Circulation Research

CARDIOVASCULAR ORGANOIDS/3D MODELS REVIEW SERIES

Blood Vessel Organoids for Development and

Disease
Kirill Salewskij , Josef M. Penninger

ABSTRACT: Despite enormous advances, cardiovascular disorders are still a major threat to global health and are responsible for
one-third of deaths worldwide. Research for new therapeutics and the investigation of their effects on vascular parameters is
often limited by species-specific pathways and a lack of high-throughput methods. The complex 3-dimensional environment
of blood vessels, intricate cellular crosstalks, and organ-specific architectures further complicate the quest for a faithful
human in vitro model. The development of novel organoid models of various tissues such as brain, gut, and kidney signified a
leap for the field of personalized medicine and disease research. By utilizing either embryonic- or patient-derived stem cells,
different developmental and pathological mechanisms can be modeled and investigated in a controlled in vitro environment.
We have recently developed self-organizing human capillary blood vessel organoids that recapitulate key processes of
vasculogenesis, angiogenesis, and diabetic vasculopathy. Since then, this organoid system has been utilized as a model
for other disease processes, refined, and adapted for organ specificity. In this review, we will discuss novel and alternative
approaches to blood vessel engineering and explore the cellular identity of engineered blood vessels in comparison to in vivo
vasculature. Future perspectives and the therapeutic potential of blood vessel organoids will be discussed.

Key Words: blood-brain barrier ◼ blood vessels ◼ endothelial cell ◼ extracellular matrix ◼ organoids ◼ vascular diseases
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T
he human vasculature is a complex system respon-
sible for maintaining oxygen and nutrient supply
to tissues, clearing waste products, or shuttling of
immune cells. It precedes the development of most other
organs as it plays a pivotal role in the proper growth and
differentiation of essentially all tissues in the developing
embryo.1–4 New blood vessels form via two distinct pro-
cesses: (1) vasculogenesis, which describes the differ-
entiation and assembly of mesodermal precursor cells
(hemangioblasts) into primitive tubular networks (capil-
lary plexus) and (2) angiogenesis, which is the process
of subsequent remodeling and branching of new ves-
sels to form a mature network.5 Broadly speaking, the
mature blood vessels can be categorized into capillar-
ies, the venous system, and the arterial system. Arter-
ies carry the oxygen-rich, high-pressure blood from the
heart, through arterioles, to tissues where the nutrient
exchange is mediated through capillaries. Deoxygenated
blood is then transported from the capillaries to venules
and via veins to the lung to be reoxygenated. Although
the vessels belonging to these categories possess clear
differences in their structure and cellular composition,
it is important to note that there are heterogeneities
between cells of the same type—on the transcriptional as
well as on the functional level. For example, endothelial
cells (ECs) form the innermost layer of all vessels. They
possess specific characteristics which are a function of
the tissue context: they control permeability of solutes,
sense shear stress, and control vasomotor tone.6,7 And
just as the functions are diverse, it has been shown that
ECs between different tissues have tremendous differ-
ences in their transcriptional identity.7,8 Through single-
cell RNA sequencing, individual tissue identities of ECs
have been identified, mapped to organs, and can now
serve as a reference for in vitro studies aiming to recre-
ate native, tissue-specific vasculature.9
Although the tubing of vessels is assembled from ECs,
the structural integrity and modification of vessel diam-
eter is controlled by mural cells, that is, pericytes and
vascular smooth muscle cells (VSMCs). During angio-
genesis, endothelial tip cells secrete PDGF-B (plate-
let-derived growth factor subunit B) which is bound by
PDGFRβ (platelet-derived growth factor receptor beta)
on the surface of developing mural cells, leading to their

Correspondence to: Josef M. Penninger, MD, Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada. Email
josef.penninger@ubc.ca
For Sources of Funding and Disclosures, see page 508
© 2023 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res

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 Salewskij and Penninger Blood Vessel Organoids for Development and Disease

CARDIOVASCULAR ORGANOIDS/
intima. The tunica externa or adventitia forms the outmost

3D MODELS REVIEW SERIES

Nonstandard Abbreviations and Acronyms layer and harbors a plethora of different cell types within
a collagen-rich ECM (extracellular matrix), including fibro-
BBB blood-brain barrier blasts, adipocytes, and multiple resident immune cells.15
bFGF basic fibroblast growth factor Besides serving as an anchor for blood vessels to the tis-
BMP-4 bone morphogenetic protein 4 sue, the adventitia contains lymphatic vessels, perivascu-
BVO blood vessel organoid lar nerves, and microvessels (vasa vasorum) that sustain
CTGF connective tissue growth factor the outer layers as luminal diffusion can be insufficient
ECM extracellular matrix
across thick vessels.16 Several studies have shown that
the adventitia is also home to multiple stem- and progen-
ECs endothelial cells
itor-like cells of unknown origin, expressing markers such
EGF epidermal growth factor
as CD34, PDGFRβ, Sca1 (mouse gene stem cell anti-
eNOS endothelial NO synthase gen-1), and Gli1 (GLI family zinc finger 1).17–19 In mouse
IL interleukin models of arteriosclerosis and atherosclerosis, these cells
LDL low-density lipoprotein reside in the adventitia of lesions,20 can acquire VSMC-
PDGF-B platelet-derived growth factor subunit B 17
and osteoblast-like19 identities, and thus contribute to
PDGFRβ platelet-derived growth factor receptor lesions such as vessel calcification. In disease, adventitial
beta fibroblasts can release inflammatory cytokines in vitro21
SCF stem cell factor and differentiate into myofibroblasts promoting calcifica-
sGC soluble guanylate cyclase tion and stenosis in vivo.22,23 Moreover, the vasa vasorum
TNF tumor necrosis factor has been shown to be involved in aneurysm development
VEGF-A vascular endothelial growth factor A and rupture,24,25 neointima formation,26 plaque vascular-
VEGFR vascular endothelial growth factor ization,27 and a proinflammatory positive feedback loop,28
receptor possibly disturbing the immune privilege of the tunica
VSMC vascular smooth muscle cell media.29 Therefore, besides ECs, special attention must
be also paid to perivascular cell types and structures.
Animal models—especially zebrafish—are commonly
recruitment, maturation, and proliferation.10 The arrange- used for vascular studies, but species-specific pathways
ment of mural cells is dependent on the type of vessel. and compensatory mechanisms complicate attempts at
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Capillaries—the smallest vessels in the body—consist
of ECs, pericytes, and an enwrapping basement mem-
brane. Arteries possess several layers of VSMCs which
are responsible for the maintenance of vascular tone
through their contraction and relaxation. A functional
crosstalk between the ECs and mural cells is important
for proper vascular homeostasis and many vascular dis-
orders are a direct result of mural cell dysfunction. As an
example, studies in rats have shown that upon injury to
the arterial wall, bFGF (basic fibroblast growth factor)
is released from dead cells and leads to proliferation of
VSMCs.11 The increase of VSMCs in turn caused intimal
thickening and promoted atherosclerotic lesions, explain-
ing the development of restenosis after angioplasty.
During thrombosis, capillaries in the brain constrict and
cause ischemia in the associated tissue.12 Even if blood
flow is restored, long-lasting reductions in the cerebral
blood flow can be observed.13 Experiments on cerebral
cortical slices revealed that upon ischemia pericytes first
constrict the capillaries and then die, leading to a long-
lasting decrease of the capillary blood flow and loss of the
blood-brain barrier integrity.14 Mural and ECs are not the
only players in pathology. Walls of larger vessels such as
veins and arteries are comprised of 3 distinct layers. The
innermost layer (tunica intima) consists of ECs forming
the tubing and the tunica media contains predominantly
layers of vascular smooth muscle cells that enwrap the
modeling disease. A particular example of this is NOTCH3
(notch receptor 3), a gene in which missense mutations
cause Cerebral Autosomal Dominant Arteriopathy with
Subcortical Infarcts and Leukoencephalopathy, which is an
inheritable vasculopathy of brain arteries that causes early
strokes and dementia.30 To better understand the function
of NOTCH3, loss of function studies have been performed
in animal models. Loss of function of notch3 in zebrafish
resulted in reduced viability, abnormal morphology of the
vasculature, and a pathological accumulation of blood in
the head and fins through compromised vessel integrity.31
However, 2 studies showed that ablation of Notch3 in mice
had no effect on the viability of the embryos.32,33 Although
the knockout mice also had a pathological vessel pheno-
type, it was overall milder, suggesting compensatory mech-
anisms in higher vertebrates. Examples like this invoked a
need for a human-based in vitro model of the vasculature
to overcome such species-specific differences.
In the past decade, many attempts have been made
to achieve this goal (Figure 1). The simplest approach to
modeling (part of) the vasculature is to culture human
vascular cells as a monolayer and examine phenotypic
or molecular changes caused by genetic perturbations
or under exposure to compounds of interest. Human
umbilical vein ECs are a commonly used primary EC
line for vascular studies. If cultured on a suitable base-
ment membrane matrix, these ECs will self-assemble into

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 Salewskij and Penninger Blood Vessel Organoids for Development and Disease
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3D MODELS REVIEW SERIES

Figure 1. General overview of different human-based models for vascular studies.

As the complexity of the model and the faithful representation of the in vivo process increases, the ease of handling and scalability usually
decrease. Endothelial cells can be cultured in 2-dimensional (2D) to form networks.34 By using scaffolds or microfluidic systems, more
complex assemblies are achieved—however, they mostly rely on primary cells.39,40 Self-organizing blood vessel organoids (BVOs) capture both
vasculogenesis and angiogenesis.41,42 Integration into microfluidic systems allows for perfusion of the vessels.43 Implant of the organoids into
immunodeficient mice can be performed to achieve larger vessels and a fully perfused human vascular tree, but throughput is low.42 Illustration
credit: Sceyence Studios.

lumenized tube structures that can be quantified and are
amendable for high-throughput analysis.34 Another inter-
esting element is the ability of ECs to sense and adapt
to flow by changing their orientation. This striking feature
can be investigated in vitro by culturing cells in a medium
of physiological viscosity and using an orbital shaker to
induce multiaxial flow.35,36 In 2015, a protocol was pub-
lished for the differentiation of induced pluripotent stem
cells into ECs and VSMCs through Wnt pathway (Wnt/β-
catenin pathway) activation by CHIR99021 and subse-
quent exposure to either VEGF-A (vascular endothelial
growth factor A) and forskolin or PDGF-BB and Activin
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In recent years, the field of organoids has vastly
expanded its repertoire of target tissues. Initially coined
in the year 1907 by Henry Van Peters Wilson based
on his observations of sponge cells regenerating into a
whole organism, followed by the observation that primary
mouse mammary epithelial cells form 3-dimensional
structures on a reconstituted basement membrane by Li
et al in 1987, the term organoid nowadays refers to a self-
organized collection of cells that developed to structurally
and functionally resemble a specific organ.44–46 In syn-
ergy with the discovery of the four Yamanaka factors to
induce stem cells from somatic cells, organoids can now

A.37 Comparison of the gene expression profile of the
mural cells demonstrated a high similarity to the respective
primary cell type. Indeed, induced ECs achieved a similar
transendothelial electrical resistance to human umbili-
cal vein ECs and could be subjected to the tube forma-
tion assay whereas VSMCs responded to stimulation by
vasoconstrictive drugs. Similarly, another protocol reported
the derivation of pericytes by magnetically enriching for
vascular-induced CD34- cells.38 Culture of these cells in
medium containing (amongst others cytokines) EGF (epi-
dermal growth factor), bFGF, and VEGF165 yielded a 95%
pure CD13+/PDGFRβ+ pericyte population that could be
expanded for at least 10 passages. These differentiation
approaches are very useful for the study of individual vas-
cular cell types and compared to primary lines have the
advantage that they can be based on patient-derived cells,
and thus faithfully represent complex disease genotypes.
However, they lack the 3-dimensional environment and
tissue assembly observed in vivo. Therefore, specifica-
tion and invasion of endothelial tip cells into extracellular
matrix, codifferentiation and recruitment of mural cells, or
vessel response to different flow parameters is difficult to
study in a 2-dimensional environment. More sophisticated
models that utilize scaffolds or exploit the self-organization
of vascular cells to form vessels exist, but they cannot fully
capture all developmental stages and thus miss potentially
important intermediary cell types39,40 (Figure 1).
be used to model genetic disorders through the utiliza-
tion of patient cells.47 In the context of vasculature, there
are two main themes for the utilization of sophisticated
3-dimensional models. (1) For development and disease,
the ability to model and observe interaction of human
vascular cells with their environment is indispensable.
During angiogenesis, the extracellular matrix is degraded
to allow for migration and infiltration of ECs into the sur-
rounding tissue. Stimulation of ECs with proangiogenic
factors leads to upregulation of matrix metalloproteinases
responsible for the degradation of collagens, gelatin, and
fibronectin.48,49 Furthermore, the vasculature is shaped
by, and responds to, different physiological and environ-
mental parameters. Organoid models can therefore (par-
tially) substitute for studies that are either unethical or
too resource-intensive to perform on live animals, such
as the effects of hypoxia or modification of flow param-
eters. (2) A major shortcoming of most organoid culture
protocols irrespective of the tissue that they represent
are the static culture conditions. As organoids surpass a
certain size, extensive cell death occurs in the core due
to the physical limits of passive diffusion of nutrients and
oxygen.50,51 One method of circumventing this limit is by
culturing organoids in microfluidic chips. The controllable
flow of nutrients not only increases the lifespan of the
organoids but also leads to an enhancement of organoid
growth.52,53 Although commercial versions are available,

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 Salewskij and Penninger
most organoid chips are specifically engineered for the
tissue of interest and scalability remains poor. Thus, the
prospect of integrating a functional, perfusable vascula-
ture into organoids of other tissues has become a key
topic. Vascularization of organoids has been shown to
contribute positively to maturation of target tissue, and
prevascularization of organoids in vitro improved engraft-
ment rates upon transplantation in vivo.54–56 For an over-
view on current strategies to vascularize organoids, we
would like to point to the review from Zhang et al.57 Our
review will focus on current blood vessel organoid (BVO)
protocols, their utilization for disease modeling, as well
as current shortcomings and approaches to solve them.
BVOS TO STUDY DEVELOPMENT AND
DISEASE
The first protocol for the derivation of a bona fide human
BVO was developed by our group in 2019.41,42 This pro-
tocol is based on differentiation of multiple vascular cell
types from a common pool of mesodermal progenitors
and the intrinsic self-organization of these vascular cells
during development. Stem cells are initially aggregated
into embryoid bodies and subsequently pushed into
mesodermal lineage via Wnt stimulation and BMP-4
(bone morphogenetic protein 4). Both mural and ECs
can be differentiated from a common mesodermal
progenitor through supplementation of VEGF-A and
forskolin. After vascular induction, the organoids still
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resemble a crude aggregate of cells with a diameter
between 100 and 200 microns. To induce sprouting of
vessels, they are embedded into a bilayered ECM sub-
stitute consisting of a collagen I/Matrigel mix. Of note,
both collagen and Matrigel are sensitive to temperature
increases and can polymerize prematurely, leading to
irregular ECM densities and inhibition of sprouting due
to uneven stiffness. Nevertheless, this approach serves
the important purpose of forming a cushion between the
organoids and the plastic of the tissue culture ware. The
density of the organoids is higher than the non-polymer-
ized surrounding matrix and without a bilayer assembly,
they sink upon plating and contact the plastic bottom,
inhibiting uniform sprouting.
Upon a few hours of embedding, first cells can be
seen infiltrating into the ECM. Following this protocol,
the organoids are cultured for further 4 to 6 days before
liberation from the Matrigel.41,42 Special attention must be
paid to the timing as liberating them too late can cause
fusions whereas removing them too early yields insuf-
ficient differentiation indicated by dense cores. The lib-
eration from the Matrigel is performed manually under a
sterile hood with fine syringe needles. Future studies on
the optimization of the protocol should focus on alternative
approaches to this step, as it is not only time-intensive but
also increases the risk of contamination. Potential solu-
tions could be found through enzymatic treatments, but
Circulation Research. 2023;132:498–510. DOI: 10.1161/CIRCRESAHA.122.321768
Blood Vessel Organoids for Development and Disease
CARDIOVASCULAR ORGANOIDS/
we are currently not aware of selective digestion options
3D MODELS REVIEW SERIES
for Matrigel. After successful liberation, the organoids
are usually maintained for approximately 3 to four weeks.
The mature organoids are comprised of ECs, pericytes,
mesenchymal stem-like cells, and a very small percent-
age of hematopoietic cells.42 Thus, for the first time, a
self-organizing human capillary network was assembled
in vitro. These vascular organoids were then used to
model the process of diabetic vasculopathy. Analysis of
the dermal vasculature of patients suffering from type
2 diabetes showed a thickened and multilayered base-
ment membrane. Accordingly, vascular organoids that
were exposed to elevated levels of glucose, TNF (tumor
necrosis factor), and IL-6 (interleukin 6) in vitro pheno-
copied the expanded basement membrane observed in
the patient vasculature. The excessive synthesis of ECM
was mediated mostly by pericytes, supporting previous
observations that mural cells produce and maintain the
basement membrane.58 Treatment with the γ-secretase
inhibitor DAPT (N-[N-(3,5-Difluorphenacetyl)-L-alanyl]-
S-phenylglycin-tert-butylester) abrogated basement
membrane thickening and restored proliferation of ECs.
Finally, pharmacological inhibition and genetic ablation
of multiple γ-secretase targets identified DLL4 (delta
like canonical notch ligand 4) and NOTCH3 as the
key mediators and potential novel targets in diabetic
vasculopathy.42
In a recent preprint, vascular organoids were
used to illustrate the importance of glycolysis in
ECs and the effects on vasculature upon metabolic
changes.59 Glycolysis in ECs is mainly regulated by the
enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase 3) and genetic ablation as well
as pharmacological targeting in mice altered vascular
sprouting and vessel formation.60 The authors showed
that upon pharmacological inhibition of PFKFB3 in
organoids, a marked decrease in EC proliferation was
observed while pericytes remained unaffected. Further-
more, the authors reported reductions in vessel den-
sity, length, and diameter after 24 hours of treatment,
indicating strong vascular remodeling upon glycolysis
inhibition in ECs. Proteomic analysis and confirmation
via quantitative polymerase chain reaction pinpointed
a reduction in CTGF (connective tissue growth factor
aka CCN2 [cellular communication network factor 2])
and subsequent reduced YAP (transcriptional coactiva-
tor YAP1) activity as a possible mechanism. Recombi-
nant CTGF was able to partially restore YAP activity and
rescued the pharmacologically induced vessel pheno-
type. The study demonstrated that our vascular organ-
oids can be indeed used to study metabolic effects on
vasculature and that perturbation of metabolism has
marked effects on human blood vessels.
Evidently, there is potential for the vascular organoids
to be used for disease research as they show phenotypic
alterations that are normally restricted to vasculature in
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CARDIOVASCULAR ORGANOIDS/
vivo. Although a great effort has been put into establish-
3D MODELS REVIEW SERIES
ing disease modeling, the developmental progression of
individual cell types and their structural context (venous
versus arterial ECs) remained unknown. In a preprint by
Nikolova et al,61 the authors tackled this problem by per-
forming a comprehensive single-cell transcriptomic analy-
sis of the BVOs at different developmental stages. After
induction of the vascular lineage, PDGFRβ+ pericytes
were detectable already at day 3 of the protocol whereas
CD31+ ECs arose at day 4, indicating that pericyte devel-
opment appears to precede maturation of ECs, at least in
this organoid model. The transcriptomic identity of ECs in
the organoid was dynamic but resembled an in vivo devel-
opmental course. A transient upregulation of the EC fate
transcription factor ETV2 (ETS variant transcription factor
2) was seen in a small cell population at day 4, followed
by upregulation of common EC markers such as CLDN5
(claudin 5) and PECAM1 (platelet and endothelial cell
adhesion molecule 1). ECs initially adapted a transcrip-
tional identity comparable to arterial ECs until day 14 of
culture, followed by an induction of venous EC markers
at day 21. The cause of this identity switch is unknown
and should be acknowledged for disease modeling. A
recent study reported a protocol for generation of human
artery- and vein-specific ECs and illustrated the conflicting
specification pathways.62 However, it also highlighted the
importance of the 2 identities in disease as two viruses
preferentially targeted arterial ECs. In our organoids, the
identity switch could be related to the observed upregula-
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tion of SULF1 (sulfatase 1) in mural cells.61 SULF1 inhibits
EC angiogenesis through attenuation of VEGF-mediated
signaling, which in turn is required for arterial specifica-
tion of ECs.63–65 Analysis of differentiation trajectories fur-
ther revealed that ECs arose from 2 different precursor
pools: although the majority developed from lateral plate
mesoderm progenitor cells as expected, late organoid
stages revealed a trajectory from mural cells into ECs.61
Pericytes are known to display a remarkable multipotency
through transdifferentiation into other mesenchymal or
immune cells.66–68 This transdifferentiation could serve as
a compensatory mechanism to increase the number of
ECs. More studies into pericyte-to-endothelial transdiffer-
entiation are required and could reveal novel mechanistic
insights in various vascular disorders where abnormal cell
proliferation and differences in pericyte versus EC num-
bers are a key element to pathogenesis.
One shortcoming of our capillary organoid protocol
is the inability to generate larger vessels in vitro.42 Inde-
pendent of culture stage, in vitro the vessels are unable
to increase complexity and form arteriole- or venule-like
structures. Thus, the organoids also do not possess a
tunica media or adventitia, confounding in vitro studies
of atherosclerotic processes in these layers. Although
it should be feasible to study certain disease hallmarks
such as changes in EC gene expression, altered pericyte
coverage, and angiogenic sprouting, current organoid
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Blood Vessel Organoids for Development and Disease
cultures cannot model excessive proliferation of VSMCs,
infiltration by macrophages, or plaque formation. This
limitation can be circumvented through transplantation of
the organoids into the renal capsule of immunodeficient
NOD/SCID (nonobese diabetic/severe combined immu-
nodeficiency) gamma mice. There, the organoids survived
for up to 6 months, anastomosed to the host vasculature,
and became fully perfusable as demonstrated through
injection of fluorescent dextran and human-specific anti-
CD31 antibodies.42 Through genetic tagging, we further
demonstrated that virtually all pericytes and ECs in these
transplants were of human origin. Although many of the
general angiogenic pathways are conserved between
species, there are fine differences in protein isoforms and
expression patterns. As an example, high doses of mouse
VEGF164, but not human VEGF165 are able to induce
angioma-like structures in SCID mice.69 Importantly, and
unlike other model systems, it appears that these trans-
planted vessels exclusively utilize human mesodermal
cells for the development and recruitment of mural cells
even to the arteries instead of relying on mouse mural
cells.42 The development into complex vessels could
enable the study of media- and adventitia-driven disor-
ders in a human context. Hopefully, future studies will be
able to present methods to develop larger vessels without
the need for an animal host, thus greatly boosting high-
throughput research for arterial- or vein-specific diseases.
In vitro, perfusion of BVOs remains a challenge. The
fact that this (and other organoids) require a trans-
plantation into an animal host for proper vasculariza-
tion and perfusion increases both cost and time while
simultaneously reintroducing potential species-specific
effects. ECs sense changes in flow-induced shear stress
through a plethora of mechanosensing membrane pro-
teins including integrins, G-protein–coupled receptors,
and junction proteins.70–72 One of the downstream media-
tors is the eNOS (endothelial NO synthase) which pro-
duces NO. NO diffuses to VSMCs and enables vascular
relaxation through activation of sGC (soluble guanylate
cyclase) and subsequent cGMP production.73 Inadequate
sensing and adaption to shear stress and in particular the
NO signaling pathway have long been associated with a
cohort of vascular disorders.74,75 For instance, mutations
in sGC are associated with Moyamoya disease—a steno-
occlusive vascular brain disorder of unknown cause.76
Besides shear stress, pressure, and associated strain,
which constitute changes in dimension or deformation,
act upon the vessel walls.77 Branching points and bifur-
cations are often subjected to haphazard hemodynamic
forces, thereby disturbing laminar flow.78 Atherosclerotic
lesions predominantly occur at these bifurcation points.79
Measurements in bovine vessels have demonstrated that
branch regions experience double the amount of strain
compared to neighboring areas.80 Modulations of blood
pressure through factors such as dietary habits, smok-
ing, or genetic predisposition can increase the already
 Salewskij and Penninger
elevated stress in these areas, leading to cumulative
tissue injury over time.78 Finally, the increased injury
and stretch could lead to augmented LDL (low-density
lipoprotein) penetration,78 supported by in vivo observa-
tions of LDL accumulation predominantly at branching
regions.81 Pressure effects, especially at vascular bifur-
cation points, are impossible to study in a 2-dimensional
system. To tackle the lack of controllable perfusion, we
are currently working on a microfluidic approach for vas-
cular organoids.43 An analysis of flow and shear rates
revealed values comparable to those observed in human
capillaries, thus allowing us to further refine the vascular
organoid model.43 As a next step, it would be interesting
to study how altered pressure influences vessel remodel-
ing, EC characteristics, or mural cell composition.
The importance of flow in vascular diseases has
been recently also addressed using induced pluripo-
tent stem cell-derived ECs from a patient carrying a
mutation in ENDOGLIN—resulting in hereditary hem-
orrhagic telangiectasia.82 Hereditary hemorrhagic tel-
angiectasia–EC networks were indistinguishable from
control ECs in 2-dimensional culture but upon imple-
mentation into a 3-dimensional and flow environment
on a chip showed reduced vascular densities, EC pro-
liferation, and pericyte coverage.
The current BVO protocols require the utilization of
Matrigel as an ECM for vessel sprouting. Matrigel is fre-
quently used as a matrix for culture of many cell types
as it is rich in ECM proteins, such as collagen IV, lam-
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inin, and entactin, as well as multiple growth factors.83,84
The exact composition, however, varies from batch to
batch, and although growth-factor–reduced versions
are manufactured, they still suffer from variability.83 To
address this problem, Schmidt et al85 newly presented
a BVO protocol that abstains from the use of Matrigel.
Instead, the authors utilized a conical agarose coating in
96-well plates for the aggregation of induced pluripo-
tent stem cells and subsequent organoid culture. Similar
to our initial protocol, a treatment with CHIR99021 and
BMP4 was performed for 3 days to induce mesoderm.
However, vascular induction was done through a single
dose of 100 ng/mL VEGF for 48 hours, after which
organoids were permanently maintained in N2B27-
medium without further vascular-specific growth factors.
This alternate culture protocol and lack of embedding in
an ECM substitute directly affected the morphology of
the organoids: while early-stage organoids consisted of
loosely connected mesenchymal cells, at day 7 a vas-
culogenic zone could be observed.85 Cells in this zone
stained positive for CD31 and remodeled after several
days by infiltrating into the other tissue layers of the
organoid. The identity of the outer tissue is unknown
but has been shown to be GATA6 negative—as opposed
to the center of the organoid. Although transplantation
into a chick chorioallantois membrane yielded perfusion
and association of SMA+ mural cells with the network,
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Blood Vessel Organoids for Development and Disease
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only few mural cells could be observed in vitro. Never-
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theless, the study demonstrated that 3-dimensional vas-
cular structures in an organoid can be achieved without
the utilization of a highly variable matrix and prolonged
subjection to proangiogenic factors, exploiting the cells’
intrinsic ability to self-organize given the correct tissue
context. Future advances should find a compromise
between both approaches, for example, through fully
synthetic hydrogels with specific stiffnesses closely
mimicking the organotypic environment of interest.
ORGANOTYPIC VASCULATURE AND
SPECIFIC FUNCTIONS
Besides transcriptional differences, the vasculature in
different organs is often highly adapted to execute spe-
cific functions and cellular organizations. One example
for this is the arrangement of ECs and EC permeabil-
ity in different organs. Brain ECs are one example for
restricted permeability as their tight junctions contribute
to the blood-brain barrier (BBB). In the kidney, a fenes-
trated endothelium can be found that allows for passage
of larger molecules and is important for glomerular filtra-
tion.86 Discontinuous endothelial layers associated with
a porous basement membrane are found in the liver,
spleen, and bone marrow.7 Specialized cell types can
also associate with the blood vessels and modulate their
function, such as astrocytes in the brain or hematopoietic
stem cells in the bone marrow. In the following section,
we will discuss efforts to develop organotypic vascular
organoids and compare their features to in vivo counter-
parts (Figure 2 and Table 1).
Brain Vasculature
The brain vasculature is highly specialized: astrocytes
associate tightly with blood vessels and in conjunction
with tight endothelial junctions form the blood-brain bar-
rier. Microglia serve as specialized immune cells, con-
tribute to neurogenesis and modulate recovery under
injury.91,92 Accordingly, these brain-specific cells can con-
tribute to cerebral vasculopathies; for example, astrocytes
play a role in the development of cerebral cavernous mal-
formations.93 Mechanistically, upon CCM3 mutations, an
endothelial-mediated elevation of NO stabilized HIF-1α
(hypoxia-inducible factor 1-alpha) in astrocytes and lead
to elevated VEGF production and initiation of a quasi-
hypoxic program, prompting abnormal angiogenesis.
In 2013, Lancaster et al50 published the first protocol
for brain organoids, and significant advances in struc-
ture and complexity have been made since. One criti-
cal aspect for brain organoids is focused on introducing
vasculature to model the BBB and allow for better nutri-
ent support and hence growth and maturation of larger
organoids.94 A study aiming for a 3-dimensional model of
the BBB exploited the self-organization ability of brain
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Figure 2. Illustration of tissues of origin for organotypic blood vessels organoids.

Boxes give information about processes of interest that are modeled by the groups that achieved the organoids. So far, brain-specific, muscle-
specific, and bone marrow–specific vascular organoids have been achieved. Illustration credit: Sceyence Studios.

vascular cells, coseeding primary brain ECs, primary
pericytes, and primary astrocytes in hanging drop culture
plates.95 All 3 cell types spontaneously self-assembled
into spheroids with a distinct cellular organization: ECs
formed the surface layer, astrocytes populated the core,
and pericytes formed an intermediate layer, separating
the ECs and astrocytes. Comparing these spheroids
to different permutations of cell mixtures, the authors
noticed that ECs are an important component in the reg-
ulation of proliferation of the other cell types. The triple-
cell spheroids arrested their cell growth after a certain
time whereas pericyte-astrocyte spheroids grew unhin-
dered until the formation of a necrotic core. Through flow
cytometry analysis, the authors demonstrated a marked
increase in surface cell adhesion molecules compared
to normal mono- and transwell-cultures, illustrating the
importance of the 3-dimensional environment and the
ability of pericytes to enhance stable EC interactions.
Yet, the authors were not able to demonstrate functional
characteristics of a BBB such as selective compound
uptake or the presence of tight junctions.
A similar protocol to model the BBB in vitro was
employed with one small but substantial change88:
VEGF-A was omitted in the spheroid culture medium
as VEGF-A is known to increase vascular permeabil-
ity by disrupting tight junctions.96,97 This small modi-
fication enabled the spheroid to acquire several BBB
characteristics, illustrating the positive and negative
effects that a singular growth factor can have on the
tissue of interest. Following VEGF-A withdrawal, these

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Table 1. Summary of Reported Blood Vessel Organoid Protocols

3D MODELS REVIEW SERIES

Type of ECM for Final medium and growth
Publication vasculature embedding General protocol factors Aim/disease modeling
Wimmer et al Nonorganotypic 75% Collagen I Embedding of mesodermal StemPro-34 SFM Diabetic vasculopathy
201941,42 capillaries solution spheroids in ECM to achieve 100 ng/mL VEGF-A
25% Matrigel sprouting and vessels
100 ng/mL FGF2
15% FCS
Schmidt et al85 Nonorganotypic None Derivation and culture of Single dose of 100 ng/mL Exploitation of intrinsic
capillaries vascular spheroids in conical VEGF-A for 48 h self-organization of vascular
agarose-coated wells N2B27 cells

Romeo et al59 Nonorganotypic 75% Collagen I Embedding of mesodermal StemPro-34 SFM Changes in vascular
capillaries solution spheroids in ECM to achieve 100 ng/mL VEGF-A structure and remodeling
sprouting and vessels upon glycolysis inhibition
25% Matrigel 100 ng/mL FGF2
15% FCS
Nikolova et al61 Nonorganotypic 75% Collagen I Embedding of microwell- StemPro-34 SFM Total characterization of
capillaries solution derived mesodermal spheroids 100 ng/mL VEGF-A development and cellular
in ECM to achieve sprouting identities in BVOs
25% Matrigel and vessels 100 ng/mL FGF2
15% FCS
Dailamy et al87 Muscle- and 2 mg/mL Collagen Individual encapsulation of StemPro-34 SFM Introduction of parenchymal
neural-tissue vascular spheroids in ECM 100 ng/mL VEGF-A cell types into vascular
capillaries organoids through genetic
20% Matrigel 100 ng/mL FGF2 drivers
15% FCS
(20 ng/mL BDNF + 20 ng/mL NT3)
Ulrich et al Brain capillaries 4% Hanging drop spheroid culture EBM -2 Development of
201395 Methylcellulose of primary brain ECs, pericytes, 50 ng/mL VEGF-A 3-dimensional in vitro model
supplementation and astrocytes for the blood-brain barrier
2% FCS
Cho et al88 Brain capillaries None Aggregation of ECs, pericytes, EGM-2/ Endothelial basal Refinement of previous
and primary astrocytes in medium 3-dimensional in vitro model
agarose-coated wells 2% FCS for the blood-brain barrier
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Sun et al54 Brain capillaries Matrigel droplet Embedding of vascular-induced N2B27 Obtaining vascularized
spheroids in Matrigel droplets/ 20 ng/mL VEGF-A brain organoids
brain organoid coculture
Giger et al89 Bone marrow None Microwell-based coculture DMEM/F12 + human endothelial Development of
vasculature of ECs and MSCs to form SFM 3-dimensional bone marrow
spheroids 1x N2 model system/establishing
a model to study leukemia
1x B27 treatments
FGF-2
20 ng/mL EGF
20 ng/mL PDGF-AB
20 ng/mL hOSM
40 ng/mL hIGF-1
15% chick embryo extract
Khan et al90 Bone marrow 30% Collagen I Simultaneous induction of STEMdiff APEL2 Medium Development of
vasculature vascular and hematopoietic 25 ng/mL VEGF-A 3-dimensional bone marrow
commitment with subsequent model system/ TGFβ-
embedding in ECM 25 ng/mL VEGF-C induced bone marrow
25 ng/mL FGF-2 fibrosis of hematological
30% Collagen IV 25 ng/mL BMP-4 cancer

25 ng/mL hSCF
25 ng/mL Flt3
25 ng/mL EPO
40% Matrigel 25 ng/mL TPO
25 ng/mL G-CSF
10 ng/mL IL-3
10 ng/mL IL-6

Organoids are grouped based on the type of vasculature that they represent. A brief description of the utilized ECM substitutes (if any) is included, as well as a description
of the protocol, the final maturation medium, and essential growth factors. The general aim of the study and the modeled diseases are listed. APEL indicates albumin
polyvinylalcohol essential lipids; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenic protein; BVO, blood vessel organoid; EBM, endothelial basal medium; EGM,
endothelial growth medium; EC, endothelial cell; ECM, extracellular matrix; EGF, epidermal growth factor; EPO, erythropoeitin; FCS, fetal calf serum; FGF, fibroblast growth
factor; Flt, fms-like tyrosine kinase; G-CSF, granuolocytic colony-stimulating factor; hIGF, human insulin-like growth factor 1; hOSM, human oncostatin M; IL, interleukin; MSC,
mesenchymal stem cell; NT, neurotrophin; PDGF, platelet-derived growth factor; SFM, serum free media; TPO, thrombopoeitin; VEGF, vascular endothelial growth factor

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 Salewskij and Penninger
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spheroids showed the formation of dense tight junc-
3D MODELS REVIEW SERIES
tions between ECs, expression of Claudin and Occlu-
din and high molecular weight dextran was not able to
diffuse inside the spheroid. The BBB-associated efflux
pump P-gp (ATP-dependent translocase ABCB1 [also
known as P Glycoprotein]) and the transcytosis recep-
tor LRP-1 (prolow-density lipoprotein receptor-related
protein) were also expressed and functional. Expres-
sion levels of P-gp, ZO-1 (tight junction protein ZO-1),
and β-Catenin were significantly higher in the spheroid
compared to a transwell model of the BBB. Importantly,
the authors demonstrated the selective uptake of drugs
using matrix-assisted laser desorption/ionization mass
spectrometry imaging.88 Although this model recapitu-
lates some aspects of the BBB, the cells assemble into
distinct compartments, partially separated too far away
from each other for uniform interaction. ECs form a layer
on the surface of the spheroid but do not form vessels.
Although BVOs with astrocytes and microglia might
be a great model, this has not yet been achieved because
of restrictive culture conditions.98 In a recently published
study, Sun et al54 instead used a coculture of vascular
organoids and brain organoids to achieve BBB-like char-
acteristics. The vascular organoids were obtained by an
initial differentiation towards mesodermal embryoid bod-
ies via Wnt signaling and subsequent vascular induction
using BMP4, bFGF, and later embedding in Matrigel.
ECs sprouted and self-assembled into lumenized struc-
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tures. Although immunofluorescent staining confirmed
the presence of pericytes, quantitative polymerase chain
reaction for PDGFRβ illustrated an upregulation at day
12 as opposed to day 3 in our original protocol.54,61
Upon coembedding of a neuroepithelial embryoid body
between 2 vascular progenitor organoids, these fusoids
were further cultured in the neurovascular maturation
medium. Prolonged culture led to acquisition of several
BBB characteristics, such as association of pericytes and
astrocytes with vessels, expression of tight junction pro-
teins CLDN5 and ZO-1, and the efflux transporter P-gp.
Similar to the BBB spheroids,88 fluorescent Angiopep-2
was taken up by the organoid, whereas the scramble
control was not, demonstrating selective transcytosis.54
The fact that these BBB characteristics developed is
surprising considering that the fusion medium contained
VEGF-A, suggesting that cellular crosstalks could over-
ride inhibition by extrinsically provided growth factors,
such as VEGF-A. Clearly, such coculture approaches will
play a very important role in the coming years as a major
organoid vascularization strategy and the initial building
block to build complex organs in vitro.
Bone Marrow Vasculature
The bone marrow is a major source of hematopoietic stem
cells in the adult human. Hematopoietic stem cells are
found in the vicinity of sinusoids—large quasicapillaries with
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Blood Vessel Organoids for Development and Disease
a discontinuous endothelium that allows passage of cells
and proteins.99,100 The fine balance between differentiating
into the different hematopoietic lineages and self-renewal
for maintenance of the stem cell pool is critical for many dis-
eases, such as acute myeloid leukemia. In essence, the bone
marrow niche represents a special type of blood vessel, and
recent studies aimed at recapitulating the development of
this environment in vitro. For instance, Giger et al89 used a
microwell-based approach where they coseeded ECs and
mesenchymal stem cells to form a spheroid. By performing
several titration experiments with regard to mesenchymal
stem cells:ECs ratios, the authors showed that the number
of self-renewing mesenchymal stem cells was higher in the
dual-cell type organoids and thus depended on the pres-
ence of ECs. The ECs self-organized into networks which
remained stable for about 1 week before showing signs
of decay. Mesenchymal stem cells from these spheroids
could differentiate into adipocytes, chondrocytes, and pre-
osteoblasts. Upon seeding of hematopoietic stem cells on 3
days old organoids, the cells homed and migrated towards
the inside of the organoid, which mimics in vivo data where
hematopoietic stem cells injected into an irradiated murine
host will home to and reconstitute the bone marrow.101
In an alternative approach, induced pluripotent stem cells
are initially aggregated and induced towards mesodermal
aggregates,90 modifying our initial BVO protocol.41,42 After
induction towards vascular and hematopoietic lineage
through addition of BMP4, FGF2, VEGF-A, SCF (stem cell
factor), and Flt3 (Fms-like tyrosine kinase 3), the organoids
were embedded in a hydrogel of collagen/Matrigel mix-
ture. These organoids showed differentiation into lumen-
ized blood vessels and association of hematopoietic stem
cells to vessels, analogous to the bone marrow. Single-cell
sequencing analysis confirmed the presence of several
hematopoietic and stromal cell subtypes. Importantly, tra-
jectory analysis revealed that the differentiated cells arose
from the major routes of myeloid differentiation that are
also found in vivo.102 Moreover, the authors showed that this
organoid system can be used to model pathological bone
marrow fibrosis that occurs during diseases, such as acute
leukemia. It would be interesting to see whether the CD34+
cells obtained from these organoids would be able to repop-
ulate and rescue the bone marrow niche of an irradiated,
immunodeficient mouse. Another approach would be a
transplantation of such organoids into for instance MISTRG
(7 modified genes in the genome of these mice: M-CSFh/h
IL-3/GM-CSFh/h SIRPah/h TPOh/h RAG2-/- IL2Rg-/-)
mice, which have been modified with human versions of
genes encoding cytokines for innate immune cell devel-
opment and support development of more immune cells
compared to conventional immunodeficient mice.103 Alter-
natively, these bone marrow organoids could be exposed to
controlled flow in microfluidic devices to test if circulation of
hematopoietic cells can be achieved. Interactions between
circulating blood cells and the vasculature are fundamental
components in the pathogenesis of many diseases such as
 Salewskij and Penninger
platelet activation following atherosclerotic plaque rupture.
Future research should focus on incorporating circulating
blood cells into in vitro vasculature. Overall, great progress
has been made to establish vascularized bone marrow
niches from human stem cells with enormous potential in
disease modeling, generation of hematopoietic progenitors
in vitro, or even controlled genetic repair of mutations that
cause hematopoietic deficiencies.
Lymphatic Vessels
Lymphatic vessels represent a specialized subtype of the
microvasculature. Much effort has been put into under-
standing the basic biology behind lymphatic vessels due to
their critical involvement in immunology, tissue drainage, and
disease processes.104 Current in vitro approaches include
cell morphology assessment under differential flow and
ECM substrates,105 assays for barrier function,106,107 trans-
port,108 permeability,109 or studies of immune responses
to different antigens.110 The process of lymphangiogen-
esis is of special interest as lymphatic vessels are criti-
cally involved in tumor metastasis, and cancer cells were
shown to secrete prolymphangiogenic factors.111 Although
the development of blood vessels depends on VEGF-A/
VEGFR (VEGF receptor)-1/-2 signaling, lymphangio-
genesis is modulated by the expression of VEGFR-3 on
lymphatic ECs, activated by the ligands VEGF-C/-D.104
Current models of lymphangiogenesis rely on the utilization
of lymphatic ECs, either in a scaffold or in coculture with
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other cells.112,113 To our knowledge, currently there is no in
vitro model that faithfully recapitulates both lymphvasculo-
genesis and lymphangiogenesis. A major hurdle for such
a model is the origin of lymphatic ECs. During embryonic
development, lymphatic ECs arise from a Prox1+(prospero
homeobox 1)/Lyve1+ EC population of veins.114–116 The
requirement of venous vessels—or at least of a venous EC
identity—raises the question of whether currently available
BVO protocols could be modified to accomplish in vitro
lymphvasculogenesis. In our BVO model, one can detect
venous EC markers upon day 21 of culture but the ves-
sels are of capillary nature and thus might not possess the
required Prox1+/Lyve1+ population.61 A recent protocol
described the derivation of human artery- and vein-specific
ECs from pluripotent stem cells.62 It would be interesting
to test whether these venous ECs have the ability to yield
lymphatic ECs and if this approach could be combined with
a 3-dimensional differentiation protocol.
Organotypism Through Genetic Drivers
One organotypic vessel organoid that we would like to dis-
cuss is based on overexpression of specific genes to drive
the development of defined cell types.87 In this study, the
authors overexpressed NEUROD1 (neuronal differentia-
tion 1) to achieve neurovascular, and MYOD1 (myogenic
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differentiation 1) + BAF60C to establish myovascular
3D MODELS REVIEW SERIES
organoids, respectively. Induction of NEUROD1 lead to
MAP2+ (human gene microtubule associated protein
2) neurons in day 15 BVOs, but they formed large clus-
ters within the organoid instead of distributing uniformly.
A similar situation was observable for the myovascular
organoids as the MYH+ (myosin heavy chain) cells clus-
tered in regions instead of distributing in a salt-and-pepper
pattern. Importantly, the authors showed an upregulation
of the neuronal markers MAP2, VGLUT2 (vesicular glu-
tamate transporter 2), BRN2 (POU class 3 homeobox 2),
and FOXG1 (forkhead box G1) upon prolonged culture
without loss of CDH5 (cadherin 5) or SMA (actin alpha
2) expression. Further characterization of the neurovas-
cular organoids showed a spontaneous firing of neurons,
demonstrating, at least using this readout, functional neu-
ronal tissue. Subcutaneous implantation of neurovascu-
lar organoids into immunodeficient mice proved that the
organoids were perfusable. The myovascular organoids
were subjected to chronic electrical stimulation on a chip
upon which the embryonic skeletal muscle myosin 3 was
upregulated.87 Thus, expression of lineage-specific driver
genes can serve as a way to introduce parenchymal tis-
sue and possibly organospecificity into vascular organoids
instead of purely relying on extrinsic growth factors. A major
challenge of this approach is—as the authors themselves
pointed out—that not all tissues have known genetic drivers
for such lineage specification. Alternative approaches such
as knockdowns or timed regulation of certain genes—as
opposed to constitutive overexpression—might be required
to engineer complex vascularized tissues of interest.
CONCLUSIONS
Vascular diseases constitute a global health burden
affecting hundreds of millions of people. Recent break-
throughs in stem cells and tissue engineering have
allowed the establishment of ECs, pericytes, and also
bona fide blood vessel organoids under controlled cul-
ture conditions. These human BVOs and engineered
blood vessel cell types can be used to study disease
processes, normal development, or screen drugs for
therapeutic effects—one of the major shortfalls of using
rodents for drug development. However, to fully explore
the potential of these organoids, several issues need to
be addressed: utilization of Matrigel, replacements, and
standardization for mid to high-throughput screens, con-
trolled perfusion, transcriptionally dynamic identities, or
lack of vessel complexity beyond capillaries. However,
several groups have already started to show that these
issues can be solved. The ability of vessels to form in
absence of Matrigel and protocols for organotypic vascu-
latures are discussed in this review. These advances and
efforts now need to be translated to human medicine
to test whether such organoids can be used for future
February 17, 2023   507
 Salewskij and Penninger
CARDIOVASCULAR ORGANOIDS/
regenerative medicine, including much-needed vascular-
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ization of other stem cell–derived tissues.
ARTICLE INFORMATION
Affiliations
Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vi-
enna (K.S., J.M.P.). Vienna BioCenter PhD Program, Doctoral School of the Uni-
versity of Vienna and Medical University of Vienna, Austria (K.S.). Department of
Medical Genetics, Life Sciences Institute, University of British Columbia, Vancou-
ver, Canada (J.M.P.).
Acknowledgments
The authors thank Astrid Hagelkrüys and Gustav Jonsson for their suggestions
and for proofreading the review.
Sources of Funding
J.M. Penninger and K. Salewskij have received funding from the Diamond-Black-
fan-Anemia Fundraising (dbaexperiment.org) and the Innovative Medicines Ini-
tiative 2 Joint Undertaking under grant agreement No 101005026. This Joint
Undertaking receives support from the European Union’s Horizon 2020 research
and innovation programme and European Federation of Pharmaceutical Indus-
tries and Associations (EFPIA). The authors would also like to thank the Leducq
Foundation for supporting their research with the Transatlantic Network of Excel-
lence grant “ReVAMP — Recalibrating Mechanotransduction in Vascular Malfor-
mations” (2022–2027).
Disclosures
J.M. Penninger declares a conflict of interest as he is named on a patent on blood
vessel organoids. Furthermore, he is founder, shareholder, and chairman of the sci-
entific advisory board of Angios Biotech. Angios Biotech is specialized in vascular
research based on blood vessel organoids. The other authors report no conflicts.
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