Blood in a dish -----in vitro synthesis of red blood cells

1. Blood in a dish-----in vitro synthesis of redblood cells Presented by :Tian Jing Co-advisor: Dr.Ma and Dr.Jiang 2012.11.25

2. Background Anemia • 2 billion people worldwide and 10% of the US population, with the highest incidence among the elderly. • Major surgery and trauma; • A common toxicity of cancer therapies; • 16 million red blood cell (RBC) transfusions every year in the United States. Source:www.hudong.com/wiki/sickle-cell%20anemia

3. Background Need for RBC transfusions • Obtained from donors • Frequent supply bottlenecks • Infectious risks ; • Requires costly screening; • Donors for rare blood types are scarce. Source:http://tupian.hudong.com/s/%

4. Background • Consequently, numerous efforts are underway to expand erythroid precursors and differentiate them in vitro into mature RBCs. • Furthermore, erythroid precursors may ultimately serve as a novel cell-based therapy providing a renewable source of RBCs.

5. Background The first cell-based therapy • The first successful blood transfusion: from one dog to another in 1665 • In 1667 , a sheep to man transfusion • The first microscopic identification of RBCs by Antonie van Leeuwenhoek in 1684. • The first successful human-to-human blood cell transfusion occurred with the treatment of postpartum hemorrhage using a husband-to-wife transfusion[1] [1]Diamond, L.K. , McGraw-Hill Book Company(1980) . Source: http://www.dohenes.com/view.asp?id=574

6. Background The first cell-based therapy • The first functional replacement therapy occurred in 1840 with whole blood transfusion treatment of hemophilia. • The discovery of blood types by Karl Landsteiner in 1901 and earned him a Nobel Prize for Medicine in 1930[2]. [2]Diamond, L.K. , McGraw-Hill Book Company(1980) . Source: http://baike.baidu.com/view/1429067.htm

7. Introduction Erythropoiesis – the synthesis of RBCs Hematopoietic stem cells (HSCs); termed burst-forming units erythroid (BFU-E); colony-forming units erythroid (CFU-E); erythroid precursors termed proerythroblasts (ProE); basophilic erythroblasts (BasoE);polychromatophilic erythroblasts (PolyE) ; orthochromatic erythroblasts (OrthoE); reticulocytes (Retic)

8. Introduction In vitro production of RBCs • This complex process of erythropoiesis, consisting of progressive phases : • (1) Progenitor expansion; • (2) Precursor amplification and maturation ; • (3) Reticulocyte remodeling into terminal RBCs.

9. Introduction In vitro production of RBCs:the 2-step erythroidculture system Twenty years ago, Fibach[3]developed a liquid culture system that included two sequential steps: • The first step contained glucocorticoids and conditioned media providing cytokines to promote erythroid‘progenitor’ proliferation ; • The second step contained EPO alone to promote survival of late-stage erythroid progenitor and maturation of erythroid precursors. dexamethasone (Dex)； erythro-myeloid progenitors (EMP) [3] Fibach, E. Haematologia (1991).

10. Introduction Improvements of 2-step erythroid culture system • The first step has been improved by the replacement of conditioned media with several defined cytokines[4]: • SCF ; • low concentrations of IL3 ; • GM-CSF; • EPO; • To expand the number of BFU-E and maintain the survival of late-stage erythroidprogenitors. [4]Malik, et al. Blood (1998) .

12. Introduction Improvements of 2-step erythroid culture system • It was also recognized that estradiol, as well as glucocorticoids, can inhibit erythroid maturation and lead to expanded numbers of erythroid ‘progenitors’ in the first phase of erythroid culture [5]. [9] Migliaccio, G. et al. Blood Cells Mol(2002).

13. Introduction Improvements of 2-step erythroid culture system • The addition of insulin and thyroid hormone to EPO [6]； • Molecules antagonistic to the action of glucocorticoids and estrogens [7]； • DMSO, ferrous citrate and transferrin[8]； • Humanized serum proteins [9]. [6] Leberbauer, C. et al. . Blood (2005). [7] Miharada, K. et al. . Nat.Biotechnol (2006) [8] Maggakis-Keleman, C. et al. Biol. Eng. Comput(2003). [9] Migliaccio G. et al . Cell Transplant (2010) .

14. Introduction Improvements of 2-step erythroid culture system • The 2-step liquid cultures of human erythroid cells have traditionally generated less than 50% enucleated RBCs. • Enucleation rates were dramatically improved by co-culture of erythroid precursors on a specific murine bone marrow (MS5) stromal cell line [10]. • Efficient enucleation has also been facilitated using feeder-free conditions [11]. • This is an important issue because the production of clinically useful RBCs in vitro will require strategies to avoid exposure of cellular products to nonhuman cells. [10] Giarratana, M.C. et al. Nat. Biotechnol (2005) . [11] Miharada, K. et al. Nat. Biotechnol (2006).

15. Introduction Improvements of 2-step erythroid culture system Culture protocol for the efficient production of enucleated red blood cells without feeder cells from hematopoietic stem/progenitor cells. Passage I∼III are the steps to expand erythroid progenitor cells. Passage IV is the step to induce enucleation of progenitor cells[12]. A MAP, mixture of D-mannitol, adenine, and disodium hydrogen phosphate dodecahydrate. B nearly 80% of RBCs were enucleated [12] Miharada, K. et al. Nat.Biotechnol (2006).

16. Introduction Improvements of 2-step erythroid culture system • Immature, multipotent hematopoietic progenitors have also been expanded in vitro by culture not only with cytokines but also by using human stromal cells transduced with hTERT[13] hTERT: human telomerase catalytic subunit gene-transduced stromal cell [13] Fujimi, A. et al. Int. J.Hematol (2008)

17. The recovery rateof RBC from the day 38 culture from filtration was 80.8 % Nearly 100% of the erythroblasts obtained from third-phase culturing with macrophages were enucleated in the medium both on day 36 and day 38 1.76 ×109 RBC were obtained from 500 CD34+ cells by the four-phase ‘‘stroma-supported macrophage co-culturing system’’ on day 38

18. Summary Ultimate goal • Enucleated RBCs ; • Oxygen delivery potential similar in vivo-generated RBCs： • Hemoglobin content, • Oxygen dissociation characteristics, • Membrane deformability, • In vivo lifespan when injected into immunodeficient mice CD71, transferrin receptor; TER119, a cell surface antigen specific for mature erythroid cells.

19. Conclusion The problem of scale • The RBC products require the ex vivo generation of cell numbers [14]; • The costs associated with ex vivo erythroid cell expansion and differentiation; • The tumorigenic potential [15]; • The establishment of an immortalized human erythroid cell line lacking the genes to produce A, B, and RhD antigens . [14] Giarratana, M.C., et al. Blood (2011). [15] H. Hentze,et al. Trends in Biotechnology, (2007).

20. Thanks for attention!