Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience

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.

Figure 1 |. Reprogramming or direct conversion to generate neural cells.

Neural cells can be generated from somatic cells through somatic tissue reprogramming, which produces induced pluripotent stem cells (iPSCs), or by direct conversion. Neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs) can be generated through the differentiation of human pluripotent stem cells (hPSCs), which can comprise human embryonic stem cells (hESCs) or iPSCs, or by direct neural conversion of somatic cells such as fibroblasts. Differentiation-derived NPCs as well as direct conversion-derived induced NPCs (iNPCs) can further be differentiated into neurons and/or glial cells and can allow the study of aspects of human neurodevelopment. When somatic cells or iPSCs are directly converted into induced neurons (which are then known as iNs or iPSC–iNs, respectively), the NPC stage is bypassed. Cultures of neurons and glia can be used for studying disease-related biology and to develop phenotypic assays and screening to evaluate patient- or disease-specific phenotypes. For example, cellular morphology, activity patterns and connectivity can be assessed. Once a distinct disease-related phenotype is identified that can be reliably monitored, drug-screening platforms can be developed to test compounds that improve cellular phenotype. New diagnostic tools and therapeutic compounds could emerge from the screenings.

Figure 2 |. Stages of neural differentiation in vitro and in vivo.

When human pluripotent stem cells hPSCs (comprising human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)) differentiate into neurons in vitro (upper row), they transit through defined stages during which they resemble distinct neural progenitor cell (NPC) populations present during in vivo neurogenesis (lower row). hPSCs resemble the inner cell mass (ICM) of the blastocyst^,. hPSCs differentiate into neuroepithelial stem cells in vitro, corresponding to the neuroepithelial NPCs that form the neural plate in vivo. During in vivo neurulation, the neural tube closes, patterning along the developmental axes takes place and the first waves of neurons are generated. In vitro, the rosette-type NPCs that can also be derived from hPSCs resemble this developmental stage^,,. During fetal and adult neurogenesis, radial glia give rise to postmitotic neurons. These correspond to the radial glia-like NPCs that are generated from the rosette-type NPCs in vitro^,.

Figure 3 |. Methods for direct iN conversion.

a | During direct conversion into induced neurons (iNs), fibroblasts progressively convert into neurons through an as yet poorly defined transient intermediate state. This process involves dramatic morphological changes but no cell division. General pro-neuronal transcription factors (TFs) such as achaete-scute homologue 1 (ASCL1) and neurogenin 2 (NGN2) act as pioneer transcription factors that trigger the expression of structural neuronal proteins such tau (which help to drive the establishment of neuronal compartments such as the axon or dendrites), neurotransmitter receptors (which are required for postsynaptic structures)^, and ion channels (which build up a neuronal membrane potential)^,. Pioneer transcription factors also open chromatin structures to allow binding of secondary transcription factors (both transgenic and endogenous) that facilitate the expression of more mature or subtype-specific proteins, such as the enzymes tyrosine hydroxylase (TH) or tryptophan hydroxylase (TPH), which are needed for dopamine or serotonin production, respectively. b | The relative timeframe for direct iN conversion using different approaches. Orange bars indicate the stage at which the respective factors or compounds are believed to be effective. General pro-neuronal pioneer transcription factors such as ASCL1 and NGN2 work to reprogramme the cells at the fibroblast stage and tend to result in the production of glutamatergic neurons (the majority) and GABAergic neurons (a minor fraction). Secondary transcription factors such as neurogenic differentiation factor 1 (NEUROD1), BRN2 and myelin transcription factor 1-like protein (MYT1L) are not sufficient to initiate iN conversion but support the process at later stages^,,. c | To facilitate neurotransmitter-specific iN conversion, cocktails of specific transcription factors can be added to shape a specific neuronal identity. These lineage-specifying transcription factors are typically well known for their essential roles during the development of the targeted neuronal subtype in vivo^,,,,–,. d | Manipulation of signal transduction pathways through growth factors and small molecules that inhibit the transforming growth factor-β (TGFβ)–ALK–SMAD pathway and glycogen synthase kinase 3β (GSK3β), as well as the promotion of cyclic AMP signalling, increases iN conversion efficiencies. Addition of other molecules (such as I-BET151, isoxazole 9 (ISX9) or protein kinase C (PKC), JUN amino-terminal kinase (JNK) or RHO-associated protein kinase (ROCK) inhibitors) to that mix facilitates direct conversion from fibroblasts without the need for transgenes^,,,. CTIP2, COUP-TF-interacting protein 2; FOX, forkhead box protein; LMX1, LIM homeobox transcription factor 1; PITX3, pituitary homeobox 3; PTB1, polypyrimidine tract-binding protein 1; REST, RE1-silencing transcription factor.

Figure 4 |. Comparing iPSC differentiation and direct iN conversion.

a | Time course and efficiency. Direct induced neuron (iN) conversion is a rapid process for the generation of neurons from donor cells. However, human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-based strategies can yield infinite numbers of neurons, whereas iN conversion is limited to the expandability of fibroblasts. b | Development and age. In contrast to iNs, human pluripotent stem cell (hPSC) differentiation follows distinct steps of human neural development and transits through cell types corresponding to the various stages of neurulation and neurogenesis. However, hPSC-derived cells transit through the embryo-like hPSC stage and, as a result, hPSC-derived neurons are regarded as rejuvenated neurons. Direct conversion skips these steps and directly transforms a fibroblast into a neuron, thus maintaining the signatures of their donors’ ages. c | Diversity and mosaicism. Human somatic cells within an individual do not have identical genomes, and this somatic mosaicism might be an important determinant for biological function of tissues and organs. During iN conversion, a genetically mosaic culture of fibroblasts is converted into a mosaic culture of neurons. By contrast, iPSC lines are clonal cell lines, leading to a culture of neurons that all arise from the same single fibroblast cell.