Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.


Figure S1 .
Figure S1.Quality control assessment of dorsal telencephalic patterning during the fourth week of organoid culture (quality checkpoint #3).Correct patterning of early stage organoids was verified by expression of FOXG1 and PAX6 in SOX2+ neuronal progenitors and expression of TBR1 in newborn neurons.For this critical quality checkpoint #3 of our culture protocol, a subset of organoids was analyzed between culture day 24 and day 28 of any given culture experiment.Images show results of immunostaining of 24-day-old organoids derived from the BIHi001-B line.Scale bars: 100 µm.

Figure S2 .
Figure S2.Examples of organoid batches that passed (upper panel) or failed to pass (lower panel) criteria of quality checkpoint #3 (assessment of dorsal telencephalic patterning).Upper panel shows positive FOXG1 immunostaining of 27-day-old organoid derived from BIHi250-A line.Lower panel shows lack of FOXG1 expression in 27-day-old organoid derived from BIHi005-A line.Note that organization of SOX2+ cells into rosette-like structures is not a sufficient criterion to verify successful generation of cerebral cortical tissue.Organoids from the non-compliant BIHi005-A batch were initially compliant with gross morphological criteria described for quality checkpoints #1 (EB formation) and #2 (neuroepithelium).Failed cortical lineage specification was only revealed by analysis of marker expression in early-stage organoids.Scale bars: 100 µm.

Figure S3 .
Figure S3.Direct neurogenesis in early-stage organoids.Distribution of TBR1+ neurons (arrows) in ventricular zone of 3.5-week-old organoids.The distribution profile of TBR1+ cells indicates that RGC divisions in the VZ produce daughter cells that directly differentiate into neurons bypassing the generation of basal neurogenic progenitors such as IPC and oRGC.When judged from prevalence of TBR1+ cells within the VZ, direct neurogenesis was prominent in organoids during culture weeks 3 and 4 and declined at later stages.Scale bars: 50 µm.

Figure S4 .
Figure S4.Sequential generation of a diverse repertoire of cortical neurons during a 10-week organoid culture recapitulates the inside-out pattern of neuronal layering.A first wave of neurogenesis between culture weeks 2 and 4 produces mainly TBR1+ neurons that do not yet express deep layer neuron markers such as CTIP2.A next wave of neurogenesis (4 to 8 weeks of culture) produces neuronal subtypes that will eventually populate the deepest layers of the cortical wall.These deep layer neurons express high levels of CTIP2.As corticogenesis proceeds further, newly generated neurons migrate radially throughout the deep neuronal layer to occupy successively the upper layers of the cortical plate.Accordingly, first expression of the upper layer neuron marker SATB2 becomes detectable during the 8th week of culture and a prominent SATB2+ neuronal layer is visible in the upper region of the cortical plate in 10-week-old organoids.Dashed lines mark the border between the ventricular zone (VZ), subventricular zone (SVZ) and cortical plate (CP).Scale bars: 20 µm.

Figure S5 .
Figure S5.Developmental expression of the upper layer neuron marker SATB2 in organoids between week (w) 5.5 and 9 of culture.SATB2 expression was undetectable in organoids before the seventh week of culture.First weak SATB2 staining was detectable in 7.5-week-old organoids.In 8.5-weekold organoids and at later stages, SATB2+ neurons were enriched in more basal regions of the CP.In human fetal cortex tissue, a corresponding onset of SATB2 expression occurs at GW12-13.Dashed lines mark the border between the ventricular zone (VZ), subventricular zone (SVZ) and cortical plate (CP).Scale bars: 50 µm.

Figure S6 .
Figure S6.Newborn TBR1+ neurons in the SVZ of 7-week-old organoids are characterized by a lower immunostaining signal intensity compared to more mature TBR1+ neurons that migrated out to the CP.Lower right image shows a magnified view of the boxed region marked in the lower left image.Dashed lines mark the border between VZ, SVZ and CP.Scale bars: 50 µm (20 µm in lower right magnified view image).