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.

GSK-3ß inhibition promotes engraftment of ex vivo-expanded hematopoietic stem cells and modulates gene expression.

Glycogen synthase kinase-3ß (GSK-3ß) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3ß, 6-bromoindirubin 3'-oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3ß provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.

Formation of an adherent hematopoietic expansion culture using fucoidan.

Expansion of transplantable cord blood (CB) progenitors using a stroma requires provision of an exogenous cell source because of the low frequency of stromal precursor cells in CB. A simpler approach from a clinical regulatory perspective would be to provide synthetic extracellular matrix. The aim of this study was to characterize the effect on hematopoietic cell culture of fucoidan. The modulation of cytokine-driven hematopoietic cell expansion by fucoidan was investigated using two-level fractional and full factorial experimental designs. Mobilized peripheral blood (PB) CD34(+) cells were grown over 10 days in various combinations of FL, SCF, TPO, G-CSF, and SDF-1. Cultures were analyzed by immunophenotype. The effect of fucoidan on the divisional recruitment of CD34(+) cells was studied by CFDA-SE division tracking. Fucoidan was adsorbed by polystyrene to tissue culture plates and promoted formation of an adherent hematopoietic culture. Factorial design experiments with mobilized PB-CD34(+) cells showed that fucoidan reduced the production of CD34(+) cells and CD34(+)CXCR4(+) ratio but did not affect the production of monocytic, granulocytic, or megakaryocytic cells. The inhibitory effect of fucoidan on expansion of CB-CD34(+) cells was greater than mobilized PB. Division tracking analysis showed that CD34(+) cell generation times were lengthened by fucoidan. Fucoidan binds growth factors via their heparin-binding domain. The formation of an adherent hematopoietic culture system by fucoidan is most likely mediated by the binding of L-selectin and integrin-αMß2 on myeloids. Fucoidan deserves further investigation as glycan scaffold that is suitable for immobilization of other matrix molecules thought to comprise blood stem cell niche.

Multi-type branching models to describe cell differentiation programs.

Cell proliferation and differentiation is described by a multi-type branching process, a probability model that defines the inheritance of cell type. Cell type is defined by (i) a repression index related to the time required for S-phase entry and (ii) phenotype as determined by cell markers and division history. The inheritance of cell type is expressed as the expected number and type of progeny cells produced by a mother cell given her type. Expressions for the expected number and type of cells produced by a multi-cellular (bulk culture) system are derived from the general model by making the simplifying assumption that cell generation times are independent. The multi-type Smith-Martin model (MSM) makes the further assumption that cell generation times are lag-exponentially distributed with phenotype transitions occurring just before entry into S-phase. The inheritance-modified MSM (IMSM) model includes the influence of generation time memory so that mother and daughter generation times are correlated. The expansion of human cord blood CD34(+) cells by haematopoietic growth factors was division tracked in bulk culture using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE). The MSM model was fitted to division tracking data to identify cell cycle length, and the rates of CD34 antigen down-regulation and apoptosis. The IMSM model was estimated for mouse granulocyte-macrophage progenitors using live cell imaging data. Multi-type branching models describe cell differentiation dynamics at both single- and multi-cell scales, providing a new paradigm for systematic analysis of stem and progenitor cell development.

Ex Vivo Expansion of Hematopoietic Stem Cells to Improve Engraftment in Stem Cell Transplantation.

The efficient use of hematopoietic stem cells (HSC) for transplantation is often limited by the relatively low numbers of HSC collected. The ex vivo expansion of HSC for clinical use is a potentially valuable and safe approach to increase HSC numbers thereby increasing engraftment and reducing the risk of morbidity from infection. Here, we describe a protocol for the robust ex vivo expansion of human CD34(+) HSC isolated from umbilical cord blood. The protocol described can efficiently generate large numbers of HSC. We also describe a flow cytometry-based method using high-resolution division tracking to characterize the kinetics of HSC growth and differentiation. Utilizing the guidelines discussed, it is possible for investigators to use this protocol as presented or to modify it for their specific needs.

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.

Ex vivo expansion of haematopoietic stem cells to improve engraftment in stem cell transplantation.

The efficient use of haematopoietic stem cells (HSC) for transplantation is often limited by the relatively low numbers of HSC collected. The ex vivo expansion of HSC for clinical use is a potentially valuable and safe approach to increase HSC numbers thereby increasing engraftment and reducing the risk of morbidity from infection. Here we describe a protocol for the robust ex vivo expansion of human CD34(+) HSC isolated from umbilical cord blood. The protocol described can efficiently generate large numbers of HSC. We also describe a flow cytometry-based method using high resolution division tracking to characterise the kinetics of HSC growth and differentiation. Utilising the guidelines discussed, it is possible for investigators to use this protocol as presented or to modify it for their specific needs.

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.

Analysis of cell differentiation by division tracking cytometry.

We propose a quantitative method to characterize growth and differentiation dynamics of multipotent cells from time series carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) division tracking data. The dynamics of cell proliferation and differentiation was measured by combining (CFDA-SE) division tracking with phenotypic analysis. We define division tracking population statistics such as precursor cell frequency, generation time and renewal rate that characterize growth of various phenotypes in a heterogeneous culture system. This method is illustrated by study of the divisional recruitment of cord blood CD34(+) cells by hematopoietic growth factors. The technical issue of assigning the correct generation number to cells was addressed by employing high-resolution division tracking methodology and daily histogram analysis. We also quantified division-tracking artifacts such as CFDA-SE degeneration and cellular auto-fluorescence. Mitotic activation of cord blood CD34(+) cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34(+) cells cycle slower than CD34(-) cells. Generation times for CD34(+) and CD34(-) cells were 24.7 +/- 0.8 h and 15.1 +/- 0.9 h (+/-SD, n = 5), respectively. The 20-fold increase in CD34(+) cell numbers at Day 6 could be attributed to a high CD34(+) cell renewal rate (91% +/- 2% per division). Although cultures were initiated with highly purified CD34(+) cells (approximately 96%), CD34(-) numbers had expanded rapidly by Day 6. This rapid expansion could be explained by their short generation time as well as a small fraction of CD34(+) cells (approximately 5%) that differentiated into CD34(-) cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics.