Petr Páral PhD thesis

The cell cycle and differentiation of haematopoietic stem and progenitor cells.
Prague, 2019

Abstract:
Haematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analysed the cell cycle and cell production rate in HSPCs in murine haematopoiesis. The labelling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled the determination of the cell cycle flow rate into the G2phase, the duration of the S-phase and the average cell cycle time in Sca-1 ⁺ and Sca-1 ^- HSPCs. The determination of cells with 2n DNA content and labelled during the preceding Sphase was used to establish the cell flow rates in the G1phase. Our measurements revealed a significant difference in how Sca1 ⁺ and Sca1 ^- HSPCs self-renew and differentiate. The division of Sca-1 ⁺ progenitors led to the loss of the Sca1 marker in about half of newly produced cells, corresponding to asymmetric cell division. In contrast both Sca-1 ^- progenitors, arising from mitotic cell division, entered a new round of the cell cycle. This corresponds to symmetric self-renewing cell division. The novel data also enabled us to estimate the cell production rates in the Sca-1 ⁺ and in three subtypes of Sca-1 ^- HSPCs. We focused on adult murine erythroid differentiation in the next part of our study. We introduced an original flow cytometry approach for identifying and studying erythroid progenitor and precursor cells. This approach is based on the changing expression of two cell surface markers, c-Kit (receptor for stem cell factor) and CD71 (transferrin receptor 1) in bone marrow cells deprived of granulocytes, monocytes, lymphocytes and Sca-1 ⁺ cells. We identified the early erythroid progenitor cells with BFUE and CFUE potentials within the cell population highly expressing c-Kit. The potential to give rise to BFU-E and CFU-E colonies was lost in the cells highly expressing CD71. Subsequently, erythroid differentiation progressed into the proerythroblasts which expressed c-Kit at a high level. Analysis of the cell cycle revealed that the differentiation of proerythroblasts into basophilic erythroblasts occurs in the course of a single cell cycle, in fact predominantly in the Sphase. During this S-phase, cells maintain the high expression of CD71 but rapidly lose the c-Kit marker and express the erythroid marker Ter119. The dual EdU-BrdU sequential labelling of DNA synthesizing cells, together with the metaphase block induced by colchicine, provide us with unique insights into the dynamics of cell proliferation and differentiation events in early erythroid cells.