Proliferation and Differentiation of Dopaminergic Neurons from Human Neuroepithelial Stem Cells Obtained from Embryo Reduction Following In Vitro Fertilization

Abstract

Background: Recent advances in stem cell technologies have rekindled an interest in the use of cell therapies to treat patients with Parkinson's disease. Although the transplantation of dopaminergic mesencephalic human fetal brain tissue has previously been reported in the treatment of patients with Parkinson's disease, this method is limited by the availability of tissue obtained from each human embryo.

Objective: Our study aimed to isolate, culture, proliferate, and differentiate dopaminergic neurons from human neuroepithelial stem cells obtained from embryo reduction procedures performed in multifetal pregnancies following in vitro fertilization.

Materials and methods: A total of 201 human embryos were dissected for isolation and culture of neuroepithelial stem cells for proliferation and differentiation into dopaminergic neurons. All embryos were obtained from embryo reduction procedures performed in multifetal pregnancies after in vitro fertilization treatments.

Results: Human neuroepithelial stem cells were isolated and cultured from embryos from 6.0 to 8.0 weeks. Neuroepithelial stem cells were successfully isolated, proliferated, and differentiated into dopaminergic neurons. The cells adhered to the surfaces of cell culture plates after 2 days and could be proliferated and differentiated into neurons within 4 days. Cultured cells expressed the dopaminergic marker tyrosine hydroxylase after 6 days, suggesting that these cells were successfully differentiated into dopaminergic neurons.

Conclusion: The successful isolation, culture, proliferation, and differentiation of human dopaminergic neurons from embryo reductions performed for multifetal pregnancies after in vitro fertilization suggests that this pathway may serve as a potential source of cell therapy materials for use in the treatment of Parkinson's disease.

Keywords: Parkinson’s disease; dopaminergic neuron; human neuroepithelial stem cells; in vitro fertilization.

Figure 1.. Isolation of neuroepithelial stem cells from human embryos. A: The number of embryos received, dissected, and from which stem cells were isolated at 6.0, 6.5, 7.0, 7.5, and 8.0 weeks. B: The average number of cells isolated from a single embryo (mean ± SEM) at 6.5, 7.0, and 7.5 weeks. C: The percentage of viable cells after isolation.

Figure 2.. Proliferation and differentiation of neuroepithelial stem cells. A: Cell adhesion on the culture plate at day 2. B: Neuronal polarization initiated at day 4. C and D: Cells proliferated and differentiated at day 6 (C) and day 9 (D). Arrows in B: branches from the cell body.

Figure 3.. Neuroepithelial stem cells differentiated into dopaminergic neurons. A: Cultured cells were visualized using an anti-vimentin antibody at day 6 (red). B: Cells were positive for Cajal silver staining at day 7 (brown). C and D: Some cells were positively stained with anti-tyrosine hydroxylase (TH) antibody at day 8 (red).

Figure 4.. Cultured cells continually differentiated into dopaminergic neurons. The numbers of tyrosine hydroxylase (TH)-positive cells at days 5, 7, and 10 in the 6.5-, 7.0-, and 7.5-week groups. *: P < 0.05; data represent the mean ± SEM.

Figure 5.. Cultured cells at day 10 expressed the morphological characteristics of neural stem cells and neurons. A: Neural stem cells; white arrows: indentations; red arrows: nucleoli. B: Neurites protruded from the cell body (yellow arrow), rough endoplasmic reticulum (*). C and D: Cultured cells have branches (* in C) and myelinated axons (* in D).