Katie E. Glen

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Current status and perspectives on stem cell-based therapies undergoing clinical trials for regenerative medicine: case studies

BACKGROUND Apart from haematopoietic stem cell transplantation for haematological disorders many stem cell-based therapies are experimental. However, with only 12 years between human embryonic stem cell isolation and the first clinical trial, development of stem cell products for regenerative medicine has been rapid and numerous clinical trials have begun to investigate their therapeutic potential. SOURCE OF DATA This review summarizes key clinical trial data, current and future perspectives on stem cell-based products undergoing clinical trials, based on literature search and author research. AREAS OF AGREEMENT It is widely recognized that the ability to stimulate stem cell differentiation into specialized cells for use as cellular therapies will revolutionize health care and offer major hope for numerous diseases for which there are limited or no therapeutic options. AREAS OF CONTROVERSY Stem cell-based products are unique and cover a large range of disorders to be treated; therefore, there is significant potential for variation in cell source, type, processing manipulation, the bioprocessing approach and scalability, the cost and purity of manufacture, final product quality and mode of action. As such there are gaps in regulatory and manufacturing frameworks and technologies, only a small number of products are currently within late phase clinical trials and few products have achieved commercialization. GROWING POINTS Recent developments are encouraging acceleration through the difficulties encountered en route to clinical trials and commercialization of stem cell therapies. AREAS TIMELY FOR DEVELOPING RESEARCH The field is growing year on year with the first clinical trial using induced pluripotent stem cells anticipated by end 2013.

A novel automated bioreactor for scalable process optimisation of haematopoietic stem cell culture.

Proliferation and differentiation of haematopoietic stem cells (HSCs) from umbilical cord blood at large scale will potentially underpin production of a number of therapeutic cellular products in development, including erythrocytes and platelets. However, to achieve production processes that are scalable and optimised for cost and quality, scaled down development platforms that can define process parameter tolerances and consequent manufacturing controls are essential. We have demonstrated the potential of a new, automated, 24×15 mL replicate suspension bioreactor system, with online monitoring and control, to develop an HSC proliferation and differentiation process for erythroid committed cells (CD71(+), CD235a(+)). Cell proliferation was relatively robust to cell density and oxygen levels and reached up to 6 population doublings over 10 days. The maximum suspension culture density for a 48 h total media exchange protocol was established to be in the order of 10(7)cells/mL. This system will be valuable for the further HSC suspension culture cost reduction and optimisation necessary before the application of conventional stirred tank technology to scaled manufacture of HSC derived products.

Integrin-substrate interactions underlying shear-induced inhibition of the inflammatory response of endothelial cells

Conditioning of endothelial cells by shear stress suppresses their response to inflammatory cytokines. We questioned whether signalling through different integrin-matrix interactions, previously associated with the pathogenic effects of disturbed flow, supported the anti-inflammatory action of steady shear. Primary human endothelial cells were cultured on different substrates and exposed to shear stress (2.0Pa) for varying periods before stimulation with tumour necrosis factor-α (TNF). Shear-conditioning inhibited cytokine-induced recruitment of flowing neutrophils. However, the effect was similar for culture on collagen, laminin or fibronectin, even when seeding was reduced to 2 hours, and shear to 3 hours before TNF treatment (to minimise deposition of endothelial matrix). Nevertheless, in short- or longer-term cultures, reduction in expression of β(1)-integrin (but not β(3)-integrin) using siRNA essentially ablated the effect of shear-conditioning on neutrophil recruitment. Studies of focal adhesion kinase (FAK) phosphorylation, siRNA against FAK and a FAK-inhibitor (PF573228) indicated that FAK activity was an essential component downstream of β(1)-integrin. In addition, MAP-kinase p38 was phosphorylated downstream of FAK and also required for functional modification. Mechanotransduction through β(1)-integrins, FAK and p38 is required for anti-inflammatory effects of steady shear stress. Separation of the pathways which underlie pathological versus protective responses of different patterns of flow is required to enable therapeutic modification or mimicry, respectively.

Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture

BACKGROUND AIMS Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. METHODS CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. RESULTS The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. CONCLUSIONS Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps.

Shear stress-induced angiogenesis in mouse muscle is independent of the vasodilator mechanism and quickly reversible.

Is modulation of skeletal muscle capillary supply by altering blood flow due to a presumptive shear stress response per se, or dependent on the vasodilator mechanism?

The productivity limit of manufacturing blood cell therapy in scalable stirred bioreactors

Manufacture of red blood cells (RBCs) from progenitors has been proposed as a method to reduce reliance on donors. Such a process would need to be extremely efficient for economic viability given a relatively low value product and high (2 × 1012) cell dose. Therefore, the aim of these studies was to define the productivity of an industry standard stirred‐tank bioreactor and determine engineering limitations of commercial red blood cells production. Cord blood derived CD34+ cells were cultured under erythroid differentiation conditions in a stirred micro‐bioreactor (Ambr™). Enucleated cells of 80% purity could be created under optimal physical conditions: pH 7.5, 50% oxygen, without gas‐sparging (which damaged cells) and with mechanical agitation (which directly increased enucleation). O2 consumption was low (~5 × 10–8 μg/cell.h) theoretically enabling erythroblast densities in excess of 5 × 108/ml in commercial bioreactors and sub‐10 l/unit production volumes. The bioreactor process achieved a 24% and 42% reduction in media volume and culture time, respectively, relative to unoptimized flask processing. However, media exchange limited productivity to 1 unit of erythroblasts per 500 l of media. Systematic replacement of media constituents, as well as screening for inhibitory levels of ammonia, lactate and key cytokines did not identify a reason for this limitation. We conclude that the properties of erythroblasts are such that the conventional constraints on cell manufacturing efficiency, such as mass transfer and metabolic demand, should not prevent high intensity production; furthermore, this could be achieved in industry standard equipment. However, identification and removal of an inhibitory mediator is required to enable these economies to be realized. Copyright

Automated image analysis with the potential for process quality control applications in stem cell maintenance and differentiation

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored, using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell‐IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony “edge,” “core periphery” or “core” cells. Conventional biomarkers, such as Oct3/4, Nanog, and Sox‐2, were shown to correspond to specific morphologies using immunostaining and flow cytometry techniques. Quantitative monitoring of these morphological attributes in‐process using the reference image libraries showed rapid sensitivity to changes induced by different media exchange regimes or the addition of mesoderm lineage inducing cytokine BMP4. The imaging sample size to precision relationship was defined for each morphological attribute to show that this sensitivity could be achieved with a relatively low imaging sample. Further, the morphological state of single colonies could be correlated to individual colony outcomes; smaller colonies were identified as optimum for homogenous early mesoderm differentiation, while larger colonies maintained a morphologically pluripotent core. Finally, we show the potential of the same image libraries to assess cell number in culture with accuracy comparable to sacrificial digestion and counting. The data supports a potentially powerful role for quantitative image analysis in the setting of in‐process specifications, and also for screening the effects of process actions during development, which is highly complementary to current analysis in optimization and manufacture.

Immobilized hematopoietic growth factors onto magnetic particles offer a scalable strategy for cell therapy manufacturing in suspension cultures

Hematopoietic therapies require high cell dosages and precise phenotype control for clinical success; scalable manufacturing processes therefore need to be economic and controllable, in particular with respect to culture medium and growth factor (GF) strategy. The aim of this work was to demonstrate the biological function, and integration within scalable systems, of a highly controllable immobilized growth factor (iGF) approach. GFs were biotinylated and attached to streptavidin coated magnetic particles. GF concentration during biotinylation, GF-biotin ratio, and GF lysine content were shown to control iGF surface concentration and enable predictable co-presentation of multiple GF on a single bead. Function was demonstrated for immobilized GMCSF, SCF, TPO and IL-3 in GF dependent cell lines TF-1 and M-07e. Immobilized GMCSF (iGMCSF) was analyzed to show sustained activity over 8 days of culture, a 2-3 order of magnitude potency increase relative to soluble factor, and retained functionality under agitation in a micro-scale stirred tank bioreactor. Further, short exposure to iGMCSF demonstrated prolonged growth response relative to soluble factor. This immobilization approach has the potential to reduce the manufacturing costs of scaled cell therapy products by reducing GF quantities and offers important process control opportunities through separation of GF treatments from the bulk media.