Development of the haematopoietic and immune systems
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Development of the Haematopoietic and Immune Systems. Embryonic origins Bone marrow transplantation as a paridigm for generating an organ from stem cells Mechanisms of stem cell renewal and differentiation Specific examples of erythrocyte and lymphocyte development .

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Development of the Haematopoietic and Immune Systems

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Development of the Haematopoietic and Immune Systems

Embryonic origins

Bone marrow transplantation as a paridigm for generating an organ from stem cells

Mechanisms of stem cell renewal and differentiation

Specific examples of erythrocyte and lymphocyte development

Development and Disease Mechanisms

Nov 11th 2004, Lecture 12

Gerald Crabtree


Overview of Environment of Embryo/FetusExtra embryonic membranes


The Developmental Origin of Blood and Immune Cells

  • Earliest Site of Haematopoiesis is the Yolk Sac (2-3 weeks) and Dorsal Aorta (AGM region) around 3-5 weeks after conception.

  • Yolk sac stem cells are not able to supply all the blood cell type.

  • True haematopoietic stem cells appear in the liver at about 6 weeks post conception


Yolk sac, transient extra-embryonic structure – initiation of blood/Hb synthesis


Bone Marrow Transplantation:Creating an Organ from a Stem Cell

  • 20,000 bone marrow transplantations per year in the US

  • Most commonly used for treatment of malignancy

  • Also used for treatment of aplastic anemia, autoimmune disorders, myleodysplastic syndromes (bone marrow failure) and exposure to toxins or radiation.

  • Rely on the ability of a small number of Haematopoietic Stem Cells (HSC) to repopulate the immune and hematopoietic systems


The Atomic Age dawned at 5:29:45 am on July 16, 1945, at Trinity Site, New Mexico


The Discovery of Stem Cells

Death due to anemia,

granulocytopenia and

thrombocytopenia

Lethal Irradiation

Transfusion of blood

or bone marrow from a

normal donor

Survival of a significant

number of irradiated

individuals

Lethal Irradiation

What does blood or bone marrow have that allows

the survival of irradiated individuals and the appearance

Of white cells, red cells and platalets?


Reconstitution of the Entire Haematopoietic System by Bone Marrow Transplantation

Transfusion of blood

or bone marrow from a

normal donor

Lethal Irradiation or

Lethal Chemotherapy

To kill all malignant cells

Death of tumor cells

And survival of patient

Donor Provides:

Red cells,

platelets,

white cells,

pulmonary alveolar macrophages,

Kupffer cells of the liver,

osteoclasts,

Langerhans cells of the skin,

and microglial cells of the brain

40,000 bone marrow

transplantations in 1998

General Reference:

F. Appelbaum

Annu. Rev. Med. 2003. 54:491–512


Can HSCs give rise to other cell types?

  • Early reports indicated that muscle, neurons, hepatocytes and cardiac muscle might derive from adult HSC.

  • More recent reports suggests that HSC fuse with other cell types and hence acquired their markers

    • Science 297, 2256, 2003

A

A


Experimental Paradigm for Study of Haematopoietic Stem Cells


Many types of cells originate from a single type of haematopoietic stem cell (HSC)


Possible Mechanisms for Maintaining a Stem Cell Population

A. Asymmetric Divisions

B. Symmetric Divisions

C. Locally Directed Divisions (Niche directs differentiation after a symmetrical division)


Symmetric and Asymmetric Divisions of Neural Stem Cells

Tuj/LeX (CD15)/DAPI

P-P

P-N

N-N

Lex (CD15) Stem cell marker

Tuj Differentiated Marker

Brg Acts Cell-Autonomously to Favor Asymmetric Divisions

Pair cell assay: E13.5 cortical culture


Maintaining Long Term Haematopoietic Stem Cells: A Major Unsolved Therapeutic Goal

  • Soluble factors that maintain HSCs:

    • SIF, Flt3L, Tpo, IL-3

    • Wnt, Notch and Sonic Hedgehog (Shh)

  • Transcription factors that increase the replication of HSC

    • HoxB4 and A9

Under the best of circumstances stem cell reconstitution

can only be sustained for 1 or 2 mouse passages

Possible problems:

In vitro creation of a stem cell niche

Telemeric shortening with sequential passage in culture;


Chromosomal Telemeres Shorten with Passage through the Cell Division Cycle

Elizabeth Blackburn

Cell 2001

A possible limitation to the sequential passage of

haematopoietic stem cells (HSC)


The Discovery of Colony Forming Units Demonstrates Self Renewal within Lineages

Implies the existence of stem cells for each class of blood cell


Sequential Steps of Blood Cell Development are Directed by Cytokines


Sequential Steps of Blood Cell Development are Directed by Cytokines

Cytokine A

Cytokine B

Cytokine C

Committed

Stem Cell

Differentiated and

Functional blood cell


Instructive Vs Selective Mechanisms of Receptor Action

  • (A and B) Selective mechanism in which two different factors (F1 and F2) allow the survival and maturation of lineage-committed progenitors generated by a cell-autonomous mechanism; “X” indicates death of the other progenitors. Erythropoietin

  • (C and D) Instructive mechanism in which the factors cause the stem cell to adopt one fate at the expense of others. Glial growth factor and BMP2


Death of an Anthropomorphism:The Instructive Hypothesis of Receptor Action

H. Lodish

And colleagues


If Cytokines Do not Give Instructive Signals…

Cytokines probably provide permissive signals that are dependent on the developmental history of a cell

_______

Developmental history is reflected by the expression of receptors, signaling molecules, transcription factors and chromatin accessibility


The Development of T Lymphocytes and Red Cells

IL# (interleukin general name for

haematopoietic growth factors

SDF-1 (stomal cell Derived factor)

FLT-3 or Flk2 (Fems like tryosine kinase

Ligand)

SCF (Stem cell factor) the product of

the White locus effects both neural crest

and haematopoietic cell development. Binds

C-kit, mutation of which has near identical

Phenotype as SCF mutations.

Epo- Erthropoietin

Tpo- thrombopoietic factor

GM-CSF granulocyte macrophage

stimulating factor

G-CSF granuloctye stimulating factor


Common

Myeloid

Progenitor

Development of Red Blood Cells

Feedback control loop

  • First red cells are produced in the yolk sac. Later red cell production shifts to the liver, spleen and then the bone marrow.

  • Feedback control of RBC Production is through Erythropoietin (Epo).

    • Necessary to prevent death and promote proliferation of committed precursors

    • Shifts non-committed progenitor cells into the erythroid lineage

    • Produced in renal tubular epithelial cells and more widely in the growing embryo

    • Feedback control targets the first committed cell in the erythroid lineage.


What regulates Erythropoietin (Epo) Production?

Semenza G.L.Cell. 2001 Oct 5;107(1):1-3

  • Epo is regulated transcriptionally by an regulatory region near the gene

  • This regulatory region binds HIF (Hypoxia Induced Factor)

  • Hypoxia regulates HIF

  • HIF also activates VEGF and induces vasculogenesis- a problem in pregnancy


If HIF-1 Controls Epo,

what Controls HIF-1?

Hypoxia Prevents Degradation of HIF-1

PHD = proline hydroxylase


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  • Anemia stimulates HIF and HIF stimulates VEGF and VEGF induces inappropriate angiogenesis and other patterning defects.


Erythropoietin: The Drug

  • Erythropoietin is given for intractable anemia

  • Best for chronic renal disease

  • Ineffective in some cases of aplastic anemia

  • Also effective for increasing blood production for preoperative storage of autologous blood.


Lymphocyte Development

Key Points

1) The role of a developmental field in

lymphocyte specification.

2) Lineage specification in T cells

is dependent on chromatin control.

3) Self vs Non-self discrimination

is dependent on decoding signal intensity


CLP (Common Lymphoid Precursor)

Pax5

Notch Inactive

Pax5 Inactive

Notch Active

B cell

T cell

Pax 5 Repression of Notch Shifts Progenitors into the B Cell Lineage

M. Busslinger and colleagues

Bone Marrow

Thymus


Local Factors Influence the Fate of HSC’s

Implies stem cells for each class of blood cell

However T cell colonies are not found in the spleen


What defines the field in which T cells develop?

Hox-1.5 essential for thymic development

And mice lacking Hox-1.5 have no:

Parathyroid

Thyroid

Submaxillary tissue

WHN (winged Helix Nude or HNF3g) mutant mice

lack a thymus

DiGeorge Syndrome 22q11.2 microdeletion

Congenital heart disease-craniofacial abnormalities

and thymic aplasia


Molecular Anatomy of the Microdeletion in DiGeorge Syndrome

  • Microdeletion of 22q11.2 occurs in 1/4000 births

  • Tbx gene implicated in congenital heart defects

  • Basis for thymic aplasia is still unknown


T Cell Development:How do lymphocytes tolerate self-antigens yet respond to foreign antigens?

Thymus

TCR

Wnt

TCR

IL-7

Reasons to Study T Cell Development

A model system for other developmental processes

Understanding autoimmune disease

If we make new organs from embryonic stem cells they will still be

rejected unless we can also control lymphocyte development.


Current View of Selection of the Immune Repertoire

J. Sprent and colleagues

High Avidity Self Antigen

Bound to self MHC

High Intensity Signal?

Low Avidity Self MHC

Low Intensity Signal?

No Signal

Signal Intensity

Default

Death

Negative Selection

Death of self reactive cells

Positive Selection

Differentiation and

Proliferation of cells able to

interact with self MHC


T Cell Development: Selection of CD4 and CD8 Cells by MHC

CD4 interacts with

MHC class II

And is required

For CD4 Cells

CD8 interacts with

MHC class I

And is required

For CD8 Cells

1

What directs the expression of CD4 and CD8?


ATP-Dependent Chromatin Remodeling Complexes (BAF) and Control T Cell Lineage Committement

Cell. 2002 Nov 27;111(5):621-33.

Nature. 2002 Jul 11;418(6894):195-9

BAF and Mi-2 complexes required

For both silencing and activation

of CD4 and CD8 genes.

CD8 Locus

CD4 Locus


Selection of the Immune Repertoire


Bone Marrow Transplantation as a Paradigm

of Therapeutics

Based on Understanding Human Developmental

  • Endocrine pancreas

  • Skin

  • Bone

  • Joint surface and articular cartilage

  • Kidney

  • Liver

  • Lung

  • Heart

  • Eye

  • Brain???


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