MICA: Single cell phenotyping platform to genetically engineer megakaryocytes to upscale platelet production for transfusion purposes

在英国，每年有25万个血小板单位被输注给患者。血小板是最小的血细胞，主要促进血液凝结。对于活动性出血(创伤或手术后)的患者，或由于遗传疾病或恶性肿瘤及其(通常)骨髓抑制治疗而导致的血小板数量严重减少的患者，血小板输注是治疗武器中的重要工具。我们完全依赖献血者来产生血小板，这造成了一系列问题。血小板的保质期很短，在5-7天之间(而红细胞单位为35天)，这使得在国家节假日、自然灾害和流行病等捐赠者可获得性发生急剧变化的情况下，血小板的供应管理变得复杂。此外，多次输血的患者或有多个孩子的妇女可能会对非自己(HLAI类抗原)产生免疫，这就需要采购HLA型匹配的血小板(英国有15%是血小板)，需要召回特定类型的捐赠者。最后，任何不匹配的血液成分都会使受血者面临通过输血传播细菌和病毒的风险(与其他血液产品不同，血小板必须保存在室温下)和病毒，这就需要严格的捐赠者选择和微生物筛查程序，这些程序必须不断适应新出现的感染源，例如最近的寨卡病毒。基础设施不稳定、缺乏集中标准化和筛查以及地方性传染病病原体的发展中国家，通过输血传播病毒的比率最高。这些问题中的大多数可以通过在体外产生血小板来解决供应、微生物安全性和同种免疫原性(例如使用基因组编辑的通用细胞)的问题。不幸的是，目前在体外产生血小板的方案比我们在体内看到的低10-100个数量级。我们认为这是因为我们培养的巨核细胞(血小板的母细胞)提供了不同程度的成熟，其中绝大多数细胞还没有准备好开始制造血小板。我们的目标是识别关键的基因组元件，使我们的培养能够以同步的方式引导我们的培养完全成熟，使我们有最好的机会扩大血小板的生产。我们将通过识别小鼠巨核细胞是终末成熟的，即产生血小板的细胞，利用它们产生长臂状突起的事实，称为前血小板，作为非终末成熟(没有前血小板)的视觉信号。来自这两组细胞的遗传物质将被分离和测序，这样就可以准备一个遗传图景。对这两个品系的比较将使我们能够识别控制遗传物质(转录因子)转录速度的差异表达因子。然后，我们将能够用这些转录因子对人类干细胞进行基因工程，以分析它们是否对MK产生、终末成熟和血小板产生有影响。

Each year 250,000 platelets units are transfused to patients in the UK. Platelets are the smallest of the blood cells and primarily promote blood clotting. Platelet transfusions represent a crucial tool in the therapeutic armament for patients who are either actively bleeding (following trauma or surgery) or for patients with a severely reduced platelet count (thrombocytopenia) as a result of genetic disorders or malignancies and the (often) myelosuppressive treatment thereof. We are totally reliant on blood donors to generate platelets which creates a range of issues. Platelets have a short shelf life, between 5-7 days (as opposed to 35 days for red blood cell units), making supply management platelet complicated in instances when there are acute changes to donor availability such as national holidays, natural disasters and pandemics. In addition, multi-transfused patients or women who have had multiple children can become immunised against non-self (HLA Class I antigens), necessitating the sourcing of HLA matched platelets (15% of the UK out in platelets) requiring the recall of specifically typed donors. Finally, any mis-matched blood component exposes the recipient to the risk of transfusion-transmitted infection with bacteria (platelets unlike other blood products have to be kept at room temperature) and viruses necessitating strict donor selection and microbiological screening programs that have to be constantly adapted to emerging new infectious agents such as recently for the Zika virus. Developing countries with unstable infrastructure, lack of centralised standardisation and screening and endemic infectious agents, have the highest rates of viral transmission through transfusions. Most of these issues may be addressed by the generation of platelets in vitro addressing issues of supply, microbiological safety and allo-immunogenicity (using for example genome edited universal cells). Unfortunately, current protocols to generate platelets in vitro are 10-100 orders of magnitude below what we see in vivo. We believe that this is because our cultures of megakaryocytes (the mother cell of platelets) offer a diverse range in levels of maturity, with the vast majority of these cells not ready to start making platelets. Our aim is to identify key genomic elements that will allow us to guide our cultures to full maturation in a synchronised manner, giving us the best opportunity to up-scale out platelet production.We will achieve this by identifying mouse megakaryocytes as terminally mature i.e. platelet producing, using the fact that they produce long arm like projections, called proplatelets, as a visual cue from non-terminally mature (no proplatelets). Genetic material from these two populations of cells will be isolated and sequenced so that a genetic landscape can by prepared. Comparison of these two lines will allow us to identify differentially expressed factors that control the rate of transcription of genetic material (transcription factors). We will then be able to genetically engineer human stem cells with these transcription factors, to analyse whether they have an effect on MK production, terminal maturation and platelet production.