Combination scaffolds for human neural stem cell and vascular tissue engineering

Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering

Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. They investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so they sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. They found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin-binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, They analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs.

Statement of significance: Interest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. They report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury1.

HNSPCs (5×104) were seeded into scaffolds of salmon, bovine, or human fibrin and cells were analyzed after 6 days. (a) More hNSPCs are present in salmon fibrin than mammalian fibrin scaffolds (nuclei detected by Hoechst staining). (b) EdU-incorporation reveals a higher percentage of cells in S-phase of the cell cycle in salmon fibrin scaffolds than mammalian fibrin. P = 0.0023 (salmon vs. bovine), P = 2.5E−05 (salmon vs. human). **P < 0.01, ***P < 0.001. Error bars represent SEM. N = 3 independent biological repeats.
Salmon fibrin encourages greater hNSPC proliferation than bovine or human fibrin

(a) RNA-seq analysis indicates hNSPC integrin gene expression levels from three biological replicates. Colors indicate the log2RPKM (reads per kilobase of transcript per million mapped reads) value for each α or β integrin, with red indicating higher expression and green lower expression. (b) The scatterplot displays average RPKM values for each integrin generated from the three replicates of hNSPCs organized from high to low expressers (error is SEM). Genes are clustered into three categories: high (>10 RPKM), moderate (1–10 RPKM), and low (<1 RPKM) expression. Dotted lines indicate 10 RPKM and 1 RPKM boundaries. For comparison, beta-actin, GAPDH, and FGFR1 fall in the highly expressed category, while FGFR2 is moderately expressed, and the muscle marker MyoD shows low to no expression. (c) Integrin binding schematic illustrating α and β integrins identified as fibrinogen-binding heterodimers; lines between α and β pairs indicate heterodimers. Red outlines denote integrins highly expressed by hNSPCs and green indicates lack of expression by hNSPCs [77]. (d) Flow cytometric analysis indicates hNSPC cell surface αV integrin expression. Histogram of Alexa-Fluor 488-positive cells reveals a shift (~54% positive cells) in hNSPCs labeled with an antibody against αV integrin compared to IgG2A isotype control.
HNSPCs express ECM-binding integrins
  1. Arulmoli, J., Wright, H. J., Phan, D., Sheth, U., Que, R. A., Botten, G. A., Keating, M., Botvinick, E. L., Pathak, M. M., Zarembinski, T. I., Yanni, D. S., Razorenova, O. V., Hughes, C., & Flanagan, L. A. (2016). Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering. Acta biomaterialia, 43, 122–138.

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