A Novel Cost-Effective Approach to Derivation of Induced Pluripotent Stem Cells from Epstein-Barr Virus Immortalized β-Lymphoblastoid Cell Lines

Poster Presentation
Friday, May 11, 2018: 5:30 PM-7:00 PM
Hall Grote Zaal (de Doelen ICC Rotterdam)
S. J. Walker1, D. Mack2 and A. Wagoner1, (1)Wake Forest University Health Sciences, Winston Salem, NC, (2)University of Washington, Seattle, WA
Background: Autism spectrum disorders (ASD) may be viewed as a collection of heterogeneous disorders that are currently diagnosed based upon DSMV criteria. The vast phenotypic differences that can exist from individual to individual suggest that the underlying etiologies are complex and likely involve multiple genetic and environmental inputs. Given that the availability of tissue, especially brain tissue, from ASD patients is extremely limited, the development of alternative tools to investigate molecular and neurobiological mechanisms is critically important. One key resource for ASD research is immortalized lymphoblastoid cell line (LCLs) banks generated from proband and family member blood samples. Induced pluripotent stem cells (iPSC), derived from these LCLs, can be to generate patient-specific neurons for use in downstream mechanistic studies.

Objectives: The objective of this study was to use LCLs to generate iPSCs for the downstream study of neurobiological aspects of ASD. The availability of a streamlined, standardized, reproducible, cost-effective, and efficient approach will render the use of well-characterized LCLs for ASD research a gold standard.

Methods: We obtained LCLs from two males (proband and sibling) from the Autism Genetics Resource Exchange (AGRE) and two males (proband and parent) from the NIMH Repository and Genomics Resource (Phelan-McDermid Syndrome [PMDS] patients). IPSCs were generated from all 4 EBV-LCLs by transfection with Epi5 Episomal iPSC reprogramming plasmids. For the first 7 days after transfection, cells were cultured on Matrigel-coated plates in N2B27 based medium. After 7 days, the reprogramming cells were cultured in Essential-8 medium until ready for passaging. PCR, RT-PCR, immunocytochemistry, and a novel assay, the Taqman® human pluripotent stem cell Scorecard™ Panel were used to fully validate endogenous pluripotency of all iPSC clones generated.

Results: IPSCs, generated via transfection with Epi5 Episomal iPSC reprogramming plasmids, were apparent as early as Day 8 post-transfection and ready for propagation as early as Day 18. IPSCs derived from LCLs obtained from the AGRE were propagated to passage 23 first, followed by the PMDS lines, which are currently at passage 10. Two clonal iSPC lines per original LCL were evaluated for normal karyotype, expression of pluripotency markers, and loss of OriP/EBNA-1 expression vectors. We have confirmed these iPSC clones are plasmid-free and EBV-free. Three of four clones had the expected expression of cell-autonomous pluripotency genes and normal karyotype. All clonal lines were allowed to spontaneously differentiate into embryoid bodies and were assayed for pluripotency markers and germline-specific transcripts using the Taqman® human pluripotent stem cell Scorecard™ Panel. Results indicated that all four iPSC lines are suitable for neuronal differentiation protocols.

Conclusions: This protocol describes a reproducible method to efficiently generate iPSCs with standardized and cost-effective reagents. IPSCs produced following this improved protocol can be used to generate and evaluate novel in vitro models to study a plethora of previously inaccessible neuronal cell types that underlie pathological mechanisms in ASD.