Hunting for the footprints of stem cells

The analysis of stem cell fate is key to a better understanding of tissue development and ultimately to develop regenerative therapies. In particular, the impact of the microenvironment on stem cell growth, division, and differentiation is far from being understood. One method to quantitatively study stem cell – microenvironment interactions under controlled conditions is automatic single cell tracking in biomimetic culture systems using time-lapse video microscopy.

HFSP Program Grant award holders Ingo Roeder and Tilo Pompe and colleagues
authored on Mon, 12 March 2012

The interplay of undifferentiated haematopoietic cells – here denoted as haematopoietic stem and progenitor cells (HSPC) – and their local growth-environment within the bone marrow is a pivotal mechanism for the organization of haematopoiesis. One of the major obstacles in achieving a comprehensive mechanistic understanding of intrinsic and extrinsic stem cell fate regulation is the current lack of sufficient amounts of high quality quantitative data on the level of individual cells. In particular, there are currently no procedures available that allow for an automated acquisition of high-content data on dynamically changing cellular parameters (such as cell cycling activity, cell growth, cell-cell contact or gene/protein expression) that can be linked to the genealogical relationship of (stem) cells. Therefore, single cell based high-content analysis tools have to be developed to be able to study stem cell fate under defined and controllable microenvironmental conditions.

Figure: Visualization of the spatio-temporal dynamics of single haematopoietic stem and progenitor cells in vitro. Shown is a three-dimensional view of the reconstructed spatio-temporal volume of cell motion, with two x-y planes representing video snapshots of the culture at two subsequent time points (t-axis). Coloured trajectories indicate the temporal development of individual cells. This visualization is complemented by two additional presentations of the data: corresponding individual cellular genealogies and a projection of all reconstructed cell paths in the spatial plane of the culture system.

We have developed a new approach to address these issues by an automated single cell tracking algorithm of dynamic stem cell features in a biomimetic in vitro setup that provides simultaneous quantification of several cellular characteristics coupled to an complete reconstruction of cellular genealogies over several days under defined conditions. For the example of HSPC we demonstrate that these tools not only allow high-resolution and high-content data to be provided, but also new insights in key features of stem cell development, such as the impact of microenvironmental cues, to be generated. Specifically, we demonstrate that sibling cells (i.e., the two offspring of one mother cell) show significantly more similar behaviour than cells that are not or only distantly related. This could not only been shown for cell cycle duration, but also for the growth kinetics or the migration profiles of these cells. Furthermore, our results provide evidence that the observed sibling similarities can be affected and even destructed by micro-environmental cues, as shown by the effect of micro-cavities that had been placed in the surface of the cell culture systems.

The presented project is a first step towards a more comprehensive study of the dynamic interactions of haematopoietic stem cells and their microenvironment. Together Cristina LoCelso, London/UK and Peter Zandstra, Toronto/Canada, Ingo Roeder, Dresden/Germany and Tilo Pompe, Leipzig/Germany (the latter two are both co-authors in the referred publication) are currently pursuing a systems biological approach within the HFSP-funded project “4D analysis of haematopoietic stem cell – niche interactions in vivo, in vitro, in silico“. In this project, 3D in vivo imaging of the murine calvarium is applied to provide a realistic picture of the spatial arrangement of haematopoietic stem cells in their natural bone marrow environment. This data, together with insights about specific regulator molecules obtained from a novel high-throughput PCR technology is used to design specific biomimetic in vitro environments. Using the automatic single cell tracking methods described above, these culture systems allow for a quantification of stem cell localization, migration, and function in response to different regulator components. The generated data will provide the basis for the development of mathematical models of defined stem cell – niche interactions that allow to quantitatively predict the dynamical properties of these interactions and the effect of spatial niche organization.

Reference

On the symmetry of siblings: automated single-cell tracking to quantify the behavior of hematopoietic stem cells in a biomimetic setup. Scherf N, Franke K, Glauche I,   Kurth I, Bornhäuser M, Werner C, Pompe T, Roeder I. Exp Haematol. 2012 Feb; 40(2):119-30.e9 (on the cover of the Feb. issue of Exp. Haematol.).

Pubmed ref