Elsevier

Biomaterials

Volume 35, Issue 28, September 2014, Pages 8123-8133
Biomaterials

Prevention of rejection of allogeneic endothelial cells in a biohybrid lung by silencing HLA-class I expression

https://doi.org/10.1016/j.biomaterials.2014.06.007Get rights and content

Abstract

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality.

Introduction

According to the World Health Organization, lung diseases rank as the 4th leading cause of death worldwide with an increasing prevalence and incidence [1]. Currently, no long-term assist device exists for the treatment of end-stage lung disease, hence lung transplantation is the only therapy option for patients with pre-terminal lung disease. However, the number of registrations on the organ waiting list by far exceeds the number of organ donors. As a result of this discrepancy, at least 20–40% of those patients will die before a suitable donor lung is available [2]. Furthermore, only 20–30% of the available donor lungs can be accepted for transplantation [3] and cold preservation time is limited to 6–8 h [4], during which 20% of the transplanted lungs develop life-threatening lung failure caused by the ischemic-reperfusion injury (IRI). Finally, the five-year survival rate after lung transplantation is only ∼56% [5].

Patients with acute respiratory failure receive artificial ventilation, but this may cause severe ventilation-associated lung injury [6], [7]. If this artificial ventilation becomes insufficient, an organ supporting system, the extracorporeal membrane oxygenation (ECMO), can be used to ensure gas exchange. However, the clinical application of ECMO systems is limited by adverse reactions, such as thrombus formation and sepsis, based on the inevitable contact between the circulating blood and the artificial surfaces [8]. With the goal of developing a biohybrid lung as a potential alternative to lung transplantation and as a long-term assist device, the physiological blood compatibility of endothelial cells (EC) was harnessed [9] to coat poly-4-methyl-1-pentene (PMP) gas exchange membranes, showing the general feasibility to endothelialize these artificial PMP, leading to an improved hemocompatibility without a loss of gas exchange capabilities [10].

One of the next challenging experimental steps is the identification of a suitable cell source for the endothelialization of these artificial PMP membranes to achieve the development of a fully implantable biohybrid lung as a clinically viable option. Autologous EC would be the ideal cell source for this purpose, but the surface area of the gas exchange membranes of lung-supporting devices is extremely large, approximately 1.3–2.5 m2. This corresponds to about 1–2 billion EC which would be required for the complete endothelialization of the gas exchange membrane. As the patients requiring biohybrid lungs are mostly older than 60 and often have several co-morbidities, it is unlikely to harvest sufficient amounts of cells to meet this need. Furthermore, it remains unclear whether EC isolated from autologous vessels and/or peripheral blood can be enriched from a single donor in the high numbers required for the endothelialization of PMP membranes. The major problem of using allogeneic EC as an alternative cell source for this approach is the high variability of the human leukocyte antigens (HLA) and the minor histocompatibility antigens (mHA). Discrepancies at the HLA loci among EC donor and recipient may trigger an allogeneic immune response and cause cell rejection [11], [12]. Previously, we have developed a RNA interference (RNAi)-based approach to stably silence the expression of HLA proteins. This strategy demonstrated a significant decrease in cell immunogenicity [13], [14], [15]. The great advantage of this approach is that there is no need for patient-specific EC. This method not only silences the expression of HLA molecules on the cell surface but also contributes to abrogate the presentation of mHA derived from polymorphic proteins.

HLA-silenced fibroblasts and platelets were shown to be capable of surviving in an allogeneic environment after transplantation by escaping allogeneic cellular and antibody-mediated immune responses. Hence, this study investigated the feasibility of using HLA class I silenced human cord blood EC (HCBECs) for the allogeneic endothelialization of PMP membranes and thus supporting the development of a biohybrid lung. The combination of both technologies (HLA silencing and bioartificial organs) may represent a new therapeutic concept in the field of cell and organ transplantation and may bring an alternative to lung transplantation for patients with end-stage lung diseases closer to reality.

Section snippets

Isolation and cell culture of human cord blood derived endothelial cells (HCBEC)

Heparinized human umbilical cord blood, obtained from healthy newborn donors after informed parental consent, was diluted 1:1 with phosphate-buffered saline [PBS; containing 2 mm ethylenediaminetetraacetic acid (EDTA)] and subjected to density gradient centrifugation [Biocoll, Biochrome AG, Germany] at 800 × g for 20 min (wo break) to isolate mononuclear cells (MNC). MNC were resuspended in endothelial growth medium [EGM; endothelial cell basal medium 2 (EBM-2); Lonza, Belgium] with 10% fetal

Silencing HLA class I expression in ECs

Previously, we have demonstrated the feasibility to permanently silence HLA class I expression in several cell types [13], [14], [16]. We have demonstrated that HLA-silenced cells effectively escape the allogeneic immune response in vitro and in vivo [15], [17]. In this study we have silenced the expression of HLA class I expression in HCBECs. Transduction efficiencies of 82%–97% were obtained in all assays (n = 10). HCBECs transduced with a vector encoding for a non-specific shRNA (shNS)

Discussion

Biohybrid organs may represent the only alternative therapeutic option for patients with pre-terminal organ disease who have lower chances of receiving an organ for transplantation. In this regard, biohybrid lungs may play a crucial role in the management of patients with pre-terminal lung disease. In the future, biohybrid lungs may constitute an efficient bridging, as well as destination therapy to circumvent the enormous discrepancy between the high number of patients waiting for a lung

Conclusion

This study shows the feasibility of silencing HLA class I expression in ECs. HLA-silenced ECs show a typical endothelial cell phenotype and are able to respond to external stimuli. Most importantly, HLA-silenced ECs are able to escape an allogeneic cellular and antibody-mediated response permitting their application in an universal manner, independently of the genetic background of the EC donor and recipient. Hence, HLA-silenced ECs may strongly contribute to the development of the biohybrid

Acknowledgments

We are grateful to Stefanie Vahlsing and Julia Janke for the excellent technical support. In addition, we are thankful to Nina McGuinness for proofreading the manuscript. This work was partly supported by funding by the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy EXC 62/1) (Unit 4.1 and 6.3), as well as by the German Center for Lung Research (DZL) (DZL: 82DZL00201), Lower Saxony Center for Biomedical Technology (NIFE) and German Research Foundation (WI

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