Download Stem cells in Hematology

Stem cells in Hematology;
from their Definition and Regulation
to their Sources and Use…
M.C. le Bousse-Kerdilès
Inserm U972, Villejuif - France
Hematopoietic Stem Cell Transplantation Training Course
ESH-EBMT
(Vienna, May 8-10, 2014)
Stem Cell Definition and Regulation
Canonical definition
(From © 2009. Teisha Rowland )
Stem cells are defined by two main properties:
(1) Self-renewal (create stem cells “indefinitely”)
(2) Differentiation into a diversity of specialized mature cell types
Definition
Regulation
Source and Use
Several types of stem cells…
Embryonic SC
Totipotent
(Generate embryonic and extra-embryonic cell types)
Ex: Fertilized egg
Endoderm
Pluripotent
Mesdoderm
Ectoderm
(Generate cells of the three germ layers)
Ex: ES cell lines
Adult SC
Multipotent
(Generate several cell types)
Ex: Hematopoietic SC
Unipotent
(Generate only one cell type/tissue)
Ex: Keratinocytes
Definition
Regulation
Source and Use
Lung
Pancreas
Muscle
Blood
Skin
Neuron
(Modified from Institute of Cell Therapy Website; Ukraine)
Stem cell hierarchy during lifespan
Loss of pluripotency and proliferative capacity
Morula
Blastocyst
Fetus
Embryonic SC
(Toti/Pluripotent)
Neonate
Fetal/Adult SC
(Multi/Unipotent)
Definition
Regulation
Source and Use
Child
Adult
Aged
Precursors
Progenitors
1%
Definition
99%
Regulation
Source and Use
Bone marrow
(Quiescent)
<< 0.01%
Blood
Stem cells
CSH/CD34+
Differentiated
cells
Differentiation
Proliferation
Hematopoietic stem cells; the archetype of
multipotent stem cells
Stemness; a hidden property!
Hoechst
SP
Functional
markers
Rhodamine
KI67
Iodure
de propidium
ALDH
NOD/SCID
Téloméres
Human
CSH/PH
Tie-2
CD34
Gata 2
CD133
Phenotypic
markers
Definition
Runx1
CD90
Regulation
Cell cycle
markers
Pyronine Y
CD38
Notch
CD117
Source and Use
Oct-4
Molecular
markers
Regulation of hematopoiesis
through extrinsic and intrinsic processes
Receptors
Cytokines
Chemokines
HSC/CD34+
Transcription
factors
Signaling
pathway
Endothelial cells
Definition
Regulation
ECM
GAGs
ECM
receptors
Adhesion molecules
Stromal cells
Source and Use
Transcription factors regulate gene expression
by binding to specific sites on DNA
Transcription
start site
Binding site
(proximal or distal)
Mechanism dependent on the 3D structure of the chromatin
that is regulated by epigenetic modifications
Definition
Regulation
Source and Use
(From Duke University website)
Molecular mechanisms mediating epigenetic
regulation
1) DNA methylation that
establishes a compact/silent
chromatin state
2) Histone post-translational
modifications
- acetylation
- methylation,
- phosphorylation
- ubiquitination/sumoylation
TF
3) Small RNAs that can regulate
the histone code
Epigenetic modifications are reversible and highly responsive to micro-environmental signals
Definition
Regulation
Source and Use
(From Alan Horsager, 2014)
The Bone Marrow,
a specific environment in adult
Endosteum
Vascular sinus
Definition
Regulation
Source and Use
Hematopoietic stem cell niches
Endosteal niche
Mesenchymal
niche
- Maintain HSC quiescence
- Trigger HSC activation
and proliferation
- Participate in HSC
mobilisation out of the BM
Vascular
niche
Definition
Regulation
- Cell-Cell interactions
- Adhesion molecules
- Cytokines/Chemokines
- Proteases
- Extra-cellular matrix
components
- Environmental factors
(Ca++, O2/perfusion..)
Source and Use
(Paul S Frenette et al, Annu, Rev, Immunol, 2013; Lapidot, ASH 2012;)
When niches become
creasy….
Homeostasy
Vacancy/Aplasia
Proliferation/Leukemia
Definition
Regulation
Source and Use
(Scadden D.T. ; Nature 441, 1075-1079, 2006 Walkley et al; Cell 2007;
Kim et al, Blood, 2008; Lataillade et al; Blood 2009;
Raaijmakers et al, Nature 2010)
Stem Cell Sources and Use
for
Cell Therapie and Regenerative Medicine
Embryonic vs. Adult stem cells
Advantages and disadvantages for cell therapies
Embryonic vs. Adults Stem cells
Advantages and disadvantages for cell therapies
Totipotent
Embryonic SC
Pluripotent
(blastocyst) Multipotent
(embryonic
ES cells
tissues)
- Transformed cell products
(ESC: in vitro derived from blastocysts)
- Destroyed source
- Pluripotent
Neonate
Adult SC
Adult
Aged
- Untransformed cell products
(HSC, MSC, EPC…)
- Undestroyed source
- Multipotent
Embryonic vs. Adults Stem cells
Advantages and disadvantages for cell therapies
Totipotent
Embryonic SC
Pluripotent
(ES cells) Multipotent
(embryonic
tissues)
- Transformed cell products
(ES cells; in vitro artifacts)
- Destroyed source
- Pluripotent
Neonate
Adult SC
Adult
Aged
- Untransformed cell products
(HSC, MSC, EPC…)
- Undestroyed source
- Multipotent
Bone marrow: major source of adult stem cells
Pluripotent somatic
stem cells ?
HSC
Hematopoietic
stem cells (1/105)
?
Neuronal
cells
EPC
Endothelial stem cells (≤1/106)
Mesoderm
Endoderm
Ectoderm
Stromal Cells
MSC
?
Hepatocytes
Osteocytes
Chondrocytes
Mesenchymal stromal cells (1-5/106)
Definition
Regulation
Sources & Use
Adipocytes
Other sources of adult stem cells…
Adipose tissue
Mobilized
peripheral blood
Liposuction
Sources of:
- HSCs
- MSCs
- EPCs…
Sources of SC capable to differentiate into cells
from meso-, ecto-, and endo-dermal lineages
In mouse model
(Baer et al., Stem Cells Int., 2012; Pitchford et al., Cell Stem Cell 2009)
Stem cells from perinatal tissues
Umbilical
Cord Blood
Sources of
- MSCs
- EPCs
- HSCs
- ES like (VSELs…)
Definition
Amniotic
membranes
Regulation
Sources & Use
Cell therapies and Regenerative medicine
Differentiated
cells
Immediate but short term
repopulating capability
(Ex: RBC, Pl)
“ Specialized”
stem cells
Long term repopulating
capability but restricted
properties
(Ex: HSC, EPC)
“Generalist”
stem cells
Multipotent properties
Action on environment
(Ex: MSC)
Definition
Regulation
Sources & Use
Mesenchymal Stroma Cell (MSC); a drug cell
for regenerative medicine
 Present in numerous organs/tissues
 Multipotential properties
 Action on environment
- Immuno-modulatory
- Anti-inflammatory
- Anti-fibrotic
- Anti-septic
 Currently used in pre-clinical
application and in some clinical
trials:
- GVHD
- HLA-mismatched HSC graft
- Osteoporosis, Bone defect
- Skin burns
- Vascular regeneration (in combination with EPCs)
Definition
Regulation
Sources & Use
(Arien-Zakay et al; Best Practice & Research Clinical Hematology;2010)
Endothelial Progenitor Cell (EPC) origin and use
for cell therapy
Bone Marrow
Peripheral
Blood
Neoangiogenesis
Umbilical
Cord Blood
Damaged ischemic tissue
Definition
Regulation
Sources & Use
HSC amplification without loosing their
multipotentiality… the quest for Holy Grail!
 Increasing HSC amplification
•
•
Awakening dormant HSC (cytokine cocktails, inhibitors of Cdki …)
Inhibiting their differentiation (activation of Notch signaling/engineered Notch
•
Using regulators of self-renewal/differentiation balance
•
Providing a niche by coculture with MSC (transplant of 2 CB units, one of which
ligands, of Wnt pathway, BMPs…)
hydrocarbon receptor antagonist, copper Chelator, PGE2, HOXB4, cytokines …)
(Aryl
ex vivo expended in MSC coculture)
 Increasing HSC homing
•
•
•
Definition
Intra-bone injection
Agents modulating chemokine/receptor axis (CXCR4…)
Agents inhibiting the peptidase CD26 …
Regulation
Sources & Use
(Dahlberg et al., 2011; Körbling and Freireich.,2011; De Lima et al., 2012)
Embryonic vs. Adults Stem cells
Advantages and disadvantages for cell therapies
Totipotent
Embryonic SC
Pluripotent
(ES cells) Multipotent
(embryonic
tissues)
Neonate
Adult SC
Adult
Aged
Use of embryonic stem cells in
regenerative medicine
- As pluripotent, it is possible to obtain all
mature cell types from ES cell differentiation
- Once differentiated, they can be used to
treat degenerative diseases of many tissues
and organs
- Transformed cell products
- Destroyed source
- Ethical and religious concerns
Back in time…
with induced Pluripotent Stem Cells (iPSC) !
Totipotent
Embryonic SC
Pluripotent
(ES cells) Multipotent
(embryonic
tissues)
iPSC
Yamanaka & Gurdon
Neonate
Adult SC
Adult
Aged
- Characteristics close
to those of ES cells
- Re-programming
adult somatic cells
to pluripotent stage
(Thomson et al; Science 2007; Yamanaka et al; Cell 2007)
Reprogramming adult somatic cells to
pluripotent state
1%
(http://www.eurostemcell.org)
Reprogramming adult somatic cells to a
pluripotent state by forced expression of
pluripotency genes or … by different
stresses such as physical squeezing or
submersion in weak acidic solution (STAP
cells “Stimulus-Triggered Acquisition of
Pluripotency” (Obokata et al, Nature; 2014))
A place for ES and iPS cells in hematology?
Experimental research and clinical applications
Drug screening
In vitro models
of human
development
In vitro models
of genetic
diseases
Cell-based
regenerative
medicine
Definition
Regulation
Sources and Use
(Schlaeger and Guan; 2009)
From human ES/iPS cells to blood cells
and…from blood cells to human iPS!
hESC
Specialization
?
hiPSC
Reprogrammation
CD34+
(Oct-4, Sox2 and Klf4, c-Myc, or Nanog, Lin28, other stimuli ?….)
Definition
Regulation
Sources & Use
(mESC: Evans & Kaufman, Nature 1981
miPSC: Yamanaka, Cell 2006 and Thomson, Cell 2007)
First clinical trial: Blood made from stem cells
will be tested in 2016-17 in UK!
2010: First generation of red blood cells from human ES/iPS
(Lapillonne et al., 2010)…. Difficulties in enucleation
(J. O’Callaghan, Scotland; MailOnline 14 April 2014)
(Scottish National Blood Transfusion Service)
2014: Biochemical environment to produce enucleated RBC
2016-2017: First in-man trial in that will probably use ES cells, but will move on to iPS
cells because it’s easier to choose the blood group (O- : “Universal donor” )…
Towards « artificial » Universal Blood?
1667
From lamb… to human ES or iPS cells!
JB Denys, physician of
King Louis XIV of France
Three and a half centuries later…
2030...?