HEMATOPOIESIS: STEM CELL AND BONE MARROW

S. Sami Kartı, MD, Prof.

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.

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

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.

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.

Introduction

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

Hierachical model of lymphohematopoiesis

Hematopoietic cells undergo an orchestrated differentiation program

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.

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.

Stem cell phenotype

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.

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

Hematopoietic growth factors- Cytokines

are glycoprotiens involved in the self-renewal of stem cells and the proliferation and differentiation of progenitor cells

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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

Effects of cytokines on hematopoietic lineages

G-CSF causes the aquisition of myeloid enzymes in granulocytes

Thrombopoietin induces the expression of platelet-spesific proteins.

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.

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.

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.

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

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.

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.

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.

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.

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.