Chimerism analysis by sex determining region Y (SRY) and major histocompatibility complex markers in non-human primates using quantitative real-time polymerase chain reaction.

The ability to discriminate and further quantify the proportion of donor and host cells is essential in hematopoietic stem cell transplant protocols. In human sex-mismatched transplants, this can be easily accomplished by the use of commercially available fluorescent in situ hybridization (FISH) probes. In many animal models, including non-human primates, this methodology is not possible due to the lack of commercially available FISH probes. In animal models, donor cell detection could be accomplished if there is a known species-specific sex determining region Y (SRY) (male) or other unique DNA sequence using either semiquantitative or quantitative real-time polymerase chain reaction (PCR). The use of real-time quantitative PCR has the obvious advantage of providing detailed enumeration of the percentage of donor cells present. We report the development of extremely sensitive primer and probe combinations for male (SRY) and major histocompatibility complex (MHC)-DQA sequences in the macaque and baboon non-human primate models. This assay has a sensitivity of a five-log range and can detect less than four target cells in the presence of 10(5) background cells (approximately 0.001%) and fetal DNA obtained from maternal serum from Macaca nemestrina. The SRY (male) primer and probe combination has similar sensitivity in Macaca fasicularis, Macaca mulatta, and Papio cynocephalus anubis.

Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: the Seattle experience.

The purpose of this study was to evaluate the role of allogeneic bone marrow transplantation (BMT) in children with myelodysplastic syndrome (MDS). In total, 94 consecutive pediatric patients with MDS received an allogeneic BMT from 1976 to 2001 for refractory anemia (RA) (n=25), RA with ringed sideroblasts (RARS) (n=2), RA with excess blasts (RAEB) (n=20), RAEB in transformation (RAEB-T) (n=14), juvenile myelomonocytic leukemia (JMML) (n=32) or chronic myelomonocytic leukemia (CMML) (n=1). The estimated 3-year probabilities of survival, event-free survival (EFS), nonrelapse mortality and relapse were 50, 41, 28 and 29%, respectively. Patients with RA/RARS had an estimated 3-year survival of 74% compared to 68% in those with RAEB and 33% in patients with JMML/CMML. In multivariable analysis, patients with RAEB-T or JMML were 3.9 and 3.7 times more likely to die compared to those with RA/RARS and RAEB (P=0.005 and 0.004, respectively). Patients with RAEB-T were 5.5 times more likely to relapse (P=0.01). The median follow-up among the 43 surviving patients is 10 years (range 1-25). We conclude that allogeneic BMT for children with MDS is well tolerated and can be curative.

Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons.

Efficient transduction of hematopoietic stem cells is a prerequisite for successful hematopoietic stem cell gene therapy. Oncoretroviral vectors are the most widely used vectors for hematopoietic gene therapy studies. However, these vectors require cell division, and thus efficient transduction of quiescent stem cells has been difficult to achieve. Lentiviral vectors can transduce non-dividing cells and therefore may be more efficient in transducing quiescent hematopoietic stem cells. We have used a competitive repopulation assay in the baboon to compare transduction of hematopoietic repopulating cells by lentiviral and oncoretroviral vectors. Baboon CD34-enriched marrow cells were transduced in the presence or absence of multiple hematopoietic growth factors using a short, 2-day, transduction protocol. Here, we show that efficient lentiviral transduction of hematopoietic repopulating cells was only achieved when cells were transduced in the presence of multiple growth factors. Using these conditions, up to 8.6% of hematopoietic repopulating cells were genetically modified by the lentiviral vector more than 1 year after transplant. Interestingly, the number of lentivirally marked cells increased over time in three of four animals. In conclusion, these results suggest that lentiviral vectors are able to tranduce multilineage hematopoietic stem cells, and thus, may provide an alternative vector system for clinical stem cell gene therapy applications.

Gene transfer into baboon repopulating cells: A comparison of Flt-3 Ligand and megakaryocyte growth and development factor versus IL-3 during ex vivo transduction.

Oncoretroviral vectors require division of target cells for successful transduction. In the case of hematopoietic repopulating cells this can be achieved by cytokine stimulation using growth factor combinations which facilitate gene transfer and maintain engraftment. Interleukin-3 (IL-3) has been widely used in growth factor combinations, although more recent data in the mouse showed reduced engraftment in the presence of IL-3. Here, we used a competitive repopulation assay to study the influence of IL-3 and the early acting cytokines megakaryocyte growth and development factor (MGDF) and Flt3-ligand (Flt3-L) on gene transfer efficiency during ex vivo transduction of hematopoietic repopulating cells. In a direct comparison, baboon CD34-enriched cells were transduced on CH-296 fibronectin fragment in the presence of either IL-6, stem cell factor (SCF), Flt3-L, and MGDF or IL-3, IL-6, and SCF. Animals were followed for up to 55 weeks, and analysis of peripheral blood leukocytes by semiquantitative polymerase chain reaction showed that both cytokine combinations achieved marking of repopulating cells. A trend toward increased gene marking, especially early after transplant (P = 0.06), was seen with the combination of IL-6, SCF, Flt3-L, and MGDF. However, the highest gene marking was achieved when IL-3 was combined with early acting cytokines, suggesting that the difference observed in this study was probably due to the addition of MGDF and Flt3-L and not due to a negative effect of IL-3 on engraftment.

Highly efficient gene transfer into preterm CD34 hematopoietic progenitor cells.

OBJECTIVE: Retrovirus-mediated gene transfer has been shown to transduce CD34(+) cells from term gestation umbilical cord blood with relatively high efficiency. The purpose of this study was to compare the efficiencies of retrovirus-mediated gene transfer into early (23-28 weeks' gestation) and term (37-41 weeks' gestation) umbilical cord blood CD34(+) hematopoietic progenitor cells. STUDY DESIGN: CD34(+) cells were purified from cyropreserved early (23-28 weeks' gestation) and term (37-40 weeks' gestation) umbilical cord blood specimens with fluorescence-activated cell sorting. The CD34(+) cells were then transduced in virus-containing medium (gibbon ape leukemia virus pseudotype vector LAPSN [PG13]) in wells coated with the recombinant human fibronectin fragment CH-296 and in the presence of multiple hematopoietic growth factors (interleukin 6, stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and megakaryocyte growth and development factor) and protamine sulfate. The LAPSN (PG13) virus-containing medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia virus pseudotype vector LAPSN (PG13) contains the human placental alkaline phosphatase and neomycin phosphotransferase (neo ) genes. The efficiency of gene transfer was assessed by histochemical staining of colony-forming cells in agar for expression of heat-stable alkaline phosphatase. RESULTS: Gene transfers, as assessed by alkaline phosphatase staining of colony-forming cells (granulocyte-macrophage colony-forming units and erythroid burst-forming units), were similar for CD34(+) hematopoietic progenitor cells from early (58.4% +/- 11.8%) and term (63.2% +/- 12.5%) gestation fetal umbilical cord blood. CONCLUSION: CD34(+) hematopoietic progenitor cells from midgestation fetal blood can be transduced with high efficiency using techniques optimized for postnatal samples with a gibbon ape leukemia virus pseudotype vector. The early fetus may be a preferable target for gene therapy because of the higher number of circulating CD34(+) and CD38(-) cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Because in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the efficiency of gene transfer into marrow-repopulating cells in vivo, studies that examine clinically applicable approaches to in utero gene therapy in appropriate animal models are still needed.

Differential engraftment of genetically modified CD34(+) and CD34(-) hematopoietic cell subsets in lethally irradiated baboons.

OBJECTIVE: To test gibbon ape leukemia virus (GALV) pseudotype vector transduction of marrow subpopulations that contribute to hematopoietic reconstitution in vivo. MATERIALS AND METHODS: Autologous CD34(+) Lin(-), CD34(+) Lin(+), and CD34(-) Lin(-) marrow cells, transduced by coculture with PG13/LN, PG13/LNX, and PG13/LNY vector-producing cells, respectively, were transplanted in three female baboons. Two female baboons also were transplanted with fresh allogeneic CD34(-)Lin(-) marrow cells from MHC-matched male siblings and, to ensure survival, with autologous CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells transduced with PG13/LN and PG13/LNX, respectively. The LN, LNX, and LNY vectors are identical except for different length sequences at the 3' end of the bacterial neomycin phosphotransferase (neo) gene. RESULTS: LN(+) and LNX(+) cells from CD34(+)Lin(-) and CD34(+)Lin(+) cells, respectively, but no LNY(+) from CD34(-)Lin(-) cells were detectable in blood and marrow of all animals after transplant. LN(+), CD34(+)Lin(-) cells contributed to reconstitution of the T, B, and myeloid lineages. LNX(+), CD34(+)Lin(+) cells contributed only to B and myeloid lineages. Male cells, CD34(-)Lin(-), were detected by polymerase chain reaction in blood and marrow of the two allogeneic transplanted animals at estimated frequencies of

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors.

Nonhuman primates are increasingly being used as models for pre-clinical assessment of retrovirus vector expression and function following stem and progenitor cell transduction. We compared the relative susceptibility of CD34+ marrow progenitors from four nonhuman primate species and humans to transduction with amphotropic pseudotyped retrovirus vectors containing the Neo gene. The rate of functional gene transfer was measured by colony formation under G418 selection. Marrow progenitors from pigtail macaques (Macaca nemestrina) were transduced at about twice the rate (19.1 +/- 4.3%) as those from rhesus (11.2 +/- 3.7%) and cynomolgus (7.6 +/- 1.9%) macaques, baboons (7.8 +/- 1.8%), and humans (9.6 +/- 1.7%). Semiquantitative RT/PCR analysis suggests this difference may be due to elevated expression of the amphotropic receptor Pit2 in pigtailed macaque CD34+ cells. Further, transduction rates increased an average 1.6 +/- 0.4-fold when the culture temperature was lowered to 33 degrees C, and 2.1 +/- 0.3-fold when the culture dishes were coated with the fibronectin fragment CH-296. The data presented here point to important differences among nonhuman primate models as well as transduction culture conditions, and suggest that pigtailed macaques may be particularly useful for assessing expression and function of therapeutic retrovirus vectors. Gene Therapy (2000) 7, 359-367.

Isolation and characterization of macaque dendritic cells from CD34(+) bone marrow progenitors.

We have developed a method for isolating and characterizing pigtailed macaque dendritic cells (DCs) generated from CD34(+) bone marrow (BM) progenitors based on methods previously developed for isolating human DCs. Macaque DCs displayed a characteristic morphology and were potent stimulators of allogeneic T cell proliferation. They expressed a set of DC-associated markers, such as MHC class II, CD1a, CD4, CD11a, CD40, CD58, CD80, CD83, CD86, and CXCR4. Macaque DCs, as well as peripheral blood CD4(+) T cells, were highly susceptible to HIV-2 infection, as detected by DNA-PCR. The expression of HIV-2 in macaque DCs was downregulated by treatment with the beta-chemokine RANTES. Macaque DCs will be useful for defining the in vivo role of DCs in HIV pathogenesis and for optimizing and testing peptide-DC vaccines or tolerizing regimens.

Engraftment of primates with G-CSF mobilized peripheral blood CD34+ progenitor cells expanded in G-CSF, SCF and MGDF decreases the duration and severity of neutropenia.

We used a primate model of autologous peripheral blood progenitor cell (PBPC) transplantation to study the effect of in vitro expansion on committed progenitor cell engraftment and marrow recovery after transplantation. Four groups of baboons were transplanted with enriched autologous CD34+ PBPC collected by apheresis after five days of G-CSF administration (100 microg/kg/day). Groups I and III were transplanted with cryopreserved CD34+ PBPC and Groups II and IV were transplanted with CD34+ PBPC that had been cultured for 10 days in Amgen-defined (serum free) medium and stimulated with G-CSF, megakaryocyte growth and development factor (MGDF), and stem cell factor each at 100 etag/ml. Group III and IV animals were administered G-CSF (100 microg/kg/day) and MGDF (25 microg/kg/day) after transplant, while animals in Groups I and II were not. For the cultured CD34+ PBPC from groups II and IV, the total cell numbers expanded 14.4 +/- 8.3 and 4.0 +/- 0.7-fold, respectively, and CFU-GM expanded 7.2 +/- 0.3 and 8.0 +/- 0.4-fold, respectively. All animals engrafted. If no growth factor support was given after transplant (Groups II and I), the recovery of WBC and platelet production after transplant was prolonged if cells had been cultured prior to transplant (Group II). Administration of post-transplant G-CSF and MGDF shortened the period of neutropenia (ANC < 500/microL) from 13 +/- 4 (Group I) to 10 +/- 4 (Group III) days for animals transplanted with non-expanded CD34+ PBPC. For animals transplanted with ex vivo-expanded CD34+ PBPC, post-transplant administration of G-CSF and MGDF shortened the duration of neutropenia from 14 +/- 2 (Group II) to 3 +/- 4 (Group IV) days. Recovery of platelet production was slower in all animals transplanted with expanded CD34+ PBPC regardless of post-transplant growth factor administration. Progenitor cells generated in vitro can contribute to early engraftment and mitigate neutropenia when growth factor support is administered post-transplant. Thrombocytopenia was not decreased despite evidence of expansion of megakaryocytes in cultured CD34+ populations.

Detection of enzootic babesiosis in baboons (Papio cynocephalus) and phylogenetic evidence supporting synonymy of the genera Entopolypoides and Babesia.

Blood smear evaluation of two baboons (Papio cynocephalus) experiencing acute hemolytic crises following experimental stem cell transplantation revealed numerous intraerythrocytic organisms typical of the genus Babesia. Both animals had received whole-blood transfusions from two baboon donors, one of which was subsequently found to display rare trophozoites of Entopolypoides macaci. An investigation was then undertaken to determine the prevalence of hematozoa in baboons held in our primate colony and to determine the relationship, if any, between the involved species. Analysis of thick and thin blood films from 65 healthy baboons (23 originating from our breeding facility, 26 originating from an out-of-state breeding facility, and 16 imported from Africa) for hematozoa revealed rare E. macaci parasites in 31%, with respective prevalences of 39, 35, and 12%. Phylogenetic analysis of nuclear small-subunit rRNA gene sequences amplified from peripheral blood of a baboon chronically infected with E. macaci demonstrated this parasite to be most closely related to Babesia microti (97.9% sequence similarity); sera from infected animals did not react in indirect fluorescent-antibody tests with Babesia microti antigen, however, suggesting that they represent different species. These results support an emerging view that the genus Entopolypoides Mayer 1933 is synonymous with that of the genus Babesia Starcovici 1893 and that the morphological variation noted among intracellular forms is a function of alteration in host immune status. The presence of an underrecognized, but highly enzootic, Babesia sp. in baboons may result in substantial, unanticipated impact on research programs. The similarity of this parasite to the known human pathogen B. microti may also pose risks to humans undergoing xenotransplantation, mandating effective screening of donor animals.

Gene transfer into fetal baboon hematopoietic progenitor cells.

We studied hematopoietic progenitors from fetal baboon blood, marrow, and liver at four time points (125, 140, 160, and 175 days) during the third trimester (gestation approximately 180 days) to determine if fetal baboons might be an appropriate model for in utero gene therapy of hematopoietic stem cells (HSCs). Cells were studied for expression of CD34, CD33, CD38, and HLA-DR, for progenitor content in colony-forming cell assays, and for susceptibility of CD34+ progenitors to retrovirus-mediated gene transfer. Throughout the third trimester, the frequency of CD34+ progenitors in blood and marrow appears to remain unchanged at approximately 0.6 and 5.0%, respectively. In liver, progenitors progressively decrease to undetectable levels by day 175. The proportion of fetal baboon bone marrow and liver CD34+ cells expressing CD38 and HLA-DR appears to increase with increasing fetal age, similar to changes reported for human cord blood CD34+ cells. In fetal baboon blood the proportion of CD34+ cells expressing CD33 appears to decrease with increasing gestational age, also similar to changes reported for human cord blood cells. Progenitors from human cord blood and baboon fetal tissues were similarly susceptible to transduction by the gibbon ape leukemia pseudotyped retroviral vector LAPSN(PG13) containing the genes for human placental alkaline phosphatase (AP) and the bacterial neomycin phosphotransferase (neo). Fetal baboon and human hematopoietic progenitor cells undergo similar phenotypic changes during the third trimester of fetal development and are similarly susceptible to retrovirus-mediated gene transfer. The fetal baboon may be a model in which approaches to mobilization and gene transfer into fetal HSCs can be studied.

Bone marrow transplantation for children less than 2 years of age with acute myelogenous leukemia or myelodysplastic syndrome.

We analyzed results of 40 infants less than 2 years of age who received bone marrow transplants (BMT) between May 1974 and January 1995 for treatment of acute myelogenous leukemia (AML; N = 34) or myelodysplastic syndrome (MDS; N = 6) to determine outcome and survival performance. Among the AML patients, 13 were in first remission, 9 were in untreated first relapse or second remission, and 12 were in refractory relapse. Patients were conditioned with cyclophosphamide in combination with either total body irradiation (TBI; N = 29) or busulfan (N = 11). Source of stem cells included 6 autologous donors, 15 HLA genotypically identical siblings, 14 haploidentical family members, and 5 unrelated donors. Graft-versus-host disease (GVHD) prophylaxis was methotrexate (MTX) for 17, MTX plus cyclosporine (CSP) for 14, or CSP plus prednisone for 3. Incidence of severe (grade 3-4) regimen-related toxicity was 10% and transplant-related mortality was 10%. Acute GVHD (grades II-III) occurred in 39% of allogeneic patients, and chronic GVHD developed in 40%. Relapse, the most significant problem for patients in this study, occurred in 1 MDS patient and 23 AML patients and was the cause of death for 19 patients. The 2-year probabilities of relapse are 46%, 67%, and 92%, respectively, for patients transplanted in first remission, untreated first relapse or second remission, and relapse. One MDS and 8 AML patients received second marrow transplants for treatment of relapse, and 5 of these survive disease-free for more than 1.5 years. All 6 MDS patients and 11 of 34 AML patients survive more than 1.5 years later. The 5-year probabilities of survival and disease-free survival are 54% and 38% for patients transplanted in first remission and 33% and 22% for untreated first relapse or second remission. None of the patients transplanted with refractory relapse survive disease-free. Outcome was significantly associated with phase of disease at transplantation and pretransplant diagnosis of extramedullary disease. Long-term sequelae included growth failure and hormonal deficiencies. Survival performance was a median of 100% (80% to 100%) and neurologic development for all survivors was appropriate for age. This study indicates that infants with AML have similar outcome after BMT compared with older children and that BMT should be performed in first remission whenever possible. In addition, allogeneic BMT provides effective therapy for the majority of infants with MDS.

Cell-surface antigen expression in early and term gestation fetal hematopoietic progenitor cells.

The objective of this study was to compare the expression of primitive cell-surface antigens on CD34+ cells from early in gestation to those from term gestations. Fetal blood samples were obtained from 10 early gestation (21.0+/-0.8 [SE] weeks) and 12 term gestation (39.3+/-0.4 weeks) fetuses. The mononuclear cell population was separated by red cell lysis. Two-color flow cytometry was used to assess cell surface antigen coexpression of CD34 with CD33, CD38, and HLA-DR as well as staining by a cocktail of monoclonal antibodies for lineage-associated (Lin) antigens (CD2, CD10, CD11b, CD19, CD20, CD33, CD36, 7B9, and Glycophorin-A). The frequency of CD34+ cells (5.5+/-0.9 versus 1.5+/-0.2, p < 0.001) was significantly higher in the early gestational age group. Within the CD34+ population, the frequency of CD34+/CD38- cells (81.8+/-9.9 versus 51.3+/-7.7, p = 0.02) and CD34+/DR- cells (15.3+/-7.4 versus 8.2+/-2.7, p = 0.05) was also higher in the early gestational age group. In contrast, CD34+/CD33- (51.8+/-10.1 versus 83.0+/-6.1, p = 0.02) and CD34+/Lin- cells (15.9+/-7.0 versus 51.8 +/-6.9, p < 0.01) were higher in the term gestation group. The high percentage of CD34+, CD34+/CD38-, and CD34+/DR- cells supports our hypothesis that early gestational age fetal blood has a higher frequency of primitive hematopoietic progenitor/stem cells than does umbilical cord blood at term. This suggests that hematopoietic progenitor/stem cells in early fetal blood may be a desirable target for in utero gene therapy. However, further studies to characterize the functional properties of CD34+ cell subsets at different stages of fetal development will be necessary to determine the appropriateness of targeting fetal hematopoietic cells for in utero gene therapy. The higher frequency of CD34+/CD33- and CD34+/Lin- cells from term gestational age fetuses was unexpected, and the significance of this finding is unclear at this time.

Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor.

We have used a competitive repopulation assay in baboons to develop improved methods for hematopoietic stem cell transduction and have previously shown increased gene transfer into baboon marrow repopulating cells using a gibbon ape leukemia virus (GALV)-pseudotype retroviral vector (Kiem et al, Blood 90:4638, 1997). In this study using GALV-pseudotype vectors, we examined additional variables that have been reported to increase gene transfer into hematopoietic progenitor cells in culture for their ability to increase gene transfer into baboon hematopoietic repopulating cells. Baboon marrow was harvested after in vivo administration (priming) of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). CD34-enriched marrow cells were divided into two equal fractions to directly compare transduction efficiencies under different gene transfer conditions. Transduction by either incubation with retroviral vectors on CH-296-coated flasks or by cocultivation on vector-producing cells was studied in five animals; in one animal, transduction on CH-296 was compared with transduction on bovine serum albumin (BSA)-coated flasks. The highest level of gene transfer was obtained after 24 hours of prestimulation followed by 48 hours of incubation on CH-296 in vector-containing medium in the presence of multiple hematopoietic growth factors (interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor). Using these conditions, up to 20% of peripheral blood and marrow cells contained vector sequences for more than 20 weeks, as determined by both polymerase chain reaction and Southern blot analysis. Gene transfer rates were higher for cells transduced on CH-296 as compared with BSA or cocultivation. In one animal, we have used a vector expressing a cell surface protein (human placental alkaline phosphatase) and have detected 10% and 5% of peripheral blood cells expressing the transduced gene 2 and 4 weeks after transplantation as measured by flow cytometry. In conclusion, the conditions described here have resulted in gene transfer rates that will allow detection of transduced cells by flow cytometry to facilitate the evaluation of gene expression. The levels of gene transfer obtained with these conditions suggest the potential for therapeutic efficacy in diseases affecting the hematopoietic system.

Gestational age changes in circulating CD34+ hematopoietic stem/progenitor cells in fetal cord blood.

OBJECTIVE: We sought to evaluate the frequency of CD34+ hematopoietic progenitor/stem cells across gestation and the proliferative response of early versus late gestational age fetal blood to growth factor stimulation. STUDY DESIGN: Fetal blood samples were obtained at 17 to 41 weeks' gestational age. The mononuclear cell population was separated by red blood cell lysis. The frequency of CD34+ cells within the lymphocyte-monocyte light scatter gate and the expression of CD34 by colony-forming cells of different types were determined by fluorescence-activated cell sorting. The growth response of colony-forming cells to varying concentrations of defined growth factors (interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin) was determined, as well as the frequency of burst-forming units erythroid, colony-forming units granulocyte-macrophage, and total colony-forming cells. Samples from 17 to 24 weeks' gestation (early) were compared with those of 39 to 41 weeks' gestation (late). RESULTS: The frequency of CD34+ cells in early fetal blood (17 to 24 weeks' gestation) was 4.9-fold higher (6.4% vs 1.3%, p < 0.002) than term gestation (37 to 41 weeks' gestation) and declined linearly with gestational age (p < 0.0001). When gestational ages were grouped into 4-week blocks (17 to 20, 21 to 24, 25 to 28, 29 to 32, 33 to 36, and >37 weeks), statistically significant changes in the frequency of CD34+ cells did not occur until after 28 weeks' gestation. By cell sorting, >99% of all colony-forming cells were contained within the CD34+ population at all gestational ages tested. Compared with term, the frequency of colony-forming cells was significantly greater in early fetal blood (burst-forming units erythroid [18.1-fold, p < 0.0001], colony-forming units granulocyte-macrophage [2.9-fold, p <0.001], and total colony-forming cells [9.4-fold, p < 0.0001]). However, when the frequency of colony-forming cells was corrected for the frequency of CD34+ cells, only the number of burst-forming units erythroid remained significantly greater in early fetal blood. The relative size of the colonies formed by individual progenitors from early fetal blood was greater than those from term samples. CONCLUSIONS: From 17 to 41 weeks' gestation the frequency of CD34+ cells in fetal blood, which includes hematopoietic progenitors and stem cells, decreases. This decline occurs during the transition from hepatic to bone marrow hematopoiesis. Early fetal blood, with a higher circulating frequency of progenitor/stem cells and proliferative capacity, may be a preferable target for gene therapy.

Characterization of monoclonal antibodies that recognize canine CD34.

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34- cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit-granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34- cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of

Prolonged allogeneic and xenogeneic microchimerism in unmatched primates without immunosuppression by intrathymic implantation of CD34+ donor marrow cells.

Engraftment of stem cell-enriched donor marrow implanted in the thymus of a foreign host might facilitate acceptance of donor-specific organ or tissue grafts. To test this hypothesis, allogeneic and xenogeneic CD34+ marrow cells from unrelated adult male baboons and humans were injected intrathymically in eight infant female baboons, both with and without standard cyclosporine-based immunosuppression. In allogeneic experiments, male (donor) cells, of both T- and B-cell lineages, were detected by PCR in the peripheral blood of all six recipients and persisted for at least 15 months in 2/4 recipients studied longtutudinally. Donor-derived skin grafts survived twice as long as third party grafts in unimmunosuppressed recipients. In xenogeneic protocols, human male (donor) cells were demonstrable for 7 and 15 months, respectively, in two baboon recipients with evidence that implanted human CD34+ cells had produced lymphoid progeny. Survival of donor-specific skin xenografts was prolonged in one of two recipients. These experiments demonstrate that the intrathymic injection of CD34+ marrow cells can result in long-lasting lymphohematopoietic microchimerism in unrelated primates even without immunosuppression and can alter donor-specific skin graft survival.

Gene transfer into marrow repopulating cells: comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons.

Many diseases might be treated by gene therapy targeted to the hematopoietic system, but low rates of gene transfer achieved in humans and large animals have limited the application of this technique. We have developed a competitive hematopoietic repopulation assay in baboons to evaluate methods for improving gene transfer and have used this method to compare gene transfer rates for retroviral vectors having an envelope protein (pseudotype) from amphotropic murine retrovirus with similar vectors having an envelope protein derived from gibbon ape leukemia virus (GALV). We hypothesized that vectors with a GALV pseudotype might perform better based on our previous work with cultured human hematopoietic cells. CD34(+) marrow cells from each of four untreated baboons were divided into two equal portions that were cocultivated for 48 hours with packaging cells producing equivalent titers of either amphotropic or GALV pseudotyped vectors containing the neo gene. The vectors contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into animals after they received a myeloablative dose of total body irradiation. Polymerase chain reaction (PCR) analysis for neo gene-specific sequences in colony-forming unit-granulocyte-macrophage from cell populations used for transplant showed gene transfer rates of 2.7%, 7.1%, <15%, and 3.9% with the amphotropic vectors and 7.1%, 11.3%, <15%, and 26.4% with the GALV pseudotyped vector. PCR analysis of peripheral blood and marrow cells after engraftment showed the neo gene to be present in all four animals analyzed at levels between 0.1% and 5%. Overall gene transfer efficiency was higher with the GALVpseudotyped vector than with the amphotropic vectors. Southern blot analysis in one animal confirmed a gene transfer efficiency of between 1% and 5%. The higher gene transfer efficiency with the GALV-pseudotyped vector correlated with higher levels of GALV receptor RNA compared with the amphotropic receptor in CD34(+) hematopoietic cells. These results show that GALV-pseudotyped vectors are capable of transducing baboon marrow repopulating cells and may allow more efficient gene transfer rates for human gene therapy directed at hematopoietic cells. In addition, our data show considerable differences in gene transfer efficiency between individual baboons, suggesting that a competitive repopulation assay will be critical for evaluation of methods designed to improve gene transfer into hematopoietic stem cells.

Normal immunologic response to a neoantigen, bacteriophage phiX-174, in baboons with long-term lymphohematopoietic reconstitution from highly purified CD34+ Lin- allogeneic marrow cells.

The CD34 antigen is thought to be expressed by hematopoietic stem cells in adult humans and nonhuman primates. We present data that baboons transplanted with highly purified allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes and myeloid cells, isolated from sex-mismatched mixed lymphocyte culture (MLC) nonreactive siblings, have maintained stable lymphohematopoietic engraftment with donor cells for greater than 4.9, greater than 6.0, and 5.0 years. Cytogenetic analysis of unfractionated marrow and peripheral blood cells at multiple time points after transplantation show virtually all donor cells in two animals and stable mixed chimerism in the third. We used polymerase chain reaction to show that colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and high proliferative potential colony-forming cells (HPP-CFC) were virtually all of donor origin in two animals and present at lower levels in the stable mixed chimera. CD20+ B-lymphoblastoid cell lines derived by Herpesvirus Papio transformation of peripheral blood cells were virtually all donor in two animals and 50% donor in the mixed chimera. CD4+ and CD8+ T cells and neutrophils purified from the peripheral blood of the two female animals also were all donor-derived. To assess immunologic function after transplantation, we immunized the three long-term chimeric animals and two normal control animals with bacteriophage phiX-174, a neoantigen that requires the interaction of antigen-presenting cells, T lymphocytes, and B lymphocytes to mount a normal antibody response. Experimental and control animals, when immunized with bacteriophage, had similar serum Ig levels. The experimental and control animals generated similar titers of antibacteriophage antibodies after primary and secondary immunizations with evidence of amplification and class switching. These findings further support the hypothesis that the CD34+ antigen is expressed on hematopoietic stem cells that can mediate stable long-term lymphohematopoiesis in vivo and, importantly, that normal immunologic function can be reconstituted in vivo after transplantation of the highly purified CD34+ Lin- cells alone.