From Human Pluripotent Stem Cells to Neurons: Promises and Pitfalls


Pluripotent stem cell‐derived neurons represent a potentially limitless supply of human neurons to model diseases, replace diseased or dysfunctional tissue and to identify novel therapeutics to treat disease. Despite the incredible potential that human stem cell‐derived neurons represent, genetic, cellular and developmental obstacles represent substantial roadblocks to realizing this potential. Current schemes used to generate neurons from pluripotent stem cells rely on incomplete knowledge of human brain development, and often generate heterogeneous and immature neuronal populations. Recent technological advances in synthetic biology, genome engineering and single cell analysis have provided new tools for biologists to advance both our understanding and use of stem cell‐derived neurons. These advances should help overcome the pitfalls of pluripotent stem cells to generate homogeneous, mature neuronal subtypes relevant to disease and to realise the therapeutic promise of human stem cell‐derived neurons.

Key Concepts

  • The ability to generate pluripotent stem cells from adult somatic cells now gives researchers unprecedented access to human neurons for disease modelling, cell replacement therapy and high throughput screening.

  • Our incomplete understanding of human brain development is an obstacle to deriving human neurons in vitro that mimic their in vivo counterparts.

  • Current differentiation schemes allow researchers to derive motor neurons, excitatory and inhibitory neurons of the human cortex and other neuronal subtypes.

  • Subtle differences in the derivation or differentiation of pluripotent stem cells can have profound functional consequences for neurons derived from them.

  • Genome editing techniques are poised to allow researchers to generate even more useful cellular tools by engineering mutations and reporters into pluripotent stem cells.

Keywords: pluripotent stem cell; neuronal differentiation; neural induction; disease models; genome editing; CRISPR; Cas9

Figure 1.

The pitfalls of human pluripotent stem cell‐derived neurons. Schematic representation of the steps required for the generation of patient‐specific neurons. iPSCs are first derived from skin cells (or other adult cells) by the addition of four transcription factors, then the stem cells are differentiated into neurons. The transcription factors or chemicals required in this process vary depending on the type of neurons desired. The resulting neuronal culture is often heterogeneous, and the neuronal subtype of interest can then be purified by FACS. The challenges faced at every step are outlined in red.

Figure 2.

Identifying and using regional specification genes to generate and purify human stem cell‐derived neurons. (a) Isolation of single neurons or single populations of neurons from human brain samples and use of whole transcriptome identification techniques such as RNA sequencing and subsequent comparison to other similarly derived neurons allows for the identification of genes that specify a neuron's regional identity. (b) The promoters of these regional specification genes can then be used to make reporter genes that control the expression of a visible marker such as green fluorescent protein (GFP). With the use of genome engineering techniques these reporters can be integrated into the human pluripotent stem cell. (c) These reporters can then be used to both optimise differentiation conditions and to isolate populations of human stem cell‐derived neurons.

Figure 3.

Generation of isogenic human pluripotent stem cell lines using site‐specific endonucleases. Two routes can be taken to generate isogenic pluripotent stem cell lines to investigate the contribution of DNA variants or mutations to cellular phenotypes. One can start with the unaffected or wild type cell line and introduce a mutation or variant using site‐specific endonucleases like CAS9/CRISPR. By contrast, one can start with the mutant or patient cell line and correct the mutation to the wild type using Cas9/CRISPR. The engineered cell line can then be differentiated into neurons and compared to the starting cell line for specific phenotypes. This workflow helps to control for natural variation in the cellular phenotype due to the cells' genetic background that might be unrelated to the disease or cellular phenotype under study.



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Further Reading

Cahan P, Li H, Morris SA et al. (2014) CellNet: network biology applied to stem cell engineering. Cell 158(4): 903–915.

Maroof AM, Keros S, Tyson JA et al. (2013) Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12(5): 559–572.

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Nehme, Ralda, and Madison, Jon M(Nov 2014) From Human Pluripotent Stem Cells to Neurons: Promises and Pitfalls. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025784]