In the last decade, induced pluripotent stem cells have opened the floodgates for studying human biology in a dish, or well, as the case may be. However, the genetic heterogeneity of human cells makes it difficult to compare findings among labs. The solution? A standardized iPSC reference line that could be widely used across the field, according to an October 5 bioRxiv preprint from the iPSC Neurodegenerative Disease Initiative (iNDI) and colleagues.

  • A stem cell consortium generated a potential iPSC reference line.
  • KOLF2.1J is stable, and reliably differentiates and responds to CRISPR editing.
  • Its genome is neutral to AD risk, making it a good comparator.

Researchers led by Florian Merkle at the University of Cambridge, U.K., Michael Ward and Mark Cookson at the National Institute on Aging in Bethesda, Maryland, and William Skarnes at the Jackson Laboratory in Farmington, Connecticut, deeply phenotyped eight different publicly available stem-cell lines. They identified one, dubbed KOLF2.1J, that exhibited excellent differentiation potential, genetic stability, and CRISPR editing efficiency. The line contained no AD-related or even neurologically harmful variants, except for one splice-site disruption in an extracellular matrix gene. In a round-robin exercise by nine independent research groups, the line compared well with their in-house iPSC lines, suggesting it will be broadly applicable.

“We believe KOLF2.1J is an excellent choice to become a commonly used iPSC line,” the authors wrote. More than 100 laboratories already do. KOLF2.1J is available from Jackson Labs.

One challenge for using iPSC lines for research is the vast genomic variability between individuals. Each person is estimated to carry around 100 distinct loss-of-function variants that may affect key biological pathways (MacArthur et al., 2012). Up until now, researchers have skirted this issue by using isogenic control lines. For example, to study the effect of an autosomal-dominant AD variant, they would create iPSCs from a carrier, and reverse-engineer a control line by correcting to the wild-type variant. However, this does nothing to tame the genetic heterogeneity that exists among lines from different labs. To help the field standardize, iNDI aims to create a suite of publicly available isogenic lines that express one of 134 genetic variants related to AD or other dementias (May 2021 news).

The first step was to anoint a reference line. Joint first authors Caroline Pantazis, Erika Lara, and Cornelis Blauwendraat at NIA, Andrian Yang at Cambridge, and Justin McDonough at JAX, combed through public repositories for lines that had broad data-sharing consents. They selected iPSCs from male donors only, due to concern that random X-chromosome inactivation could skew gene expression in female lines. Then they analyzed each line’s karyotype, pluripotency, differentiation efficiency, and cell morphology. They checked for genetic stability through multiple generations, and after CRISPR editing. Under this scrutiny, all but KOLF2.1J fell by the wayside, eliminated by their instability, slow growth, or incomplete editing.

In addition to these tests, the scientists subjected each line to whole-genome sequencing to find any variants that might interfere with Alzheimer’s research. KOLF2.1J carried no AD factors, and a polygenic score showed average risk for the disease. Overall, the line contained few potentially deleterious variants.

One occurred in DEDD, a caspase-interacting gene unrelated to disease, and another in SHOX, a gene active in skeletal cells. More relevant for neurological research, KOLF2.1J carries a splice-site disruption in the COL3A1 gene that encodes type III collagen, an essential component of the extracellular matrix (ECM). The disruption would likely create a nonfunctional protein, causing cells to make only half the normal amount of type III collagen. In people, this haploinsufficiency weakens blood vessels, causing one type of the connective tissue disorder Ehlers-Danlos syndrome. In the cell line, less type III collagen might affect studies of cell-ECM interactions, the authors noted. A fourth harmful mutation, in the DNA-binding protein ARID2, was present in the parent KOLF2 line, but was corrected by CRISPR to create KOLF2.1J.

Despite the line's relatively healthy genome, the researchers acknowledge some limitations. KOLF2.1J comes from a white man of European ancestry, so it may not reflect all the biology of diverse populations. Lines from women and other ancestries should also be developed, the authors noted. One such line already in use is WTC11, derived from a Japanese man and generated at the Gladstone Institutes in San Francisco. This line is publicly available from the Allen Cell Collection, which has created a series of WTC11 reporter strains (Roberts et al., 2017; Roberts et al., 2019). 

“We hope the workflow described in this study can serve as a blueprint to enable other groups to identify and promote additional reference iPSC lines,” the authors wrote. Data from the study is available, after registration, at the Alzheimer’s Disease Workbench, a global data-sharing resource maintained by the Alzheimer’s Disease Data Initiative. The genetic analysis is described online at NIH.—Madolyn Bowman Rogers


  1. We sincerely applaud this tour de force performed in the bioRXiv preprint by Pantazis, Yang, Lara, McDonough, Blauwendraat, and Peng et al., who have taken on the tremendous effort of deeply characterizing eight well-established iPSC lines for the identification of a single reference line that could be used across the dementia research community. Although the need for such a Rosetta line, akin to the selected subset of mouse strains being used in CNS research, has been called for in the past (Volpato and Webber, 2020), it is great to see that the first explicit selection of a single reference iPSC line has now been put forward and has been made easily accessible. It will be up to the dementia community to scrutinize the utility of this line over the years to come, but we foresee that the inclusion of such a Rosetta line across iPSC experiments could substantially aid in the inter- and cross-laboratory comparison of phenotypic outcomes of induced variants or perturbation experiments.

    Given the adoption of the KOLF2.1S line within the UK-DRI and other laboratories as a reference line, side-by-side comparison of the genetic and phenotypic differences with the newly established KOLF2.1J is now warranted. In that sense, it was a pity that the authors did not directly include this line in their study. Additionally, and as also alluded to in the manuscript, we would like to urge for expansion of a single reference line to a panel of reference lines, including genetically female lines and those of other ethnicities. Equality, diversity, and inclusion have every place in this panel design as genetically diverse reference panels will be pivotal in truly appreciating how genetic diversity can influence cell states, functional phenotypes, and enable the broadest translations. Given the polygenic nature of most neurodegenerative diseases, we may only be able to capture the precise (genetic) circumstances under which risk genes for dementia may exert their effect if we study them in a wide range of genetic backgrounds and sift out the drivers of genetic risk from the modulators. 

    —Caleb Webber is a co-author of this comment.


    . Addressing variability in iPSC-derived models of human disease: guidelines to promote reproducibility. Dis Model Mech. 2020 Jan 17;13(1) PubMed.

  2. This is a remarkable study by design, with extraordinary rigorous attention to detail and definition of genomic properties of cell lines to identify as neutral a cell line as is possible. The proposed reference cell line will be extremely useful to compare phenotypes and to generate -omic data for various differentiation, developmental, and gene-editing studies across laboratories. However, many studies on phenotypes related to late-onset disorders will likely require further extensive genetic and/or culture perturbations (e.g., stress, age acceleration, etc.), which, again, will introduce layers of variability. It should prove to be easier to interpret these against a well-characterized and neutral genetic “starting” background than for individual investigators to simply use cell lines that are at hand.

    It will be interesting to know whether this reference cell line is neutral against other late-onset neurodegenerative disorders, e.g., Parkinson’s Disease or ALS. It is challenging to try to study commonalities and differences between AD and other neurodegenerative disorders, and a reference cell line could be very helpful.

    For certain questions, e.g., to study the impact of aging on mid- or late-life disorders such as AD, or to study sex differences, this cell line may be insufficient. Several laboratories have published on iPSCs from sporadic late-life AD, with interesting findings, one noting that they have identified “person-specific processes” (e.g., Mertens et al., 2021Lagomarsino et al., 2021). However, pooling genomic or other data across studies of sporadic AD iPSC lines may be challenging, and such studies might be strengthened by this reference cell line as a control in experiments where skin biopsies or iPSCs from a variety of donors are to be studied. This comment has some parallels with mouse models. For example, the C57BL mouse, which has been extensively sequenced, is used as a wild-type or background strain for many genetic and breeding manipulations. However, genetic diversity in mouse strains can help to understand aging-related phenomena, as with the Mighty Mouse Models of Memory and it is likely that studies of iPSCs derived from different human donors will provide knowledge to shed light on complexities of AD.


    . Age-dependent instability of mature neuronal fate in induced neurons from Alzheimer's patients. Cell Stem Cell. 2021 Sep 2;28(9):1533-1548.e6. Epub 2021 Apr 27 PubMed.

    . Stem cell-derived neurons reflect features of protein networks, neuropathology, and cognitive outcome of their aged human donors. Neuron. 2021 Nov 3;109(21):3402-3420.e9. Epub 2021 Sep 1 PubMed.

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News Citations

  1. iNDI Aims to Standardize Human Stem Cell Research

Paper Citations

  1. . A systematic survey of loss-of-function variants in human protein-coding genes. Science. 2012 Feb 17;335(6070):823-8. PubMed.
  2. . Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. Epub 2017 Aug 16 PubMed.
  3. . Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs. Stem Cell Reports. 2019 May 14;12(5):1145-1158. Epub 2019 Apr 4 PubMed.

External Citations

  1. WTC11
  2. Allen Cell Collection
  3. Alzheimer’s Disease Workbench
  4. Alzheimer’s Disease Data Initiative
  5. NIH

Further Reading

Primary Papers

  1. . A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell Stem Cell. 2022 Dec 1;29(12):1685-1702.e22. PubMed. bioRxiv