HEMOPOIESIS Arnold D Rubin, MD
Cancer Institute of New Jersey
Jan,2009
HEMOPOIESIS• Maintaining the optimal numbers of formed elements
in the circulation: RBC for oxygen carrying,WBC for defense and platelets for liquidity without hemorrhage.
• Elements in constant turnover: RBC-120 days,WBC-few days ( except for lymphocytes), platelets-short time.
• Production must react to rapid changes in environment to provide homeostasis.
• Only mature elements gain access to the circulation.• Plasticity.
How Can You Fulfill these Requirements?
Only a mechanism involving stem cells could provide the plasticity and a life long rapid response to the ever changing inflammation, hemostatic and oxygen needs of the organism
CELL DEVELOPMENT
STEM
STEM
STEM
STEM
STEM
Hierarchic
Stochastic
Hemopoiesis
Tumors
STEM CELLS
• A stem cell must be capable of self renewal and should not be “used up”
• It must be capable of differentiating into a mature functioning cell
• Committed stem cell can only differentiate into one type of cell
• Pluripotential stem cell can differentiate into more than one type of cell
• Totipotential stem cell can differentiate into any type of cell ( like an embryonic cell )
Definition of a Hemopoietic Stem Cell
1 Repopulate the marrow of a lethally irradiated 1 Repopulate the marrow of a lethally irradiated animalanimal
2 Have the capacity for self renewal and2 Have the capacity for self renewal and expansionexpansion
3 Have the capacity to differentiate into all of the 3 Have the capacity to differentiate into all of the lymphomyeloid serieslymphomyeloid series
Replenish old or Replenish old or damaged tissue by damaged tissue by limited proliferation and limited proliferation and maturationmaturation
Stem cellStem cell
Differentiation Differentiation leading to leading to senescence and senescence and apoptosisapoptosis
THE FATE of the STEM CELLTHE FATE of the STEM CELL
Mutation leading to neoplasia: ie failure to differentiate or undergo apoptosis
Genetics of Differentiation
TF
Silent HOX DNA
RNA transcribed
Translation into proteins to drive differentiation in to specific tissues
Dormant or Dividing Stem Cells
Epithelial Cell
With differentiation mitotic potential is limited or ceases Cell ages and dies True stem cells are ageless
Identification and Function of Stem Cells
Cell Surface Markers
Functional Assays
Concept of Stem CellsHemopoietic
lethallethalradiationradiation
BM cellsBM cells
spleen spleen coloniescolonies
mousemouse Till- McCullogh PreparationTill- McCullogh Preparation
CFU-SCFU-S8 day 8 day (committed)(committed)12 day12 day( multipot.)( multipot.)
Maintainance of “Stemness”
The length of the telomer determines the ability of the stem cell to keep from differentiating and aging. Thus, stem cells contain a lot of telomerase
M. Pera, ASH Education,2002
M. Pera, ASH Eduation,2002
M. Pera, ASH Education,2002
Hemopoietic Stem Cells- Surface Characteristics
CD34-/loCD34+
Sca-1+ Sca-1+Lin- Lin-
C-kit+ C-kit+
Low self renewel Multilineage Progenitors
True stem cells
Long term repopulation
SKL Cells
Characteristics of Stem CellsPropertiesPropertiesCD34++CD34++Thy-1+Thy-1+
SSC-lowSSC-lowc-kit+c-kit+HLADR-HLADR-CD45RA-CD45RA-CD38-CD38-lin-lin- CD13-CD13- CD33-CD33-
in vitroin vitro Assays Assays in vivo in vivo AssaysAssays
LTC-IC Tx sheep LTC-IC Tx sheep in uteroin uteroCAFCCAFCCFU-Blast SCID miceCFU-Blast SCID mice SCID-HuSCID-Hu PrimatesPrimates Human BMTHuman BMT
Rh-123lowRh-123lowFSC-lowFSC-low
Culture of Clonogenic Progenitors
nutrientsnutrients
mediummedium
““gamisch”:gamisch”:
CSF’s lineageCSF’s lineagespecificspecific
InterleukinsInterleukins
semisolid suspensionsemisolid suspension
test cellstest cellscolonies(CFU’s):colonies(CFU’s):Blast, GEMM, GMBlast, GEMM, GMG,M,Meg, E,BFU etcG,M,Meg, E,BFU etc
NOD-SCID Mouse
Inject human stem cells
CD38
CD45
Blood for FACS to show human cells grafted
Assay for Human Stem Cells
Ontogeny of Hemopoiesis
• Primitive – transient in yolk sac, fetal Hb RBC -7.5 dpc (mouse)
• Migration to fetal liver and spleen• By birth nearly all in bone marrow• Cell of origin hematogenic endothelium
or hemangioblasts?
Orkin,S, Cell,2008
Orkin,S, Cell,2008
Other Cells and their Products Interact with Stem Cells
THE JACK SPRAT RIDDLE
• Marrow cells from the w/w mouse will not engraft a normal mouse
• This mouse will accept a marrow graft from normal mice
• The sl/sl mouse will not accept a marrow cell graft from normal or w/w mice, but its marrow cells will engraft normal and w/w mice
• What is going on here?
The Steel Factor-Stem Cell Stromal Interaction
Bone Marrow StromaBone Marrow Stroma
SLFSLF SLFSLF
normalnormal w/ww/w sl/slsl/sl
c-kitc-kit
c-kitc-kit
(
((
c-kitc-kit
c-kitc-kit
((
((
Stem CellsStem Cells
anemiasanemias
The Big Questions
• Infused marrow cells know to go directly to the bone marrow
• No matter how many stem cells infused, the circulating mature cell numbers are “just right”
• Pure CD34 positive stem cells are insufficient to restore a lethally irradiated bone marrow
• WHY? HOW?
The Concept of a Hemopoietic NICHE
• BONE NICHE – Maintenance of long term quiescience of hemopoietic stem cells in appropiate numbers for blood cell homeostasis
• VASCULAR NICHE – Proliferation of hemopoietic stem cells, their differentiation into mature blood cells and delivery to the circulation
Mutant Mice and the Bone Niche
Runx-2-/- mutant lacks osteoblast formation.
Consequently no hemopoiesis is found in the bone marrow, hemopoiesis is present in the liver and yolk sac.
cbfb-/- mutant lacks hemopoiesis in fetal liver
Migration to bone marrow depends on vascular factors such as vegf and tel .
Orkin,S, Cell,2008
Copyright ©2006 American Society for Clinical InvestigationYin, T. et al. J. Clin. Invest. 2006;116:1195-1201
Hematopoiesis of bone cells and marrow stromal cells
Marrow Stroma HSC BMP Interaction
HSCSC
SCF c-kit
Jagged1 Notch
Ang1 Tie-2
Cadherin βcatenin
VCAM integrin
Osteopentin βintegrin
Other Factors Affecting Interaction and Proliferation of HSC
BMP
Wnt
Hedgehog
Il-3
Il-6
TPO
PTH
Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.
Taichman, R. S. Blood 2005;105:2631-2639
Figure 1. Model of support for hematopoietic stem cells by osteoblasts
Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.
Taichman, R. S. Blood 2005;105:2631-2639
Figure 2. Model of secreted and cell-associated factors produced by osteoblasts that influence HSCs
Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.
Taichman, R. S. Blood 2005;105:2631-2639
Figure 3. Model of HSC-OB adhesion-ligand pairs
Zhang,Nature,2003
Zhang,Nature,2003
The Vascular Niche
• More support for proliferation and differentiation of HPC’s by sinusoidal endothelial cells
• Mobilization and homing of HPC’s
• Backup in spleen when bone marrow is not functioning
Factors Involved in the Vascular Niche
• SDF-1 is a homing factor, inducing HSC’s to transendothelial migration mediated by E- and P-selectins.
• HSC receptor is CXCR4, aided by Rac family
• G-CSF increases SDF-1, elastase, cathepsin-G etc in circulation to facilitate mobilization
Copyright ©2006 American Society for Clinical Investigation
Yin, T. et al. J. Clin. Invest. 2006;116:1195-1201
The osteoblastic and vascular niches in bone
Zhu,Oncogene,2002
Transcription FactorsThese factors play a vital role in both embryogenesis and lineage restricted differentiation of hemopoietic stem cells.
Curiously most of these factors are subjected to mutations resulting in the development of hematologic malignancies. e.g. MLL, RUNX1,TEL/ETV6, SCL/tal1, LMO2
Detection is by knock-out and “knock-in” experiments in various model systems.
MicroRNA’s, a new field, probably are also vital
Orkin, SH, Cell, 2008
Orkin,SH, Cell, 2008
Blood Cell Development
Lympho-myeloid growth depends on a hierarchic array of Lympho-myeloid growth depends on a hierarchic array of clonogenic cells with widely varying potential for clonogenic cells with widely varying potential for replication and differentiationreplication and differentiation
Hierarchy of Myeloid Cell Production
SS SS
gemmgemm gemmgemm
GMGM GMGM GMGM GMGMStromalStromalCellCell
MacrophageMacrophage
T-cellT-cellCD34CD34++ Sca Sca++ Lin Lin- - Thy-1Thy-1++
))
))
((
))((
L L eessss
ddIIvvIIssIIoonn
MMoorree
mmaattuurree
IL’sIL’s
CSF’sCSF’s
Hemopoietic Cytokines
Metcalf designed the system to demonstrate the existence and specificity of the various cytokines important in hemopoiesis
Dr Metcalf
Culture of Clonogenic Progenitors
nutrientsnutrients
mediummedium
““gamisch”:gamisch”:
CSF’s lineageCSF’s lineagespecificspecific
InterleukinsInterleukins
semisolid suspensionsemisolid suspension
test cellstest cellscolonies(CFU’s):colonies(CFU’s):Blast, GEMM, GMBlast, GEMM, GMG,M,Meg, E,BFU etcG,M,Meg, E,BFU etc
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Metcalf, D. Blood 2008;111:485-491
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Metcalf, D. Blood 2008;111:485-491
Passegue,PNAS,03
Bruno,Mol & Cell Biol,2004
Stem Cell Transplantation(BMT)
BMT can be used to replace a failed marrow or correct certain genetic defects.
The adoptive immune system growing out of a BMT may be used to destroy hematologic malignancies resistant to chemotherapy
BMT can be the vehicle to deliver a therapeutic gene in a viral vector.
BMT may contain stem cells with multipotential plasticity for organ repair
ANNUAL NUMBERS OF BLOOD AND MARROW TRANSPLANTS
WORLDWIDE1970-2002
NU
MB
ER
OF
TR
AN
SP
LA
NT
S
YEAR
1970 1975 1980 1985 1990 1995
Autologous
Allogeneic
20000
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
1
stem cellsstem cells
T cellsT cells
stromal stromal
myeloid progenitorsmyeloid progenitors
cancer cellscancer cellsother cellsother cells
Bone Marrow TransplantationBone Marrow Transplantation
BMT is composed of disparate cellsBMT is composed of disparate cells
There are cells contributing to:There are cells contributing to: maturing myeloid cellsmaturing myeloid cells myeloid expansionmyeloid expansion support for myeloid growthsupport for myeloid growth immune activityimmune activity GVHDGVHD reintroduction of malignant cellsreintroduction of malignant cells
These may be conflictingThese may be conflicting
Sources of Stem CellsSourceSource
Bone MarrowBone Marrow
Stimulated Blood 1/4-1/5 8-9Stimulated Blood 1/4-1/5 8-9
Cord Blood slightly increased 20-46Cord Blood slightly increased 20-46
Fetal Liver/BM rich source similar to CBFetal Liver/BM rich source similar to CB
Content of StemContent of StemCells relative to BMCells relative to BM
----------- 21-30----------- 21-30
Days to ANCDays to ANCOf 500Of 500
in vitro in vitro Expansion concentrated no faster than PBExpansion concentrated no faster than PB
Nonmyeloid Stem Cells
in vitro in vitro expansion of CD34expansion of CD34++, CD45RA, CD45RAlow low CD71CD71low low By IL-3, IL-6 SLF & EPO provides an expansion of :By IL-3, IL-6 SLF & EPO provides an expansion of :
5x for marrow5x for marrow
187x for UC blood187x for UC blood
2125x for fetal liver2125x for fetal liver
More primitive by ontogeny yields stem cells more active in More primitive by ontogeny yields stem cells more active in proliferation because telomeric DNA has not been lost yet.proliferation because telomeric DNA has not been lost yet.
Stem Cells from Sources Other than Bone Marrow
• Bood has 10-100 fold fewer stem cells as Bood has 10-100 fold fewer stem cells as compared to marrow. But stimulation by compared to marrow. But stimulation by cytotoxins &/or CSU’s results in peripheralization cytotoxins &/or CSU’s results in peripheralization of CSF’s & to a lesser extent LTC-IC’sof CSF’s & to a lesser extent LTC-IC’s
• Umbilical cord (UC) blood has more LTC-IC’s Umbilical cord (UC) blood has more LTC-IC’s than marrow. But it takes 13-46 days for than marrow. But it takes 13-46 days for leukocyte recovery.leukocyte recovery.
• UC blood is easy to recover but limited in volume. UC blood is easy to recover but limited in volume. Need >2X10Need >2X1077/kg nuc. cells/kg nuc. cells
Adult Stem Cell PlasticityAdult Stem Cell Plasticity
Hemic Stem cellsHemic Stem cells
Mesenchymal Stem CellsMesenchymal Stem Cells
LiverLiver
Neurons, glial Neurons, glial cellscells
CardiacCardiac
Fibroblasts Fibroblasts Osteoblasts Osteoblasts Adipocytes Adipocytes ChondroblastsChondroblasts
C. M. Verfaillie, ASH Education, 2002
ex vivo Stem Cell Expansion• Efficiency of harvest. At times this might be Efficiency of harvest. At times this might be
the only way to obtain sufficient cells.the only way to obtain sufficient cells.• As cells may be in synchronous cycle, gene As cells may be in synchronous cycle, gene
transfer may be facilitated.transfer may be facilitated.• With appropriate cytokines, one could design With appropriate cytokines, one could design
grafts according to specifications.grafts according to specifications.
• This may be a means of obtaining grafts more This may be a means of obtaining grafts more
likely to be free of tumorlikely to be free of tumor cells. cells.
ex vivo Expansion of Stem Cells
• Incubate with stroma. Porcine stroma supports 5 fold Incubate with stroma. Porcine stroma supports 5 fold expansion in 5 weeks. Maybe negative influence as expansion in 5 weeks. Maybe negative influence as well.well.
• Incubate with mixtures of cytokines without stroma Incubate with mixtures of cytokines without stroma yield limited growth. There is no LTC-IC expansion.yield limited growth. There is no LTC-IC expansion.
• Biorectors are very promising but only 10% of cells Biorectors are very promising but only 10% of cells
are responsible for growth.are responsible for growth.
• No real success because niche can not be replicted
Retroviral Vectors Used to Transduce Stem Cells
helper DNAhelper DNAgag polgag polenvenv
gag polgag polenv env vectorvector
viralviral
RNARNAproteinsproteins
helper virushelper virus
RNARNAproteinsproteins
empty virionsempty virions
RNARNAproteinsproteins
vector in virionsvector in virions