Highlights
- •hPSC maintenance conditions influence the quality of cortical organoid formation
- •Identification of an intermediate pluripotency state optimal for cortical organoids
- •MEF feeder support enhances activation of diverse TGFβ signaling pathways
- •The organoid-promoting effects of MEFs can be mimicked by a TGFβ factor mixture
Summary
Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.
Introduction
The emergence of methods to direct the formation of neurons from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)—collectively hPSCs—provides unprecedented opportunities for investigating mechanisms behind healthy human brain development and neurological disease. Attention has most recently turned to the creation of tridimensional structures termed organoids or spheroids, which display spatial organization of neural progenitors and diverse populations of neurons and glial cells that better approximate the features of the developing human brain in vivo than two-dimensional cultures in vitro (Chiaradia and Lancaster, 2020; Velasco et al., 2020). Thus far, organoids have been used to model a variety of disorders impacting brain growth such as microcephaly and lissencephaly, and efforts to model more complex neurological disorders that principally impact neural circuit formation and function such as schizophrenia, autism spectrum disorders, and degenerative conditions are under way (Amin and Pasca, 2018; Samarasinghe et al., 2021; Zhang et al., 2021). Success in these endeavors, however, depends on the reproducible creation of well-structured organoids in which neural networks can be assembled.
Several studies have suggested that organoid reproducibility is best accomplished using protocols that direct the formation of specific brain regions such as the cerebral cortex and basal ganglia (Watanabe et al., 2017; Qian et al., 2018; Sloan et al., 2018; Xiang et al., 2018) reflected by a high degree of organoid consistency seen at the transcriptomic level (Qian et al., 2016; Watanabe et al., 2017; Velasco et al., 2019; Yoon et al., 2019). However, the cytoarchitecture of the organoids, particularly the laminar organization of the cortex, can still markedly vary (Bhaduri et al., 2020a, Bhaduri et al., 2020b). Some of these differences could arise from variabilities inherent to the PSC lines used in a given experiment (Ortmann and Vallier, 2017) and the lack of standardization in protocols used for neuronal differentiation (Anderson et al., 2021). The importance of organoid quality has been further raised by recent studies suggesting that in vitro culture can impart metabolic stress and thereby negatively impact progenitor maturation, cell type specification, and developmental trajectories (Bhaduri et al., 2020b). Given the importance of anatomical structure for neural circuit formation and function in vivo, organoids with irregular structure could serve as poor models for studying the mechanisms behind healthy human brain development and disease.
Previously, we established efficient and reproducible cerebral organoid methods that exhibited improved structural features, including marked expansion of the outer subventricular zone (SVZ), abundant formation of basal progenitors and astrocytes, and enhanced production of upper-layer neurons (Watanabe et al., 2017). We further documented that the developmental trajectory of these organoids was strikingly like that of the human fetal brain in vivo. Our methods also allowed use of developmental patterning signals to create distinct forebrain regions such as the ganglionic eminences (GE), permitting the formation of cortex-GE fusion organoids in which excitatory and inhibitory neurons can intermix to form functional neural networks that mimic activities seen in the human fetal cortex (Samarasinghe et al., 2021). However, in conducting these experiments, we observed that optimal results were achieved when hPSCs were maintained under certain culture conditions, suggesting that even with a well-established organoid protocol, variability could still arise from changes inherent to the starting stem cell population.
While there are many potential contributors to PSC variance, one feature that has been gaining recognition is their state of pluripotency: naive, primed, or a transition phase termed formative (Smith, 2017; Kinoshita et al., 2020). Naive and primed states reflect the developmental progression of pre- to post-implantation epiblast populations in vivo. Formative PSCs have intermediate properties of the two extremes, exhibiting features of the early post-implantation embryonic stage in vivo in which lineage-specific gene expression is minimal and cells are still capable of adopting early developmental fates such as germline stem cells (Kalkan et al., 2017; Smith, 2017; Rostovskaya et al., 2019; Kinoshita et al., 202). Naive and primed PSC states are interconvertible in vitro (Weinberger et al., 2016); thus, the different ways in which hPSCs are maintained could potentially alter their capacity to effectively form brain organoids.
Here, we define variances in the transcriptional state of hPSCs maintained under different conditions including certain mouse embryonic fibroblast (MEF) feeder-supported and feeder-free (FF) conditions, which were associated with markedly different differentiation outcomes with respect to the efficiency and structural quality of cerebral organoids. We further show that some of the major differences are the expression and activity of transforming growth factor β (TGFβ) superfamily signals derived from MEFs and the hPSCs themselves, which modulate the state of hPSC pluripotency along the naive-to-primed trajectory. The quality of organoids produced by MEF-supported hPSCs was reduced by inhibition of TGFβ signaling, while outcomes from FF hPSCs was improved by pre-treatment of these cells with a mixture of four TGFβ growth factors. Collectively, these studies identify the state of hPSC pluripotency influenced by TGFβ superfamily signaling as a major source of variability that can impact brain organoid formation and illustrate a strategy for modulating this state to help ensure reliability and consistency in organoids to support developmental studies and disease research.
Results
hPSC growth conditions influence their capacity to form well-structured cortical organoids
