Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

In vivo haematopoietic potential of human neural stem cells.

The fetal sheep model was used to compare the in vivo haematopoietic potential of human neural stem cells (NSC) versus bone marrow (BM)-derived haematopoietic stem cells (HSC). To this end, sheep were transplanted with either 8 x 10(5) NSC (n = 11) or HSC, CD34(+)Lin(-) (n = 5), and subsequently analysed for haematopoietic chimaerism. While HSC-transplanted sheep displayed robust donor-derived haematopoiesis starting at less than 2 months post-transplant, NSC recipients exhibited haematopoietic engraftment at much later time points. Nevertheless, chimaerism persisted in both groups throughout the course of this study. Transplantation of secondary recipients with human CD45(+)/HLA-DR(+) cells from the BM of NSC primary recipients at 14 and 16 months post-transplant demonstrated that long-term engrafting HSC were present in these animals. At 6 months post-transplant, both NSC- and HSC-transplanted sheep were mobilised with granulocyte colony-stimulating factor. In contrast to HSC-transplanted animals, levels of human blood cells in peripheral blood of NSC-transplanted sheep remained low throughout mobilisation. Our results show that, although human NSC were able to give rise to multilineage haematopoiesis in our model, the levels, timing of blood cell production and the ability to respond to cytokine mobilisation were different, suggesting that human NSCs latent haematopoietic potential is inherently different from that of true HSC.

Modelling of ex vivo expansion/maintenance of hematopoietic stem cells.

In this study, we described the modelling of the expansion/maintenance of human hematopoietic stem/progenitor cells from adult human bone marrow. CD 34(+)-enriched cell populations from bone marrow were cultured in the presence and absence of human stroma in serum-free media containing bFGF, SCF, LIF and Flt-3 ligand for several days. The cells in the culture were analysed for expansion and phenotype by flow cytometry. Although significant expansion of bone marrow cultures occurred in the presence and absence of human stroma, the results of expansion were effectively better in the presence of a stromal layer. In both situations the phenotypic analysis demonstrated a great expansion of CD 34(+)38(-) cells. The differentiative potential of bone marrow CD 34(+) cells co-cultured with human stroma was primarily shifted towards the myeloid lineage with the presence of CD 15 and CD 33.

Adult stem cell plasticity and methods of detection.

The ability to selectively produce one or more differentiated cell types at will from totipotent stem cells would be of profound clinical importance, as it would enable the specific replacement of damaged/dysfunctional cell types within the body, potentially curing numerous diseases. Until recently, it was thought that the only cells that possessed sufficient immaturity to be capable of giving rise to more than one tissue type in vitro and in vivo were the embryonic stem cells. However, recent studies have now provided compelling evidence that the adult bone marrow, brain and skeletal muscle contain stem cells that possess the remarkable ability to trans-differentiate and give rise to progeny of alternate embryologic derivations. These recent findings have shattered the existing dogma that the stages of embryologic development are irreversible. In this review, we present a brief summary of the most significant findings in the field of stem cell plasticity, emphasizing studies involving the hematopoietic system, discussing the models used thus far, and finishing with our findings on human stem cell plasticity using the fetal sheep model.

The monoclonal antibody W7C5 defines a novel surface antigen on hematopoietic stem cells.

We recently raised a monoclonal antibody, termed W7C5, against a surface antigen that is expressed at low levels on bone marrow and peripheral blood CD34+ stem/progenitor cells but at high levels on fetal liver CD34+ cells. A reasonable staining intensity was achieved using magnetofluorescent liposome conjugates to analyze expression of W7C5 antigen on CD34+CD38- bone marrow (BM) cells. Flow cytometric analyses revealed that W7C5 detects about 50% of immature CD34+CD38- BM cells that coexpressed the differentiation antigens CD164, CD133, and CD172a (SIRP alpha). In addition, W7C5 also recognized a CD34- BM fraction. These cells were negative for CD117 and CD133, but expressed CD45 and moderate levels of CD164. Injection of selected CD34+W7C5+ and CD34-W7C5+ cells into 55-60-day-old fetal sheep resulted in an engraftment of both fractions. Partial amino acid sequence analysis of affinity-purified lysates of KU-812 cells revealed that W7C5 detects a novel membrane protein. Together, W7C5 defines a novel molecule that is expressed on CD34+ as well as on CD34- stem cell subsets.

Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses.

The successful transduction of hematopoietic stem cells and long-term (28 months) transgene expression within the hematopoietic system following the direct injection of high-titer retroviral vectors into preimmune fetal sheep was previously demonstrated. The present studies extended these analyses for 40 months postinjection and evaluated whether the longevity of transgene expression in this model system was the result of induction of prenatal tolerance to the transgene product. The intraperitoneal injection of retroviral vectors into preimmune sheep fetuses transduces thymic epithelial cells thought to present antigen and thus define self during immune system development. To directly demonstrate induction of tolerance, postnatal sheep were boosted with purified beta-galactosidase and showed that the peripheral blood lymphocytes from in utero-transduced sheep exhibited significantly lower stimulation indices to transduced autologous cells than did control animals and that the in utero-transduced sheep had a reduced ability to mount an antibody response to the vector-encoded beta-galactosidase protein compared with control sheep. Collectively, our results provide evidence that the direct injection of retroviral vectors into preimmune sheep fetuses induces cellular and humoral tolerance to the vector/transgene products and provide an explanation for the duration and stability of transgene expression seen in this model. These results also suggest that even relatively low levels of gene transfer in utero may render the recipient tolerant to the exogenous gene and thus potentially permit the successful postnatal treatment of the recipient. (Blood. 2001;97:3417-3423)

Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system.

This report describes stroma-based and stroma-free cultures that maintain long-term engrafting hematopoietic cells for at least 14 days ex vivo. Umbilical cord blood (UCB) CD34(+) cells were cultured in transwells above AFT024 feeders with fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + interleukin 7 (IL-7), or FL + thrombopoietin (Tpo). CD34(+) progeny were transplanted into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice or preimmune fetal sheep. SCID repopulating cells (SRC) with multilineage differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo containing cultures for up to 28 days. Marrow from mice highly engrafted with uncultured or expanded cells induced multilineage human hematopoiesis in 50% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep. Day 14 expanded cells, although engrafting primary and to a lesser degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary recipients were maintained for at least 14 days in medium containing glycosaminoglycans and cytokines found in stromal supernatants. This is the first demonstration that ex vivo culture in stroma-noncontact and stroma-free cultures maintains "long-term" engrafting cells, defined by their capacity to engraft secondary or tertiary hosts. (Blood. 2001;97:3441-3449)

Evaluation of serum-free culture conditions able to support the ex vivo expansion and engraftment of human hematopoietic stem cells in the human-to-sheep xenograft model.

To develop culture conditions devoid of serum that would support the ex vivo expansion and maintenance of hematopoietic stem cells (HSC) with engraftment capability, we performed in vitro studies in which phenotypic and functional expansion of putative HSC populations were evaluated. We then used the human-sheep xenograft model to evaluate the engraftment potential of the ex vivo expanded cells. Adult human bone marrow CD34+-enriched cells were cultured in QBSF-60 for 14 days with or without fetal bovine serum (FBS) in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), and analyzed at days 0, 3, 7, and 14 for expansion, phenotype, clonogenic ability, and cell cycling status. Although there was a progressive expansion of numbers of cells in both groups, the group cultured with serum exhibited more than twice the expansion seen in the group without serum at all time points. The phenotypic analysis of the cultured cells showed an increase in the absolute numbers of CD34+ cells in both groups. However, when we evaluated the presence of CD34+CD38- cells, this population persisted in significantly higher numbers in the group cultured without serum, with maximal output of CD34+CD38- cells seen at 3 and 7 days. A higher total clonogenic potential was found in the serum-free cultures. To evaluate the in vivo engraftment potential of these cultured cells, 19 sheep fetuses were each injected i.p. with 9 x 10(5) cells either fresh or cultured in the conditions described above. Although all the transplanted fetal sheep showed the presence of human cells in their bone marrow (BM), the highest levels of long-term engraftment in primary recipients were obtained with the fraction of cells cultured for 3 days followed by 7 days in the absence of serum. In the secondary sheep recipients, the highest level of long-term engraftment was also achieved in sheep that received cells from primary recipients that had received cultured cells in serum-free conditions for 3 days.

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow.

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.

Neonatal gene therapy. transfer and expression of exogenous genes in neonatal sheep following direct injection of retroviral vectors into the bone marrow space.

We investigated whether gene transfer into hematopoietic cells could be achieved by direct injection of retroviral vector supernatant into the bone marrow space of newborn sheep. Six sheep (5 weeks old) were injected bilaterally with either 1 mL of G1nBgSvNa8.1 vector supernatant (titer: 1 x 10(7)) in each hip (n = 5) or with 3 mL of the same vector preparation/hip (n = 1). In addition, one 3-month-old sheep was injected unilaterally with 1 mL of the same vector preparation. Blood and marrow of these animals were analyzed for the transgene before injection and at intervals thereafter. At 1 week postinjection, an average of 11.6% of the lymphocytes and 25.5% of the granulocytes/monocytes in the marrow, and an average of 0.9% of the lymphocytes and 1.8% of the granulocytes/monocytes in the blood contained and expressed the LacZ gene. The presence/expression of the transgene has persisted for at least 13 months within the blood and bone marrow of these animals. These findings demonstrate that the direct injection of small volumes of high-titer retroviral supernatant into the bone marrow of newborn sheep results in transduction of hematopoietic cells that persists for at least 13 months postinjection.

Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation.

Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human-sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.

In utero transfer and expression of exogenous genes in sheep.

OBJECTIVE: We have previously reported that directly injecting low-titer retroviral vector supernatant into pre-immune sheep fetuses resulted in the transfer and long-term expression of the bacterial NeoR gene within the hematopoietic system of these animals for over 5 years. In the present studies, we investigated whether using a higher titer vector would enable more efficient transduction and expression of the transgenes within the hematopoetic cells in sheep injected in utero. MATERIALS AND METHODS: Sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatant encoding the bacterial NeoR and LacZ genes (titer: 1x10(7) cfu/mL). RESULTS: Over the 2-year time course of these studies, the presence and expression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained > or =6% transduced peripheral blood lymphocytes. Vector distribution was widespread without any detectable pathology. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered. CONCLUSIONS: These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving long-term expression of the transgenes within the recipient's hematopoietic cells. Furthermore, expression of the NeoR gene from these studies was higher than that reported in our previous study in which a lower titer vector was used.

In vitro infection of megakaryocytes and their precursors by human cytomegalovirus.

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34(+) cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42(+) MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the beta-galactosidase (beta-gal) gene. Using chromogenic beta-gal substrates, HCMV was detected during MK differentiation of infected CD34(+) cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV. (Blood. 2000;95:487-493)

Prospects for in utero human gene therapy.

Gene therapy for the treatment of disease in children and adults is being actively pursued at many medical centers. However, a number of genetic disorders result in irreversible damage to the fetus before birth. In these cases, as well as for those with genetic diseases who may benefit from therapy before symptoms are manifested, in utero gene therapy (IUGT) could be beneficial. Although some successes with in utero gene transfer have been reported in animals, significant questions remain to be answered before IUGT clinical trials would be acceptable. This review analyzes the state of the art and delineates the studies that still need to be performed before it would be appropriate to consider human IUGT.

Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero.

Although promising, clinical and experimental efforts at in utero hematopoietic stem cell (HSC) transplantation currently are limited by minimal donor cell engraftment and lack of early donor cell expression after transplantation. We reasoned that cotransplantation of stromal elements (ST) might condition the fetal microenvironment for the engraftment of donor HSC and facilitate precocious bone marrow (BM) hematopoiesis. In this study we cotransplanted sheep ST, derived from adult or fetal BM, with either adult or fetal HSC, into preimmune fetal sheep. We analyzed donor cell chimerism in BM and peripheral blood and compared levels of chimerism achieved with recipients of HSC alone. In all experimental groups, stromal cotransplantation markedly increased the level of peripheral blood donor cell expression at 60 days after transplantation relative to controls. Adult BM-derived stroma cotransplanted with adult HSC provided the highest levels of circulating donor cells, whereas fetal-derived stroma was less effective. In addition, ST cotransplantation resulted in increased donor cell engraftment in the BM and led to significantly increased levels of donor hematopoiesis for over 30 months after transplant. Cotransplantation of stroma may represent a valuable clinical strategy for optimal application of in utero HSC transplantation.

Homing of human cells in the fetal sheep model: modulation by antibodies activating or inhibiting very late activation antigen-4-dependent function.

The mechanisms by which intravenously (IV)-administered hematopoietic cells home to the bone marrow (BM) are poorly defined. Although insightful information has been obtained in mice, our knowledge about homing of human cells is very limited. In the present study, we investigated the importance of very late activation antigen (VLA)-4 in the early phases of lodgment of human CD34(+) progenitors into the sheep hematopoietic compartment after in utero transplantation. We have found that preincubation of donor cells with anti-VLA-4 blocking antibodies resulted in a profound reduction of human cell lodgment in the fetal BM at 24 and 48 hours after transplantation, with a corresponding increase of human cells in the peripheral circulation. Furthermore, IV infusion of the anti-VLA-4 antibody at later times (posttransplantation days 21 to 24) resulted in redistribution or mobilization of human progenitors from the BM to the peripheral blood. In an attempt to positively modulate homing, we also pretreated human donor cells with an activating antibody to beta1 integrins. This treatment resulted in increased lodgment of donor cells in the fetal liver, presumably for hemodynamic reasons, at the expense of the BM. Given previous involvement of the VLA-4/vascular cell adhesion molecule (VCAM)-1 adhesion pathway in homing and mobilization in the murine system, our present data suggest that cross-reacting ligands (likely VCAM-1) for human VLA-4 exist in sheep BM, thereby implicating conservation of molecular mechanisms of homing and mobilization across disparate species barriers. Thus, information from xenogeneic models of human hematopoiesis and specifically, the human/sheep model of in utero transplantation, may provide valuable insights into human hematopoietic transplantation biology.

KDR receptor: a key marker defining hematopoietic stem cells.

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.