Xenotransplantation最新文献

Soft tissue repair patches of decellularized extracellular matrices (ECM) with inherently preserved structural components and biomacromolecules are desirable in regenerative applications. This study characterizes three detergent-based decellularization methods to fabricate acellular porcine liver matrices to remove antigenic determinants without compromising the structural integrity, glycosaminoglycans (GAG) content, and bound growth factors within the resulting ECM. Three detergents chosen for decellularization were sodium dodecyl sulfate (SDS), SDS with sodium deoxycholate (SDS + SDC-combinatorial method), and Triton X-100 followed by SDS. Combinatorial detergent decellularization effectively removed cellular components and retained intact collagenous structure with minimal residual DNA and protein. It also preserved significantly higher amounts of GAG, HGF, and bFGF. TX100 decellularization was highly destructive with the least preservation of GAG and GFs. The SDS method showed an intermediate level of preservation of biomolecules. The correlation obtained between GAG and GFs revealed quantification of GAG to be an indirect way of estimating the bound GFs preserved within the ECM. In vitro experiments revealed the noncytotoxic nature of the scaffolds. The study revealed that, among the three methods of decellularization, ECM scaffold fabricated by combinatorial detergent decellularization is extremely promising for use as a soft tissue repair patch with inherent bioactive molecules for scaffold-based regenerative therapy.

Background: Transplant programs preparing to initiate first-in-human pig kidney xenotransplant clinical trials must be especially careful when obtaining participants' informed consent. Little is known about the kind of information patients want for making an informed decision about trial participation.

Methods: We conducted semi-structured telephone interviews with waitlisted kidney transplant patients about information needs regarding participating in a first-in-human pig kidney xenotransplant trial, which guided development of a prototype consent form. Subsequent usability testing interviews sought patient feedback on the consent form. We analyzed qualitative data by thematic analysis and quantitative data by descriptive statistics.

Results: Twenty-eight patients participated in semi-structured interviews; 16 patients participated in usability testing interviews. Most interview participants were male (68%, 56%), White (54%, 56%), or Black (36%, 31%), respectively. Interview participants identified five types of information needs: (1) the potential for infection contraction and transmission; (2) risks, benefits, and impact of xenotransplant trials; (3) xenotransplant clinical trial and recipient experience; (4) clinical trial logistics; and (5) the pig and its kidney. Usability testing participants suggested adding details to the prototype. Participants' preparedness to make a decision about participating in a xenotransplant trial increased after reviewing the prototype (12.5% vs. 31.3%, n.s.).

Conclusion: We identified multiple unique types of information patients desired to make informed decisions about pig kidney xenotransplant trial participation. Transplant programs initiating xenotransplant trials should be prepared to address patients' information needs to optimize informed decision-making for trial participation. The prototype consent form may support a patient-centered approach to informed consent.

This review highlights groundbreaking progress from July to December 2024, including developments in gene-edited pigs, cellular therapies, organ preservation, and transplantation techniques. Recent advancements, particularly in gene-editing technologies and immunosuppressive protocols, have brought the field closer to clinical application. While significant ethical, immunological, and societal challenges remain, this progress underscores the immense potential of xenotransplantation to revolutionize transplantation medicine.

Background: Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of an exogenous complement.

Results: There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement-mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.

Conclusion: The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.

Advancements in xenotransplantation intersecting with modern machine perfusion technology offer promising solutions to patients with liver failure providing a valuable bridge to transplantation and extending graft viability beyond current limitations. Patients facing acute or acute chronic liver failure, post-hepatectomy liver failure, or fulminant hepatic failure often require urgent liver transplants which are severely limited by organ shortage, emphasizing the importance of effective bridging approaches. Machine perfusion is now increasingly used to test and use genetically engineered porcine livers in translational studies, addressing the limitations and costs of non-human primate models. Current reports about artificial and bioartificial liver support combined with xenografts showcase the potential in ex vivo xenogeneic perfusion. Breakthroughs, such as the perfusion of genetically modified porcine liver with FDA-approved machine perfusion systems connected to human blood circulation, underscore the interest and potential feasibility of a "liver dialysis" bridge to allotransplantation or recovery. This review provides an overview of the past and current research in the field of ex vivo pig liver xenoperfusion.

Background: Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of exogenous complement.

Results: There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.

Conclusion: The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.

Background: The removal of preformed antibodies with cleaving enzyme like IdeS (Imlifidase) has demonstrated therapeutic potential in organ transplantation for sensitized recipients. However, preformed xenoreactive antibodies (XAbs) against porcine glycans are predominantly IgM and considered detrimental in pig-to-human xenotransplantation.

Methods: Recombinant IceM, an endopeptidase cleaving IgM, was generated in Escherichia coli. Four maximally MHC-mismatched rhesus macaques underwent two serial skin transplantations to model allosensitized patients awaiting xenotransplantation. IceM was administered IV in allosensitized animals at 28 and 56 days after the first skin transplantation to assess in vivo IgM cleavage. Total IgG and IgM were quantified with western blot, and anti-pig (xenoreactive) IgG/IgM were evaluated using flowcrossmatch. B cell and its subpopulations were assessed using flow cytometry.

Results: IceM selectively cleaved human IgM, while showing no cleavage activity toward other isotypes including IgG, IgA, IgD, and IgE. Additionally, IceM cleaves only human and non-human primate IgM in vitro, but not in sera from other species. At a dose of 0.5 mg/kg, IceM reduced xenoreactive IgM levels to 13.76% ± 4.98% of baseline (B cell flow crossmatch) at 24 h post-administration, with baseline levels restored approximately 2 weeks after treatment. Additionally, animals showed similar kinetics of xenoreative IgM degradation with the repeated dose of IceM.

Conclusion: In this study, we report a recombinant bacterial enzyme that selectively cleaves IgM in human and non-human primate sera. Repeat administration of IceM in macaques enables selective, robust clearance of circulating xenoreactive IgM. This approach will be useful in treating preformed natural and rebound IgM in xenotransplantation.

Patients with fulminant liver failure ineligible for transplantation have a high mortality rate. With recent progress in genetic modifications and clinical achievements, using pig livers as a bridge-to-transplant has regained popularity. Preclinical testing has been done in small cohorts of nonhuman primates (NHP), and maximum survival is limited to 1-month. We conducted a systematic review and comparative outcomes analysis of NHP-liver xenotransplantation and gathered 203 pig-to-NHP and NHP-to-NHP transplants reported in 23 studies. Overall, NHP survival after pig-liver xenotransplantation was limited (1, 3, 4 weeks: 18.0%, 5.6%, 1.1%), compared to NHPs after allotransplantation (1, 3, 4 weeks: 60.6%, 47.4%, 45.4%). A focus on pigs with genetic modifications evidenced some short-term survival benefits (1, 3, 4 weeks: 29.1%, 9.1%, 1.8%). The use of the auxiliary transplant technique was also associated with better short-term results (1, 3, 4 weeks: 40.9%, 9.1%, 4.5%). Causes of graft and animal loss were mostly rejection and liver failure in allotransplants, while bleeding, liver, and respiratory failure predominated in xenotransplants. Notably, the 1-month survival rate for NHP-allotransplants was significantly lower than the national > 98% rate for human liver transplants. This data confirms the short-term improvements brought by genetic modifications and auxiliary implantation in the NHP model, which remains imperfect.

First-in-human pig xenotransplant clinical trials may soon begin, which raises ethical concerns about patients' decision-making to participate in such trials. We assessed kidney transplant candidates' attitudes and hypothetical decision-making about participating in xenotransplant trials through semi-structured telephone interviews and an online survey. We analyzed qualitative data by thematic analysis and quantitative data by descriptive statistics. Twenty-eight patients participated in interviews; 142 patients participated in the survey. Most interview and survey respondents were male (68%, 56%), White (54%, 61%), or Black (36%, 22%). Although interview participants appreciated xenotransplantation research's potential to advance science, they expressed concerns about infection transmission and graft function. Few survey respondents were willing to participate in a pig kidney trial to test the safety of pig kidneys (12.6%) or pig kidney function (16.9%). Interview participants would be more likely to participate in a first-in-human pig kidney trial if receiving a human kidney was unlikely and their health status declined. Willingness would also depend on how long the pig kidney would function. Most interview participants were receptive to long-term monitoring, but not to family monitoring. Transplant programs planning xenotransplant trials should anticipate these types of concerns for optimizing human subject protections and conducting a robust informed consent process.