Whole organ perfusion decellularization has been proposed as a promising method to generate non-immunogenic organs from allogeneic and xenogeneic donors. However, the ability to recellularize organ scaffolds with multiple patient-specific cells in a spatially controlled manner remains challenging. Here, we propose that replacing donor endothelial cells alone, while keeping the rest of the organ viable and functional, is more technically feasible, and may offer a significant shortcut in the efforts to engineer transplantable organs. Vascular decellularization was achieved ex vivo, under controlled machine perfusion conditions, in various rat and porcine organs, including the kidneys, liver, lungs, heart, aorta, hind limbs, and pancreas. In addition, vascular decellularization of selected organs was performed in situ, within the donor body, achieving better control over the perfusion process. Human placenta-derived endothelial progenitor cells (EPCs) were used as immunologically-acceptable human cells to repopulate the luminal surface of de-endothelialized aorta (in vitro), kidneys, lungs and hind limbs (ex vivo). This study provides evidence that artificially generating vascular chimerism is feasible and could potentially pave the way for crossing the immunological barrier to xenotransplantation, as well as reducing the immunological burden of allogeneic grafts.

Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine‐kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second‐line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib‐resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.

Perfusion decellularization has been proposed as a promising method for generating nonimmunogenic organs from allogeneic or xenogeneic donors. Several imaging modalities have been used to assess vascular integrity in bioengineered organs with no consistency in the methodology used. Here, we studied the use of fluoroscopic angiography performed under controlled flow conditions for vascular integrity assessment in bioengineered kidneys. Porcine kidneys underwent ex vivo angiography before and after perfusion decellularization. Arterial and venous patencies were defined as visualization of contrast medium (CM) in distal capillaries and renal vein, respectively. Changes in vascular permeability were visualized and quantified. No differences in patency were detected in decellularized kidneys compared with native kidneys. However, focal parenchymal opacities and significant delay in CM clearance were detected in decellularized kidneys, indicating increased permeability. Biopsy‐induced leakage was visualized in both groups, with digital subtraction angiography revealing minimal CM leakage earlier than nonsubtracted fluoroscopy. In summary, quantitative assessment of vascular permeability should be coupled with patency when studying the effect of perfusion decellularization on kidney vasculature. Flow‐controlled angiography should be considered as the method of choice for vascular assessment in bioengineered kidneys. Adopting this methodology for organs premodified ex vivo under normothermic machine perfusion settings is also suggested.

IntroductionExtracorporeal photopheresis (ECP) is considered an effective treatment for patients with chronic graft vs host disease (cGVHD) and demonstrates efficacy in ameliorating GVHD. The mechanism by which ECP acts against cGVHD is not fully understood. Preliminary observations have hinted at the potential involvement of neutrophil extracellular traps (NETs) formation in the pathogenesis of cGVHD. We aimed to assess the influence of ECP on the formation of NETs in patients with cGVHD as a potential mechanism in this setting.MethodsPatients treated with ECP for cGVHD at the Rabin Medical Center were included in this study. Blood samples were obtained at three different time points: before starting an ECP cycle, at the end of the first day of treatment, and 24 h following the initiation of the ECP treatment cycle. Neutrophils were harvested from all blood samples. NET formation was assessed by measurement of NET‐bound specific neutrophil elastase activity and by immunofluorescence staining.ResultsSix patients (two females and four males) with cGVHD were included in the study. We observed a significant increase in NET formation among all six patients following ECP. Net‐bound specific neutrophil elastase activity was elevated from a median value of 2.23 mU/mL (interquartile range [IQR] 2.06‐2.47 mU/mL) at baseline to a median value of 13.06 mU/mL (IQR 10.27‐15.97 mU/mL) immediately after the treatment and to a peak median value of 14.73 mU/mL (IQR 9.6‐22.38 mU/mL) 24 h following the initiation of the ECP cycle. A qualitative assessment of NET formation using immunofluorescence staining has demonstrated markedly increased expression of citrullinated histone H3, a marker of NET formation, following ECP treatment.ConclusionsOur preliminary data indicate that ECP induces NET formation among patients with cGVHD. The contribution of increased NET formation to the therapeutic effect of cGVHD should be further investigated.

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