1. HEMATOPOIESIS: STEM CELL AND BONE MARROW
S. Sami Kartı, MD, Prof.

2. Introduction
Lymphohematopoesis is a tightly regulated system in which production of various blood cell types responds to specific functional demands:
red cell production in response to hypoxia,
granulocyte/monocyte production in response to infection,
lypmhocyte production in response to antigen cahallenge,
platelet production in response to hemorrhage.

3. Introduction
In this system (tissue), early multipotent stem cells with extensive proliferative potential reside largeley in the bone marrow cavity.
These stem cells with appropriate inductive signals differantiate and give rise to populations of progenitor cells which differentiate to spesific cell lineages
Lymphocytes
Erythrocytes
Platelets
Granulocytes
Monocytes

4. Introduction
This event occur continously with a large turnover of differentiated cells, as illustrated by the blood lifespan of
human erythrocytes (120 days),
platelets (10 days),
granulocytes (9 hours).
Lymphocyte (T and B cells) lifespans vary from hours to years.

5. Introduction
The production of active blood cell types occurs predominantly in the bone marrow.
However, the spleen, lymph nodes, and accessory lymphoid tissues are also ongoing sites of cell production, predominantly lymphoid; under stress, myeloid cell production also accur at these sites.

6. Introduction
The end cells produced in the marrow are released into the blood stream under various stimuli and circulate in the blood.

11. Hierachical model of lymphohematopoiesis

12. Hematopoietic cells undergo an orchestrated differentiation program

14. Stem Cell
Early multipotent stem cells are present in the yolk sac and in the mesenchymal tissues.
These stem cells subsequently traffic predominantly to the liver, followed by the establishment of marrow as the major site of active hematopoiesis.
The earlier in ontogeny stem cells are harvested, the greater their proliferative potential, as illustrated by the proliferative and growth potential of fetal liver and cord blood cells in clinical transplantation.

15. Stem Cell
The hematopoietic cell has been characterized as to surface proteins and physical, metabolic, and cell cycle characteristics; and these characteristics have been utilized physically to purify stem cells.
Early stem cells do not express differentiated lineage markers, but they express certain classes of receptor proteins of other markers; in humans, CD34 and c-kit have been proposed as stem cell-spesific markers.

16. Stem cell phenotype

17. Stem Cell
The most primitive stem cells tend to be dormant in G0 or prolonged G1 phases and also to show strong activity of the p170 pump, the multidrug resistance pump that exudes certain chemotherapeutic agents and the dyes of rhodamine and Hoechst, from cells.
Thus the most primitive stem cells are characterized by very low staining with rhodamine and Hoechst.

18. Regulation and cytokines
A large number of cytokines have been characterized and shown to affect lymphohematopoiesis.
Regulation of lymphohematopoiesis is based on a large number of circulating and membrane based cytokines.
Over 70 cytokines maintain, stimulate, or inhibit various aspects of hematopoiesis

19. Hematopoietic growth factors- Cytokines
are glycoprotiens involved in the self-renewal of stem cells and the proliferation and differentiation of progenitor cells

20. Characteristics of lymphohematopoietic cytokines
Glycoproteins
Act on cell surface receptors
Initiate complex second messenger and transcriptional and post-transcriptional regulation
May act on stem cell, progenitor cell, and differentiated cells of the same lineage
May act on multiple different lineages (e.g. erythroid, granulocyte, and lymphoid)
Stimulate or inhibit proliferation, apoptosis, differentiation, or function

21. Cytokines
Cytokines exert a wide variety of actions on diverse cell types both within and outside spesific differentiation lineages, but many have predominant actions.
Erythropoietin, Granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) are examples of cytokines with a high degree of specificity.

22. Cytokines However, most of the cytokines have multiple actions.
Interleukin (IL)-6, which acts on primitive stem cells as well as lymphoid, granulocyte, megakaryocyte, and macrophage lineages,
IL-3 effects all lineages.
IL-1 induces many other cytokines.

23. Production of the cytokines
These cytokines are produced by a large variety of tissues and cell types.
Most cells produce multiple cytokines, which can be differentially induced by various stimuli, including other cytokines, such as IL-1.

24. Production of the cytokines
Monocytes, T lymphocytes, endothelial cells, fibroblasts and marrow stromal cells are important sources of lymphohematopoietic cytokines.
Erythropoietin production is an exception, because it is largely produced in the kidney in response to hypoxia

25. Production of the cytokines
Stimuli that induce white blood cell formation are related to exposure foreign or noxious agents
whereas platelet production occurs in response to hemorrhage, anemia, and thrombocytopenia.

26. Cytokine receptors
Each cytokine has its private receptor, but different cytokines may share class-spesific signal transducers.
Many receptors dimerize on cytokine binding and then activate thyrosine kinase, promoting phosphorylation of intracellular proteins; other receptors do not have intrinsic enzymatic activity but induce protein phosphorylation through associated non-receptor-type tyrosine kinase activities, such as JAK-2, Fes, and Lyn.

27. Cytokine receptors
Receptors are expressed in low numbers and do not exceed a few hundred per cell.
The multipotent repopulating stem cell possesses receptors for most cytokines, but more mature cells have a more restricted distribution of receptors.

28. Cytokine receptors Two major receptor families have been described.
The hematopoietic receptor family includes receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, G-CSF, granulocye-macrophage CSF (GM-CSF), and erythropoietin..
The tyrosine kinase receptor family includes receptors for FLT3-ligand, steel factor (c-kit ligand), CSF-1, and thrombopoietin.
Receptors not fitting into these families include those for IL-1 and IL-8.

29. Cytokine receptors
Signaling through these receptors activates transcription factors
Progenitor cells differentiate toward spesific lineages.
For example, GATA-1 and FOG promote erythroid and megakaryocyte differentiation, whereas SCL, AML-1, and GATA-2 regulate primitive stem cell differentiation.

30. Adhesion Molecules Adhesion molecules function
both to bind cells or extracellular matrix
and as signaling molecules.
Steel factor, IL-3, and GM-CSF activate very late antigens 4 and 5 (VLA-4 and VLA-5), which are adhesion molecules expressed on CD34-positive cells.
This activation results in promotion of the ability of VLA-4 and VLA-5 to bind fibronectin.
Stromal or microenvironmental cells are major regulators of hematopoiesis, both by positioning stem/progenitor cells and by signaling with secreted and membrane-based cytokines.

31. Effects of cytokines on hematopoietic lineages
An important effect of erythropoietin on erythroid progenitors and precursors is to prevent apoptosis and thus maintain the viability of these cells.
Erythropoietin induces erythroid hemoglobinization

32. Effects of cytokines on hematopoietic lineages
G-CSF causes the aquisition of myeloid enzymes in granulocytes
Thrombopoietin induces the expression of platelet-spesific proteins.

33. CYTOKINES IN THERAPY
The first successful use of a cytokine in the therapy for a cytokine deficiency state was that of erythropoietin (epo) to treat the anemia of chronic renal failure.
Administration of epo to patients with chronic renal failure corrects or partially corrects the anemia and restores a better state of well-being.

34. CYTOKINES IN THERAPY Epo may also be used in;
myelodysplastic syndrome,
anemia of inflammation (anemia of chronic disease),
Autologous blood collection,
chemotherapy-induced anemia.
Interleukin 2 and -interferon have been used successfully in therapy for solid tumors.

35. CYTOKINES IN THERAPY
G-CSF has a role in stem cell mobilization and appears effective in certain severe chronic neutropenic states, including Kostmann’s neutropenia and cyclic neutropenia.
G-CSF and GM-CSF stimulate a wide variety of cell types of both hematopoietic and non-hematopoietic origin and, not surprisingly, have a wide variety of side effects, including bone pain, fever and chills, pleural and pulmonary effusions, vasculitis, splenic enlargement, and proteinuria.
Agents that stimulate in vivo platelet production such as IL-11 and thrombopoietin have been used in clinical trials in thrombocytopenic patients.

36. Mobilization of stem/progenitor cells
Stem cells reside in bone marrow
These stem cells can be easily mobilized in to the peripheral blood by a number of cytokines, including G-CSF, steel factor, FLT-3, IL-11, IL-12, IL-3, IL-8, IL-7, MIP-1 and erythropoietin.
In addition, previous exposure to cyclophosphamide or other cytotoxic agents also mobilizes stem cells from the bone marrow to the blood

37. Mobilization of stem/progenitor cells
Pretreatment with cyclophosphamide, followed by treatment with G-CSF, may be the most potent regimen for mobilizing stem/progenitor cells.
In general mobilized stem/progenitor cells appear to restore hemopoiesis more rapidly than unstimulated marrow derived stem cells.

38. Stem/progenitor cell expansion
The ability to expand lymphohematopoietic stem cells in vitro has immediate implications for strategies of repetitive transplant, immunotherapy, and gene therapy.
A large number of studies have established that exposure of marrow cells in liquid culture to a variety of cytokines leads to differentiated and progenitor cell expansion.

39. Homing and engrafment of stem cells
Homing to marrow appears complete within 20 hours, and engrafted stem cells rapidly enter cell cycle after intravenous infusion.
The blood stream clears quickly of stem cells, and there appears to be virtually no primary thymic engrafment
Later secondary engrafment of thymus occurs.

40. THERAPEUTIC USES OF STEM CELLS
Marrow transplantation represents one of the major therapeutic advances of the past 30years.
It has been successfully used to cure marrow deficiency states, genetic marrow diseases and leukemias.
To restore deficient cell production after otherwise lethal damage by high-dose radiotherapy/chemotherapy is used to eliminate malignant cell population.

41. THERAPEUTIC USES OF STEM CELLS
Now it is known that a graft-versus-tumor effect is a major component of the theraputic efficiency of allogeneic brother/sister or mathched unrelated transplantations, thus indicating that transplantation may work in part by mediating a cellular immune attack against cancer cells.
Marrow cells were initial source of stem cells for transplantation, but pheresis of peripheral blood stem cells has supplemented marrow.
Umblical cord blood is also becoming a major source of stem cells, especially for unrelated transplantations.