Differentiation of neural lineage cells from human pluripotent stem cells

Abstract

Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: (1) starting material, (2) induction of loss of pluripotency, (3) neural induction, (4) neural maintenance and expansion, and (5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

Figure 1

Schematic of the stages in the differentiation of PSCs. The starting material, undifferentiated PSCs (1), progresses through four transitions, each time becoming more lineage restricted. The first transition is loss of pluripotency (stage 2), which is usually accomplished by the formation of embryoid bodies. At stage (3), neural rosettes have formed. These are the anlage of the nervous system and can be thought of as consisting of neural stem cells with the greatest degree of multipotentiality. At stage (4), a certain degree of lineage restriction, within the neural lineage, has taken place. This is indicated by a broken ring. These cells tend to further differentiate toward a particular mature neural lineage, although under some circumstances a residual plasticity can still be demonstrated. At stage (5), neural cells of a particular lineage have been produced and there is little interchangeability among them, again illustrated by the breaks in the circle. These include oligodendroglia, astroglia and neurons of anterior, posterior, or retinal derivation.

Figure 2

Gene expression microarray data from two PSC cultures grown under STD conditions, plotted against each other. Note the rather loose grouping of the data. The data points in gray (shaded area at the bottom-left of the graph) represent those transcripts whose expression levels fell below the limit of detection. Although classic markers of undifferentiated PSCs can easily be detected, such as OCT4, Nanog, and teratocarcinoma derived growth factor 1 (cripto 1), markers of more differentiated stem cells and their progeny are also seen, as described in the text. These data represent two pairs of four different cultures of the same PSC line (H9).

Figure 3

Immunocytochemical staining of doublecortin(DCX)-positive cells (inset) overlaid on the differentiation schema of Figure 1. The inset-left panel (10x) shows DXC+ cells (green, arrows) at the juncture of two colliding PSC colonies (unstained, hESCs), growing under STD conditions. The inset-right panel (40x) shows DCX+ cells (green, arrows) with typical morphologies found in the MEFs (unstained, MEFs) surrounding the colonies. These photomicrographs illustrate that, even under conditions favoring pluripotency, some cells progress through stages 2 and 3 to stage 4 and show, therefore, the propensity of PSCs to differentiate down the neural lineage.

Figure 4

ePSCs in culture. Photomicrographs are taken through the phase-contrast microscope at 40x (left panel), 100x (center panel), and 400x (right panel) magnification. These colonies of ePSCs, as is commonly the case, are grown in the presence of a feeder layer of cells (black arrowheads), in this case mouse embryonic fibroblasts. Even when grown under conditions that do not favor differentiation, spontaneous differentiation occurs and is seen as groups of less tightly packed cells emanating from the sides of the ePSC colonies (white arrowheads).

Figure 5

Formation of embryoid bodies induces a dramatic shift in gene expression in PSCs. The line of identity (thick line) and the bounds of 2-fold expression differences or less (thin lines) are shown. The expression of many gene products falls well outside the boundaries. Note the increased spread of the data compared to that in Figure 2. In this particular case, EBs express some 617 genes at significantly higher levels (p<0.0001) compared to PSCs, while PSCs express some 1305 genes at higher levels than EBs. The data represent pooled mRNA from the H1, H7, H9, BG01, BG02, BG03, and BG01v cell lines.

Figure 6

bNSC gene expression versus that of eNSCs. The former were derived from human brain[103] and the latter were derived from ePSCs[23]. Both cell types were cultured in bFGF/EGF-containing media. In the graph, a select few stem cell-specific (red) and neural cell-specific (green) genes are highlighted. The darker gray dots are those gene products that are significantly different between the two cell populations (p<0.0001). What is clear is that there is a wide digression of gene expression between the two cell types: there are both a large scatter of the data as well as a lack of identity of stem cell-specific and neural cell-specific genes.