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Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience

Nature Reviews Neuroscience volume 17, pages 424–437 (2016)Cite this article

Subjects

Key Points

  • Since the discovery of induced pluripotent stem cell (iPSC) reprograming, many protocols to generate human neural cells, including specific neuronal subtypes, astrocytes and other glial cells have emerged.

  • Guided differentiation protocols for the production of specific brain cell subtypes from human pluripotent stem cells (hPSCs) follow known developmental pathways. The precise combination of molecular cues allows scientists to guide neural commitment, early regionalization and cell type specification to develop cultures enriched for specific neuronal subtypes.

  • The generation of functional neurons and glia from hPSCs can be markedly improved by controlling the cellular environment in vitro, and advances in developing optimized culture scaffolds, culture media and 3D neural organoid cultures enhance our ability to provide more sophisticated tissue models of the brain. Authentic models of physiological human neuronal tissue in vitro will provide novel means to study human neuronal development and disease.

  • Functional neural cell cultures can also be obtained by converting primary human somatic cells, such as skin fibroblasts, directly into neurons or glia. Direct conversion into induced neurons (iNs) is typically achieved through the overexpression of pro-neuronal transcription factors; alternative methods based on RNA interference or small molecules are also being developed.

  • Direct conversion of human somatic cells into defined neuronal subtypes can be achieved through combined overexpression of general pro-neuronal drivers and secondary lineage-specifying transcription factors.

  • The increasing number of available methods to generate different neural cell types from human skin cells demands a careful consideration of practical as well as conceptual differences between hPSC differentiation and direct conversion methods. Practical concerns may include duration, difficulty, costs, efficiency, available cell numbers and subtypes that can be generated by a certain method; conceptual differences encompass the influence of the respective reprogramming method on the genomic identity of the derived cell culture (for example, clonal or mosaic), their epigenetic state (for example, rejuvenation), as well as whether a certain method recapitulates relevant neurodevelopmental and maturation.

Abstract

The scarcity of live human brain cells for experimental access has for a long time limited our ability to study complex human neurological disorders and elucidate basic neuroscientific mechanisms. A decade ago, the development of methods to reprogramme somatic human cells into induced pluripotent stem cells enabled the in vitro generation of a wide range of neural cells from virtually any human individual. The growth of methods to generate more robust and defined neural cell types through reprogramming and direct conversion into induced neurons has led to the establishment of various human reprogramming-based neural disease models.

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Figure 1: Reprogramming or direct conversion to generate neural cells.
Figure 2: Stages of neural differentiation in vitro and in vivo.
Figure 3: Methods for direct iN conversion.
Figure 4: Comparing iPSC differentiation and direct iN conversion.

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Acknowledgements

The authors thank M. L. Gage for editorial comments and J. R. Herdy and S. T. Schafer for helpful discussions. This work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, the JPB Foundation, Ipsen Pharma, The Leona M. and Harry B. Helmsley Charitable Trust grant #2012-PG-MED002, the Paul G. Allen Foundation, Janssen, and the American Federation for Aging Research.

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  1. Jerome Mertens and Maria C. Marchetto: These authors contributed equally to this work.

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  1. Salk Institute for Biological Studies, Laboratory of Genetics, La Jolla, 92037, California, USA

    Jerome Mertens, Maria C. Marchetto, Cedric Bardy & Fred H. Gage

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Glossary

Induced pluripotent stem cells

(iPSCs). Pluripotent stem cells created from differentiated somatic cell types, such as fibroblasts, by reprogramming with a set of transcription factors or other approaches.

Direct conversion

This term (also known as direct cell fate conversion, lineage conversion or transdifferentiation) describes the forced identity change of a somatic cell — for example, a fibroblast — directly into another related or unrelated somatic cell — such as a neuron — by means of transcription factors or other approaches.

Epigenetic

Describes changes in gene function that occur through changes to the genome that do not involve altering the DNA sequence. Examples of epigenetic events include DNA methylation, histone acetylation or X-chromosome inactivation. Epigenetic changes control biological processes such as differentiation, cell type identity or ageing.

Human pluripotent stem cells

(hPSCs). Stem cells that have the potential to form any cell type of the human body. They include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). All hPSCs have the same ability to differentiate into cells of distinct lineages, such as neurons.

Human embryonic stem cells

(hESCs). Pluripotent stem cells that are derived from the inner cell mass of the developing blastocyst.

Mitogens

Proteins or chemical compounds that induce cell division by triggering mitosis. Proliferation of stem cells during development in vivo and in vitro depends on mitogen-induced signalling, whereas mitogen-deprivation induces stem cell differentiation.

Morphogens

Proteins or chemical compounds that define the relative position of stem and progenitor cells in the developing organ in vivo and thereby lay out the pattern for the spatial organization of cellular subtypes within the same tissue. In vitro, morphogens are used to pattern stem and progenitor cells such as neural progenitor cells (NPCs) to promote the generation of a desired cellular subtype that is associated with a certain tissue region in vivo.

Neural progenitor cells

(NPCs). A broad term describing stem and progenitor cells of the nervous system. There are many types of NPCs during neural development and in the adult brain that all share the characteristics of proliferation and multipotency, meaning that they can give rise to enlarged numbers of differentiated neurons and glia.

Neural rosette

The developmental signature of neural progenitors in cultures of differentiating human pluripotent stem cells; rosettes are radial arrangements of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube.

Radial glial cells

A subtype of neural progenitor cells that span the radial axis of the developing cortex and serve as precursors or guides for newly born postmitotic neurons on their way into the mantle zone.

Embryoid bodies

Aggregates of pluripotent stem cells that are in the process of differentiation. The 3D structure of embryoid bodies is thought to provide a cellular environment that promotes the differentiation of pluripotent cells into desired cell types in several differentiation protocols.

Short interfering RNA

(siRNA). Short double-stranded RNA molecules that silence gene expression in a sequence-specific manner by a process termed RNA interference.

Genetic mosaicism

The presence of a variety of genetically distinct populations of cells within one individual. Differences in the cellular genotypes may comprise single-nucleotide polymorphisms (SNPs), indels, copy number variations and loss of heterozygosity. Such genetic changes can be caused by viral insertions, endogenous retrotransposition, DNA damage or repair and other mechanisms and may arise during development or later in life.

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Mertens, J., Marchetto, M., Bardy, C. et al. Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience. Nat Rev Neurosci 17, 424–437 (2016). https://doi.org/10.1038/nrn.2016.46

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