Journal: Journal of Neurotrauma

Article Title: Transplantation of Neural Progenitors and V2a Interneurons after Spinal Cord Injury

doi: 10.1089/neu.2017.5439

Figure Lengend Snippet: Generation and aggregation of neural progenitor cells (NPCs) and V2a interneurons (INs). (A) NPCs isolated from E13.5 rat spinal cords were stored frozen and thawed 1 day prior to aggregation. (B) Chx10-Puro embryonic stem cells (ESCs) expressing TdTomato were induced using a 2-/4+ protocol followed by 24 h of puromycin selection for V2a INs. (C) Schematic showing aggregation protocol. Populations of NPCs were mixed at a 1:1 ratio with TdTomato-V2a cells and seeded into an AggreWell plate. After 2 days of culture, spherical aggregates form in the dish (green fluorescent protein/TdTomato, NPC/V2a INs, respectively shown in D-F), which are then resuspended in fresh media and washed in Hank's Balanced Salt Solution prior to transplantation (F). Scale bars are as indicated. (G) Experimental timeline of the study.

Article Snippet: Two-tailed Student's t -test: * p < 0.0001 and # p < 0.05 compared with FSC group. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Host Antigen Concentration Vendor Catalogue number Primary antibodies Chicken GFP 1:700 IHC Abcam Ab13970 Mouse NeuN 1:200 IHC Millipore MAB377 Mouse Nestin 1:1000 IHC BD Pharmigen 556309 Mouse GFAP 1:400 IHC Millipore MAB360 Mouse Synaptophysin 1:1000 IHC Synaptic Systems 101 011 Mouse GAD67 1:500 IHC Millipore MAB5406 Mouse vGlut1 1:1000 IHC Millipore MAB5502 Mouse ENCAM 1:2 ICC DSHB Mouse A 2 B 5 1:2 ICC DSHB Rabbit PRV 1:10,000 IHC Courtesy of Lynn Enquist, Princeton University Rabbit NeuN 1:500 IHC Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab177487","term_id":"62867256","term_text":"AB177487"}} Ab177487 Rabbit Ki67 1:200 IHC ThermoFischer PA5-16785 Rabbit ßIII Tubulin 1:1000 ICC Abcam Ab18207 Sheep CHX10 1:200 ICC Millipore AB9016 Secondary antibodies Anti-chicken Alexa Fluor 488 1:200 IHC ThermoFischer A-11039 Anti-mouse Alexa Fluor 647 1:200 IHC Molecular Probes A31571 Anti-mouse DyLight 405 1:200 IHC Jackson ImmunoResearch 715-475-150 Anti-rabbit DyLight 405 1:200 IHC Jackson ImmunoResearch 711-475-152 Anti-mouse DyLight 594 1:400 ICC; 1:200 IHC Jackson ImmunoResearch 715-515-150 Anti-rabbit Alexa Fluor 488 1:400 ICC Invitrogen/ ThermoFischer A21206 Anti-sheep Alexa Fluor 594 1:400 ICC Jackson ImmunoResearch 713-585-003 Open in a separate window GFP, green fluorescent protein; IHC, immunohistochemistry; GFAP, glial fibrillary acidic protein; ICC, immunocytochemistry.

Techniques: Isolation, Expressing, Selection, Transplantation Assay

Journal: Journal of Neurotrauma

Article Title: Transplantation of Neural Progenitors and V2a Interneurons after Spinal Cord Injury

doi: 10.1089/neu.2017.5439

Figure Lengend Snippet: Transplanted neural progenitor cells (NPCs)/V2a interneuron (IN) aggregates differentiate into mature neurons and glia. Horizontal section through the lesion/transplant epicenter of a NPC/V2a IN aggregate-transplant showing TdTomato positive V2a INs (A), GFP positive NPCs (B), stained with NeuN (C) to visualize mature neurons, and glial fibrillary acidic protein (GFAP; D) to visualize astrocytes. Overlay of (A-D) is depicted in (E). Black arrowhead points to a mature (NeuN positive) neuron from NPCs, while white arrowheads point to mature (NeuN positive) V2a INs. Red arrowheads point to NeuN negative, TdTomato positive V2a INs. Quantification of the number of V2a INs that were also positive for NeuN as percent of total number of V2a INs counted (F) and as the total number of NeuN positive neurons within GFP positive transplant (G). (H-K) shows a similar section of NPC/V2a IN aggregate, stained for Ki67 (white in J; blue in K), showing proliferative capacity of NPCs, but not V2a INs (white arrowheads in J and K). (L) Confocal orthogonal image of a host (GFP and TdTomato negative) synapse (synaptophysin, blue), onto V2a IN (TdTomato, red). Scale bars are as indicated.

Article Snippet: Two-tailed Student's t -test: * p < 0.0001 and # p < 0.05 compared with FSC group. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Host Antigen Concentration Vendor Catalogue number Primary antibodies Chicken GFP 1:700 IHC Abcam Ab13970 Mouse NeuN 1:200 IHC Millipore MAB377 Mouse Nestin 1:1000 IHC BD Pharmigen 556309 Mouse GFAP 1:400 IHC Millipore MAB360 Mouse Synaptophysin 1:1000 IHC Synaptic Systems 101 011 Mouse GAD67 1:500 IHC Millipore MAB5406 Mouse vGlut1 1:1000 IHC Millipore MAB5502 Mouse ENCAM 1:2 ICC DSHB Mouse A 2 B 5 1:2 ICC DSHB Rabbit PRV 1:10,000 IHC Courtesy of Lynn Enquist, Princeton University Rabbit NeuN 1:500 IHC Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab177487","term_id":"62867256","term_text":"AB177487"}} Ab177487 Rabbit Ki67 1:200 IHC ThermoFischer PA5-16785 Rabbit ßIII Tubulin 1:1000 ICC Abcam Ab18207 Sheep CHX10 1:200 ICC Millipore AB9016 Secondary antibodies Anti-chicken Alexa Fluor 488 1:200 IHC ThermoFischer A-11039 Anti-mouse Alexa Fluor 647 1:200 IHC Molecular Probes A31571 Anti-mouse DyLight 405 1:200 IHC Jackson ImmunoResearch 715-475-150 Anti-rabbit DyLight 405 1:200 IHC Jackson ImmunoResearch 711-475-152 Anti-mouse DyLight 594 1:400 ICC; 1:200 IHC Jackson ImmunoResearch 715-515-150 Anti-rabbit Alexa Fluor 488 1:400 ICC Invitrogen/ ThermoFischer A21206 Anti-sheep Alexa Fluor 594 1:400 ICC Jackson ImmunoResearch 713-585-003 Open in a separate window GFP, green fluorescent protein; IHC, immunohistochemistry; GFAP, glial fibrillary acidic protein; ICC, immunocytochemistry.

Techniques: Staining