TGFβ superfamily signaling regulates the state of human stem cell pluripotency and competency to create telencephalic organoids

Abstract

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are emerging as an effective system to study the distinct features of the developing human brain and the underlying causes of many neurological disorders. While progress in organoid technology has been steadily advancing, many challenges remain including rampant batch-to-batch and cell lineto-cell line variability and irreproducibility. Here, we demonstrate that a major contributor to successful cortical organoid production is the manner in which hPSCs are maintained prior to differentiation. Optimal results were achieved using fibroblast-feeder-supported hPSCs compared to feeder-independent cells, related to differences in their transcriptomic states.Feeder-supported hPSCs display elevated activation of diverse TGFβ superfamily signaling pathways and increased expression of genes associated with naïve pluripotency. We further identify combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enable reproducible formation of brain structures suitable for disease modeling. HIGHLIGHTS• hPSC maintenance conditions influence outcomes in cortical organoid formation • Identification of an intermediate pluripotency state optimal for cortical organoids • Feeder support involves activation of diverse TGFβ signaling pathways • The organoid-promoting effects of feeders can be mimicked by a TGFβ factor mixture 3 KEYWORDS brain organoid, neurogenesis, neural stem cell, embryonic stem cell, pluripotent stem cell, differentiation, neural development, stem cell heterogeneity, pluripotency, cerebral cortex, ganglionic eminence, hippocampus 4

Identification of neural oscillations and epileptiform changes in human brain organoids

“…In the absence of Shh signaling, organoids predominantly exhibited cortical character including expression of the apical and basal radial glial progenitor marker PAX6, the intermediate progenitor marker TBR2 (EOMES), deep cortical plate markers including TBR1, CTIP2 (BCL11B), and BHLHB5 (BHLHE22), and superficial layer markers such as SATB2, and BRN2 (POU3F2) (Figs. 1b and 3, see also 17, 25 ). Shh pathway-stimulated organoids by contrast expressed canonical GE progenitor and migratory interneuron markers such as NKX2.1, DLX1, DLX2, and OLIG2.…”

Identification of neural oscillations and epileptiform changes in human brain organoids

“…Without Shh signaling, organoids exhibited cortical characteristics including expression of the apical and basal radial glial progenitor marker PAX6, the intermediate progenitor marker TBR2 (EOMES), deep cortical plate markers including TBR1, CTIP2 (BCL11B), and BHLHB5 (BHLHE22), and superficial layer markers such as SATB2, and BRN2 (POU3F2) (Fig. 1b, see also 17,25 ). Shh pathway-stimulated organoids by contrast expressed canonical GE progenitor and migratory interneuron markers such as NKX2.1, DLX1, DLX2, and OLIG2.…”

“…Over time in culture, many neurons within GE organoids expressed GABAergic inhibitory neuron markers such as GAD65 (GAD2) and GABA along with a variety of interneuron subtype markers (Fig. 1b, see also 17,25 ).…”

Patient-Derived Midbrain Organoids to Explore the Molecular Basis of Parkinson's Disease

“…Furthermore, the way in which hiPSC cells are cultured in the lab may have an important effect on their ability to generate reproducible organoid structures. Indeed, a recent study from Watanabe et al ( 118 ) revealed that commonly used feeder-free hiPSC culture conditions (compared to fibroblast-supported), reduced their ability to generate reproducible high-quality cortical organoids by altering their pluripotency state. The authors however showed that these defects could be alleviated through the use of TGFß superfamily agonists, which increase the quality of organoid differentiation toward different brain areas as well as reproducibility across cell lines.…”