(P 10) GENERATION OF A CHROMOSOME 8 GAIN;NF1-/- HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED SCHWANN CELL PRECURSOR MODEL

Objective: Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by a germline mutation in the NF1 gene, and somatic loss of the second copy of NF1 within Schwann cell precursors (SCPs) contributes to the formation of plexiform neurofibromas (PNs), benign tumors which occur in 30-50% of patients with NF1. PNs possess the potential to progress into malignant peripheral sheath tumors (MPNSTs). MPNSTs are an aggressive form of cancer and a leading cause of mortality in NF1 patients. The current treatment modalities for MPNSTs typically involve a combination of surgery, radiation therapy, and chemotherapy. Although the outlook for MPNSTs remains difficult, identifying the particular cellular lineage responsible for tumor development is crucial for improving treatment approaches. To explore the genomic complexity of MPNSTs, our lab previously analyzed a collection of NF1-MPNST patient-derived xenografts (PDX). Chromosome 8 gain was identified as a nearly universal event in MPNSTs through our analyses of copy number variation, whole exome sequencing, and RNA sequencing. This genomic alteration was also associated with lower overall survival rates. In order to investigate and gain a comprehensive understanding of the impact of Chr8 gain, we employed an isogenic human induced pluripotent stem cell (hiPSC) system, generating four distinct cell lines and differentiating them directly into SCPs.
Our objectives in this research are: 1) to investigate the impact of Chr8 gain on the formation of malignant peripheral nerve sheath tumors (MPNSTs) after successful differentiation of human induced pluripotent stem cells (hiPSCs) into Schwann cell precursors (SCPs). 2) we aim to assess cell proliferation and survival in our cell lines with and without Chr8 gain. 3) in the future, we plan to evaluate the tumor volume progression in vivo to assess the effect of Chr8 gain.

Methods: A mosaic population of fibroblasts with trisomy 8 was obtained from Coriell and subsequently single-cell cloned to establish both wildtype (WT) and trisomy 8 (Chr8 gain) populations. The Genome Engineering & Stem Cell Center (GESC@MGI) at Washington University assisted in reprogramming these cells into human induced pluripotent stem cells (hiPSCs). NF1 knockout lines were also generated by GESC@MGI using synthetic gRNA, and CRISPR/Cas9 to introduce NF1 mutations into these cells. WT, Chr8 gain, NF1-/-, and Chr8 gain; NF1-/- iPSC cell lines were differentiated into Schwann cell precursors (SCPs) using a differentiation protocol that included TGF-beta inhibitor (SB431542), GSK-3 inhibitor (CT099021), N2, and B27 supplements. NRG-1 was added at day 6 of the protocol. The iPSC and SCP states of the cell lines were verified with qPCR after the completion of the differentiation protocol. Cell survival and proliferation of SCPs were assessed by Incucyte cell survival and CellTiter-Glo® 2.0 Cell Viability Assay.

Results: First, we validated the iPSCs with FISH and karyotyping. High expression of iPSC markers OCT3/4 and SOX2 was observed in all four iPSC lines. Second, after completing the differentiation protocol, we observed high expression of SCP markers GAP43 and ITGA4 in WT, NF1-/-, and Chr8 gain;NF1-/- SCP lines compared to their iPSC state, consistent with their morphology. However, the Chr8 gain iPSC line was unable to survive the differentiation process to SCPs, consistent with detrimental effects of aneuploidy in the absence of other oncogenic changes. Third, we observed that Chr8 gain; NF1-/- SCPs proliferate faster compared to WT and NF1-/- SCPs, and have enhanced survival compared to WT. Taken together, these findings support that Chr8 gain improves proliferation and cell survival in the setting of NF1 loss.

Conclusion: We have successfully generated isogenic NF1-/- and WT hiPSC cell line system with and without Chr8 gain.
Validated differentiation of hiPSCs into SCPs for all four genotypes.
Began characterization of all four cell lines: Chr8 gain improves proliferation and cell survival in the setting of NF1 loss.