Human Hematopoietic Stem Cells

1
Human Hematopoietic Stem Cells
Human donor bone marrow
Isolate CD34/Lin-/CD38- cells
Stromal cells and cytokines provide the growth
and differential signal for HSC.
Culture single cells on bone marrow stromal cells
with cytokines
Analyze the progeny cells
cytokines
DCdendritic cells M myeloid cells DM DCM
NK natural killer cells B B cells.
Different cytokines promote the differentiation
of different cell types.
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Common Lymphoid Progenitor (CLP)
HSC
Common Lymphoid Progenitor (CLP)
Common Myeloid Progenitor (CMP)
Monocytes, Granulocytes
B cells, T cells
CLP Lin-IL7RaThy1.1-Sca-1-c-Kitlo HSC
Lin-IL7Ra-Thy1.1loSca-1c-Kit
HSC
CLP
CLP
CLP
HSC
HSC
3
CLP differentiates into B and T cells, but not
myeloid cells.
Competitive Reconstitution
2 x 103 CLP (Ly5.2)
Lethally irradiated
2 x 105 total bone marrow (BM) cells (Ly5.1)
Analyze blood cells
Ly5.2cells (CLP-derived)
Chimerism
Total (CLPBM)
4
Common Myeloid Progenitor (CMP)
Bone marrow
Lin-IL-7R?
Lin-IL-7R?
CLP
Sca-1
Sca-1-
HSC
CD34Fc?Rlo
CD34-Fc?Rlo
CD34Fc?Rhi
Assay for differentiation
5
Granulocyte (G) Macrophage (Mac) Granulocyte and
Macrophage (GM)
Single cell
Erythrocyte (E) Megakaryocyte (Me)
Culture in media with cytokines
Colonies with differentiated cells
Analyze cells in the colony
FcRhiCD34
FcRloCD34
FcRloCD34-
GMP
MEP
CMP
G, Mac
E, Me
G, Mac, E, Me
Megakaryocyte Erythrocyte progenitor
Granulocyte Macrophage progenitor
Common myeloid progenitor
Cytokines that induce differentiation into
various cell types.
6
B cells are derived from CLP.
B cells
B220
CLP
Lin-
T cells
CD4, CD8
HSC
Granulocytes
Gr-1
Lin-
GMP
Monocytes
Lin-
Mac-1
CMP
Erythrocytes
Lin-
Ter-119
MEP
Lin-
Megakaryocytes
7
B cell maturation in bone marrow leads to the
surface expression of IgM (B-cell receptor, BCR)
Bone marrow
HSC
CLP
Surface IgM (sIgM) B-cell receptor (BCR)
pre-pro-B cell
B220IgM-
pro-B cell
B220IgM-
pre-B cell
B220IgM-
B cell
Immature B cell
B220IgM
Exit from bone marrow
8
Hematopoietic precursors are associated with
stromal cells. Stromal cells provide growth and
differentiation factors.
HSCs are enriched in endosteum.
Osteoblasts in endosteum support the growth of
HSCs.
CXCL12 (SDF-1)
CXCR4 (pre-pro-B cells)
Stromal cells
IL-7
IL7R (pro-B cells, pre-B cells)
9
IL-7 is important for B and T cell maturation.
FACS analysis of bone marrow cells
B cell maturation is blocked in the absence of
IL-7.
No IgM B cells are generated in the absence of
IL-7.
The number of B and T cells in spleen are
severely reduced in the absence of IL-7.
10
Signal transduction of IL-7/IL-7R
IL-7 induces dimerization of IL-7R ? and ?
subunits.
?
IL7
?
IL-7R
Jak
Jak
Activation of Jak (Janus family tyrosine kinase)
Phosphorylation and dimerization of Stat
Stat translocates into the nucleus
nucleus
Stat activates transcription of target genes
11
Common themes in receptor-mediated signal
transduction
Ligand-induced receptor dimerization or
oligomerization
PAMP
bacteria
TLR
IL7
?
?
IL-7R
FcR
IRAK1 IRAK4
JaK
JaK
Src family of tyrosine kinases
Activation of receptor-associated kinases
Phosphorylation cascade
12
Activation of latent transcription factors
FcR
TLR
Ca2-Calmodulin
IL-7R
Calcineurin
I?B
(phosphatase)
p65
p50
stat
stat
NF-AT
p65
Nuclear localization signal revealed
p50
Translocation into the nucleus
NF-AT
p65
p50
NF-AT
stat
stat
Activate transcription
13
Immunoglobulin (Ig) genes are assembled from gene
segments during B cell maturation.
Ig locus in human
Constant region
Joining
Variable
Diversity
9 CH
65 VH
27 DH
6 JH
Immunoglobulin heavy chain (IgH)
40 V?
5 J?
1 C?
Ig?
Immunoglobulin light chain (IgL)
30 V?
4 J?-C?
Ig?
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The coding sequence for IgH variable region is
assembled from VH, DH, JH gene segments by V(D)J
recombination.
The gene segments within each cluster are
similar, but not identical.
JH3
JH1
JH2
DH gene segments
JH gene segments
VH gene segments
C
Germline configuration
D-J joining
Intervening sequences deleted
V-DJ joining
Rearranged configuration
Intervening sequences deleted
15
Productive V(D)J recombination leads to the
expression of immunoglobulin heavy chain.
Rearranged IgH gene
D
J
C
V
transcription
promoter
Primary transcript
Splicing and polyadenylation
AAAAA
IgH mRNA
translation
Ig heavy chain
C?1
VH
C?4
C?2
C?3
Membrane anchor
Ig light chain is not produced at this point.
16
The joining of different VH, DH, and JH gene
segments generates diverse VH regions.
6
27
65
DH gene segments
JH gene segments
VH gene segments
C
Random combination of different VH, DH, JH
segments
Ig heavy chain
65 x 27 x 6 10,530 different VH region
C?1
VH
C?4
C?2
C?3
17
V, D, J gene segments are flanked by
recombination signal sequence (RSS).
12-23 rule V(D)J recombination only occurs
between a 12-bp RSS and a 23-bp RSS.
12-bp RSS
23-bp RSS
Nonamer
heptamer
Nonamer
heptamer
5-CACAGTG-12bp spacer-ACAAAAACC-3
5-GGTTTTTGT-23bp spacer-CACTGTG-3
DH recombines with JH.
D
V
J
C
VH recombines with DH.
23-bp RSS
12-bp RSS
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V(D)J recombination is catalyzed by Rag-1 and
Rag-2 proteins.


Rag1
Rag2
Intervening sequences loop out.


Rag1 and Rag2 synapse a pair of 12-bp and 23
bp-RSS.


Rag1 and Rag2 cut at the junction between RSS and
DH and JH segments.
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ACAAAAACC
CACAGTG
D
12bp
Intervening sequence

GTGTCAC
TGTTTTTGG
CACTGTG
GGTTTTTGT

23bp
J
Intervening sequence
CCAAAAACA
GTGACAC
Rag-1 Rag-2
nick
OH
ACAAAAACC
CACAGTG
D
12bp
Intervening sequence

GTGTCAC
TGTTTTTGG
CACTGTG
GGTTTTTGT

23bp
J
Intervening sequence
CCAAAAACA
GTGACAC
OH
nick
Rag-1 Rag-2
Coding end
Signal end
hairpin
ACAAAAACC
CACAGTG
D
12bp
Intervening sequence

GTGTCAC
TGTTTTTGG
CACTGTG
GGTTTTTGT

23bp
J
Intervening sequence
CCAAAAACA
GTGACAC
hairpin
20
D
3
5
3
3
5
5
Hairpin coding end
Rag-1,2
Rag-1,2
nick
H2O
OH
OH
Signal end
RSS
5
5
5
3
3
3
21
The processing of hairpin introduces additional
diversity.
Hairpin can be cut at different locations.
3
5
Artemis (hairpin cutting nuclease) DNA-dependent
protein kinase (DNA-PK)
TC
D
2
3
AG
1
1
3
2
P-nucleotide
P-nucleotide
5
5
3
TC
GA
3
T
D
5
D
3
TC
3
3
AGC
5
D
5
3
AG
5
Terminal deoxynucleotide transferase (TdT)
Non-templated addition of nucleotides
N-nucleotide
N-nucleotide
5
5
TC
GAGGCT
TCCTAA
D
D
3
3
AG
5
5
22
The ends are joined by non-homologous ending
joining (NHEJ) system
5
TC
GAGGCT
3
D
J
TA
3
5
AT
AGGC
5
5
3
J
TA
TC
GAGGCT
D
3
AT
AGGC
5
5
Polymerase fills gap.
NHEJ
Ligase joins the strands.
5
3
J
TA
TC
GAGGCTCCG
D
3
AT
AGGC
CTCCG
5
5
Gain of nucleotides (P- and N-nucleotides) at the
coding joint
Imprecise coding joint introduces additional
diversity.
23
DH gene segments
JH gene segments
VH gene segments
C
D-J joining
Imprecise coding joint
V-DJ joining
Imprecise coding joint
D
J
C
V
Transcription, translation
Imprecise coding joints coincides with CDR3 and
diversifies antigen binding specificity.
C?1
C?3
C?4
C?2
CDR3
24
Each B cell expresses only one IgH allele.
Paternal allele
Both alleles Undergo DH-JH joining
JH
C
DH
VH
Maternal allele
One allele undergoes VH-DHJH joining earlier
than The other allele.
The choice is random (stochastic).
GAGGATGCTCC
GTGGACTAGG
T
GAGGATGCTCC
GTGGACTAGG
Asp
Stop
Glu
Ala
Pro
Val
Asp
Trp
Glu
Asp
Pro
Thr
Arg
Ala
Complete open reading frame from V to C
No IgH protein
Non-productive V(D)J recombination
IgH protein
Productive V(D)J recombination
25
IgH associates with surrogate light chain (SLC)
to form pre-B cell receptor (pre-BCR).
VH
VH
Vpre-B
C?1
C?1
?5
C?2
C?2
Surrogate light chain
Surface IgM (sIgM) B-cell receptor (BCR)
C?3
C?3
pre-B cell receptor
C?4
C?4
Membrane anchor
B cell
pre-B cell
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Allelic exclusion
Ligand?
Maternal IgH
Paternal IgH
One IgH allele undergoes V(D)J recombination.
Pre-BCR
Ig?
Ig?
ITAM

productive
Non-productive
Shut off expression of Rag-1 and Rag-2
The second allele excluded
The other allele rearranges
Prevent V(D)J recombination of the other IgH
allele
Allelic exclusion

Apoptosis
productive
27
Allelic exclusion ensures that each B cell
produces one kind of Antibody.
Allelic exclusion
No allelic exclusion
pathogen
pathogen
Not self- reactive
Self- reactive
Self-reactive antibodies are also produced.
Only antibodies against pathogen are produced.
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Questions
What are HSC, CLP, and CMP? Which cell types can
they differentiate into?
How is HSC function demonstrated?
What is the differentiation pathway for B cells?
How does V(D)J recombination work?
How does imprecise joining contribute to the
diversity of V region?
What is pre-BCR?
What is allelic exclusion? What is the function
of allelic exclusion?
Relevant parts in textbook
P28-30, p161-162, p312-314, p271-275, p115-125