Cytotune 2.0 kit

PBMCs or fibroblasts obtained either from human fetal liver tissues or human adult skin were isolated after collagenase dissociation and reprogrammed into iPSCs with the integration-free based Sendai virus (Cytotune 2.0 kit catalog #A16517 from Life Technologies). Fibroblasts were used at low population doubling (<5) to insure high efficiency of reprogramming. Emerging hiPSC colonies were manually picked and cultured under feeder-free conditions in Essential 8 medium on Geltrex-coated dishes (Life Technologies). hiPSC clones were maintained in Essential 8 Flex medium (Life Technologies) in feeder-free conditions and passaged at least 15 times to increase stable pluripotency. hiPSC generation and characterization were performed in the iPSC cell reprogramming core facility of CHU Sainte-Justine. hiPSC colonies were stained with antibodies for anti-human SSEA-4, Sox2, OCT4, and TRA1-60 followed by incubation with appropriate ALEXA-conjugated secondary antibodies using the pluripotent Stem Cell 4-Marker Immunocytochemistry Kit following the manufacturer's instructions (catalog #A24881 from Life Technologies). Karyotypes were produced by G-banding and analyzed by the CHU Sainte-Justine Cytogenetic Department.

One control and one GRN (GRN #3) fibroblast line were reprogrammed into iPSCs using the Cytotune 2.0 Kit (Life Technologies) per the manufacturer’s protocol. In brief, early passage fibroblast (P < 10) were grown to approximately 50–80 % confluency in fibroblast medium consisting of 10 % ES-qualified FBS (Life Technologies, 0.1 mM NEAA, 55 μM β-mercaptoethanol, high glucose DMEM (Life Technologies). On Day 0, fibroblasts were transduced with three Sendai viruses encoding Klf4–Oct3/4–Sox2 (KOS), hc-Myc, and hKlf4, each at an MOI of 5). Cells were fed with fibroblast medium every other day for 7 days. On day 7, cells were passaged onto vitronectin (Life Technologies) coated dishes at a density of 2.5 × 10⁵ - 5.0 × 10⁵ cells/well. Beginning on day 8, cells were fed every day in Essential 8 medium (Life Technologies). Resulting iPS colonies were manually picked and transferred to a dish coated with either vitronectin or Matrigel (BD). iPSCs were maintained on Matrigel coated dishes and mTesR1 medium (Stem Cell Technologies) and passaged every 5–7 days.

Total RNA was extracted using RNeasy® columns and DNAse-treated using RNase-free DNase (Qiagen). For quantitative PCR, first-strand complementary DNAs (cDNAs) were generated using 500 ng of RNA, M-MLV reverse transcriptase (Invitrogen), 25 µg/ml polydT and 9.6 µg/ml random primers (Invitrogen).
To quantitate transcripts, absolute quantitative PCR was performed on a Viia 7 (Applied Biosystems) using power SYBR green PCR master mix (Applied Biosystems), for genes listed in the primers table (Supplementary Table 3). For each sample, the ratio of specific mRNA level relative to GAPDH levels was calculated. Experimental results are shown as levels of mRNA relative to the highest value.
All quantitative real-time PCR primers have a hybridization temperature of 60 °C and their sequences are listed in Supplementary Table 3. All amplicons span two adjacent exons. SeV primers are from Life Technologies (cytotune 2.0 kit).

Fibroblasts were first isolated either from human fetal liver tissues or human skin after collagenase dissociation. Single cell fibroblasts cultures were then reprogrammed into hiPSCs using integration‐free Sendai virus (Cytotune 2.0 kit catalog # A16517 from Life Technologies). Fibroblasts were used at low population doubling (between 5 and 10) to increase efficiency of reprogramming. Emerging colonies from transduced cells were manually picked and cultured under feeder‐free conditions in Essential 8 and Essential 8 Flex medium on Geltrex‐coated dishes (Life Technologies). hiPSC clones were passaged at least 15 times to increase stable pluripotency. hiPSC generation and characterization were done in the iPSC‐cell reprogramming core facility of CHU Sainte‐Justine. hiPSC colonies were stained with the antibodies for anti‐human SSEA‐4, Sox2, OCT4, and TRA1‐60 overnight at 4°C using the pluripotent Stem Cell 4‐Marker Immunocytochemistry Kit (catalog # A24881 from Life Technologies), followed by incubation with an ALEXA secondary antibodies for 30 minutes at room temperature. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Karyotypes were produced by G‐banding and analyzed by the CHU Ste‐Justine cytogenetic department.

Peripheral blood samples from patients were obtained from the IRCCS Neuromed of Pozzilli through informed consent approved by the local Ethical Committee (Clinical Trials: #NCT03682458; ID: CGM-02). Primary peripheral blood mononuclear cells (PBMCs) were isolated from the blood withdrawals and reprogrammed by non-integrating Sendai viruses expressing the four Yamanaka’s factors using the CytoTune-2.0 kit (Thermo Fisher) at the IRCCS San Raffaele Hospital in Milan. iPSC lines were maintained in feeder-free conditions in mTeSR1 (Stem Cell Technologies) and expanded in HESC-qualified Matrigel (Corning)-coated six-well plates.