Umbilical cord blood (CB) stem cells have been proposed for cell-based therapeutic applications for diverse diseases of the CNS. We hypothesized that tissue-engineering strategies may extend the efficacy of these approaches by improving the long-term viability and function of stem cell-derived neuronal progenitors. To test our hypothesis, we explored the survival and differentiation of human CB-derived neuronal progenitors (HUCBNP) in a three-dimensional (3D) collagen construct. In contrast to two-dimensional culture conditions, the cells survived in 3D for an extended period of time of more than 2 months. Under 3D conditions, HUCBNP underwent spontaneous neuronal differentiation, which was further enhanced by treatment with neuronal conditioned medium (CM) and nerve growth factor (NGF). Neurite outgrowth, quantified by assessing the fractal dimension (D f) of the complex neuronal networks, was significantly enhanced under 3D conditions in the presence of CM/NGF, concomitant with a reduced expression of the early neuronal marker nestin (1.9-fold), and increased levels of mature neuronal markers such as MAP-2 (3.6-fold), β-tubulin (1.5-fold), and neuronal specific enolase (6.6-fold) and the appearance of the synaptic marker synaptophysin. To assess the feasibility for clinical usage, HUCBNP were also isolated from frozen CB samples and cultured under 3D conditions. The data indicate the essential complete preservation of neurotrophic (survival) and neurotropic (neurite outgrowth) properties. In conclusion, 3D culture conditions are proposed as an essential step for both maintenance of CB neuronal progenitors in vitro and for investigating specific features of neuronal differentiation towards future use in regenerative therapy.
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Acknowledgments This study was supported by the Israel Ministry of Science and Technology (PL). PL holds the Jacob Gitlin Chair in Physiology and is affiliated with and supported by the David R. Bloom Center for Pharmacy and the Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at the Hebrew University of Jerusalem, Israel. PIL is the Laura H. Carnell Professor of Regenerative Engineering at Temple University’s Department of Bioengineering. The authors thank Dr. Marck Tarshish, Dr. Ezra Rachamim, and Ms. Zahava Cohen for their technical support.
- Collagen gel
- Human umbilical cord blood progenitors
- Long-term survival
- Neuronal differentiation
- Three-dimensional construct