Both BMP and CNTF promote the differentiation of hGPCs into GFAP-positive astrocytes expressing S100b but with otherwise distinct morphological and antigenic phenotypes. hGDAs^BMP express lower levels of GFAP and exhibit a more compact morphology. hGDAs^CNTF have a more elongated morphology and expressed high levels of GFAP. hGDAs^CNTF also expressed high levels of neurite-outgrowth inhibitory chondroitin sulfate proteoglycans, phosphacan and CSPG4, as well as the transcription factor Olig2 - all of which have been found to be upregulated in glial scar associated astrocytes. (A–C) Human GPCs grown in bFGF (A) were induced to differentiate into astrocytes using BMP-4 (B) or CNTF (C). Labeling with anti-GFAP (Alexa-488) demonstrates that both BMP4 and CNTF induce differentiation of human glial precursors into GFAP-expressing astrocytes, while Olig2 expression (Alexa-568) is repressed in hGDAs^BMP. Scale bar = 50 µm. (D) RT-QPCR analysis of hGPC, hGDA^BMP and hGDA^CNTF populations reveals induction of AQP4 and S100β in both hGDAs^BMP and hGDAs^CNTF. Induction of CX43, GLT1, AKAP12 and GDNF however are restricted to hGDAs^BMP. Average fold change and SD of expression levels is shown for three independent experiments using 9W-1 hGPCs. (E and F) Phosphacan and CSPG4 remain elevated in hGDAs^CNTF and are reduced in hGDAs^BMP derived from both 9W-1 (E) and 9W-2 (F) glial precursors. Mean relative protein expression and SD from three independent experiments are shown. Values were normalized to β-actin and expression in hGPCs.

Immuno-staining for human mitochondrial marker (red channel) of histological cross sections at sites of injury revealed hGDAs^BMP (A, C, E) and hGDAs^CNTF (B, D, F) transplant masses spanning the dorsal-ventral and lateral-medial margins of injury sites. Arrowheads in C and D indicate accumulations of hGDAs^BMP and hGDAs^CNTF respectively at the pial surface of lateral funiculus white matter (see also Fig. 4 A, B). Co-labeling for neurofilament (NF: green) and human mitochondrial marker (hMito: red) shows a markedly higher density of axons within hGDAs^BMP treated injury sites (A, E) compared to hGDAs^CNTF treated injury sites (B, F). Greater numbers of NF+ axons within hGDAs^BMP transplants aligned with the normal rostral/caudal trajectory of DLF white matter (E) compared to NF+ axons within hGDAs^CNTF transplants (F). Survival = 5 weeks post injury/transplantation. Scale bars: A, B, C, D = 200 µm; E, F = 100 µm.

Images of hMito+ (red) and GFAP (green) immuno-reactivity within hGDAs^BMP (A, C, E, F) and hGDAs^CNTF (B, D, G, H) transplants at the center of injury sites showing comparable densities of GFAP+ intermediate filaments (green) within hMito immuno-positive cell bodies and process of both types of hGDAs. Arrowheads indicate some examples of GFAP+/hMito+ hGDA cell bodies at low and high magnification. Images are maximum projections of apotome (Zeiss) optical sections captured through a depth of 3.5 µm of tissue. Survival = 5 weeks post injury/transplantation. All scale bars = 20 µm.

(A, B) High power images showing hMito+ hGDAs^BMP in the process of migrating and accumulating at the pial surface within lateral funniculus white matter. Note the elongated radially orientated processes displayed by some hMito+ hGDAs^BMP within white matter (B: arrowheads), a glial morphology indicative of tangential migration of these cells towards the adjacent pial surface (hMito: red; NF+ axons: green). (C) hGDAs^BMP displaying typical astrocytic “stellate” arrangements of their processes within white matter immediately ventral to the injury site. Survival = 5 weeks post injury/transplantation. Scale bars: A, B = 100 µm; C = 20 µm.

Graphs show the average number of mistakes per experimental group made during Grid walk testing of locomotor recovery at 1 day before injury to 28 days after injury. In two separate experiments, hGDA^BMP transplanted animals (closed circles) performed significantly better than hGDA^CNTF (A) or hGPC (B) transplanted animals at all time points from 7 to 28 days post injury/transplantation. Note that the performance of hGDA^CNTF or hGPC transplanted animals was not significantly different from control injured rats at all time points (two-way repeated measures ANOVA, *p< 0.05).

Montaged images of NeuN immuno-histochemistry at the C5 spinal level of normal (A) and untreated injured (control) spinal cords (B) show that the unilateral DLF transection injury causes loss of NeuN+ neurons in multiple spinal cord laminae adjacent to the transected white matter. Transplantation of hGDAs^BMP (C) promotes significant protection of neurons in laminae 7, 8, and 9 at the injury center. In contrast, transplantation of hGDAs^CNTF (D) or hGPCs (E) did not promote significant levels of neuroprotection. (F) Schematic showing gray matter laminae at the C5 level of the rat spinal cord (adapted from [39]). All scale bars = 200 µm.

(A) hGDA^BMP transplantation led to significant increases in numbers of NeuN+ neurons counted in a 1.8mm length of spinal cord encompassing the injury site. Graphs show percentage changes in numbers of NeuN+ neurons in laminae 4 to 9; laminae 4, 5, and 6; 7; and 8 and 9 in spinal cords from animals that received transplants of 9W2 or 9W1 hGDAs^BMP, hGDAs^CNTF or hGPCs and untreated control injuries. (B) Analysis of neuron survival within laminae immediately adjacent to the site of injury shows that hGDA^BMP transplantation promoted significant protection of neurons when all laminae were considered (4 to 9), with the most robust increases in neuron numbers in intermediate (7) and ventral (8 and 9) gray matter laminae. Numbers of NeuN+ neurons in spinal cords of rats transplanted with GDAs^CNTF or hGPCs were not significantly different from each other or untreated spinal cord injuries. * ANOVA and Pairwise Multiple Comparison (Holm-Sidak), p<0.05. + t-test comparison with untreated spinal cord injuries, p<0.05.