In Vitro Hematopoietic Lineage Interconversion from Human Bone Marrow Stem and Progenitor Cells.

Abstract

The cell surface antigen, CD133, marks a fraction of hematopoietic stem and progenitor cells and has been successfully used to study their differential biology. To evaluate the differentiating capacity of stem/progenitor cells, we cultivated purified normal human bone marrow CD133 selected cells for 2 weeks with erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF) to induce erythroid or myeloid differentiation, respectively. After the second week of cultivation, we reversed the seeding environment of the two populations by placing EPO treated cells into a G-CSF environment and G-CSF treated cells into an EPO environment for an additional 2-week culture. The cells produced in the culture were phenotypically defined by morphology and flow cytometry, and genotypically by RNA and proteomic analyses. Three-color flow cytometry was used for identifying CD133+ progenitors, CD36+ erythroid and CD13+ myeloid cells, as shown in Table 1. The morphology of the cultured cells, assessed by Wright-Giemsa staining, is consistent with the conversion of cellular specific markers. Rapid analysis of gene expression demonstrated co-expression of 76% of 266 genes analyzed among the erythroid and myeloid lineages. Furthermore, proteomic analysis exhibited the sharing of 33% of 9518 expressed protein spots assayed in the two populations after the first 2-week culture, and 32% after 2 weeks of the switch culture. Our data clearly demonstrate that the committed erythroid and myeloid precursors are able to change their fate and can switch into the opposite cell type by a conversion pathway under a specifically defined condition. We termed this switch as interconversion, considering conversion of hematopoietic cells to non-hematopoietic cells. Furthermore, the observations presented in this study show that cytokines used can improve the conversion. We are developing a mathematical model describing the kinetics of hematopoietic stem/progenitor cell transitions into specific lineages, along with the conversion of committed cells based on multiple potential energy wells corresponding to different cell states and cytokines.Table 1. Expression of cell surface markers after 4-week cultureD01 week2 weeks4 weeksCD expression (%)EGEGE2w →G2w→ G2w E2wCD133+96.1915.7413.60.240.360.010.63CD36+060.3727.3996.3725.8745.4168.54CD13+0.4335.4157.2924.4192.185.8737.76CD133+ / CD36+0.4422.2415.970.120.181.557.65CD133+ / CD13+1.2419.4313.360.361.0913.3114.92CD36+ / CD13+0.0941.2517.8023.6954.146.6079.41Data are presented as a mean of at least 2 experiments. E: EPO; G: G-CSF; E2w or G2w: EPO or G-CSF treatment for two weeks.