Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors

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A non-contact suspension culture approach to the culture of osteogenic cells derived from a CD49elow subpopulation of human bone marrow-derived cells

We demonstrate that adult human bone marrow (BM) contains a population of mesenchymal stromal cells (MSCs) that can be expanded in non-adherent, cytokine-dependent, suspension culture conditions for at least 42 days. The cells generated during suspension culture lacked detectable levels of gene expression associated with differentiated mesenchymal cell types, including bone, muscle and fat, suggesting that suspension culture maintains MSCs in an uncommitted state. However, when these undifferentiated cells were taken out of suspension culture and placed in adherent osteogenic conditions, osteogenic genes were upregulated and morphologically identifiable bone matrix was elaborated. Flow cytometric analysis of uncultured, density gradient-separated human BM revealed that colony forming unit-fibroblast (CFU-F) and CFU-osteoblast (CFU-O) activity was associated with a CD45(-) CD49e(low) phenotype. Importantly, suspension-grown MSCs, capable of CFU-F and CFU-O development, maintained the CD45(-)CD49e(low) phenotype whereas MSCs directly cultured under adherent conditions rapidly upregulated CD49e expression and were associated with a CD45(-)CD49e(high) phenotype. Tracking the CD49e(low) expression under suspension culture conditions provides a mechanism to isolate an expanding suspension-grown MSC population with osteogenic potential. This could provide a potential strategy to isolate populations of MSCs, with functional osteogenic capacity, in a scalable and controllable culture system for therapeutic applications.     

Concentration-dependent internalization of a cytokine/cytokine receptor complex in human hematopoietic cells

Soluble steel factor (SF) is a potent stimulator of hematopoietic progenitor cell proliferation in vitro, and cytokine combinations that include SF can support extensive expansions of hematopoietic cells. Recently, we showed that very primitive progenitor cells from normal human bone marrow require exposure to very high concentrations of cytokines to maintain their primitive status while proliferating. These cells also display higher cell-specific cytokine uptake rates than more differentiated types of hematopoietic cells. As a first step toward identifying the mechanisms involved in mediating such cytokine dose-dependent effects, we have now investigated the kinetics of SF receptor (c-kit) internalization by human Mo7e cells exposed to different extracellular concentrations of soluble SF. Transfer of Mo7e cells to a higher concentration of SF caused an initially rapid downregulation of cell surface c-kit which was accompanied by a rapid depletion of extracellular SF. Confocal microscopy showed a concomitant increase in the number and intensity of intracellular c-kit aggregates. After the first 30 min, the cells continued to deplete SF from the medium but at a much slower rate. During this period, there was a gradual recovery of expression of c-kit on the cell surface. A mathematical analysis of bulk medium to cell-surface SF-mass transport indicated that the cytokine-depletion rates measured were not likely to have significantly depleted the SF concentration in the microenvironment of the cells. Taken together, these results underscore the importance of monitoring and appropriately regulating cytokine concentrations in hematopoietic cell expansion cultures. They may also help to explain the different biological responses exhibited by primitive hematopoietic cells exposed to different types and concentrations of cytokines for periods of days.     

Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.     

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Culture development for human embryonic stem cell propagation: molecular aspects and challenges

Basic fibroblast growth factor and members of the transforming growth factor-beta superfamily are important regulators of human embryonic stem cell (hESC) self-renewal. Extensive cross-talk between the intracellular signaling pathways activated by these factors contributes to maintenance of the undifferentiated hESC state. Understanding the molecular regulation of hESC self-renewal will facilitate the design of improved systems for hESC propagation and provide a foundation for strategies to direct the differentiation of hESCs to clinically relevant cell types.     

Development of a perfusion fed bioreactor for embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of cardiomyocyte output

Cell transplantation is emerging as a promising new approach to replace scarred, nonfunctional myocardium in a diseased heart. At present, however, generating the numbers of donor cardiomyocytes required to develop and test animal models is a major limitation. Embryonic stem (ES) cells may be a promising source for therapeutic applications, potentially providing sufficient numbers of functionally relevant cells for transplantation into a variety of organs. We developed a single-step bioprocess for ES cell-derived cardiomyocyte production that enables both medium perfusion and direct monitoring and control of dissolved oxygen. Implementation of the bioprocess required combining methods to prevent ES cell aggregation (hydrogel encapsulation) and to purify for cardiomyocytes from the heterogeneous cell populations (genetic selection), with medium perfusion in a controlled bioreactor environment. We used this bioprocess to investigate the effects of oxygen on cardiomyocyte generation. Parallel vessels (250 mL culture volume) were run under normoxic (20% oxygen tension) or hypoxic (4% oxygen tension) conditions. After 14 days of differentiation (including 5 days of selection), the cardiomyocyte yield per input ES cell achieved in hypoxic vessels was 3.77 +/- 0.13, higher than has previously been reported. We have developed a bioprocess that improves the efficiency of ES cell-derived cardiomyocyte production, and allows the investigation of bioprocess parameters on ES cell-derived cardiomyogenesis. Using this system we have demonstrated that medium oxygen tension is a culture parameter that can be manipulated to improve cardiomyocyte yield.