Porcine Microvascular Endothelial Cells Research Articles

Bone marrow CD34+ cells expanded on human brain endothelial cells reconstitute lethally irradiated baboons in a variable manner

Increased cell dose has a positive impact on the therapeutic outcome of bone marrow (BM) hematopoietic stem cell (HSC) transplant. However, methods to successfully expand BM HSCs have yet to be achieved. It has been shown previously that ex vivo expansion of BM cells using porcine microvascular endothelial cells can rescue a baboon from a lethal dose of radiation. However, in a prior study, baboons that received CD34+ cell doses less than 4 × 106 cells/kg body weight failed to achieve hematopoietic reconstitution. In our present study we used human brain endothelial cells (HUBECs) and cytokines to expand BM cells, and examined their ability to provide hematopoietic reconstitution in three lethally irradiated baboons following autologous transplant as a surrogate preclinical model. After ex vivo culture, the grafts represented a 1.8- to 2.1-fold expansion of CD34+ cells, a 3.7- to 13.2-fold increase of colony-forming cells, and a 1.9- to 3.2-fold increase of cobblestone area-forming cells, in comparison to the input cell numbers. Despite transplanting CD34+ cell grafts displaying a comparable degree of expansion, there was an obvious variability in the kinetics of hematopoietic reconstitution. The variation in hematopoietic reconstitution cannot be fully explained by the properties tested in expanded CD34+ cells, and warrant caution against taking into account such attributes as cell dose, expression of adhesion molecules, and migration as a measure of successful expansion of HSCs.

Bone Marrow CD34+ Cells Expanded On Human Brain Endothelial Cells Reconstitutes Lethally Irradiated Baboons in a Variable Manner.

Abstract 3214Poster Board III-151Increased cell dose has a positive impact on the therapeutic outcome of bone marrow (BM) transplantation. However, methods to successfully expand hematopoietic stem cells (HSC) from BM have yet to be achieved. It has been shown previously that ex vivo expansion of BM cells using porcine microvascular endothelial cells can rescue a baboon from a lethal dose of radiation (Brandt et al. Blood 1999). However, in prior studies baboons that received CD34+ cell doses less than 4 × 106 cells/kg body weight failed to achieve hematopoietic engraftment. In our current studies we have used human brain endothelial cells (HUBECs) and cytokines to expand BM cells and examined their ability to provide hematopoietic reconstitution in three lethally irradiated baboons following autologous transplantation. After ex vivo culture, the grafts represented a 1.8- to 2.1-fold expansion of CD34+ cells, a 3.7-fold to 13.2-fold increase of colony-forming cells (CFC), and a 1.9-fold to 3.2-fold increase of cobblestone area-forming cells (CAFC) in comparison to the input cell numbers. The animal (PA6873) which received expanded product of a suboptimal dose of CD34+ cells (1.6 × 106/kg) achieved only myeloid engraftment (day 24). Out of 3 baboons transplanted two displayed myeloid engraftment, one animal achieved both myeloid and platelet engraftment and the third animal (PA6888) failed to achieve engraftment. The animal (PA6893) which received the expanded product of 4.9 × 106/kg CD34+ cells achieved myeloid engraftment (WBC > 500/ml blood) by day 8 and platelet engraftment (>20,000/μl) by day 39. The WBC recovery of this baboon was comparable but the platelet recovery was delayed (31 day vs. 39 day) in comparison to that experienced by an animal that received a large number of unexpanded CD34+ cells (26 × 106 /kg). Interestingly, despite the grafts of all three animals having a similar degree of CD34+ cell expansion, similar progenitor cell (CFC, CAFC) expansion, a similar pattern of cell adhesion molecule expression and similar migration capacity across an SDF1 gradient, the hematopoietic reconstitution capacity of each graft differed greatly. Prior studies using human BM cells in non-contact HUBEC co-cultures in NOD/SCID mice demonstrated no such variability. Our current data indicates three possibilities for the variations in hematopoietic reconstitution observed in a baboon model. One possibility is that there might be cell autonomous biological variation of HUBEC expanded BM grafts. The second possibility is the expanded graft may have contained variable numbers of contaminating endothelial cells (HUBEC) contributing to variations in hematopoietic reconstitution. Transplantation of vascular endothelial cells without HSC rescue has been shown to enhance hematopoietic recovery following radiation in mice (Chute et al. Blood 2007). Biological variation amongst the hosts is unlikely since identical pre-transplant conditioning and post-transplant care was provided to all three baboons. In addition, since only non-adherent cells were harvested from the co-culture, it is possible that more primitive repopulating HSCs embedded within the endothelial monolayer might have been excluded from the graft. Taken together, our findings highlight inherent differences in the hematopoietic reconstitution capacity of expanded BM grafts in xenotransplantation studies and large animal models. DisclosuresNo relevant conflicts of interest to declare.

Ex vivo culture rescues hematopoietic stem cells with long-term repopulating capacity following harvest from lethally irradiated mice

Objective High-dose ionizing radiation can cause lethal myeloablation in exposed individuals. We examined whether ex vivo culture could rescue hematopoietic stem cells with repopulating capacity following harvest from lethally irradiated animals. Methods We exposed B6.SJL mice to 1050 cGy, harvested their irradiated bone marrow (BM), and examined whether ex vivo culture of the irradiated BM mononuclear cells (MNC) with porcine microvascular endothelial cells (PMVEC) or cytokines alone could rescue hematopoietic cells with in vitro colony-forming activity, in vivo radioprotective capacity, and long-term repopulating potential. Results PMVEC coculture supported the recovery of fourfold and 80-fold greater numbers of total cells and colony-forming cells (CFC) compared to cyokines alone following 1050 cGy irradiation. All control mice irradiated with 1050 cGy died by day 30, as did mice transplanted with 1050 cGy–irradiated BM MNC. In contrast, transplantation of 1050 cGy–irradiated/PMVEC-cultured BM was fully radioprotective in 12 of 16 recipient mice (75%) exposed to 1050 cGy. Six of the 12 CD45.2 + mice (50%) transplanted with 1050 cGy–irradiated/PMVEC-cultured cells showed long-term (>6 months) multilineage repopulation derived from irradiated donor CD45.1 + cells. Surprisingly, transplantation of identical doses of 1050 cGy–irradiated/cytokine-cultured BM was also radioprotective in 50% of irradiated recipient mice and 50% of these mice demonstrated donor-derived repopulation. Conclusions Fully functional BM stem and progenitor cells can be rescued following harvest from lethally irradiated animals via ex vivo culture with PMVEC or cytokines alone. This method can serve as a model for the rapid ex vivo rescue and transplantation of autologous BM progenitors in the treatment of victims of radiation injury.

Rescue of Hematopoietic Stem Cells Following High-Dose Radiation Injury Using Ex Vivo Culture on Endothelial Monolayers

Military personnel are at significant risk for potentially lethal myeloablative injury secondary to nuclear accident, nuclear attack, or chemical weapons attack. In an attempt to develop culture conditions in which hematopoietic stem cells might be rescued from the effects of radiation, we irradiated (1,000 cGy split dose) 6-week-old C57BL/6 (Ly 5.1) and syngeneic C57BL/6 (Ly 5.2) mice and tested whether bone marrow mononuclear cells (MNCs) harvested postirradiation could be rescued via coculture in porcine microvascular endothelial cell (PMVEC) monolayers. We found that a subpopulation of bone marrow MNCs exposed to 1,000 cGy could be maintained and expanded over 10 days in a PMVEC culture (3.8-fold expansion), whereas liquid suspension culture did not maintain a significant number of hematopoietic cells postirradiation. Colony-forming assays demonstrated that murine MNCs exposed to 1,000 cGy did not give rise to granulocyte/macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-e), or CFU-Mix in 14-day cultures, whereas irradiated MNCs that were subsequently cultured in PMVEC-generated CFU-GM, BFU-e, and CFU-Mix at cloning efficiencies of 1.3%, 0.2%, and 0.2%, respectively. In survival studies, we found that 78% of mice that were irradiated and then transplanted with irradiated/PMVEC-expanded MNCs were alive at day 50, compared with 18% of irradiated control mice (p < 0.05). We also observed that mice transplanted with irradiated/PMVEC-expanded MNCs showed complete hematologic recovery. At 8 weeks post-transplant, we found evidence of Ly 5.1 donor cells in both the bone marrow and the spleen of the transplanted animals, but the levels of engraftment were low (range, 0-5.1%; mean, 1.9%). These results demonstrate that a subpopulation of bone marrow stem cells are capable of surviving the effects of high-dose radiation if these cells are placed in coculture with endothelial cell monolayers.

Characterization of human killer cell reactivity against porcine target cells: differential modulation by cytokines.

The cytotoxic cell response to porcine cells by human lymphocytes, and the modulation of cytolytic cellular activity by human cytokines were investigated. Human peripheral blood mononuclear cells (PBMC) and purified lymphocyte subsets were co-cultured with fresh irradiated porcine stimulator cells and examined for the development of lytic activity and for their proliferative response. Porcine target cells included a new cell line, MS-PBMC-J2 (designated J2; SLA-DR+MHC class I+CD2+CD3 CD8+CDI6+CD45+), aortic and microvascular endothelial cells. Initial results showed that natural killer (NK) cells were fivefold more efficient in killing porcine target cells compared with T cells. IL-12 augmented the killing of porcine target cells by human NK cells beyond that induced by stimulation with cells alone. In contrast, IL-2 and IL-15 often induced substantial human NK cell mediated killing of porcine target cells, including endothelial cells in the case of IL-2 where such targets were examined, even in the absence of stimulator cells. Finally, neither IL-18 nor IL-8 had any effect beyond background on NK cell mediated killing of porcine target cells. These findings show that cytokines that would be produced in a xenograft setting clearly modulate the ability of human cytolytic cells to kill porcine targets. In addition, fresh unstimulated human NK cells lysed J2 and porcine aortic endothelial cells, but not porcine microvascular endothelial cells, suggesting the possibility of rapid attack of xenografts by NK cells, and differential susceptibility of endothelial cells from different vascular structures to this attack.

Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation.

Although there is strong evidence that plasma HDL levels correlate inversely with the incidence of coronary artery disease, the precise mechanism(s) for the protective effect of HDLs remains unclear. We recently showed that HDLs inhibit endothelial cell expression of cytokine-induced leukocyte adhesion molecules in vitro. Our study therefore sought to test the hypothesis that elevating the level of circulating HDLs would inhibit endothelial cell activation in vivo. We used a porcine model of inflammation previously established in our laboratory, in which the level of vascular endothelial cell expression of E-selectin in interleukin (IL)-1alpha-induced skin lesions was measured by the uptake of a radiolabeled anti-E-selectin antibody (1.2B6). Porcine plasma HDL levels were elevated by use of a bolus injection of reconstituted discoidal HDL (recHDL). These particles resemble nascent HDL particles in shape and contain apolipoprotein A-I as the sole protein and soybean phosphatidylcholine as the sole phospholipid. We found that recHDLs inhibited the expression of IL-1alpha-induced E-selectin by porcine aortic endothelial cells in vitro, confirming that the inhibitory effect is conserved with synthetic HDLs and demonstrating that the phenomenon is not restricted to human endothelial cells. In vivo, elevating the circulating level of HDLs approximately 2-fold led to significant inhibition of basal and IL-1alpha-induced E-selectin expression by porcine microvascular endothelial cells. These observations demonstrate the potential anti-inflammatory action of HDLs and provide support for the further investigation of the mechanisms underlying the inhibitory effects of HDLs on endothelial cell activation.

Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells.

Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.

An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells

The marrow repopulating potential (MRP) of different sources of human hematopoietic stem cells (HSCs) was directly compared using an in vivo assay in which severe combined immunodeficient disease (SCID) mice were implanted with human fetal bones. HSCs from 2 human lymphocyte antigen (HLA)-mismatched donors were injected individually or simultaneously into the fetal bones of a 3rd distinct HLA type and donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB) and adult bone marrow (ABM) CD34+ cells as well as grafts of each type expanded ex vivo. Equal numbers of CB and ABM CD34+ cells injected individually demonstrated similar abilities to establish multilineage hematopoiesis. However, when CB and ABM cells were transplanted simultaneously, the engraftment of CB cells was markedly superior to ABM. CB and ABM CD34+ cells were expanded ex vivo using either a porcine microvascular endothelial cell (PMVEC)-based coculture system or a stroma-free expansion system. Primary CB CD34+ cells or CD34+ cells expanded in either culture system demonstrated a similar ability to engraft. However, the MRP of expanded grafts simultaneously injected with primary CB cells was uniformly inferior to primary CB cells. CD34+ cell grafts expanded in the stroma-free system, furthermore, outcompeted CD34+ cells expanded using the PMVEC coculture system. The triple HLA-mismatched SCID-hu model represents a novel in vivo stem cell assay system that permits the direct demonstration of the functional consequences of ex vivo HSC expansion and ontogeny-related differences in HSCs.

An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells

AbstractThe marrow repopulating potential (MRP) of different sources of human hematopoietic stem cells (HSCs) was directly compared using an in vivo assay in which severe combined immunodeficient disease (SCID) mice were implanted with human fetal bones. HSCs from 2 human lymphocyte antigen (HLA)-mismatched donors were injected individually or simultaneously into the fetal bones of a 3rd distinct HLA type and donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB) and adult bone marrow (ABM) CD34+ cells as well as grafts of each type expanded ex vivo. Equal numbers of CB and ABM CD34+ cells injected individually demonstrated similar abilities to establish multilineage hematopoiesis. However, when CB and ABM cells were transplanted simultaneously, the engraftment of CB cells was markedly superior to ABM. CB and ABM CD34+ cells were expanded ex vivo using either a porcine microvascular endothelial cell (PMVEC)-based coculture system or a stroma-free expansion system. Primary CB CD34+ cells or CD34+ cells expanded in either culture system demonstrated a similar ability to engraft. However, the MRP of expanded grafts simultaneously injected with primary CB cells was uniformly inferior to primary CB cells. CD34+ cell grafts expanded in the stroma-free system, furthermore, outcompeted CD34+ cells expanded using the PMVEC coculture system. The triple HLA-mismatched SCID-hu model represents a novel in vivo stem cell assay system that permits the direct demonstration of the functional consequences of ex vivo HSC expansion and ontogeny-related differences in HSCs.

Cocultivation of umbilical cord blood cells with endothelial cells leads to extensive amplification of competent CD34 +CD38 − cells

Objective In this report, methods to expand the number of human cord blood hematopoietic stem cells were explored. Materials and Methods CD34 + cord blood cells were expanded in the presence of various cytokine combinations in either a stroma-free cell culture system or a preformed porcine microvascular endothelial cell layer. After 7 to 21 days, stem cell number and function were monitored. In addition, the replicative history of stem cells was tracked using the fluorescent dye, PKH26. Results With the addition of various cytokine combinations, total cellular expansion was equivalent for both culture systems, although the endothelial cell–based system contained statistically greater numbers of CD34 + cells. By day 21, the endothelial-based system receiving the FLT3L, SCF, IL-6, and GM-CSF cytokine combination contained five-fold greater numbers of CD34 + than the stroma cell–free culture cell system. Endothelial-based cultures receiving these four cytokines plus megakaryocyte growth and development factor produced a 640-fold expansion of CD34 + CD38 − cells as compared to a four-fold expansion in the stroma-free system. The number of progenitor cells generated was similar with both systems, yet the greatest degree of expansion of cobblestone area–forming cells was observed in the endothelial based cultures (11-fold vs four-fold). Virtually all CD34 + and CD34 + CD38 + cells expanded in the presence of endothelial cells had undergone self replication by day 10, yet stromal cell–free cultures contained a significant number (4.8%) of quiescent cells. Identical numbers of re-isolated cord blood CD34 + cells expanded in both systems exhibited a similar ability to engraft and generate cells belonging to multiple hematopoietic lineages in human fetal bones implanted in immunodeficient mice. Conclusions These results suggest that the use of preformed endothelial cell monolayers might permit the ex vivo generation of sufficient numbers of cord stem cells to serve as successful grafts for adult transplant recipients.

Ex Vivo Expansion of Autologous Bone Marrow CD34+ Cells With Porcine Microvascular Endothelial Cells Results in a Graft Capable of Rescuing Lethally Irradiated Baboons

Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow repopulation. Our group has demonstrated that human HSC expanded ex vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, our laboratory has established a myeloablative, fractionated total body irradiation conditioning protocol for autologous marrow transplantation in baboons. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 43, and 59 days postirradiation, respectively. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in PMVEC coculture with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone area-forming cells (CAFC) over input. Both baboons became transfusion independent by day 23 posttransplant and achieved absolute neutrophil count (ANC) >500/microL by day 25 +/- 1 and platelets >20,000/microL by day 29 +/- 2. This hematopoietic recovery was delayed in comparison to two animals that received either a graft consisting of freshly isolated, unexpanded CD34(+) cells or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergone cycling, and that the population of cells expressing a CD34(+) CD38(-) phenotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell division during ex vivo expansion in the presence of endothelial cells to provide a graft capable of rescuing a myeloablated autologous host.

A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34 + cells cultured in stroma-free versus porcine microvascular endothelial cell cultures

Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34 +CD38 − cells that are capable of multilineage engraftment within the bone marrow of a secondary host. CD34 +CD38 − cells contain the self-renewing, long-term culture-initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments. Previous experiments attempting retroviral infection of CD34 +CD38 − cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34 + cells and the CD34 +CD38 − subset before and after ex vivo expansion on PMVECs. Purified human CD34 + cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage–colony-stimulating factor (GM-CSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand. The total CD34 + population and the CD34 +CD38 − subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit–granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells. After 7 days of coculture with PMVECs, 44% of the CD34 +CD38 + subset were found to be in G 1, and 51% were in G 2/S/M phase of the cell cycle. More remarkably, 53% of the CD34 +CD38 − subset were in G 1, and 17% were in G 2/S/M phase after 7 days of PMVEC coculture. In contrast, only 22% of the CD34 +CD38 − subset remaining after 7 days of stroma-free culture were in G 1, and 6% were in G 2/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L selectin) increased after ex vivo expansion. In contrast, CD34 + cells expanded on stroma-free cultures showed lower and more variable expression of CD62L and CD15s. These findings demonstrate that the primitive CD34 +CD38 − subset of marrow progenitor cells can be induced to enter cell cycle and can be significantly expanded ex vivo on a hematopoietic supportive microenvironment (PMVECs) while preserving the expression of cell adhesion molecules that may be important in stem cell homing and engraftment.

COSTIMULATORY PATHWAYS ARE ACTIVE IN XENOGENEIC IMMUNE RESPONSES

36 Costimulatory blockade has been shown in this and other laboratories to prevent allograft rejection. Recent advances in the control of the innate humoral response to discordant xenografts have focused attention on potential means of preventing subsequent cellular and acquired humoral reactivity. A variety of costimulatory pathway blockers have been tested in mixed lymphocyte reactions (MLR) for their ability to prevent human peripheral blood mononuclear cell (PBMC) activation when stimulated by irradiated porcine splenocytes. The results that human CTLA4-Ig is capable of attenuating the xenoreactive PBMC proliferation by 60%, anti-human CD40L(5C8) by 52%, anti-human CD80 (1F1) by 13%, and anti-human CD86 (3D1) by 28%. Furthermore, combination of these reagents proves to have synergistic effects. The combination of hCTLA4-Ig/5C8 attenuates the response by 76% and the 1F1/3D1/5C8 combination by 72%. Consistent with this observation, LPS stimulated porcine splenocytes have been shown by flow cytometry to specifically bind hCTLA4-Ig, 1F1, and 3D1. A panel of 12 anti-human CD80 and CD86 antibodies have been screened with only 1F1 and 3D1 exhibiting cross reactivity to pig. In addition, using irradiated human PBMCs as stimulators and porcine PBMC's as the responding cells revealed that the combination of 5C8 and hCTLA4-Ig is also capable of blocking pig cell proliferation. It would thus appear that costimulatory ligands are sufficiently homologous, such that they support cross species ligand/receptor interaction which is important in the xenoreactive response between human and porcine cells. Preliminary evidence also suggests that blockade of CD86 on the porcine microvascular endothelial cell (PMVEC) inhibits their killing by human NK cells. This is consistent with the constitutive expression of CD86 on PMVEC's that we have observed. Furthermore, we have demonstrated that CD86 is upregulated by exposure to human T and NK cell and human T cell conditioned media. We conclude that costimulatory molecules play an important role in cellular xenoreactivity and that the same reagents used to prevent allograft rejection may function in the prevention of xenograft rejection. These results and the known effects of 5C8 on isotype switching and maturation of acquired humoral responses efficacy of these reagents in modulating both the humoral and cellular immune response. We are now in the process of initiating these studies in a pig to primate model.

COSTIMULATORY PATHWAYS ARE ACTIVE IN XENOGENEIC IMMUNE RESPONSES.

36 Costimulatory blockade has been shown in this and other laboratories to prevent allograft rejection. Recent advances in the control of the innate humoral response to discordant xenografts have focused attention on potential means of preventing subsequent cellular and acquired humoral reactivity. A variety of costimulatory pathway blockers have been tested in mixed lymphocyte reactions (MLR) for their ability to prevent human peripheral blood mononuclear cell (PBMC) activation when stimulated by irradiated porcine splenocytes. The results indicate that human CTLA4-Ig is capable of attenuating the xenoreactive PBMC proliferation by 60%, anti-human CD40L (5C8) by 52%, anti-human CD80 (1F1) by 13%, and anti-human CD86 (3D1) by 28%. Furthermore, combinations of these reagents proves to have synergistic effects. The combination of hCTLA4-Ig/5C8 attenuates the response by 76% and the 1F1/3D1/5C8 combination 72%. Consistent with this observation, LPS stimulated porcine splenocytes have been shown by flow cytometry to specifically bind hCTLA4-Ig, 1F1, and 3D1. A panel of 12 anti-human CD80 and CD86 antibodies have been screened with only 1F1 and 3D1 exhibiting cross reactivity to pig. In addition, using irradiated human PBMCs as stimulators and porcine PBMC's as the responding cells revealed that the combination of 5C8 and hCTLA4-Ig is also capable of blocking pig cell proliferation. It would thus appear that costimulatory ligands are sufficiently homologous, such that they support cross species ligand/receptor interaction which is important in the xenoreactive response between human and porcine cells. Preliminary evidence also suggests that blockade of CD86 on the porcine microvascular endothelial cell (PMVEC) inhibits their killing by human NK cells. This is consistent with the constitutive expression of CD86 on PMVEC's that we have observed. Furthermore, we have demonstrated that CD86 is upregulated by exposure to human T and NK cell and human T cell conditioned media. We conclude that costimulatory molecules play an important role in cellular xenoreactivity and that the same reagents used to prevent allograft rejection may function in the prevention of xenograft rejection. These results and the known effects of 5C8 on isotype switching and maturation of acquired humoral responses suggests efficacy of these reagents in modulating both the humoral and cellular immune response. We are now in the process of initiating these studies in a pig to primate model.

Soluble factor(s) alone produced by primary porcine microvascular endothelial cells support the proliferation and differentiation of human CD34 + hematopoietic progenitor cells with a high replating potential

Soluble factor(s) alone produced by primary porcine microvascular endothelial cells support the proliferation and differentiation of human CD34 + hematopoietic progenitor cells with a high replating potential

CONDITIONED MEDIUM FROM PRIMARY PORCINE ENDOTHELIAL CELLS ALONE PROMOTES THE GROWTH OF PRIMITIVE HUMAN HAEMATOPOIETIC PROGENITOR CELLS WITH A HIGH REPLATING POTENTIAL: EVIDENCE FOR A NOVEL EARLY HAEMATOPOIETIC ACTIVITY

The authors have recently shown that direct contact with primary porcine microvascular endothelial cell monolayers (PMVECs) in combination with haematopoietic growth factors enhances the expansion of primitive human haematopoietic CD34+bone marrow progenitor cells. It is now demonstrated that serum-free conditioned medium (PMVEC CM, concentrated 70× for proteins >30 kDa) from untreated PMVECs contains haematopoietic growth factor activity that enhances the in vitro proliferation, haematopoietic cell production, and colony cell formation of primitive human haematopoietic progenitor cells. In combination with exogenously added human growth factors such as interleukin 3 (IL-3), granulocyte–-macrophage colony-stimulating factor (GM-CSF) and EPO, PMVEC CM enhances the proliferation and colony growth of human haematopoietic CD34+cells. In contrast, PMVEC CM has no significant synergistic activity on either stem cell factor (SCF) of flt3-ligand-induced CD34+cell proliferation, cell production or colony formation. Blocking mAbs against the c-kit receptor have no effect on PMVEC CM-induced CD34+cell proliferation at titres that completely suppress SCF-induced proliferation. Moreover, it is shown that this haematopoietic growth factor supports the proliferation and colony formation of murine, non-human primate, and porcine marrow progenitor cells without any apparent species-specific restrictions in its activity. These finding suggest that PMVEC CM contains a novel early haematopoietic activity.

Porcine Brain Microvascular Endothelial Cells Support the In VitroExpansion of Human Primitive Hematopoietic Bone Marrow ProgenitorCells With a High Replating Potential: Requirement for Cell-to-CellInteractions and Colony-Stimulating Factors

Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).