Continuous Single Cell Observation Proves Lineage Instruction by Hematopoietic Cytokines.

Abstract 3607

Poster Board III-543

The significance of cytokines in hematopoiesis is irrevocable, but their contribution in lineage choice of multipotent progenitors is still controversial. Can late acting cytokines actively induce lineage decisions (instructive model), or do they only allow the survival and proliferation of already unilineage-restricted precursors, which have been differentiated by cell-autonomous mechanisms (selective/stochastic model)?

A major reason for contradictory results is the fact that hematopoiesis is usually followed by analyzing populations of cells - rather than individual cells - at very few time points of an experiment and without knowing their individual identities. Both models can only be distinguished by constantly observing the fate of each cell and its progeny, but not by endpoint analyses of whole populations. Therefore, real-time tracking of individual cells in culture, tissues or whole organisms is an extremely powerful approach to fully understand the exact effect of cytokines on fate decisions.

A computer aided culture and imaging system was developed to follow the fate of individual cells over long periods of time with high temporal and spatial resolution. New software modules were written, which help to record, display and analyze the divisional history, position, properties etc. of individual cells.

Purified progenitor cells from mouse bone marrow of lineage-reporter mice were cultivated in defined media with the addition of selected cytokines. Video-time-lapse experiments enabled the tracking of individual cells and their progeny over many days. Pedigrees of hundreds of progenitor cells under each condition were generated and statistically analyzed. This novel single cell tracking technology revealed an unexpected instructive action of M-CSF and G-CSF directing lineage decisions of granulocyte-macrophage progenitors (GMPs) to differentiate into the monocytic or granulocytic lineage, respectively. Additionally, both late acting cytokines accelerated the differentiation process of GMPs. First results indicate an instructive in vivo action of these cytokines on progenitor lineage choice.