Most strict definition of an HSC: ability to serially

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Most strict definition of an HSC ability to
serially
reconstitute hematopoiesis (HSC loses ability to
do this after it has self-renewed several times)
HSC home to marrow are quiescent for up to 48
hrs
Serial transplantation to 2ndary host
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Plasticity of hematopoietic stem cells
Circulating HSCs that have capacity to give rise
to neurons, muscle (smooth, cardiac, skeletal),
intestine, liver, skin, and lung tissue. Saul
Sarkis lab at Hopkins performed serial long-term
reconst. with one HSC Therapeutic implications
HSCs with ability to differentiate into
epithelial cells target cells for gene therapy or
source for organ reconstitution repair
Lung bronchi epithelium
FISH for Y chromosome
Krause et al, 2001 Cell 105369
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Plasticity of (adult) hematopoietic stem cells

• Hematopoietic stem cells may differentiate into
  three major types of brain cells (neurons,
  oligodendrocytes, and astrocytes) skeletal
  muscle cells cardiac muscle cells and liver
  cells.
• Bone marrow stromal cells may differentiate
  into cardiac muscle cells and skeletal muscle
  cells.
• Brain stem cells may differentiate into blood
  cells and skeletal muscle cells

From NIH Stem Cell Primer at http//www.nih.gov/n
ews/stemcell/scireport.htm
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• Cytokines play a major role in influencing the
  direction in which differentiation proceeds
• Support the survival and/or proliferation of
  certain subsets of cells in the differentiation
  pool
• Exert a wide variety of biological functions on
  various cell types (e.g., IL-6 B cell
  maturation, T cells, hematopoietic progenitor
  cells, hepatocytes, neuronal cells).
• Can be redundant overlapping

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Cytokines

• Progenitor cells require a minimum of 2
  cytokines to grow effectively express
  receptors for multiple cytokines COMBINATORIAL
  EFFECT
• Committed precursor cells can grow in
  response to a single cytokine (progressive
  focusing of cytokines receptors on cells as
  cells move toward commitment to a terminally
  differentiated cell type) UNILATERAL EFFECT

TPO
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2 examples of common clinically utilized
cytokines which affect terminal
differentiation G-CSF A) sustains
myelopoiesis expansion of committed myeloid
precursor cells. Also, increases sensitivity of
cycling stem cells to other cytokines and
increases their entry into cell cycle. Used in
patients who are neutropenic after chemotherapy
to speed recovery of normal granulocyte counts.
B) Induces mobilization of CD34 stem cells
prior to HSC transplantation. Erythropoietin
sustains erythropoiesis. Treatment of
chemotherapy-induced anemia in cancer patients,
other anemias. Illegal blood doping in
athletes.
Blood doping is bad, Mkay?
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Cytokines signal through specific receptors
Most cytokine receptors consist of a multisubunit
protein complex - unique and specific ligand
binding subunit - signal transducing subunit,
which may be structurally similar to other
members of the cytokine receptor superfamily.
(ligand-binding)
(signal-transducing)
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Modular nature of cytokine receptors For a
number of receptors, proximal cytoplasmic region
is sufficient for proliferation, distal sequences
are required for differentiation Most cytokine
receptors lack intrinsic tyrosine kinase
activity, and signal through cytoplasmic
partners They must recruit cellular tyrosine
kinases to mediate their downstream signal
transduction pathways. Several different
pathways, including shc/ras, shc/PI3K, JAK/STAT
pathway.
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JAK

• Interaction of ligand with receptor leads to
  dimerization of receptor and recruitment
  activation of Janus kinases (JAKs)
• JAKs phosphorylate receptors cytoplasmic domain
  on tyrosine (Y) residues
• Phosphorylated tyrosine residues serve as docking
  sites for STATs
• JAKS phosphorylate STATs (signal transducers and
  activators of transcription)
• STATs dissociate from receptor, dimerize, and
  translocate to nucleus
• STATs activate transcription of genes (e.g.,
  those involved in terminal differentiation)

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Stat-3 activation is required for normal
G-CSF-dependent proliferation and granulocytic
differentiation McLemore et. al., Immunity,
14193-204 (2001)
G-CSF the principal hematopoietic cytokine
regulating the production of neutrophils Effects
of G-CSF are mediated solely through its
interaction with the G-CSFR. STAT-3 is the
principal STAT protein activated by G-CSF, with
STAT-5 and STAT-1 to a lesser degree what is
the contribution of STAT-3 to the signaling
function of G-CSF??
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Background

• G-CSF induces (activates) Stat3
• Mice lacking G-CSF or G-CSF receptor do form
  neutrophils, though in significantly reduced
  numbers (severe neutropenia) accumulate immature
  myeloid precursors in marrow, and granulocyte
  differentiation is severely impaired
• Expression of DN-Stat3 (which cant bind DNA)
  blocks G-CSF-mediated differentiation in cell
  lines get sustained G-CSF mediated proliferation
  without granulocytic differentiation
• This group had previously generated a targeted
  mutation of G-CSFR in mice with the distal 98 AAs
  deleted (deleting 3 of 4 Ys), leaving only one
  terminal Y at AA 715 intact. These mice had
  normal granulopoiesis and activated STAT-3 in
  response to G-CSF.

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wt (813aa)
Y Y Y Y
Generates neutrophils, activates Stat3
d715
Y
Generates neutrophils, activates Stat3
d715F
F
Hypothesized that this would not activate Stat 3
(cant dock at phenylalanine)
G-CSFR MUTANT MICE USED IN McLEMORE STUDY
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JAK

• Interaction of ligand with receptor leads to
  activation of Janus kinases (JAKs)
• JAKs phosphorylate receptors cytoplasmic domain
  on tyrosine (Y) residues
• Phosphorylated tyrosine residues serve as docking
  sites for STATs
• STATs (signal transducers and activators of
  transcription) are phosphorylated by JAKs
• STATs dissociate from receptor, dimerize, and
  translocate to nucleus
• STATs activate transcription of genes (e.g.,
  those involved in terminal differentiation)

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Assay for activation of STAT-3 EMSA with
radioactive DNA oligo specifically bound by
STAT-3 in nuclear extracts
BM isolated from mice given 10 min. treatment w/
cytokine (G-CSF in comparison to IL-6)
Assay for activation of STAT-5
(G-CSF in comparison to IL-3)
Mice had 7 days treatment w/ G-CSF, BM isolated
4 hrs. after last injection
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In vivo response to G-CSF D715F mice had normal
promyelocytes, increased myelocytes, and
decreased bands and neutrophils
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Example of differentiation from the precursor
stage onward myeloblast to granulocyte
(neutrophil maturation)
Terminal cell division
Increasing development of granuoles
(antibacterial and phagocytic) Increasing
phagocytic function pseudopodia extend around
microorganisms and fuse to form a phagosome into
which granuole contents are released
Expansion of cell number occurs as cells in the
mitotic or proliferative pool replicate The
post-mitotic pool can no longer divide but
continues to mature into terminally
differentiated cells In the setting of
infection or stress, maturation time may be
shortened, divisions may be skipped, and cells
may be released into the bloodstream earlier
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Hematopoietic progenitor assays ability of BM
(progenitor cells within) to form differentiated
colonies in vitro
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Can STAT3 rescue d715F mutants from block in
difftn?
MSCV control empty vector
Expression of constitutively activated Stat3
(Stat3C contains 2 Cys in its SH2 domain, which
causes spontaneous dimerization) restores some
G-CSF responsiveness (induction of colonies)
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MSCV murine stem cell virus (empty vector)
Neutrophils bands
Cultured 7 days in presence of G-CSF
G-CSF induced differentiation is enhanced by
Stat3C transfection of mutant mice bone marrow
cells
21
Conclusions Stat activation, particularly of
Stat3 and Stat5, play an important role in
myeloid differentiation and survival Common
theme that mutant cytokine receptors lacking Stat
recruitment sites fail to induce differentiation
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Transcription factors
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Granulocyte colony-stimulating factor regulates
myeloid differentiation through
CCAAT/enhancer-binding protein e (C/EBP
e) Nakajima and Ihle, BLOOD, 98, 897-905 (2001) e
Background C/EBP e is a (granulocyte)
lineage-specific transcription factor upregulated
at the promyelocyte or myelocyte stage and
expressed thereafter. KO mice revealed a
critical role in mid-late stage of granulocytic
differentiation (produce mature granulocytes but
they are morphologically and functionally
abnormal). Using 32Dc13 cells an IL-3
dependent murine myeloid line that can be induced
to differentiate to neutrophils or macrophages
using G-CSF or GM-CSF, respectively.
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G-CSF stimulation
Nakajima, Blood 2001
Northern results agree with those of Western blots
IL-3 negative control (dont differentiate to
granulocytes)
NFS60 do not differentiate, but rather
proliferate in response to G-CSF
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C/EBP e Stable transfectant
Nakajima, Blood 2001
Also showed constitutive MPO expression in stable
transfectant
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Is Tyr 703 (same as Y at 715 previous study) of
G-CSFR important for induction of C/EBP e by
G-CSF?
YES Might suggest a role for STAT-3
Nakajima, Blood 2001 98897
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Check to see if STAT-3 activates C/EBP e
Using wt G-CSFR, but mutant STAT3
Lacks activation domain
Morphological features of DN-STAT3 transfected
cells show differentiation block
BUT, expression of C/EBP e and MPO in DN-STAT3
transfected cells indicates normal induction
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Therefore, C/EBP e and STAT3 work in different
pathways and C/EBP e is regulated by a signaling
molecule other than STAT-3 Previous mutant mice
were generated in which the cytoplasmic domain of
G-CSFR was replaced with that of EPO-R , and had
no apparent defect in lineage commitment
suggests that signaling to induce C/EBP e
generated from G-CSFR can be replaced by Epo
signaling and should be a common pathway used by
both Epo and G-CSF.