Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System

Abstract

SummaryHuman pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4–5 months, even without astrocyte feeder layers.

Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease

“…Human iPSC were derived, maintained, and differentiated into MNs following previously described protocols (Yang et al, 2013, Rigamonti et al, 2016) and specific modifications are included in Supplemental Experimental Procedures. For astrocyte co-cultures, mouse astrocytes were prepared from P0-P2 CD1 mouse cortices as previously described (Schildge et al, 2013).…”

The future is now: cutting edge science and understanding toxicology

“…Neurodegenerative diseases are predicted to become an increasing burden on society, and iPSCs can be used to generate disease models for compound screening to identify new drug candidates, such as Parkinson's disease and, notably, Zika virus infection (Kikuchi et al 2017;Xu et al 2016). Devine and Patani also describe the latest progress in scaling up the production of therapeutically relevant cell types derived from iPSCs for cell therapy applications, for example, developing 3D cultures that allow the harvesting of billions of uniform, mature neurons from a single flask (Rigamonti et al 2016). The most recent approaches for iPSC-based cell therapy were also described, such as the intracerebral injection of human iPSC-derived oligodendrocytes to treat primate models of multiple sclerosis (Thiruvalluvan et al 2016).…”

Neural organoids for disease phenotyping, drug screening and developmental biology studies

“…In contrast to the manipulation of adherent colonies or single cells outlined in the aforementioned proof-of-concept reports, Rigamonti et al, demonstrate that by beginning with 3D spheres of hESCs and hiPSCs, rather than adherent colonies, EBs can be generated simply by changing the growth medium (Rigamonti et al, 2016). Moreover, the EBs they describe are more uniform in both size and shape, do not require transition through a cell intermediate or matrigel droplet manipulation, and can be directed to either functionally mature cortical or motor neuron fates by changing the growth factor additives (Rigamonti et al, 2016). The authors conclude that this protocol is capable of yielding subtype specific populations of neurons in a highly reproducible fashion with minimal experiment-to-experiment variation.…”

“…Thus, it is not clear whether current protocols generate organoids with fundamentally identical cell compositions from organoid-toorganoid or batch-to-batch. Although Rigamonti et al, argue that starting with 3D hPSC spheres generates neurons of distinct phenotypes in “a highly reproducible manner, with minimal experiment-to-experiment variation” (Rigamonti et al, 2016), their study only focused on well characterized hPSC lines and thus it remains to be seen how this and other protocols will scale across large cohorts with multiple clones.…”

“…To date, assays of neural organoid connectivity and neuronal activity have not typically been performed in situ ; electrophysiology, calcium imaging and immunofluorescence techniques are generally performed in either slice culture or using dissociated and re-plated organoids (Mariani et al, 2015; Paşca et al, 2015; Qian et al, 2016). Combining advanced optical techniques, such as two-photon microscopy (Truong, Supatto, Koos, Choi, & Fraser, 2011) and light-sheet microscopy (Ahrens, Orger, Robson, Li, & Keller, 2013) with approaches such as optical clearing for immunofluorescence analysis (Azaripour et al, 2016; Chung & Deisseroth, 2013; Renier et al, 2014) will provide a level of analysis in whole intact 3D neural organoids as recently demonstrated (Rigamonti et al, 2016). Moreover, organoid analyses using advanced genetically encoded indicators (Hochbaum et al, 2014) or reporter cell lines driven by cell type specific promoters (Shcheglovitov et al, 2013), together with in situ gene expression analysis (Lee et al, 2015) and connectivity mapping (Cai, Cohen, Luo, Lichtman, & Sanes, 2013; Markram et al, 2015) will also greatly benefit phenotyping.…”