | ThermoFisher Scientific | 12-1069-42; RRID: AB_10854126 |
| Mouse anti-ZAAT | Boreletal., 201728 | Umassmed.edu/muellerlab/contact/reagent-request/ |
| Rabbit polyclonal anti-AAT | Proteintech | 16382-1-AP; RRID: AB_10641185 |
| Goat polyclonal anti-mouse IgG, Alexa Fluor 647 conjugated | Jackson ImmunoResearch | 115-605-003; RRID: AB_2338902 |
| Donkey polyclonal anti-mouse IgG, Alexa Fluor 488 conjugated | ThermoFisher Scientific | A-21202; RRID: AB_141607 |
| Donkey polyclonal anti-rabbit IgG, Alexa Fluor 488 conjugated | ThermoFisher Scientific | A-21206; RRID: AB_2535792 |
| Mouse IgG1 isotype, APC-conjugated | ThermoFIsher Scientific | MG105; RRID: AB_2921170 |
| Mouse IgG1, kappa isotype, PE-conjugated (clone MOPC-21) | BD Pharmingen | 559320; RRID: AB_397218 |
| Mouse IgG2a, kappa isotype, PE-conjugated | StemCell Technologies | 60071PE |
| Mouse IgG1 isotype | Santa Cruz | sc-3877; RRID: AB_737222 |
| Rabbit IgG isotype | Abcam | ab172730; RRID: AB_2687931 |
|
| ||
| Chemicals, peptides, and recombinant proteins | ||
|
| ||
| Human embryonic stem cell (hESC)-qualified matrix, LDEV free | Corning | 354277 |
| Y-27632 dihydrochloride | Tocris | 1254 |
| Primocin | InvivoGen | ant-pm |
| Trypsin-EDTA (0.25%), phenol red | ThermoFisher Scientific | 25200114 |
| ClaI restriction enzyme | New England BioLabs | R0197 |
| N-2 Supplement | ThermoFisher Scientific | 17502048 |
| B27 Supplement, minus vitamin A | ThermoFisher Scientific | 12587010 |
| Ascorbic Acid | MilliporeSigma | A4544 |
| 1-Thioglycerol (MTG) | MilliporeSigma | M6145 |
| BSA 7.5% Stock | ThermoFisher Scientific | 15260037 |
| Calcein Blue | ThermoFisher Scientific | C1429 |
| Hoechst 33342 | ThermoFisher Scientific | H3570 |
| Prolong diamond antifade mountant | ThermoFisher Scientific | P36965 |
| Recombinant human BMP-4 | R&D Systems | 314-BP-01M |
| Recombinant human FGF basic | R&D Systems | 233-FB-025 |
| Recombinant human VEGF | R&D Systems | 293-VE-010 |
| Recombinant human EGF | R&D Systems | 236-EG-200 |
| Recombinant human TGF-alpha | R&D Systems | 239-A-100 |
| Recombinant human HGF | R&D Systems | 294-HG |
| Dexamethasone | MilliporeSigma | D4902 |
| Recombinant human oncostatin M | ThermoFisher Scientific | PHC5015 |
| Phylloquinone (K1) | MilliporeSigma | 47773 |
| InSolution γ-secretase inhibitor X-Calbiochem | MilliporeSigma | 565771 |
| Dimethyl sulfoxide | MilliporeSigma | D2650 |
| Elastase, human neutrophil | MilliporeSigma | 324681 |
| N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide | MilliporeSigma | M4765 |
| MitoTracker deep red FM | ThermoFisher Scientific | M22426 |
| Triton X-100 Solution | MilliporeSigma | 93443 |
| Tween20 | MilliporeSigma | P9416 |
| EXPRE36S35S Protein Labeling Mix, [35S-]-,14mCi (518MBq), 50mM Tricine (pH7.4), 10mM 2-mercaptoethanol | PerkinElmer | NEG072014MC |
| Methanol | Fisher Scientific | A456-1 |
| Chloroform | Fisher Scientific | A461-1 |
| L-Phenylalanine-d8 | MilliporeSigma | 749885 |
| L-Valine-d8 | Cambridge Isotope Laboratories | DLM-7784-PK |
| Acetonitrile, HPLC | J.T. Baker | 9012-03 |
| Ammonium Acetate | MilliporeSigma | 17836 |
| Ammonium hydroxide Solution | Millipore-Sigma | 17837 |
| Gluteraldehyde | Ladd Research | 20100 |
| Osmium Tetroxide | Polysciences | 0223D |
| Uranyl Acetate | Electron Microscopy Sciences | 22400 |
| Embed 812 | Electron Microscopy Sciences | 14120 |
|
| ||
| Critical commercial assays | ||
|
| ||
| STEMdiff Definitive Endoderm Kit | StemCell Technologies | 05110 |
| RNeasy mini kit | QIAGEN | 74104 |
| QIAzol lysis reagent | QIAGEN | 79306 |
| P3 primary cell 4D-nucleofector X kit L | Lonza | V4XP-3024 |
| Lactate assay kit | MilliporeSigma | MAK064 |
| Pyruvate assay kit | MilliporeSigma | MAK071 |
| Human alpha-1 antitrypsin ELISA kit | Genway Biotech | GWB-5428A0 |
| Chromium next GEM single cell 3’ kit v3.1 | 10× Genomics | 1000269 |
| C1 single-cell mRNA seq HT IFC (10–17μm) | Fluidigm | 101-4981 |
| C1 single-cell mRNA seq HT reagent kit v2 | Fluidigm | 101-3473 |
| Seahorse XF cell mito stress test kit | Agilent | 103015-100 |
|
| ||
| Deposited data | ||
|
| ||
| RNA sequencing PiZZ6 syngeneic ZZ, MZ, and MM iHeps. | This paper | GEO: GSE198233 |
| scRNA sequencing PiZZ1 syngeneic (ZZ, MZ, MM) and PiZZ6 ZZ iHeps using 10X Genomics Chromium Controller. |
This paper | GEO: GSE198234 |
| scRNA sequencing PiZZ100 ZZ and MM iHeps using the Fluidigm C1. | This paper | GEO: GSE198234 |
| RNA sequencing ZZ and MM syngeneic iHeps | Segeritz et al., 20181 | ArrayExpress: E-MTAB-6781 |
| scRNA sequencing of primary human hepatocytes | MacParland et al., 20182 | GEO: GSE115469 |
|
| ||
| Experimental models: Cell lines | ||
|
| ||
| Human: PiZZ AATD donor iPSC line (PiZZ1ZZ) | Wilson Lab, Kaserman et al., 202021 | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line (PiZZ6ZZ) | Wilson Lab, Kaserman et al., 202021 | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line (PiZZ100ZZ) | Wilson Lab, Wilson et al., 201525 | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line mono-allelic correction (PiZZ1MZ) | This paper | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line mono-allelic correction (PiZZ6MZ) | This paper | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line mono-allelic correction (PiZZ100MZ) | This paper | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line bi-allelic correction (PiZZ1MM) | Wilson Lab, Kaserman et al., 202021 | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line bi-allelic correction (PiZZ6MM) | This paper | https://stemcellbank.bu.edu/Catalog/Item/Home |
| Human: PiZZ AATD donor iPSC line bi-allelic correction (PiZZ100MM) | Wilson Lab, Kaserman et al., 202021 | https://stemcellbank.bu.edu/Catalog/Item/Home |
|
| ||
| Oligonucleotides | ||
|
| ||
| SERPINA1 Z mutation gRNA | IDT | 5′ GTGCTGACCATCGACAAGAA3’ |
| MAAT ssODN donor template | IDT | 5′ TCTAAAAACATGGCCCCAGCAGCTTCAGTACCTTTTTCATCGATGGTCAGCACAGCCTTATGCACGGCCT 3’ |
| ZAAT ssODN donor template | IDT | 5′ TCTAAAAACATGGCCCCAGCAGCTTCAGTCCCTTTTTTATCGATGGTCAGCACAGCCTTATGCACGGCCT 3’ |
| SERPINA1 Z mutation Primer Forward | IDT | 5′ GCAGACGTGGAGTGACGATG 3’ |
| SERPINA1 Z mutation Primer Reverse | IDT | 5′ CCTGGATTCAAGCCCAGCAT 3’ |
|
| ||
| Recombinant DNA | ||
|
| ||
| pSpCas9(BB)-2A-GFP | Addgene | 48138; RRID:Addgene_48138 |
|
| ||
| Software and algorithms | ||
|
| ||
| FlowJo software | Becton, Dickinson & Company |
https://www.Flowjo.com; RRID:SCR_008520 |
| Prism software | Graphpad Software |
https://www.Graphpad.com; RRID:SCR_002798 |
| ImageJ | National Institutes of Health |
https://ImageJ.nih.gov/ij/; RRID:SCR_00370 |
| Cell Ranger, version 3.0.2 | 10X Genomics |
https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome; RRID:SCR_017344 |
| Seurat, version 3 | Satija Lab, NYU |
https://satijalab.org/seurat; RRID:SCR_007322 |
|
| ||
| Other | ||
|
| ||
| mTeSR-1 | StemCell Technologies | 05850 |
| Gentle cell dissociation reagent | StemCell Technologies | 07174 |
| ReLeSR | StemCell Technologies | 05872 |
| Seahorse XF base medium minimal DMEM | Agilent | 103334–100 |
| Seahorse XF calibrant solution | Agilent | 100840–000 |
| Sodium pyruvate (100mM) | ThermoFisher Scientific | 11360070 |
| Glucose solution | ThermoFisher Scientific | A2494001 |
| Hank’s buffered saline solution (HBSS; no calcium, no magnesium, no phenol red) | ThermoFisher Scientific | 14175095 |
| Ham’s F12 medium | MilliporeSigma | N6658 |
| Iscove’s modified dulbecco’s medium (IMDM) | ThermoFisher Scientific | 12440053 |
| Glutamax | ThermoFisher Scientific | 35050061 |
| Amicon Ultra-4 Centrifugal Filter Unit | MilliporeSigma | UFC8030 |
| X-Bridge BEH Amide Column, 130A, 3.5 μm, 4.6 mm × 30 mm, 1/pkg | Waters | 186004866 |