Programmatic introduction of parenchymal cell types into blood vessel organoids - PubMed (original) (raw)

Programmatic introduction of parenchymal cell types into blood vessel organoids

Amir Dailamy et al. Stem Cell Reports. 2021.

Abstract

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.

Keywords: differentiation; human pluripotent stem cells; myo-vascular organoids; neuro-vascular organoids; reprogramming; vascularized organoids.

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Construction and characterization of inducible cell lines (A) Schematic of PiggyBac transposon-based inducible overexpression vector. (B) Schematic of cell line generation and validation process. (C) Inducible NEUROD1 (iN) cell line validation at 3 weeks post induction via (1) qRT-PCR analysis of signature neuronal markers MAP2, TUBB3, VG LUT 2, and VGAT; data represent the mean ± SD (n = 4 independent experiments); (2) immunofluorescence micrograph of MAP2_+ cells (scale bars, 50 μm); and (3) representative spike plots from MEA measurements of spontaneously firing iN cells. (D) Inducible ASCL1+DLX2 (iAD) cell line validation at 3 weeks post induction via (1) qRT-PCR analysis of signature neuronal markers MAP2, TUBB3, VG LUT 2, and VGAT; data represent the mean ± SD (n = 4 independent experiments); (2) immunofluorescence micrograph of MAP2_+ cells (scale bars, 50 μm); and (3) representative spike plots from MEA measurements of spontaneously firing iAD cells. (E) Inducible MYOD1+BAF60C (iMB) cell line validation at 2 weeks post induction via (1) qRT-PCR analysis of signature skeletal muscle markers MYH8, TNNC1, and RYR; data represent the mean ± SD (n = 3 independent experiments); and (2) immunofluorescence micrograph of MYH+-, MYOG+-, and SAA+-labeled cells (scale bars, 50 μm). (C–E) ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001; ns, not significant.

Figure 2

Figure 2

Generation of iN-VOs and iMB-VOs (A) General strategy for the generation of vascularized organ tissues via introduction of parenchymal cell types in VOs. (B) Schematic of iN-VO and iMB-VO culture protocol. (C) Immunofluorescence 100-μm z stack, maximum projection, confocal micrographs of _MAP2_- and _CDH5_-labeled uninduced (iN-VO, −Dox) and induced (iN-VO, +Dox) day 15 iN-VO organoids (scale bars, 50 μm). (D) qRT-PCR analysis of signature endothelial genes CDH5 and VEPTP; signature smooth muscle gene SMA; and signature neuronal genes MAP2, VG LUT 2, BRN2, and FOXG1 at day 15 of culture for iN-VO organoids. Data represent the mean ± SD (n = 7 organoids, from three independent experiments). (E) Immunofluorescence 100-μm z stack, maximum projection, confocal micrographs of _MYH_- and _CDH5_-labeled uninduced (iMB-VO, −Dox) and induced (iMB-VO, +Dox) day 15 iMB-VO organoids (scale bars, 50 μm). (F) qRT-PCR analysis of signature endothelial genes CDH5 and VEPTP; signature smooth muscle gene SMA; and signature skeletal muscle genes MYOG, MYH8, TNNC1, and RYR at day 15 of culture for iMB-VO organoids. Data represent the mean ± SD (n = 7 organoids, from three independent experiments). (G) Pan-organoid tile-scan immunofluorescence confocal micrograph of a _CDH5_- and _MAP2_-labeled day 15 neuro-vascular organoid (scale bars, 500 μm). Image is a 200-μm z stack maximum intensity projection. (H) Pan-organoid tile-scan immunofluorescence confocal micrograph of _CDH5_- and _MYH_-labeled day 15 myo-vascular organoid (scale bars, 500 μm). Image is a 200-μm z stack maximum intensity projection. (D and F) ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001; ns, not significant.

Figure 3

Figure 3

Molecular and functional characterization of iN-VOs (A) Outline of long-term cultured iN-VO characterization. (B) qRT-PCR analysis of signature endothelial genes CDH5 and VEPTP, signature smooth muscle gene SMA, and signature neuronal genes MAP2 and BRN2 at day 30 of iN-VO culture. Data represent the mean ± SD (n = 7 organoids, from three independent experiments). (C) Immunofluorescence 100-μm z stack, maximum projection, confocal micrographs of MAP2+- and CDH5+-induced day 30 iN-VOs (scale bars, 100 μm). (D) Immunofluorescence100-μm z stack, maximum projection, confocal micrographs of PDGFR+-and CDH5+-induced day 30 iN-VOs (scale bars, 50 μm). (E) Uniform manifold approximation and projection (UMAP) visualization of cell types from day 45 iN-VOs. Two independent induction conditions, along with one non-induction condition. (F) Cluster-specific expression of marker genes in day 45 iN-VOs. (G) Experimental validation of iN-VO perfusibility in vivo by subcutaneous implantation of iN-VO, showing immunofluorescence micrographs of intravital Dextran, CDH5, and overlay (scale bar, 25 μm). Representative image from two independent experiments.

Figure 4

Figure 4

Electrical characterization and stimulation of organoids (A) Schematic of iN-VO electrical characterization via MEA recordings. (B) Image of day 30 iN-VOs plated on microelectrode array (scale bars, 500 μm). (C) Representative spike plots from MEA measurements of spontaneously firing iN-VOs and corresponding raster plot. Representative image and plot from two independent experiments. (D) Schematic of iMB-VO in vitro maturation by electrical stimulation. (E) qRT-PCR analysis of skeletal muscle myosins: MYH2, MYH3, MYH7, and MYH8, and genes involved in calcium handling, CASQ1, CASQ2, SERCA1, SERCA2, and RYR for stimulated versus unstimulated iMB-VOs. Data represent the mean ± SD (n = 6 organoids, from two independent experiments). ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001; ns, not significant.

References

    1. Albini S., Coutinho P., Malecova B., Giordani L., Savchenko A., Forcales S.V., Puri P.L. Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres. Cell Rep. 2013;3:661–670. -PMC -PubMed
    1. Butler A., Hoffman P., Smibert P., Papalexi E., Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 2018;36:411–420. -PMC -PubMed
    1. Cakir B., Xiang Y., Tanaka Y., Kural M.H., Parent M., Kang Y.-J., Chapeton K., Patterson B., Yuan Y., He C.-S. Engineering of human brain organoids with a functional vascular-like system. Nat. Methods. 2019;16:1169–1175. -PMC -PubMed
    1. Clevers H. Modeling development and disease with organoids. Cell. 2016;165:1586–1597. -PubMed
    1. Daniel E., Cleaver O. Vascularizing organogenesis: lessons from developmental biology and implications for regenerative medicine. Curr. Top. Dev. Biol. 2019;132:177–220. -PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources