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Pluripotent Stem Cell Derived "Minibrains" to Aid Research

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Original article from Nature

The generation of cerebral organoids (or "mini-brains") from pluripotent stem cell sources is one of the most exciting and widely reported recent research articles.   This study from the laboratory of Juergen A. Knoblich at the Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna , Austria could have huge importance in the study of human brain development and the study of related diseases (Lancaster et al).   Herein, we briefly describe the details behind the headline.

The group began by generating neuroectoderm from embryoid bodies from human pluripotent stem cells (EBs) which were then transferred to a spinning bioreactor which mediated enhanced nutrient absorption. Interestingly, SMAD inhibitors, which are commonly used to promote neuroectoderm and suppress mesoderm and endoderm derivatives (Chambers et al), were not used in this study. However, this technique allowed for the observation of neural cell identity within 10 days, followed by the detection of defined brain regions between days 20 to 30. In these early stages of organoid development, large ventricular-like structures of continuous neuroepithelia were apparent, which is in contrast to 2D growth conditions in which small rosette-like neuroepithelial aggregates are observed.   At maximal size (up to 4mm) at 2 months of differentiation, analysis of organoids revealed regions reminiscent of cerebral cortex, choroid plexus, retina and meninges with similar results seen between human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

mRNA analysis of distinct marker genes confirmed the diminution of OCT4 and NANOG and the upregulation of neural identity markers (SOX1 and PAX6). During organoid development , forebrain (FOXG1 and SIX3) and hindbrain (KROX20 and ISL1) markers were apparent, PAX6 was expressed in several regions of forebrain identity, while OTX1 and OTX2 expression marked forebrain/midbrain identity adjacent to regions reminiscent of the early mid–hindbrain boundary (GBX2, KROX20 and PAX2). Further in depth analysis of these regions found FOXG1+EMX1+ cells indicating a dorsal cortical identity.   Cortical lobe markers (AUTS2, TSHZ2, LMO4) were also observed in neurons labelling distinct regions of dorsal cortex suggesting sub-specification of cortical lobes. While hippocampal and ventral forebrain markers were evident in cells, the typical in vivo cytoarchitecture was not present, although some evidence of interneuron (Calretinin+) migration from the ventral forebrain to the dorsal cortex was observed. Additionally, structures such as the choroid plexus and immature retina were also observed, suggesting that organoid production in this manner can generate a variety of brain regions.

The dorsal cortical region is dramatically different between human and rodents; and so this region was studied in detail and found to display typical zone organization in organoids. Radial glial stem cells (RGs) and newborn neurons formed layers akin to the ventricular zone and there was evidence of the development of neural identity and radial migration to the developing pre-plate and the presence of intermediate progenitors adjacent to the ventricular zone. Additionally RGs displayed mitotic division at the apical surface of the ventricular zone and had basal cellular processes typical of RGs. Using GFP to track RGs; their morphology was further shown to be akin to that observed during development; early pseudostratified neuroepithelium, then bipolar morphology with extended apical and basal processes.   RGs were also shown to be able to divide symmetrically and asymmetrically.

Marker staining also found evidence of a basal neural layer reminiscent of the pre-plate, the origin of the cortical plate. Reelin+ cells, indicative of Cajal–Retzius cells which are important to cortical plate architecture, were also observed. Evidence of distinct layer identities was demonstrated through CTIP2+ neurons adjacent and internal to the pre-plate at early stages and separation of neurons into early-born deep layer (CTIP2+) and a late-born superficial layer (SATB2+BRN2+) which became more distinct with time. However, full recapitulation of mature layer organization was not observed suggesting a lack of signals required for this event to occur. Dorsal cortical neurons were also present and displayed complex branching and growth cone behaviour and projected long-range axons in a manner reminiscent of axon bundling. Furthermore, Ca2+ oscillation analysis suggested that the neurons within cerebral organoids were active.

The final stage of structural analysis examined cortical organization. SOX2+ progenitors were found to be displaced from the apical surface, similar to that observed for outer radial glia, and were separated from the apical ventricular zone by a TUJ1+ inner fibre layer (IFL) somewhat akin to that observed in vivo. Outer radial glia had basal process but no apical processes, divided asymmetrically, and were found in a vertical orientation, all similar to the in vivo status (LaMonica et al).

The final part of this exciting study analysed whether such organoids could be used for models of neurodevelopmental disorders. To this end, skin fibroblasts from a patient with severe microcephaly were reprogrammed and the resultant iPSCs used to generate organoid cultures. A greater number of disease-harboring cells was required for EB formation compared with unaffected cells, and subsequent organoid analysis revealed smaller neuroepithelial tissues and a large degree of neuronal outgrowth with few progenitor rich regions compared with control tissues. This underdevelopment was studied over time and at 22 days of development patient-derived organoids presented with only occasional neuroepithelial regions, decreased RGs and increased neurons suggestive of premature neural differentiation. Furthermore, not all RGs were horizontally oriented which could affect the symmetric expansion of neural stem cells (Yingling et al).

Human pluripotent cells have previously been utilised to create self-organizing structures that can develop into recognizable three-dimensional tissues (Sasai) and now, the application of an improved protocol and detailed analysis has allowed the generation of brain organoids in vitro which recapitulate many important developmental processes occurring in the human brain and give rise to primitive brain structures. This system may enable the detailed analysis of early human brain development as well as the development of neurological disease. Unfortunately, as the authors note, the organoids however do not fully model the organization of the human brain, with the random location of brain regions and lack of overall structure. However, this study represents a launch pad for the refinement of this technique to allow better organization of cerebral tissues towards the recapitulation of a more complete final product. 

References

Chambers, S.M. et al.
Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.
Nat. Biotechnol. 27, 275–280 (2009).

LaMonica, B. E. et al.
Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.
Nature Commun 4, 1665 (2013)

Sasai, Y.
Next-Generation Regenerative Medicine: Organogenesis from Stem Cells in 3D Culture.
Cell Stem Cell12, 520–530 (2013)

Yingling, J. et al.
Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division.
Cell 132, 474–486 (2008)

 

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.