GENERATION OF IPSCS DISEASE MODEL DERIVED FROM THREE ALSP PATIENTS WITH CSF1R MUTATION

Abstract

Background & Aim: Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is an autosomal dominant inherited rare neurodegenerative disease that causes lesions in the white matter of the brain and presents with dementia-like symptoms such as memory loss, motor impairment, depression, and personality changes. Colony-stimulating factor 1 receptor (CSF1R) is mainly expressed in microglia, and it is known that mutation in the tyrosine kinase of this gene results in dysfunction of microglia and causes ALSP. However, the specific pathomechanism is still unknown. Induced pluripotent stem cells (iPSCs) are pluripotent cells that transduce specific factors (Oct4, Sox2, Klf4, C-myc) into adult somatic cells. Since human-derived iPSCs have the same genetic information as donors and can differentiate into a specific cell type, they can be used as disease modeling to search for the pathomechanism or as drug screening tool to test potential treatment of ALSP. In this study, we aimed to establish an iPSC disease model derived from three ALSP patients and generate CSF1R gene-corrected isogenic iPSC lines. Methods, Results & Conclusion: We isolated peripheral blood mononuclear cells (PBMCs) from 3 patients with ALSP and 1 normal control. The mutation sites of three ALSP patients were NM_005211.3(CSF1R):c.2546_2548delTCT, NM_005211.3(CSF1R):c.1765G>A, and NM_005211.3(CSF1R):c.2345G>A. To generate iPSC lines, PBMCs were transduced with reprogramming factors using Sendai virus. In addition, we edited the CSF1R mutation to a normal genotype by using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. To characterize the iPSC lines, we confirmed that each iPSC line had normal karyotype and expressed pluripotent stem cell (PSC) markers such as OCT4, SOX2, SSEA4, and Tra-1-60. We also investigated the potential of iPSC lines to form embryoid bodies and differentiate into 3 germ layers by staining with specific markers (ectoderm-SOX1, mesoderm-BRACHYURY, endoderm-GATA-4). Each iPSC line had the same CSF1R mutation as the PBMC donor and Sendai virus was no longer detected. The iPSC lines from ALSP patients were corrected to normal CSF1R gene and no off-target effects have been observed. In this study, we successfully establish the ALSP-iPSCs disease model and CSF1R gene-corrected isogenic iPSCs using PBMC from three patients. In further studies, theses iPSC lines can be used as a platform to study the pathological mechanisms of ALSP.