Xenogeneic Cells Research Articles

Lack of CD47 on Donor Hepatocytes Promotes Innate Immune Cell Activation and Graft Loss: A Potential Barrier to Hepatocyte Xenotransplantation

We have previously shown that interspecies incompatibility of CD47 plays an important role in triggering rejection of xenogeneic hematopoietic cells by macrophages. However, whether CD47 incompatibility also induces rejection of nonhematopoietic cellular xenografts remains unknown. Herein, we have addressed this question in a mouse model of hepatocyte transplantation in which CD47(-/-) hepatocytes were used to resemble xenografts for CD47 incompatibility. We show that intrasplenic transplantation of CD47(-/-), but not wild-type (WT) hepatocytes, into partially hepatectomized syngeneic WT mice resulted in a rapid increase in Mac-1(+) cells with an activation phenotype (i.e., Mac-1(+)CD14(+) and Mac-1(+)CD16/32(high)), compared to nontransplant controls. In addition, CD47(-/-) hepatocytes were more severely damaged than WT hepatocytes as indicated by the greater AST and ALT serum levels in these mice. Furthermore, long-term donor hepatocyte survival and liver repopulation were observed in mice receiving WT hepatocytes, whereas CD47(-/-) hepatocytes were completely rejected within 2 weeks. These results suggest that CD47 on donor hepatocytes prevents recipient myeloid innate immune cell activation, hence aiding in graft survival after hepatocyte transplantation. Thus, CD47 incompatibility is likely to present an additional barrier to hepatocyte xenotransplantation.

Theological‐ethical legitimization of xenotransplantation and challenges regarding the hospital chaplaincy including the pastoral psychological companionship

BackgroundIn contrast to allotransplantation, xenotransplantation has thus far attracted lit‐tle interest within the field of theological ethics [6]. This can essentially be attributed to the fear of considerable problems regarding the medical feasibility due to possible immune reac‐tions and the risk of infection [10,11]. Consequently, at the beginning of the 21st century a recommendation was issued to stop all relevant research; this also led to a retardation of theo‐logical‐ethical reflections on xenotransplantation.Ethical questions: Irrespective of technical feasibility, the question whether xenotransplanta‐tion is ethically justifiable or not can systematically be divided into three areas [10]: (i) Ethics regarding animals and nature are concerned with the significance of non‐human rights; (ii) ethics in the areas of medicine, biomedicine and research deal with the importance of human rights; (iii) within the frame of a social‐ethical perspective, these rights need to be debated within the context of distributive justice. The prevalent ethical discussions regarding xeno‐transplantation focus on the following issues [2, 10]: protection of animals, risks of infection and informed consent. These have been the main reasons for the reluctance regarding human experiments [3, 5, 8]. Another range of concerns centers on the creation of human/animal chimeras by Xenotransplantation and the psychosocial and sociocultural acceptance of such procedures. These reservations are especially strong where the heart is concerned, since it has a special value in terms of personal and cultural identity, and a strong symbolic meaning. The morality of breeding transgenic pigs as donor animals has also recently been questioned, rais‐ing the question of whether it is legitimate to transgress the human‐animal boundary [1].Since chimerism raises questions regarding its psychosocial and sociocultural acceptance, the basic levels of the bioethical discourse that underlie the specific normative regulations are addressed. This concerns the anthropological question of human self‐conception and ques‐tions regarding fundamental interpretations of life and essential beliefs regarding the meaning of life. To the extent that the last mentioned levels are inevitably incorporated into the norma‐tive judgments of applied ethics and relate to the psychosocial and sociocultural acceptance of the xenotransplantation of the heart, it is necessary to bear in mind the fundamental anthropo‐logical and ideological implications [4, 9]. These implications are reflected by theological ethics. From a perspective of theological ethics the question of identity also has to be debated within a theological anthropological framework.Most ethical perspectives on xenotransplantation refer explicitly or implicitly to the question of the psychosocial and sociocultural acceptance of Xenotransplantation. As a consequence, the question of personal [7], social and cultural identity as well as the pivotal question con‐cerning the significance of the acceptance category for the ethical argumentation and ethical legitimization of standards are addressed. According to the ethical standards definition, validi‐ty of a standard is also characterized by an opportunity to achieve social recognition and ac‐ceptance.Research areas: The relationship between psychosocial and sociocultural acceptance and ethical legitimization shall be discussed within the context of xenotransplantation, and the relevance of public opinion shall be mapped out with the goal of achieving social acceptance and thereby facilitating the success of xenotransplantation. With reference to the psychosocial conditions for acceptance and success, the relevance as well as the tasks of pastoral compan‐ionship and pastoral‐psychological support for potential organ recipients shall be debated and guidelines shall be drafted. In light of the identity‐related challenges, an ethical and theologi‐cal request for psychosocial counseling and pastoral companionship. Furthermore, special focus shall be given to the particular importance of the spiritual dimension of counseling.This project is funded by DFG (TR CRC 127 “Biology of xenogeneic cell, tissue and organ transplantation – from bench to bedside”).

Evaluating the functionality of xeno‐relevant transgenes

With the scientific progress made in recent years, clinical application of xenotransplantation has become a realistic goal. Besides optimizing immunosuppressive regimens, the development of geneticallytailored donor pigs contributed to this success. Within the CRC 127 “Biology of xenogeneic cell, tissue and organ transplantation – from bench to bedside” we aim at developing novel transgenic donor pigs for islet transplantation, and providing existing transgenic donor lines to German as well as international collaboration partners.Regarding novel donor models, we have established transgenic pigs carrying the x‐linked inhibitor of apoptosis (XIAP) under the control of the porcine insulin promoter to achieve beta‐cell specific expression, as apoptosis has been postulated to play a major role in islet loss during isolation, in vitro cultivation and engraftment. Expression analyses of the first transgenic founders are presently underway. In addition, an advanced donor pig aiming at the temporary expression of vascular endothelial growth factor (VEGF) for improved engraftment of islets is being established. The model is based on the TetOn principle which requires two independent transgenes. First litters of founder pigs carrying a construct for beta‐cell specific expression of the transactivator (TA) have been delivered and qPCR analysis of the pancreas revealed different levels of TA expression in individual animals. Primary cells from a highly expressing founder will be transfected with a construct carrying the regulated VEGF gene under the control of the TA response element.For detailed pre‐clinical evaluation of already characterized transgenic pigs, we have established multitransgenic lines in collaboration with Revivicor Inc. carrying either aGalTKO/CD46/hTM, aGalTKO/CD46/INS‐LEA or aGalTKO/CD46/HLA‐E. We provide these animals, in addition to single‐transgenic INS‐LEA pigs, to the groups of P.Brenner and J.Seissler within the consortium. Furthermore, multi‐transgenic animals are provided to the groups of M.Mohiuddin, D.K.C. Cooper and R. Pierson by D. Ayares and islets of INS‐LEA expressing pigs are supplied to B. Hering in collaboration with J. Seissler.The ongoing evaluation of existing transgenes or their combination will reveal more detailed insight into the complexity of rejection mechanisms and, thus, support the design of optimized future transgenic approaches.

Hypomethylation of perforin regulatory elements in CD4+T cells from rat spleens contributes to the development of autoimmune emphysema

It is widely accepted that perforin regulatory elements are hypomethylated in CD4+ T cells from patients with active lupus, but whether this is the case in autoimmune emphysema is not known. Twenty rats were randomly divided into a normal control group and an emphysema group. Rat models of emphysema were established by intraperitoneal injection with xenogeneic endothelial cells. The levels of tumour necrosis factor-α, interleukin-8 and matrix metalloproteinase (MMP)-9 in bronchoalveolar lavage fluid (BALF) were measured, lung mean linear intercept and destructive index measured. Mean methylation of perforin gene promoter in CD4+ T cells and the expression of perforin were investigated. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling methods were used to examine the percentage of apoptotic cells in the alveolar septa. The levels of MMP-9 in BALF were higher in emphysema group than in control group (P < 0.05). The mean linear intercept and destructive index were higher in emphysema group than in control group (P < 0.05). The mean perforin gene promotor methylation of emphysema group was significantly decreased as compared with control group, while the expression levels of perforin gene were relatively higher (P < 0.05). There were more terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive cells in the alveolar septa in control group than in emphysema group. Hypomethylation of perforin regulatory elements in CD4+ T cells may result in the lung septal cell apoptosis associated with the development of experimental autoimmune emphysema. MMP-9 may play an important role in the pathogenesis of this kind of disease.

Effects of human adipose-derived stem cells on the viability of rabbit random pattern flaps

Background aimsFlap necrosis is the most commonly encountered outcome influencing the effect of operations in clinical practice. The advent of cytotherapy and regenerative medicine with stem cells, especially adipose-derived stem cell therapy, appears to be a promising approach in providing multi-lineage differentiating cells. However, autologous stem cells are limited in both quantity and quality in aging individuals. Hence, xenogenic stem cell therapy was used in this study. MethodsRandom pattern flaps (6 cm × 2 cm) were prepared in a rabbit model transplanted either with 4 × 105 human adipose-derived stem cells at five sites or equal volumes of Dulbecco's modified Eagle's medium. At 7 days after operation, the viability of the flaps from both groups was evaluated. We determined the numbers of locally infiltrating T cells, whereas the CD4/CD8 ratio, interferon, interleukin (IL)-2, IL-4 and IL-10 in the serum were determined to evaluate the immunological response of the rabbit. Moreover, Dil labeling was administrated to trace the homing of the transplanted stem cells. ResultsBoth the survival areas and the capillary number of the flaps that were injected with human adipose-derived stem cells significantly increased as compared with the control group (P < 0.05). Additionally, no significant difference in the immune response was detected between the groups. Dil-labeled stem cells were found to participate in the formation of tubular structures, which were further shown to be CD31+, although not predominantly. ConclusionsHuman adipose-derived stem cells could be used therapeutically to improve the viability of random pattern flaps without detection of serious immune rejection of stem cells.

Review: Macro-Encapsulation of Islets in Polyvinyl Alcohol Hydrogel

Diabetes mellitus (DM) can be cured by adequate insulin secretion from a relatively small volume of cells. Cell encapsulation enables allo- and even xeno-geneic cell therapy without immunosuppression. Micro-encapsulated islets used in recent clinical trials are not fully retrievable after transplantation. This paper summerizes the development of retrievable and theoretically replaceable macro-encapsulated islets using polyvinyl alcohol (PVA) hydrogel. An aqueous solution of PVA becomes a gel through micro crystallization by freezing and thawing. Utilizing this feature, PVA-macro-encapsulated islets (PVA-MEIs) were developed. Rat islets suspended in Euro-Collins solution containing 3% PVA were encapsulated in a mesh-reinforced PV A hydrogel sheet by freezing and thawing. The feasibility of PVA-MEIs for DM therapy was tested in vitro and in vivo. PVA-MEIs showed glucose-responsive insulin secretion in vitro even after 14-day culture. Rat PVA-MEIs cultured in media containing fresh human plasma showed no morphological changes and maintained insulin content. Intra-peritoneal transplantation of PVA-MEIs containing 750 rat islets ameliorated hyperglycemia in streptozotocin (STZ)-induced diabetic mice to nearly normal levels for up to 30 days with a consistent increase in body weight. Transplantation of PVA-MEIs also prevented metabolic and renal disorders in STZ-induced diabetic mice. PVA-MEIs cryo-preserved for 1, 7, and 30 days showed similar function in vitro and corrected hyperglycemia after intra-peritoneal transplantation in STZ-induced diabetic mice. Intra-peritoneal transplantation of PVA-MEIs containing iso- or allo-geneic islets (approx. 2,000 islets) ameliorated hyperglycemia in STZ-induced diabetic rats in a similar manner for almost half a year although the efficacy gradually decreased with time. Transplantation of PVA-MEIs ameliorated hyperglycemia in diabetic mice and rats without immunosupression. Retrievable and theoretically replaceable PVA-MEIs that can secure cell entrapment can mitigate the potential risks associated with xeno-geneic cells and cells made from undifferentiated cells. Therefore, PVA-MEIs are a promising modality for future DM therapy.

Clinical trials of immunogene therapy for spontaneous tumors in companion animals.

Despite the important progress obtained in the treatment of some pets' malignancies, new treatments need to be developed. Being critical in cancer control and progression, the immune system's appropriate modulation may provide effective therapeutic options. In this review we summarize the outcomes of published immunogene therapy veterinary clinical trials reported by many research centers. A variety of tumors such as canine melanoma, soft tissue sarcomas, osteosarcoma and lymphoma, feline fibrosarcoma, and equine melanoma were subjected to different treatment approaches. Both viral and mainly nonviral vectors were used to deliver gene products as cytokines, xenogeneic tumor associated antigens, specific ligands, and proapoptotic regulatory factors. In some cases autologous, allogenic, or xenogeneic transgenic cytokine producing cells were assayed. In general terms, minor or no adverse collateral effects appeared during this kind of therapies and treated patients usually displayed a better course of the disease (longer survival, delayed or suppressed recurrence or metastatic spread, and improvement of the quality of life). This suggests the utility of these methodologies as standard adjuvant treatments. The encouraging outcomes obtained in companion animals support their ready application in veterinary clinical oncology and serve as preclinical proof of concept and safety assay for future human gene therapy trials.

Stem Cells Take Part in Regulation of Prooxidant Activity and Immunity at Liver Fibrosis

It has been explored the possibility of correction of prooxidant-antioxidant parameters at rats with experimental liver fibrosis and immunological status of these animals using xenogeneic embryonic stem cells. It is shown that the threefold sequential administration of copper sulfate in dose 1 mg / 100 g of body weight, which amounted to 30% of the lethal dose in 48 hour intervals between administrations induced liver fibrosis in 24 hours after the last administration to 3 month animals. It was found that the development of fibrosis, was accompanied 50 per cent increase in TBA-active products in the mitochondria and 25% of the increase in the microsomes of liver cells. Content of Ig A, IgM, circulating immune complexes (CIC) and the average molecular weight peptides was increased to 2-3 times in the same time. Introduction of xenogenic embryonic stem cells in animals with experimental fibrosis accompanied by decrease in the amount of TBA-active products to almost intact control. It coincided with the 60% increase in the activity of glutathione peroxidase in the mitochondria. Introduction of stem cells to experimental animals provide a normalization of the content of Ig A, Ig M, the CiC and the average molecular weight of the peptides.

Immunological characteristics of human umbilical cord mesenchymal stem cells and the therapeutic effects of their transplantion on hyperglycemia in diabetic rats

Islet transplantation involves the transplantation of pancreatic islets from the pancreas of a donor to another individual. It has proven to be an effective method for the treatment of type 1 diabetes. However, islet transplantation is hampered by immune rejection, as well as the shortage of donor islets. Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HUMSCs) are an ideal cell source for use in transplantation due to their biological characteristics and their use does not provoke any ethical issues. In this study, we investigated the immunological characteristics of HUMSCs and their effects on lymphocyte proliferation and the secretion of interferon (IFN)-γ, and explored whether direct cell-to-cell interactions and soluble factors, such as IFN-γ were important for balancing HUMSC-mediated immune regulation. We transplanted HUMSCs into diabetic rats to investigate whether these cells can colonize in vivo and differentiate into pancreatic β-cells, and whether the hyperglycemia of diabetic rats can be improved by transplantation. Our results revealed that HUMSCs did not stimulate the proliferation of lymphocytes and did not induce allogeneic or xenogeneic immune cell responses. qRT-PCR demonstrated that the HUMSCs produced an immunosuppressive isoform of human leukocyte antigen (HLA-I) and did not express HLA-DR. Flow cytometry revealed that the HUMSCs did not express immune response-related surface antigens such as, CD40, CD40L, CD80 and CD86. IFN-γ secretion by human peripheral blood lymphocytes was reduced when the cells were co-cultured with HUMSCs. These results suggest that HUMSCs are tolerated by the host in an allogeneic transplant. We transplanted HUMSCs into diabetic rats, and the cells survived in the liver and pancreas. Hyperglycemia of the diabetic rats was improved and the destruction of pancreatic cells was partly repaired by HUMSC transplantation. Hyperglycemic improvement may be related to the immunomodulatory effects of HUMSCs. However, the exact mechanisms involved remain to be further clarified.

Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion.

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human NK cell activation through the recruitment of Siglec-7. Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy.

Functional Chimeric Antigen Receptor-Expressing Natural Killer Cells Derived From Human Pluripotent Stem Cells

Natural killer (NK) cells are a key part in the innate immune system and have the ability to recognize diverse types of tumors and virally-infected targets. NK cells represent an attractive cell population for adoptive immunotherapy due to their ability to kill target cells in a human leukocyte antigen (HLA) non-restricted manner and without prior sensitization. Clinical studies using IL-2 activated NK cells demonstrate significant anti-tumor effects when adoptively transferred into patients with refractory leukemia (mainly AML). However, there has been a more limited response observed in clinical trials for the treatment of ovarian cancer and other solid malignancies. Chimeric antigen receptors (CARs) consist of an antigen-specific single chain antibody variable fragment fused to intracellular signaling domains derived from receptors involved in lymphocyte activation. CARs targeting various tumor-associated antigens have been developed and tested via expression in primary T cells with promising clinical results. However, engineering these T cells must be done on a patient-specific basis, thus limiting the number of patients who can be treated.In order to produce a potential targeted, “off-the-shelf” product suitable to treat patients with diverse tumors or chronic infections, we have generated human pluripotent stem cells with stable CAR expression. Previous studies by our group demonstrate that human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable, and homogenous starting cell population to develop NK cells. We use a combined approach using “Spin-EB”- mediated differentiation of hESCs/iPSCs, followed by co-culture with artificial antigen presenting cells (aAPCs) that express mbIL-21. Using this strategy, we can generate 109 NK cells from a population of approximately 106 undifferentiated hESCs or iPSCs. This GMP compatible method is fully defined, without xenogeneic stromal cells or serum.Here, we have expressed both an anti-CD19 (targeting B cell malignancies) and an anti-mesothelin CAR (targeting ovarian cancer cells and other adenocarcinomas) in both hESCs and iPSCs. Using the Sleeping Beauty transposon system, both hESCs and iPSCs have been genetically engineered to express 3rd generation CARs, which express a single chain antibody fragment recognizing either CD19 or mesothelin, a CD8α hinge region, the transmembrane protein CD28, a co-stimulatory protein 4-1BB, and the activating domain CD3ζ. NK cells derived from hESCs/iPSCs with or without CAR expression are phenotypically similar to NK cells isolated from peripheral blood. These NK cells are CD56+, CD94+/CD117-, Nkp44+, Nkp46+, NKG2A+, NKG2D+, and KIR+. In 51Cr release assays against tumor targets expressing either CD19 or mesothelin, NK cells expressing the corresponding CAR show an enhanced killing ability. In cell lines lacking CD19 or mesothelin expression, the engineered cell lines exhibit equal activity compared to their non-engineered counterparts. Specifically, at a 10:1 effector:target ratio, anti-CD19 CAR+ iPSC-NK cells kill 58% of Lax7R cells (a CD19+ ALL cell line) compared to just 5% cell killing by CAR- iPSC-NK cells. Anti-CD19 CAR+ iPSC-NK cells also killed 2 other CD19+ ALL cell lines (018Z and Raji) better than CAR- iPSC-NK cells killing 63% vs 18% and 61% vs 8%, respectively. Similar results are seen against the mesothelin+ ovarian tumor line A1847. Here, anti-mesothelin CAR+ iPSC-NK cells kill 39% vs 14% for CAR- iPSC-NK cells. Currently, CAR-expressing NK cells derived from hESCs and iPSCs are being tested in vivo against both mesothelin+ ovarian tumor lines and CD19+ leukemia cells. Together, these studies demonstrate engineering hESCs and iPSCs with tumor-specific receptors provides a novel strategy to produce targeted NK cells suitable for immune therapies against refractory malignancies. Disclosures:No relevant conflicts of interest to declare.

Engraftment Of Human Polycythemia Vera CD34+ Cells In hSIRPα-Transgenic-Human-TPO-Expressing RAG2-/-, IL2Rγ-/- Immunodeficient Mice

IntroductionXenotransplantation of human hematopoietic malignancies into immunodeficient mice represents the most appropriate in vivo model system for human malignant hematopoiesis. While a diversity of immunodeficient mouse strains on the NOD/SCID and RAG2-/-/IL2Rγ-/--BALB/C background are described for the most aggressive human malignancies like acute leukemias, xenotransplantation models of less aggressive human hematologic disorders like myeloproliferative neoplasms and myelodysplastic syndromes show only limited engraftment levels. We recently developed next generation mouse strains expressing human cytokines and key factors of xenogeneic cell acceptance (e.g. hSIRPα to inactivate mouse macrophage activation by human cells) and hypothesized that these would represent suitable models for the assessment of human less aggressive hematologic disorders in vivo by providing an optimized “humanized” microenvironment. MethodsPeripheral blood (PB) and bone marrow (BM) samples of polycythemia vera (PV) patients were collected after informed consent at the Division of Hematology, Zurich University Hospital. Human CD34+ cells were isolated by density gradient centrifugation followed by immunomagnetical selection using anti-CD34 coupled beads. Purity of magnetical selection process was confirmed by FACS analysis. Newborn (24h-48h old) hSIRPα-tg-hTPO-knockin mice on the RAG2-/-/IL2Rγ-/--BALB/C background received sublethal irradiation (split dose of 2x1.5 Gy) and were transplanted intra-hepatically 24 hours later. Transplanted cell dose was dependent on availability of CD34+ stem and progenitor cells isolated from one phlebotomy sample (∼400ml of PB) of the respective patient. Mice were bled 4 weeks after transplantation and chimerism in peripheral blood was analyzed by flow cytometry using a panel of antigens (mCD45.2, hCD45, hCD33, hCD34, hCD3, hCD19). Mice showing positive chimerism in PB (i.e.>0.1% hCD45+ of total MNCs) at week 4 were sacrificed between week 8-16 and engraftment in BM, spleen and PB was analyzed by flow cytometry. To verify engraftment of human malignant hematopoiesis we quantified allele-burden of JAK2V617F point mutation in mouse BM using allele specific (AS)-PCR for the pathognomonic point mutation of the JAK2 gene. ResultsBy transplantation of 4-10x105 CD34+ cells into newborn hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could detect engraftment of hCD45+ cells in PB at week 4 (median 0.68%, range 0.12-23.8%). At week 8, BM engraftment of hCD45+ cells ranged 0.88-54.1% (median 4.43%) with a high proportion of human myeloid cells detected by hCD45/hCD33 co-staining (median 3.07 %, range 0.6-17.6% of total MNCs). We could detect engraftment until week 16, the latest timepoint assessed. Since all transplanted PV patient samples were positive for the common point mutation JAK2V617F, AS-PCR was used to quantify human malignant hematopoiesis. In tested BM samples of engrafted mice we found JAK2V617F positive alleles with a frequency of 2-12% (median 8%). To further assess the clonal composition of the engrafted population we established single cell sorting of primary and engrafted human PV-CD34+ cells in a 96 well format followed by liquid culture expansion and AS-PCR. In pilot studies we could show the clonal composition of a BM engrafted CD34+ population that split into 80% JAK2 WT expressing, 10% JAK2V617F heterozygous and 10 % JAK2V617F homozygous clones. We are currently extending these findings by side by side comparison of the clonal composition of primary vs. xenografted human PV-CD34+ cells of the same patient to test for the influence of a xenogeneic humanized microenvironment on maintenance of malignant cells in vivo. ConclusionsBy using hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could show engraftment of PV-CD34+ cells that extends previous reported engraftment levels in other model organisms. To our knowledge this is the first study assessing the clonal composition of human PV engrafted cells in the xenogeneic environment aiming at identifying components that are critical for the maintenance of human malignant hematopoiesis in vivo. This model will thus be a useful tool to test targeted therapies in vivo. Disclosures:No relevant conflicts of interest to declare.

The Effect of Donor Variation and Senescence on Endothelial Differentiation of Human Mesenchymal Stromal Cells

Application of autologous cells is considered for a broad range of regenerative therapies because it is not surrounded by the immunological and ethical issues of allo- or xenogenic cells. However, isolation, expansion, and application of autologous cells do suffer from variability in therapeutic efficacy due to donor to donor differences and due to prolonged culture. One important source of autologous cells is mesenchymal stromal cells (MSCs), which can differentiate toward endothelial-like cells, thus making them an ideal candidate as cell source for tissue vascularization. Here we screened MSCs from 20 donors for their endothelial differentiation capacity and correlated it with the gene expression profile of the whole genome in the undifferentiated state. Cells of all donors were able to form tubes on Matrigel and induced the expression of endothelial genes, although with quantitative differences. In addition, we analyzed the effect of prolonged in vitro expansion on the multipotency of human MSCs and found that endothelial differentiation is only mildly sensitive to expansion-induced loss of differentiation as compared to osteogenic and adipogenic differentiation. Our results show the robustness of the endothelial differentiation protocol and the gene expression data give insight in the differences in endothelial differentiation between donors.

Investigation of the immune response to autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses

Mesenchymal stem cells have demonstrated immunomodulatory capabilities as well as modest efficacy in animal models of joint injury, warranting further study as a potential treatment of joint disease. The goal of the study was to investigate the blood and synovial immune and histologic response to intra-articular injection of autologous, allogeneic, and xenogeneic bone marrow-derived mesenchymal stem cells (MSC) in horses. The study group consisted of 6 five-year-old Thoroughbred mares that had been injected previously with 15 million, genetically modified autologous, allogeneic, or xenogeneic MSC into the fetlock joints. One group of autologous cells was genetically modified to permit MSC biolocalization in the synovium. To assess response to the injection, synovial biopsies were obtained via arthroscopy 60 days after MSC injection for gross, histologic and molecular analyses. Peripheral blood mononuclear cells were isolated from each horse 120 days after MSC injection and co-cultured with a monolayer of each MSC group to permit quantification of activated CD4+ lymphocytes and cytokine release (ELISA) upon re-exposure to MSC.Arthroscopic examination revealed normal synovium with no grossly detrimental effect to the synovium or cartilage. Intra-articular MSC produced a persistent mononuclear infiltrate for at least 60 days, mostly perivascular, identified as CD3+ lymphocytes. An immune response (significant increase in CD4+ lymphocytes) was detected upon re-exposure to xenogeneic but not to allogeneic or autologous MSC. An inflammatory cytokine release from peripheral blood mononuclear cell/MSC co-cultures was present in all MSC groups but was significantly greater in the xenogeneic group. In conclusion, intra-articular injection of MSC, regardless of cell origin, incited a persistent mononuclear synovitis demonstrating a sustained biologic influence of these cells. Allogeneic cells did not elicit a detectable immune response upon re-exposure using our methods.

Identification of the Tetraspanin CD82 as a New Barrier to Xenotransplantation

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galβ1,4GlcNAc-R (Galα1,3-Gal)-dependent and -independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal-independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal-independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal-independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major "xenogenicity sensor" and open new avenues of intervention to making xenotransplantation a clinical reality.

Human T cells upregulate CD69 after coculture with xenogeneic genetically-modified pig mesenchymal stromal cells

Mesenchymal stromal cells (MSC) obtained from α1,3-galactosyltransferase gene knock-out pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pMSC) suppress in vitro human anti-pig cellular responses as efficiently as allogeneic human MSC. We investigated the immunoregulatory effects of GTKO/CD46 pMSC on human CD4+ and CD8+ T cell proliferation in response to pig aortic endothelial cells (pAEC). pMSC efficiently suppressed T cell proliferation, which was associated with downregulation of granzyme B expression. No induction of CD4+CD25+Foxp3hi regulatory T cells or T cell apoptosis was documented. In correlation with T cell proliferation, CD25 expression was upregulated on T cells in response to pAEC but not to pMSC. In contrast, CD69 expression was upregulated on T cells in response to both pMSC and pAEC, which was associated with a significant increase in the phosphorylation of STAT5. GTKO/CD46 pMSC possibly regulate human T cell responses through modulation of CD69 expression and STAT5 signaling.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

Combinatorial biomatrix/cell-based therapies for restoration of host tissue architecture and function.

This Progress Report reviews recent advances in the utility of extracellular matrix (ECM)-mimic biomaterials in presenting and delivering therapeutic cells to promote tissue healing. This overview gives a brief introduction of different cell types being used in regenerative medicine and tissue engineering while addressing critical issues that must be overcome before cell-based approaches can be routinely employed in the clinic. A selection of five commonly used cell-associated, biomaterial platforms (collagen, hyaluronic acid, fibrin, alginate, and poly(ethylene glycol)) are reviewed for treatment of a number of acute injury or diseases with emphasis on animal models and clinical trials. This article concludes with current challenges and future perspectives regarding foreign body host response to biomaterials and immunological reactions to allogeneic or xenogeneic cells, vascularization and angiogenesis, matching mechanical strength and anisotropy of native tissues, as well as other non-technical issues regarding the clinical translation of biomatrix/cell-based therapies.

Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: Effect on spinally grafted human neural precursor survival

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague–Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6weeks to 3months after cell grafting. In animals implanted with TAC pellets (3.4mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1±4.9ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4–5.1mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats.

Effects of Sertoli cells Implantation on Type 2 Diabetes in Nonhuman Primates

Transplanted allo‐ and xenogeneic Sertoli cells, encapsulated in alginate, have been shown to successfully treat endocrine and neurodegenerative dysfunctions in experimental models with efficacy in the diabetic rodents. In order to determine the translational potential for therapeutic efficacy in humans, we have investigated its application in the spontaneously obese nonhuman primate with naturally‐occurring type 2 diabetes. Isolated Sertoli cells 0.05 to 0.1 × 109 cells/kg from outbred prepubertal piglets were encapsulated in alginate and injected (ip) into insulin‐treated diabetic monkeys. One month following transplantation there was a reduction in fasting plasma glucose, HbA1c decreased by 0.5–1.4% accompanied by a reduction of mean AM nonfasting glucose levels without change in daily insulin therapeutic doses (by design). Results suggest that this novel encapsulated cell transplantation treatment protocol utilizing xenogeneic Sertoli cells may have resulted in a potential positive therapeutic effect on the metabolic status of the diabetic condition which occurred without an apparent inflammatory response. Due to the therapeutic evidence and safety of Sertoli cell transplantation to provide clinical relief in models of diabetes, including the monkey, these data support additional studies in nonhuman primates to further establish potential for successful treatment of diabetes in human.