Hematopoietic Stem Cells

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Hematopoietic Stem Cells

• Warren S. Pear
• 611 BRB II/III
• 573-7764
• wpear_at_mail.med.upenn.edu

3/17/2008
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Question 1 What is this?
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Hematopoietic Stem Cells

• One of the best characterized stem cells
• Used clinically in bone marrow transplant and
  gene therapy
• Well established purification methods
• Development into downstream progeny is well
  understood

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Hematopoietic Stem Cells

• Definition
• Overview of hematopoiesis
• Identification of HSCs (phenotype)
• Identification of HSCs (function)
• Development
• Microenvironment the Stem Cell Niche
• Hematopoietic Stem Cell Gene Therapy

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Stem cellA single cell

• Ability to differentiate into multiple cell types
• Maintain self-renewal capacity

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Pluripotent vs Multipotent Stem Cells

• Pluripotent
• Able to differentiate into cells of all 3 germ
  layers endoderm, ectoderm, and mesoderm
• Examples Embryonic stem cells (ES)-derived from
  inner cell mass of the blastocyst, capable of
  indefinite in vitro culture, used to generate
  gene-targeted mice
• Multipotent
• Lineage specific
• May be isolated from various tissues in fetal and
  adult animals
• Examples Hematopoietic stem cells (HSCs),
  Neuronal stem cells, hepatic stem cells

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Stem Cells Pluri- vs. Multi-potent
Weissman 2000 Science 2871442
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Daily Production 4 x 106 cells / sec, 200 x 109
RBCs / day (this is 238 cells or theoretically 38
cell divisions from a single stem cell), 50 x 109
granulocytes / day
Hematopoietic Stem Cells (HSCs)
Weissman 2000 Science 2871442
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The understanding of hematopoiesis continues to
evolve
Hock Orkin, Nature 2005435, 573
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The understanding of hematopoiesis continues to
evolve
ETP
Allman, Bhandoola, et al., Nat. Immunol. 4 2003
Hock Orkin, Nature 2005435, 573
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Hematopoiesis
Made by a single HSC
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Bone Marrow and Peripheral Blood
Bone Marrow (hi)
Bone Marrow (low)
PB
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Hematopoietic cells undergo an orchestrated
differentiation program
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Why HSCs

• Need to produce a large number of cells from a
  relatively small space (proliferation)
• Cell acquire lineage specific characteristics or
  functions that define it (differentiation)

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Therapeutic Uses

• Bone marrow transplant
• Gene therapy
• Transdifferentiation into other lineages (liver,
  muscle) unlikely

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Existence of the HSC

• Radiation experiments-hi dose leads to death
  (1945), one cause is bone marrow failure
• 1949 shielding spleen provides rescue
• 1951 injection of spleen or BM cells can rescue
• What is the phenotype
• Injection of BM cells into irradiated mice led to
  clonogenic mixed spleen colonies (granulocytes,
  macrophages, erythrocytes) (Till McCulloch)
• CFU-S Assay Day 12 can rescue 2ndary recipients
  (day 8 lack this ability)

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Existence of HSC CFU-S Assay
Till and McCulloch. 1961. Rad Research 14213
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CFU-S Each colony is derived from a single stem
cell
Till and McCulloch. 1961. Rad Research 14213
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CFU-S Assay Breakthrough to identify HSCs

• 1961- Till McCulloch Spleen Colony Forming
  Units (CFU-S) - Theorize that different types of
  cells arise from proposed colony forming units
• 1963- Radiation Hybrid Experiments - All cells in
  colony are clonal.
• 1964 - Lewis and Trobaugh - Colonies re-generate
  colony forming cells whatever their morphology
  (colonies split and 1/2 injected into 2nd
  recepient).
• 1965 - Becker, et.al. - Stem cells are
  insensitive to thymidine suicide, i.e. they are
  mitotically inactive.

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Genetic Markers to detect HSCs-chromosomal
translocations
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How to identify HSCs Finding the one in a
million (0.3-1.2 cells/million)

• Assays
• Self-renew and clonally differentiate to form all
  mature blood cell types
• Generate d12 CFU-S
• Rescue lethally irradiated recipients long term
• Competitive reconstitution assays
• Enrich by phenotypes
• Size fractionation density gradients,
  elutriation
• Cell cycle status-?resting
• Vital Dye exclusion
• FACS (fluorescence activated cell
  sorting)-current method of choice

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Use of vital dyes to identify HSCs
Goodell, Nature Med 3 1377
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Flow Cytometry
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Immunophenotype of the HSC

• Adult mouse bone marrow
• Lin-, c-kit, Sca-1, Flt3-
• Single cells capable of long-term multilineage
  reconstitution and self-renewal in irradiated
  mice
• Offspring have limited self-renewal-express Flt3
• 1/5 cells from young mice capable of long term
  reconstitution
• Human HSCs-enriched in CD34 fraction
• Mice LT HSCs-low expression of CD34

Weissman 2000 Science 2871442 Adolfsson 2005
Cell 121 295
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Immunophenotype of HSCs
Then, inject purified cells into lethally
irradiated mouse and assay for multi-lineage and
long term (gt3 months) engraftment. Can also look
at specific subsets in colony assays.
Hock, H et al., Nature 4312004
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Rescue of irradiated mice with purified HSCs
Closed circle-100 HSCs Open Square-10,000 HSCs
Weissman 2000 Science 2871442
Clinical Application Purified human HSCs
restore long term hematopoiesis in patients
receiving hi dose radiation/chemotherapy.
Increased dose shortens engraftment
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Serial Transplantation to detect HSC activity
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Identifying genes associated with HSC identity
Kiel et al. 2005 Cell 1211109

• Compared highly purified populations of LT-HSC to
  MPP using Affymetrix oligonucleotide arrays
• Identified 27 genes that were expressed more
  highly in HSCs (1.9-fold cutoff)
• One transcript identified was SLAM (CD150)-a cell
  surface receptor not known to be expressed in
  stem cells

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CD150 cells are enriched in the LT-HSC fraction
and give rise to long term reconsititution
Only CD150 cells were capable of serial
reconstitution
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CD150 HSCs are localized near osteoblasts and/or
sinusoidal endothelium
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HSCs from CD150-/- knockout mice are normal

• What does this mean?

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Human HSCs LTC-IC Assay
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NOD-SCID mice can be used to functionally read
out human HSCs
Non-obese diabetic, severe combined
immunodeficient mice can engraft human tissues,
including HSCs
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Types of HSCs

• Adult-bone marrow, circulating
• Fetal liver
• Placenta
• Cord blood
• Yolk sac
• AGM (aorta-
• gonad-
• metanephros
• region)

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Fetal Hematopoiesis A dynamic process

• E7.5 blood and endothelial progenitors emerge in
  AGM blood islands
• Is there a common precursor?
• Notch1 may be required for HSC
• E10-12 colonization of fetal liver from AGM and
  yolk sac (?placenta), Notch1 not required
• Unique markers AA4.1, Mac1
• Unique cell types B1 B cells-majority of B cells
  in the newborn
• Higher capacity than adult HSCs for long-term
  engraftment
• E16-17 fetal liver HSCs migrate to fetal bone
  marrow
• Shortly after birth BM is predominant site of
  HSC activity
• Long term reconstituting ability declines with age

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Dividing HSCs have 4 developmental choices

• Self-renewal
• Differentiation
• Programmed Cell Death/Survival-bcl2
• Emigration-GM-CSF mobilization

The frequency of HSCs in hematopoietic organs is
regulated by the fraction of HSCs that choose one
of these fates
Weissman, IL, Science 2872000
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Effect of manipulating self-renewal

• Serial transplant experiments showed that the
  number of murine HSCs increased upon each round
  of transfer and then plateaued (4 rds of transfer
  8400-fold increase, Iscove Nawa, Curr Biol
  97, 1997)
• In vitro expansion of human HSCs by cytokines is
  much less (6-fold in optimal growth factors)
• How can one obtain large numbers of human HSCs
  for therapeutic manipulation ex vivo?

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HoxB4 provides yolk sac HSCs with the ability to
engraft adult BM

• Adult-bone marrow, circulating
• Fetal liver
• Cord blood
• Yolk sac
• unable to engraft lethally irradiated adults
• May require education in fetal liver or other
  hematopoietic organs
• HoxB4 expression sufficient to engraft adults
  (Kyba et al., Cell, 2002)

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HoxB4

• DNA binding homeobox protein
• binds in cooperation with Pbx1 and Meis1
• Capable of promoting self-renewing divisions of
  BM HSCs in vitro and expanding HSCs in vivo
• Only transient expression required in yolk sac to
  confer adult engraftment
• Induces expression of markers of definitive
  hematopoiesis
• CXCR4-chemokine receptor, Tel-transcription
  factor both may be important for finding the
  stem cell soil
• What are the key targets?

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Developmental regulators of HSC expansion (1)

• Notch and Notch ligands
• Expression of activated Notch or stimulation of
  Notch through ligands can maintain and expand
  hematopoietic progenitors in culture (not sure if
  LT-HSC) and expand HSCs in vivo (2-fold).

Varnum-Finney et al., Nat Med 62000 Calvi et
al., Nature 4252003
CoR
CSL
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Notch signaling HSCs

• Notch signals are required to establish the
  earliest fetal hematopoietic stem cell
• How about adults
• Integration of Notch and Wnt signaling in
  hematopoietic stem cell maintenance.
• Duncan et al., Nature Immunology 2005
• A fundamental question in hematopoietic stem cell
  (HSC) biology is how self-renewal is controlled.
  ... we identify Notch signaling as a key factor
  in inhibiting differentiation. ... Inhibition of
  Notch signaling led to accelerated
  differentiation of HSCs in vitro and depletion of
  HSCs in vivo. ...

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DNMAML reveals the site of Notch action during
early T cell development
Sambandam, Maillard et al., Nat Immunol 2005
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Normal frequency of LSK progenitors in
Notch-deprived bone marrow (retroviral
transduction approach)
Maillard et al., Cell Stem Cell, in the press
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How does absence of Notch affect competitive
repopulation by HSCs
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Competitive repopulation potential of
Notch-deprived progenitors in secondary recipients
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Limiting dilution competitive repopulation
Transduction MigR1 vs. DNMAML1
12 wks
GFP LSK (500, 150, 50) Host-type BM (2x105)
16 wks
Analyze reconstitution
BMT
BMT
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A role for Notch in the HSC environment?
Ø
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Developmental regulators of HSC expansion (2)

• Wnt
• Purified Wnt3a synergized with growth factors to
  induce proliferation of HSCs in single-cell
  cultures (Willert et al., Nature 4232003)
• Wnt activation in vivo led to an 8-80-fold
  increase in HSCs and Wnt inhibition blocked
  proliferation (Reya et al., Nature 4232003)
• Wnt may regulate Notch and/or Hoxb4

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Developmental regulators of HSC expansion (3)

• Gfi-1
• Frequent site of proviral integration in T-cell
  leukemia in mice
• Encodes a zinc finger, transcriptional repressor
• KO mice
• Increased long term HSCs (by phenotype)
• Unable to serial transplant, recips die at 6-8
  wks
• Severely compromised in chimeras
• Increased proliferation

Stay tuned for March 24
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Lineage Commitment

• In general, development from pluripotential
  progenitors to mature, specialized cells involves
  the progressive loss of developmental potential
  to other lineages
• When does irreversible commitment occur?

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Transcription Factors are important for
hematopoietic development
Notch1
?
Hardy, Curr Op Immunol, 152003 (revised)
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How to explain Pax5

• Is Pax5 a commitment factor for B cells?
• Pax5 downregulates myeloid (c-fms) and T cell
  specific genes (Notch) while activating B cell
  specific (CD19)
• Pro-B cells from Pax5 knockout mice can develop
  into all hematopoietic lineages-is this
  dedifferentiation?
• True commitment may require epigenetic changes in
  the chromatin

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The Stem Cell Niche
Hanahan Weinberg, Cell 100 57, 2000
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Where is the Stem Cell Niche
But HSCs can also exist outside of the bone marrow
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You have knocked out a gene that results in a HSC
defect. Suggest an experiment to determine
whether this is cell autonomous
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Factors regulating HSC homeostasis
Kiel, MJ Morrison, SJ, Nature Rev Immunology,
2008
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Potential HSC niches in bone marrow spleen
Kiel, MJ Morrison, SJ, 2008
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Cells that may regulate HSCs in the niche
Kiel, MJ Morrison, SJ, Nat Rev Imm, 2008
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Potential mechanisms for endosteal regulation of
HSCs
Kiel, MJ Morrison, SJ, Nat Rev Imm, 2008
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Potential mechanisms for perivascular regulation
of HSCs
Kiel, MJ Morrison, SJ, Nat Rev Imm, 2008
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Where is the BM niche-Models
Kiel, MJ Morrison, SJ, Nat Rev Imm, 2008
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HSCs Gene Therapy
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HSCs Advantages for gene therapy

• property of self-renewal and multilineage
  differentiation, allowing long term and widely
  distributed production of the transgene from a
  single engineered cell
• numerous inherited and acquired disorders that
  disrupt the normal functions of HSC and its
  descendents
• extensive clinical experience with bone marrow
  transplantation
• ? potential to produce non-hematopoietic cells
  that home to correct sites

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Diseases for HSC Gene Tx
Kohn, DB, Nature Reviews Cancer, 32003
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Diseases for HSC Gene Tx
Kohn, DB, Nature Reviews Cancer, 32003
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Gene Tx using HSCs
Baum et al., Blood 101 2003
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X-linked Severe Combined Immunodeficiency Syndrome

• Lack of gamma-c chain with consequent lack of
  cytokine receptors needed for T cell function
  (and T, B, NK development)
• 1/80,000 live births, most common SCID
• Death within 1st year of life from recurrent
  infections
• Tx BMT of HLA-identical BM gt90 survival, but
  most patients lack ideal HLA-match and survival
  is 50-70
• Gene Tx Restore function to autologous cells

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Retroviral-mediated Gene Tx for SCID
Autologous BM
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X-SCID Gene Tx
Kohn, DB, Nature Reviews Cancer, 32003
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Gene Tx for X-SCID

• In French trial
• 9/10 showed evidence of immune reconstitution
  following gene tx, kinetics similar to
  HLA-matched BMT
• Gene tx is successful
• 4 years later
• T cell leukemia development in 3/9.
• Currently being treated with chemotx
• 1 child has died from T-ALL
• Side effects of gene tx treatment have
  substantial risk

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What would you do?

• Continue trial or Halt trial?
• How about other retroviral gene tx trials in
  progress?
• How about new trial applications?
• What additional information would you want to
  know?

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Retroviral Insertional Mutagenesis

• Retrovirus inserts randomly into chromosomal DNA
• Has the potential to activate transcription,
  create a dominant negative, or inactivate an
  allele (heterozygous)
• When delivering a gene, has the potential to
  induce a 2nd hit.

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The provirus caused oncogene activation

• Both had a single proviral integration into
  chromosome 11
• Led to expression of the LMO2 gene, a known
  oncogene in T cell leukemia
• How can you determine when the malignant clone
  first emerged?
• Is this an integration hotspot or selection?

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What is the future of HSC gene tx?

• Need to evaluate each disease?
• No reports of leukemia in other gene tx trials
  (250 pts in 40 trials)
• Only 1 reported example in mice (AML in NGFR gene
  marking led to EVI-1 expression)
• HIV lentivirus does not seem to cause leukemia
• Will it be necessary to analyze integration sites
  prior to cell transfer? What methodology must be
  developed?
• Design of vectors capable of inducing cellular
  suicide, inactivation of the transgene,
  site-specific integration, or prevention of
  insertional activation

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Designer Stem Cell Therapy
Happy St. Patricks Day
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