title: High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal.
abstract: To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (>1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.
authors.affiliation: Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada V5Z 4E6.
authors.forename: Brad
authors.initials: B
authors.lastname: Dykstra
authors.forename: John
authors.initials: J
authors.lastname: Ramunas
authors.forename: David
authors.initials: D
authors.lastname: Kent
authors.forename: Lindsay
authors.initials: L
authors.lastname: McCaffrey
authors.forename: Erin
authors.initials: E
authors.lastname: Szumsky
authors.forename: Liam
authors.initials: L
authors.lastname: Kelly
authors.forename: Kristen
authors.initials: K
authors.lastname: Farn
authors.forename: April
authors.initials: A
authors.lastname: Blaylock
authors.forename: Connie
authors.initials: C
authors.lastname: Eaves
authors.forename: Eric
authors.initials: E
authors.lastname: Jervis
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D019070:Cell Lineage; D049109:Cell Proliferation; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018715:Microscopy, Video / Q000379:methods*; D013997:Time Factors
chemical_list: D017493:Leukocyte Common Antigens