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

Blood in a dish
-----in vitro synthesis of red
blood cells
Presented by :Tian Jing
Co-advisor: Dr.Ma and Dr.Jiang
• 2 billion people worldwide and 10% of the US
population, with the highest incidence among the
• Major surgery and trauma;
• A common toxicity of cancer therapies;
• 16 million red blood cell (RBC) transfusions every
year in the United States.
Need for RBC transfusions
• Obtained from donors
• Frequent supply bottlenecks
• Infectious risks ;
• Requires costly screening;
• Donors for rare blood types are
• 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.
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) .
The first cell-based therapy
• The first functional replacement therapy occurred in
1840 with whole blood transfusion treatment of
• The discovery of blood types by Karl Landsteiner in
1901 and earned him a Nobel Prize for Medicine in
[2]Diamond, L.K. , McGraw-Hill Book Company(1980) .
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)
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.
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 latestage erythroid progenitor and maturation of erythroid precursors.
dexamethasone (Dex); erythro-myeloid progenitors (EMP)
[3] Fibach, E. Haematologia (1991).
Improvements of 2-step erythroid
culture system
• The first step has been improved by the
replacement of conditioned media with several
defined cytokines[4]:
low concentrations of IL3 ;
• To expand the number of BFU-E and maintain the survival of
late-stage erythroid progenitors.
[4]Malik, et al. Blood (1998) .
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).
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) .
Improvements of 2-step erythroid
culture system
• The 2-step liquid cultures of human erythroid cells
have traditionally generated less than 50% enucleated
• Enucleation rates were dramatically improved by coculture 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).
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% of
RBCs were enucleated
[12] Miharada, K. et al. Nat.Biotechnol (2006).
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: human
telomerase catalytic
subunit gene-transduced
stromal cell
[13] Fujimi, A. et al. Int. J.Hematol (2008)
The recovery rate of
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
macrophage co-culturing
system’’ on day 38
Ultimate goal
• Enucleated RBCs ;
• Oxygen delivery potential similar in vivogenerated 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.
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).
Thanks for attention!

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