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Megakarocyte formation in vitro to expand and explore
van den Oudenrijn, S.
Publication date
2001
Link to publication
Citation for published version (APA):
van den Oudenrijn, S. (2001). Megakarocyte formation in vitro to expand and explore.
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Chapter 1
Scope of this thesis
Scope of this thesis
Platelets are small anucleated particles whose main function is the formation of a plug in response to vascular injury. Platelets are produced upon fragmentation of their precursor cell, the megakaryocyte (Chapter 2). Megakaryocyte formation is mainly regulated by thrombopoietin (Tpo), the most important growth factor of megakaryocyte formation and platelet production. Tpo stimulates the stem cell to proliferate and differentiate via a megakaryocyte progenitor cell into a mature megakaryocyte, which will result in the release of platelets. The whole process of platelet formation from a hematopoietic stem cell takes approximately ten days and the life span of platelet in the circulation is also about ten days.
High-dose chemotherapy, frequently used to treat various malignancies, is a myeloablative therapy that leads to neutropenia (severe decrease in neutrophil counts) and thrombocytopenia (severe decrease in platelet counts). To accelerate hematologic recovery, hematopoietic stem cell transplantations are given to the patient. In the past, bone marrow was the major source of stem cells, but currently there is an increase in the use of peripheral blood-derived stem cells for transplantation. Peripheral blood-derived stem cells are harvested by leukocytapheresis, after treatment of the patients with Granulocyte Colony-Stimulatmg-Factor(G-CSF), with or without chemotherapy, to mobilize stem cells from the bone marrow into the peripheral blood. Cord blood is another source of stem cells, but the limited number of stem cells restricts the use of cord blood stem cells to children.
Despite the use of stem-cell transplantations after myeloablative therapy, the period of thrombocytopenia is considerable. This seems to be due to the fact that reinfused stem cells still have to undergo the whole proces of differentiation and maturation from stem cell to megakaryocyte before platelets are released. Thrombocytopenia can lead to life-threatening bleeding tendencies. To overcome the period of thrombocytopenia, patients are treated with platelet transfusions. Repeated platelet transfusions carry the risk of alloantibody formation by the patient, which can lead to a severely decreased survival time of transfused platelets and to refractoriness to platelet transfusions. Moreover, there is risk of transmission of blood-borne infections. Alternative treatments to reduce the period of chemotherapy-induced thrombocytopenia became available with the cloning of thrombopoietin (Tpo) (Chapter 2). Administration of recombinant Tpo after myeloablative therapy did not result in a satisfactory reduction in the period of platelet transfusion dependency (Chapter 2). Apparently, Tpo is not able to shorten the time needed for megakaryocyte formation. An alternative approach might therefore be to expand ex vivo autologous CD34+ hematopoietic stem cells into
megakaryocyte progenitors and megakaryocytes. Stem cell transplants contain
Chapter 1
variable numbers of megakaryocyte committed cells. In the past it has been shown that higher numbers of megakaryocyte progenitors (represented by cells expressing both CD41 and CD34) or colony-forming-unit megakaryocyte (CFU-Meg) reduces the time to platelet recovery{21,65,66,68}. Reinfused ex vivo expanded megakaryocytes and megakaryocyte progenitors can directly produce platelets, because they do not have to undergo the whole proces of megakaryocyte differentiation. In this way sufficient platelets may be produced during the time bone-marrow reconstitution takes place, thereby reducing the period of severe thrombocytopenia.
The aim of this thesis was to define optimal culture conditions for the ex vivo expansion of megakaryocytes. For this purpose, a liquid culture system was developed. Different growth-factor combinations and culture media were tested to determine the most suitable and clinical applicable culture conditions (Chapter 3 and 5). Moreover, the outgrowth of CD34+ stem cells from various sources into the
megakaryocytic lineage was compared (Chapter 4).
In addition, the developed megakaryocyte culture system was used to analyse the capacity of hematopoietic progenitor cells from patients with various forms of thrombocytopenia to form megakaryocytes (Chapter 7). In patients with congenital amegakaryocytic thrombocytopenia mutations in the gene encoding the Tpo-receptor, c-mpl, were found as the likely cause of this disorder (Chapter 6). In Chapter 8, a functional analysis of the mutations that did not directly predict loss of Mpl function or expression is described.
