Download Manufacture of Red Blood Cells from Stem Cells

The Generation of Red Cells
from Stem Cells.
Dave Anstee
Director, NIHR Blood and Transplant Research Unit Red Cell
Products
University of Bristol
The process
Multipotential
haematopoietic stem
cell
106 CD34+ HPCs from a
single apheresis cone, buffy
coat or cord blood unit
Proerythroblast
Basophilic
erythroblast
21 days
Polychromatic
erythroblast
Orthochromatic
erythroblast
Mature reticulocytes –
leucocyte filtration
Reticulocyte
Erythrocyte
Cultures in spinner vessels
Mature functional human reticulocytes can be generated
in a liquid culture system from peripheral blood
CD34+ cells.
• α and ß Globins
• Reversible O2 binding
• Normal expression of blood group active
proteins and cell surface antigens
•Absence of T and Tn antigens
• Deformability
• Proteome
Griffiths et al.Blood 2012;119(26):6296, Wilson et al.Mol Cell Proteomics 2016;15(6):1938
Reticulocytes reduce surface area and volume
and remove residual organelles by
expulsion of autophagic vesicles.
Mankelow et al. Blood 2015; 126(15): 1831
Role of the Spleen . in Vesicle Removal
4
% cells with vesicles
PS
3
BRIC163
BRIC155
2
Elevated levels of vesicles on circulating
red cells in splenectomised patients suggests
the spleen is important for their removal.
1
0
Control 1
Patient 1
Control 2
Patient 2
.
Mankelow et al. Blood 2015;126(15):1831
.
3D
Adult Cultured Reticulocytes
remodel in vivo (mouse).
10 min
24 hours
Becky Griffiths
BUT
•
Need a sustainable supply of cultured red cells of
desired blood group phenotype
Generation of the First Adult Erythroid Progenitor Cell Lines
Adult
BM CD34+
D0
1o medium
D1
D2-4
Transduce with Tet- 1o medium
inducible HPV16 E6/E7
D5
Transfer to Expansion
medium + doxycycline
BEL-A Dif. d4
Dif. d10
Dif. d18
Filtered retics
Adult Dif. d4
Dif. d14
Dif. d18
Filtered retics
“Tom” Trakarnsanga
Method of Kurita et al. Plos One 2013;8(3):e59890
Live imaging of filtered reticulocytes showing
presence of autophagic vesicles
PS
PS
BRIC163
BRIC155
Adult Peripheral
Blood
BELA
Becky Griffiths
BELA Reticulocytes in vivo (mouse)
10 min
3D
24 hours
Becky Griffiths
Why do we need cultured red cells?
For patients with rare blood
groups.
For patients with Sickle Cell
Disease
•
.
Based on data from Dr.Fiona Regan
New Technologies applied in Transfusion Medicine
2015
2000
1975
1945
Gene Editing
Molecular Diagnostics
Development of Monoclonal Antibody
Technology
Development of Antiglobulin
test
Genome editing BELA’s using CRISPR
Number of cells
Glycophorin A Knockdown
Basigin Knockdown
RHAG Knockout
RhD and RhCE knockdown
APC Fluorescence Intensity (arb. units)
Wild type BEL-A
IgG control
CRISPR Knockdown
Joe Hawksworth, Tim Satchwell and Ash Toye
Autophagic Vesicles from Reticulocytes remain in the Blood of
Patients with Sickle Cell Disease
BRIC163
BRIC155
% cells with vesicles
SCD patient
PS
5
4
3
2
1
0
PS
BRIC163
BRIC155
Mankelow T et al. Blood 2015;126(15):1831
Current Challenges
•
Induce maturation of cultured reticulocytes to
erythrocytes in vitro.
•
Develop technology for scale up to therapeutic doses.
Sabine Taylor
Nicky Cogan
Develop Technology for
Scale Up - two
approaches
Functionalised Scaffolds
Genetic Manipulation
Use of synthetic scaffolds for ex vivo
culture
T75 with scaffold and
40ml expansion medium
1.6 liters in
Spinner Flask
Severn et al. Scientific Reports 2016;6:32149
Summary
• Erythroid progenitor cell lines provide a sustainable
supply of cultured reticulocytes with the same properties
as those obtained from cultures of CD34+ cells.
• Gene Editing of Blood Group Genes will allow the
manufacture of cultured reticulocytes with different rare
blood group phenotypes from a single founder erythroid
progenitor cell line.
• Further work is required to effect maturation of cultured
reticulocytes to erythrocytes in vitro and to manipulate
erythroid progenitor cell lines to maximise yield of
reticulocytes but the technology required to do this is
now available.
The work is funded by National Institute of Health Research
(NIHR)
NHS Blood and Transplant
Wellcome Trust
Key Collaborators: Yukio Nakamura, Riken Institute,Tokyo
Sara Trompeter,UCLH, London
NIHR Blood and Transplant Research Unit University of Bristol 2016