Self-Contained Induction of Neurons from Human Embryonic Stem Cells

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.

Pluripotent stem cell-derived neural stem cells: From basic research to applications

“…The neural stem sphere (NSS) method is a simple neural differentiation method using only astrocyte-conditioned medium (ACM) prepared from serum-free medium under free floating conditions [40][41][42] . In brief, ES cell colonies formed on MEF feeder layers at clonal density are mechanically picked.…”

Cell Therapy for Parkinson’s Disease

“…In our method, astrocytes derived from ES cells in a chemically de fi ned medium can be used for continuous generation of neurons [ 38 ] (Fig. 15.2 ).…”

Increase of sensitivity to mechanical stimulus after transplantation of murine induced pluripotent stem cell–derived astrocytes in a rat spinal cord injury model

“…24 Human neurons, generated from ESCs by the NSS method, have been shown to survive in murine brains following xenotransplantation. 34 Nakayama and colleagues 29 reported that the NSS method was very efficient because each ESC generated 1000 NSCs in an 11-day period. Moreover, serumfree induction is possible because chemically defined N-2 medium can generate NSS without having to resort to rodent ACM.…”

“…Moreover, serumfree induction is possible because chemically defined N-2 medium can generate NSS without having to resort to rodent ACM. 34 We hypothesized that the NSS method could be applied to iPSC differentiation. In the present study, we transferred iPSC colonies to floating cultures, and differentiated NSCs on the surface of the NSS.…”

“…Okuno et al 34 demonstrated that human ESCs could differentiate into neural cells within 1 month when using the NSS method. We emphasize that the NSS method could be quite rapid if applied clinically.…”