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. 2021 Jul 10;24(8):102827. doi: 10.1016/j.isci.2021.102827

Figure 3.

Figure 3

Encapsulating purified SMN-OPC co-cultures

(A) Overview of differentiation protocol used to generate OPCs through neuromesodermal progenitor (NMP) and then multipotent spinal cord neural stem cell (scNSC) stages. Small molecules and growth factors used in caudo-ventral patterning and maturation are listed.

(B) Immunofluorescence images of OPC differentiation. Left-to-right: Pax6 (day 6); Nestin/OLIG2 (day 20); Nkx-2.2 (day 25); O4 (day 36). Cells counterstained with DAPI (D, nuclei).

(C) GFP-OPC MACS separation scheme (magnetic cell sorting). GFP-OPCs were isolated from differentiating mixed cultures using magnetic beads functionalized with O4 antibody. Enriched GFP-OPCs were plated for further differentiation or seeded onto SMN cultures.

(D) Adherent GFP-OPCs after MACS enrichment.

(E) GFP-OPC co-culture with SMNs prior to encapsulation.

(F) SMN-OPC connected networks were encapsulated within neural ribbons as a dual-component system. GFP and SMI312 immunostaining of neural ribbons is shown at day 7 post-encapsulation. High magnification image provided on right. Scale bars are 50 μm, and 10 μm in high magnification panel in (D).