Generating platelets in vitro for the clinic: optimisation and added clinical efficacy

Every year, 280,000 pools of blood platelets are transfused in the UK for patients whose platelet count is too low and are therefore at risk of bleeding. The provision of these platelets relies entirely on altruistic donations. Unfortunately, sourcing platelets from donors has implications such as precarious supply and the risk of infection. That is why donors are tested for, amongst others, HIV and hepatitis. Finally, there is the difficulty of "finding a match" for each patient.
These issues could be tackled if organs and blood cells could be derived from stem cells in the laboratory. Production in the laboratory would guarantee a safe infection-free constant supply. Also, by choosing the stem cell source carefully it is possible to provide perfectly matched products for each patient.
Pluripotent stem cells are a unique type of stem cell that can be grown in the laboratory and from which any kind of tissue can be derived. While they are ideal candidates to create platelets (as well as organs and other blood cells) in the laboratory, they also have their drawbacks.
Making platelets from pluripotent stem cells requires the formation of the platelet mother cell, called megakaryocytes (MKs). Doing this with traditional methods is a complex, inefficient and costly process. Therefore, it is difficult to translate into large scale manufacturing for the treatment of patients.
To overcome this, Eto in Japan and Ghevaert in the UK have developed novel methodologies to produce MKs. Eto creates MK "early cells" (called progenitors) and make them immortal so that their numbers can be expanded for long periods of time. Ghevaert has used "forward programming" which works by putting additional proteins in the stem cells called transcription factors. These turn on "switches", called genes, in the stem cells to efficiently guide them to become a specific type of cell type, in this case an MK.
However both methods of differentiation present common problems, so the project will allow both groups to exchange reagents and cells and promote collaboration to address the following issues:
1. Which stem cell line to choose. There are a lot of pluripotent stem cell lines to choose from and each will produce MKs efficiently (or not), according to its own nature. We have as yet, no idea why some lines are good producers or not. We will therefore collate data generated with 55 lines in between each lab to identify what makes a cell line a good producer or not, thereby guiding future choice to make platelets at large scale for clinical applications.
2. Promoting the number of MKs produced. Both methods of production rely on the generation of MK progenitors and their subsequent growth and expansion. We will therefore try to understand how the progenitors are made and what markers can be used to identify amongst the more mature cells in the culture. Using both these data we will be able to modify the early stage of culture when the progenitors are made from the stem cells and the subsequent culture so that these progenitors can be maintained for long periods of time without having to make them immortal (which may represent issues of final product integrity).
3. Test soluble factor that increase platelet release. By studying platelet donors Ghevaert has identified some small molecules which in the blood of donors promote the production of platelets from their mother cell MKs. We will test some of these in a bioreactor developed by Eto to see whether we can produce more than 100 platelets per MK (the current maximum reached). We know each MK can release more than 1000 platelets in donors.
4. Platelets contain granules which they release when they become activated. We can therefore use platelets as delivery vehicles to promote tissue repair or even fight infections. We will therefore generate platelets that will be supercharged with specific proteins and test them in 3 settings: active bleeding, bone fracture and infection.

The project is divided in 4 workpackages
WP1. Identification of efficient hPSC lines.
Ghevaert and Eto have tested between them 55 iPSC lines to identify good/bad MK producers. We will carry out whole genome expression and epigegetic study to pinpoint markers that identify good cell lines to facilitate the choice of clinical grade starting material.
WP2. Identification of MKProgs identity and optimisation of culture
The culture method used by both Ghevaert and Eto rely on the generation of MK progenitors. To maximise their formation and subsequently maintain their expansion in culture we will:
a. Identify markers to be able to quantify MK progenitor formation and culture monitoring using single cell sequencing./phenotyping
b. Understand the pathway of differentiation though dynamic analysis of the culture using single cell sequencing
c. Look at transcription regulatory networks during differentiation and what drives cell fate lineage
d. Use knowledge developed in a. to c. in order to optimise two key step of the culture: formation of MKProgs in the early stage and MKProgs maintenance without exhaustion in the expansion phase.
WP3. We will test soluble small molecules that promote platelet formation in platelet donors in the bioreactor developed by Eto (VerMES) to increase platelet formation in vitro and subsequently test the functionality of the platelets released in the presence of these small molecules.
WP4. We will supercharged platelets by targeting chosen proteins to the alpha granules using an expression vector already in use by Ghevaert. We will overexpress FVIIa, BMP-2/-4 and LL-34/-37 and test the supercharged platelets for increased haemostasis, bone repair and control of staphylococcal infection respectively.

This project is part of a much broader programme of research aimed at translating hPSC technologies into clinically relevant cell therapies, in this case focusing on platelets for transfusion.
1. Academic beneficiaries.
The main impact of current proposal is likely to be in the field of regenerative medicine. Both the cellular biology technique and statistical analyses developed as part of this project are eminently applicable to the production of any somatic cells from hPSCs. Both Japan and the UK are at the forefront of this regenerative medicine revolution and therefore the impact of this collaboration between 2 groups based in Japan and the UK will be substantial. The programme of work translating the production of platelets in vitro to the clinic is already on the way (Eto carried out PoC study in a first patient over the last 12 months) and therefore the learnings from this will be prime exemplars for the whole of the field. Both Eto and Ghevaert have a string of high impact publications and frequent oral communications which will facilitate the spreading of the knowledge developed here to the academic world.
2. Patients.
The potential advantages of in vitro- over donor-derived blood cells/organs are numerous: no risk of transmission of donor-acquired infections, a continuous supply and in particular, better tissue matching. Both CiRA (Japan) and the Cambridge Stem Cell Institute have active programmes of engagement with both patients and end users. This will continue during the lifetime of this grant and will be key to the future promotion of the products and commercialization.
Although the demand for blood products is currently being met through donation, the in vitro production of blood cells opens avenues for the production of blood cells with improved antigen matching (such as "HLA null" platelets or red cells lacking antigens that are relevant to the treatment of haemoglobinopathy patients from ethnic minorities).
In addition, platelets have been shown to play biological roles well beyond pure haemostasis, including in tissue repair and immunology/infection control. Our ability to "supercharged" platelets to emphasize this added clinical benefit, as proposed in this programme, is likely to lead to the development of new cellular therapies with applications beyond transfusion.
3. Commercial partners.
A robust manufacturing protocol for the generation of platelets from hPSCs has a global market of $300m. Based on figures from other hPSC regenerative medicine licensing deals, the ROI is expected to be at least 10-fold. Platelets produced in vitro are expected to obtain a large share of the market due to advantages in providing consistent cell supply from a validated, pathogen-free source. Both Eto and Ghevaert have linked to commercial partners (Megakaryon and Platelet Biogenesis) who have licenced some of the technologies developed in the academic laboratories. This will continue during the lifetime of the grant and facilitate transition from pure academia to large-scale production and delivery of a product to the health services.