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.
Highlights
According to the World Health Organization, end-stage lung diseases (ELD) are one of the leading causes of death worldwide with an increasing prevalence and incidence.More than 210 million patients already suffer from chronic obstructive pulmonary disease, of which about 3 million patients die per year [1], while reliable and long-lasting therapy options for ELD are limited
We investigated whether induced pluripotent stem cells (iPSCs)-endothelial cells (ECs) in cultures on the fibronectin-coated PMP membrane are present in their non-activated state, and whether they are able to show a physiologic pro-coagulative, pro-thrombogenic and pro-inflammatory response, comparable to the control cells
For these experiments we used flat fibronectin-coated PMP membranes made of the same material as the hollow fiber membranes (HFM), which are used in membrane oxygenators
Summary
More than 210 million patients already suffer from chronic obstructive pulmonary disease, of which about 3 million patients die per year [1], while reliable and long-lasting therapy options for ELD are limited. Lung transplantation (LTx) as the only current curative therapy option can only be offered to a few patients due to the increasing mismatch between potential organ donors to organ recipients. We aim for the development of the implantable biohybrid lung (BHL) as alternative to LTx and as final destination therapy, based on the technology of extracorporeal membrane oxygenation (ECMO). In order to achieve this, ECMO is made up of a multitude of gas exchange hollow fiber membranes (HFM), in which an oxygen-rich gas atmosphere is supplied while on the outside the blood passes by for the gas exchange via the partial pressure gradient
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