Generation of 3X FLAG-tagged human embryonic stem cell (hESC) line to study WNT-induced β-catenin DNA interactions (HVRDe009-A-2)


In the canonical WNT signaling pathway, active WNT signaling results in the nuclear translocation of β-catenin where it regulates target gene expression. As a tool to understand these β-catenin DNA interactions, we used a CRISPR/Cas9 based approach to engineer a human embryonic stem cell line (hESC) harboring a 3X FLAG sequence fused to the C-terminus of β-catenin. Engineered cells displayed a characteristic hESC morphology, expressed pluripotency-associated markers, retained tri-lineage differentiation potential, and had a normal euploid karyotype. This cell line represents a valuable tool to dissect the transcriptional mechanisms by which WNT signalling regulates pluripotent cell fate.

Cutts, J., Kostes, W., & Brafman, D. A. (2021). Generation of 3x flag-tagged human embryonic stem cell (hesc) line to study wnt-induced β-catenin DNA interactions (HVRDE009-A-2). Stem Cell Research, 57, 102586.

APOE2 mitigates disease-related phenotypes in an isogenic hiPSC-based model of Alzheimer’s disease


Genome-wide association studies (GWAS) have identified polymorphism in the Apolipoprotein E gene (APOE) to be the most prominent risk factor for Alzheimer’s disease (AD). Compared to individuals homozygous for the APOE3 variant, individuals with the APOE4 variant have a significantly elevated risk of AD. On the other hand, longitudinal studies have shown that the presence of the APOE2 variant reduces the lifetime risk of developing AD by 40 percent. While there has been significant research that has identified the risk-inducing effects of APOE4, the underlying mechanisms by which APOE2 influences AD onset and progression have not been extensively explored. In this study, we utilize an isogenic human induced pluripotent stem cell (hiPSC)-based system to demonstrate that conversion of APOE3 to APOE2 greatly reduced the production of amyloid-beta (Aβ) peptides in hiPSC-derived neural cultures. Mechanistically, analysis of pure populations of neurons and astrocytes derived from these neural cultures revealed that mitigating effects of APOE2 are mediated by cell autonomous and non-autonomous effects. In particular, we demonstrated the reduction in Aβ is potentially driven by a mechanism related to non-amyloidogenic processing of amyloid precursor protein (APP), suggesting a gain of the protective function of the APOE2 variant. Together, this study provides insights into the risk-modifying effects associated with the APOE2 allele and establishes a platform to probe the mechanisms by which APOE2 enhances neuroprotection against AD.

Brookhouser, N., Raman, S., Frisch, C., Srinivasan, G., & Brafman, D. A. (2021). APOE2 mitigate disease-related phenotypes in an isogenic hipsc-based model of alzheimer’s disease. Molecular Psychiatry, 26(10), 5715-5732.

A transient reporter for editing enrichment (TREE) in human cells


Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.

Standage-Beier, K., Tekel, S. J., Brookhouser, N., Schwarz, G., Nguyen, T., Wang, X., & Brafman, D. A. (2020). A transient reporter for editing enrichment (tree) in human cells. Nucleic Acids Research, 48(3), 1602–1602.

Generation and characterization of two human induced pluripotent stem cell (hiPSC) lines homozygous for the Apolipoprotein e4 (APOE4) risk variant-Alzheimer’s disease (ASUi005-A) and healthy non-demented control (ASUi006-A)


Although the majority of late-onset Alzheimer’s disease (AD) patients are labeled sporadic, multiple genetic risk variants have been identified, the most powerful and prevalent of which is the e4 variant of the Apolipoprotein E (APOE) gene. Here, we generated human induced pluripotent stem cell (hiPSC) lines from the peripheral blood mononuclear cells (PBMCs) of a clinically diagnosed AD patient [ASUi005-A] and a non-demented control (NDC) patient [ASUi006-A] homozygous for the APOE4 risk allele. These hiPSCs maintained their original genotype, expressed pluripotency markers, exhibited a normal karyotype, and retained the ability to differentiate into cells representative of the three germ layers. Resource table.

Brookhouser, N., Zhang, P., Caselli, R., Kim, J. J., & Brafman, D. A. (2018). Generation and characterization of two human induced pluripotent stem cell (hipsc) lines homozygous for the apolipoprotein E4 (APOE4) risk variant—alzheimer’s disease (ASUI005-A) and healthy non-demented control (ASUI006-A). Stem Cell Research, 32, 145–149.

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