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

blood cells

Presented by :Tian JingCo-advisor: Dr.Ma and Dr.Jiang

2012.11.25

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.

Background

Source:www.hudong.com/wiki/sickle-cell%20anemia

Need for RBC transfusions

• Obtained from donors• Frequent supply bottlenecks

•Infectious risks ;•Requires costly screening;•Donors for rare blood types are

scarce.

Background

Source:http://tupian.hudong.com/s/%

• 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.

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) .

Background

Source: http://www.dohenes.com/view.asp?id=574

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].

Background

[2]Diamond, L.K. , McGraw-Hill Book Company(1980) .

Source: http://baike.baidu.com/view/1429067.htm

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)

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.

Introduction

In vitro production of RBCs: the 2-step erythroid culture 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.

[3] Fibach, E. Haematologia (1991).

Introduction

dexamethasone (Dex) ； erythro-myeloid progenitors (EMP)

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 erythroid progenitors.

[4]Malik, et al. Blood (1998) .

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).

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) .

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).

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].

MAP, mixture of D-mannitol, adenine, and disodium hydrogen phosphate dodecahydrate.

nearly 80% ofRBCs were enucleated

[12] Miharada, K. et al. Nat.Biotechnol (2006).

Introduction

A

B

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]

[13] Fujimi, A. et al. Int. J.Hematol (2008)

hTERT: human telomerase catalytic subunit gene-transducedstromal cell

Introduction

The recovery rate of RBC from the day 38 culture from filtration was 80.8 %

1.76 ×109 RBC were obtained from 500 CD34+ cells by the four-phase ‘‘stroma-supportedmacrophage co-culturing system’’ on day 38

Nearly 100% of the erythroblasts obtained from third-phase culturing with macrophages were enucleated in the medium both on day 36 and day 38

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.

Summary

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).

Conclusion

Thanks for attention!