Tissue processing and cell culture
Tissue samples
De-identified human tissue samples were collected with previous patient consent in strict observance of the legal and institutional ethical regulations. Protocols were approved by the Human Gamete, Embryo and Stem Cell Research Committee (Institutional Review Board) at the University of California, San Francisco.
The Primate Center at the University of California, Davis, provided four specimens of cortical tissue from PCD60 (n = 1), PCD75 (n = 1) and PCD80 (n = 2) rhesus macaques. All animal procedures conformed to the requirements of the Animal Welfare Act, and protocols were approved before implementation by the Institutional Animal Care and Use Committee at the University of California, Davis.
Cell culture and lentiviral transduction
For cryopreservation, tissue samples were cut into small pieces, placed in Bambanker (NIPPON Genetics, BB02), frozen in CoolCell at −80 and transferred to liquid nitrogen within 2 weeks. For single-cell dissociation, each cryovial was thawed in 37 °C warm water and placed in a vial containing a pre-warmed solution of Papain (Worthington Biochemical Corporation, catalogue no. LK003153) solution supplemented with 5% trehalose (Fisher Scientific, catalogue no. BP268710) that was prepared according to the manufacturer protocol for 10 min at 37 °C. After approximately 30 min incubation, tissue was triturated following the manufacturer protocol. Cells were plated on a 24-well tissue culture dish coated with 0.1% PEI (Sigma, catalogue no. P3143), 5 μg ml−1 Biolamina LN521 (Invitrogen, catalogue no. 23017-015) and at a density of 500,000 cells cm−2. Expansion medium contained insulin (Thermo Fisher, catalogue no. A1138IJ), transferrin (Invitria, catalogue no. 777TRF029-10G), selenium (Sigma, catalogue no. S5261-10G), 1.23 mM ascorbic acid (Fujifilm/Wako, catalogue no. 321-44823), 1% polyvinyl alcohol (PVA) (Sigma, catalogue no. P8136-1KG), 100 μg ml−1 primocin (Invivogen, catalogue no. ant-pm-05), 20 ng ml−1 fibroblast growth factor 2 (Preprotech, catalogue no. 100-18B) and 20 ng ml−1 EGF (Preprotech, catalogue no. AF100-15) in DMEM-F12 (Corning, catalogue no. MT10092CM), supplemented with ROCK inhibitor CEPT cocktail105. For lentiviral transduction, CRISPRi and STICR lentivirus was added to culture media on day 5 at roughly 1:500 and 1:5,000 dilution, respectively. After 6 h, the virus-containing medium was removed and replaced with fresh medium. Seven days after infection, expansion medium was removed and replaced with differentiation medium containing insulin-transferrin-selenium, 1.23 mM ascorbic acid, 1% PVA, 100 μg ml−1 primocin, 20 ng ml−1 BDNF (Alomone Labs, catalogue no. B-250) in DMEM-F12. At 7 days after differentiation, cultures were dissociated using papain supplemented with 5% trehalose and GFP-positive or GFP and mCherry co-positive cells were isolated by fluorescence-activated cell sorting (FACS) on BD Aria Fusion, resuspended in 0.2% bovine serum albumin (BSA) in PBS and captured with 10x Chromium v.3.1 HT kit (catalogue no. PN-1000348) or Illumina Single Cell CRISPR Library kit (Illumina Single Cell CRISPR Prep, T10, catalogue no. 20132435).
Organotypic slice culture and lentiviral transduction
For organotypic slice culture experiments, samples were embedded in 3% low-melting-point agarose (Fisher, catalogue no. BP165-25) and then cut into 300-μm sections perpendicular to the ventricle on a Leica VT1200S vibrating blade microtome in oxygenated artificial cerebrospinal fluid containing 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 and 1.25 mM NaH 2 PO 4 . Slices were cultured slice medium containing in insulin-transferrin-selenium, 1.23 mM ascorbic acid, 1% PVA, 100 μg ml−1 primocin, Glutamax (Invitrogen, catalogue no. 35050061), 1 mg ml−1 BSA, 15 μM uridine (Sigma, catalogue no. U3003-5G), 1 μg ml−1 reduced glutathione (Sigma), 1 μg ml−1 (+)-α-tocopherol acetate (Sigma, catalogue no. t3001-10g), 0.12 μg ml−1 linoleic (Sigma, catalogue no. L1012) and linolenic acid (Sigma, catalogue no. L2376), 10 mg ml−1 docosahexaenoic acid (Cayman, catalogue no. 10006865), 5 mg ml−1 arachidonic acid (Cayman, catalogue no. 90010.1), 20 ng ml−1 BDNF in DMEM-F12. CEPT cocktail was added on the first day. Lentiviral transduction was performed on the following day locally at the germinal zone to preferentially label neural progenitor cells. CRISPRi and STICR lentivirus was added at 1:20 and 1:100 dilution, respectively. At 24 h after transduction, virus-containing medium was replaced with fresh medium and daily half-medium replacement was performed. At 12–14 days after transduction, cultures were dissociated using papain supplemented with 5% trehalose, and GFP- and mCherry co-positive cells were isolated by FACS and captured with 10x Chromium v.3.1 HT kit (catalogue no. PN-1000348).
Immunocytochemistry
Cells were fixed in 2% paraformaldehyde for 15 min at room temperature and then in ice cold 90% methanol for 10 min. After a 1-h incubation in blocking solution (5% BSA, 0.3% Triton-X in PBS), cells were incubated with primary antibodies with the following dilution in blocking solution at room temperature for 1 h: mouse-EOMES (Thermo Fisher, catalogue no. 14-4877-82) 1:500, rabbit-NEUROD2 (Abcam, catalogue no. ab104430) 1:500, goat-SOX9 (R&D, catalogue no. AF3075) 1:300, mouse-KI67 (BD, catalogue no. 550609) 1:500, mouse-DLX2 (Santa Cruz Biotechnology, catalogue no. sc-393879) 1:200. The cells were then washed with PBS three times and incubated with secondary antibodies in blocking solution for 1 h at room temperature, and counterstained with 4′,6-diamidino-2-phenylindole. Finally, after PBS washes, digital image acquisition was performed using an Evos M7000 microscope and Evos M7000 Software.
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