Manufacture of red blood cells from stem cells

Although the current system of blood transfusion is relatively safe and established within the UK, periodic shortages of certain blood groups and residual risks of emerging transfusion transmitted infections (TTIs) make an industrial manufacture process for the generation of red blood cells more desirable. The generation of red blood cells from human embryonic stem cells has been completed in vitro but the major challenge lies in making the process highly scalable and economically viable. Initially human embryonic and induced pluripotent stem cells were trialled for use on the project however these were found to be inconsistent, a major issue in cellular therapies. They were replaced with CD34+ cord blood stem cells which are morphologically and physiologically divergent. In order to assess their suitability for a GMP-complaint manufacturing process an ultra-scale down (microfluidic) approach was taken to assess the cells’ reactions to the changeable physical environment associated with scale-up procedures. Cellular responses to hypoxia and shear stress were evaluated at successive time-points in the step-wise haematopoietic differentiation process and recommendations made for optimum scale-up conditions. Conversely further challenges in the manufacture of red blood cells from stem cells were uncovered regarding the differences between stem cell derived red blood cells and their adult equivalents

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells

Engineering rFVIIa-loaded platelets; a novel approach to treating acute bleeding

Haemorrhage remains a leading cause of mortality around the world, resulting from both trauma and surgery. Current treatments for acute haemorrhage include blood products such as fresh frozen plasma, as well as platelet transfusion and recombinant clotting factors such as rFVIIa. However, the use of recombinant clotting factors is limited due to cost and adverse side effects resulting from increased thrombosis, such as increased rate of myocardial infarction and cerebrovascular accidents. Platelets are small anucleate cells that exist in very large quantities in our blood and, along with cross-linked fibrin from the coagulation cascade, are involved in forming a haemostatic plug upon exposure to damaged endothelium in a wound. They are metabolically active and undergo a process of platelet activation when stimulated by pro-thrombotic agonists such as thrombin resulting from the coagulation cascade. This then triggers a process whereby the contents of their granules are released locally to facilitate coagulation. This thesis explores the possibility of loading these platelet granules with recombinant clotting factors, in this case rFVIIa which has already been shown in clinical trials to result in a significant decrease in mortality from acute haemorrhage. The targeted delivery of this drug is explored, both through endocytosis and genetic engineering of megakaryocytes differentiated in vitro from induced pluripotent stem cells (iPSCs). These are evaluated with in vitro assays to model the process of clot formation, as well as an in vivo model of haemostasis to compare their efficacy to conventional treatments. Overall, this serves as a proof of concept of the engineering of platelet granules as a novel drug delivery system.Cambridge School of Clinical Medicine, the Rosetrees Trust and the Frank Edward Elmore Fun

The Cellular and Molecular Characterization of Essential Hypertension Using Innovative Pathology Models

Essential hypertension, characterized by elevated blood pressure levels, is a primary risk factor for other cardiovascular diseases. There has been a drastic rise in the amount of people who have essential hypertension; trends predict that 1.56 billion people worldwide will be living with essential hypertension by 2025. Anti-hypertensive drugs lower elevated blood pressure levels, but do not eliminate the disease itself or reverse the structural damage to arteries. This study aims to elucidate hypertension-induced endothelial cell (EC) dysfunction using a blood vessel tissue engineering approach as a pathology model. In order to characterize EC dysfunction, the FlexCell® system was used to induce a dynamic 140 mmHg transmural pressure onto EC monocultures and endothelial cell–smooth muscle cell (EC-SMC) co-cultures over the course of four days; control samples were subjected to 120-mmHg. Cell-matrix adhesion and cell-cell interaction were evaluated by EC culture studies and vasoregulation was the main focus for the EC-SMC co-culture studies. Immunofluorescence and western blots were used to detect integrin β1, FAK, and VE-cadherin, as well as eNOS, endothelin, and angiotensin. Additionally, a novel bioreactor platform was developed to simulate and control hypertension values. Cell-seeded vascular scaffolds were mounted into a bioreactor system that induced a flow rate of 150 mL/min and a hypertensive pressure profile, which matched that of a femoral artery found in the human body. Preliminary results showed that cells were able to attach and that the arterial structure remained intact. However, dynamic rotational seeding should be implemented for future studies for better cell attachment on arteries. This system could be tailored towards studying other vascular diseases, or modified to produce clinically relevant tissue engineered blood vessels, resistant to high blood pressure

Using Genome Editing to Engineer Universal Platelets

Genome editing technologies such as Zinc Finger nucleases, TALENS and CRISPR/Cas9 have recently emerged as tools with the potential to revolutionise cellular therapy. This is particularly exciting for the field of regenerative medicine, where the large-scale, quality controlled editing of large numbers of cells could generate essential cellular products ready to move towards the clinic. This review details recent progress towards generating HLA Class-I null platelets using genome editing technologies for beta-2-microglobulin deletion, generating a universally transfusable cellular product. In addition, we discuss various methods for megakaryocyte (MK) production from human pluripotent stem cells and subsequent platelet production from the MKs. As well as simply producing platelets, differentiating MK cultures can enable us to understand megakaryopoeisis in vivo and take steps towards ameliorating bleeding disorders or deficiencies in MK maturation in patients. Thus by intersecting both these areas of research, we can produce optimised differentiation systems for the production of universal platelets, thus offering a stable supply of platelets for difficult-to-match patients and providing areas with transmissible disease concerns or an unpredictable supply of platelets with a steady supply of quality controlled platelet units.UK Regenerative Medicine Platform and the Pluripotent and Engineered Cell Hub. Research in the laboratory is supported by core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institut

Culture-Derived Human Platelets for Clinical Transfusion

Platelets are a vital component of human blood due to their role in clotting. Despite the incredible importance of platelets for human survival, there is very little supply of platelets to meet the clinical demand. Currently, platelets are only supplied from donations, whether through whole blood or apheresis. Therefore, there is a need for ex vivo means of producing platelets at a price that is competitive with the current market price. This project seeks to meet 65% of the demand for platelets in Philadelphia by producing platelets ex vivo in a process that utilizes shear stress to induce platelet formation from megakaryocytes. To begin this batch process, induced pluripotent stem cells derived from bone marrow are proliferated over the course of 10 days. During a 20-day differentiation phase that occurs in 2 parallel 2000 L stirred single-use bioreactors, a number of growth factors are supplied to the stem cell culture to induce differentiation of the cells into megakaryocytes. The megakaryocytes, once mature, are exposed to shear stress in a specially developed microfluidic device, inspired by the work done in Dr. Daeyeon Lee’s lab, to induce platelet formation. Once platelets have been generated, they are processed over the course of 5 days to be separate, washed, and concentrated in a resuspension solution via a series of centrifugation and mixing steps, after which they will be packaged into platelet units and transported to surrounding hospitals for clinical transfusion. This process design results a yearly production of 69,550 300 mL platelet units each year, with a platelet count of 5.5x1010 platelets in each unit. For this process, the IRR is 28.99%, the NPV is 14.4 million in the year 2030, and ROI is 23.21%

A versatile platform for three-dimensional dynamic suspension culture applications

In the last decades, the rapid upgrading in cell biological knowledge has bumped the interest in using cell-based therapeutic approaches as well as cell-based model systems for the treatment of diseases. Given the rapid translation towards cell-based clinical treatments and the consequent increasing demand of cell sources, three-dimensional (3D) suspension cultures have demonstrated to be an advantageous alternative to monolayer techniques for large scale expansion of cells and for the generation of three-dimensional model systems in a scale-up perspective. In this scenario, a versatile bioreactor platform suitable for 3D dynamic suspension cell culture under tuneable shear stress conditions is developed and preliminarily tested in two different biotechnological applications. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits a laminar hydrodynamics, enabling dynamic cell suspension in an environment favourable to mass transport. Technically, the bioreactor is conceived to produce dynamic suspension cell culture under tuneable shear stress conditions without the use of moving components (from ultralow to moderate shear stress). A multiphysics computational modelling strategy is applied for the development and optimization of the suspension bioreactor platform. The in silico modelling is used to support the design and optimization phase of the bioreactor platform, providing a comprehensive analysis of its operating principles, also supporting the development/optimization of culture protocols directly in silico, and thus minimizing preliminary laboratory tests. After the technical assessment of the functionality of the device and a massive number of in silico simulations for its characterization, the bioreactor platform has been employed for two preliminary experimental applications, in order to determine the suitability of the device for culturing human cells under dynamic suspension. In detail, the bioreactor platform has been used to culture lung cancer cells for spheroid formation (Calu-3 cell line) under ultralow shear stress conditions, and for human induced pluripotent stem cell (hiPSC) dynamic suspension culture. The use of the bioreactor platform for the formation of cancer cell spheroids under low shear stress conditions confirms the suitability of the device for its use as dynamic suspension bioreactor. In fact, compared to static cell suspension, after 5 days of dynamic suspension culture the bioreactor platform preserves morphological features, promotes intercellular connection, increases the number of cycling cells, and reduces double strand DNA damage. Calu-3 cells form functional 3D spheroids characterized by more functional adherence junctions between cells. Moreover, the computational model has been used as a tool for assisting the setup of the experimental framework with the extraction of the fluid dynamic features establishing inside the bioreactor culture chamber. As second proof of concept application, the bioreactor platform has been tested for the dynamic suspension of hiPSCs. Starting from the ‘a priori’ knowledge gained by the development of the in silico culture protocol, the agglomeration of human induced pluripotent stem cells has been modulated by means of the combination of moderate intermittent shear stress and free-fall transport within the bioreactor culture chamber. The inoculation of single cells suspensions inside the bioreactor chamber promotes cell-cell interaction and consequently the formation of human induced pluripotent stem cell aggregates. In conclusion, the impeller-free functioning principle characterizing the proposed bioreactor platform demonstrates to be promising for human cell dynamic suspension culture. In the future, this bioreactor platform will be further optimized for the realization of impeller-free dynamic suspension bioreactors dedicated and optimized to specific applications in stem cell and cancer cell culture

Bioreactor-Based Bone Tissue Engineering

The aim of this chapter is to describe the main issues of bone tissue engineering. Bone transplants are widely used in orthopedic, plastic and reconstructive surgery. Current technologies like autologous and allogenic transplantation have several disadvantages making them relatively unsatisfactory, like donor site morbidity, chronic pain, and immunogenicity and risk hazard from infectious disease. Therefore, regenerative orthopedics seeks to establish a successful protocol for the healing of severe bone damage using engineered bone grafts. The optimization of protocols for bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries. We discuss mesenchymal stem cells as the most suitable cell type for bone tissue engineering. They can be isolated from a variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal. Mechanical support can be provided by different cell scaffolds based on natural or synthetic biomaterials, as well as combined composite materials. Three-dimensional support is enabled by bioreactor systems providing several advantages as mechanical loading, homogeneous distribution of cells and adequate nutrients/waste exchange. We also discuss the variety of osteoinductive signals that can be applied in bone tissue engineering. The near future of bone healing and regeneration is closely related to advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries

Osteochondral Tissue Engineering: The Potential of Electrospinning and Additive Manufacturing

The socioeconomic impact of osteochondral (OC) damage has been increasing steadily over time in the global population, and the promise of tissue engineering in generating biomimetic tissues replicating the physiological OC environment and architecture has been falling short of its projected potential. The most recent advances in OC tissue engineering are summarised in this work, with a focus on electrospun and 3D printed biomaterials combined with stem cells and biochemical stimuli, to identify what is causing this pitfall between the bench and the patients' bedside. Even though significant progress has been achieved in electrospinning, 3D-(bio)printing, and induced pluripotent stem cell (iPSC) technologies, it is still challenging to artificially emulate the OC interface and achieve complete regeneration of bone and cartilage tissues. Their intricate architecture and the need for tight spatiotemporal control of cellular and biochemical cues hinder the attainment of long-term functional integration of tissue-engineered constructs. Moreover, this complexity and the high variability in experimental conditions used in different studies undermine the scalability and reproducibility of prospective regenerative medicine solutions. It is clear that further development of standardised, integrative, and economically viable methods regarding scaffold production, cell selection, and additional biochemical and biomechanical stimulation is likely to be the key to accelerate the clinical translation and fill the gap in OC treatment