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Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons

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Abstract

Understanding neurological diseases requires tractable genetic systems, and engineered three-dimensional (3D) neural tissues are an attractive choice. Yet how the cellular transcriptomic profiles in these tissues are affected by the encapsulating materials and are related to the human brain transcriptome is not well understood. Here, we report the characterization of the effects of different culturing conditions on the transcriptomic profiles of induced neuronal cells and developed a method for the rapid generation of 3D co-cultures of neuronal and astrocytic cells from the same pool of human embryonic stem cells. By comparing the gene-expression profiles of neuronal cells in culture conditions relevant to the developing human brain, we found that modifying the degree of crosslinking of composite hydrogels can tune expression patterns so that they correlate with those of specific brain regions and developmental stages. Moreover, single-cell-sequencing results showed that our engineered tissues recapitulate transcriptional patterns of cell types in the human brain. Analyses of culturing conditions will inform the development of 3D neural tissues for use as tractable models of brain diseases.

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Fig. 1: 3D cultures and co-cultures of hESC-derived human iN cells within Matrigel show enriched neuronal processes compared with 2D cultures and co-cultures.
Fig. 2: Incorporating HA within Matrigel leads to enriched non-neuronal biological processes in 3D co-cultured human iN cells and decreases the gene expression correlation to the human brain developmental transcriptome.
Fig. 3: CHs modulate the expression levels of individual neuronal genes in 3D co-cultured human iN cells.
Fig. 4: CHs alter the correlation of gene expression profiles in 3D co-cultured human iN cells to the human brain developmental transcriptome and tune the expression levels of individual neuronal genes with varying amounts of crosslinker.
Fig. 5: Global comparison of effects of culture conditions on human iN cells and mechanical properties of encapsulating hydrogels.
Fig. 6: Generation of 3D neural tissues composed of human iN and astrocytic cells.
Fig. 7: scRNA-seq reveals that cells in 3D neural tissues reflect their counterparts in the human brain and in human brain organoids.

Change history

  • 25 June 2018

    In the version of this Article originally published, the acronym ‘CHs’ was not defined; the acronym should have been included in the sentence beginning “The effects of the addition of hyaluronic acid (HA) and the formation of composite hydrogels (CHs) of Matrigel and alginate...” This has now been corrected.

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Acknowledgements

We thank R. Macrae for critical reading of the manuscript and R. Belliveau for technical support. We thank R. Langer for input during the preparation of the manuscript and the entire Zhang Laboratory for assistance in the laboratory and helpful discussions. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by the following grants and institutes: NIH grant nos. 1R01-HG009761, 1R01-MH110049 and 1DP1-HL141201; the Howard Hughes Medical Institute; the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and J. and P. Poitras, R. Metcalfe, and D. Cheng. J.Z.L and J.Q.P. are supported by The Stanley Center for Psychiatric Research at the Broad Institute. We thank the Klarman Cell Observatory for supporting experiments using the 10× Chromium Instrument. Reagents are available through Addgene and codes can be accessed via GitHub.

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Contributions

H.T. and F.Z. conceived the study. H.T. designed the experiments, developed 3D and 2D cultures and 3D co-cultures of iN cells and the method to derive astrocytic cells, analysed the RNA-seq data, performed qPCR, immunostaining and imaging. S.S. filtered mouse reads from bulk RNA-seq data of iN cells co-cultured with mouse astrocytes. B.C. and H.T. performed and interpreted the comparisons between the transcriptomes of 3D cultures and co-cultures of iN cells and the human brain developmental transcriptome. X.A., C.C.H. and J.Z.L. constructed bulk RNA-seq libraries and performed sequencing. D.D. constructed scRNA-seq libraries and C.C.H. performed sequencing. J.Z.L aligned the scRNA-seq data to reference genomes. S.S. and H.T. analysed and interpreted the scRNA-seq data. L.G., S.R.C. and M.H. cloned the DNA constructs. L.G. and H.T. tested single guide RNAs in HEK cells. S.R.C. produced the AAVs. H.T. developed 3D tissues of iN cells and astrocytic cells, performed AAV infection of iN cells in 3D tissues and isolated targeted iN cells by FACS. L.G. performed next-generation sequencing and indel analyses. A.G. and J.Q.P. performed electrophysiology experiments. V.Y. and H.T. performed the mechanical characterization of the hydrogels. N.E.S. and X.S. developed hESCs with inducible expression of NGN1 and NGN2. C.L. isolated and expanded mouse glia. H.T. wrote the paper with input from all authors.

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Correspondence to Halil Tekin.

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H.T. and F.Z. are co-inventors in a patent application relating to work in this manuscript. The remaining authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary text, methods, figures, tables captions, video captions and references.

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Supplementary Tables

Differentially expressed genes, gene rankings and qPCR probes.

Supplementary Video 1

Z-section sequences of a 3D neural tissue of iN cells cultured in 4.6 mg ml–1 Matrigel for 40 days stained for MAP2 (green, MAP2) (using 10× objective).

Supplementary Video 2

Z-section sequences of a 3D neural tissue of iN cells cultured in 4.6 mg ml–1 Matrigel for 40 days stained for MAP2 (green, MAP2) (using 20× objective).

Supplementary Video 3

Z-section sequences of a 3D neural tissue of iN cells cultured in 4.6 mg ml–1 Matrigel for 40 days stained for MAP2 (green, MAP2) (using 63× objective).

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Tekin, H., Simmons, S., Cummings, B. et al. Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons. Nat Biomed Eng 2, 540–554 (2018). https://doi.org/10.1038/s41551-018-0219-9

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