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Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues

Abstract

The generation of organoids and tissues with programmable cellular complexity, architecture and function would benefit from the simultaneous differentiation of human induced pluripotent stem cells (hiPSCs) into divergent cell types. Yet differentiation protocols for the overexpression of specific transcription factors typically produce a single cell type. Here we show that patterned organoids and bioprinted tissues with controlled composition and organization can be generated by simultaneously co-differentiating hiPSCs into distinct cell types via the forced overexpression of transcription factors, independently of culture-media composition. Specifically, we used such orthogonally induced differentiation to generate endothelial cells and neurons from hiPSCs in a one-pot system containing either neural or endothelial stem-cell-specifying media, and to produce vascularized and patterned cortical organoids within days by aggregating inducible-transcription-factor and wild-type hiPSCs into randomly pooled or multicore-shell embryoid bodies. Moreover, by leveraging multimaterial bioprinting of hiPSC inks without extracellular matrix, we generated patterned neural tissues with layered regions composed of neural stem cells, endothelium and neurons. Orthogonally induced differentiation of stem cells may facilitate the fabrication of engineered tissues for biomedical applications.

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Fig. 1: OID of stem cells for patterning vascularized organoids and bioprinted tissues.
Fig. 2: Programmable differentiation of pluripotent stem cells via OID under identical media conditions.
Fig. 3: Programmable vascularization of cortical organOIDs.
Fig. 4: Multicore-shell cortical organOIDs.
Fig. 5: Multicellular neural tissues via 3D bioprinting coupled with OID.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Source data for the figures are provided with this paper. Raw and processed sequence data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through the GEO series accession number GSE193149. Flow cytometry data, RT-qPCR data and processed bulk RNA-sequencing data are available at https://github.com/churchlab/Vascularized-Organoids. Source data are provided with this paper.

Code availability

The custom code for measuring multicore-shell organoid cell distributions and the code for measuring cell migration in printed tissues is available at https://doi.org/10.5281/zenodo.5823604. The scripts for the analysis of processed single-cell and bulk RNA-sequencing data are available at https://github.com/churchlab/Vascularized-Organoids.

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Acknowledgements

This research was sponsored by the National Human Genome Research Institute of the NIH under award number RM1HG008525, NIH Brain Initiative under award number R01MH123977-01, and by the Vannevar Bush Faculty Fellowship Program, sponsored by the Basic Research Office of the Assistant Secretary for Defense for Research and Engineering through the Office of Naval Research Grants N00014-16-1-2823 and N00014-21-1-2958. A.L. received fellowship support from the Charles Stark Draper Laboratory. We thank T. Ferrante and the Harvard Center for Biological Imaging for microscopy infrastructure and support; the Bauer Core Facility at Harvard University for Illumina sequencing of organoid single-cell libraries and flow cytometry; the Harvard Biopolymers Facility for RNA sequencing support; S. Uzel, S. Han, M. Mata and Z. Niziolek for experimental assistance; and J. Coppeta and J. Aach for useful discussions.

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Authors

Contributions

M.A.S.-S., J.Y.H., A.L. and J.A.L. designed the research. M.A.S.-S., J.Y.H., A.L., T.D., S.L., L.L.N. and S.D performed the research and analysed data. All authors contributed to manuscript writing.

Corresponding authors

Correspondence to Mark A. Skylar-Scott or Jennifer A. Lewis.

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The authors have submitted a patent application associated with this work (U.S Provisional Application Serial No. 63/048,502, filed July 6, 2020).

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Nature Biomedical Engineering thanks Jeremy Crook and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Pooled hiPSCs enable the formation of cohesive embryoid bodies with tailorable cellular composition.

a, Left, 50% WT-eGFP and 50% RFP HUVEC EBs in microwell arrays cultured for 1 day. Right, 50% WT-eGFP and 50% RFP HUVEC EBs cultured for 3 days. b, Left, 50% WT-eGFP and 50% iEndo-mKate2 EBs in microwell arrays cultured for 1 day. Right, WT-eGFP and 50% iEndo-mKate2 EBs cultured for 3 days. c, Different seeded proportions of WT-eGFP and iEndo-mKate2 hiPSC aggregates in microwell arrays 3 days before suspension culture. Left, 100% iEndo-mKate2. Middle-left, 67% iEndo-mKate2 and 33% WT-eGFP. Middle-right 33% iEndo-mKate2 and 67% WT-eGFP. Right, 100% WT-eGFP. d, WT-eGFP and iEndo-mKate2 EBs 1 day before suspension culture. Left, 100% iEndo-mKate2. Middle-left, 67% iEndo-mKate2 and 33% WT-eGFP. Middle-right 33% iEndo-mKate2 and 67% WT-eGFP. Right, 100% WT-eGFP. Scale bars: 500 μm in a (left), b (left), and c; 100 μm in a (right), b (right), and d.

Extended Data Fig. 2 Programmable vascularized cortical organOIDs.

a, Immunostaining of SOX2 and nestin in 100% WT organoids cultured for 10 days. b, Immunostaining of SOX2 and nestin in 67% WT and 33% iEndo organOIDs cultured for 10 days. c, Immunostaining for SOX2 and UEA1 for 67% WT and 33% iEndo organoid cultured for 10 days. d, Immunostaining of SOX2 and CD31 in 100% WT organoids cultured for 25 days. e, Immunostaining of SOX2 and CD31 in 67% WT and 33% iEndo organOIDs cultured for 10 days. f, Immunostaining of SOX2, NeuN, and CD31 for 100% WT organoids cultured for 45 days. g, Immunostaining for SOX2, NeuN, and CD31 in a 67% WT and 33% iEndo organOIDs cultured for 45 days. Scale bars: 200 μm in a, b, d, f (left), and g (left), 100 μm in c and e, and 50 μm in f (right) and g (right).

Extended Data Fig. 3 Large ventricular architectures in programmable multicore–shell cortical organOIDs.

a, Immunostaining of SOX2 in 10-day-old WT-only organoids. b, Immunostaining of SOX2 in 10 day-old randomly pooled organOIDs c, Immunostaining of SOX2 in 10-day-old multicore-shell organOIDs. d, Immunostaining of SOX2 and NCAD in 10-day old WT-only organoids. e, Immunostaining of SOX2 and NCAD in 10-day old randomly pooled organOIDs. f, Immunostaining of SOX2 and NCAD in 10-day old multicore-shell organOIDs. g, Lengths of neuroepithelium within ventricles as measured by the length of NCAD expression within ventricles. Data represents mean ± s.e.m. * P = 0.0169 between WT-only organoids and randomly pooled organOIDs, * P = 0.0286 between randomly pooled and multicore-shell organOIDs (n = 72 WT ventricles, n = 10 randomly pooled ventricles, n = 30 multicore-shell ventricles, unpaired two-tailed t-test). Scale bars: 100 μm in a-f.

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Supplementary information

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Supplementary figures, tables and video captions.

Reporting Summary

Video 1

Formation of a vascular network and neural rosettes in co-culture over a 4 d period on a Matrigel-coated surface in dual-SMAD inhibiting medium.

Video 2

Confocal z-stack of iDISCO-cleared WT and WT + iEndo organoids cultured for 25 d.

Video 3

Bioprinting of a pseudo-Hilbert curve using a densely cellular matrix-free hiPSC ink on a transwell membrane.

Video 4

Confocal z-stack of a printed iNeuron-derived neuron filament in neural induction medium cultured for 6 d.

Video 5

Bioprinting of a layered cortical construct using WT and iNeuron bioinks.

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Skylar-Scott, M.A., Huang, J.Y., Lu, A. et al. Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues. Nat. Biomed. Eng 6, 449–462 (2022). https://doi.org/10.1038/s41551-022-00856-8

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