Small-Caliber Tissue-Engineered Vascular Grafts Based on Human-Induced Pluripotent Stem Cells: Progress and Challenges

Abstract

Small-caliber tissue-engineered vascular grafts (TEVGs, luminal diameter <6 mm) are promising therapies for coronary or peripheral artery bypassing surgeries or emergency treatments of vascular trauma, and a robust seed cell source is required for scalable manufacturing of small-caliber TEVGs with robust mechanical strength and bioactive endothelium in future. Human-induced pluripotent stem cells (hiPSCs) could serve as a robust cell source to derive functional vascular seed cells and potentially lead to generation of immunocompatible engineered vascular tissues. Up to date, this rising field of small-caliber hiPSC-derived TEVG (hiPSC-TEVG) research has received increasing attention and achieved significant progress. Implantable, small-caliber, hiPSC-TEVGs have been generated. These hiPSC-TEVGs displayed rupture pressure and suture retention strength approaching to those of human native saphenous veins, with vessel wall decellularized and luminal surface endothelialized with monolayer of hiPSC–endothelial cells. Meanwhile, a series of challenges remain in this field, including functional maturity of hiPSC-derived vascular cells, poor elastogenesis, suboptimal efficiency of obtaining hiPSC-derived seed cells, and relative low ready availability of hiPSC-TEVGs, which are waiting to be addressed. This review is conceived to introduce representative achievements and challenges in small-caliber TEVG generation using hiPSCs, and encapsulate the potential solution and future directions.

Impact statement

Generation of small-caliber tissue-engineered vascular grafts (TEVGs) is a promising field, and may significantly benefit clinical treatments of a series of severe cardiovascular diseases or trauma. Application of human-induced pluripotent stem cells (hiPSCs) as seed cell source may further overcome hurdles in the current TEVG generation using primary cells, and lead to efficient manufacturing and application of small-caliber hiPSC-derived TEVGs (hiPSC-TEVGs) in future. This review is dedicated to summarizing representative achievements and scientific hurdles in small-caliber hiPSC-TEVG generation, to provide readers an overall picture of the progress, challenges, and potential future directions of the field.

Introduction

A huge number of patients suffer from cardiovascular obstructive diseases (coronary artery diseases and below-knee peripheral artery diseases) and peripheral vascular trauma, leading to the worldwide demand for small-caliber vascular grafts (luminal diameter [LD] <6 mm).^1–4

In clinic, autologous blood vessels, including saphenous veins and internal mammary arteries, are routinely used as native vascular grafts. However, severe vascular lesions or injuries may make these native vessels not qualified or unavailable for transplantation. Vascular prosthesis can be efficiently manufactured using synthetic polymers, but small-caliber synthetic grafts remain challenging due to limited biocompatibility and hemocompatibility of synthetic materials.

Tissue-engineered vascular graft (TEVG) is a promising alternative strategy to address the issues above, and to date TEVG technologies have achieved remarkable progresses.^5–7 In the recent decade, human-induced pluripotent stem cell (hiPSC) technologies have been utilized as seed cell source to empower the generation of small-caliber TEVGs.^8,9 This review focuses on generation of small-caliber, hiPSC-derived TEVGs and elaborates representative progress, challenges, and potential future directions.

Architectural and Biophysical Properties of Artery

Arteries with LDs <6 mm contain intima, media, and adventitia. Intima is an endothelial cell (EC) monolayer (endothelium) on luminal surface. Endothelium can sense shear stress from blood flow, prevent thrombus formation, and regulate functions of vascular mural cells.^10,11 Media layer is composed of contractile vascular smooth muscle cells (VSMCs) and extracellular matrices (ECMs); adventitia involves abluminal connective tissues that contribute to immunological surveillance and regeneration of injured vascular wall.^12–14

Blood vessel wall is circumferentially stretched by pulsatile blood flow in vivo. Collagen and elastin deposited by VSMCs guarantee strength and compliance of blood vessels to respond to cyclic tensile stress.^15,16 Moreover, laminar blood flow exerts shear stress on endothelium. Exposure to arterial laminar shear stress (∼10–20 dyn/cm² in human) promotes ECs into antithrombogenic stages,^17,18 while turbulent flow with disrupted direction and abnormal shear stress drives ECs into proinflammatory states.^17,19

Design Philosophies of Small-Caliber TEVGs

TEVGs should be designed to achieve necessary functions, optimal efficacy, and convenient application. First, as a fundamental requirement, mechanical properties of TEVGs, such as rupture pressure and suture retention strength, should be comparable with those of native vascular grafts.^4,20 Second, TEVGs should be patent without severe occlusion after long-term engraftments.

Due to relatively low flow rate and high downstream resistance in narrow vessels, small-caliber TEVGs are inclined to thrombogenesis after engraftment, therefore high hemocompatibility is an essential requirement.⁴ Third, TEVGs should be biocompatible and nonimmunogenic. Fourth, TEVGs should be capable of promoting efficient tissue remodeling and allow vascular regeneration. Fifth, TEVGs should be off-the-shelf therapy for emergency intervention.²¹

Approaches of TEVG Generation

Fundamental components for developing engineered vascular conduits

Seed cells

Primary cells were commonly used for TEVG generation due to their mature functions. Human VSMCs from organ donor's aortic tissues are utilized in constructing TEVGs.^4,6 Dermal fibroblasts are also used as seed cells due to their high accessibility and outstanding ECM synthesis capacities.^7,22

To endothelialize TEVGs, autologous ECs were obtained from recipient's vascular tissues and seeded onto luminal surface of TEVGs.^23,24 However, primary cells display finite expandability, making it difficult to culture at large scale for tissue fabrication. Moreover, primary cells may display donor-to-donor variation in cellular functions, which may lead to inconsistency of TEVG quality.⁴ It is important to find a more robust seed cell source for TEVG production.

Scaffolds

Fibrin hydrogel and nonwoven polyglycolic acid (PGA) mesh are commonly used scaffolds for TEVG generation, and effectively support cell growth and tissue fabrication, generally due to their high accessibility, outstanding porosity, and rapid degradability.^7,22,25–29 Moreover, scaffold-free, cell-derived matrix-based method was developed^23,30 using cultured cell sheets, which were highly enriched in ECMs. These sheets were peeled off, wrapped around a mandrel, and let maturate to generate tubular tissues.

Culture conditions

Blood vessels are constantly exposed to circumferential stretching from blood flows. Tensile stress can be sensed by cells through mechanosensors, triggers intracellular signaling such as transformation growth factor-β1 (TGFβ1)-SMAD pathways, promotes contractile phenotype and ECM deposition of mural cells.^9,31 Therefore, TEVGs were usually cultured in the presence of cyclic, circumferential stretching to improve ECM deposition.^26,31

A variety of bioactive molecules are supplemented in culture medium to promote ECM synthesis and cell proliferation. Vitamin C is commonly applied to enhance collagen deposition.^25,26,32 TGFβ1 is also frequently used due to its high potency to stimulate ECM synthesis and improve functional maturity of mural cells.^9,33,34

TEVG endothelialization

Since small-caliber TEVGs are inclined to thromboformation and neointima hyperplasia, it is crucial to perform endothelialization and obtain atheroprotective luminal surface.

In vivo endothelialization

Endothelialization of vascular grafts can be potentially achieved by promoting inward migration of host ECs at anastomotic sites or recruiting circulating EC progenitors to the luminal surface of grafts.³⁵ As such, methods of chemical modifications of luminal surface have been studied to enhance in vivo EC-recruiting capacity of TEVGs. Heparin-based coating has been intensively studied due to heparin's antithrombotic and EC-recruiting nature. It was reported that 4-cm–long TEVGs with heparin coating achieved in vivo endothelialization within 3 months postimplantation.³⁶

Similarly, integrin-binding ligands or endothelialization-promoting growth factors could be immobilized to the luminal surface to accelerate recruitment and growth of ECs or EC-like cells.^37,38 Smith et al. immobilized vascular endothelial growth factor (VEGF) to graft's luminal surface, successfully captured circulating CD14⁺/CD163⁺ monocytes in blood stream and differentiate them into EC-like phenotype.³⁹

Besides, renovating ECMs on luminal surface of grafts may favor attraction of host ECs. Rohringer et al. applied ECM hydrogel derived from decellularized human chorion to coat luminal surface of vascular grafts, which effectively promoted EC adhesion.⁴⁰

However, biological mechanism of in vivo endothelialization of vascular grafts remains blurry. MacDonald et al. demonstrated that endothelial regeneration in mouse aortic endothelial injury model depended on proliferation and migration of ECs flanking denuded area, rather than circulating ECs.⁴¹ This indicates that recipients' migrating ECs at anastomotic sites are major contributors of in vivo endothelium regeneration. However, endothelial injury area in this study was 2.5–3.0 mm in length, while TEVGs could be several centimeters in length, which dramatically escalates difficulties for rapidly completing endothelialization by endothelial migration.³⁵

A previous study showed that 7-cm–long vascular grafts were implanted into rat abdominal aorta, while rat ECs only migrated inward for no >2 cm from anastomotic sites after 6-month implantation, leaving large areas of luminal surface without endothelialization.⁴² Hence, in vivo endothelialization might be suboptimal for long segments of small-caliber TEVG applications.

In vitro endothelialization

Alternatively, endothelium of TEVGs can be constructed by seeding a layer of primary human ECs on graft lumen surface before implantation.²⁹ During in vitro culture, basic fibroblast growth factor and VEGF are supplemented in medium to promote EC proliferation and functions.^4,43–45 Atheroprotective shear stress through intraluminal medium flows could be applied to ECs to mimic in vivo hemodynamics.^29,46 Fluid shear stress can be sensed by mechanosensors such as integrins, VE-cadherin, and CD31,^43,47,48 causing the activation of downstream enzymes such as PI3K and ERK and transcription factors such as KLF2 and KLF4 in ECs.^18,49–51

Laminar shear stress promotes ECs aligning to the direction of flow, enhances expression of anti-inflammatory genes, and reduces expression of prothrombotic genes.^18,29,52,53 Therefore, priming with arterial shear stress in vitro can enhance atheroprotective functions of ECs and reduce acute stress response when ECs were suddenly exposed to physiological blood flow after transplantation.⁵⁴

Notably, nonimmunogenic ECs such as autologous ECs should be utilized for endothelializing TEVGs, but invasive procedure is unavoidable when harvesting patients' ECs. Moreover, due to aging or vascular lesions, autologous ECs may be less functional to meet requirements for TEVG endothelialization. It took ∼21 days from collecting patients' ECs to completion of TEVG endothelialization,^4,55 further making autologous ECs a less practical choice. Alternative source of hypoimmunogenic ECs is needed for developing small-caliber TEVGs.

Current Progress of hiPSC-TEVG Technologies

Using hiPSCs as seed cell source for developing TEVGs

As mentioned above, primary seed cells display low accessibility, robust expandability, and donor–donor functional variation, which may hinder TEVG manufacturing efficiency and clinical application.^4,56,57 Alternatively, hiPSCs can be potentially applied as a robust source of seed cells. hiPSCs, produced by cellular reprogramming by ectopic expression of Yamanaka factors, closely resemble human embryonic stem cells, particularly in terms of morphology, pluripotency marker expression profile, and differentiation capacity.⁵⁸ Therefore, hiPSCs are self-renewable and can be committed toward various somatic cell lineages, including VSMCs and ECs.^9,18,59–64 Representative progress to date in deriving VSMCs and ECs from hiPSCs is listed in Table 1.

Table 1.

Recent Representative Progress in Vascular Cell Differentiation from Human-Induced Pluripotent Stem Cells

Representative hiPSC-EC differentiation methods
Publication	Method description	Outcome	Strength	Limitation
Nature Biotechnology, 2014⁶¹	hiPSCs were differentiated into endothelial lineage, and the hiPSC-ECs with strong CD31 and NRP1 expression were sorted and subcultured.	hiPSC–endothelial colony-forming cells were highly proliferative. These cells have been applied to endothelialize acellular hiPSC-TEVGs.⁸	These NRP1⁺ hiPSC-ECs were highly proliferative and could be subcultured for 18 passages, which suggested that these cells can be produced at large scale and are potentially suitable for vascular tissue engineering.	The procedure of differentiation contains multiple steps and was complicated, including cell sorting and colony picking. Moreover, the yield of NRP⁺/CD31⁺ hiPSC-ECs appeared to be less robust, which may hamper the wide spreading of this method.
Nature Cell Biology, 2015⁶⁰	hiPSCs were first differentiated into vascular progenitors in monolayer and further allow to be committed to ECs using high dosage of VEGF.	hiPSC-ECs were derived under chemically defined condition. hiPSC-ECs derived through this protocol have been applied to endothelialize acellular vascular grafts.¹⁸	This endothelial differentiation method is easy to be applied, since it basically relied on biochemical induction using growth factors and small molecules. Moreover, this protocol appeared to be efficient, since it was mentioned in original report that 25–30 million hiPSC-ECs could be generated within 6 days as described in the publication.	Although the original report mentioned that this protocol could promote endothelial differentiation of hiPSCs with efficiencies between 61.8% and 88.8%, Wang et al. reported that the efficiency of this method was ∼5% to 40% among hiPSCs with different donor origins.⁶³ This suggests that the reproducibility of this method might be suboptimal for variant hiPSC lines.
Stem Cell Report, 2014¹²⁵	hiPSCs were differentiated into ECs through mesodermal differentiation using media with simple ingredients.	Functional hiPSC-ECs could be effectively derived.	The formula of culture medium for inducing effective endothelial differentiation was simple and cost effective.	Cellular functions of hiPSC-ECs derived through this protocol, such as the response to arterial shear stress, antithrombogenic capacity and long-term expandability, should be further analyzed.
Science Advances, 2020⁶³	hiPSCs were first subjected to mesodermal differentiation and further transiently introduced with mRNAs coding ETV2 to allow effective EC differentiation.	Functional hiPSC-ECs could be effectively derived with high efficiency and high consistency among cell lines.	Due to the high efficiency and outstanding reproducibility of hiPSC-EC derivation, this method offered a promising strategy for large-scale production of vascular seed cells for TEVG endothelialization.	This method is less economically attractive due to the dependence on transient introduction of modified mRNAs encoding ETV2.
Science Advances, 2017¹²⁶	hiPSCs were differentiated into ECs on compliant PDMS substrates.	Substrate compliance effectively promoted mesoderm differentiation from hiPSCs and enhanced EC commitment.	Results suggested the high efficiency of this protocol, as compliant substrates could enhance mesoderm induction and endothelial maturation, even under serum-free, cytokine-free condition.	The cellular functions of hiPSC-ECs generated using this protocol, such as the response to arterial shear stress, antithrombogenic capacity and long-term expandability, should be comprehensively analyzed.
Acta Biomaterialia, 2020 ¹⁸ Stem Cell Research, 2015 ¹²²	hiPSCs were differentiated into ECs through mesodermal induction under xenogeneic-free or chemically defined conditions.	hiPSC-ECs derived under xeno-free or chemically defined conditions presented typical endothelial marker expression and functions.	Xeno-free and chemically defined conditions made these methods potentially cGMP regulations.	Application of human serum or commercialized serum-free medium might escalate the economic costs. Moreover, the reproducibility of these methods in hiPSC lines with distinct parental cell origins should be further characterized.

Representative hiPSC-VSMC differentiation methods
Publication	Method description	Outcome	Strength	Limitation
Arteriosclerosis, Thrombosis, and Vascular Biology, 2007⁵⁹ Stem Cell Reports, 2016¹¹⁴	hiPSCs were subjected to spontaneous differentiation through embryoid body formation assay, and further cultured in VSMC-specific medium to derive functional hiPSC-VSMCs.	hiPSC-VSMCs at proliferative stage could be derived and could be further induced into contractile status. The hiPSC-VSMCs derived through optimized methods have been applied to develop TEVGs.^8,9	It was reported that this method with slight optimization could lead to the generation of ∼180 million hiPSC-VSMCs.⁹ This method is to date one of the most effective methods for obtaining large amount of seed cells for constructing engineered tissue conduits as TEVGs.	The germ layer origins of hiPSC-VSMCs should be clearly identified, since this method begins with spontaneous differentiation through embryoid body formation and the undefined developmental origin of these cells may lead to low reproducibility of this method. Moreover, differentiation took >4 weeks, which is less efficient. In addition, elastin expression level in hiPSC-VSMCs derived from this method was low compared with that of human primary VSMCs.⁷⁷
Nature Biotechnology, 2012¹²⁷ Nature Protocol, 2014¹²⁸	hiPSCs were cultured in monolayer and induced to neuroectodermal, lateral plate mesodermal and paraxial mesodermal differentiation, and further differentiated into VSMC lineages.	Mature hiPSC-VSMCs were derived through different embryonic lineages and displayed distinct functions.	Chemically defined culture conditions were established, which enhanced the reproducibility of this method.	The hiPSC-VSMCs derived from this method presented highly mature phenotype and might be less proliferative for tissue engineering application. Expansion of these hiPSC-VSMCs still required the presence of fetal bovine serum.
Nature Cell Biology, 2015⁶⁰ Cell Stem Cell, 2020¹²⁹	hiPSCs were first differentiated into vascular progenitors in monolayer and further allowed to be committed to VSMCs using PDGF-BB and activin A.	Mesodermal hiPSC-VSMCs with mature contractility could be generated. Toyohara et al. generated functional hiPSC-VSMCs from healthy donors and diabetic patients as disease models by using this method.¹²⁹	This method applied chemically defined medium to derive hiPSC-VSMCs, which suggests potentially high reproducibility. Moreover, this protocol appears to be highly efficient, since it was mentioned in original report that 25–30 million hiPSC-VSMCs could be generated within 6 days as described in the article.	The potential of long-term expandability, ECM synthesis capacity, and feasibility in tissue engineering of these hiPSC-VSMCs were not well characterized.
Cardiovascular Research, 2013⁶² Stem Cell Reports, 2014⁹⁰	hiPSCs were cultured in monolayer and induced to VSMC differentiation in the presence of TGFβ1 and PDGF-BB.	Three types of hiPSC-VSMCs could be derived, including immature, synthetic, and contractile hiPSC-VSMCs.	This protocol could lead to hiPSC-VSMCs with defined status of maturity. The functions of hiPSC-VSMCs at each stage have been comprehensively characterized. Moreover, the expression level of elastin gene in contractile hiPSC-VSMCs appeared to approach that of human aortic VSMCs.	This method could be relatively time-consuming, as it took ∼30 days to derive synthetic hiPSC-VSMCs when using this method.

cGMP, compliant with good manufacturing practice; EC, endothelial cell; ECM, extracellular matrix; hiPSC-TEVG, human-induced pluripotent stem cell-derived tissue-engineered vascular graft; PDGF, platelet-derived growth factor; TGFβ1, transformation growth factor-β1; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cell.

More importantly, allogeneic “universal” hiPSCs with minimal or no immunogenicity can be generated through eliminating expression of human leukocyte antigen,^65,66 and functional VSMCs and ECs could be obtained from universal hiPSCs.^9,18,65 Therefore, universal hiPSCs provide a powerful seed cell source for developing hypo- or nonimmunogenic hiPSC-derived TEVGs (hiPSC-TEVGs) at large scale in future.⁹ Representative progress to date in developing hiPSC-TEVGs has been listed in Figure 1 and Table 2.

FIG. 1.

Strategy of generation and application and small-caliber hiPSC-TEVG and the representative progress and potential challenges in the field. hiPSC-TEVG, human-induced pluripotent stem cell-derived tissue-engineered vascular graft. Color images are available online.

Table 2.

Representative Progress in Generation of Human-Induced Pluripotent Stem Cell-Derived Tissue-Engineered Vascular Grafts

Publication	Donor origin of hiPSCs	Methods of vascular differentiation	Luminal diameters (LD) and thickness	Scaffolds	Essential biochemical and biophysical cues during TEVG culture	Decellularization	Rupture pressure	Suture retention strength	Young's modulus	Collagen and elastin ECM accumulation	Endothelialization	Animal implantation
Stem Cell Translational Medicine, 2014⁷⁶	Two hiPSC lines derived from lung fibroblasts and foreskin tissues	To generate hiPSC-derived mesenchymal stem cells, hiPSCs were first cultured with TGFβ1 inhibitor and bFGF to initiate neural crest induction, and further induced into mesenchymal lineage in commercialized MesenCult Medium.	LD: ∼2 mm; Thickness: ∼250 μm	Nonwoven PGA	bFGF, PDGF-BB, TGFβ1 and 5% cyclic stretching	N/A	700 mmHg	30 g	N/A	45% of dry weight through hydroxyproline assay; No data of elastin deposition	N/A	N/A
Biomaterials, 2016⁷⁷	hiPSC line derived from peripheral blood monocytes	To generate hiPSC-VSMCs, hiPSCs were first subjected to embryoid body formation and further induced into VSMC lineage in commercialized Smooth Muscle Growth Medium.	LD: 2 and 3 mm; Thickness: N/A	Nonwoven PGA	PDGF-BB, TGFβ1 and static culture	N/A	500 mmHg	70 g	N/A	8–12% of dry weight through hydroxyproline assay; No sign of apparent elastin deposition through EVG staining	N/A	TEVGs were implanted into rat abdominal aorta for 14 days. Implanted TEVGs were patent and without rupture. However, obvious thrombosis and dilation was found at day 7 and 14 post implantation.
Acta Biomaterialia, 2019⁷⁸	hiPSC line derived from peripheral blood monocytes	To generate hiPSC-VSMCs, hiPSCs were first subjected to embryoid body formation and further induced into VSMC lineage in commercialized Smooth Muscle Growth Medium; To generate hiPSC-ECs, hiPSCs were first subjected to embryoid body formation, dissociated into single cells and subjected to cell sorting. VE-cadherin⁺ cells were sorted and further cultured in commercialized endothelial medium.	LD: 0.7 mm; Scaffold thickness: 0.5 mm	Nonwoven PGA meshes coated with poly-4-hydroxybutyrate were used as basic scaffold, and the cells were further mixed with fibrin hydrogel and seeded onto scaffold.	Commercialized Smooth Muscle Growth Medium supplemented with Vitamin C was used as culture medium. Dynamic culture condition was applied using pulsatile flow with a constant flow rate at 2.5 ml/min.	N/A	N/A	N/A	N/A	After 9 weeks of dynamic culture, collagen content have reached ∼500 μg per mg of TEVG's dry weight. There was no data of elastin deposition.	N/A	N/A
Cell Stem Cell, 2020 ⁹	hiPSC line derived from peripheral blood monocytes	To generate hiPSC-VSMCs, hiPSCs were first subjected to embryoid body formation through optimized method and further induced into VSMC lineage in commercialized Smooth Muscle Growth Medium.	LD: 3.2 mm; Thickness: 496 ± 48 μm	Nonwoven PGA	TGFβ1 and cyclic stretching with incremental stretching (frequency at 110–120 beats per minute and maximum strain at 3%)	N/A	1419 ± 174 mmHg	157 ± 16 g	∼1 MPa	8–12% of dry weight through hydroxyproline assay; No sign of apparent elastin deposition through EVG staining	N/A	TEVGs were implanted into rat abdominal aorta for 30 and 60 days. Implanted TEVGs were patent and without rupture or apparent deformation.
Circulation Research, 2022⁸	hiPSC line derived from peripheral blood monocytes were used in generated hiPSC-VSMCs.	hiPSC-VSMCs were obtained through same methods in Cell Stem Cell, 2020; hiPSC-ECs were derived according to method reported in Nature Biotechnology, 2014.⁶¹	LD: 3.2 mm; Thickness: N/A	Nonwoven PGA	TGFβ1 and cyclic stretching with incremental stretching (frequency at 110–120 beats per minute and maximum strain at 3%) were applied during TEVG construction.	hiPSC-TEVGs before endothelialization were decellularized using CHAPS, SDS and serum.	1203 ± 116 mmHg	123 ± 12 g	N/A	8–12% of dry weight through hydroxyproline assay; No sign of apparent elastin deposition through EVG staining	Commercialized endothelial medium and luminal shear stress at 58.4 dyn/cm² were applied during in vitro endothelialization using hiPSC-ECs. Endothelialized TEVGs reached endothelial coverage at almost 100%.	Decellularized hiPSC-TEVGs without endothelialization were implanted into rat abdominal aorta for 30 days. Implanted TEVGs were patent and without rupture or aneurysmal deformation; Decellularized rat aorta endothelialized with hiPSC-ECs were implanted into rat abdominal aorta for 30 days. Implanted grafts were patent with minimal or no thrombosis.

bFGF, basic fibroblast growth factor; LD, luminal diameter; PGA, polyglycolic acid.

Potential tumorigenesis by hiPSCs' derivatives is a major concern. Evolution of cellular reprogramming technologies has largely addressed this issue. Nonintegrative reprogramming methods have been developed and commercialized to guarantee the safety of hiPSCs.^67–72 Selective elimination of remaining pluripotent cells in engineered tissues can be performed as an alternative method. Engineering hiPSCs with drug-inducible suicide genes regulated by promoter of core pluripotency gene^73,74 or treating hiPSC-TEVGs with small molecules such as PluriSIn-1 can selectively kill undifferentiated cells.⁷⁵

Progress in generating TEVG conduit using hiPSCs

In the study by Sundaram et al.,⁷⁶ hiPSCs were differentiated into mesenchymal progenitors, further induced to VSMC lineage on PGA scaffold, and cultured in the presence of platelet-derived growth factor (PDGF)-BB and cyclic stretching to develop hiPSC-TEVGs. Seed cells within TEVG expressed essential VSMC markers, but the rupture pressure of these grafts ranged in 672–827 mmHg, which was significantly weaker than that of saphenous veins.⁴

Gui et al. utilized hiPSC-VSMCs to generate TEVGs in the presence of TGFβ1 and PDGF-BB under static condition.⁷⁷ hiPSC-TEVGs were highly cellularized with apparent collagen deposition, while rupture pressure was ∼500 mmHg. Moreover, collagen weight composed of 8–12% of the dry weight of hiPSC-TEVGs, while collagen content was 36% in human umbilical arteries.⁷⁷ hiPSC-TEVGs were next implanted into abdominal aorta of nude rats for 14 days.

Not surprisingly, aneurysmal dilation of hiPSC-TEVGs was observed due to low mechanical strength. Therefore, enhancing collagen deposition was the top priority for developing mechanically strong hiPSC-TEVGs. Generali et al. utilized hiPSC-derived vascular cells and PGA scaffold, and successfully developed small-caliber hiPSC-TEVGs through dynamic culture.⁷⁸ Collagen content of these hiPSC-TEVGs was >40%, but the mechanical properties and implantability were not characterized.

Luo et al. reported the construction of hiPSC-TEVGs under dynamic culture.⁹ Results suggested that TGFβ1, but not PDGF-BB, was favorable for survival and collagen deposition of hiPSC-VSMCs in three-dimensional culture. Moreover, authors found that cyclic stretching of vessel wall with optimized strain and frequency was essential for the success of hiPSC-TEVG culture. As a result, hiPSC-TEVGs demonstrated collagen content at ∼43.1% and mechanical strength approaching that of human native saphenous vein (rupture pressure: 1419 mmHg).

Moreover, hiPSC-TEVGs implanted into nude rat aortic models maintained stable LD for ∼1 month. Recently, Luo and colleagues made these hiPSC-TEVGs “off-the-shelf” by decellularizing them.⁸ Decellularization unavoidably disrupted histological structure and decreased mechanical strength of hiPSC-TEVGs, but rupture pressures were still maintained at considerably high levels (∼1203.5 mmHg).

Progress in endothelializing TEVGs using hiPSC-ECs

The feasibility of utilizing hiPSC-ECs to endothelialize small-caliber TEVGs has been evaluated in recent studies. Dr. George Truskey's group has developed a method of building up small-caliber tissue-engineered blood vessels (TEBVs), and a layer of hiPSC-ECs was applied to the lumen.^79–82 Luo et al. reported a xenogeneic-free method for deriving hiPSC-ECs and applied these hiPSC-ECs to endothelialize decellularized human arteries.¹⁸

Recently, colony-forming hiPSC-ECs were applied to endothelialize acellular TEVGs.^8,61 Endothelialized vascular grafts exhibited efficient luminal coverage and were further implanted into nude rats for 30 days. Compared with those without endothelialization, no appreciable thrombogenesis was found, and rapid replacement of hiPSC-derived endothelium by host ECs was observed postimplantation.

Challenges and Future Directions in Developing Small-Caliber hiPSC-TEVGs

Despite the progress in developing small-caliber hiPSC-TEVGs, a number of challenges await to be addressed (Fig. 1).

Functional maturity of hiPSC-derived vascular cells

Considering tremendous condition differences between embryonic development and in vitro differentiation, hiPSC-ECs may not completely recapitulate cellular functions of primary ECs.^83,84 Antithrombogenicity of hiPSC-ECs is essential for TEVG generation.

ECs prevent thromboformation through multiple approaches, including synthesizing nitric oxide, accumulating heparin-enriched glycocalyx, presenting protein C receptors, upregulation of antithrombotic regulatory genes (e.g., eNOS, KLF2, and KLF4) and downregulated expression of surface adhesion molecules (e.g., VCAM-1 and ICAM-1).^85–87 However, current studies basically focused on expression of fundamental pan-EC markers, including CD31, VE-cadherin, vWF, and KDR, and functional test was largely limited to evaluations of elementary EC functions, including tube formation assay, low-density lipoprotein-uptake assay, endothelial activation assay, and basic evaluation of response to shear stress.^18,60,61

Comprehensive examination of antithrombogenic-associated markers and functions in hiPSC-ECs was usually missing. In future study, hiPSC-EC monolayers can be preconditioned with arterial shear stress and subjected to in vitro evaluation of antithrombotic marker expression and functions, such as expression of abovementioned antithrombotic and surface adhesion markers, resistance to adhesion of platelets, macrophages, and whole blood.

It should be noted that Zhang et al. established a novel in vitro model to evaluate endothelium functions in engineered vascular tissues⁷⁹: functional tissue-TEBVs were constructed with primary VSMCs and ECs, and then perfused with medium containing monocytes or whole blood under physiological shear stress. When medium or blood was supplemented with prothrombotic reagents, TEBVs displayed EC activation, monocyte accumulation, and expression of proinflammatory cytokines. In contrast, removal of prothrombotic reagents or treatment with antithrombogenic drugs led to recovery of endothelial functions.

A similar model could be utilized as an effective platform to evaluate antithrombogenic functions of hiPSC-ECs in future. Moreover, biological identity of hiPSC-ECs should be carefully evaluated. In recent study, transcriptomic analysis suggested that many current protocols for generating hiPSC-derived brain microvascular ECs produced epithelial cells, while overexpression of ETS transcription factors could largely restore endothelial phenotypes and key functions.⁸⁸ This suggests that the expression of master regulators for endothelial development, such as ETV2 and FLI1, should be tightly monitored and manipulated during hiPSC-EC differentiation.^63,88,89

Similarly, studies have suggested that hiPSC-VSMCs might not reach complete functional maturation in comparison with primary human VSMCs. For instance, expression of essential ECMs, such as collagen type III and elastin, was much lower than that of primary VSMCs.^62,77,90 Moreover, previous study suggested that cyclic stretching should be incrementally exerted to hiPSC-TEVGs at 110–120 beats per minute,⁹ while abrupt cyclic stretching with 2–3% of strain and higher frequency induced disintegration of hiPSC-TEVGs.^9,91

In contrast, primary VSMC-based TEVGs were directly stretched at strain of 2.5% and frequency of 2.75 Hz.⁴ Abrupt exertion of stretching with high distension may activate stress-induced pathways such as p38-MAPK and killed hiPSC-VSMCs.⁹² The requirement of lower stretching frequency indicated that hiPSC-VSMCs might need more time to respond after being stretched by pulse of intraluminal flow, and might not be contractile enough to restore the original shape before the arrival of next pulse. hiPSC-VSMCs are developed statically on stiff plastic plates without cyclic tensile stress.

The distinct conditions of cellular differentiation in vivo and in vitro may diverge functional maturation levels of primary and hiPSC-derived VSMCs. As such, hiPSC-VSMCs can be cultivated in the presence of appropriate mechanical stimulation in future. It was reported that cyclic stretching or low levels of shear stress could significantly favor functional maturation and ECM synthesis of hiPSC-VSMCs.^93,94

In vivo retention of hiPSC-derived endothelium

A number of studies reported poor retention of human endothelium in TEVGs after animal implantation. Luo et al. endothelialized TEVGs using hiPSC-ECs and implanted them into abdominal aorta of nude rats.⁸ Preseeded hiPSC-ECs gradually disappeared in 7 days postimplantation and were completely replaced by host ECs on day 30. Similarly, Elliott et al. implanted vascular grafts endothelialized with human primary ECs into immunodeficient mice, while only few human ECs retained by the first week of implantation.⁹⁵

In a recent study, retention of hiPSC-ECs rapidly decreased within 1 week after vascular graft implantation into canine models.⁹⁶ In contrast to abovementioned xenoimplantation, autologous, GFP-labeled porcine primary ECs or circulating endothelial progenitor cells (EPCs) were used to endothelialize TEVGs with an endothelial coverage rate of 64% ± 9%.²⁹ After grafts being implanted into donor pigs' carotid arteries for 30 days, GFP⁺ ECs were found in four of five EC-seeded grafts, and the average luminal coverage rate remained as high as 35%.

According to the studies above, it appeared that blood-touching endothelium with human origin may undergo acute xenogeneic immunoresponse after implantation into animal models, even inside immunodeficient animals, and consequently decreased retention of human endothelium.

Previous study has suggested the potential of immunodeficient animal models in generating xenoreactive responses.⁹⁷ If there is any pre-existing residuals of xenoreactive antibodies in animal models, complement cascade can be rapidly triggered upon vascular graft implantation, resulting in endothelial damage.⁹⁸ Future efforts should be made to investigate the potential xenogeneic response against human cells in animal models for TEVG implantation.

It should be noted that compared with terminally differentiated ECs, EPCs might present longer residency.^95,99 Previous study showed that preseeded ovine EPCs covered 80% of luminal surface of vascular grafts at week 2 postimplantation into ovine model, and the retention rate was maintained at 10% after 18 weeks.⁹⁹ Therefore, it is worthwhile testing the in vivo performance of hiPSC-derived EPCs or other endothelial progenitor-like cells such as endothelial colony-forming cells.⁶¹ In addition, considering distinct cardiovascular physiologies (shear stress, pulse rate, etc.) between human and animals, endothelialized TEVGs could be strictly preconditioned with hemodynamics mimicking that of animal models.

Elastogenesis of hiPSC-VSMCs

Elastin is an essential matrix component of arteries besides collagens, comprising ∼30% dry weight.¹⁰⁰ Sufficient compliance contributed by elastin mitigates instantaneous high shear stress exerted on endothelium and allows the vessel wall to recoil when being impacted by pulsatile blood pressure. Disruption of elastin fibers can lead to aneurysm, intimal hyperplasia, and thrombosis.^101–103 Therefore, elastin deposition is critical for small-caliber TEVG's functions.

Elastin fiber synthesis begins with coacervation of soluble tropoelastins encoded by ELN gene. Tropoelastin coacervates are deposited along microfibrils and crosslinked by lysyl oxidase to form insoluble, functional elastic fibers.^104,105 Elastin expression is quite apparent in primary VSMCs but dramatically lower in hiPSC-VSMCs.^77,93,94 Studies by Gui et al. and Luo et al. both suggested that no apparent elastin deposition was observed in hiPSC-TEVGs.^9,77

A number of studies have attempted to augment elastin expression in hiPSC-derived cells. Wanjare et al. showed that extra biomechanical stimulation could raise intrinsic ELN expression of hiPSC-VSMCs.⁹³ Ellis et al. overexpressed ELN in hiPSC-VSMCs and enhanced elastin deposition in cell culture.¹⁰⁶ Eoh et al. generated hiPSC-derived smooth muscle tissue in the presence of shear stress. Mechanical stimulation substantially increased in elastin production in engineered tissue, but elastin only comprised of ∼6% of the total mass, which was insufficient to provide compliance of the tissues.⁹⁴

Future efforts should be made to understand the mechanisms of poor ELN expression in hiPSC-VSMCs, accordingly optimize VSMC differentiation methods, and further evaluate if these hiPSC-VSMCs may improve elastin deposition and compliance of hiPSC-TEVGs.

In fact, efficient elastogenesis is a critical issue for the whole field of tissue engineering. Research progresses on augmenting elastin synthesis of primary cells in vitro may enlighten the improvement of elastin deposition of hiPSC-TEVGs in future: In terms of biomechanics, biaxial stretching and cyclic hydrostatic pressure were reported to promote elastin deposition in primary cell-based engineered tissues.^31,107 From a biochemical perspective, dual presence of BMP4 and TGFβ1, microRNA-29a inhibitor, and polyphenol treatments could promote expression and protein assembly of elastin in primary cells.^108–112

In addition, the presence or absence of certain ECM components may regulate elastin deposition. A recent study found that collagen type VIII deficiency significantly increased elastic deposition by rat cells.¹¹³

Efficiency of seed cell derivation

Derivation of hiPSC-derived seed cells at large scale usually takes long time. For example, based on an embryoid body-driven VSMC differentiation method, it took 4–6 weeks to obtain sufficient amount of hiPSC-VSMCs for launching TEVG construction.^9,77,114 Similarly, although only ∼6 days were needed to generate hiPSC-ECs, the efficiency of endothelial differentiation reached only ∼30%,^18,63 and the expandability of these hiPSC-ECs was limited within several passages.¹⁸

In addition, it has been reported that endothelial differentiation efficiencies of hiPSC lines derived from dermal fibroblasts, umbilical ECs, and epithelial cells presented dramatic variation,⁶³ potentially due to the variation of epigenetic patterns in donor cells.^115,116 Therefore, low efficiency and poor reproducibility of hiPSC differentiation methods will lead to laborious cell sorting and expansion and unpredictable outcomes, which will eventually cause massive consumption of reagents, including specialized culture media, ECMs for surface coating, growth factors, and serum.

A growing body of studies have indicated that manipulating the expression of key transcription factors for lineage commitment during hiPSC differentiation could be a potential path to efficient derivation of vascular seed cells. A typical example is that Wang et al. delivered modified mRNAs encoding ETV2, a master regulator of endothelial development,⁸⁹ in hiPSCs at mesodermal differentiation stage. This method allowed highly reproducible endothelial differentiation with efficiency at ∼89% to 95% among several hiPSC lines with distinct donor origin.⁶³

In comparison, conventional EC differentiation methods based on small molecules and growth factors reached efficiency at ∼3% to 70% with inconsistencies. This strategy may allow scalable derivation of functional hiPSC-ECs from previously expanded hiPSCs in short period of time.

Ready availability of hiPSC-TEVGs with live endothelium

Peripheral vascular injuries require emergency operations to re-establish blood supply in below-the-knee hind limbs using small-caliber vascular grafts.¹¹⁷ Therefore, it is expected that hiPSC-TEVGs with live endothelium can be readily available for emergency treatments. However, the presence of live cells could reduce ready availability of TEVGs. It required 4–5 days to precondition hiPSC-derived endothelium with shear stress, which remain suboptimal for emergency treatments.⁸ Therefore, future efforts should be made to seek for strategies to make endothelialized hiPSC-TEVGs completely off-the-shelf products.

Progress in cryopreservation may help in long-term storage of in vitro-endothelialized hiPSC-TEVGs. Several studies have reported the preservation of EC monolayers by using improved cryoprotectants.^118,119 Recently, a novel method of thawing vitrified tissues by inductive heating of magnetic nanoparticles has been invented,¹²⁰ which may offer opportunity for application of cryopreserved hiPSC-TEVGs. Despite these promising progresses, antithrombogenic functions and responses to shear stress of cryopreserved hiPSC-endothelium and the effect of cryopreservation on mechanical properties of acellular hiPSC-TEVGs should be carefully evaluated.

Regulatory path for hiPSC-TEVG production

Manufacturing TEVGs containing live hiPSC-ECs requires extensive quality control (QC). QC standards for hiPSC manufacturing have been established,¹²¹ which can be referred to cover the validation of critical facets, including safety, identity, and potency of hiPSC-EC production. Moreover, TEVG manufacturing process should be in compliance with Good Manufacturing Practice (GMP) regulations. Accordingly, xenogeneic-free and chemically defined methods for deriving hiPSC-ECs have been established.^18,122

Moreover, Humacyte, a biotechnology company developing human acellular vessels, established LUNA200 systems, which allows scalable, GMP-level TEVG production.¹²³ These achievements have set the foundation for future production of endothelialized TEVGs with even quality.

In addition, safety and immunogenicity of universal hiPSC's derivatives should be intensively evaluated. In vivo evaluation of universal hiPSC-ECs was merely limited to humanized mice.⁶⁵ To extend the evaluation to animal models resembling human physiology, immunologically universal porcine iPSCs can be established to allow preclinical evaluation of universal iPSC-TEVGs in future.¹²⁴

Footnotes

Acknowledgments

Due to space limitation, we were not able to collect all important papers in related fields. We apologize to those researchers whose papers we omitted here. We thank Drs. Yibing Qyang and Laura Niklason (Yale University) for their support.

Authors' Contributions

All authors in this work contributed to data collection, figure and table preparation, and article writing and editing.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by start-up fund of School of Life Science and Technology, ShanghaiTech University.

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