Ann Dahlberg

Ex vivo expansion of human hematopoietic stem and progenitor cells

Despite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood-derived stem cell. Finally, we discuss some of the challenges still facing this field.

Cord-Blood Transplantation in Patients with Minimal Residual Disease

BACKGROUND The majority of patients in need of a hematopoietic-cell transplant do not have a matched related donor. Data are needed to inform the choice among various alternative donor-cell sources. METHODS In this retrospective analysis, we compared outcomes in 582 consecutive patients with acute leukemia or the myelodysplastic syndrome who received a first myeloablative hematopoietic-cell transplant from an unrelated cord-blood donor (140 patients), an HLA-matched unrelated donor (344), or an HLA-mismatched unrelated donor (98). RESULTS The relative risks of death and relapse between the cord-blood group and the two other unrelated-donor groups appeared to vary according to the presence of minimal residual disease status before transplantation. Among patients with minimal residual disease, the risk of death was higher in the HLA-mismatched group than in the cord-blood group (hazard ratio, 2.92; 95% confidence interval [CI], 1.52 to 5.63; P=0.001); the risk was also higher in the HLA-matched group than in the cord-blood group but not significantly so (hazard ratio, 1.69; 95% CI, 0.94 to 3.02; P=0.08). Among patients without minimal residual disease, the hazard ratios were lower (hazard ratio in the HLA-mismatched group, 1.36; 95% CI, 0.76 to 2.46; P=0.30; hazard ratio in the HLA-matched group, 0.78; 95% CI, 0.48 to 1.28; P=0.33). The risk of relapse among patients with minimal residual disease was significantly higher in the two unrelated-donor groups than in the cord-blood group (hazard ratio in the HLA-mismatched group, 3.01; 95% CI, 1.22 to 7.38; P=0.02; hazard ratio in the HLA-matched group, 2.92; 95% CI, 1.34 to 6.35; P=0.007). Among patients without minimal residual disease, the magnitude of these associations was lower (hazard ratio in the HLA-mismatched group, 1.28; 95% CI, 0.51 to 3.25; P=0.60; hazard ratio in the HLA-matched group, 1.30; 95% CI, 0.65 to 2.58; P=0.46). CONCLUSIONS Our data suggest that among patients with pretransplantation minimal residual disease, the probability of overall survival after receipt of a transplant from a cord-blood donor was at least as favorable as that after receipt of a transplant from an HLA-matched unrelated donor and was significantly higher than the probability after receipt of a transplant from an HLA-mismatched unrelated donor. Furthermore, the probability of relapse was lower in the cord-blood group than in either of the other groups.

Enhanced generation of cord blood hematopoietic stem and progenitor cells by culture with StemRegenin1 and Delta1 Ext-IgG

Enhanced generation of cord blood hematopoietic stem and progenitor cells by culture with StemRegenin1 and Delta1 Ext-IgG

Angiopoietin-like proteins stimulate HSPC development through interaction with Notch receptor signaling.

Angiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34+ cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets. DOI: http://dx.doi.org/10.7554/eLife.05544.001

Donor derived T-cell large granular lymphocyte leukemia after cord blood transplant for pediatric T-cell lymphoblastic leukemia

Large granular lymphocyte (LGL) leukemia is characterized by a clonal expansion of either CD3(+) cytotoxic T or CD3(−) NK cells. LGL leukemia most commonly affects the elderly with a median age of 60, although cases in younger adults and very rarely pediatric patients have been reported [1–4]. In most cases T-LGL leukemia is associated with an indolent clinical course characterized by cytopenias and autoimmune phenomena, with infections due to neutropenia being among the most common presenting symptoms [4–7]. LGL leukemia mortality is mainly due to severe infections, although this occurs in <10% of patients and the overall survival at 10 years is approximately 70% [8, 9]. Donor-derived T-LGL leukemia has been reported after hematopoietic stem cell transplant (HSCT) with both bone marrow and peripheral blood donor sources [4, 6]. T-LGL leukemia is a rare post-HSCT neoplasm that is rarely diagnosed in the pediatric population. Here we present the case of a female who underwent an unrelated umbilical cord blood transplant for T-cell acute lymphoblastic leukemia (T-ALL) and was subsequently diagnosed with donorderived T-LGL at 11 years of age. At 6 years of age, the patient was diagnosed with TALL, and had refractory disease with 25% T lymphoblasts in the bone marrow following induction chemotherapy. She then underwent salvage chemotherapy and achieved complete remission without detection of minimal residual disease by flow cytometry. She subsequently received an unrelated, 4/6 matched cord blood transplant (CBT) with single mismatches at HLA-B and DRB1. The preparative transplant conditioning regimen contained fludarabine, cyclophosphamide and total body irradiation (13.2 Gy) with cranial boost. Graft versus host disease (GVHD) prophylaxis included cyclosporine and mycophenolate mofetil. Early post-transplant, the patient developed acute skin grade IIB GVHD which was treated with high-dose prednisone. Following completion of an initial prednisone taper, she developed delayed acute gastrointestinal GVHD grade IIA that was treated with lower dose prednisone, beclomethasone, and budesonide. The patient developed a mild, intermittent skin rash which was treated with topical steroid ointment as needed. She also developed transaminitis 9 months after transplant in the setting of her cyclosporine taper that was presumed liver GVHD. She was transitioned to sirolimus and low-dose prednisone was restarted with good response. She ultimately completed systemic immune suppression approximately 14 months after transplant and aside from mild skin flares treated with topical immune suppression only, had been without signs or symptoms of chronic GVHD since that time. Two years following CBT, the patient presented with sepsis, pancytopenia and adrenal insufficiency. Bone marrow evaluation revealed no leukemia and appropriate cellularity and no other abnormalities. The patient received 1 week of empiric antibiotioic therapy and recovered blood counts within 1–2 weeks. Subsequently at 3 years following CBT she again presented with pancytopenia. She recovered to normal blood counts in <1 week but 2 months later developed neutropenia. A bone marrow exam at the time demonstrated no evidence of leukemia, approximately 50% marrow cellularity, and her chimerisms demonstrated 100% donor for CD3, CD33, CD56 and CD19 cells by fractionated chimerism analysis of peripheral blood leukocytes using amplified fragment length polymorphism on the sorted populations. The patient was seen by Infectious Disease and Immunology specialists for further workup for etiologies of her cytopenias. Testing for CMV and HHV-6 from peripheral blood were negative, EBV was detected at low level of 126 International Units (IU)/mL, and parvovirus * Tyler G. Ketterl tyler.ketterl@seattlechildrens.org

Risk Factors for Subsequent Central Nervous System Tumors in Pediatric Allogeneic Hematopoietic Cell Transplant: A Study from the Center for International Blood and Marrow Transplant Research (CIBMTR)

Survivors of hematopoietic cell transplantation (HCT) are at risk of subsequent solid tumors, including central nervous system (CNS) tumors. The risk of CNS tumors after HCT in pediatric HCT recipients is not known. We evaluated the incidence and risk factors for CNS tumors in pediatric recipients of allogeneic HCT reported to the Center for International Blood and Marrow Transplant Research between 1976 and 2008. A case control design was used. There were no CNS tumors in the nonmalignant cohort (n = 4543) or in those undergoing HCT for solid tumors (n = 26). There were 59 CNS tumors in 8720 patients transplanted for hematologic malignancies. In comparison with the general population, pediatric HCT recipients with hematologic malignancies had a 33 times higher than expected rate of CNS tumors (95% confidence interval, 22.98 to 45.77; P < .0001). The cumulative incidence of subsequent CNS tumors was 1.29% (95% confidence interval .87 to 1.87) at 20 years after HCT. Significant risk factors in the entire cohort were having an unrelated donor (HR, 3.35; P = .0002) and CNS disease before HCT for both acute lymphoblastic leukemia (HR, 8.21; P = .0003) and acute myeloid leukemia (HR, 6.21; P = .0174). Analysis of the matched cohort showed having an unrelated donor transplant (HR, 4.79; P = .0037), CNS disease before HCT (HR, 7.67; P = .0064), and radiotherapy exposure before conditioning (HR, 3.7; P = .0234) to be significant risk factors. Chronic graft-versus-host disease was associated with a lower risk (HR, .29; P = .0143). Survivors of HCT for nonmalignant diseases did not show an increased incidence of CNS tumors, whereas survivors of hematologic malignancies have a markedly increased incidence of CNS tumors that warrants lifelong surveillance.

Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy

BACKGROUND The intensive chemotherapy regimens used to treat acute myeloid leukaemia routinely result in serious infections, largely due to prolonged neutropenia. We investigated the use of non-HLA-matched ex-vivo expanded cord blood progenitor cells to accelerate haemopoietic recovery and reduce infections after chemotherapy. METHODS We enrolled patients with a diagnosis of acute myeloid leukaemia by WHO criteria and aged 18-70 years inclusive at our institution (Fred Hutchinson Cancer Research Center) into this phase 1 trial. The primary endpoint of the study was safety of infusion of non-HLA-matched expanded cord blood progenitor cells after administration of clofarabine, cytarabine, and granulocyte-colony stimulating factor priming. The protocol is closed to accrual and analysis was performed per protocol. The trial is registered with ClinicalTrials.gov, NCT01031368. FINDINGS Between June 29, 2010, and June 26, 2012, 29 patients with acute myeloid leukaemia (19 newly diagnosed, ten relapsed or refractory) were enrolled. The most common adverse events were fever (27 [93%] of 29 patients) and infections (25 [86%] of 29 patients). We observed one case of acute infusional toxicity (attributed to an allergic reaction to dimethyl sulfoxide) in the 29 patients enrolled, who received 42 infusions of expanded progenitor cells. The following additional serious but expected adverse events were observed (each in one patient): grade 4 atrial fibrillation, grade 4 febrile neutropenia, lung infection with grade 4 absolute neutrophil count, colon infection with grade 4 absolute neutrophil count, grade 4 changed mental status, and one death from liver failure. No unexpected toxicity or graft-versus-host disease was observed. There was no evidence of in-vivo persistence of the expanded progenitor cell product in any patient beyond 14 days or induced alloimmunisation. INTERPRETATION Infusion of the expanded progenitor cell product seemed safe and might provide a promising treatment method for patients with acute myeloid leukaemia. FUNDING Biomedical Advanced Research and Development Authority in the US Department of Health and Human Services and Genzyme (Sanofi).

ArticlesInfusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy: a phase 1 trial

BACKGROUND The intensive chemotherapy regimens used to treat acute myeloid leukaemia routinely result in serious infections, largely due to prolonged neutropenia. We investigated the use of non-HLA-matched ex-vivo expanded cord blood progenitor cells to accelerate haemopoietic recovery and reduce infections after chemotherapy. METHODS We enrolled patients with a diagnosis of acute myeloid leukaemia by WHO criteria and aged 18-70 years inclusive at our institution (Fred Hutchinson Cancer Research Center) into this phase 1 trial. The primary endpoint of the study was safety of infusion of non-HLA-matched expanded cord blood progenitor cells after administration of clofarabine, cytarabine, and granulocyte-colony stimulating factor priming. The protocol is closed to accrual and analysis was performed per protocol. The trial is registered with ClinicalTrials.gov, NCT01031368. FINDINGS Between June 29, 2010, and June 26, 2012, 29 patients with acute myeloid leukaemia (19 newly diagnosed, ten relapsed or refractory) were enrolled. The most common adverse events were fever (27 [93%] of 29 patients) and infections (25 [86%] of 29 patients). We observed one case of acute infusional toxicity (attributed to an allergic reaction to dimethyl sulfoxide) in the 29 patients enrolled, who received 42 infusions of expanded progenitor cells. The following additional serious but expected adverse events were observed (each in one patient): grade 4 atrial fibrillation, grade 4 febrile neutropenia, lung infection with grade 4 absolute neutrophil count, colon infection with grade 4 absolute neutrophil count, grade 4 changed mental status, and one death from liver failure. No unexpected toxicity or graft-versus-host disease was observed. There was no evidence of in-vivo persistence of the expanded progenitor cell product in any patient beyond 14 days or induced alloimmunisation. INTERPRETATION Infusion of the expanded progenitor cell product seemed safe and might provide a promising treatment method for patients with acute myeloid leukaemia. FUNDING Biomedical Advanced Research and Development Authority in the US Department of Health and Human Services and Genzyme (Sanofi).