Generation of 3 patient induced Pluripotent stem cell lines containing SORD mutations linked to a recessive neuropathy

The SORD neuropathy has been identified as the most common autosomal recessive inherited neuropathy, occurring in thousands of patients worldwide. Fibroblast lines from 3 different patients containing the c.753delG; p.Ala253GlnfsTer27 SORD mutations were reprogrammed into induced Pluripotent Stem Cell (iPSC) lines. These iPSC lines demonstrate an apparent normal karyotype and have positive expression of pluripotency markers. These iPSC lines also stain positively for Ectoderm, Endoderm and Mesoderm markers following Embryoid body differentiation. These lines pose to serve as a valuable disease modeling resource for studying the SORD neuropathy, including studying disease phenotype and treatment efficacy.


Resource Details
The SORD neuropathy is caused by biallelic mutations to the gene SORD, encoding sorbitol dehydrogenase, leading to the arrest of the polyol pathway and the development of a peripheral neuropathy (Cortese, 2020).This recessive neuropathy is also the focus on an ongoing clinical trial looking at the efficacy of Aldose Reductase Inhibitor (ARI) treatment in patients (NCT05397665).Currently there are several thousand patients identified with these mutations and with the ongoing trial, the need to study and understand cellular phenotypes is ever growing.
The 3 patient fibroblast lines containing the biallelic SORD mutations were plated into duplicate 6-well plates and grown to around 60 % confluency.The duplicate plates were counted prior to the reprogramming protocol to calculate for virus titers.The fibroblasts were transduced using the Sendai Virus 2.0 Reprogramming kit, containing vectors encoding KLF4, C-Myc, OCT3/4 and SOX2.After 7 days, cells were replated onto 10 cm 2 plates and allowed to grow with daily media changes.Following approximately 3-4 weeks of culturing and allowing colonies to form, several potential colonies were then chosen for each line and expanded (Table 2).A single colony for each line was then selected and characterized.Brightfield imaging of iPSC lines shows apparent normal iPSC morphology (Fig. 1, Panel A).Pluripotency staining for NANOG, SSEA3, OCT4 and SOX2 was performed, with each line showing successful expression of the markers (Fig. 1, Panel B).Each of these markers shows a high rate of staining efficiency (Fig. 1, Panel C), indicating successful reprogramming.
Karyotyping performed by Cell Line Genetics shows all iPSC lines have an apparent normal karyotype (Fig. 1, Panel D, Supplementary File 1).STR analysis was performed on the lines and confirmed their origins from the patient fibroblast lines (Supplementary File 2).All 3 lines were sequenced for the SORD gene and the mutations were confirmed present in each patient line (Fig. 1, Panel E). iPSC lines underwent embryoid body differentiation to demonstrate differentiation potential.Each of the lines show successful staining for Ectoderm (TUBB3, PAX6), Mesoderm (TBXT, SMA), and Endoderm (FOXA2, AFP) markers, demonstrating their potential to be differentiated.(Fig. 1, Panel F).
RT-PCR of iPSC lines for SeV based vectors shows successful removal of SeV vectors from the 3 iPSC lines (Fig. 1, Panel G), using an early passage (passage 3) clone as a positive PCR control.Mycoplasma testing of the lines confirmed that no mycoplasma species are present (Fig. 1, Panel H).
In ongoing studies in our lab, these cells have been successfully differentiated into Lower Motor Neurons reproducibly, following an in-house differentiation protocol (Saporta 2015), further demonstrating their differentiation potential and usefulness as a resource in studying the SORD neuropathy.The differentiated cells also replicate an important disease phenotype, increased intracellular sorbitol content, that is seen in patients and animal models (Zhu, 2023).

Reprogramming from human fibroblasts
Donor fibroblasts containing the same biallelic SORD mutations were grown to 60 % confluency in 6-well plate format using DMEM containing 10 % FBS.These fibroblasts were transduced using the Cytotune 2.0 Sendai Virus kit, with wells plated in parallel used to calculate appropriate viral titer.Cells were then replated into 10 cm 2 dishes and cultured until colony formation was noticed.Colonies were selected based off morphology and plated into dishes coated with Matrigel.Media was transitioned to mTeSR + .Cells were passaged using 0.5 mM EDTA at a 1:5 ratio.Cells were grown at 37°C and 5 % CO 2 .

Immunocytochemistry
iPSCs (passage 13) were fixated using 4 % paraformaldehyde at room temperature for 15 min, permeabilized using 0.2 % PBS-T (Triton X-100) and blocked with 5 % BSA in PBS.Primary antibodies were diluted in 5 % BSA and incubated at 4C overnight.Secondary antibodies were diluted in 5 % BSA and incubated for 1 h at room temperature.DAPI was applied for 15 min and cells were then covered with PBS prior to imaging.Cells stained for AFP and SMA were fixated using cold methanol for 15 min prior to permeabilization.

Karyotype and STR analysis
Cells at passage 14 were shipped to Cell Line Genetics for karyotype analysis.iPSC cells were pelleted from culture, along with the parent fibroblast lines, with DNA extraction and STR analysis being performed by the University of California Berkeley DNA Sequencing Facility.

Analysis of mutations
DNA was isolated using the Qiagen DNeasy kit and an in-house PCR protocol for the SORD gene was performed to amplify fragments.PCR products were sent to Eurofins Scientific for Sanger Sequencing, with data being analyzed in our lab.We were able to confirm that all iPSC lines contained the biallelic SORD mutations.

Embryoid body differentiation
iPSCs (passage 15) were grown to 60-70 % confluency in 10 cm 2 plates before being split into untreated 10 cm 2 plates to allow a single cell suspension.Cells were then grown in Embryoid Body media (KO DMEM, 20 % KOSR, 1 % Glutamax, 1 % NEAA) with 10 μM Rock Inhibitor Y27632 and 100 μM Beta-mercaptoethanol, with media being changed every 48 h.This was done until Day 8, where the cell culture was pelleted and split into 96well plates treated with Matrigel for attachment, with the plates being fixed with 4 % paraformaldehyde after 48 h.

Mycoplasma testing
iPSC lines (passage 15) were plated in 6-well format and media was collected following 72 h of culture.Media was tested using the MycoAlert PLUS Mycoplasma Detection Kit (LONZA) including the MycoAlert Assay Control Set as a positive control.

Table 1
Characterization Details.
Stem Cell Res.Author manuscript; available in PMC 2024 August 01.