Segmentation of human functional tissue units in support of a Human Reference Atlas.

The Human BioMolecular Atlas Program (HuBMAP) aims to compile a Human Reference Atlas (HRA) for the healthy adult body at the cellular level. Functional tissue units (FTUs), relevant for HRA construction, are of pathobiological significance. Manual segmentation of FTUs does not scale; highly accurate and performant, open-source machine-learning algorithms are needed. We designed and hosted a Kaggle competition that focused on development of such algorithms and 1200 teams from 60 countries participated. We present the competition outcomes and an expanded analysis of the winning algorithms on additional kidney and colon tissue data, and conduct a pilot study to understand spatial location and density of FTUs across the kidney. The top algorithm from the competition, Tom, outperforms other algorithms in the expanded study, while using fewer computational resources. Tom was added to the HuBMAP infrastructure to run kidney FTU segmentation at scale-showcasing the value of Kaggle competitions for advancing research.

Organization of the human intestine at single-cell resolution.

The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health1. The intesting has a length of over nine metres, along which there are differences in structure and function2. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.

Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP).

The Human BioMolecular Atlas Program (HuBMAP) aims to create a multi-scale spatial atlas of the healthy human body at single-cell resolution by applying advanced technologies and disseminating resources to the community. As the HuBMAP moves past its first phase, creating ontologies, protocols and pipelines, this Perspective introduces the production phase: the generation of reference spatial maps of functional tissue units across many organs from diverse populations and the creation of mapping tools and infrastructure to advance biomedical research.

Robotic donor nephrectomy: optimizing outcomes beyond the limitations of laparoscopy.

BACKGROUND: Robotic donor nephrectomy (RDN) has emerged as a safe alternate to laparoscopic donor nephrectomy (LDN), offering improved visualization, instrument dexterity and ergonomics. There is still concern about how to safely transition from LDN to RDN. METHODS: We performed a retrospective review of 150 consecutive living donor operations (75 LDN and 75 RDN) at our center, comparing the first 75 RDN's with the last 75 LDN's performed prior to the initiation of the robotic transplant program. Operative times and complications were used as surrogates of efficiency and safety, respectively, to estimate the learning curve with RDN. RESULTS: RDN was associated with a longer total operative time (RDN 182 vs LDN 144 min; P < 0.0001) but a significantly shorter post-operative length of stay (RDN 1.8 vs LDN 2.1 days; P = 0.0213). Donor complications and recipient outcomes were the same between both groups. Learning curve of RDN was estimated to be about 30 cases. CONCLUSIONS: RDN is a safe alternate to LDN with acceptable donor morbidity and no negative impact on recipient outcomes even during the early part of the RDN learning curve. Surgeon preferences for the robotic approach compared to traditional laparoscopy will require further scrutiny to improve ergonomics and operative efficiency.

Circulating exosomes from brain death and cardiac death donors have distinct molecular and immunologic properties: A pilot study.

BACKGROUND AND AIMS: Comparison of donation after brain death (DBD) and donation after cardiac death (DCD) lung tissue before transplantation have demonstrated activation of pro-inflammatory cytokine pathway in DBD donors. The molecular and immunological properties of circulating exosomes from DBD and DCD donors were not previously described. METHODS: We collected plasma from 18 deceased donors (12 DBD and six DCD). Cytokines were analyzed by 30-Plex luminex Panels. Exosomes were analyzed for liver self-antigen (SAg), Transcription Factors and HLA class II (HLA-DR/DQ) using western blot. C57BL/6 animals were immunized with isolated exosomes to determine strength and magnitude of immune responses. Interferon (IFN)-γ and tumor necrosis factor-α producing cells were quantified by ELISPOT, specific antibodies to HLA class II antigens were measured by ELISA RESULTS: We demonstrate increased plasma levels of IFNγ, EGF, EOTAXIN, IP-10, MCP-1, RANTES, MIP-ß, VEGF, and interleukins - 6/8 in DBD plasma versus DCD. MiRNA isolated from exosome of DBD donors demonstrated significant increase in miR-421, which has been reported to correlate with higher level of Interleukin-6. Higher levels of liver SAg Collagen III (p = .008), pro-inflammatory transcription factors (NF-κB, p < .05; HIF1α, p = .021), CIITA (p = .011), and HLA class II (HLA-DR, p = .0003 and HLA-DQ, p = .013) were detected in exosomes from DBD versus DCD plasma. The circulating exosomes isolated from DBD donors were immunogenic in mice and led to the development of Abs to HLA-DR/DQ. CONCLUSIONS: This study provides potential new mechanisms by which DBD organs release exosomes that can activate immune pathways leading to cytokine release and allo-immune response.

Integrated analysis of genomic and transcriptomic data for the discovery of splice-associated variants in cancer.

Somatic mutations within non-coding regions and even exons may have unidentified regulatory consequences that are often overlooked in analysis workflows. Here we present RegTools ( www.regtools.org ), a computationally efficient, free, and open-source software package designed to integrate somatic variants from genomic data with splice junctions from bulk or single cell transcriptomic data to identify variants that may cause aberrant splicing. We apply RegTools to over 9000 tumor samples with both tumor DNA and RNA sequence data. RegTools discovers 235,778 events where a splice-associated variant significantly increases the splicing of a particular junction, across 158,200 unique variants and 131,212 unique junctions. To characterize these somatic variants and their associated splice isoforms, we annotate them with the Variant Effect Predictor, SpliceAI, and Genotype-Tissue Expression junction counts and compare our results to other tools that integrate genomic and transcriptomic data. While many events are corroborated by the aforementioned tools, the flexibility of RegTools also allows us to identify splice-associated variants in known cancer drivers, such as TP53, CDKN2A, and B2M, and other genes.

COVID-19 Vaccination Status and Operative Outcomes after Kidney Transplantation.

BACKGROUND: Kidney transplantation remains the best available treatment for end-stage renal disease. However, promoting graft longevity and preventing allosensitization requires strict adherence with a stringent immunosuppression regimen. The COVID-19 pandemic has offered new challenges for kidney transplant patients and many transplant centers are denying transplantation to unvaccinated patients. The aim of this study was to evaluate whether unvaccinated patients had inferior adherence after kidney transplantation along with a reduction in graft survival. STUDY DESIGN: Patients undergoing a deceased donor kidney transplantation at a single academic medical center from February 2021 to May 2022 were retrospectively reviewed. February 2021 was chosen as the start date for record review because it was 3 months after the first COVID-19 vaccination was authorized for emergency use. Patients were considered to be vaccinated if they received at least 1 dose of any mRNA vaccine by their transplantation date. RESULTS: Of the 301 patients who met study criteria, 234 were vaccinated and 67 were unvaccinated. Cohorts stratified by vaccination status were well matched. Younger age was an independent risk factor for nonvaccination. Interestingly, unvaccinated patients had worse postoperative adherence with a greater average number of missed postoperative clinic visits (p = 0.03) and a strong trend toward missing 3 or more postoperative clinic visits (p = 0.07). Finally, unvaccinated patients had statistically more subtherapeutic tacrolimus troughs (p = 0.01). CONCLUSIONS: Patients not vaccinated against COVID-19 had higher rate of postoperative nonadherence in key areas of immunosuppression monitoring and clinic visit attendance. Providers should be cognizant that an unvaccinated status may be a harbinger for poor adherence; therefore, stricter strategies for patient outreach are critical to ensure graft success in this vulnerable patient population.

Mitochondrial Autophagy in Ischemic Aged Livers.

Mitochondrial autophagy (mitophagy) is a central catabolic event for mitochondrial quality control. Defective or insufficient mitophagy, thus, can result in mitochondrial dysfunction, and ultimately cell death. There is a strong causal relationship between ischemia/reperfusion (I/R) injury and mitochondrial dysfunction following liver resection and transplantation. Compared to young patients, elderly patients poorly tolerate I/R injury. Accumulation of abnormal mitochondria after I/R is more prominent in aged livers than in young counterparts. This review highlights how altered autophagy is mechanistically involved in age-dependent hypersensitivity to reperfusion injury.

Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.

Improving Liver Graft Function Using CD47 Blockade in the Setting of Normothermic Machine Perfusion.

BACKGROUND: Toward the goal of using more livers for transplantation, transplant centers are looking to increase the use of organs from "marginal" donors. Livers from these donors, however, have been shown to be more susceptible to preservation and reperfusion injury. METHODS: Using a porcine model of donation after circulatory death, we studied the use of antibody-mediated CD47 blockade to further improve liver graft function undergoing normothermic machine perfusion. Livers from 20 pigs (5 per group) were brought under either 30 or 60 min of warm ischemia time followed by the administration of CD47 monoclonal antibody (CD47mAb) treatment or immunoglobulin G control antibodies and 6 h of normothermic extracorporeal liver perfusion. RESULTS: After 6 h of normothermic extracorporeal liver perfusion, CD47mAb-treated livers with 30 or 60 min warm ischemia time had significantly lower alanine transaminase levels and higher bile production compared with their respective control groups. Blockade of the CD47 signaling pathway resulted in significantly lower thrombospondin-1 protein levels, lower expression of caspase-3, and higher expression of phosphorylated extracellular signal-regulated kinase. CONCLUSIONS: These findings suggested that CD47mAb treatment decreases ischemia/reperfusion injury through CD47/thrombospondin-1 signaling downregulation and the presence of necrosis/apoptosis after reperfusion and could increase liver regeneration during normothermic perfusion of the liver.

NRF2 assessment in discarded liver allografts: A role in allograft function and salvage.

Antioxidant defence mechanisms, such as the nuclear factor-erythroid 2-related-factor-2 (NRF2) axis, are integral to oxidative stress responses and ischemic injury. Hepatic antioxidant capacity is contingent on parenchymal quality, and there is a need to develop new insights into key molecular mechanisms in marginal liver allografts that might provide therapeutic targets. This study examines the clinical relevance of NRF2 in donor livers and its response to normothermic machine perfusion (NMP). Discarded donor livers (n = 40) were stratified into a high NRF2 and low NRF2 group by quantifying NRF2 expression. High NRF2 livers had significantly lower transaminase levels, hepatic vascular inflammation and peri-portal CD3+ T cell infiltration. Human liver allografts (n = 8) were then exposed to 6-h of NMP and high NRF2 livers had significantly reduced liver enzyme alterations and improved lactate clearance. To investigate these findings further, we used a rat fatty-liver model, treating livers with an NRF2 agonist during NMP. Treated livers had increased NRF2 expression and reduced transaminase derangements following NMP compared to vehicle control. These results support the association of elevated NRF2 expression with improved liver function. Targeting this axis could have a rationale in future studies and NRF2 agonists may represent a supplemental treatment strategy for rescuing marginal donor livers.

Anatomical structures, cell types and biomarkers of the Human Reference Atlas.

The Human Reference Atlas (HRA) aims to map all of the cells of the human body to advance biomedical research and clinical practice. This Perspective presents collaborative work by members of 16 international consortia on two essential and interlinked parts of the HRA: (1) three-dimensional representations of anatomy that are linked to (2) tables that name and interlink major anatomical structures, cell types, plus biomarkers (ASCT+B). We discuss four examples that demonstrate the practical utility of the HRA.

Dual Lactate Clearance in the Viability Assessment of Livers Donated After Circulatory Death With Ex Situ Normothermic Machine Perfusion.

Perfusate lactate clearance (LC) is considered one of the useful indicators of liver viability assessment during normothermic machine perfusion (NMP); however, the applicable scope and potential mechanisms of LC remain poorly defined in the setting of liver donation after circulatory death. METHODS: The ex situ NMP of end-ischemic human livers was performed using the OrganOx Metra device. We further studied the extracellular signal-regulated kinases (phospho-extracellular signal-regulated kinase1/2 [pERK1/2]) pathway and several clinical parameters of these livers with successful LC (sLC, n = 5) compared with non-sLC (nLC, n = 5) in the perfusate (<2.2 mmol/L at 2 h, n = 5, rapid retrieval without normothermic regional perfusion). RESULTS: We found the pERK1/2 level was substantially higher in the nLC livers than in the sLC livers (n = 5) at 2- and 6-h NMP (P = 0.035 and P = 0.006, respectively). Immunostaining showed that upregulation of pERK1/2 was in both the hepatocytes and cholangiocytes in the nLC livers. Successful LC was associated with a marginally higher glycogen restoration than nLC at 2 h NMP (n = 5, P = 0.065). Furthermore, bile lactate levels in all sLC livers were cleared into the normal range at 6 h NMP, whereas in the nLC group, only 2 livers had lower bile lactate levels, and the other livers had rising bile lactate levels in comparison with the corresponding perfusate lactate levels. The necrosis scores were higher in the nLC than in the sLC livers (n = 5) at 0- and 6-h NMP (P = 0.047 and P = 0.053, respectively). CONCLUSIONS: The dual LC in perfusate and bile can be helpful in evaluating the hypoxic injury of hepatocytes and cholangiocytes during the NMP of donation after circulatory death in liver donors.

A Novel Multidrug Combination Mitigates Rat Liver Steatosis Through Activating AMPK Pathway During Normothermic Machine Perfusion.

BACKGROUND: Hepatic steatosis is now the leading cause of liver discards in deceased donors. Previous studies [Yarmush formula (Y) defatting] have successfully reduced the fat content by treating rat steatotic livers on extracorporeal normothermic machine perfusion (NMP) with a multidrug combination including the GW compounds that were linked to an increased risk of carcinogenesis. METHODS: We developed a novel multidrug combination by replacing the GW compounds with 2 polyphenols, epigallocatechin-3-gallate (E) and resveratrol (R). Sixteen rat livers were placed on NMP and assigned to control, Y defatting, Y + E + R defatting, or Y'-GW + E + R defatting groups (Y'-GW = 90% dose-reduced Y defatting, n = 4/group). RESULTS: All livers in defatting groups had significant decreases in hepatic triglyceride content at the end of the experiment. However, livers treated with our novel Y'-GW + E + R combination had evidence of increased metabolism and less hepatocyte damage and carcinogenic potential. Our Y'-GW + E + R combination had increased phosphorylation of AMP-activated protein kinase (P = 0.019) and acetyl-CoA carboxylase (P = 0.023) compared with control; these were not increased in Y + E + R group and actually decreased in the Y group. Furthermore, the Y'-GW + E + R group had less evidence of carcinogenic potential with no increase in AKT phosphorylation compared with control (P = 0.089); the Y (P = 0.031) and Y + E + R (P = 0.035) groups had striking increases in AKT phosphorylation. Finally, our Y'-GW + E + R showed less evidence of hepatocyte damage with significantly lower perfusate alanine aminotransferase (P = 0.007) and aspartate aminotransferase (P = 0.014) levels. CONCLUSIONS: We have developed a novel multidrug combination demonstrating promising defatting efficacy via activation of the AMP-activated protein kinase pathway with an optimized safety profile and reduced hepatotoxicity during ex vivo NMP.

Organ Procurement Organization Run Department of Motor Vehicle Registration and Drivers Licensing Offices Leads to Increased Organ Donor First Person Authorization Registrations.

BACKGROUND: More people who have personally consented to organ donation via first person authorization (FPA) registration before death become organ donors than those not personally consenting. The majority of registrations occur at state-specific department of motor vehicle (DMV) and licensing offices, where people register their vehicles and obtain driver's licenses. METHODS: One organ procurement organization (OPO) ran 3 DMV offices and implemented an intervention: a donor-centric approach, including employee education, office decoration with donation materials, and customer experience improvements. Data about registry enrollment was collected before and during the 4-year OPO licensing office contract. A linear mixed model and interrupted time series analyses were performed to evaluate whether the intervention improved rates of registration. RESULTS: Preintervention registry enrollment rates per month were 10%-50%. Having the offices run by an OPO was associated with more enrollments independent of the increasing trend of enrollment (P < 0.001). Also, the DMV office with the lowest preimplementation registration rates had an immediate increase in enrollments after the intervention leading to higher registration rates (P < 0.001). CONCLUSIONS: A donor-centric OPO-managed DMV experience increases FPA registration, especially at offices with low initial registration rates. However, even at the office with the highest percentage of FPA registrations, rates were only 65% at intervention conclusion. The transplant community should consider other opportunities for FPA registration.

Enhanced immunosuppression improves early allograft function in a porcine kidney transplant model of donation after circulatory death.

It remains controversial whether renal allografts from donation after circulatory death (DCD) have a higher risk of acute rejection (AR). In the porcine large animal kidney transplant model, we investigated the AR and function of DCD renal allografts compared to the non-DCD renal allografts and the effects of increased immunosuppression. We found that the AR was significantly increased along with elevated MHC-I expression in the DCD transplants receiving low-dose immunosuppression; however, AR and renal function were significantly improved when given high-dose immunosuppressive therapy postoperatively. Also, high-dose immunosuppression remarkably decreased the mRNA levels of ifn-g, il-6, tgf-b, il-4, and tnf-a in the allograft at day 5 and decreased serum cytokines levels of IFN-g and IL-17 at day 4 and day 5 after operation. Furthermore, Western blot analysis showed that higher immunosuppression decreased phosphorylation of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells-p65, increased phosphorylation of extracellular-signal-regulated kinase, and reduced the expression of Bcl-2-associated X protein and caspase-3 in the renal allografts. These results suggest that the DCD renal allograft seems to be more vulnerable to AR; enhanced immunosuppression reduces DCD-associated AR and improves early allograft function in a preclinical large animal model.