A comprehensive human leukocyte antigen analysis of 36 782 cord blood units stored in the Australian Public Cord Blood Banking Network.

BACKGROUND AND AIMS: The network of public cord blood banks (CBBs) in Australia, known as AusCord, comprises CBBs located in Brisbane, Sydney and Melbourne. A novel comprehensive analysis has been performed to determine whether the cryopreserved, searchable cord blood unit (CBU) inventory of approximately 36 000 units share similar tissue types or haplotypes. METHODS: Human leukocyte antigen (HLA) data was analysed using Microsoft Excel following standardisation of typing data. RESULTS: The analysis has found that the majority of stored, searched and released CBU exhibit a tissue type that is unique within and between the CBBs. Therefore, each collection performed by the CBBs is likely to comprise a tissue type that is not already stored among the total AusCord inventory. HLA alleles (HLA-A*34, HLA-B*56, HLA-DRB1*08:03), which are uncommon in European populations, were associated with Pacific Islander and/or Indigenous Australian populations and confirmed to be more frequent among donors who, when screened, self-identified as these ethnicities. CONCLUSIONS: These data indicate that (i) continued addition of CBU to existing inventories is likely to further increase the HLA diversity and (ii) screening donors for ethnicity or strategically locating collection sites where ethnic minorities reside can successfully result in collection of rare HLA associated with ethnic minority groups for whom finding donors might otherwise be more difficult.

Identification and Re-consent of Existing Cord Blood Donors for Creation of Induced Pluripotent Stem Cell Lines for Potential Clinical Applications.

We aim to create a bank of clinical grade cord blood-derived induced pluripotent stem cell lines in order to facilitate clinical research leading to the development of new cellular therapies. Here we present a clear pathway toward the creation of such a resource, within a strong quality framework, and with the appropriate regulatory, government and ethics approvals, along with a dynamic follow-up and re-consent process of cord blood donors from the public BMDI Cord Blood Bank. Interrogation of the cord blood bank inventory and next generation sequencing was used to identify and confirm 18 donors with suitable HLA homozygous haplotypes. Regulatory challenges that may affect global acceptance of the cell lines, along with the quality standards required to operate as part of a global network, are being met by working in collaboration with bodies such as the International Stem Cell Banking Initiative (ISCBI) and the Global Alliance for iPSC Therapies (GAiT). Ethics approval was granted by an Institutional Human Research Ethics Committee, and government approval has been obtained to use banked cord blood for this purpose. New issues of whole-genome sequencing and the relevant donor safeguards and protections were considered with input from clinical genetics services, including the rights and information flow to donors, and commercialization aspects. The success of these processes has confirmed feasibility and utility of using banked cord blood to produce clinical-grade iPSC lines for potential cellular therapies.

Antileukemic potency of CD19-specific T cells against chemoresistant pediatric acute lymphoblastic leukemia.

Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy.

Small-molecule inhibitor of glycogen synthase kinase 3ß 6-Bromoindirubin-3-oxime inhibits hematopoietic regeneration in stem cell recipient mice.

Small-molecule inhibitors of glycogen synthase kinase 3ß (GSK3ß) have demonstrated strong anti-leukemia effects in preclinical studies. Here, we investigated the effect of GSK3ß inhibitor 6-Bromoindirubin-3-oxime (BIO) previously shown to inhibit leukemia cell growth in vitro and of animal models on hematopoietic regeneration in recipients of stem cell transplant. BIO administered to immunocompromised mice transplanted with human hematopoietic stem cells inhibited human stem cell engraftment in the bone marrow (BM) and peripheral blood. BIO reduced CD34(+) progenitor cells in the BM, and primitive lymphoid progenitors re-populated host thymus at later stages post-transplant. The development of all T-cell subsets in the thymus was suppressed in BIO-treated mice. Human cell engraftment was gradually restored after discontinuation of BIO treatment; however, T-cell depletion remained until the end of experiment, which correlated with the attenuated thymic function in the host. BIO delayed CD34(+) cell expansion in stroma-supported or cytokine-only cultures. BIO treatment delayed progenitor cell divisions and induced apoptosis in cultures with sub-optimal cytokine support. In addition, BIO inhibited B- and T-cell development in co-cultures with MS5 and OP9-DL1 BM stroma cells, respectively. These data suggest that administration of GKS3ß inhibitors may act to delay hematopoietic regeneration in patients who received stem cell transplant.

GSK-3ß inhibition preserves naive T cell phenotype in bone marrow reconstituted mice.

Hematopoietic stem cell transplantation (HSCT) is used in the treatment of hematologic and nonhematologic disorders. PostHSCT immunologic reconstitution is a critical component for successful outcome. Pretransplant conditioning impairs thymic function, leading to delayed T cell regeneration. Thymus-independent T cell expansion is associated with defective generation of naive T cells and memory T cell skewing, resulting in decreased diversity in the T cell repertoire, thus attenuating the immune responses and increasing the risk of opportunistic infections and leukemia relapse. Wingless (Wnt) signaling has been identified as an important regulator of T cell development and function. Activated Wnt signaling inhibited differentiation of mature T cells in transgenic mouse models. The effect of Wnt activation on T cell regeneration following HSCT was not investigated. In this study, we demonstrate that the GSK-3ß inhibitor 6-bromoindirubin 3'-oxime (BIO) activates Wnt/ß-catenin signaling, elevates the proportion of naive T cells, and delays T cell differentiation during homeostatic T cell expansion in lymphodepleted mice transplanted with human hematopoietic stem cells. In vitro BIO-treatment promoted naive T cell expansion following mitogenic stimulation and improved proliferative responses of T cells to allogeneic stimuli. Treatment with BIO acts to expand the IL7Rα(+) subset of naive T cells, suggesting the potential mechanism driving T cell expansion during IL-7-dependent T cell proliferation. BIO downregulated expression of genes activated during effector cell differentiation and preserved naive T cell gene expression. We propose that administration of GSK-3ß inhibitor increases the potency of T cells in recipients of HSCT by expansion of naive T cell subsets with a diverse T cell receptor repertoire.

GSK3 inhibition prevents lethal GVHD in mice.

Graft-versus-host disease (GVHD) is a major contributor to transplant-related mortality and morbidity after allogeneic stem cell transplantation. Despite advancements in tissue-typing techniques, conditioning regimens, and therapeutic intervention, the incidence rate of GVHD remains high. GVHD is caused by alloreactive donor T cells that infiltrate and destroy host tissues (e.g., skin, liver, and gut). Therefore, GVHD is prevented and treated with therapeutics that suppress proinflammatory cytokines and T-cell function (e.g., cyclosporine, glucocorticoids). Here we report that the small molecule inhibitor of glycogen synthase kinase 3, 6-bromoindirubin 3'-oxime (BIO), prevents lethal GVHD in a humanized xenograft model in mice. BIO treatment did not affect donor T-cell engraftment, but suppressed their activation and attenuated bone marrow and liver destruction mediated by activated donor T cells. Glycogen synthase kinase 3 inhibition modulated the Th1/Th2 cytokine profile in vitro and suppressed activation of signal transducers and activators of transcription 1 and 3 signaling pathways both in vitro and in vivo. Importantly, human T cells derived from BIO-treated mice were able to mediate anti-tumor effects in vitro, and BIO did not affect stem cell engraftment and multilineage reconstitution in a mouse model of transplantation. These data demonstrate that inhibition of glycogen synthase kinase 3 can potentially abrogate GVHD without compromising the efficacy of transplantation.

Glycogen synthase kinase--3ß inhibitors suppress leukemia cell growth.

OBJECTIVE: The objective of this study was to investigate the effect of small molecule inhibitors of glycogen synthase kinase-3ß (GSK-3ß) on leukemia cell growth and survival. MATERIALS AND METHODS: Analysis of cytotoxicity and cell proliferation was conducted using the MTS assay, cell-cycle analysis, and division tracking. Apoptosis was investigated by Annexin-V/7-aminoactinomycin D and caspase-3 expression. The effect of GSK-3ß inhibitors was also tested in vivo in an animal model of leukemia. Gene expression analysis was performed to identify the genes modulated by GSK-3ß inhibition in leukemia cells. RESULTS: GSK-3ß inhibitors suppress cell growth and induce apoptosis in seven leukemia cell lines of diverse origin, four acute myeloid leukemia, one myelodysplastic syndrome, and one acute lymphoblastic leukemia samples. GSK-3ß inhibitors are cytotoxic for rapidly dividing clonogenic leukemia blasts, and higher doses of the inhibitors are needed to eliminate primitive leukemia progenitor/stem cells. Slow cell-division rate, low drug uptake, and interaction with bone marrow stroma make leukemia cells more resistant to apoptosis induced by GSK-3ß inhibitors. Global gene expression analysis combined with functional approaches identified multiple genes and specific signaling pathways modulated by GSK-3ß inhibition. An important role for Bcl2 in the regulation of apoptosis induced by GSK-3ß inhibitors was defined by expression analysis and confirmed by using pharmacological inhibitors of the protein. In vivo administration of GSK-3ß inhibitors delayed tumor formation in a mouse leukemia model. GSK-3ß inhibitors did not affect hematopoietic recovery following irradiation. CONCLUSIONS: Our data support further evaluation of GSK-3ß inhibitors as promising novel agents for therapeutic intervention in leukemia and warrant clinical investigation in leukemia patients.