Human pluripotent-stem-cell organoids can be vascularized by co-culture with mesodermal progenitor cells (MPCs), yielding organoid-intrinsic vessels with hierarchical organization, mural-cell recruitment, and — after transplantation of tumor organoids to the chick chorioallantoic membrane — rapid connection to a host circulation; in vitro, however, these pre-formed vascular networks remain unperfused and therefore only partially mature. The open problem, stated in the source work and still current, is to establish stable, physiologically relevant long-term perfusion of organoid-intrinsic vascular networks — by transplantation-mediated anastomosis to a living host's circulation or by connection to an engineered perfusable vascular circuit — sufficient to drive final endothelial and mural-cell maturation of the vessel wall, together with the generation of more uniform vascularized neural organoids across experiments and induced pluripotent stem cell (iPSC) lines. The source work further envisions that stably perfused in vitro networks could be live monitored and developed into standardized high-throughput platforms for drug testing. Although substantial partial progress has been made on every component, no published work yet demonstrates the coupled objective, and the lack of functional perfused vasculature and perfusion-dependent maturation remains a central bottleneck of organoid technology.

Background

Organoids are self-organizing three-dimensional tissues differentiated from human pluripotent stem cells (hPSCs, comprising embryonic stem cells and induced pluripotent stem cells, iPSCs, which are adult cells reprogrammed to pluripotency). They mimic aspects of organ development, but most organoids lack vasculature: without perfused blood vessels, nutrients and oxygen reach the interior only by diffusion, which restricts organoid size, induces central necrosis, and blocks maturation beyond fetal-like stages. Blood vessels are also more than endothelial tubes: a mature vessel wall comprises endothelial cells lining the lumen, mural cells (pericytes on small vessels and vascular smooth muscle cells on larger ones), a basement membrane, and cell-cell junctions. Full maturation of this wall is driven in vivo by shear stress — the mechanical force of pulsatile blood flow on the endothelium — which triggers adaptive responses in endothelial and mural cells; static in vitro culture cannot supply this signal.

Wörsdörfer et al. (Scientific Reports, 2019) introduced a developmental approach: mixing or co-culturing organoids with iPSC-derived mesodermal progenitor cells (MPCs, the embryonic mesoderm-derived precursors that give rise to endothelial, mural, and some hematopoietic lineages). The MPCs self-organize into vessel networks inside tumor and neural organoids, delivering hierarchically organized vessels with mural cells, basement membrane, endothelial junctions, and microglia-like immune cells in neural organoids. After transplantation of tumor organoids onto the chick chorioallantoic membrane (CAM, the vascularized extraembryonic membrane of the chick embryo used as an in vivo vascularization assay), the organoid-derived vessels connect to the host circulation within days. A parallel protocol paper (Wörsdörfer et al. 2020) standardized the method. Related but distinct strategies from the same period include: transcription-factor engineering, in which ETV2 (a master regulator of endothelial fate) is induced in neural organoids to produce vascular-like networks (Cakir et al. 2019); transplantation of unvascularized organoids into mouse brain, where host vessels invade the graft (Mansour et al. 2018); self-organizing human blood vessel organoids that engraft and perfuse in mice (Wimmer et al. 2019); and kidney organoids cultured under flow on millifluidic chips, where perfusion-like shear expands endogenous endothelial pools and matures the tissue (Homan et al. 2019).

The MPC method leaves a stated gap that defines this problem: the pre-formed organoid vessels are not perfused in vitro, so their final wall maturation is incomplete; generating more uniform vascularized neural organoids across iPSC lines and connecting the networks to a physiologically relevant circulation for the long term (ideally via an engineered, perfused artificial vascular network that can be anastomosed to organoid vessels) remain to be achieved. The source further envisions that such in vitro models would allow easy manipulation of the system and live monitoring, and that high-throughput platforms for drug testing could be developed. Subsequent work has addressed pieces of this gap: transplanted cortical organoids with intrinsic vessels form functional chimeric human-mouse vessels in the mouse brain (Shi et al. 2020); months-long host vascularization of transplanted cortical organoids supports neuronal maturation (Revah et al. 2022); a kidney organoid-on-chip achieves lumen-on-lumen anastomoses between organoid endothelium and a perfused engineered macrovessel (Kroll et al. 2024); vascular organoids gain hierarchical mural coverage only after host-blood-flow perfusion on the CAM (Kowalski et al. 2025); and bioprinted MPC-derived 'mother vessel' constructs interconnect with pre-vascularized organoids and withstand bioreactor flow (Dogan et al. 2026). A functional, stably perfused, mature vasculature — reproducible across lines and scalable — is still a missing capability of organoid models.

Problem Statement

Establish a reproducible way to perfuse, over the long term, the pre-formed vascular networks of in vitro-generated human vascularized organoids, such that the perfusion is sufficient to complete vessel-wall maturation, and the resulting capability is uniform and reproducible. Concretely, the source work's stated challenges remain to be solved: (1) generate more uniform and reproducible vascularized neural organoids across experiments and iPSC lines; (2) achieve stable long-term perfusion of the organoid-intrinsic vascular networks — whether by transplantation-mediated anastomosis to a physiologically relevant host circulation or by connecting the organoid vasculature to an engineered perfused artificial vascular network under controlled hemodynamic (shear) conditions; and (3) demonstrate that this sustained perfusion drives the final maturation of the endothelium and mural cells of the organoid-derived vessels, including final vessel-wall organization. These are coupled aspects of one objective — a stably perfused, mature, reproducible vascularized organoid platform — not independent questions: the perfusion (aspect 2) is required for the maturation (aspect 3), and the uniformity (aspect 1) for reproducibility of both. The deliverable is a concrete, executable program — an experimental vascularization route, the perfusion means appropriate to that route (for the engineered route a perfusion set-up with its controlled hemodynamic (shear) regime; for the transplantation route the characterized host shear regime), and the read-outs that would establish final vessel-wall maturation and uniformity across experiments and iPSC lines — whose execution would realize this objective; an executed wet-lab demonstration is not required. The source work additionally envisions that the resulting perfused in vitro models could be live monitored and developed into standardized, high-throughput platforms for drug testing and disease modelling; this platform translation is the end use envisioned for the resulting capability, not a further component of the stated challenges.

References

  1. Wörsdörfer P, Dalda N, Kern A, Krüger S, Wagner N, Kwok CK, Henke E, Ergün S. Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells. Scientific Reports 9 (2019). DOI: 10.1038/s41598-019-52204-7. https://doi.org/10.1038/s41598-019-52204-7
  2. Wörsdörfer P, Rockel A, Alt Y, Kern A, Ergün S. Generation of Vascularized Neural Organoids by Co-culturing with Mesodermal Progenitor Cells. STAR Protocols 1:100041 (2020). DOI: 10.1016/j.xpro.2020.100041. https://doi.org/10.1016/j.xpro.2020.100041
  3. Cakir B, Xiang Y, Tanaka Y, Kural MH, Parent M, Kang Y-J, Chapeton K, Patterson B, Yuan Y, He C-S, Raredon MSB, Dengelegi J, Kim K-Y, Sun P, Zhong M, Lee S, Patra P, Hyder F, Niklason LE, Lee S-H, Yoon Y-S, Park I-H. Engineering of human brain organoids with a functional vascular-like system. Nature Methods 16 (2019). DOI: 10.1038/s41592-019-0586-5. https://doi.org/10.1038/s41592-019-0586-5
  4. Mansour AA, Gonçalves JT, Bloyd CW, Li H, Fernandes S, Quang D, Johnston S, Parylak SL, Jin X, Gage FH. An in vivo model of functional and vascularized human brain organoids. Nature Biotechnology 36 (2018). DOI: 10.1038/nbt.4127. https://doi.org/10.1038/nbt.4127
  5. Wimmer RAE, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M, Taubenschmid J, Hämmerle M, Esk C, Bagley JA, Lindenhofer D, Chen G, Boehm M, Agu CA, Yang F, Fu B, Zuber J, Knoblich JA, Kerjaschki D, Penninger JM. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565 (2019). DOI: 10.1038/s41586-018-0858-8. https://doi.org/10.1038/s41586-018-0858-8
  6. Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nature Methods 16 (2019). DOI: 10.1038/s41592-019-0325-y. https://doi.org/10.1038/s41592-019-0325-y
  7. Shi Y, Sun L, Wang M, Liu J, Zhong S, Li R, Li P, Guo L, Fang A, Chen R, Ge W-P, Wu Q, Wang X. Vascularized human cortical organoids (vOrganoids) model cortical development in vivo. PLOS Biology 18:e3000705 (2020). DOI: 10.1371/journal.pbio.3000705. https://doi.org/10.1371/journal.pbio.3000705
  8. Revah O, Gore F, Kelley KW, Andersen J, Sakai N, Chen X, Li M-Y, Birey F, Yang X, Saw NL, Baker SW, Amin ND, Kulkarni S, Mudipalli R, Cui B, Nishino S, Grant GA, Knowles JK, Shamloo M, Huguenard JR, Deisseroth K, Pașca SP. Maturation and circuit integration of transplanted human cortical organoids. Nature 610 (2022). DOI: 10.1038/s41586-022-05277-w. https://doi.org/10.1038/s41586-022-05277-w
  9. Zhang S, Wan Z, Kamm RD. Vascularized organoids on a chip: strategies for engineering organoids with functional vasculature. Lab on a Chip 21 (2021). DOI: 10.1039/d0lc01186j. https://doi.org/10.1039/D0LC01186J
  10. Tan SY, Feng X, Cheng LKW, Wu AR. Vascularized human brain organoid on-chip. Lab on a Chip 23 (2023). DOI: 10.1039/d2lc01109c. https://doi.org/10.1039/D2LC01109C
  11. Kroll KT, Homan KA, Uzel SGM, Mata MM, Wolf KJ, Rubins JE, Lewis JA. A perfusable, vascularized kidney organoid-on-chip model. Biofabrication 16 (2024). DOI: 10.1088/1758-5090/ad5ac0. https://doi.org/10.1088/1758-5090/ad5ac0
  12. Boutom SM, Silva TP, Palecek SP, Shusta EV, Fernandes TG, Ashton RS. Central nervous system vascularization in human embryos and neural organoids. Cell Reports 43:115068 (2024). DOI: 10.1016/j.celrep.2024.115068. https://doi.org/10.1016/j.celrep.2024.115068
  13. Nwokoye PN, Abilez OJ. Blood vessels in a dish: the evolution, challenges, and potential of vascularized tissues and organoids. Frontiers in Cardiovascular Medicine 11:1336910 (2024). DOI: 10.3389/fcvm.2024.1336910. https://doi.org/10.3389/fcvm.2024.1336910
  14. Kowalski WJ, Vatti S, Sakamoto T, Li W, Odutola SR, Liu C, Chen G, Boehm M, Mukouyama YS. In vivo transplantation of mammalian vascular organoids onto the chick chorioallantoic membrane reveals the formation of a hierarchical vascular network. Scientific Reports 15 (2025). DOI: 10.1038/s41598-025-91826-y. https://doi.org/10.1038/s41598-025-91826-y
  15. Gong L, Zhang Y, Zhu Y, Lee U, Luo AC, Li X, Wang X, Chen D, Pu WT, Lin R-Z, Ma M, Cui M, Chen K, Wang K, Melero-Martin JM. Rapid generation of functional vascular organoids via simultaneous transcription factor activation of endothelial and mural lineages. Cell Stem Cell 32 (2025). DOI: 10.1016/j.stem.2025.05.014. https://doi.org/10.1016/j.stem.2025.05.014
  16. Chen H, Liang Y, Sun X, Xiong W, Yang T, Liang Y, Ye X, Li X, Wang W, Gu J, Zhang J, Chen L, Chan HF, Chen J. Generation of vascularized retinal organoids containing microglia based on a PDMS microwell platform. Science Advances 11 (2025). DOI: 10.1126/sciadv.ady6410. https://doi.org/10.1126/sciadv.ady6410
  17. Dogan LE, Chicaiza-Cabezas NA, Kleefeldt F, Wörsdörfer P, Groll J, Ergün S. Generation of Self-Organizing Macrovascular Constructs by Bioprinting Human iPSC-Derived Mesodermal Progenitor Cells. Advanced Science (2026). DOI: 10.1002/advs.76018. https://doi.org/10.1002/advs.76018