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Biomedical subjects

E D Zanjani

Publications and source records attributed to E D Zanjani.

At least 19 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed2000

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)

Antigens, CD↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Adult↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Engraftment and multilineage expression of human bone marrow CD34- cells in vivo.

The fetal sheep competitive engraftment model of human hematopoietic stem cells (HSC) was used to evaluate the in vivo engraftment potential of human bone marrow CD34- Lin- cells. Transplantation of CD34- Lin- cells into primary hosts resulted in the long-term (> 1 year) engraftment and multilineage donor cell/progenitor expression with production of significant numbers of CD34+ cells. Secondary transplantation and limiting dilution studies confirmed the presence in human CD34- fraction of HSC with in vivo long-term engraftment and multilineage differentiation potentials.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses.

We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.

Animals↗
PubMed1998

In utero transplantation for thalassemia.

In utero hematopoietic stem cell transplantation is a promising approach for the treatment of a variety of congenital hematologic diseases. Although the approach has been successful for immunodeficiency syndromes, attempts thus far to treat the hemoglobinopathies have failed. In most of these cases the late gestational age at transplantation, source of donor cells, or procedure-related complications, provide an explanation for failure. Nevertheless the biology of thalassemia, in the context of prenatal transplantation, requires examination. In contrast to postnatal bone marrow transplant regimens, engraftment after in utero transplantation requires donor cells to effectively complete for developing receptive sites in the recipient hematopoietic microenvironment. Effective prenatal treatment of thalassemia will depend on the ability of normal cells to engraft and complete in the thalassemic microenvironment. Clinical observations after bone marrow transplantation of amelioration of anemia in beta-thalassemia by relatively low degrees of mixed chimerism, and the apparent selective advantage observed for donor erythropoiesis, suggest prenatal transplantation could succeed. Prenatal strategies involving multiple transplants, donor-specific tolerance induction, and postnatal same-donor transplants should be considered.

Anemia, Sickle Cell↗
PubMed1998

Engraftment of cultured human hematopoietic cells in sheep.

In an effort to expand human hematopoietic progenitors and stem cells in vitro, we cultured human CD34(+)c-kitlow bone marrow cells in suspension in the presence of KIT ligand, FLK2/FLT3 ligand, interleukin-6 (IL-6), and erythropoietin with or without IL-3 and tested their engrafting capabilities by injecting them into sheep fetuses. As markers for engraftment, we analyzed CD45(+) cells and karyotypes of the colonies grown in methylcellulose culture. In three separate experiments, day-60 engraftment in the bone marrow was seen with both fresh cells and cells cultured in the presence or absence of IL-3. When fetuses were allowed to be born and analyzed for CD45(+) cells, no long-term engraftment was seen with cultured cells. We then pooled the CD45(+) cells of the fetal samples and transplanted them into secondary recipient fetuses. Day-60 engraftment in the secondary recipients was again noted when transplantation in the primary recipients was initiated with fresh cells. There were 3 cases in which cultured cells showed signs of engraftment in the secondary recipients, but the remaining 24 cases showed no signs of engraftment. These data documented that suspension culture for 2 weeks of enriched adult human bone marrow cells can maintain short-term (2 months) engrafting cells, but may not maintain longer term engrafting cells. This sheep/human xenograft model may serve as an excellent method for the evaluation of the engraftment potential of in vitro-expanded cells.

Animals↗