Xenogeneic Stem Cells Research Articles

Effects of various xenogenic mesenchymal stem cell secretome fractions on the regenerative capacity of the liver in vitro

Liver diseases cause many deaths worldwide and wreak havoc on the economy. The main hepatoprotectors are plant substances and peptides. Regenerative medicine based on mesenchymal stem cells (MSCs) can offer a new set of biologically active substances for liver regeneration, which are part of their secretome. The work applied the methods of cell isolation and cultivation, ultrafiltration for fractionation of secretome components, and organotypic culture model. The study’s results indicate an increase in the regenerative capacity of the liver under the impact of the components of the MSC secretome; the fraction below 10 kDa shows the most significant activity. Keywords: organotypic liver culture; xenogenic stem cells; fractions of secretome; absorption spectrum, ultrafiltration

Xenogeneic Stem Cell–Induced Cardiac Progenitor Cells Regenerated Infarcted Myocardium in Rat Model

Xenogeneic Stem Cell–Induced Cardiac Progenitor Cells Regenerated Infarcted Myocardium in Rat Model

Xenogeneic stem cell transplantation: Research progress and clinical prospects.

Organ transplantation is the ultimate treatment for end-stage diseases such as heart and liver failure. However, the severe shortage of donor organs has limited the organ transplantation progress. Xenogeneic stem cell transplantation provides a new strategy to solve this problem. Researchers have shown that xenogeneic stem cell transplantation has significant therapeutic effects and broad application prospects in treating liver failure, myocardial infarction, advanced type 1 diabetes mellitus, myelosuppression, and other end-stage diseases by replacing the dysfunctional cells directly or improving the endogenous regenerative milieu. In this review, the sources, problems and solutions, and potential clinical applications of xenogeneic stem cell transplantation will be discussed.

Effects of canine adipose-derived mesenchymal stem cells on the epithelialization of rabbits’ skin autograft (Oryctolagus cuniculus)

ABSTRACT: The present study aimed to evaluate the effects of mesenchymal stem cells derived from canine adipose tissue in the healing process of full-thickness mesh skin grafts in rabbits. The stem cells were collected from young dogs; and, after characterization, remained in cryopreservation, in independent doses containing 2 x 106 cells. The mesh distal limb graft technique was performed in 60 rabbits, divided into three groups, CG (Control Group), GT1 (Intralesional Stem Cell Treated Group), and GT2 (Intravenous Stem Cell Treated Group), containing 20 animals each. After grafting, each group was randomly divided into four subgroups according to euthanasia time 3, 7, 14, and 30 days, containing five animals in each group. Animals of GT1_14, GT1_30, and GT2_14, GT2_30 subgroups received a second dose of xenogeneic cells on the seventh day. Meanwhile, animals from GT1_30 and GT2_30 received the third dose of xenogeneic cells on day 14. The groups treated with xenogeneic stem cells positively affected type III collagen re-epithelialization and deposition, and possibly GT1 had a controlled inflammatory response. However, no effect on angiogenesis. Thus, it was possible to demonstrate tolerance and therapeutic action of mesenchymal stem cells from canine adipose tissue in skin grafts in rabbits.

Effects of antioxidants and antifreeze proteins on cryopreservation of blue catfish (Ictalurus furcatus) spermatogonia

Long-term storage of spermatogonial stem cells (SSCs) represents the next step for advancing aquaculture research by providing valuable genetic resources for genetic enhancement programs and xenogenesis technologies. Hybrid catfish, the cross between channel catfish, Ictalurus punctatus ♀ and blue catfish, I. furcatus ♂, are in high demand by the US aquaculture industry, but production can be limited by the availability of blue catfish gametes. Therefore, xenogeneic stem cell transplantation is a powerful method for generating blue catfish gametes within faster growing and maturing channel catfish hosts. These technologies could be facilitated by having cryopreserved SSCs from testicular tissue on-hand in gene banks. Cryopreservation protocols for blue catfish testicular tissues and for other fish species are still being optimized, and currently, data is lacking on the effects antioxidants and antifreeze proteins (AFPs) have on cellular post-thaw viability of SSCs. The objective of this study was to analyze the individual and combined effects of antioxidants (catalase, hypotaurine, and ascorbic acid) and AFPs (I and III) on post-thaw type A spermatogonia cell production and viability from testicular tissue. In this study, there were no improvements with individual antioxidant or AFP treatments. However, when the antioxidants hypotaurine and catalase were tested in combination with different AFPs, three treatments had higher type A spermatogonia cell production than the control, including hypotaurine 7 mM + AFPI 1 μg/mL, hypotaurine 3.5 mM + AFPI 0.1 μg/mL, and hypotaurine 7 mM + AFPIII 0.1 μg/mL. These treatments along with hypotaurine 7 mM + AFPI 0.1 μg/mL and catalase 100 IU + AFPI 0.1 μg/mL had higher viability than the control. From these results, we recommend adding these antioxidant-AFP combinations to future cryopreservation protocols. Overall, this is the first study showing that combining specific antioxidants and AFPs can improve spermatogonia cryopreservation for a fish species.

Xenogeneic mesenchymal stem cell transplantation for mandibular defect regeneration.

It is commonly accepted that xenogeneic stem cell transplantation for tissue engineering is faced with host immune rejection. Using a rat critical-size mandibular defect model, this study examined whether the immune rejection can be evaded by diminishing T-cell immunity. To examine donor cell survival and host immune reaction, pig bone marrow-derived mesenchymal stem cells (BM-MSCs) were labeled with CM-DiI, loaded onto gelatin sponge (5×106 cells/scaffold), and transplanted into 5-mm mandibular defects of immunocompetent and T cell-deficient athymic rats. To examine the effects of xenogeneic BM-MSCs on bone regeneration, athymic rats undergone the same surgeries were terminated at post-operative weeks 1, 3, and 6. Control rats underwent the same jaw surgery without BM-MSC transplantation. The density of CM-DiI-labeled BM-MSCs decreased with time in both strains of rats. Although it was substantially higher in athymic rats than in immunocompetent rats at post-operative day 1, by day 3-7 the density became comparable between the two strains of rats. Apoptosis reflected by cleaved Caspase-3 staining was low in both strains. Stronger infiltration of neutrophils, macrophages, B cells and CD8+ T cells was found in MSC-treated animals. In athymic rats, infiltration of neutrophils and macrophages was strong, but it occurred later than that in immunocompetent rats. While bone volume fraction significantly increased with time (P<.001), no difference was found between MSC-treated and control groups. Even in hosts with deficient T-cell immunity, xenogeneic BM-MSC transplantation into mandibular critical-sized defects still faces challenges from host innate immunity, which compromises their regenerative efficacy.

Xenogeneic Mesenchymal Stem Cells in the Formation of Hyaline Cartilage in Osteochondral Goat Failure

Background: Osteochondral knee failures are among the most common causes of disability among the elderly human population and animal athletes. The xenogeneic transplantation of mesenchymal stem cells is a questionable therapeutic alternative that, despite the low expression of Major Histocompatibility Complex type II by these cells, still has relevantuncertainties about the safety and clinical efficacy. The main objective of the present study was to investigate whether the xenogeneic transplantation of mesenchymal stem cells induces hyaline cartilage formation, without histopathological evidence of rejection, in osteochondralfailures of goats.Materials, Methods &amp; Results: Five female goats were used, submitted to three surgical osteocondral failures in the right knee, treated with xenogenic mesenchymal stem cells of dental pulp, xenogenic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen, respectively. The lesions were evaluated after 60 days of treatment, aiming to identify thepresence of hyaline cartilage or fibrocartilage and the subchondral bone pattern (regenerated or disorganized). Transplantation of xenogenic mesenchymal stem cells induced predominant formation of hyaline cartilage (P &lt; 0.05), with no histopathological evidence of inflammationwhen compared to the other treatments. Therapies with xenogeneic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilage, with no significant difference between them (P &gt; 0.05). Macroscopically, the lesions of the stem cell treated group showed formation of firm repair tissue, opaque staining, integrated with adjacent cartilage and with the failure filling almost completely. The groups treated with PRP and hemostatic sponge of hydrolyzed collagen presented, on average, partial filling of the lesion, with irregular shape and darkened coloration.Discussion. The absence of macroscopic and histopathological evidences of an inflammatory process on the surface and in the internal portion of the osteochondral lesions treated with xenogeneic stem cells, probably due to the low expression of Major Histocompatibility Complex type II by these cells, which would theoretically induce low rejection response. Such observations are of great importance, since graft-versus- host disease syndrome is a serious condition, responsible for the low therapeutic efficacy with transplantation of cells or grafts in humans. The formation of fibrocartilage, although without macro and microscopic evidence of degeneration or necrosis, in the osteochondral failures treated with PRP and hemostatic collagen sponge suggest that paracrine factors of the local microenvironment of the osteochondral failure are possibly responsible for the formation of fibrocartilaginous tissue or by inhibition of normal cartilage formation. The fibrocartilage formed in the Plasmaand Control groups, contributed to the commitment in the filling of the lesion, contrasting with the almost complete fill of the lesions treated with stem cells. The xenotransplantation of mesenchymal stem cells induced formation of hyaline cartilage and did not promote histopathological evidence of rejection in osteochondral lesions of goat knees. The treatments with PRP and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilaginous surface in the osteochondral failures.

Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

Reconstruction of cranial cruciate ligament in rabbits using polyester implants saturated with PRP, antlerogenic stem cells MIC-1 and their homogenate

ABSTRACTAim of the study: The attempt to limit the negative effects of polyester implants on the articular cavity by using preparations containing growth factors. Materials and Methods: Polyester implants used for the reconstruction of a rabbit’s cranial cruciate ligament (CCL) were saturated with autogenic platelet-rich plasma (PRP), antlerogenic stem cells MIC-1 and their homogenate prior to the surgery. Six months after CCL reconstruction, morphological, and biochemical blood tests were carried out, including proteinogram and acute phase proteins. The knee joints were also examined macro- and microscopically. Results: The results, compared to the control group, showed a favorable effect of the PRP and homogenate of antlerogenic cells on limiting the inflammation caused by the presence of polyester implant in the knee joint. The addition of growth factors caused covering the implant faster with the recipient’s connective tissue, thus contributing to reducing the inflammatory reaction of the articular capsule to the presence of polyester. At the same time, no enhanced local or general reaction of the rabbit organism was observed to the presence of xenogenic antlerogenic stem cells MIC-1 homogenate which, like the PRP, may provide an easily available source of growth factors, increasingly often used in regenerative medicine. Conclusions: Applying antlerogenic stem cells, their homogenate or PRP increases the volume of connective tissue that surrounds and intertwines polyester CCL implant, separating it from synovial cavity environment.

Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived from Different Species.

Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose‐derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction. Stem Cells Translational Medicine 2017;6:22–30

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1(-/-).IL-2rγ(-/-) mice transplanted with human CD34(+) cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

Activation of Expression of Brain-Derived Neurotrophic Factor at the Site of Implantation of Allogenic and Xenogenic Neural Stem (Progenitor) Cells in Rats with Ischemic Cortical Stroke

Ischemic stroke was modeled in the sensorimotor zone of the brain cortex in adult rats. Rat embryonic nervous tissue, neural stem cells from human olfactory epithelium, and rat fibroblasts (cell control) were implanted into the peri-infarction area of rats of different groups immediately after stroke modeling. Expression of BDNF mRNA was analyzed 7 days after surgery by real-time PCR. BDNF expression in cell preparation before their implantation was minimum. The expression of BDNF mRNA increased by 5-6 times in the areas of implantation of rat fibroblasts and human olfactory epithelium and by 23 times in the area of implantation of rat embryonic nervous tissue compared to periinfarction areas without cell implantation. These findings confirm the possibility of realization of the therapeutic effects of neural stem cells via expression of trophic factors.

Changes in Host Blood Factors and Brain Glia Accompanying the Functional Recovery after Systemic Administration of Bone Marrow Stem Cells in Ischemic Stroke Rats

In this study, we examined the effects of systemic administration of rat or human bone marrow stromal stem cells (MSC) at early and later times following middle cerebral artery occlusion (MCAO) on blood cytokines/growth factors, brain glia, and motor behavior in rats. Rats were tail vein injected with rat (r) and human (h) MSCs at 1 or 7 days post-MCAO. In some rats (N = 4) MSCs isolated from transgenic GFP rats were used to track the migration of cells peripherally and centrally at 2.5 and 28 days. Motor behavior was assessed using the modified Neurological Severity Score/climbing test at various time points before and after MCAO and transplantation. Prior to sacrifice at 1, 7, or 28 days post-MCAO, blood serum was collected for cytokine array analysis. Brains were analyzed for markers of activated microglia (CD11) and reactive astrocytes (GFAP). Administration of either allogeneic (rMSCs) or xenogeneic (hMSCs) stem cells produced a significant recovery of motor behavior after MCAO, with cells delivered at 1 day having greater effect than those at 7 days. Correlated with recovery was an amplification in activated microglia, reactive astrocytes, and new blood vessels in the infarct region, resulting in greater preservation in brain integrity. Concomitantly, expression of blood cytokines/chemokines (IL-13, MMP2, MIP) and growth factors/receptors (VEGF, neuropilin, EPOR, TROY, NGFR, RAGE) were modified following MSC administration. Because only rare GFP-labeled MSCs were observed in the brain, these effects did not depend on the central incorporation of stem cells. The early systemic administration of allogeneic or xenogeneic MSCs soon after experimental stroke produces a structural/functional recovery in the brain which is correlated with an increase in activated brain glia and changes in circulating cytokines and growth factors. Stem cells therefore induce an important neuroprotective and/or regenerative response in the host organism.

Morphofunctional Study of the Therapeutic Efficacy of Human Mesenchymal and Neural Stem Cells in Rats with Diffuse Brain Injury

We studied the effect of transplantation of human stem cells from various tissues on reparative processes in the brain of rats with closed craniocerebral injury. Combined treatment with standard drugs and systemic administration of xenogeneic stem cells had a neuroprotective effect. The morphology of neurons rapidly returned to normal after administration of fetal neural stem cells. Fetal mesenchymal stem cells produced a prolonged effect on proliferative activity of progenitor cells in the subventricular zone of neurogenesis. Adult mesenchymal stem cells had a strong effect on recovery of the vascular bed in ischemic regions.

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans.

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good long-term engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

Application of xenogeneic stem cells for induction of transplantation tolerance: present state and future directions.

Xenotransplantation using pig organs provides a possible solution to the severe shortage of allogeneic organ donors, one of the major limiting factors in clinical transplantation. However, because of the greater antigenic differences that exist between different species than within a species, the immune response to xenografts is much more vigorous than to allografts. Thus, tolerance induction is essential to the success of clinical xenotransplantation. Tolerance induced by mixed hematopoietic chimerism across the MHC barrier is remarkably robust, but its ability to induce tolerance across highly disparate xenogeneic barriers remains poorly studied. None of the current available regimens of host conditioning, which permit hematopoietic stem cell engraftment and chimerism induction in allogeneic or closely related (concordant) xenogeneic combinations, has been demonstrated to be effective in establishing porcine hematopoietic chimerism in a discordant xenogeneic species. Unlike bone marrow transplantation within the same species, the innate immune system and the species specificity of cytokines and adhesion molecules essential to hematopoiesis pose formidable obstacles to the establishment of donor hematopoiesis across discordant xenogeneic barriers. The genetic incompatibility between species may also impede xenograft tolerance induction by mixed chimerism. While we remain far from achieving tolerance in clinical xenotransplantation, recent studies using a transgenic mouse model have proven the principle that mixed hematopoietic chimerism may induce mouse and human T cell tolerance to porcine xenografts. This review article focuses on the barriers to porcine hematopoietic engraftment in highly disparate xenogeneic species and the possible application of mixed hematopoietic chimerism to xenograft tolerance induction.

129IN UTERO TRANSPLANTATION OF HUMAN HEMATOPOIETIC STEM CELLS (HSC) INTO FETAL GOATS OFFERS A NEW APPROACH TO STUDIES OF HSC EXPANSION AND DEVELOPMENT

In utero transplantation of xenogeneic stem cells into animals (such as mouse, sheep, goats) is a new approach to the studies of the engraftmen, expansion and development of foreign cells in vivo. In addition, such an approach is able to offer a model to clinical potential for correcting or replacing damaged tissues in a variety of disorders, including many that are inherent as well as acquired. Recently, we generated a human/goat hematopoietic stem cell (HSC) xenogeneic model to investigate the engraftment, expansion and development of human hematopoietic cells at an in vivo level. Human HSCs (lin-cells) were isolated and purified from fresh cord blood, and then injected intraperitoneally into pre-immune fetal goats at 55–65 gestation days (term: 145 days). A total of 50 fetal goats were injected. The growth and development of the recipient goats were monitored by B-type ultrasound scan. Ten goats suffered miscarriage due to GVH response; 39 recipients survived a complete gestation and were born alive. All these live-born goats were healthy and grew very well. They could produce offspring normally. In order to determine whether human cells were present longterm in the circulatory blood of the recipient goats, FACS, real-time PCR, RT-PCR, and Southern-blot hybridization as well as FISH were used to analyze the genotype and phenotype up to 11 months after birth. The results showed that hematopoietic chimerism (on average, &amp;gt;108 human cells per goat) was documented in 90% (35/39) of live-born goats that had been originally transplanted with 105 human HSCs. The regenerated human cells included myeloid, B-lymphoid and erythroid lineages as well as more primitive cells as shown by specific positive staining for human CD14, CD20, glycophorin A (GPA) and CD34, and the detection of human-specific DNA sequences and human GPA and CD34 transcripts in the blood of the transplanted goats. No human T or NK-lineage cells were detected. These studies demonstrate the feasibility of using in utero-transplanted fetal goats as a new approach for evaluating the longterm engraftment potential, expansion and development of human hematopoietic cells in vivo.

Porcine hematopoiesis on primate stroma in long-term cultures: enhanced growth with neutralizing tumor necrosis factor-alpha and tumor growth factor-beta antibodies.

Donor hematopoiesis is at a competitive disadvantage when bone marrow transplantation is across species barriers. This could present major limitations to xenogeneic stem cell transplantation as an approach to tolerance induction. An in vitro model of xenogeneic engraftment was established to identify inhibitors of porcine hematopoiesis in a primate environment. Porcine bone marrow cells (BMC), in the presence or absence of primate CD34+ positive cells, were cultured for 4-6 weeks on primate stroma with porcine cytokines. Cellularity and growth of colony-forming cells were indicators of hematopoietic growth. Effects of soluble factors were determined by using Transwell inserts to separate porcine cells from stroma. Neutralizing antibodies for human transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) were added to cultures. Porcine hematopoiesis can be maintained in long-term cultures on primate stroma with pig cytokines. Adding BMC to the stroma below Transwell-containing porcine cells dramatically inhibited porcine hematopoiesis, showing an inhibitory role for soluble factors. Neutralizing antibodies against TNF-alpha or TGF-beta caused a modest enhancement of porcine hematopoiesis; however, the combination of both led to a substantial increase. Inhibitory effects of these cytokines were confirmed by adding TNF-alpha and TGF-beta to porcine cultures. Porcine cells may be more sensitive to inhibitory effects of TNF-alpha and TGF-beta than primate cells and are at a disadvantage when in a primate environment. Potential implications of this observation are discussed in the context of establishing specific immune tolerance via mixed chimerism to facilitate xenotransplantation.

Enhancement of swine progenitor chimerism in mixed swine/human bone marrow cultures with swine cytokines

Objective: The induction of transplantation tolerance across xenogeneic barriers by bone marrow transplantation holds great promise, but engraftment of xenogeneic stem cells has been difficult to achieve. Part of this difficulty is due to species-specific differences in regulatory cytokines and elements of the stromal microenvironment, which we studied here. Materials and Methods: We developed a system where fresh bone marrow cells from swine and human are cultured on human bone marrow stroma in order to study these limiting factors in a clinically relevant species combination. Results: We report here the ability of recombinant swine interleukin (IL)-3 and c-kit ligand (KL) to specifically enhance swine hematopoietic chimerism in this system. In the absence of exogenous swine cytokines, there were about half as many swine progenitors as human progenitors at 1, 2, and 4 weeks of culture. When used alone, swine IL-3 led to a notable but transient increase in the relative ratio of swine progenitors, while addition of swine KL increased the ratio of swine progenitors only modestly and only at later time points. In contrast, when swine IL-3 and KL were added together, there was a two- to fourfold increase in the ratio of swine to human progenitors at all times tested. Conclusion: These data demonstrate that both swine IL-3 and KL are needed for prolonged enhancement of swine progenitor chimerism under these conditions, and suggest that the species specificity of either one or both of these cytokines may represent an important barrier to prolonged engraftment of swine bone marrow in humans.