1. Development of the Haematopoietic and Immune Systems Development and Disease Mechanisms Nov 11th 2004, Lecture 12 Gerald Crabtree 1.Embryonic origins 2.Bone marrow transplantation as a paridigm for generating an organ from stem cells 3.Mechanisms of stem cell renewal and differentiation 4.Specific examples of erythrocyte and lymphocyte development

3. Overview of Environment of Embryo/Fetus Extra embryonic membranes

4. 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

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

7. 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

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

9. The Discovery of Stem Cells Lethal Irradiation Transfusion of blood or bone marrow from a normal donor Death due to anemia, granulocytopenia and thrombocytopenia Lethal Irradiation Survival of a significant number of irradiated individuals What does blood or bone marrow have that allows the survival of irradiated individuals and the appearance Of white cells, red cells and platalets?

10. 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 40,000 bone marrow transplantations in 1998 General Reference: F. Appelbaum Annu. Rev. Med. 2003. 54:491–512 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

11. 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

12. Experimental Paradigm for Study of Haematopoietic Stem Cells

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

14. 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)

15. Pair cell assay: E13.5 cortical culture P-P Tuj/LeX (CD15)/DAPI P-NN-N Symmetric and Asymmetric Divisions of Neural Stem Cells Brg Acts Cell-Autonomously to Favor Asymmetric Divisions Lex (CD15) Stem cell marker Tuj Differentiated Marker

16. 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 Possible problems: 1)In vitro creation of a stem cell niche 2)Telemeric shortening with sequential passage in culture; Under the best of circumstances stem cell reconstitution can only be sustained for 1 or 2 mouse passages

17. Chromosomal Telemeres Shorten with Passage through the Cell Division Cycle A possible limitation to the sequential passage of haematopoietic stem cells (HSC) Elizabeth Blackburn Cell 2001

18. Implies the existence of stem cells for each class of blood cell The Discovery of Colony Forming Units Demonstrates Self Renewal within Lineages

19. Sequential Steps of Blood Cell Development are Directed by Cytokines

20. Cytokine ACytokine BCytokine C Committed Stem Cell Differentiated and Functional blood cell

21. 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

22. Death of an Anthropomorphism: The Instructive Hypothesis of Receptor Action H. Lodish And colleagues

23. 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

24. 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

25. Development of Red Blood Cells Common Myeloid Progenitor 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. Feedback control loop

26. What regulates Erythropoietin (Epo) Production? 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 Semenza G.L.Cell. 2001 Oct 5;107(1):1-3

27. Hypoxia Prevents Degradation of HIF-1 If HIF-1 Controls Epo, what Controls HIF-1? PHD = proline hydroxylase

28. Leadville, CO- The birth defect capital of the United States It is with great pride that we invite you to share Leadville's spectacular scenery with majestic mountains, rushing streams, alpine lakes, wildflowers in vivid colors, and sky so blue it appears retouched. And that doesn't include winter thrills. Leadville was incorporated in 1878 and is the highest incorporated city in the continental United States at 10,152 feet above sea level. –From the Leadville Chamber of Commerce Anemia stimulates HIF and HIF stimulates VEGF and VEGF induces inappropriate angiogenesis and other patterning defects.

29. 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.

30. Lymphocyte Development 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 Key Points

31. Pax 5 Repression of Notch Shifts Progenitors into the B Cell Lineage CLP (Common Lymphoid Precursor) B cellT cell Pax5 Notch Inactive Pax5 Inactive Notch Active M. Busslinger and colleagues Thymus Bone Marrow

32. Implies stem cells for each class of blood cell However T cell colonies are not found in the spleen Local Factors Influence the Fate of HSC’s

33. 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

34. 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

35. T Cell Development: How do lymphocytes tolerate self-antigens yet respond to foreign antigens? Wnt IL-7 TCR Thymus 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.

36. Current View of Selection of the Immune Repertoire Low Avidity Self MHC Low Intensity Signal? High Avidity Self Antigen Bound to self MHC High Intensity Signal? Positive Selection Differentiation and Proliferation of cells able to interact with self MHC Negative Selection Death of self reactive cells Signal Intensity Default Death No Signal J. Sprent and colleagues

37. T Cell Development: Selection of CD4 and CD8 Cells by MHC 1 What directs the expression of CD4 and CD8? CD4 interacts with MHC class II And is required For CD4 Cells CD8 interacts with MHC class I And is required For CD8 Cells

38. ATP-Dependent Chromatin Remodeling Complexes (BAF) and Control T Cell Lineage Committement CD4 Locus CD8 Locus 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.

39. Selection of the Immune Repertoire

40. 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???