Human apob

Human induced pluripotent stem cell (iPSC)-derived motor neurons were purchased from iXCells Biotechnologies and plated at a density of ∼86 000 cells/cm² in 8-well chamber slides coated with 80 µg/µL Matrigel in DMEM/F-12. Motor neurons were cultured in Motor Neuron Maintenance Medium (iXCells Biotechnologies) for 8 days. On Day 8, human ApoB (Millipore), human MOG (Sigma-Aldrich), human haptoglobin (abcam), human ApoC-III (Athens Research & Technology), and human ApoE (Novus Biologicals) proteins were applied at 0.01 µg/µL or 0.05 µg/µL in media, or 50% sALS CSF and ApoB-depleted sALS CSF diluted in media were applied. In a separate ApoB titration experiment, lower doses of 0.005 ng/µL or 0.01 ng/µL ApoB were applied. At 24 h pre- and post-treatment, cells were visualized using a 10X objective on an Olympus IX71 microscope and bright field images were captured on a Hamamatsu Orca-Spark camera using Cell Sens Dimension Programme. Motor neurons were fixed with 4% PFA for immunocytochemistry at 24 h post-treatment. Using ImageJ, areas of ChAT⁺ motor neuron clusters were quantified from three images per well and then averaged.

Adult female C57BL/6J mice (aged 8–12 weeks) purchased from The Jackson Laboratory (Bar Harbor, ME) were used in all in vivo experiments. All procedures were approved by the Institutional Animal Care and Use Committee at Mispro Biotech Services (New York). Prior to surgery, mice were anaesthetized with a ketamine (110 mg/kg) and xylazine (10 mg/kg) cocktail and received subcutaneous injections of 0.1 mg/kg buprenorphine, 2.5 mg/kg baytril and 1 ml 0.9% saline. Laminectomies at cervical levels 4 (C4) and 5 (C5) were performed to expose the underlying spinal cord. A 32-gauge Hamilton syringe was inserted underneath the dura mater and 3 µL saline, total CSF, or individual human CSF proteins were slowly injected into the subarachnoid space. Proteins injected included the following: human ApoB purified from plasma (Millipore), human MOG protein 35–55 (Sigma-Aldrich), native human haptoglobin (abcam), human ApoC-III purified from plasma (Athens Research & Technology), recombinant human ApoE (Novus Biologicals) and recombinant human CHIT1 (ABclonal). A minimum of three mice were injected per unpooled, individual patient CSF sample or protein sample. Mice were assigned to different treatment groups in a randomized manner.