Specification of positional identity in forebrain organoids


Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

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Fig. 1: Establishment of dorso-ventral and antero-posterior developmental axes in SHH-organoids.
Fig. 2: In vivo-like topographic organization of major forebrain subdivisions in SHH-organoids.
Fig. 3: Characterization of long-range SHH signaling mechanism.
Fig. 4: Statins perturb tissue growth and SHH signaling.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.


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We would like to thank S. Anderson for providing the LHX6-citrine hPSC line. M. Ross, S. Irion, M. Tomishima and members of the Studer lab for their valuable input on experimental design and feedback on manuscript. This work was supported in part through NYSTEM contract C030137 (L.S.) and through the NIH Cancer Center support grant P30 CA008748. G.C. is supported by a Ruth L. Kirschstein F30 M.D./Ph.D. pre-doctoral fellowship (F30 MH113343–01A1) and a training grant from the National Institute of General Medical Sciences (T32GM007739) to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program.

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G.Y.C.: Conception and study design, Organoid protocol development, hPSC cell line engineering, differentiation and characterization assays and writing of manuscript. L.S.: Conception and study design, data analysis and interpretation, writing of manuscript. J.J.A.: SHH protein analysis. M.D.R.: SHH protein analysis. J.T. SHH protein analysis, hPSC cell line engineering. R.M.W.: Organoid protocol development and analysis. D.C.: cell line engineering.

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Correspondence to Lorenz Studer.

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Competing interests

The Memorial Sloan-Kettering Cancer Center has filed a provisional patent application (US PRO 62/538,350) on the methods described in the manuscript with G. Cederquist and L. Studer listed as inventors. L.S. is a scientific co-founder of Bluerock Therapeutics.

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Supplementary Figure 1 Additional data related to Fig. 1.

(a) Schematic of [I125]Iodopalmitate labeling experiment (left). SHH produced from the iSHH line is palmitoylated at levels proportional to the amount of SHH protein expressed, suggesting that overexpression does not saturate processing machinery. (b) Reproducibility of organizer plating and formation of SHH-H9 spheroids. 1,000 iSHH cells are plated in low-attachment microwells and allowed to aggregate overnight. 10,000 wildtype hPSCs are plated on top of the iSHH organizer cells. (c) Spheroids are embedded in matrigel, and the day of embedding is critical to efficient neuroepithelial growth. SHH organoids (no dox) embedded on day 5 exhibit no neuroepithelial growth (“Failed,” N = 3 organoids), while SHH organoids embedded on day 6 exhibit efficient neuroepithelial formation (–DOX N = 3 organoids; +DOX N = 4 organoids). (d) Typically, the iSHH organizer remains clustered at one pole during differentiation, though in ~ 25% of instances the organizer can split into multiple distinct clusters. Scale bars: 200 μm.

Supplementary Figure 2 Quantification method for characterizing SHH-organoid topography.

(a) SHH-organoids are quantified using a grid of regions of interest (ROIs), and each ROI is associated with an X and Y coordinate. The origin is defined for each section by calculating the “center of mass” of the organizer signal. The grid is then used to define ROIs that are positive for regional markers (e.g. PAX6). The linear distance from all positive ROIs to the origin is calculated, and these data can be plotted as a frequency histogram. (b) Organoids that did not express FOXG1 (7/16 batches), or that (c) had a split organizer (10/40 organoids) were not included in the quantification. Scale bars: 200 μm.

Supplementary Figure 3 Topographic patterning in additional hPSC lines (MEL1, HUES6, HUES8) and iPSC lines (J1 and 348).

(a) The iSHH organizer can induce distinct regional domains that emerge in the anatomically correct topographic order. However, the size of domains and overall growth rate of organoids may differ between lines. Without doxycycline all lines are predominantly PAX6 and FOXG1 positive. Sparse induction of NKX2.1 and NKX2.2 was observed in MEL1 and 348 lines. +Doxycycline, N = 8 organoids, 2 batches for all lines; - Doxycycline, N = 4 organoids, 1 batch for all lines. (b) Pluripotency analysis for 348 iPSC line. Scale bars: 200 μm (a); 100 μm (b).

Supplementary Figure 4 Additional data related to Fig. 2.

(a) iSHH organizer cells (red) at least partially express NKX2.1 (green) and are negative for FOXG1, suggesting hypothalamic identity. N = 8 organoids, 2 batches. (b) SHH-dependent topographic patterning can be achieved without matrigel embedding. N = 8 organoids, 2 batches. (c) MGE-like neuroepithelium (FOXG1+/NKX2.1+) acquires a circular, rosette-like structure suggesting a radial organization. N = 6 organoids, 2 batches. Scale bars: 50 μm (a), 200 μm (b, c).

Supplementary Figure 5 Characterization of maintenance of SHH-organoid topography.

(a) OTX2 and FOXG1 regions remain largely distinct over time, and appear to retain their orientation with respect to the organizer. In some instances, the organizer tissue disperses throughout the organoid, making it difficult to determine orientation. –Dox N = 4 organoids, 1 batch; +Dox N = 8 organoids, 3 batches. (b, c) Cerebral cortex-like tissue consisting of radially organized bands of PAX6, TBR2, and TBR1 emerge in regions distal to the organizer. –Dox N = 4 organoids, 1 batch; +Dox N = 5 organoids, 2 batches. (d) Striatum-like tissue expressing DARPP32 emerges in regions distal to the organizer. LHX6 + cells are found more proximal to the organizer. –Dox N = 4 organoids, 1 batch; +Dox N = 6 organoids, 2 batches. (e, f) Hypothalamic-like cells, which express OTP, POMC, and TH, are found in the immediate vicinity of the organizer. –Dox N = 4 organoids, 1 batch; +Dox N = 3 organoids, 2 batches. Scale bars: 50μm (high magnification), 200μm (low magnification).

Supplementary Figure 6 Characterization of interneuron diversity in SHH-organoids using LHX6-citrine line.

(a) LHX6 + cells emerge in regions proximal to the organizer, and co-express NKX2.1. N = 1 organoid. (b) A subset of LHX6 + cells expresses FOXG1, consistent with striatal or cortical interneuron identity. N = 6 organoids, 2 batches. (c) Some FOXG1+/LHX6 + cells have a leading process morphology, characteristic of migrating cortical interneurons. (df) Diverse interneuron populations expressing somatostatin N = 3 organoids, 2 batches (d), parvalbumin N = 3 organoids, 2 batches (e), and calretinin N = 6 organoids, 2 batches (f) are observed. Parvalbumin + cells do not express LHX6, suggesting a non-MGE source of these cells. Scale bars: 50 μm (high magnification), 100 μM (intermediate magnification), 200 μm (low magnification).

Supplementary Figure 7 Additional data related to Fig. 4.

(a) AY9944 and lovastatin treated organoids retain NKX2.1 and OTX2 expression within the organizer at day 20. No Drug N = 12 organoids, 3 batches; Lova N = 11 organoids, 3 batches; AY9944 N = 12 organoids, 3 batches; Cyclopamine N = 8 organoids, 2 batches. (b) Absolute and relative quantification of organizer size in drug treated organoids at day 20. Graphs depict mean ± S.D., dots represent individual organoids. One-way ANOVA with Dunnett Test. **P=0.0038, ****P = 0.0001. No drug N = 14 organoids; Lova N = 13 organoids; AY9944 N = 16 organoids; Cyclopamine N = 14 organoids. All samples from 3 batches. (c) AY9944 strongly inhibits induction of NKX2.2 in day 6 organoids at all concentrations tested. Graph depicts mean ± S.D., dots represent individual organoids. No Drug N = 18 organoids, 3 batches; AY9944 0.31μM N = 14 organoids, 2 batches; AY9944 0.62μM N = 16 organoids, 2 batches; AY9944 1.25μM N = 15 organoids, 2 batches; Lova 2μM, 5μM, 20μM, N = 12 organoids, 2 batches each. (d) Western blot for SHH protein from day 4 hPSC-derived neural differentiations shows appropriate processing of SHH peptide length. Representative image of N = 3 experiments. Scale bars: 100 μM.

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Cederquist, G.Y., Asciolla, J.J., Tchieu, J. et al. Specification of positional identity in forebrain organoids. Nat Biotechnol 37, 436–444 (2019). https://doi.org/10.1038/s41587-019-0085-3

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