Efficient Differentiation of Human Induced Pluripotent Stem Cells into Endothelial Cells under Xenogeneic-free Conditions for Vascular Tissue Engineering

Abstract

Tissue engineered vascular grafts (TEVGs) represent a promising therapeutic option for emergency vascular intervention. Although the application of small-diameter TEVGs using patient-specific primary endothelial cells (ECs) to prevent thrombosis and occlusion prior to implantation could be hindered by the long time course required for in vitro endothelialization, human induced pluripotent stem cells (hiPSCs) provide a robust source to derive immunocompatible ECs (hiPSC-ECs) for immediate TEVG endothelialization. To achieve clinical application, hiPSC-ECs should be derived under culture conditions without the use of animal-derived reagents (xenogeneic-free conditions), to avoid unwanted host immune responses from xenogeneic reagents. However, a completely xenogeneic-free method of hiPSC-EC generation has not previously been established. Herein, we substituted animal-derived reagents used in a standard method of xenogeneic hiPSC-EC differentiation with functional counterparts of human origin. As a result, we generated xenogeneic-free hiPSC-ECs (XF-hiPSC-ECs) with similar marker expression and function to those of human primary ECs. Furthermore, XF-hiPSC-ECs functionally responded to shear stress with typical cell alignment and gene expression. Finally, we successfully endothelialized decellularized human vessels with XF-hiPSC-ECs in a dynamic bioreactor system. In conclusion, we developed xenogeneic-free conditions for generating functional hiPSC-ECs suitable for vascular tissue engineering, which will further move TEVG therapy toward clinical application.

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.