Additional file 3 of Human ex vivo spinal cord slice culture as a useful model of neural development, lesion, and allogeneic neural cell therapy
Creators: name: Lin, Chenhong
name: Calzarossa, Cinzia
name: Fernandez-Zafra, Teresa
name: Liu, Jia
name: Li, Xiaofei
name: Ekblad-Nordberg, Åsa
name: Vazquez-Juarez, Erika
name: Codeluppi, Simone
name: Holmberg, Lena
name: Lindskog, Maria
name: Uhlén, Per
name: Åkesson, Elisabet
Publisher: name: figshare
Year: 2020
Subjects: Medicine
Cell Biology
Molecular Biology
Neuroscience
Biotechnology
Biological Sciences not elsewhere classified
Developmental Biology
Cancer
Science Policy
Hematology

description: Additional files 3 : Supplementary Figure 1. Phase contrast images for hOC. (A) A Representative BS-SC longitudinal slice deriving from 6-8.5w. and (B) 9-10.5 w., both at 21 DIV. In cases &lt;8.5w., tissue architecture of BS and SC were preserved over time in culture. However, in older cases (&gt;8.5w.) BS partly lost tissue integrity and tissue thickness was reduced to few cell layers, edges were uneven, and dark spots disappeared from BS as seen in (B). Arrows: dark spots; arrowheads: dark aggregates. (C) A representative SC cross sectional slice at 21 DIV, deriving from 7.5-8w. Arrows: dark spots; arrowhead: dark aggregates. (D) A SC slice from 5.5w. at 21 DIV. Tissue maintained its appearance but at this stage studied landmarks were not fully developed. (E) SC slice deriving from 6w at 21 DIV. Fibers appeared from 7 DIV and they grew creating a network in culture as seen in (D, E). (F) In vitro phase contrast micrograph and (G) Cresyl violet stained hOC SC slices cultured under serum and glucose deprivation for one week. The in vitro slices lost tissue integrity, edges were uneven and was becoming very thin. Bar=0.6 mm. Abbreviations: PO, pons; MO, medulla oblongata; SC, spinal cord; WM: white matter; DF, dorsal funiculi and or dorsal septum; VF, ventral median fissure and or ventral funiculi; DH, dorsal horns or alar plate; VH, ventral horns or basal plate; CC, central canal and or extra canalicula. Supplementary Figure 2. Flow cytometric quantification of proliferation, apoptosis, glial cells, microglia on BS-SC and SC slices. Flow cytometric quantification of proliferation (A, B), apoptosis (C, D) and GFAP expression (E, F) and CD11b+CD45low expressing cells (G, H) in BS-SC (A, C, E, G) and SC (B, D, F, H) slices cultures, grouped depending on original weeks post conception. (A, B) proliferation increased significantly from 7DIV to that after 21 DIV in slices derived from 5-6.5w. in both BS-SC (A; p&lt;0.01) and SC (B; p&lt;0.05) slice cultures. At 21 DIV, BS-SC slices derived from 5-6.5w. presented double the percentage of proliferating cells compared to that at 9-10.5w. (A; p&lt;0.05). (C, D) In the slices, the amount of apoptotic cells was relatively stable during cultures from 7DIV to 21 DIV, while the percentages of caspase-3+ cells at 14 and 21 DIV were often significantly higher compared to that in situ (p&lt;0.05). At 7 DIV the proportion of apoptotic cells was higher in 9-10.5w. compared to 5-6.5w. (p&lt;0.05). (E, F) No significant differences were detected by flow cytometry in the percentage of GFAP+ cells among groups at same DIV or over time. Values are presented as mean ± SEM. *p&lt;0.05; **p&lt;0.01. Supplementary Figure 3. Immunostaining of proliferating and apoptotic cells in BS-SC and SC slices. (A-L) Representative images of Ki-67 (red), caspase-3 (green) and DAPI (blue) immunofluorescent staining on SC (A, C, D, G, H and K) and BS-SC (B, E, F, I, J and L) slices of different time points (in situ, 7 DIV, 14 DIV and 21 DIV). For the in situ and 21 DIV images of Ki-67, please see Fig. 1. Supplementary Figure 4. HLA-DR quantification and representative dot plots of the flow cytometric analysis. (A-B) Representative images of HLA-DR immunofluorescent staining of BS-SC slices of 7 DIV (A) and 14 DIV (B). (C) Quantification of HLA-DR+ cells. The image analysis was based on BS-BC slices 7 DIV and 14 DIV (3-4 sections per condition). Images were randomly taken in both conditions. DAPI+ cells were counted automatically by ImageJ, with the same filter setting for all sections. HLA-DR+DAPI+ cells were considered as HLA-DR+ cells. Values are presented as mean ± SEM. Bars=0.1mm. (D-E) Representative dot plots of the flow cytometric analysis of glial cell populations. (F) Representative dot plots over the hematopoietic cell populations, macrophages and monocytes. Gating is set from the negative isotype controls. Gating strategy: (Da, Db) microglia, CD11b+/ CD45low; (Da, Db, Dc) activated microglia, CD11b+/CD45low/HLA-DR+; (Fa, Fb) macrophages, CD11b+/CD45high; (Fa, Fb) monocytes, CD11b-/CD45+ cells. Abbreviations: Iso, mouse IgG isotype control for the respective fluorochromes. Supplementary Figure 5: Phase contrast images of contusion/cut SCI with hfNPC grafts. Data description please see respective figure legends. (A-I) “Donor” allogeneic hfNPCs grafted to “host” slices (G-I) subjected to contusion SCI and compared to contusion SCI alone (D-F) or to sham control slices (A-C). (J-W) GFP-hfNPC graft in spinal cord slices. After culture for 9DIV, a mechanical cut in the dorso-lateral spinal cord was performed. 1 hour later, GFP-hfNPCs were grafted at the injury site. Images taken with phase contrast and/or fluorescence microscope at 1h, 3 or 7 days postinjury. J-M) sham control; N-Q) cut SCI; R-U) cut SCI+ GFP-hfNPCs. V and W) represent green GFP-hfNPC grafted in the slices imaged in T and U with increased magnification. Bar: a-l) 1mm; m-n) 0.5mm. Size of slices is presented in mm. Score represents the total hOC Score.