Self-Contained Induction of Neurons from Human Embryonic Stem Cells

BACKGROUND: 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). 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 FINDINGS: We 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/SIGNIFICANCE: By 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

RT-PCR analysis and differentiation of hES cells into dopaminergic neurons in xeno-free ES-ACM.

<p>(A): RT-PCR analysis of hES cells, neural stem cells and mature neurons. RNA was isolated from clones of undifferentiated hES cells, from neural stem cells, and from mature neurons which had been cultured for 8 weeks in ES-ACM and analyzed for expression of marker genes. The expression levels of each gene were normalized to GAPDH gene expression level. hES, undifferentiated hES cells; NSC, neural stem cells; Neuron; mature neurons. (B): Differentiation of hES cells into dopaminergic neural cells in xeno-free ES-ACM. Neural stem cells induced by ES-ACM were subcultured onto PLL coated plate and cultured for 8 weeks in ES-ACM. Immunostaining with antibody TH and expression of hrGFP showed that the subcultured neural stem cells had differentiated into dopaminergic neurons. Expression of hrGFP (green), DAPI (blue) and TH (red) staining profiles. Bar = 100 µm.</p

Differentiation of hES cells into astrocytes in a chemically defined medium.

<p>(A): Neural stem cells induced by N2 medium were cultured for 14 days after removal of the core of spheres with a glass pipette and change of medium to G5 medium. The proliferated cells were subcultured onto a PLL/LAM coated plate and cultured for 14 days in G5 medium. Immunostaining with antibody to GFAP showed that most of the subcultured cells had differentiated into astrocytes. DAPI (blue) and GFAP (green) staining profiles. Neural stem cells induced by xeno-free ES-ACM were subcultured onto PLL coated plate and cultured for 6 weeks in ES-ACM. (B): Immunostaining with antibody to MAP2 showed that the subcultured NSCs had differentiated into mature neurons. DAPI (blue) and MAP2 (red) staining profiles. Bar = 100 µm.</p

Selective induction of hES cells into neurons and astrocytes.

<p>(A, B): Neural stem cells that had migrated from floating spheres in NSCM were subcultured onto a PLL coated plate and cultured for 14 days in P-ACM. Immunostaining with antibody to Tuj1 and MAP2 showed that the subcultured neural stem cells had differentiated into neurons. Expression of hrGFP (green), (A) Tuj1 (red), and (B) MAP2 (red) staining profiles. (C): Neural stem cells were cultured for 14 days after removal of the core of spheres with a glass pipette and change of medium to G5 medium. The proliferated cells were subcultured onto PLL/LAM coated plate and cultured for 14 days in G5 medium. Immunostaining with antibody to GFAP showed that the subcultured cells had differentiated into astrocytes. Expression of hrGFP (green) and GFAP (red) staining profiles. Bar = 100 µm.</p

Survival of transplanted neural stem cells <i>in vivo</i>.

<p>(A): Most of the donor cells were confirmed to be Nestin-immunoreactve neural stem cells before transplantation. Anti-Nestin staining (green) and DAPI (blue). Bar = 50 µm. (B): Transplantation site. Grafted cells expressing hrGFP can be seen in the striatum. Bar = 500 µm. (C): High power magnification view of a white box in panel A. Some of the hrGFP-positive cells display a morphology similar to that of neurons. Bar = 50 µm. (D): Merged image of hrGFP expression (green) and immunostaining of anti-Tuj1 (red). Bar = 20 µm. (E): Merged image of hrGFP expression (green) and immunostaining of anti-Ki-67 (red). Bar = 50 µm.</p