Megakaryopoiesis and Ex Vivo Differentiation of Stem Cells into Megakaryocytes and Platelets N. Pineault1,2& G. J. Boisjoli1 1Center for Innovation, Canadian Blood Services, Ottawa, ON, Canada 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada

About ISBT The International Society of Blood Transfusion (ISBT) is a scientific society that was founded in 1935. Since that time the ISBT has grown in to an international society where transfusion medicine professionals from across the globe come together and do the one thing they do best: share knowledge to improve the safety of blood transfusion worldwide. Our strategic vision is: 'Facilitating knowledge about transfusion medicine to serve the interests of donors and patients'. The ISBT Central Office is located in the center of Amsterdam, the Netherlands.

Contents 1 2 3 4 5 Concise overview of megakaryopoiesis Production of megakaryocytes and platelets ex vivo 3 Morphological and functional properties of platelets 4 Production of megakaryocytes and platelets from iPSCs 5 Clinical transfusion of PC and CDP

Background Blood agencies worldwide face the same recurring problem of enrolling healthy donors to produce platelet concentrates (PC) to meet the fluctuating demand. PCs are highly susceptible to bacterial contamination due to their storage conditions at 22 ± 2°C under constant agitation for up to 5 days. production of culture-derived platelets (CDP) raising the possibility of producing donor-independent platelets.

Concise overview of megakaryopoiesis 0.05% of bone marrow cells bone marrow sinusoids Megakaryocyte 20-100μm, 2N-64N release 2000–5000 platelets

Production of megakaryocytes and platelets ex vivo Bone marrow Mobilized PB Cord Blood iPS

Production of megakaryocytes and platelets ex vivo Thrombopoietin (TPO) Successful cloning by Yoshiaki Sohmain 1994. High purity and low expansion In vivo, decreases in platelet levels lead to increased TPO concentration resulting in the promotion of megakaryocyte development and platelet production. Stem cell factor(SCF) & Flt-3 ligand (FL) Powerful cytokines for the expansion of HSPCs and most myeloid cells. Interleukin (IL)-3 and IL-6 Pleiotropic factors that promote the survival of a wide range of progenitors . IL-9, IL-1b and IL-11

Production of megakaryocytes and platelets ex vivo Microenvironment Elaborate regulatory signals coming from stromal cells Interactions of cell adhesion molecules with the extracellular matrix constituents such as fibronectin and vitronectin. Modulating physical culture parameters PH pO2 Temperature (39℃) 3D Genetically modified HSPCs HLA-low platelets

Morphological and functional properties of platelets produced in vitro CDPs were more heterogeneous in morphology and in size than blood platelets Some were much bigger (mean size of 4.4 ± 1.6 μm in length vs. 2.8 ± 0.6 μm) Contained mitochondria, alpha and dense granules, open canalicular systems CD42b loss on CDPs (partially inhibited by addition of GM6001 to culture) Difficulty of producing sufficient number of CDPs CDPs could recapitulate platelet properties and functions Respond normally to common platelet agonists such as ADP, collagen and thrombin and P-selectin (CD62P) extracellular expression Undergo aggregation following activation

Production of megakaryocytes and platelets from iPSCs

Platelet transfusion 国内外统计发现临床使用血小板的增长速度远远超过红细胞输注的增长速度,如广州2000年血小板的用量(12303 袋) ,是1995年用量的70 倍; 美国输血协会公布美国血小板的用量从1971年至1980年增长了598% ,而同期红细胞输血只上升了58%。 2003年血小板使用量较1998年上升了214% , 而同期红细胞的使用量只上升了105%

生理性止血的基本过程 1 血管收缩：小血管损伤后立即收缩，限制血流。 2 血小板血栓形成：血小板附着、释放、聚集 形成不牢固、松软血小板血栓（白血栓）达到初步止血。 3 血液凝固：启动凝血系统，血液凝固形成二期止血，纤维蛋白原变为纤维蛋白，形成牢固止血栓（红血栓，牢固）达到永久性止血。

【适应证】 （1)治疗性血小板输注： （2）预防性血小板输注 ①血小板＜20×109/L(再障、白血病、DIC、脾亢、同种免疫或药物介导的血小板破坏等），伴严重出血 ②血小板不少，但功能障碍，伴严重出血 ③大量输血所致的血小板稀释减少 ④急性特发性血小板减少性紫癜（ITP)，伴严重出血、或血小板减少而需手术时 （2）预防性血小板输注 ① 应慎重选择，因反复输注血小板，可发生同种免疫或有病毒感染的危险，血小板减少如无严重出血不需作预防性血小板输注 ②血小板＜5×109/L，无论有无出血，都应作预防性血小板输注

【剂量】 一、手工分离浓缩血小板制品： 二、单采血小板： 以采200ml的1U全血分离制备的血小板定义为1U浓缩血小板，国家表准要求：血小板含量≥2×1010/L，容积为25 ～ 35ml 【使用剂量】通常成人单次输注12U；2～3天输一次 二、单采血小板： 即采用血细胞分离机采集的单个供血者的浓缩血小板（SDPC），以袋为计量单位，国家表准要求每袋SDPC中，血小板含量≥2.5×1011/L 【使用剂量】：每次1袋，严重时2袋，因血小板只能存活5天，故2 ～ 3天可再输1次，直到出血停止

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