TY  - JOUR
AB  - Primary adrenal insufficiency is a life-threatening disorder, which requires lifelong hormone replacement therapy. Transplantation of xenogeneic adrenal cells is a potential alternative approach for the treatment of adrenal insufficiency. For a successful outcome of this replacement therapy, transplanted cells should provide adequate hormone secretion and respond to adrenal physiological stimuli. Here, we describe the generation and characterization of primary porcine adrenal spheroids capable of replacing the function of adrenal glands in vivo. Cells within the spheroids morphologically resembled adult adrenocortical cells and synthesized and secreted adrenal steroid hormones in a regulated manner. Moreover, the embedding of the spheroids in alginate led to the formation of cellular elongations of steroidogenic cells migrating centripetally towards the inner part of the slab, similar to zona Fasciculata cells in the intact organ. Finally, transplantation of adrenal spheroids in adrenalectomized SCID mice reversed the adrenal insufficiency phenotype, which significantly improved animals' survival. Overall, such adrenal models could be employed for disease modeling and drug testing, and represent the first step toward potential clinical trials in the future.
AU  - Malyukov, M.*
AU  - Gelfgat, E.*
AU  - Ruiz-Babot, G.*
AU  - Schmid, J.*
AU  - Lehmann, S.*
AU  - Spinas, G.*
AU  - Beuschlein, F.*
AU  - Hantel, C.*
AU  - Reisch, N.*
AU  - Nawroth, P.*
AU  - Bornstein, S.R.*
AU  - Steenblock, C.*
AU  - Ludwig, B.
C1  - 68028
C2  - 54506
CY  - 111 River St, Hoboken 07030-5774, Nj Usa
TI  - Transplantation of porcine adrenal spheroids for the treatment of adrenal insufficiency.
JO  - Xenotransplantation
VL  - 30
IS  - 5
PB  - Wiley
PY  - 2023
SN  - 0908-665X
ER  - 

TY  - JOUR
AB  - BACKGROUND: Islet xenotransplantation is a promising concept for beta-cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long-term survival, and graft failure support the optimization of transplantation strategies. Near-infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP. METHODS: CAG-iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS-selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin-diabetic immunodeficient NOD-scid IL2Rgnull (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model. RESULTS: CAG-iRFP transgenic NPIs were functionally equivalent with wild-type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000-NPI compared to 1000-NPI grafts. Ten weeks after transplantation, the fluorescence intensity of the 4000-NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose-dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose-dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6 mm) than after submuscular (depth ∼15 mm) placement of the NPIs. CONCLUSIONS: Islets from CAG-iRFP transgenic pigs are fully functional and accessible to long-term monitoring by state-of-the-art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno-islets.
AU  - Kemter, E.*
AU  - Citro, A.*
AU  - Wolf-van Buerck, L.*
AU  - Qiu, Y.*
AU  - Böttcher, A.
AU  - Policardi, M.*
AU  - Pellegrini, S.*
AU  - Valla, L.*
AU  - Alunni-Fabbroni, M.*
AU  - Kobolak, J.*
AU  - Kessler, B.*
AU  - Kurome, M.*
AU  - Zakhartchenko, V.*
AU  - Dinnyes, A.*
AU  - Cyran, C.C.*
AU  - Lickert, H.
AU  - Piemonti, L.*
AU  - Seissler, J.*
AU  - Wolf, E.*
C1  - 63885
C2  - 51782
CY  - 111 River St, Hoboken 07030-5774, Nj Usa
TI  - Transgenic pigs expressing near infrared fluorescent protein-A novel tool for noninvasive imaging of islet xenotransplants.
JO  - Xenotransplantation
VL  - 29
IS  - 1
PB  - Wiley
PY  - 2022
SN  - 0908-665X
ER  - 

TY  - JOUR
AB  - Background: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step. Methods: Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an α1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs. Results: At age 6 months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion: GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.
AU  - Hinrichs, A.*
AU  - Riedel, E.O.*
AU  - Klymiuk, N.*
AU  - Blutke, A.
AU  - Kemter, E.*
AU  - Längin, M.*
AU  - Dahlhoff, M.*
AU  - Keßler, B.*
AU  - Kurome, M.*
AU  - Zakhartchenko, V.*
AU  - Jemiller, E.M.*
AU  - Ayares, D.*
AU  - Bidlingmaier, M.*
AU  - Flenkenthaler, F.*
AU  - Hrabě de Angelis, M.*
AU  - Arnold, G.J.*
AU  - Reichart, B.*
AU  - Fröhlich, T.*
AU  - Wolf, E.*
C1  - 60685
C2  - 49442
CY  - 111 River St, Hoboken 07030-5774, Nj Usa
TI  - Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies.
JO  - Xenotransplantation
PB  - Wiley
PY  - 2020
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Böttcher, A.
AU  - Tritschler, S.
AU  - Yang, K.
AU  - Theis, F.J.
AU  - Lickert, H.
AU  - Wolf, E.*
AU  - Kemter, E.*
C1  - 57135
C2  - 47562
CY  - 111 River St, Hoboken 07030-5774, Nj Usa
TI  - Studying in vivo beta cell maturation in pigs by scRNAseq.
JO  - Xenotransplantation
VL  - 26
IS  - 5
PB  - Wiley
PY  - 2019
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Cohrs, C.M.
AU  - Kemter, E.*
AU  - Ludwig, B.
AU  - Bornstein, S.R.
AU  - Wolf, E.*
AU  - Speier, S.
C1  - 57137
C2  - 47560
CY  - 111 River St, Hoboken 07030-5774, Nj Usa
TI  - Transplantation of porcine neonatal islet like cell clusters display rapid engraftment and the capability to adequately respond to hyperglycemia.
JO  - Xenotransplantation
VL  - 26
IS  - 5
PB  - Wiley
PY  - 2019
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Kemter, E.*
AU  - Seissler, J.*
AU  - Böttcher, A.
AU  - Dobler, M.*
AU  - Kurome, M.*
AU  - Kessler, B.*
AU  - Zakhartchenko, V.*
AU  - Claussen, Y.*
AU  - Qiu, Y.*
AU  - Burton, N.*
AU  - Cyran, C.*
AU  - Wolf, E.*
C1  - 57139
C2  - 47558
CY  - 111 River St, Hoboken 07030-5774, Nj Usa
TI  - The iRFP720 pig as a novel tool for non-invasive fluorescence monitoring of NPIs transplant engraftment and growth in preclinical animal model in vivo.
JO  - Xenotransplantation
VL  - 26
IS  - 5
PB  - Wiley
PY  - 2019
SN  - 0908-665X
ER  - 

TY  - JOUR
AB  - BackgroundRegulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo-organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen-specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non-human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno-specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. MethodBaboon tolerogenic dendritic cells (tolDC) were generated in 3days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti-inflammatory cytokines. After loading with porcine-specific (PS) in vitro-transcribed RNA (ivtRNA), tolDC were used to induce CD4(+) T cells to become porcine-specific Treg (PSTreg) in cocultures supplemented with IL-2 and rapamycin for 10days. Anti-inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine-specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. ResultsTolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL-10 and TGF-1, whereas IL-12p40 and IFN- were not expressed. PSTreg were successfully generated in cocultures of CD4(+) T cells and PS ivtRNA-loaded tolDC. They exhibited a CD3(+)CD4(+)CD25(+)CD127(low/-)CD45RA(low)Foxp3(+) phenotype and were characterized by high expression of IL-10 and TGF-1 mRNA and protein. They showed upregulated expression of EBI3 and GARPmRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL-10 and TGF-1 cytokine upon interaction with PSTeff and suppressing IFN- expression on PSTeff. ConclusionIn this study, a fast 3-day method to generate baboon-derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine-antigen specificity and expression of IL-10 and TGF-1. Porcine-specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.
AU  - Li, M.*
AU  - Eckl, J.
AU  - Abicht, J.M.*
AU  - Mayr, T.*
AU  - Reichart, B.*
AU  - Schendel, D.J.
AU  - Pohla, H.
C1  - 52772
C2  - 44149
CY  - Hoboken
TI  - Induction of porcine-specific regulatory T cells with high specificity and expression of IL-10 and TGF-1 using baboon-derived tolerogenic dendritic cells.
JO  - Xenotransplantation
VL  - 25
IS  - 1
PB  - Wiley
PY  - 2018
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Ludwig, B.
AU  - Ludwig, S.*
AU  - Steffen, A.
AU  - Knauf, Y.*
AU  - Zimmerman, B.*
AU  - Avni, Y.*
AU  - Bornstein, S.R.
C1  - 51999
C2  - 43637
CY  - Hoboken
TI  - Successful islet xenotransplantation without immunosuppression in a non-human-primate model of diabetes.
JO  - Xenotransplantation
VL  - 24
IS  - 5
PB  - Wiley
PY  - 2017
SN  - 0908-665X
ER  - 

TY  - JOUR
AB  - Background: The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT). Methods: Goettingen minipigs of younger age (1 year) and retired breeder animals (3.5 years) were studied. Pancreata were harvested according to the standards of human organ retrieval including in situ cold perfusion with either Custodiol®-HTK or Belzer® UW solution. Pancreatic tissue was characterized by quantification of apoptotic cells. Islet isolations were performed according to a modified Ricordi method, and isolation outcome was assessed by determining islet particle numbers (IP), islet equivalents (IEQ), and isolation factor (IF). Isolated islets were cultured for 24 and 48 h for the assessment of in vitro survival. Results: Islet viability was significantly higher in Custodiol®-HTK preserved pancreas organs compared to Belzer® UW. Furthermore, organs harvested from retired breeder preserved in Custodiol®-HTK resulted in stable islet isolation yields even after prolonged CIT and showed superior survival rates of islets in vitro compared to the Belzer® UW group. Younger porcine donor organs resulted generally in lower islet yield and survival rates. Conclusions: In summary, Custodiol®-HTK solution should be preferred over Belzer® UW solution for the preservation of pancreata from porcine origin. Custodiol®-HTK allows for maintaining islet viability and promotes reproducible isolation outcome and survival even after longer CIT. The usage of retired breeder animals over young animals for islet isolation is highly advisable to yield high quality and quantity.
AU  - Steffen, A.
AU  - Kiss, T.*
AU  - Schmid, J.*
AU  - Schubert, U.*
AU  - Heinke, S.*
AU  - Lehmann, S.*
AU  - Bornstein, S.R.
AU  - Ludwig, B.
AU  - Ludwig, S.*
C1  - 50486
C2  - 42501
CY  - Hoboken
TI  - Production of high-quality islets from goettingen minipigs: Choice of organ preservation solution, donor pool, and optimal cold ischemia time.
JO  - Xenotransplantation
VL  - 24
IS  - 1
PB  - Wiley
PY  - 2017
SN  - 0908-665X
ER  - 

TY  - JOUR
AB  - Background: Safe and reliable diabetes models are a key prerequisite for advanced preclinical studies on diabetes. Chemical induction is the standard model of diabetes in rodents and also widely used in large animal models of non-human primates and minipigs. However, uncertain efficacy, the potential of beta-cell regeneration, and relevant side effects are debatable aspects particularly in large animals. Therefore, we aimed to evaluate a surgical approach of total pancreatectomy combined with splenectomy for diabetes induction in an exploratory study in Goettingen minipigs. Methods: Total pancreatectomy was performed in Goettingen minipigs (n = 4) under general anesthesia and endotracheal intubation. Prior to surgery, a central venous line was established for drug application and blood sampling. After median laparotomy, splenectomy was performed and the lobular pancreas was carefully dissected with particular attention to the duodenal vascular arcade. Close monitoring of blood glucose was initiated immediately after surgery by standard glucometer measurement or continuous glucose monitoring systems (CGMS). Exogenous insulin was given by multiple daily subcutaneous (s.c.) injections or via insulin pump systems (CSII). Complete endogenous insulin deficiency was confirmed by intravenous glucose tolerance test (ivGTT) and measurement of c-peptide. For establishing a suitable regimen for diabetes management, the animals were followed for 4-6 weeks. Results: Following pancreatectomy and splenectomy, the animals showed a quick recovery from surgery and initial analgetic medication and volume substitution could be terminated within 24 h. A rapid increase in blood glucose was observed immediately following pancreatectomy necessitating insulin therapy. The induced exocrine insufficiency did not cause any clinical symptoms. Complete insulin deficiency could be confirmed in all animals by determination of negative c-peptide during glucose challenge. The two regimen of insulin treatment (multiple daily injections (MDI) and continuous subcutaneous insulin infusion (CSII)) were both feasible with respect to acceptable glycemic control whereas CSII was considerably advantageous in comfort and popularity for both animals and care takers. Conclusions: Surgical pancreatectomy in combination with splenectomy to facilitate access to the pancreas is a feasible model for efficient diabetes induction in minipigs. The procedure itself and postoperative animal care could be performed without complications in this exploratory study. Nevertheless, this approach requires well-equipped infrastructure, experienced and skilled surgeons and anesthesiologists and dedicated animal care takers. The impact of total pancreatectomy in combination with splenectomy on the digestive and immune system must be considered in the design and definition of end points of experimental diabetes and transplantation studies.
AU  - Heinke, S.*
AU  - Ludwig, B.
AU  - Schubert, U.*
AU  - Schmid, J.*
AU  - Kiss, T.*
AU  - Steffen, A.
AU  - Bornstein, S.R.
AU  - Ludwig, S.*
C1  - 50233
C2  - 42231
CY  - Hoboken
SP  - 405-413
TI  - Diabetes induction by total pancreatectomy in minipigs with simultaneous splenectomy: A feasible approach for advanced diabetes research.
JO  - Xenotransplantation
VL  - 23
IS  - 5
PB  - Wiley-blackwell
PY  - 2016
SN  - 0908-665X
ER  - 

TY  - JOUR
AB  - BackgroundXenotransplantation using pig cells, tissues or organs may be associated with the transmission of porcine zoonotic micro-organisms. Hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine endogenous retroviruses (PERVs) are potentially zoonotic micro-organisms which do not show clinical symptoms in pigs and which are due to the low expression level difficult to detect. Gottingen Minipigs (GoMP) are often used for biomedical investigations and they are well characterized concerning the presence of numerous bacteria, fungi, viruses and parasites and therefore may be used for islet cell transplantation. MethodsIslet cells derived from three GoMP were transplanted into four healthy, non-diabetic cynomolgus monkeys using a macroencapsulation device. PCR, nested PCR, real-time PCR, real-time RT-PCR and Western blot analyses were used to estimate the presence of PERV, PCMV and HEV in the donors and recipients. ResultsUsing sensitive detection methods, no HEV was found in the donor pigs and in the pig islet cell preparations. Antibodies against PERV, PCMV and HEV were not found in all cynomolgus monkeys with exception of one monkey showing an immune response against HEV. Using real-time PCR, no PCMV and HEV were found in the sera of all monkeys. ConclusionAlthough the donor islet cells and the recipients were negative for HEV using PCR and Western blot analysis, in one recipient, antibodies against HEV were found, indicating infection in a single case. All recipients were negative for antibodies against PERV, and all were negative for PCMV, indicating absence of infection. As HEV was not detected in the donor pig before transplantation, a more complex and regular screening of the animals using highly sensitive methods is required to avoid virus transmission.
AU  - Morozov, V.A.*
AU  - Ludwig, S.*
AU  - Ludwig, B.
AU  - Rotem, A.*
AU  - Barkai, U.*
AU  - Bornstein, S.R.
AU  - Denner, J.*
C1  - 49713
C2  - 40864
CY  - Hoboken
SP  - 320-327
TI  - Islet cell transplantation from Gottingen minipigs to cynomolgus monkeys: Analysis of virus safety.
JO  - Xenotransplantation
VL  - 23
IS  - 4
PB  - Wiley-blackwell
PY  - 2016
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Kemter, E.*
AU  - Cohrs, C.M.
AU  - Wolf, A.*
AU  - Wuensch, A.*
AU  - Kurome, M.*
AU  - Kessler, B.*
AU  - Zakhartchenko, V.*
AU  - Loehn, M.*
AU  - Ivashchenko, Y.*
AU  - Speier, S.
AU  - Schulte, A.*
AU  - Wolf, E.*
C1  - 47511
C2  - 40631
SP  - S62-S63
TI  - INS-eGFP transgenic piglets provide a novel tool for studying maturation and vascularization of Neonatal Islet Cell Clusters (NICCs) in vivo.
JO  - Xenotransplantation
VL  - 22
PY  - 2015
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Ludwig, B.
AU  - Lehmann, S.*
AU  - Schmid, J.*
AU  - Schubert, U.*
AU  - Steffen, A.
AU  - Bornstein, S.R.
C1  - 47510
C2  - 40630
SP  - S75-S76
TI  - Attenuation of immunosuppression related negative effects on islet grafts by Growth Hormone Releasing Hormone (GHRH) agonist.
JO  - Xenotransplantation
VL  - 22
PY  - 2015
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Ludwig, B.
AU  - Ludwig, S.*
AU  - Steffen, A.
AU  - Zimerman, B.*
AU  - Schmid, J.*
AU  - Schubert, U.*
AU  - Heinke, S.*
AU  - Knauf, Y.*
AU  - Kaup, F.*
AU  - Goldman, T.*
AU  - Barkai, U.*
AU  - Rotem, A.*
AU  - Bornstein, S.
C1  - 47515
C2  - 40635
SP  - S19-S20
TI  - Preclinical studies on porcine islet macroencapsulation in non-human primates.
JO  - Xenotransplantation
VL  - 22
PY  - 2015
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Steffen, A.
AU  - Lehmann, S.
AU  - Balyura, M.
AU  - Bornstein, S.R.
AU  - Ludwig, B.
C1  - 47513
C2  - 40633
SP  - S111-S112
TI  - The influence of age on islet size distribution in male and female rats.
JO  - Xenotransplantation
VL  - 22
PY  - 2015
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Steffen, A.
AU  - Ludwig, S.*
AU  - Schmid, J.
AU  - Schubert, U.
AU  - Weitz, J.*
AU  - Bornstein, S.R.
AU  - Ludwig, B.
C1  - 47514
C2  - 40634
SP  - S119
TI  - Pig pancreas preservation and the influence on islet isolation outcome.
JO  - Xenotransplantation
VL  - 22
PY  - 2015
SN  - 0908-665X
ER  - 

TY  - JOUR
AU  - Steffen, A.*
AU  - Mueller, A.
AU  - Bornstein, S.
AU  - Ludwig, B.
C1  - 47516
C2  - 40636
SP  - S177
TI  - The influence of previous pregnancy on the functionality of smaller and larger rat islets.
JO  - Xenotransplantation
VL  - 22
PY  - 2015
SN  - 0908-665X
ER  -