PINE-TREE enables highly efficient genetic modification of human cell lines.

Prime editing technologies enable precise genome editing without the caveats of CRISPR nuclease-based methods. Nonetheless, current approaches to identify and isolate prime-edited cell populations are inefficient. Here, we established a fluorescence-based system, prime-induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE), for real-time enrichment of prime-edited cell populations. We demonstrated the broad utility of PINE-TREE for highly efficient introduction of substitutions, insertions, and deletions at various genomic loci. Finally, we employ PINE-TREE to rapidly and efficiently generate clonal isogenic human pluripotent stem cell lines, a cell type recalcitrant to genome editing.

A microcarrier-based protocol for scalable generation and purification of human induced pluripotent stem cell-derived neurons and astrocytes.

Here, we describe a protocol for a microcarrier (MC)-based, large-scale generation and cryopreservation of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes. We also detail steps to isolate these populations with a high degree of purity. Finally, we describe how to cryopreserve these cell types while maintaining high levels of viability and preserving cellular function post-thaw. For complete details on the use and execution of this protocol, please refer to Brookhouser et al. (2021).

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 (Aß) 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 Aß 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.

Human Autopsy-Derived Scalp Fibroblast Biobanking for Age-Related Neurodegenerative Disease Research.

The Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program at Banner Sun Health Research Institute (BSHRI) is a longitudinal clinicopathological study with a current enrollment of more than 900 living subjects for aging and neurodegenerative disease research. Annual clinical assessments are done by cognitive and movement neurologists and neuropsychologists. Brain and body tissues are collected at a median postmortem interval of 3.0 h for neuropathological diagnosis and banking. Since 2018, the program has undertaken banking of scalp fibroblasts derived from neuropathologically characterized donors with Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Here, we describe the procedure development and cell characteristics from 14 male and 15 female donors (mean ± SD of age: 83.6 ± 12.2). Fibroblasts from explant cultures were banked at passage 3. The results of mRNA analysis showed positive expression of fibroblast activation protein, vimentin, fibronectin, and THY1 cell surface antigen. We also demonstrated that the banked fibroblasts from a postmortem elderly donor were successfully reprogramed to human-induced pluripotent stem cells (hiPSCs). Taken together, we have demonstrated the successful establishment of a human autopsy-derived fibroblast banking program. The cryogenically preserved cells are available for request at the program website of the BSHRI.