Next-generation vascular organoids: multi-lineage and immune integration for disease modeling and regenerative medicine

Recapitulating the perivascular niche: mural cells and basement membrane assembly

Functional vascular maturation requires precise spatiotemporal coordination and heterotypic signaling between ECs and mural lineages^[14]. In late-stage angiogenesis, pericytes stabilize nascent endothelial sprouts^[15], strengthen tight junctions through paracrine factors and direct contact^[16], and deposit basement membrane components^[17]. Heterocellular gap junctions such as Connexin 43 (Cx43) mediate these interactions and stabilization events^[8]. Mural cell recruitment thus directly determines microvascular integrity, compliance, and selective permeability.

To recreate this niche in vitro, stem cell bioengineering employs two main strategies.

• Spontaneous co-differentiation vs. directed sequential induction: This approach mimics embryogenesis by guiding mesodermal progenitors through wingless-related integration site (WNT) and transforming growth factor-beta (TGF-β) signaling modulations to generate distinct endothelial and mural fates^[18]. Early protocols by Patsch et al.^[19] and Orlova et al.^[20] demonstrated the concurrent expansion of hiPSC-derived ECs with VSMCs or pericytes, respectively. Beyond soluble factor cocktails, the biophysical and spatial cues of the surrounding microenvironment actively instruct endothelial fate determination. Zhang et al. demonstrated that a 3D differentiation protocol not only enhances the efficiency of hiPSC-EC generation but also preserves a stable endothelial phenotype, highlighting that spatiotemporal 3D context - rather than biochemical signals alone - is a critical determinant of EC specification^[21]. More recently, the identification of shared mesenchymoangioblast (MAB) progenitors has accelerated pericyte differentiation^[22,23]. PDGFRβ⁺ CD271⁺CD73^- progenitors derived from MABs further differentiate into pericytes {cluster of differentiation 274 [CD274]+ capillary or delta-like homolog 1 [DLK1]+ arteriolar}, VSMCs and stromal cells. Self-assembling vessel organoids differentiated via WNT activation and BMP4 stimulation exhibit endothelial lumens, covered by a continuous basement membrane and interact closely with pericytes^[6,24].

• Orthogonal transcription factor (TF) reprogramming: To bypass conflicting morphogenetic signals, recent studies have turned to direct TF-driven fate engineering^[25]. Gong et al. demonstrated that orthogonal activation of TF ETV2 (endothelial) and NKX3.1 (mural) using Dox-inducible or modified mRNA (modRNA) systems induces balanced co-differentiation within five days^[26]. Delivering these TFs via chemically modified mRNA produces footprint-free, physiologically relevant organoids, offering a scalable platform for disease modeling^[27]. However, forced TF overexpression may bypass normal developmental checkpoints^[28,29], raising concerns about incomplete epigenetic remodeling and off-target effects^[29]. For ETV2/NKX3.1 systems, independent control of endothelial and mural compartments remains challenging due to the shared trigger, and absent perfusion limits maturation^[26].

Parallel to lineage specification, accommodating these multicellular networks requires animal-origin-free extracellular matrices (ECMs) to replace batch-variable Matrigel preparations^[30]. Recombinant vitronectin, engineered fibrin and human-derived collagen hydrogels match Matrigel in driving sprouting and capillary morphogenesis^[30]. Paired with ultra-low attachment U-bottom or “sitting drop” configurations, these platforms enable standardized, high-throughput biofabrication of biomimetic vascular architectures^[31] [Table 2].

Integrating the immune compartment: leukocyte trafficking and vascular-immune crosstalk

Beyond the physical barrier, the vascular endothelium functions as a dynamic immunological interface coordinating homeostasis, repair, and inflammatory cascades^[32,33]. Under inflammatory conditions or altered shear stress, ECs upregulate adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), capturing circulating leukocytes for rolling, adhesion, and transendothelial migration (TEM)^[13].

Single-cell transcriptomics revealed that aged splenic endothelial subpopulations acquire immune-like intermediate states with antigen-presentation signatures^[34]. This vascular-immune coupling is most evident at the blood-brain barrier (BBB), where ECs collaborate with pericytes and astrocytes to regulate central nervous system (CNS) immunosurveillance^[33].

Modern vascular organoid models incorporate immune compartments by co-differentiating or embedding myeloid and lymphoid lineages into 3D constructs.

• Myeloid integration (monocytes/macrophages): hiPSC-ECs closely mimic primary human umbilical vein endothelial cells (HUVECs) in inflammatory responsiveness. In microfluidic systems exposed to tumor necrosis factor-alpha (TNF-α), hiPSC-ECs support increased monocyte adhesion, capturing leukocyte extravasation^[13]. Once recruited via endothelial chemokines, monocytes and macrophages drive ECM remodeling, endothelial sprouting, and lumen anastomosis by secreting Matrix Metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF)^[9,35].

• Lymphoid integration [T cells/ natural killer (NK) cells]: Modeling adaptive immune cell trafficking through 3D vascularized matrices is essential for evaluating cancer immunotherapies and chronic inflammation^[36]. Perfused microfluidic platforms with arterial-venous ECs wrapped by pericytes allow real-time tracking of T-cell rolling and TEM after cytokine stimulation^[37]. These systems provide quantitative models to test the homing, extravasation efficiency, and tumor-penetration kinetics of engineered cell therapies, including chimeric antigen receptor (CAR) T cells.

• Organ-specific immune microenvironments: Replicating specialized pathology requires tailored multicellular design. Modeling the neurovascular unit, for instance, requires the integration of microglia, the brain’s resident myeloid population. Co-culturing hiPSC-derived brain microvascular ECs with astrocytes induces mature BBB phenotypes with tight junctions and restrictive permeability^[38,39]. Adding microglia enables precise dissection of heterocellular communication and cellular dynamics under neuroinflammatory conditions^[40].

Synergistic homeostasis: the triadic crosstalk driving vascular maturation

Mural and immune integration are functionally interdependent. Mural cells regulate immune trafficking by controlling vessel diameter, tone, and basal permeability, governing leukocyte TEM kinetics^[16]. Conversely, integrated macrophages secrete pro-angiogenic factors (e.g., VEGF) to direct tip-cell sprouting while recruiting and anchoring pericytes.

Tri-culture systems (EC + mural + immune) have been reported to enhance network complexity, basement membrane assembly, and long-term stability^[41-43]. Defining and exploiting this triadic crosstalk is key to next-generation organoids, though translating these advances into therapeutic applications will require overcoming substantial barriers in scale, safety, and functional validation^[44-46].

Recapitulating vascular inflammation and cytokine dynamics

Leukocyte rolling, adhesion, TEM, and secondary leakage are hallmarks of inflammatory conditions^[47]. Next-generation organoid systems move beyond static end-point measurements and capture these shear-dependent cellular dynamics in real time.

In advanced BBB models exposed to ischemia/reperfusion, the introduction of circulating monocytes triggers a classic biphasic barrier breakdown via upregulation of pro-inflammatory cytokines, including TNF-α, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β), driving MMP release and basement membrane degradation^[48,49]. Notably, monocyte migration across the damaged barrier has been documented in inflammatory BBB models, where chemokine gradients and endothelial adhesion molecules orchestrate leukocyte diapedesis^[50]. Real-time imaging shows monocytes migrating across destabilized junctions toward the abluminal space. These emergent properties materialize only when endothelium, perivascular stroma, and immune cells interact simultaneously^[51].

Deconstructing the tumor-immune-vascular interface

In tumor microenvironments, aberrant, leaky vasculature sustains immunosuppression, impairing adoptive immunotherapy delivery. Coupling vascular organoids with microfluidic chips provides a controllable system to deconstruct this interface^[52].

When hiPSC-ECs are cultured in 3D matrices exposed to tumor-conditioned medium, they assemble into perfusable networks that adopt tumor-like transcriptional and structural signatures. Here, perivascular mural cells (pericytes and VSMCs) directly determine vessel permeability and tight junction integrity, thereby controlling how effectively immune cells penetrate the matrix. These platforms allow quantitative tracking of the homing, rolling, adhesion, and transendothelial extravasation efficiency of engineered cellular therapeutics, including chimeric antigen receptor T (CAR-T) cells or NK cells^[53-56]. By capturing the interplay between tumor-derived angiogenic factors [e.g., vascular endothelial growth factor-A (VEGF-A), Angiopoietin-2] and the vessel wall, these models help clarify why immunotherapies fail in solid tumors and serve as screening tools for vascular normalization drugs.

Modeling infectious disease pathology and barrier invasion

Vascular networks serve as both primary target tissues and physical barriers for diverse pathogens, which makes multi-lineage organoids valuable for infectious disease modeling.

In human vascular organoids infected with SARS-CoV-2, complement factor D (CFD) has been identified as a critical driver of microvascular endothelitis and immunothrombosis, providing a potential link between viral infection, complement cascade activation, and vessel occlusion^[57]. Similarly, dynamic BBB-on-a-chip models have been employed to replicate the stepwise passage of diverse blood-borne pathogens across restrictive human endothelia. In bacterial meningitis models, pathogens disrupt tight junction proteins and activate local endothelial cells^[58]. These platforms also capture fungal neurotropism^[59] and viral encephalitis pathology^[60].

Vascularized organoid platforms extend infection modeling to tissue interfaces beyond endothelium. In vascularized colon organoids-on-a-chip models, STEAP3 depletion increased viral signals within the vascular lumen, demonstrating host-factor facilitation of viral dissemination^[61]. Likewise, vascularized human placental organoids microphysiological systems (MPS) maintain trophoblast viability, proliferation, and differentiation while enabling the study of vertical transmission at the trophoblast-vascular interface^[62].

Modeling hereditary and vascular disorders

Multi-lineage vascular organoids are also advancing the study of genetic vasculopathies and metabolic disorders.

• Hereditary hemorrhagic telangiectasia (HHT): Organoids generated from endoglin (ENG)-haplo-insufficient hiPSCs display microvascular wall thickening, disrupted endothelial-smooth muscle interactions, and reduced cell survival. Single-cell transcriptomics in these models identified dysregulated Notch signaling as a core driver of the phenotype^[63].

• Cerebral small vessel disease (CSVD): Vascular organoids carrying the NOTCH3 p.R141C mutation, modeling the most common monogenic cerebral small vessel disease, exhibit reduced capillary density, premature sprouting, and stunted overall organoid growth. Transcriptomic profiling indicates widespread downregulation of genes associated with cell adhesion, ECM assembly, and vessel maturation^[64].

• Atherosclerosis: In vitro models of atherosclerosis combine iPSC-derived vessel organoids with fluid shear stress, low-density lipoprotein (LDL) exposure, pro-inflammatory cytokines, and circulating monocytes. This setup captures key pathological events that require both mural and immune components, including endothelial activation, monocyte-to-foam cell transition, fibrous plaque formation, and calcification^[65].

• Other vascular disorders: Capillary malformation organoids show increased EC/smooth muscle cell (SMC) density, elongated branches, and endothelial-to-mesenchymal transition features^[66]. Moyamoya disease organoids (RNF213-mutant) exhibit reduced branching with ECM dysregulation^[67,68]. Lamin A/C (LMNA)-mutant organoids model progeroid vasculopathy via serum response factor (SRF) pathway repression^[69]. Finally, diabetic vasculopathy organoids reproduce basement membrane thickening, excessive ECM deposition, and microvascular rarefaction under hyperglycemia^[7,70-72].

Modeling neurodegenerative and metabolic disease

Vascularized neuroimmune organoids containing neurons, microglia, astrocytes, and blood vessels develop Amyloid-beta (Aβ) plaque-like and tau tangle-like aggregates, neuroinflammation, synaptic pruning, and network dysfunction within four weeks of exposure to brain extracts from patients with sporadic Alzheimer’s disease. Notably, lecanemab reduced amyloid burden but increased vascular inflammation, with direct drug-safety implications ^[73,74]. Liver-vascular co-culture shows that effluent from steatotic hepatic organoids induces foam cell formation, revealing a liver-vascular axis in metabolic dysfunction-associated steatotic liver disease (MASLD) and providing a system to test interventions such as sirolimus^[75,76].

Together, these models illustrate the generality of the multi-lineage concept: wherever a pathogen must cross a tissue barrier to reach the bloodstream, an organoid that reconstructs both the barrier and its vascular exit route becomes a uniquely informative platform. Combining multi-lineage organoids with single-cell multi-omics and CRISPR gene editing establishes a precise, human-relevant framework to map how cell-cell interactions drive disease phenotypes and to identify target-directed therapeutics.

Solving “where”: spatial organization and biomaterial architecture

Recreating native microvasculature requires micro-scale spatial control: mural cells must wrap the abluminal surface of endothelial capillaries, whereas immune cells require functional routes from luminal fluid into perivascular tissues.

• Layer-by-layer bioprinting: Modern 3D bioprinting addresses this spatial challenge by depositing diverse cell types embedded in customized bioinks with micrometer-level precision. For example, in skin-on-a-chip models, multi-layered extrusion printing embeds perfusable microvascular networks within dermal equivalents, which are subsequently capped with stratified epidermal layers to build functional, organotypic barrier models^[78].

• Matrix chemistry and integrin signaling: These spatial approaches are complemented by biocompatible hydrogels, such as gelatin methacryloyl (GelMA) and alginate derivatives, engineered with tunable mechanical stiffness and viscoelasticity to mirror native tissue compliance and guide cell morphodynamics^[79,80]. Functionalizing these matrices with basement membrane proteins, such as laminin and type IV collagen, engages cellular integrins (e.g., αvβ3/α5β1), triggering focal adhesion kinase (FAK) signaling to direct endothelial polarization and leukocyte extravasation^[81].

• Translational ECM formulations: Non-swelling, degradable hydrogels matching the viscoelastic properties of brain tissue provide physical space for organoid expansion without imposing compressive mechanical stress. Replacing batch-variable Matrigel with defined recombinant human vitronectin and engineered fibrin matrices provides standardized, animal-origin-free scaffolds compatible with clinical-grade, high-throughput biofabrication^[30,82].

Solving “how”: dynamic microenvironments and biomechanical force regimes

Vascular network assembly depends on physical forces. The incorporation of cellular complexity into 3D matrices must therefore be paired with microfluidic technologies that provide physiological fluid dynamics.

• Mechanotransduction and junctional tightening: Perfusable microchannels generate physiological pressure gradients and fluid shear stress, both of which are essential for endothelial maturation and barrier permeability regulation^[83]. Shear stress stimulates endothelial mechanosensors, driving downstream activation of the TFs, which regulate tight junction proteins. When pericytes and perivascular fibroblasts are introduced at the abluminal surface under flow, these systems induce the self-assembly of stable, non-leaky networks with robust basement membrane deposition^[84,85].

• Perfusion dynamics and live-cell diagnostics: Microfluidic architectures with perfusable channels generate uniform cytokine gradients and steady shear stress profiles across the organoid network. These perfusion systems enable real-time imaging of reactive oxygen species (ROS) levels^[86], leukocyte recruitment, rolling, adhesion, and TEM under controlled hemodynamic regimes^[87].

• On-chip multi-omics and multi-organ linkage: Beyond mechanical stimulation, microfluidic platforms with integrated automated processing modules can isolate high-viability single-cell suspensions directly from dense 3D matrices without conventional enzymatic digestion. When coupled with on-chip single-cell RNA transcriptomics, these platforms track dynamic phenotypic drift and spatial heterogeneity in integrated immune cells in real time^[88-90]. At a systemic level, multi-organ-on-a-chip setups connect distinct tissue modules (e.g., cardiac, hepatic, osseous, and cutaneous compartments) via endothelialized fluidic channels, maintaining organ-specific endothelial phenotypes while modeling human pharmacokinetic profiles and systemic immune trafficking^[91-94].

Solving “what”: defining cell sources and unifying genetic identity via fate engineering

A persistent challenge in engineering third-generation vascular networks is sourcing scalable, donor-matched, and immune-compatible endothelial, mural, and immune cell lineages^[4]. Precision gene editing and TF driven reprogramming are increasingly addressing this limitation.

• CRISPR editing and single-cell droplet selection: CRISPR-Cas9 genome editing allows precise targeted modification of hiPSCs before directed differentiation into discrete vascular or hematopoietic branches^[95-97]. Encapsulating these edited hiPSCs within microfluidic droplet platforms enables single-cell clonal selection and rapid line validation^[98].

• TF-directed reprogramming: The transition from empirical cytokine cocktails to TF-guided fate engineering is informed by single-cell multi-omics, which maps the molecular checkpoints governing vascular lineage decisions. Large-scale perturbation screens and single-cell multi-omic analyses have identified TFs governing early mesodermal-to-endothelial fate commitment^[99]. Furthermore, TF-directed reprogramming has enabled the generation of organ-specific endothelial subtypes, including brain-specific barrier phenotypes with functional efflux transporter activity^[99].

• Isogenic multicellular assembly: By temporally modulating the VEGF-Notch signaling axis, hiPSC-ECs can be directed toward specific arterial (e.g., EphrinB2+) or organ-specific barrier states^[100]. Recent advances have demonstrated the feasibility of generating endothelial, mural, and immune compartments from a single hiPSC donor line, providing an isogenic platform for disease modeling and therapeutic screening^[101]. This shared genetic background reduces the risk of confounding allogeneic immune responses, providing an isogenic system for disease modeling and therapeutic screening.

Single-cell and spatial multi-omics for quality assessment and mechanistic dissection

As vascular organoids increase in complexity, bulk analysis methods become insufficient. Emerging single-cell and spatial multi-omics are essential for evaluating organoid quality, maturity, and physiological relevance.

Navigating biochemical antagonism, media incompatibility, and metabolic paradoxes

A central biological obstacle in constructing complex tissue models arises from the distinct, and often conflicting microenvironmental, biochemical, and metabolic requirements of different cell lineages.

• Divergent lineage dependencies: Under normal homeostasis, ECs, mural cells, and immune populations operate in tight synchrony. In co-culture, however, their survival and maturation requirements diverge^[106]. Quiescent ECs require continuous laminar fluid shear stress and baseline VEGF signaling to maintain barrier integrity. Conversely, perivascular mural cells rely on specific ECM cues and TGF-β signaling to sustain a contractile, non-fibrotic phenotype^[107]. These divergent dependencies can be partially reconciled through targeted modulation of shared intracellular signaling nodes^[108]. Adding tissue-resident macrophages introduces another layer of complexity, because their functional plasticity and survival depend on localized cytokines that can directly oppose the anti-inflammatory, quiescent state required for a baseline endothelial barrier^[109].

• Diffusion limits and nutrient gradients: When these cell types are assembled at physiological densities, nutrient and oxygen diffusion limits emerge, causing necrotic core formation and network regression^[110,111]. While microfluidic channels help partition lineages and establish nutrient or oxygen gradients, dynamically balancing these signals, such as promoting angiogenic sprouting without triggering hyper-inflammatory immune responses, remains difficult. This is further complicated by donor-specific transcriptomic variation in hiPSC-derived cells^[18].

• Integrated multi-omic solutions: Resolving these limitations requires single-cell multi-omics (transcriptomics, metabolomics, and fluxomics) to map the metabolic consumption and signaling networks of each cell subpopulation in real time. These datasets can inform the design of switchable, computer-controlled perfusion media and precision delivery systems^[106].

Scaling, automation, and the transition to predictive biomanufacturing

To move multi-lineage vascular organoids from small-scale laboratory prototypes to standardized platforms compliant with cGMP guidelines, the field must overcome limitations in scalability and batch-to-batch variability.

• Artificial intelligence (AI)-driven robotic assembly: Manual and semi-automated fabrication methods suffer from inconsistent embryoid body size, variable cellular aggregation, and uneven ECM crosslinking. Automated, AI-guided paradigms, such as “pick-place-perfuse” (Bio-P3), enable robotic systems to assemble pre-formed microtissue modules (e.g., ring- or honeycomb-shaped perivascular aggregates) with high spatial accuracy while maintaining continuous luminal perfusion to preserve cell viability^[112].

• Systemic multi-organ linkage: When coupled with microfluidic chips, these platforms may help establish biomimetic branching, hierarchy, and shear-induced maturation under physiological flow conditions. Interconnecting distinct organoid modules (e.g., heart, liver, bone, and skin) via vascularized channels helps preserve organ-specific endothelial identities over extended durations (exceeding four weeks) while capturing systemic pharmacokinetic and pharmacodynamic profiles^[113].

• Closed-loop feedback systems: Combining solid-state sensors with deep-learning models enables continuous tracking of dissolved oxygen, pH, and metabolite levels (e.g., lactate, glucose). Autonomous, closed-loop adjustments convert vascular organoid engineering from empirical optimization to predictive biomanufacturing^[114].

Immunological compatibility and immune cell integration challenges

For clinical applications, such as using vascularized constructs as therapeutic grafts for ischemic disease, allogeneic rejection remains a critical hurdle^[115]. Additionally, immune cell integration introduces distinct biological challenges.

• Universal hypoimmunogenic master lines: Patient-specific hiPSCs offer an autologous source, but are time- and cost- prohibitive for acute interventions such as myocardial infarction or stroke^[116]. Universal off-the-shelf allogeneic cell lines are being developed via CRISPR-Cas9 genome editing^[117]. Disruption of human leukocyte antigen (HLA) class I and II complexes {via β-2-microglobulin [B2M] and class II transactivator [CIITA] knockouts} combined with CD47 overexpression (“don’t-eat-me” signaling) helps constructs evade both adaptive T cell responses and innate NK-cell surveillance^[118].

• Biomaterial encapsulation and immunomodulation: Semi-permeable, immune-protective biomaterials allow nutrient exchange while shielding constructs from immunoglobulins and cytotoxic T cells^[119]. Additionally, embedding tolerogenic subsets, such as regulatory T cells or M2-polarized macrophages, can promote local immune tolerance and facilitate integration with the host microvasculature after transplantation^[120].

• Immune cell maturation and maintenance: induced pluripotent stem cell (iPSC)-derived macrophages often exhibit fetal-like phenotypes with immature cytokine profiles^[121]. Maintaining long-term function remains difficult since niche signals, such as colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and TGF-β, are often absent in conventional organoid culture medium, limiting chronic disease modeling^[122].

• Context-dependent effects: The impact of immune cells is highly context-dependent. In a 3D tri-culture model, pro-inflammatory (M1) microglia inhibited neuronal differentiation and vascular development, while anti-inflammatory (M2) microglia supported neurovascular maturation via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) signaling axis^[40]. While appropriately polarized immune cells can support vascular maturation, uncontrolled or pro-inflammatory immune populations may actively disrupt organoid development and homeostasis.

• Tissue-specific immune phenotypes: Organ-specific macrophages (microglia, Kupffer cells, alveolar macrophages) require tissue-specific cues to instruct appropriate immune cell differentiation, making recapitulation of tissue-specific inflammatory or infectious pathologies challenging^[123,124].

• Immune disruption of vascular homeostasis. While quiescent macrophages support vascular homeostasis and remodeling, their activation, whether by pathogens, or damage-associated signals, can rapidly destabilize the vascular network^[125]. SARS-CoV-2 infection activates inflammatory macrophages and upregulates interferon signaling, driving cytokine release and vascular damage^[126]. Balancing baseline stability with on-demand inflammatory responses remains a critical challenge.

Table 4 summarizes these challenges and potential solutions.

AI-driven quality control and phenotypic screening

Deep learning models and computer vision pipelines are transforming quality control and phenotypic screening in vascular bioengineering^[127].

• Label-free image analysis: Because multi-lineage organoids exhibit high structural complexity, manual quantification of network connectivity, capillary diameter, and spatial organization is inefficient^[128]. Convolutional neural networks, such as the Vessel Connectivity Network (VC-Net), enable label-free, automated classification of normal versus disrupted vascular networks directly from brightfield or phase-contrast images^[129].

• High-throughput sandbox for precision medicine: Applying these AI vision systems during manufacturing provides real-time quality control, flagging defective batches and guiding protocol adjustments. Combining patient-derived multi-lineage organoids with microfluidic chips under physiological flow creates a robust platform for precision medicine. These integrated platforms allow researchers to study how hemodynamics influence patient-specific disease phenotypes and drug responses, connecting genetic variation to functional human pathophysiology.

Beyond the current blueprint: engraftment, missing lineages, and aging

Several frontiers remain largely unexplored. First, in vivo functionality: although hypoimmunogenic master lines address immune rejection, the decisive translational test is whether transplanted vascular constructs anastomose with the host circulation and sustain perfusion; engraftment, inosculation, and long-term patency should become standard reporting criteria. An alternative or complementary approach to circumvent the engraftment and rejection challenges is the use of cell-free therapeutics. Wei et al. recently demonstrated that nanovesicles derived from hiPSC-ECs effectively promote angiogenesis and restore perfusion in ischemic limb disease models, suggesting that the paracrine/vesicular payload of vascular organoids - rather than the cells themselves - may offer a safer, more scalable route to clinical translation^[130]. Second, the current multilineage blueprint omits perivascular nerves (regulating tone beyond the neurovascular unit) and lymphatic endothelium (coordinating fluid homeostasis and immune trafficking); integration of these lineages may define the next level of physiological completeness. Third, aging: organoids derived from progeria patients^[69] and aged endothelial populations^[34] indicate that vascular organoids can serve as tunable models of vascular aging, opening a route to test senolytic and rejuvenation strategies.