Towards Biohybrid Lung: Induced Pluripotent Stem Cell Derived Endothelial Cells as Clinically Relevant Cell Source for Biologization

Abstract

In order to provide an alternative treatment option to lung transplantation for patients with end-stage lung disease, we aim for the development of an implantable biohybrid lung (BHL), based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. Complete hemocompatibility of all blood contacting surfaces is crucial for long-lasting BHL durability and can be achieved by their endothelialization. Autologous endothelial cells (ECs) would be the ideal cell source, but their limited proliferation potential excludes them for this purpose. As induced pluripotent stem cell-derived ECs enable the generation of a large number of ECs, we assessed and compared their capacity to form a viable and confluent monolayer on HFM, while indicating physiologic EC-specific anti-thrombogenic and anti-inflammatory properties. ECs were generated from three different human iPSC lines, and seeded onto fibronectin-coated poly-4-methyl-1-pentene (PMP) HFM. Following phenotypical characterization, ECs were analyzed for their thrombogenic and inflammatory behavior with or without TNFα induction, using FACS and qRT-PCR. Complementary, leukocyte- and platelet adhesion assays were carried out. The capacity of the iPSC-ECs to reendothelialize cell-free monolayer areas was assessed in a scratch assay. ECs sourced from umbilical cord blood (hCBECs) were used as control. iPSC-derived ECs formed confluent monolayers on the HFM and showed the typical EC-phenotype by expression of VE-cadherin and collagen-IV. A low protein and gene expression level of E-selectin and tissue factor was detected for all iPSC-ECs and the hCBECs, while a strong upregulation of these markers was noted upon stimulation with TNFα. This was in line with the physiological and strong induction of leukocyte adhesion detected after treatment with TNFα, iPSC-EC and hCBEC monolayers were capable of reducing thrombocyte adhesion and repopulating scratched areas. iPSCs offer the possibility to provide patient-specific ECs in abundant numbers needed to cover all blood contacting surfaces of the BHL with a viable, non-thrombogenic and non-inflammatory monolayer. iPSC-EC clones can differ in terms of their reendothelialization rate, and pro-inflammatory response. However, a less profound inflammatory response may even be advantageous for BHL application. With the proven ability of the seeded iPSC-ECs to reduce thrombocyte adhesion, we expect that thrombotic events that could lead to BHL occlusion can be avoided, and thus, justifies further studies on enabling BHL long-term application.

Towards Biohybrid Lung Development—Fibronectin-Coating Bestows Hemocompatibility of Gas Exchange Hollow Fiber Membranes by Improving Flow-Resistant Endothelialization

“…Further, endothelial proteins that reside on the endothelial cells (EC) will actively control hemostasis for optimized hemocompatibility [14]. For this approach, MHC silenced human cord blood derived ECs [15] and ECs derived from induced pluripotent stem cells (iPSC-ECs) [16] were identified as clinically relevant cell sources, which can be gained in sufficient numbers, while being immunologically invisible. Additional key findings within BHL development were the identification of potential surface coatings, enabling EC adhesion to the otherwise hydrophobic artificial material [17].…”

Towards Biohybrid Lung Development: Establishment of a Porcine In Vitro Model

“…Moreover, different studies demonstrated that the neo-endothelium, established on the HFM, remains viable, anti-thrombogenic, non-inflammatory and resilient under clinically relevant flow conditions that were simulated with endothelial cell culture medium even in long-term evaluation [ 14 , 17 ]. Furthermore, we identified two potential candidates for EC sources that could be safely applied for future clinical use of the BHL without the risk of acute rejection, i.e., ECs derived from induced pluripotent stem cells [ 21 ] and MHC-Class-I silenced human ECs [ 22 ].…”

“…Heparin and Albumin (H/A) coated and 1.8 × 3.2 cm (40 parallel aligned hollow fibers) sized patches were trimmed from the original HFM of the iLA membrane ventilator (Xenios, Heilbronn, Germany). After sterilization with ethylene oxide, the patches were endothelialized as described previously [ 21 ]. In brief, HFM patches were sandwiched between two custom made polycarbonate frames and three patches each were placed into a 50 mL syringe (B. Braun, Melsungen, Germany), which was filled with 23 mL EC suspension (9 × 10 5 ECs/mL in EGM2).…”

“…Hereinafter, the viable, flow-resistant, anti-inflammatory and anti-thrombogenic endothelial monolayer could be maintained for several weeks [ 19 ] without any significant gas exchange impairment [ 20 ]. Focusing on appropriate cell sources, induced pluripotent stem cell-derived ECs [ 21 ] and MHC-silenced ECs were used for PMP-HFM endothelialization, preventing allogenic cell rejection [ 22 ]. Finally, for clinically highly relevant “off-the-shelf” use of the BHL, hypothermic storability was shown [ 23 ].…”

Trends, Advantages and Disadvantages in Combined Extracorporeal Lung and Kidney Support From a Technical Point of View

“…Therefore, it is primordial that future advances in the research field focus on the development of better anticoagulant molecules and ECLS circuits that better mimic the physiologic conditions for extended ECLS use (79). Moreover, research in new ECLS bio-hybrid materials with the use of an endothelization technique is an important step toward the creation of complete biocompatible materials (79,(82)(83)(84)(85)(86).…”