Efficient Differentiation of Human Induced Pluripotent Stem Cells into Endothelial Cells under Xenogeneic-free Conditions for Vascular Tissue Engineering
Graphical abstract
Introduction
Readily available vascular grafts are urgently needed for treating acute vascular injuries. Native vessels and synthetic prosthesis are widely used as vascular grafts in the clinic, though their application can be significantly hindered by the restricted access to suitable native vessel segments and the risks of infection and thrombosis. Alternatively, tissue engineered vascular grafts (TEVGs) offer a non-synthetic approach for readily available vascular therapy. To develop TEVGs, primary or human induced pluripotent stem cell (hiPSC)-derived cells are cultured on a bioabsorbable scaffold within dynamic bioreactors to form vessel-like tissue engineered conduits [1,2]. TEVGs can be further decellularized as “off-the-shelf” acellular vascular grafts for emergency vascular intervention [1], [2], [3]. To date, acellular, large diameter (6 mm of inner diameter) TEVGs have led to promising results in phase 2 clinical trials for hemodialysis access [4]. However, for small diameter (< 6 mm) application, endothelial cells (ECs) are required to cover the luminal surface of acellular TEVGs to prevent blood clotting and thrombogenesis [1]. To endothelialize TEVGs using autologous primary ECs, an average of 23 days is required to complete the process from obtaining patient ECs to finalizing TEVG endothelization [1], which makes this approach for small diameter acellular TEVGs less practical for urgent interventions. Additionally, previous studies described that primary EC seeding onto the lumen of decellularized TEVGs resulted in an average percent coverage of 64% or lower with a wide variation [1,5], indicating that previously described methods of TEVG endothelialization may need further optimization for more effective therapeutic efficacy.
Fortunately, human induced pluripotent stem cell (hiPSC) technology may offer a unique opportunity to address the above issue. hiPSCs can be derived from somatic cells via the ectopic expression of essential stem cell transcription factors [6]. hiPSCs are self-renewable and can differentiate into various types of somatic cells including ECs (hiPSC-ECs), which make hiPSCs an unlimited reservoir for obtaining vascular cells. More importantly, by modulating the expression of human leukocyte antigens (HLAs), it is possible to establish minimal- or non-immunogenic “universal” hiPSCs [7], [8], [9], [10], which when differentiated into hiPSC-ECs could be immunocompatible to any patient. Further, these cells could be mass produced and cryopreserved as a robust cell source for TEVG endothelialization. Therefore, future application of hiPSC-ECs could eliminate the need for autologous primary ECs to endothelialize TEVGs, effectively expedite the endothelialization process, and dramatically enhance the readiness of TEVG therapy for emergency vascular treatments.
A growing body of research has described approaches to derive functional hiPSC-ECs [11], [12], [13], [14], [15], [16], [17], [18]. To meet clinical requirements, hiPSC-ECs should be generated under xenogeneic-free conditions, since the use of animal-derived reagents may cause xenogeneic immune responses in human recipients and transmit life-threatening zoonotic diseases [19]. Establishment and expansion of hiPSCs has been accomplished under xenogeneic-free conditions [20], while a completely xenogeneic-free approach for EC differentiation from hiPSCs has yet to be established (Table S1). It is worth noting that chemically-defined methods for generating ECs from human pluripotent stem cells have been reported [11,21]. However, reagents with animal-derived components (mouse tissue-derived Matrigel, mTeSR1 medium, StemPro-34 etc.) were commonly employed in these protocols [11,21]. Therefore, it is essential to establish a completely xenogeneic-free approach to obtain functional hiPSC-ECs for endothelializing vascular grafts for future therapeutic applications.
Herein, we aimed to establish a xenogeneic-free method to derive ECs from hiPSCs for acellular vascular graft endothelialization. Based on a standard xenogeneic protocol for obtaining functional hiPSC-ECs, we have replaced animal-derived reagents with their functional counterparts of human origin, including serum supplements, serum albumin, recombinant growth factors, and extracellular matrix proteins (Fig. 1A). By using our approach, we derived xenogeneic-free hiPSC-ECs (XF-hiPSC-ECs) readily responsive to shear stress and suitable for endothelializing decellularized human vessels. Our studies have established a robust approach to attain xenogeneic-free hiPSC-ECs suitable for vascular graft endothelialization, which sets the stage for future production of promptly available endothelialized vascular grafts for potential human clinical application.
Section snippets
Cultivation of human induced pluripotent stem cells (hiPSCs)
As previously described [22], unedited, wildtype human neonatal fibroblasts derived from a healthy female donor were reprogrammed into hiPSCs using non-integrative Sendai viral particles that encode the OCT4, KLF4, SOX2, and c-MYC genes. A human leukocyte antigen-C (HLA-C) retained (HLA-A-, HLA-B-, CIITA-knockout) hiPSC line (HLA-C-retained hiPSCs; named as 585A1 hiPSCs in previously published study) was derived from peripheral blood mononuclear cells isolated from a male donor via episomal
Endothelial differentiation of hiPSCs under xenogeneic-free conditions
A method for endothelial differentiation from hiPSCs using reagents derived from animals (e.g. mTeSR1 medium and Matrigel) has been previously established [11]. However, the inclusion of animal-derived reagents during EC derivation may trigger xenogeneic immune responses and convey severe zoonotic diseases [19], thereby hampering the potential therapeutic application of hiPSC-ECs. To obtain hiPSC-ECs under xenogeneic-free conditions (XF-hiPSC-ECs), we significantly modified the previous
Discussion
In this study, we established a xenogeneic-free method based on the culture conditions including growth factors and small molecules in a previous xenogeneic hiPSC-EC differentiation approach, and derived functional XF-hiPSC-ECs suitable for endothelializing vascular grafts [11]. We have replaced all the materials of animal origin in the original protocol with new reagents of human origin, and hiPSC-ECs derived under such xenogeneic-free conditions presented typical EC marker expression
Conclusion
To our knowledge, we have established the first xenogeneic-free method for deriving functional hiPSC-ECs suitable for endothelializing decellularized human vessels. By substantially modifying a standard xenogeneic hiPSC-EC derivation approach and replacing all animal-derived materials with new reagents of human origin, we generated XF-hiPSC-ECs with effective endothelial marker expression and function comparable to those of human primary ECs and XG-hiPSC-ECs. These XF-hiPSC-ECs were readily
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank the members of Qyang group for their feedback for this research. We thank the supports from DOD W81XWH1910557, R01HL116705, and R01HL150352 (all to YQ), The American Heart Association (AHA) Postdoctoral Fellowships 19POST34450100 (to JL), 20POST35210709 (to YY), and 19POST34381048 (to MHK), China Scholarships Council 201706370156 (to XS) and 201806280209 (to YL), Ruth L. Kirschstein Predoctoral Individual National Research Service Award NIH 5F31HL143928-03 (to CWA) and NIH Grants
References (36)
- et al.
Bioengineered human acellular vessels for dialysis access in patients with end-stage renal disease: two phase 2 single-arm trials
Lancet
(2016) - et al.
Induction of pluripotent stem cells from adult human fibroblasts by defined factors
Cell
(2007) - et al.
Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling
Stem Cell Reports
(2014) - et al.
Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor
Biomaterials
(2015) - et al.
Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells
Cell Stem Cell
(2014) - et al.
Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells
Stem Cell Res
(2015) - et al.
Tissue-Engineered Vascular Rings from Human iPSC-Derived Smooth Muscle Cells
Stem Cell Reports
(2016) - et al.
Vascular smooth muscle cells derived from inbred swine induced pluripotent stem cells for vascular tissue engineering
Biomaterials
(2017) - et al.
Modular design of a tissue engineered pulsatile conduit using human induced pluripotent stem cell-derived cardiomyocytes
Acta Biomater
(2020) - et al.
Endothelial Basement Membrane Laminin 511 Contributes to Endothelial Junctional Tightness and Thereby Inhibits Leukocyte Transmigration
Cell Rep
(2017)
Substrate stiffness regulates arterial-venous differentiation of endothelial progenitor cells via the Ras/Mek pathway
Biochim Biophys Acta Mol Cell Res
Allogeneic human tissue-engineered blood vessel
J Vasc Surg
Fas ligand and nitric oxide combination to control smooth muscle growth while sparing endothelium
Biomaterials
cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications
Stem Cell Reports
Readily available tissue-engineered vascular grafts
Sci Transl Med
A completely biological "off-the-shelf" arteriovenous graft that recellularizes in baboons
Sci Transl Med
Tissue-Engineered Vascular Grafts with Advanced Mechanical Strength from Human iPSCs
Cell Stem Cell
Decellularized tissue-engineered blood vessel as an arterial conduit
Proc Natl Acad Sci U S A
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Contributed equally to this paper.