Isolation of Hematopoietic Stem Cell Colonies for Subsequent Analyses
Hematopoietic stem and progenitor cells (HSPCs) are responsible for the maintenance of the hematopoietic system by giving rise to myeloid and lymphoid lineages. HSPCs derived from bone marrow, cord blood or peripheral blood are studied to understand hematopoiesis and leukemogenesis.
A widely used in vitro assay for the study of HSPCs is the colony forming cell (CFC) assay.
When cultured in semi-solid medium like methylcellulose supplemented with nutrients and cytokines, HSPCs proliferate and differentiate into colonies of discrete morphology. The amount of colonies as well as their morphology provides information about the proliferation and differentiation capacity of the input cells under defined conditions.
For further study of the colonies like enumeration of myeloid vs. erythroid colonies, assessment of their differentiation state as well as analyzing gene expression profiles or gene mutations colonies are imaged, detected and picked from the semi-solid medium. The CellCelector facilitates the counting and picking of individual HSPC colonies in an automated and fully documented process providing a precise count of myeloid vs. erythroid colonies, images of each individual colony as well as an overview image of all colonies. Detected colonies can then be picked and analyzed by molecular biological methods. This workflow is used in new approaches for gene therapy and regenerative medicine.
Overview scan (brightfield) of a colony forming cell (CFC) assay in methylcellulose based media. The automatically detected colonies (based on their morphological and gray value data) are green marked.
All images kindly provided by Nina Friesgaard Øbro, Wellcome-MRC Cambridge Stem Cell Institute and Department of Haematology, University of Cambridge, Cambridge, UK.
Target cell colony after detection
Target cell colony before picking
Image after picking