Pakharenko M. V. Heterogeneity of hematopoietic progenitor cells in cell culture in vitro and in vivo in norm and in myelodysplastic syndrome. – Qualifying scientific work as a manuscript.
Thesis for the degree of Doctor of Philosophy in the field of knowledge 09 "Biology" in the speciality 091 "Biology". National University of “Kyiv-Mohyla Academy”, Kyiv, 2023.
Experimental data from recent years suggest that malignant stem cells can exist as independent populations relative to cells that are at rest and do not respond at all to conventional cellular toxic agents. It is known that most stem cells in an adult organism are in the G0 state, which allows cells to stay in hematopoietic niches for a long time and, if necessary, proceed to proliferation. Due to being outside the cell cycle, stem cells are more resistant to DNA damage. This feature of "dormant" stem cells makes it possible to preserve the "gold reserve" from all sorts of stressful events and thereby promote treatment. At the same time, there is a population of hematopoietic progenitor cells, at the level of which the processes of proliferation and differentiation occur, since they are sensitive to the action of cytokines, which are ejected in the event of a shortage of blood cells in the periphery. The role of hematopoietic progenitor cells is underestimated, and it turned out to be more significant than previously thought. Although more evidence of leukemia-initiating stem cells is emerging through the use of mouse models, less is known about changes at the level of stem cell compartment in patients with myelodysplastic syndrome (MDS). Although it is assumed that MDS is a “stem cell disease”, there is still no strong evidence of this statement, in addition, the level of damage to hematopoietic progenitor cells in patients with MDS, which constitute a whole group of progenitors that differ in their distance from the stem cell, remains unknown. In addition, despite the described chromosomal abnormalities, mutations and epigenetic changes in MDS observed in progenitors, the stages of development at which pathogenic events occur have not yet been determined. To solve these issues, new methodological approaches are needed to identify different types of progenitor cells and solve questions that can be answered through phenotyping and/or culturing early hematopoietic cells.
The expediency of determining the morphological and functional parameters of hematopoietic progenitor cells from the bone marrow of mice, rats and humans is due to the need to deepen knowledge about the peculiarities of their functioning, to address the issue of heterogeneity of progenitor cells in different mammals and humans, as a general identification of the biological nature and potentials in cell culture in vitro and in vivo in normal and disorders of hematopoiesis. Therefore, the aim of the dissertation was to determine the heterogeneity of hematopoietic progenitor cells in vitro and in vivo to clarify their role in the formation of the pathological process in MDS-IB.
To answer these questions, we studied the population of progenitor cells in patients with MDS-IB subtype. We studied hematopoietic stem cell cultures in vitro and in vivo. For the first time, a heterogeneous group of colony types (CFU-GEMM, CFU-G, CFU-GM and CFU-MM) was identified, the early representatives of which were characteristic of MDS-IB, and the later ones – of normal hematopoiesis. The optimal terms of cultivation of hematopoietic progenitor cells for humans (12-14 days), mice (10 days) and rats (8 days) were established. The response of progenitor cells to high doses of cytokines (G-CSF, GM-CSF, IL-3) and their combination in culture was analyzed, and it was found that the combination of these factors has the best effect, significantly increasing the efficiency of colony formation. The study of the cultural characteristics of progenitor cells in MDS-IВ demonstrated a reduced ability to colony formation of the latter compared to the norm, where the number of cells in a clone did not exceed 50, and in the norm – 500. In addition, the qualitative composition of the colonies was characterized by the presence of chaotically arranged capricious cell forms.
For the first time, it was demonstrated that the presence of a stromal substrate of healthy bone marrow cells does not affect the defectiveness of hematopoietic progenitor cells in MDS-IВ.
Key words: heterogeneity, myelodysplastic syndrome, method of cultivation in vitro and in vivo, kinetics of pathological process development in culture, cell mechanisms, cell morphology, growth factors, migration of progenitor cells, blood system cells, hematopoiesis, proliferation, stem cells, bone marrow, mesenchyme, cytokines.
