Robert G. Andrews

Antigen CD34+ marrow cells engraft lethally irradiated baboons.

The CD34 antigen is present on 1-4% of human marrow cells including virtually all hematopoietic progenitors detected by in vitro assays. Since the anti-CD34 monoclonal antibody 12-8 reacts with a similar marrow population in baboons, it was possible to test whether this antigen is expressed by stem cells responsible for hematopoietic reconstitution in vivo. CD34+ cells were enriched from marrows of five baboons using avidin-biotin immunoadsorption. After lethal irradiation, the five animals were given 15-27 X 10(6) autologous marrow cells (3.2-4.4 X 10(6) cells/kg) containing 65-91% CD34+ cells. All animals achieved granulocyte counts greater than 1,000/mm3 and platelet counts greater than 20 X 10(3)/mm3 by 13-24 d posttransplant and subsequently developed normal peripheral blood counts. Two additional animals received 184 and 285 X 10(6) marrow cells/kg depleted of CD34+ cells. One animal died at day 29 without engraftment, while the other had pancytopenia for greater than 100 d posttransplant. The data suggest that stem cells responsible for hematopoietic reconstitution are CD34+.

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.

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

Summary: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.

In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells.

In utero transplantation of hematopoietic stem cells is a promising treatment for immune and hematologic diseases of fetuses and newborns. Unfortunately, there are limited data from nonhuman primates and humans describing optimal transplantation conditions. The purpose of this investigation was to determine the effect of T‐cell number on engraftment and the level of chimerism after in utero transplantation in nonhuman primates. CD34+ allogeneic adult bone marrow cells, obtained from the sire after G‐CSF and stem cell factor administration, were transplanted into female fetal recipients. The average CD34+ cell dose was 3.0 × 109/kg (range, 9.9 × 108 to 4.4 × 109) and the T‐cell dose ranged from 2.6 × 105 to 1.1 × 108/kg. Chimerism was determined in peripheral blood subsets (CD2, CD13, and CD20) and in progenitor cell populations by using polymerase chain reaction. Chimerism was noted in seven of eight live‐born animals. The level of chimerism in the progenitor population was related to the fetal T‐cell dose (r = 0.64, p < 0.02). At the lowest T‐cell dose (2.6 × 105/kg), no chimerism was detected. As the T‐cell dose increased to 106–7/kg, the level of chimerism increased. Adjusting the T‐cell dose to 1.1 × 108/kg resulted in fatal graft‐versus‐host disease (GVHD). The results of this study emphasize the importance of T cells in facilitating donor cell engraftment and in producing GVHD in fetal nonhuman primates. Some animals achieved levels of chimerism in the marrow hematopoietic progenitor cell population that would likely have clinical relevance. However, the levels of chimerism in peripheral blood were too low for therapeutic benefit. Further studies are needed to test methods that are likely to enhance donor cell engraftment and peripheral blood levels of donor cells.

Cutaneous and extracutaneous neutrophilic infiltrates (Sweet syndrome) in three patients with Fanconi anemia

Three patients with coexistent Fanconi syndrome and Sweet syndrome (neutrophilic dermatosis) are presented. These sterile skin lesions responded to systemic corticosteroid therapy in all three cases, and recurred when treatment was discontinued. The association in children of Sweet syndrome with malignancy has previously been recognized; it has not been reported in the premalignant phase of Fanconi anemia. This report expands the differential diagnosis of the neutrophilic dermatoses. Children with Sweet syndrome and anemia should be examined for Fanconi anemia by diepoxybutane cytogenetic studies.

Fetal hematopoietic stem cell transplantation: a challenge for the twenty-first century.

Successful in utero hematopoietic stem cell transplantation will likely represent a major step forward in the management of patients with congenital hematological, metabolic, and immunological disorders. We review the naturally occurring models of hematopoietic chimerism in animals and humans, as well as available experimental animal data and human clinical attempts of fetal transplantation. Data available from naturally occurring models and experimental models of fetal transplantation suggest that this technique should be translatable to the human fetus. However, to date, the success of human fetal hematopoietic stem cell therapy has been limited to fetuses with severe immunologic defects. Evaluation of successful attempts of human transplantation, the ontogeny of fetal immune development, and data available from animals provide insights into innovative approaches to fetal therapy that may bring the reality of successful fetal transplantation closer.

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 μg/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 ηg/ml. Group III and IV animals were administered G‐CSF (100 μg/kg/day) and MGDF (25 μg/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/μL) 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.

Fetal Immune Suppression as Adjunctive Therapy for In Utero Hematopoietic Stem Cell Transplantation in Nonhuman Primates

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow‐derived, CD34+‐enriched donor cells. Animals treated postnatally received either donor‐derived T cell–depleted or CD34+‐enriched marrow cells. Chimerism was determined by traditional and real‐time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune‐suppressed animals relative to controls (11.3% ± 2.7% and 5.1% ± 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence‐activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at ≥ 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune‐suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6‐month follow‐up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long‐term chimerism also was not improved with postnatal donor cell infusion.

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.

Recombinant Human Ligand for MPL, Megakaryocyte Growth and Development Factor (MGDF), Stimulates Thrombopoiesis in Vivo in Normal and Myelosuppressed Baboons

Megakaryocyte growth and development factor (MGDF) is a ligand for c‐mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD 34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5‐fluorouracil (5‐FU). rHuMGDF was administered to normal baboons as single s.c. injection at doses of 1, 10, 25 and 50 μg/kg/day for 10 days and, as a control, heat‐inactivated MGDF was administered at a dose of 10 μg/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat‐inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7‐, 3.4‐, 5.1‐ and 4.0‐fold above baseline in animals administered 1, 10, 25 and 50 μg/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 μg/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in circulation.