Table 1.
Characterization and validation.
| Classification | Test | Result | Data |
|---|---|---|---|
| Morphology | Photography | Typical primed pluripotent human stem cell morphology. | Fig. 1 Panel A |
| Pluripotency status evidence for the described cell line | Qualitative analysis | Immunocytochemistry showed expression of pluripotency markers: OCT4, SOX2, NANOG, SSEA4. | Fig. 1 Panel D |
| Quantitative analysis | Compared to DAPI, % of positive cell (ULLi002-A-49, ULLi002-A-51) OCT4: 98%, 98%; SOX2: 99%, 98%; NANOG: 97%, 95%; SSEA-4: 97%, 98%. RT-PCR showed highly express OCT4, SOX2, NANOG, KLF4. | Fig. 1 Panel D and F | |
| Karyotype | Karyotype (G-banding) and resolution | 46, XY, Resolution 400 | Fig. 1 Panel C |
| Genotyping for the desired genomic alteration/allelic status of the gene of interest | PCR across the edited site and deep sequencing analysis | Heterozygous or homozygous GAG deletion (c.907–909) in TOR1A gene. | Fig. 1 Panel B and Supplementary Fig. S1A and B. |
| Transgene-specific PCR | N/A | N/A | |
| Verification of the absence of random plasmid integration events | PCR/Southern | Off Target Analysis of gRNA showed 100% minus a weighted sum of off-target hit-scores in the target genome. | N/A |
| Parental and modified cell line genetic identity evidence | STR analysis, microsatellite PCR (mPCR) or specific (mutant) allele seq | STR analysis of 18 loci, all matched. | Supplementary Fig. S1C |
| Mutagenesis/genetic modification outcome analysis | Sequencing (genomic DNA PCR or RT-PCR product) | The sequencing results of genomic DNA all matched with the parental line. | Fig. 1 Panel B and Supplementary Fig. S1A and B |
| PCR-based analyses | The sequencing results PCR products all matched with the parental line. | Fig. 1 Panel B and Supplementary Fig. S1A and B | |
| Southern Blot or WGS; western blotting (for knock-outs, KOs) | N/A | N/A | |
| Off-target nuclease analysis | PCR across top 5/10 predicted top likely off-target sites, whole genome/exome sequencing | N/A | N/A |
| Specific pathogen-free status | Mycoplasma | Tested by MycoAlert PLUS kit: Negative | Fig. S1D |
| Multilineage differentiation potential | Embryoid body formation, RT-PCR | Upregulation of trilineage markers PAX6, OTX1 (ectoderm), DCN, IGF2, GATA2 (mesoderm), and SOX7, SOX17 (endoderm). | Fig. 1 Panel G |
| Donor screening (OPTIONAL) | HIV 1 + 2 Hepatitis B, Hepatitis C | N/A | N/A |
| Genotype additional info (OPTIONAL) | Blood group genotyping | N/A | N/A |
| HLA tissue typing | N/A | N/A |