Generating human neural diversity with a multiplexed morphogen screen in organoids

Neal D. Amin, Kevin W. Kelley, Jin Hao, Yuki Miura, Genta Narazaki, Tommy Li, Patrick McQueen, Shravanti Kulkarni, Sergey Pavlov, Sergiu P. Paşca

Preprint posted on 1 June 2023 https://doi.org/10.1101/2023.05.31.541819

How do morphogen signals instruct stem cells to acquire a specific neuronal identity? This preprint by Amin, Kelley, and team provides us with a roadmap. 🧫 🛣️ 🧠

Selected by Monica Tambalo

Categories: cell biology, developmental biology, neuroscience

Background

During embryogenesis the formation and regionalization of the neural tube involves a series of finely orchestrated steps, ultimately leading to the development of different domains of the nervous system (i.e., forebrain, midbrain, hindbrain, spinal cord). Cells receive precise instructions through morphogen signalling, which lead them to acquire specific brain identities and differentiate into the diverse cell types found in the brain [1, 2]. Although these processes have been extensively studied in various animal models, both mammalian and non-mammalian, our understanding of the role of morphogen signalling in human brain development is still limited due to technical and ethical reasons [3, 4].

The knowledge gained from studying brain development in animal models has played a crucial role in the establishment of in vitro protocols. Researchers were first able to generate human neurons in two-dimensional cultures, [5] and later with the emergence of neural organoid and assembloids protocol [6-8], it has become possible to reproduce specific brain areas in 3D and study their interactions [3, 9]. However, the use of morphogen pathways in establishing such in vitro systems has relied on a trial-and-error-based approach and there has been a lack of systematic screening of morphogen-directed differentiation. This missing knowledge has prompted Amin and colleagues to systematically uncover the capacity of morphogen signalling to orchestrate human neurodevelopment [10].

Key findings

Starting with human induced pluripotent stem cells (hiPSCs), Amin, Kelley, and colleagues used a dual SMAD inhibition protocol to promote neural induction (Fig.1A), followed by a morphogen screen. An ad hoc platform was designed to allow standardized screening of 8 morphogen pathways (WNT, SHH, growth factors, retinoic acid, BMPs, TGFb, SMAD, Notch) stimulated with 14 molecules in 46 unique combinations. 1500 individual organoids were grown and assessed over time for size, morphological features, neural differentiation, and transcriptional profiling by single-cell RNA-sequencing (Fig.1B).

The study yielded important insights into the effects of tested morphogens and identified general rules governing human neurodevelopment (Fig.1C):

Retinoic acid + SHH agonist (SAG) generated the highest number of oligodendrocyte cells.
Exposure to FGF8, along with FGF4, positively correlated with organoids size and the presence of ventricular-like regions (rosettes).
Glutamatergic and GABAergic neurons were generated under many conditions.
Dopaminergic neurons were generated after exposure to SAG+CHIR+FGF8.
The choroid plexus developed after exposure to BMP+CHIR [11].
FGF2 exposure in the presence of N2 supplement led to the development of cerebellar cellular types (e.g., cerebellar Purkinje cells, cerebellar interneurons, granule neurons), as also previously reported [12]. Importantly, this was specific to FGF2 and not shared by FGF8 or FGF4.
WNT signalling activation, using CHIR, functioned in a narrow time window to generate Cajal-Retzius (CR) neurons of cortical hem (CH) origins.
A population of primate-specific striatal forebrain interneurons (TAC3-INs) was identified in organoids grown with CHIR (1.5 µM) and SAG (1000-2000nM).
The expression of many genes associated with neuropsychiatric disorders (autism spectrum disorder (ASD), and schizophrenia (SCZ)) was found to be ubiquitous across neuronal and non-neuronal cell types, while others were associated with unique cell types originating from specific morphogen stimulation.

**Figure 1.** Roadmap for human neuronal diversity: from pluripotent stem cells to multiple human neuronal linages. Adapted from Amin, Kelley et al. [10].

Why I like this preprint

As a researcher working with neural organoids, I am constantly faced with the challenge of generating reproducible organoids in my need to differentiate several brain regions to model human diseases. The absence (so far) of a roadmap that is able to guide scientists in obtaining precise and reproducible differentiation of region-specific brain organoids has been a limitation for this field of research. Thus, this systematic attempt to decode the ability of morphogens to orchestrate human neurodevelopment – when either administered alone or in combination with others, at different concentrations, and/or at different time points – is a highly valuable guideline to help researchers develop new protocols and interpret their results. Furthermore, the identification of a rare and unique class of interneurons in primates once again underlines the potential of human neural organoids in studying unique human traits and shedding light on evolutionary innovations.

Questions for the authors

Q1) Did all tested hiPSC lines exhibit the same response to morphogen exposure, or were there some unique responses? If differences were observed, what do you speculate is the main driving force?

Q2) What was the rationale behind the conditions tested in terms of starting concentrations, timings and combinatorial use of the morphogens?

Q3) I am curious about the neuropsychiatric-associated genes that were found only in specific subsets of cell types, is their expression in vitro in line with their physiological expression in the developing human embryo?

References

Sagner, A. and J. Briscoe, Morphogen interpretation: concentration, time, competence, and signaling dynamics. Wiley Interdiscip Rev Dev Biol, 2017. 6(4) DOI: 10.1002/wdev.271
Wurst, W. and L. Bally-Cuif, Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci, 2001. 2(2): p. 99-108 DOI: 10.1038/35053516
Kelley, K.W. and S.P. Pasca, Human brain organogenesis: Toward a cellular understanding of development and disease. Cell, 2021 DOI: 10.1016/j.cell.2021.10.003
Lodato, S. and P. Arlotta, Generating neuronal diversity in the mammalian cerebral cortex. Annu Rev Cell Dev Biol, 2015. 31: p. 699-720 DOI: 10.1146/annurev-cellbio-100814-125353
Chambers, S.M., et al., Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol, 2009. 27(3): p. 275-80 DOI: 10.1038/nbt.1529
Eiraku, M., et al., Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell, 2008. 3(5): p. 519-32 DOI: 10.1016/j.stem.2008.09.002
Lancaster, M.A., et al., Cerebral organoids model human brain development and microcephaly. Nature, 2013. 501(7467): p. 373-9 DOI: 10.1038/nature12517
Pasca, A.M., et al., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat Methods, 2015. 12(7): p. 671-8 DOI: 10.1038/nmeth.3415
Pasca, S.P., et al., A nomenclature consensus for nervous system organoids and assembloids. Nature, 2022. 609(7929): p. 907-910 DOI: 10.1038/s41586-022-05219-6
Amin, N.D., et al., Generating human neural diversity with a multiplexed morphogen screen in organoids. bioRxiv, 2023 DOI: 10.1101/2023.05.31.541819
Pellegrini, L., et al., Human CNS barrier-forming organoids with cerebrospinal fluid production. Science, 2020. 369(6500) DOI: 10.1126/science.aaz5626
Muguruma, K., et al., Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep, 2015. 10(4): p. 537-50 DOI: 10.1016/j.celrep.2014.12.051

Tags: morphogen, organoids, stem cells

Posted on: 20 July 2023

doi: https://doi.org/10.1242/prelights.35166

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Author's response

Neal D. Amin, Kevin W. Kelley, Sergiu P. Paşca shared

Q1)

hiPS cells from different individual or derivation methods are thought to have differences in in vitro differentiation. These differences have been speculated to be due in part to open and closed chromatin states that vary between hiPS lines and intrinsic differences in proliferation rates. Some organoid differentiation protocols are highly robust and generate the intended cell types across most hiPS lines tested, such as the forebrain organoid protocol characterized by the Pasca Lab [ref: PMID 30573846]. Other protocols may be more prone to variability between hiPS lines, or even between differentiations. This can be due to a variety of reasons, some of which we controlled for in our study. For example, larger organoids might not receive the same morphogen signals as smaller organoids due to differences in molecule diffusion. Thus, we ensured that organoids started off at the same size and were physically separated to prevent fusion. For our large screen, we used one hiPS line to identify principals of morphogen action. We have validated a key protocol that we highlighted in this manuscript in multiple hiPS lines and other protocols with additional hiPS lines that will be included in subsequent versions of the preprint.

Q2)

Our goal in selecting conditions was to include positive controls (protocols known to generate a specific cell populations) and original protocols so that we could assess the validity of our approach. Indeed, conditions with SAG+CHIR+FGF8 generated midbrain neurons, and SAG+RA+CHIR+FGF2 generated spinal neurons, serving as positive controls. We then split the remainder of conditions into several categories – (1) individual molecules, (2) timing and concentrations of CHIR and SAG, and (3) combinations of molecules. We anticipated that this set of conditions would maximize the sampling of the “morphogen space” to assess how all of these features might relatively contribute to cell identity. Indeed, each of these factors were critical for different cell types. WNT timing controlled Cajal-Retzius generation, SAG concentration altered TAC3 neuron generation, FGF2 alone (with N2 in all conditions) generated cerebellum, and combinations such as FGF8+CHIR+SAG generated midbrain neurons.

Q3)

We have not systematically investigated the expression of neuropsychiatric-associated disease genes in primary human brain data. However, the regionalized organoid expression of disease genes highlighted in Figure S6D are broadly recapitulated in the human fetal brain with EBF3 expressed in Purkinje cells; GATA3 expressed in midbrain/hindbrain neurons; and NR4A2 expression in midbrain dopaminergic neurons.

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Generating human neural diversity with a multiplexed morphogen screen in organoids

Background

Key findings

Why I like this preprint

Questions for the authors

References

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Also in the cell biology category:

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