Abstract
Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.
Highlights
Human embryonic stem cells, which are derived from the inner cell mass (ICM) of blastocysts, are pluripotent cells that can proliferate indefinitely and still maintain their pluripotency and capability to differentiate into cells of all three germ layers: ectoderm, mesoderm, and endoderm [1, 2]
Under correct maintenance conditions, these cells can be programmed to differentiate into neural stem cells, which naturally are capable of self-renewal and can be further generated into lineages such as mixed neuronal cultures, astrocytes, oligodendrocytes, and cholinergic neurons [7]
Immunocytochemistry was used to confirm the direction of cell fates into neurons, astrocytes, oligodendrocytes, and cholinergic neurons after three weeks of terminal differentiation (Figure 4). β-Tubulin III was used to visualize the presence of neurons, GFAP used for identification of astrocytes, GALC
Summary
Human embryonic stem cells (hESCs), which are derived from the inner cell mass (ICM) of blastocysts, are pluripotent cells that can proliferate indefinitely and still maintain their pluripotency and capability to differentiate into cells of all three germ layers: ectoderm, mesoderm, and endoderm [1, 2]. Their limitless capacity for self-renewal along with the potential of differentiation has made hESCs an effective model for regenerative medicines (Klimasnkaya, et al 2014).
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