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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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.
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 text, methods, figures, tables captions, video captions and references.
Differentially expressed genes, gene rankings and qPCR probes.
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).
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).
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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