Concise Review: Stem Cell-Based Approaches to Red Blood Cell Production for Transfusion Presenter：Yu Zhang 2015.11.4 Presenter ：Zhangyu 2015.11.4

1. Introduction www.who.int/worldblooddonorday/en/

Human Erythroid Massive Amplification (HEMA) culture conditions, a two phase liquid culture system, the first phase of which is designed to promote expansion and is stimulated with SCF, suboptimal concentration of IL-3 (to promote proliferation while limiting myeloid differentiation), EPO, DXM and ES while the second phase is stimulated with EPO and insulin and sustains maturation.4 Anna Rita Migliaccio, et al. Blood Reviews 26 (2012) 81–95.

Luc Douay Institut National de la Transfusion Sanguine, Paris, Lutetia Parisorum, Île-de-France, France Article: Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model. Haematologica 06/2012; · 6.42 Impact Factor Article: Banking of pluripotent adult stem cells as an unlimited source for red blood cell production: potential applications for alloimmunized patients and rare blood challenges. Transfusion medicine reviews 03/2011; 25(3):206-16. · 3.61 Impact Factor Article: Human fetal liver: an in vitro model of erythropoiesis. Stem cells international. 01/2011; 2011:405429. Article: Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica 10/2010; 95(10):1651-9. · 6.42 Impact Factor Article: Unimpaired terminal erythroid differentiation and preserved enucleation capacity in myelodysplastic 5q(del) clones: a single cell study. Haematologica 10/2009; 95(3):398-405. · 6.42 Impact Factor Article: Ex vivo expansion does not alter the capacity of umbilical cord blood CD34+ cells to generate functional T lymphocytes and dendritic cells. Stem Cells 10/2006; 24(9):2150-7. · 7.78 Impact Factor Article: Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nature Biotechnology 02/2005; 23(1):69-74. · 23.27 Impact Factor Article: Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nature Biotechnology 06/2002; 20(5):467-72. · 23.27 Impact Factor

2. Hematopoiesis & Erythropoiesis hESCs向各种血液细胞分化方法的突破为研究 造血发生提供了良好的模式。然而, hESCs/hiPSCs 向血液细胞分化是一个复杂而漫长的过程, 需要跨 越多胚层发育阶段并经由中胚层进入造血发生、发 育过程。在一个成体, 所有功能成熟的血细胞均来 源于骨髓里的HSC。而在胚胎阶段, 造血的早期发 育是一个随着时间和空间发展变化的复杂过程。在 哺乳类动物体内, 血液发生在时序上可以观察到一 个原始造血(primitive hematopoiesis)向成体造血(definitive hematopoiesis)演变的过程。由于受伦理和 法律的限制, 不能使用活的人类胚胎作为研究对象, 我们对早期造血发育的大部分理论是从小鼠实验积 累得来的[50-51]。以小鼠为例(图3), 随着胚胎发育, 造 血发生起始于卵黄囊, 后转移至主动脉/性腺/中肾 (aorta/gonad/mesonephros, AGM)区域, 在妊娠中期, 胎儿肝脏和脾脏成为血细胞产生的主要位置, 出生 后造血中心转移到骨髓并终身定居于此。 Fig.1　The developmental process of hematopoiesis in early embryo of mouse. Mao Bin, et al. Chinese Journal of Cell Biology 2012, 34(11): 1067–1079

2. Hematopoiesis & Erythropoiesis going through decreasing cell volume, increased hemoglobin synthesis, and nuclear condensation on the way 很多银子细胞-细胞，细胞间基质参与调控。 500ml -- 1/10 血循环量，每天1%血量更新。网织红细胞进入血液1~2天成熟脱核，丢掉多余胞质，体积再缩小。120天被脾和肝脏的网状内皮系统清除。A unit of blood contains numbers of RBC equivalent to one-tenth of those present in the circulation (2.5 × 1012 RBC). Since under normal circumstances, humans make 2 × 1011 RBC per day, the production of 2.5 × 1012 RBC in vivo requires at least 12– 13 days and occurs in compartmentalized areas of the marrow, the erythroid island, that, by minimizing cell interactions, allows production of great cell numbers in relatively small volumes. In vitro, progression from HSC/HPC to RBC also occurs within 12–13 days, but the cells mature in a disorganized environment that is permissive for cell interaction. To reduce the inhibitory effects exerted by negative regulatory factors (including TRAIL) released by erythroblasts as they mature [16, 17], cell interactions are minimized in vitro by maintaining the cells at densities <106 cells per milliliter. Therefore, production of 2.5 × 1012 RBC requires >2,500 l of culture media and growth factors in milligram quantities [14]. Fig.2　A current model of erythropoiesis. Siddharth Shah, et al. Stem cells translational medicine,2014,3:346-355.

3. Ex vivo Production of Human RBCs Source of stem cells for the production process 去白细胞处理产生的副产品，用人源化培养基方法可以产生和 少量脐带血相同量的 红血球 It has been calculated that HSCs derived from human ESC (hESC) and iPSC generate 500 erythroblasts each versus 104–105 erythroblasts generated by one CB/AB-derived CD34pos cell. Thus

3. Ex vivo Production of Human RBCs Summary of current protocols for in vitro RBC generation. 去白细胞处理产生的副产品，用人源化培养基方法可以产生和 少量脐带血相同量的 红血球 It has been calculated that HSCs derived from human ESC (hESC) and iPSC generate 500 erythroblasts each versus 104–105 erythroblasts generated by one CB/AB-derived CD34pos cell. Thus Vimal K. Singh, et al. Cell and developmental bilolgy. 2014, (1),1-18.

Cell size & Surface makers 4. The biological principles of ex-vivo erythroblasts (EBs) expansion Cell size & Surface makers Ann Zeunera, et al. Stem Cells. 2012 August ; 30(8): 1587–1596. Anna Rita Migliaccio, et al. Blood Reviews 26 (2012) 81–95.

4. The biological principles of ex-vivo EB expansion Hemoglobin (Hb) 1、组成 2、种类 胚型血红蛋白ε、ζ，胎儿型血红蛋白γ, 成体型血红蛋白δ、β 正常成人红细胞中有3种血红蛋白： 成人血红蛋白：HbA（α2β2) 96%-98% 成人次要血红蛋白:HbA2（α2δ2) 1%-3.1% 胎儿血红蛋白:HbF （α2γ2) 2%以下 胚胎血红蛋白: HbG1( ζ2ε2) HbG2 (α2ε2　) <5周 HbP( ζ2γ2) 3周-12周 α链基因和β链基因的内含子1的长度约为120个碱基对，而对内含子2，β链很长（例如人的α链基因之一的α2，内含子2含140个碱基对，而β链基因含849个碱基对）。珠蛋白基因在哺乳类是数次重复的结构，形成一种多重基因群。例如人的拟β链珠蛋白基因（β-like globin genes）在整个65000个碱基对程度的长度上，从5′末端按顺序连锁地存在ψβ2、ε、Gy、Aγ、ψβ1、δ和β7个基因。其中ε在胚期表达，γ在胎儿期表达，β和δ在成年时表达，ε在量上极少。ψβ2和ψβ1为假基因状态，无表达。而人的拟α链珠蛋白基因（α-like glo-bin genes）最少有ξ、ψα1、α1、α2 4个，ξ在胎儿时期表达。α1及α2在成年时期表达。ψα1为假基因。有证据说明这些重复结构基因是同时进化的。（参见多重基因群）。ζ和εare considered embryonic globin chains, γas fetal, and β as an adult chain HBF也能改善镰刀细胞病人的贫血症状。 Fig.A. Fig.3 The oxyhemoglobin dissociation curve

4. The biological principles of ex-vivo EB expansion Hb analysis & Oxygen transport capacity & Deformability measurements Figure 3. Hemoglobin analyses and functionality of erythroid cells generated from hiPSCSCD. Ladan Kobari ,et al. Hematopoiesis & Hematopoietic stem cells 2012;97(12):1795-1803.

4. The biological principles of ex-vivo EB expansion Erythrocyte-specific gene expression Honglian Jin, et al. BioMed Research International,2014,1-9

4. The biological principles of ex-vivo EB expansion Enucleation Mechanisms 红细胞脱核过程与多种分子和细胞路径相关[69], 包括肌动蛋白、胞浆移动、细胞凋亡、细胞吞噬作用、微环境、小分子物质、囊泡运输等, 新近研究的热点主要集中于以下几个方面。(1)囊泡运输(vesicle trafficking) (2)微环境微环境主要包括巨噬细胞与红细胞的相互作用、细胞外基质等, 对红细胞成熟脱核过程有着重要的影响[71]。4）组蛋白脱乙酰作用 Fig.5 Key events in cytokinesis and enucleation.

4. The biological principles of ex-vivo EB expansion In vivo maturation of cRBC in the NOD/SCID mouse. Fig.6 In vivo maturation of cRBCs in the NOD/SCID mouse model. Marie-Catherine Giarratana, et al. Blood, 2011, 118: 5071-5079

Introduction-Aim 10 million RBCs (the equivalent of 2 ml of blood) were generated ex vivo under good manufacturing practice (GMP) conditions from CD34pos cells obtained by apheresis from an adult volunteer. The survival in vivo of these expanded autologous RBC labeled with 51Cr (the only method accepted by the U.S. Food and Drug Administration) was comparable to that of native RBC. 5天后，这些人造血细胞中的至少94%仍在这位捐献者的体内循环。26天后，41%到63%的血细胞仍然存活，天然血液的存活率也大抵如此。

Siddharth Shah, et al. Stem cells translational medicine,2014,3:346-355.

Ann Zeunera, et al. Stem Cells. 2012 August ; 30(8): 1587–1596.

6. Prospective time table for clinic application of ex-vivo expanded erythrocytes. Anna Rita Migliaccio, et al. Blood Reviews 26 (2012) 81–95.

Discussion Thank you！