A reference human induced pluripotent stem cell line for large-scale collaborative studies

Pantazis CB, Yang A, Lara E, McDonough JA, Blauwendraat C, Peng L, Oguro H, Kanaujiya J, Zou J, Sebesta D, Pratt G, Cross E, Blockwick J, Buxton P, Kinner-Bibeau L, Medura C, Tompkins C, Hughes S, Santiana M, Faghri F, Nalls MA, Vitale D, Ballard S, Qi YA, Ramos DM, Anderson KM, Stadler J, Narayan P, Papademetriou J, Reilly L, Nelson MP, Aggarwal S, Rosen LU, Kirwan P, Pisupati V, Coon SL, Scholz SW, Priebe T, Öttl M, Dong J, Meijer M, Janssen LJM, Lourenco VS, van der Kant R, Crusius D, Paquet D, Raulin AC, Bu G, Held A, Wainger BJ, Gabriele RMC, Casey JM, Wray S, Abu-Bonsrah D, Parish CL, Beccari MS, Cleveland DW, Li E, Rose IVL, Kampmann M, Calatayud Aristoy C, Verstreken P, Heinrich L, Chen MY, Schüle B, Dou D, Holzbaur ELF, Zanellati MC, Basundra R, Deshmukh M, Cohen S, Khanna R, Raman M, Nevin ZS, Matia M, Van Lent J, Timmerman V, Conklin BR, Johnson Chase K, Zhang K, Funes S, Bosco DA, Erlebach L, Welzer M, Kronenberg-Versteeg D, Lyu G, Arenas E, Coccia E, Sarrafha L, Ahfeldt T, Marioni JC, Skarnes WC, Cookson MR, Ward ME, Merkle FT
Source: Cell Stem Cell
Publication Date: (2022)
Issue: 29(12): 1685-1702.e22
Research Area:
Cancer Research/Cell Biology
Stem Cells
Basic Research
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Culture Media:
4D-Nucleofector® X-Unit

Genetic correction of the KOLF2-C1 cell line

CRISPR-Cas9 editing of KOLF2-C1 human iPSCs was performed to correct a 19bp pathogenic deletion in one copy of the ARID2 gene.16 Cells were grown in a 5% CO2, 37C incubator in StemFlex media (Thermo Fisher) on Synthemax-treated wells (Synthemax II; Corning) and dissociated to single cells with Accutase (Stemcell Technologies). In a volume of 0.1 mL P3 Primary Cell P3 buffer (Lonza), 8 x 10^5 KOLF2-C1 cells were nucleofected using the Lonza Amaxa 4D nucleofector (program CA-137) with 20 µg Cas9 protein (HiFi v3; IDT), 16 µg single guide RNA targeted to the deleted ARID2 allele (50 -AAAAGATCACTTGCTAATGCCGG . -3’; chemically-modified; Synthego), and 200 pmol of a 120-mer oligonucleotide repair template.

Cortical neuron differentiation

To generate iPSCs with a stable tet-inducible NGN2 construct, 2.75µg PiggyBac transposase and 2.75µg tet-NGN2 (both from Michael Ward) were nucleofected using a Nucleofector I (Lonza, protocol A-23) and the Human Stem Cell Nucleofector Kit 1 (Lonza). To improve survival after nucleofection, mTeSR plus was supplemented with CET (50nM Chroman 1, MedChem Express; 5uM Emricasan, Selleckchem; 0.7uM Trans-ISRIB, Tocris).

Motor neuron differentiation

 Cell culture. KOLF2.1J iPSCs were differentiated to motor neurons by inducible expression of 3 transcription factors in the hNIL transgenic system. Briefly, 350,000 KOLF2.1J iPSCs were electroporated (Lonza 4D nucleofector, setting DS-138; Amaxa P3 Primary Cell 96-well Nucleofector kit) with plasmids containing the i3 LMN hNIL inducible construct (pUCM-CLYBL-hNIL, Addgene) and TALENs targeting the CLYBL safe harbor locus (pZT-C13-R1, Addgene; pZT-C13-L1, Addgene).

Macrophage differentiation

Cell culture. Human iPSCs were maintained and differentiated as described previously with minor modifications.92 In summary, iPSCs were maintained in mTeSR1 Plus media (Stem Cell Technology) in plates coated with 10 mg/mL of Cell Adhere Laminin 521. (Stem Cell Technology) diluted in Dulbecco’s PBS (DPBS) containing calcium and magnesium (Gibco). Embryoid body (EB) generation was performed by dissociating the iPSCs colonies and plating 10^4 cells/well into low adherence 96-well plates (Corning) using mTeSR1 Plus media containing 50 ng/mL human bone morphogenetic protein 4 (BMP-4, Fisher Scientific), 50 ng/mL human vascular endothelial growth factor (VEGF, PeproTech), 20 ng/mL human stem cell factor (SCF, PeproTech), and 10uM ROCK inhibitor (Fisher Scientific). Plated cells were centrifuged at 800 rpm for 3 min and half-medium change was performed on day 2. For myeloid maturation, EBs were gently dislodged from the wells and plated on 6 well plates coated with growth factor reduced matrigel (Corning) diluted in KnockOut DMEM/F-12 (Gibco). EBs were maintained in X-VIVO 15 media (Lonza) supplemented with 1% penicillin/streptomycin (Gibco), 1X GlutaMAX (Gibco), 55 mM 2-mercaptoethanol (Gibco), 100 ng/mL human macrophage colony stimulating factor (M-CSF, PeproTech), and 25 ng/mL human interleukin- 3 (IL-3, PeproTech). Macrophage progenitors were collected weekly during medium change and terminally differentiated into unpolarized iPSC-derived macrophages by culture for seven days in X-VIVO 15 supplemented with 1% penicillin/streptomycin, 1X GlutaMAX, and 100 ng/mL M-CSF at a density of 100,000 cells per cm2 . Halfmedium change was performed every 3 days. 


Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.