Computational Tools for Quantifying Concordance in Single-Cell Fate.

Cells are dynamic biological systems that interact with each other and their surrounding environment. Understanding how cell extrinsic and intrinsic factors control cell fate is fundamental to many biological experiments. However, due to transcriptional heterogeneity or microenvironmental fluctuations, cell fates appear to be random. Individual cells within well-defined subpopulations vary with respect to their proliferative potential, survival, and lineage potency. Therefore, methods to quantify fate outcomes for heterogeneous populations that consider both the stochastic and deterministic features of single-cell dynamics are required to develop accurate models of cell growth and differentiation. To study random versus deterministic cell behavior, one requires a probabilistic modelling approach to estimate cumulative incidence functions relating the probability of a cell's fate to its lifetime and to model the deterministic effect of cell environment and inheritance, i.e., nature versus nurture. We have applied competing risks statistics, a branch of survival statistics, to quantify cell fate concordance from cell lifetime data. Competing risks modelling of cell fate concordance provides an unbiased, robust statistical modelling approach to model cell growth and differentiation by estimating the effect of cell extrinsic and heritable factors on the cause-specific cumulative incidence function.

Analysis of cardiac stem cell self-renewal dynamics in serum-free medium by single cell lineage tracking.

Cardiac colony forming unit-fibroblasts (cCFU-F) are a population of stromal cells residing within the SCA1+/PDGFRα+/CD31- fraction of adult mouse hearts, and which have functional characteristics akin to bone marrow mesenchymal stem cells. We hypothesise that they participate in cardiac homeostasis and repair through their actions as lineage progenitors and paracrine signaling hubs. However, cCFU-F are rare and there are no specific markers for these cells, making them challenging to study. cCFU can self-renew in vitro, although the common use of serum has made it difficult to identify cytokines that maintain lineage identity and self-renewal ability. Cell heterogeneity is an additional confounder as cCFU-F cultures are metastable. Here, we address these limitations by identifying serum-free medium (SFM) for growth, and by using cCFU-F isolated from PdgfraGFP/+ mice to record fate outcomes, morphology and PDGFRα expression for hundreds of single cells over time. We show that SFM supplemented with basic fibroblast growth factor, transforming growth factor-ß and platelet-derived growth factor, enhanced cCFU-F colony formation and long-term self-renewal, while maintaining cCFU-F potency. cCFU-F cultured in SFM maintained a higher proportion of PDGFRα+ cells, a marker of self-renewing cCFU-F, by increasing Pdgfra-GFP+ divisions and reducing the probability of spontaneous myofibroblast differentiation.

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis.

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level.

Ex vivo expansion of cord blood progenitors impairs their short-term and long-term repopulating activity associated with transcriptional dysregulation of signalling networks.

OBJECTIVES: Cord blood (CB) has been established to be an alternative source of haematopoietic stem/progenitor cells (HPC) for transplantation. The number of HPC per CB unit is limited, which results in engraftment delay. Ex vivo expansion of HPC improvement must overcome this. MATERIALS AND METHODS: Flow cytometry was used to extensively phenotype HPC pre- and post-expansion and CFDA-SE staining was used to track cell divisions. The NSG mouse model was employed in transplantation studies to determine long and short term repopulation in human cells. Gene array analysis was used to evaluate signalling pathways regulated following ex vivo expansion of HPC. RESULTS: expansion of CD34(+) HPC impaired their regenerative function. In this xenograft transplantation model we showed that repopulating activity of CB cells declined following expansion. Expanded HPC had delayed engraftment at early and late stages post-transplant. High resolution division tracking revealed that the cultured HPC had reduced expansion and self-renewal probability and increased differentiation rate compared to non-expanded cells. Gene expression analysis exposed significant modulation of a complex network of genes and pathways that normally maintain HPC proliferation and limit their differentiation. CONCLUSIONS: The decline in short-term engraftment is consistent with the loss of rapid SCID repopulating ability r(SRA) by expanded CD34(+) CD38(+) cells recently reported. Our data raise concerns for future clinical applications of expanded HPC alone in transplantation.

Biological performance of a novel synthetic furanone-based antimicrobial.

Infection of medical devices causes significant morbidity and mortality and considerable research effort has been directed at solving this problem. The aim of this study was to assess the biological performance of a novel furanone compound that has potential as an anti-infective coating for medical devices. This study examined in vitro leukocyte response following exposure to the antibacterial 3-(1'-bromohexyl)-5-dibromomethylene-2(5H)-furanone and assessed the tissue response following subcutaneous implantation of the furanone compound covalently bound to polystyrene (PS). Peripheral human blood was exposed to furanones in solution for 1h and flow cytometry used to analyse viability and changes in expression of surface receptors CD11b/CD18 and CD44. Flow cytometry results from propidium iodide stained cell suspensions suggested that the leukocytes were viable after exposure to furanones in whole blood. No significant difference was found in the expression of CD11b/CD18 and CD44 between the furanone exposed samples and the negative control for neutrophils suggesting that the furanones themselves do not activate these leukocytes. The positive control lipopolysaccharide significantly up-regulated CD11b/CD18 and slightly down-regulated CD44 on both PMNs and monocytes. In vivo studies of the tissue response to furanone covalently bound to PS showed that there was no significant difference in cellularity of capsules surrounding the disk and no significant increase in myeloperoxidase expression. These results demonstrate negligible acute inflammatory response to synthetic brominated antibacterial furanones. Future studies will focus on chronic responses and examination of in vivo efficacy.

Prolonged ex vivo culture of cord blood CD34(+) cells facilitates myeloid and megakaryocytic engraftment in the non-obese diabetic severe combined immunodeficient mouse model.

A clinical goal for ex vivo expansion of cord blood (CB) CD34(+) cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34(+) cells should support the continued production of neutrophils and platelets in the 6-week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45(+) cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non-obese diabetic severe combined immunodeficient mouse (NOD-SCID) following 7 or 14 d of ex vivo expansion of CB CD34(+) cells. Mice transplanted with 14 d cells showed greater percentages of human CD45(+) cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34(+) cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34(+), CD33(+), CD61(+) and CD19(+) cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45(+), CD34(+) and CD19(+) cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.

Prior cryopreservation of ex vivo-expanded cord blood cells is not detrimental to engraftment as measured in the NOD-SCID mouse model.

Cytokine-mediated expansion has been proposed and successfully used to facilitate engraftment post transplantation. This study examined whether cryopreservation following expansion has a detrimental effect on the ability of cells to engraft, using the NOD-SCID mouse model. Cord blood (CB) CD34(+) cells were incubated for 7 days with stem cell factor (SCF), flt-3 ligand (FL), and megakaryocyte growth and development factor (MGDF). Expanded CD34(+) cells were transplanted into NOD-SCID mice either fresh or following cryopreservation and thawing. After thawing, recovery of nucleated cells was 94%, of CD34 cells was 63%, and of day-14 progenitors was 17%. The loss of day-14 progenitor cells among the thawed expanded cells did not influence the kinetics of human engraftment in the mouse. Bone marrow (BM) of mice transplanted with thawed expanded CD34(+) cells (14 +/- 3.9%) showed significantly higher levels of human engraftment than mice transplanted with fresh expanded CD34(+) cells (1.5 +/- 0.5%, p = 0.0064). Thawed expanded CD34(+) cells had significantly higher SCID Engrafting Potential (SEP) than freshly expanded CD34(+) cells (p < 0.001). Results suggest that prior cryopreservation does not prevent expanded cells engrafting in NOD-SCID mice.

Characterization of cytokine interactions by flow cytometry and factorial analysis.

BACKGROUND: Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. METHODS: Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(7-3)(IV)) or full factorial (2(4)) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. RESULTS: In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. CONCLUSIONS: Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level.

Analysis of growth kinetics by division tracking.

Cell division tracking using fluorescent dyes, such as carboxyfluorescein diacetate succinimidyl ester, provides a unique opportunity for analysis of cell growth kinetics. The present review article presents new methods for enhancing resolution of division tracking data as well as derivation of quantities that characterize growth from time-series data. These include the average time between successive divisions, the proportion of cells that survive and the proliferation per division. The physical significance of these measured quantities is interpreted by formulation of a two-compartment model of cell cycle transit characterized by stochastic and deterministic cell residence times, respectively. The model confirmed that survival is directly related to the proportion of cells that enter the next cell generation. The proportion of time that cells reside in the stochastic compartment is directly related to the proliferation per generation. This form of analysis provides a starting point for more sophisticated physical and biochemical models of cell cycle regulation.

High-resolution cell division tracking demonstrates the FLt3-ligand-dependence of human marrow CD34+CD38- cell production in vitro.

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high-resolution procedure for tracking 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)- labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE-fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that the addition of Flt3-ligand (FL) to a cytokine cocktail consisting of Steel factor, IL-3, IL-6 and G-CSF increased the proportion of CD34+ (CD45RA/CD71)-, but not CD34+(CD45RA/CD71)+, human marrow cells initially recruited into division in vitro, shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34+CD38- population through several (up to four) cell generations. These studies also showed that during the first 4d there was no detectable apoptosis among the progeny of the CD34+(CD45RA/CD71)- cells generated in the presence of this four-cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.

Design of hollow fiber modules for uniform shear elution affinity cell separation.

Large-scale monoclonal antibody based systems for the selection of cell subsets will play a prominent role in the development of hematotherapy and graft engineering. Hollow fiber systems for affinity cell separation rely on the generation of uniform fluid shear stress at the lumenal attachment interface. Potential mechanisms for nonuniformity of lumenal wall shear stress are fiber wall permeation fluxes driven by the pressure gradient along individual fibers and the influence of inlet header dynamic pressure on the radial distribution of axial flow within the fiber module. Dimensional analysis and numerical solution of the flow field within the lumen of a hollow fiber module illustrate the main physical criteria for design of hollow fiber modules. There will be a nearly uniform distribution of flow within the fiber bundle provided that the dynamic inlet pressure is small in comparison with the pressure drop along fibers. Fiber wall permeation fluxes will have a negligible effect on axial flow rate for nonporous membranes such as Cuprophan.

Hollow-fibre affinity cell separation system for CD34+ cell enrichment.

A hollow-fibre immunoadsorption system has been developed for the purification of CD34+ cells from mononuclear cells. This cell separation technique is based on the use of uniform surface fluid shear stress to fractionate cells that attach to the inside surface of hollow fibres. Monoclonal antibody to the CD34 antigen was covalently coupled to the lumenal surface of cuprophan minidialysers (surface area 220 cm2). After the selective adsorption of CD34+ cells (28 min), a depleted fraction was collected at 5 dynes/cm2 followed by washes at 10 and 25 dynes/cm2. Antigen-positive cells were recovered after incubation with chymopapain. The device was tested by using peripheral blood mononuclear cells from seven patients who had received granulocyte colony-stimulating factor and chemotherapy. The average number of cells processed was 1.3 +/- 0.2 x 10(8) (+/- S.E.M.), and the preselection incidence of CD34+ cells ws 1.6 +/- 0.6% (range 0.21-4.13%; n = 7). The enrichment purity was 94.4 +/- 3.1%, and 61 +/- 9% of input CD34+ cells were recovered in the enriched fraction (n = 4). Enrichment resulted in a 3.3 +/- 0.1% log10 depletion of CD34- cells (n = 4). Hollow-fibre affinity cell separation has potential as a medium to large-scale cell enrichment technology.

Ex vivo manipulation of cell subsets for cell therapies.

Large-scale cell separation and ex vivo expansion technologies will form the basis for development of new cellular products for the treatment of cancer and fatal viral diseases. The cell subsets that are likely to play a significant role in cellular therapy include hematopoietic stem cells, platelet and granulocyte precursors, cytotoxic lymphocytes, and genetically modified hematopoietic or lymphoid precursors. Cell enrichment techniques are required to eliminate tumor cells from autologous stem cell grafts and to reduce the size of culture systems required for expansion or gene transfection. The consumption of expensive culture components such as cytokines and serum may be reduced by the use of perfusion bioreactor devices. Methods that have been developed for the production of cell subsets for cellular therapy are reviewed.

An experimental model of affinity cell separation.

Cell affinity separations are based on the selective attachment of cell phenotype using antibody or lectins specific for cell surface markers. The major physicochemical factors which influence ligand-mediated cell adhesion dynamics and the efficiency of cell affinity separation have been examined. Uniform cell detachment forces were generated with a parallel-plate flow cell (plate separation 100 microns, surface area 3 cm2). Hydrodynamic shear stress was used to measure cell adhesion strength and to separate cells on the basis of surface affinity. Human cell lines grown in tissue culture were separated on a flat derivatised glass immunoadsorbent which formed the floor of the flow chamber. Flow-cell residence time, detachment shear stress, temperature, and ligand density were shown to influence cell attachment probability. An understanding of the physical basis of ligand-mediated cell adhesion provided a rationale for optimisation of affinity cell separation. At room temperature attachment of positive cells was rapid (< 2 min) and adhesion strength was directly related to immunoadsorbent ligand density. Purity and recovery of enriched fractions were dependent on the separation shear stress and could be optimised using this parameter. Enrichment factors were greater than 100-fold, with at least 90% of positive cells recovered in enriched fractions. Enrichment purity and yields did not decline at higher loading densities (10(5) cells/cm2). Selective immunoadsorbent surface chemistry is a prerequisite for efficient affinity cell separation. Purity and recovery may be optimised by fractionating enriched and depleted cell populations with uniform fluid shear stress.