The in vivo
survival and differentiation of transplanted ESNPCs was evaluated in a subacute model of SCI. The transplantation of ESNPCs in fibrin scaffolds containing NT-3 and PDGF increased overall cell survival and proliferation by X fold over 2 weeks. Furthermore, the transplantation of ESNPCs in fibrin scaffolds containing GF and HBDS increased the number of ESNPC-derived NeuN+ neurons (1.2×105
NeuN(+) cells in the 10EB + GF + DS group compared to < 0.25×105
NeuN(+) cells in the groups without GF). Others have found that NPC transplantation into the injured spinal cord results in poor cell survival and differentiation into a predominately glial fate (7
). Previously, ESNPCs induced using the same 4-/4+ RA protocol, were injected into a crush model of SCI 9 days after injury and found to promote a modest increase in functional recovery (21
). However, McDonald et al.
found that cell survival was poor (~10%), and few of the surviving cells differentiated into neurons (~8%). The findings in our current study are significant because they demonstrate the ability to enhance the survival and differentiation of NPCs into neurons after transplantation into the injured spinal cord.
ESNPCs in fibrin scaffolds containing GF exhibited robust survival and proliferation at 2 weeks following implantation into a subacute model of SCI. In vitro
studies evaluating NT-3 and PDGF, at the same total doses used in this study, found that the combination resulted in increased survival of ESNPCs at 2 weeks (32
). Consistent with these in vitro
findings, our study found that the stereological count of ESNPCs with GF (with or without HBDS) was higher than either ESNPCs implanted directly into the lesion site or in fibrin without GF after transplantation. The stereological counts estimate that the addition of GF increased the total cell number approximately 10 fold over the initial estimated cell number in 2 weeks. In contrast, those ENSPCs embedded in fibrin scaffolds alone only increased roughly 2 fold over the same time period. Our findings are consistent with other studies that have shown that the combination of NT-3 and PDGF promotes NPC survival in models of CNS disease (14
), and PDGF specifically promotes NPC proliferation (11
The incorporation of the HBDS in fibrin scaffolds containing GF and ESNPCs resulted in an increase in the number of ENSPC-derived NeuN+ cells. These findings are consistent with in vitro
studies that found NT-3 and PDGF delivered from the HBDS increased the percentage ESNPCs that differentiated into neurons (32
). It has been shown that soluble NT-3 promotes the differentiation of human ESCs into neurons in culture and treatment of NPCs with NT-3 results in the formation of bipolar neurons (19
). The stereological count of NeuN+ neurons derived from ESNPCs in fibrin with GF and no HBDS was not different from the other groups. These results suggest that controlled delivery of NT-3 and PDGF using the HBDS was required for the increase in neuronal differentiation compared groups without GF.
The addition of the HBDS could have affected the differentiation of the ESNPCs. Heparin alone in the culture medium of ESCs and NPCs has been shown to increase proliferation through secondary interactions with fibroblast growth factor (FGF) that enhance FGF's mitotic activity (5
). Willerth et al.
found that using the same concentration of heparin found in the HBDS resulted in a decrease in astrocytes differentiation (32
). In our study, the HBDS had no effect on astrocytes differentiation of ESNPCs, and thus it is unlikely that direct interaction between heparin and the ESNPCs resulted in an increase in NeuN positive neurons. As with heparin, the presence of the heparin-binding peptide from the HBDS could have an effect on ESNPCs differentiation. Others have demonstrated the ability of cell adhesion sequences from laminin to promote differentiation of NPCs (28
). However, evaluation of the HBDS in vitro
showed that the affinity peptide alone in culture had no effect on neuronal differentiation (32
). Based on these previous results, it is unlikely that interaction of ESNPCs with the heparin-binding peptide in the HBDS accounted for the increase in NeuN+ neurons. A more likely explanation for the observed increase in NeuN+ neurons with the HBDS is its ability to sequester NT-3 and PDGF to the fibrin scaffold (29
). Furthermore, the HBDS could also have increased the residence time of other endogenously produced GFs within the fibrin scaffolds.
A method to analyze the differentiation of transplanted ESNPCs was developed to compliment the stereological analysis performed in this study. With the exception of NeuN, the cell differentiation markers used in this study were cell surface (SSEA-1/O4) or intracellular proteins (nestin, Tuj1, GFAP) that are expressed in different locations within the cell. As a consequence, if the cell of interest is adjoining another cell with the same marker it is difficult to delineate the boundary between each independent cell and thus impossible to make an accurate stereological count. A program was written that evaluates the total area of pixels that are positive for both green fluorescent protein (GFP) and the neural marker of interest. While this method of analysis does not indicate a specific number of cells stained positive for a given marker, it does quantify the average area of expression for each given marker. The area of expression can then be compared between experimental groups to determine a difference in expression between groups. To assure that the method correlated to the stereological counts of the GFP and NeuN positive cells, we analyzed the amount of GFP expression for transplanted ESNPCs and Tuj1 for neurons. The analysis of average area of expression for GFP and Tuj1 predicted the same trends obtained with the stereological counts for transplanted ESNPCs that expressed the neuronal marker NeuN and GFP, thus for a given marker a greater area of expression correlated with a greater number of cells, as expected. However, this method does not allow correlations between markers (e.g. area of Tuj1 versus GFAP) that could be performed with absolute cell number counts from stereology.
By analyzing the average area of GFP+ O4 expression using this method, we found that ESNPC-derived cells in fibrin with GF and the HBDS showed increased O4 expression compared to all other groups. The combination of the two GF and the HBDS was found to increase oligodendrocyte differentiation in vitro
). The increased O4 expression seen in the 10EB+DS+GF group suggests that the presence of the controlled delivery of GF promotes oligodendrocyte differentiation. ESNPCs in fibrin containing GF with or without HBDS showed an increase in the average area of GFAP expression compared to groups without GF. In vitro
studies found that the addition of the HBDS decreased astrocytes differentiation compared to controls that were cultured without the GF (32
). While the addition of the GF resulted in increased GFAP and O4 staining, the neuronal markers were more widely expressed than glial markers on ESNPC-derived cells after transplantation.
Staining with the mouse ESC marker SSEA-1 revealed a moderate amount of expression of the ESC marker by the transplanted cells. This is not surprising considering the RA induction protocol only results in a population of cells that are 70% Nestin positive ESNPCs (31
). Quantification of the average area of SSEA-1 expression revealed differences between experimental groups. The 10EB+DS+GF group average are of SSEA-1 expression was greater than all other experimental groups. Previously it has been shown that PDGF can enhance the survival and proliferation of SSEA-1 positive mouse ESCs in in vitro
). It follows that in this study the presence of the growth factors and the HBDS enhanced the survival and proliferation of SSEA-1 positive mouse ESCs.
Analysis of the average area of marker expression is not the same as stereological counts of cells. As mentioned above, the quantification of proteins that are located in different places within the cell make comparison of areas between different antibodies inaccurate (eg. area positive for Tuj1 vs. GFAP). Likewise, the summation of the different average areas of expression (Tuj1, GFAP, Nestin, and SSEA-1) are not expected to add up to the average area of expression of GFP positive transplanted ESNPCs and they do not. It is possible that the differences in area are a result of the quantification method and it is possible that some of the GFP positive cells are expressing markers other than those assayed in this study. Based on the type of analysis that was utilized in this study, it is impossible to tell. However, the goal of the analysis was to look at the differential effects these growth factors have on differentiation of the ESNPCs into the three neural subtypes (neurons, oligodendrocytes, and astrocytes)The foreign body response to a transplanted material can drastically affect the function of the material in vivo
. We analyzed the intensity of macrophage/microglia staining surrounding the implants assess any adverse response to the ESNPC containing scaffolds. In previous studies, we have found that fibrin scaffolds alone do not induce an adverse response in the surrounding host tissue based on the presence of macrophage/microglia (17
). In this study we found that three of the four groups that were treated with ESNPCs had a decrease in the intensity of macrophage staining surrounding the lesion site compared to the control group (received the same surgical procedures but no treatment). Transplanted NPCs have been found to be neuroprotective and increase the viability of neural tissue during and following injury to the CNS (1
). Similarly, if the transplanted ESNPCs increase survival of host tissue, then that may have led to the decrease in macrophage/microglia staining surrounding the lesion.
Here we have investigated the feasibility of transplanting ESNPCs within fibrin scaffolds containing GF and a HBDS to increase cell survival and direct their differentiation following transplantation into the injured spinal cord. Transplantation of the ESNPCs in fibrin scaffolds containing NT-3 and PDGF enhanced their survival and proliferation 2 weeks after transplantation. Also the addition of a HBDS resulted in an increase in the number of ESNPCs that differentiated into NeuN+ neurons. These results are the first step toward a larger goal of enhancing functional recovery following SCI by repopulating the damaged cord with neurons and oligodendrocytes that can increase local plasticity. Finally, the method of implantation outlined in this work could be used to increase the survival and differentiation of transplanted cells for other cell therapies.