Impact of oxygen levels on human hematopoietic stem and progenitor cell expansion

Oxygen levels are an important variable during the in vitro culture of stem cells. There has been increasing interest in the use of low oxygen to maximize proliferation and, in some cases, effect differentiation of stem cell populations. It is generally assumed that the defined pO2 in the incubator reflects the pO2 to which the stem cells are being exposed. However, we demonstrate that the pO2 experienced by cells in static culture can change dramatically during the course of culture as cell numbers increase and as the oxygen utilization by cells exceeds the diffusion of oxygen through the media. Dynamic culture (whereby the cell culture plate is in constant motion) largely eliminates this effect, and a combination of low ambient oxygen and dynamic culture results in a fourfold increase in reconstituting capacity of human hematopoietic stem cells compared with those cultured in static culture at ambient oxygen tension. Cells cultured dynamically at 5% oxygen exhibited the best expansion: 30-fold increase by flow cytometry, 120-fold increase by colony assay, and 11% of human CD45 engraftment in the bone marrow of NOD/SCID mice. To our knowledge, this is the first study to compare individual and combined effects of oxygen and static or dynamic culture on hematopoietic ex vivo expansion. Understanding and controlling the effective oxygen tension experienced by cells may be important in clinical stem cell expansion systems, and these results may have relevance to the interpretation of low oxygen culture studies.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bonemarrowfailure and immunodeficiencies. The currentmethods forHSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell- free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellularmatrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and itmay also provide insights into the constitution of the niche in which these cells reside in the bone marrow.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Expansion of human hematopoietic stem/progenitor cells on decellularized matrix scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.

Quantitation of ascorbic acid in Arabidopsis thaliana reveals distinct differences between organs and growth phases

Optimal plant growth is the result of the interaction of a complex network of plant hormones and environmental signals. Ascorbic acid (AsA) is a crucial antioxidant in plants and is involved in the regulation of cell division, cell expansion, photosynthesis and hormone biosynthesis. Quantitative analysis of AsA in Arabidopsis thaliana organs was conducted using HPLC with d -isoascorbic acid (Iso-AsA) as an internal standard. Analysis revealed Àuctuations in the levels of AsA in different organs and growth phases when plants were grown under standard conditions. AsA concentrations increased in leaves in direct proportion to leaf size and age. Young siliques (seed set stage) and Àowering buds (open and unopened) showed the highest levels of AsA. A relationship was found between the level of AsA and indole acetic acid (IAA) in leaves, stems, Àowers, and siliques and the highest level of IAA and AsAwere found in the Àowers. In contrast, the lowest level of the plant hormone, salicylic acid, was found in the Àowers and the highest quantity measured in the leaves. Consequently, AsA has been found to be a multifunctional molecule that is involved as a key regulator of plant growth and development.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Y704, Y729, Y744, Y764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation and cell survival. However, it is unclear whether these tyrosines are equally important under more physiological conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated GCSF- R deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (mO), single tyrosine "add back" mutants or wild type (WT) receptors. G-CSFinduced responses were determined in primary colony assays, serial replatings and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Y764 strongly enhanced proliferativeresponses, which was reverted by inhibition of ERK activitity. Y729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing mO gradually dropped compared to WT. The presence of Y729, but also Y704 and Y744, both involved in activation of STAT3, further reduced replating
efficiencies. Conversely, Y764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a >104–fold increase of colony forming cells over mO after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.

Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals

Using a milk-cell cDNA sequencing approach we characterised milk-protein sequences from two monotreme species, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) and found a full set of caseins and casein variants. The genomic organisation of the platypus casein locus is compared with other mammalian genomes, including the marsupial opossum and several eutherians. Physical linkage of casein genes has been seen in the casein loci of all mammalian genomes examined and we confirm that this is also observed in platypus. However, we show that a recent duplication of β-casein occurred in the monotreme lineage, as opposed to more ancient duplications of α-casein in the eutherian lineage, while marsupials possess only single copies of α- and β-caseins. Despite this variability, the close proximity of the main α- and β-casein genes in an inverted tail-tail orientation and the relative orientation of the more distant kappa-casein genes are similar in all mammalian genome sequences so far available. Overall, the conservation of the genomic organisation of the caseins indicates the early, pre-monotreme development of the fundamental role of caseins during lactation. In contrast, the lineage-specific gene duplications that have occurred within the casein locus of monotremes and eutherians but not marsupials, which may have lost part of the ancestral casein locus, emphasises the independent selection on milk provision strategies to the young, most likely linked to different developmental strategies. The monotremes therefore provide insight into the ancestral drivers for lactation and how these have adapted in different lineages.

Replication of hematopoietic stem cell niche

This thesis models the hematopoietic stem cell (HSC) niche by using decellularized extracellular matrix (ECM) scaffolds prepared by MS-5 cell line. The ECM replicates many of the properties of HSC niches in vivo, providing insights into expansion of HSCs that may have several applications in translational medicine.

Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34+ hematopoietic progenitor cells

Background aims. Cord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.

Methods. This is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34 hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.

Results. Optimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.

Conclusions. This study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.

Tissue engineered, biomimetic acellular matrices for expansion of hematopoietic progenitors

This thesis discusses a novel strategy for ex vivo expansion of human HSPC in a cell free culture system and it suggests methods to improve the functional properties of stromal cell derived ACMs to support ex-vivo HSPC growth.

Enhanced Ex Vivo expansion of human hematopoietic progenitors on native and spin coated acellular matrices prepared from bone marrow stromal cells

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion.