Hematopoiesis - University of Medicine and Dentistry of New ...

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

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



Figure 2. Model of secreted and cell-associated factors produced by osteoblasts that influence HSCs



Figure 3. Model of HSC-OB adhesion-ligand pairs

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


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


empty virionsempty virions


vector in virionsvector in virions

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