Early Blood Cell Formation
“in sickness and health, ‘till death do us part”
Kees-Jan Pronk
Sektion för Immunologi&
Hematopoietisk Stamcell Laboratoriet,
Medicinska fakulteten, Lunds Universitet
Barn- & Ungdomssjukhuset, Lund
VPH möte
Stockholm 100204
…. Erytrocytens dag….
Mentor: David Bryder
Co-mentor: Sten Eirik Jacobsen
Mogen blodcellsbildning … allt ifrån en cell
HSC
(hematopoietiska
stamcellen)
Vad händer i “ the black box”?
… under fysiologiska betingelser,
hos sjuka
och vid åldrandet …
Vad händer i “ the black box”? (immature hematopoiesis)
HSC (hematopoetiska stamcellen)
Multi-potent
Oligo-potent/
Oligo-primed
Uni-potent
Stamcellskonceptet Olika funktioner
• självförnyelse (identisk kopia).
• differentiering i >1 typ av dotterceller
• Kan (funktionellt) ersätta ett organ …. reservdel
Olika typer
•
embryonala stamceller
•
cancer stamceller
•
vuxna (somatiska) stamceller
Toti-, pluri-, multi-, bi-, unipotent
home.earthlink.net/~zaptos/website/media/differentiation.jpg
www.nih.gov/news/stemcell/primer.htm
Cancer stamceller
Varför är cancerstamceller viktiga?
1. Infrekventa celler.
2.
Ansvarar för uppkomsten och ‘underhåll’ av
tumören (vissa/alla?).
3. Ofta terapiresistenta
Cancer stamceller
Illustrerad vetenskap, nr 8 2007
Efter celldelningen får inte bara den
nya stamcellen utan också
mellanformerna och senare de färdiga
vävnadscellerna skador - i form av
aneuploidi och/eller mutationer
Embryonala stamceller
www.nih.gov/news/stemcell/primer.htm
’ny’ DNA
’egen’ DNA
Nuclear transfer ( --- Kloning)
ES cell culture
Embryonala stamceller
Ian Wilnut och Dolly (1997)
iPS - induced Pluripotent Stem cells
Illustrerad
vetenskap,
Nr 14, 2008
…. iPS
Även i blodsystemet:
Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.
Hanna J, et al. Science 2007
Overview of included studies
Hematopoietic stem cell (HSC)
Article I (TNF)
• Most studied and well defined organ restricted stem cell
• Resides in adult bone marrow (BM) in HSC niches
• Resides in BM at low frequencies (1/10.000-20.000)
• Can functionally repopulate tissue of origin
• Potential to produce all blood cell lineages …. Life-long!!!
• Widely used in clinical practice
• Responsible for cellular output of 5.000.000 cells/sec in man
Article II (Aging)
Article III&IV (subfractionation)
13.500.000.000 blodceller under denna föreläsning
Article III&IV
Elucidation of the phenotypic, functional, and molecular topography
of a myeloerythroid progenitor cell hierarchy.
Pronk, C.J.*, Rossi, D.J.*, Mansson, R., Attema, J.L., Norddahl, G.L., Chan, C.K., Sigvardsson,
M., Weissman, I.L., and Bryder, D.
Deciphering developmental stages of adult myelopoiesis.
Pronk, C.J., Attema, J., Rossi, D.J., Sigvardsson, M., and Bryder, D.
Articles III&IV: lite bakgrund
Articles III + IV study the developmental stages of hematopoiesis and regulators
involved in these processes
Why is this important?
1. To better understand normal
biology
2. To better understand abnormal
blood cell production, f.i.
• Aging: lineage skewing
• Cancer: block in differentiation
Articles III&IV: some background …. The black box ..
Bryder et al. ‘06
Articles III&IV: experimental assay
FACS (fluorescent assisted cell sorting)
105
26.3
104
103
73.6
Sort green cells
0
0
103
104
105
Run samples for analysis
• Functional evaluation
• Gene expression evaluation
• Etc.
Articles III&IV: Immunophenotypic analysis of myeloerythroid progenitors
MkP:
Pre GM:
GMP
Pre MegE
Pre CFU-E
CFU-E:
Megakaryocytic progenitor
pre Granulocyte/Macrophage
Granulocyte/macrophage progenitor
pre Megakaryocyte/Erythrocyte
pre Colony Forming Unit - Erythrocyte
Colony Forming Unit - Erythrocyte
Articles III&IV: Functional evalutation
In vitro investigation
of erythroid,
megakaryocytic,
granulocytic and
monocytic lineage
potentials
In vivo investigation
of erythroid, lineage
potentials
Articles III&IV: Gene expression analysis
Population based or single cell
evaluation of gene activity,
both global gene profile and a
selected set of genes
Articles III&IV: Some conclusions
1.
Phenotypic and functional identification of
Pre MegE, Pre CFU-E and Pre GM cells
2.
No evidence for the existence of the
previously identified CMP
3.
Hierarchical and lineage relationship of
newly and previous identified cellular
subsets
4.
Closer developmental relationship of the
innate (“G/M”) and adaptive (“lymphoid”)
immune system than previously assumed
Composite model of early hematopoiesis
Lineage- (Ter119- B220- CD4- CD8- C3- CD11b- Gr1-)
KLS-
KLS
Itga2b ??
Itga2b+
Endoglin+
Slamf1+
?
LTHSC
MkP*
Slamf1+
?
Itga2bEndoglin+/-
Ikaros-/Pu.1-
STHSC
Endoglin+
Pu.1-
pre
MegE
Gata1+
pre CD71+/CFU-E
CFU-E
Slamf1+
Slamf1-
Endoglin+/-
Ikaros+/Pu.1+
CD34
LMPP/
GMLP
Meg*
pre
Gata1- GM
Pu.1+
VCAM+
FcgR+
Flt3-
GMP
Slamf1-
Flt3
IL7Ra+
VCAM-
CLP
K+/-LS+/B
prog
T
prog
NK
prog
Proliferation capaMulticity
potentiality
Hur kan vi nu använda dessa resultat?
Exempel i “den röda linjen”
4000
CD105
CD150
ckit
FSC-A
2000
10 4
10 3
10 3
ii.
10 3
0
0
10 4
iii. iv.
v.
10 4
10 3
i.
1000
0
10 5
10 5
10 5
10 4
CD71
10 5
10 4
CD71
10 5
3000
vi.
vii.
10 3
0
0
0
0
10 2
10 3
10 4
Lineage
10 5
0
10 4
Sca1
More immature
...
10 3
10 5
0
10 2
10 3
10 4
CD41
10 5
pre MegE*
ii.
10 3
10 4
CD150
10 5
0
iii.
CFU-E
10 3
10 4
Ter119
10 5
0
10 3
10 4
Ter119
10 5
Late Baso&Chro EB
Pro EB
pre CFU-E*
i.
0
iv.
v.
Baso EB
vi.
vii.
Orth EB
Retikulocyt
Kartläggning av hela den röda linjen
Erythrocyt
Hur kan vi nu använda dessa resultat?
Exempel i “den röda linjen”
Gamla möss
0.12
0.06
0.10
0.05
0.08
0.04
0.06
0.15
0.10
0.05
0.00
Mindre erytroid
0.04
0.02
Young
Old
0.00
Young
Old
Frequency in bone marrow (%)
0.35
0.30
0.25
0.20
Mer myeloid +
megakaryocyt
GM
MkP
Pre CFU-E
0.03
0.02
0.01
0.00
7.00
6.00
5.00
4.00
3.00
Young
Old
Basophilic
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
CFU-E
Young
Old
Late Baso- &
Chromatophilic
Äldre människor
- Anemi
2.00
- Blodpropp sjukdomar
1.00
0.00
Young
- Myeloida sjukdomar (MDS, leukemier)
Young
Old
- Rel mindre lymfoida leukemier jmf barn (+dålig Ak-svar)
+ samma translokationer … olika leukemier hos barn jmf vuxna!!
…… studier i människor
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
3.00
Proerythroblasts
Young
Old
Orthochromatic
Normoblasts
2.50
2.00
1.50
1.00
0.50
Old
0.00
Young
Old
Hur kan vi nu använda dessa resultat?
Exempel i “den röda linjen”
Mitokondrial dysfunktion - prematurt åldrande
Block i utmognaden
… inblandning av mitokondrier i leukemier/erytrocytära sjkd??
Hur kan vi nu använda dessa resultat?
Exempel i “den röda linjen”
TEC?
Block i utmognaden
Sista slide
Min framtidsförhoppning vid en “abnormal röd linje (erytrocytär sjkd) …”
… inga kroniska transfusioner, utan:
• bot, eller
• expansion av kroppens egna celler
TACK!!
