Differentiation profiles of NSPCs are impacted by dbcAMP treatment.

<p><b>A–L</b>) Representative images of tissue samples demonstrating NSPC differentiation profile of (A–C) nestin-positive progenitor cells, (D–F) BetaIII-positive neurons, (G–H) CC1-positive oligodendrocytes, and (J–L) GFAP-positive astrocytes. Scale bar represents 50 µm. <b>M</b>) Quantification of NSPC differentiation profile for the various treatment groups. Mean ± standard deviation are plotted, n = 3 to 5; significant differences noted with an asterisks, p<0.05. <b>N</b>) Deconvoluted confocal image of betaIII-positive NSPC-derived neurons (arrows) 6 weeks post-transplantation. Scale bar represents 50 µm.</p

Channel implantation after spinal cord transection facilitates tissue bridging, NSPC survival, and behavioural improvement over time.

<p><b>A</b>) Photograph of the surgical implantation of fibrin-filled chitosan channels. <b>B</b>) Tissue bridges obtained from animals 2 weeks after implantation. <b>C,D</b>) Longitudinal section of tissue bridge demonstrating NSPC survival after 6 weeks in an animal receiving dbcAMP pre-treatment (dbcAMP, 4div). Boxed area in (C) is magnified in (D). <b>E</b>) NSPC survival after 2 and 6 weeks for various treatment groups. <b>F</b>) Assessment of functional recovery using the BBB locomotor scale. After 6 weeks, rats receiving transplants of dbcAMP-pre-treated NSPCs show a statistically significant increase in hindlimb function relative to untreated animals (*, p<0.05). Mean ± standard deviation shown for n = 4 to 6.</p

<i>In vivo</i> treatment groups.

<p><i>In vivo</i> treatment groups.</p

Microsphere-loaded channels effectively release dbcAMP <i>in vitro</i>.

<p><b>A</b>) Cumulative release profiles of dbcAMP from free-floating microspheres and microsphere-loaded channels. The process of embedding microspheres into channel walls is likely responsible for early degradation of PLGA and faster drug release from channels. <b>B</b>) Schematic of the entubulation strategy. NSPCs are seeded on fibrin scaffold within a chitosan channel. Drug-loaded PLGA microspheres release the differentiation factor dibutyryl cyclic-AMP in a local and sustained manner, influencing NSPCs to preferentially differentiate into neurons. <b>C</b>) Viability of NSPCs in a three-dimensional fibrin scaffold. Simultaneous staining of CalceinAM (green) and Ethidium homodimer (red) for live and dead cells respectively show good cell viability of NSPCs in fibrin scaffolds at 1 week. Scale bar represents 100 µm. <b>D–G</b>) Immunostaining of NSPCs for DAPI-nuclear stain and betaIII-tubulin with various dbcAMP treatments. Scale bar represents 100 µm. <b>H</b>) Quantification of betaIII-tubulin immunostained NSPCs with various dbcAMP treatments. <b>I,J</b>) Quantitative RT-PCR data for (I) betaIII tubulin and (J) nestin mRNA expression with various dbcAMP treatments, normalized to housekeeping gene HPRT. Data represented as mean ± standard (n = 3 to 6). Statistical differences denoted by *, p<0.05.</p

NSPCs respond to dbcAMP <i>in vitro</i>.

<p><b>A</b>) Dose response curve of dbcAMP on neuronal differentiation of NSPCs after 7 days in culture. <b>B</b>) Differentiation profile of NSPCs after 7 days. NSPCs were treated with media containing 1 mM dbcAMP for 0, 1 or 7 days. Only sustained exposure to dbcAMP resulted in increased number of BetaIII-positive neurons. <b>C–J</b>) Representative images of NSPCs after 7 days in culture for markers of progenitors cells (nestin), neurons (BetaIII), oligodendrocytes (RIP), and astrocytes (GFAP). Scale bar represents 100 µm. <b>K</b>) Cell numbers at 1, 3, and 7 days in culture with or without 1 mM dbcAMP. <b>L</b>) Ki67 staining for proliferating cells after 3 days. <b>M</b>) Cell differentiation over time with or without 1 mM dbcAMP treatment. Data represented as mean ± standard (n = 3 to 9). Statistical differences denoted by *, p<0.05.</p

The regenerated bridge tissue contains host axons, blood vessels, and fibroblasts.

<p><b>A</b>) Representative image of endogenous axonal regeneration into the tissue bridge based on betaIII tubulin staining. <b>B</b>) Evidence of association between betaIII-positive endogenous axons with surviving GFP-positive NSPCs at six weeks. Synaptophysin staining is observed at the interface (inset). <b>C</b>) RECA1 staining for endothelial cells show blood vessel formation throughout the tissue bridge at 2 weeks. <b>D</b>) Prolyl-4-hydroxylase (rPH) staining of bridge tissue indicates that the majority of cells are collagen producing fibroblasts.</p

<i>Vsx2-5.3-PRE-Cre</i> is specifically localized to Vsx2-expressing neurons of the inner nuclear layer in the mature retina.

<p>(A–C) The vast majority of Vsx2-labeled nuclei (A) in the inner nuclear layer (boundaries indicated by broken lines) co-label for Cre (B). Arrowheads in (C) indicate Vsx2-positive/Cre-negative nuclei outlined in (B). (D–F) Sox9-positive Müller glial nuclei (D) do not express Cre. Arrowheads (F) show examples of Cre-negative/Sox9-positive nuclei outlined in (E). (G–I) Horizontal cells labelled with Calbindin-D28k (G) do not label for Cre (H). Sections are from >6 week old mice. Scale bar  = 10 µm.</p

Summary of <i>Vsx2-5.3-PRE-Cre</i> transgenic mouse lines.

<p>*“+” indicates Cre immunolabeling restricted to postmitotic presumptive bipolar cells and/or mature retinal bipolar cells</p><p>**“+” indicates reporter expression in the mature retina in bipolar cells and a subset of photoreceptor cells</p

<i>Vsx2-5.3-PRE-Cre</i> is expressed in a large subset of bipolar neurons in the adult retina.

<p>Immunolabeling for Cre-recombinase under control by the <i>Vsx2-5.3-PRE</i> transgene is localized in nearly all PKCα expressing rod bipolar cells (A–C); a large subset of CaBp5-expressing Type-III a/b, Type-V ON, or rod bipolar cells (D–F); <i>mGlur6-lacZ</i> expressing ON bipolar cells (G–I); and a large subset of PKARIIβ-expressing Type-IIb and OFF bipolar neurons (J–L). Bhlhb5 expressing Type-II OFF bipolar cells do not express Vsx2-5.3-PRE-Cre (M–O). Arrows indicate co-localized cells; solid arrowheads indicate Cre-only cells; open arrowheads indicate Cre-negative cells. Scale bar (N)  = 10 µm.</p

Cre expression precedes expression of the mature bipolar markers PKCα and Cabp5.

<p>At postnatal day 3 (P3) Cre-positive nuclei do not co-localize to Cabp5 (A–C), PKCα (D–F), or with the early bipolar fate marker Bhlhb5 (M–O). Insets (G–I) are high magnification view of Cre-positive (solid arrowhead) and Cre-negative (open arrowhead) nuclei located apical to the Bhlhb5-positive layer (arrow for example). Scale bar (O)  = 20 µm.</p