E-MTAB-6057:Foetal19wkGFPplus1_p
NextSeq 500 paired end sequencing; RNA-seq of isolated human foetal and hiPSC L/M-opsin cone photoreceptor populations labelled using the AAV2/9.pR2.1.GFP reporter
RNA-seq of isolated human foetal and hiPSC L/M-opsin cone photoreceptor populations labelled using the AAV2/9.pR2.1.GFP reporter
Foetal19wkGFPplus1_p
Human foetal eyes were obtained from the Joint Medical Research Council UK (grant # G0700089)/Wellcome Trust (grant # GR082557) Human Developmental Biology Resource (http://www.hdbr.org/. Human foetal retinae were dissected under sterile conditions to remove all surrounding ocular tissue using stainless steel 8mm blade curved micro scissors and 0.1mm straight forceps (World Precision Instruments). Human foetal retinal explants and iPSC-derived retinal differentiation cultures were dissociated enzymatically into a single cell suspension using the papain dissociation system (Worthington Biochemical, Lorne Laboratories) following manufacturer's protocol. For large foetal retinae (16-22pcw), samples were cut into smaller pieces (5mm x 5mm) before the addition of papain (approx. 100µl per piece). For retinal differentiation cultures, tissue was cut into smaller pieces using micro dissection scissors before adding 1ml of papain per well of 6 well culture plate. 70µm cell strainers were used to ensure cell single density of cell suspension. For GFP cell isolation, cells were resuspended in a final volume of 500µl blocking solution (1% (w/v) BSA in PBS), whereas cells for immunocytochemistry were resuspended in 1ml of blocking solution and counted using a haemocytometer before being diluted to 1x10⁶ cells per 100µl.  A human induced pluripotent stem cell line (NCUS:7) was generated by Cambridge BRC hiPSC Core Facility from skin fibroblasts from an individual with normal ophthalmological examination. iPSCs were cultured on irradiated mouse embryonic fibroblast layer (GlobalStem) in knockout serum replacement medium (KSR; DMEM/F12 1:1, Knockout Serum Replacement (KOSR), MEM non-essential amino acids, glutamax, β-mercaptoethanol) with 4ng/ml fibroblastic growth factor (FGF; Peprotech). The retinal differentiation protocol for transcriptome profiling stem cell-derived cones was based on Meyer et al., 2009 (Protocol 1). iPSC colonies were enzymatically lifted using dispase (Life Technologies) on Day 0 and transfer into T25 flasks containing KSR medium without FGF to induce the formation of embryoid bodies (EBs). Medium was changed daily before substituting into neural induction medium (NIM) containing DMEM/F12 1:1 (Gibco), MEM non-essential amino acids (Gibo), N2 supplement (Gibo), heparin (Sigma) on Day 4. To promote neural rosette formation, EBs were transferred onto 30% laminin (Sigma) coated plates in NIM on Day 6, which appeared by differentiation day 8/9. Cultures were then fed every 2 days with NIM until Day 16, where the media was substituted for retinal differentiation media (RDM; DMEM (Gibo), F12 (Gibo) and B27 supplement (Gibo). Optic vesicle structures were visible from Day 18-20 onwards and media was changed every 2-3 days. 10% FBS (Gibo) was added to cultures at week 14 for late stage cultures. Intact human foetal retina were cultured free floating in 12 well plates with retinal differentiation media (RDM) containing DMEM-F12 (Gibo), Glutamax (Gibo), 1x N2 and 1x B27 neural supplements (Invitrogen), 10% FBS (Invitrogen) and 1.5x penicillin/streptomycin (Invitrogen). Cell culture media was changed every 2 days. AAV2/9.pR2.1:GFP production and application: 293T cells were cultured in D10 medium containing DMEM+Glutamax, 10% (v/w) FBS and 1x pencillin/streptomycin in 150cm plates and grown to 80% confluency. pd10/pR2.1.L/Mopsin-eGFP construct, pHGT1 helper vector, AAV2/9 caspid were transfected into 293T cells using polyethylenimine (PEI), before harvesting cells after 72 hours and performing four thaw-vortex-freeze cycles for viral release. pR2.1.L/M-opsin promoter was originally generated by the Jeremy Nathans group (Wang et al., 1992). Virus lysate underwent benzonase treatment and was prepared for purification through multiple centrifugation steps and sequential filtering with 5µm, 0.45µm and 0.22µm PES membranes. Virus was purified via ion exchange chromatography using AKTA Prime (GE Life Sciences) and concentrated using a Vivaspin 4 concentrator (Vivaproducts). Viral genomic titer was determined through qRT PCR and the virus titer used in this study ranged from 1.48x1013-1.01x1014. Virus was added to human foetal retinal explant and iPSC-derived retinal differentiation cultures at a MOI of 40,000-45,000 through addition to culture media. Human foetal and iPSC-derived retinal explants were cultured for 7 days to allow for pR2.1:GFP expression to be visualised. On day 7, human foetal retinal explants and iPSC-derived retinal differentiation cultures were dissociated enzymatically into a single cell suspension using the papain dissociation system (Worthington Biochemical, Lorne Laboratories) following manufacturer's protocol. Total retina without pR2.1:GFP transfection were dissected and dissociated into single cell suspension using the same method. For large foetal retinae (16-22pcw), samples were cut into smaller pieces (5mm x 5mm) before the addition of papain (approx. 100µl per piece). For retinal differentiation cultures, tissue was cut into smaller pieces using micro dissection scissors before adding 1ml of papain per well of 6 well culture plate. 70µm cell strainers were used to ensure cell single density of cell suspension. For GFP cell isolation, cells were resuspended in a final volume of 500µl blocking solution (1% (w/v) BSA in PBS). Cell populations (GFP+ and GFP-) were collected into eppendorf tubes containing 500µl of 50% v/v FBS (DMEM). After sorting, cell populations were collected via centrifugation for 20 minutes at 200 x g; the supernatant was subsequently removed and RNA extraction was performed on cell pellets. mirVana miRNA Isolation Kit used for all extractions. Uses organic extraction followed by purification on a glass fiber filter under specialised binding and wash conditions collect all RNAs. RNA-seq samples were amplified using the SMART-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories) and cDNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina).
UCL GOS Institute of Child Health
E-MTAB-6057:Foetal19wkGFPplus1_p
Experimental Factor: developmental stage
late embryo
Experimental Factor: age
19
Experimental Factor: progenitor cell type
not applicable
Experimental Factor: cell type
retinal cone cell
Experimental Factor: phenotype
GFP (L/M-opsin) positive