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Abstract
BackgroundNeurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins.Methodology/Principal FindingsWe have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation.Conclusion/SignificanceBy inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.
Highlights
Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, are pluripotent cells that can differentiate into a variety of cell types including neural cells [1,2]
When cultured in P-ACM containing fibroblast growth factor-2 (FGF-2) under free-floating conditions, colonies of undifferentiated human ES (hES) cells gave rise to floating spheres composed of neural stem cells and undifferentiated cells, which gradually increased in size during the culture
After 12 days of culture, the spheres were plated onto a poly-L-Lysine/Laminin coated dish and cultivated in neural stem cell medium (NSCM) containing high concentrations of FGF-2 and epidermal growth factor (EGF)
Summary
Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, are pluripotent cells that can differentiate into a variety of cell types including neural cells [1,2]. Neural stem cells and neurons can be directly differentiated from ES cells without forming embryoid bodies by culturing ES cells on mousecultured stroma cells (PA-6) [5], or under chemically defined lowdensity culture conditions [6] All of these procedures, are time consuming and require highly complicated processes to generate many neurons. We have previously reported an efficient method to prepare transplantable neural cells from mouse ES cells using a conditioned medium collected from rat primary-cultured astrocytes (P-ACM) [7,8,9]. Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). The use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins
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