High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal

Department of Chemical Engineering, University of Waterloo, Ватерлоо, Ontario, Canada
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2006; 103(21):8185-90. DOI: 10.1073/pnas.0602548103
Source: PubMed


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.

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    • "Of additional interest is the observation that the time taken for mitogenically stimulated ESLAM cells to complete a first mitosis is positively associated with the likelihood that at least one of their two daughter cells will retain HSC functionality. This is consistent with previous evidence that longer cell-cycle transit times correlate with the most primitive HSCs (Dykstra et al., 2006; Lutolf et al., 2009; Yamazaki et al., 2009). Such associations suggest the possibility that cell-cycle control, like retention of GM differentiation potential, may be mechanistically linked to DSR competence in adult mouse BM HSCs. "
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    ABSTRACT: Hematopoietic stem cells (HSCs) are identified by their ability to sustain prolonged blood cell production in vivo, although recent evidence suggests that durable self-renewal (DSR) is shared by HSC subtypes with distinct self-perpetuating differentiation programs. Net expansions of DSR-HSCs occur in vivo, but molecularly defined conditions that support similar responses in vitro are lacking. We hypothesized that this might require a combination of factors that differentially promote HSC viability, proliferation, and self-renewal. We now demonstrate that HSC survival and maintenance of DSR potential are variably supported by different Steel factor (SF)-containing cocktails with similar HSC-mitogenic activities. In addition, stromal cells produce other factors, including nerve growth factor and collagen 1, that can antagonize the apoptosis of initially quiescent adult HSCs and, in combination with SF and interleukin-11, produce >15-fold net expansions of DSR-HSCs ex vivo within 7 days. These findings point to the molecular basis of HSC control and expansion.
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    • "SCs—and other adult stem cells—rapidly lost their myogenic ability (Dykstra et al., 2006) so that they cannot be used in clinical trials (Farini et al., 2009). As Kuang and collaborators suggested, the balance among the signals deriving from the various components of the niche is necessary to maintain the myogenic potential of the SCs (Kuang et al., 2008). "
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    • "Tunable synthetic polymer platforms offer the flexibility to study stem cell fate in response to simple or complex combinations of putative niche parameters. In addition, these systems are highly amenable to time-lapse microscopy analysis and with recently developed strategies to automatically analyze cell behavior and lineage relationships, it is now feasible to evaluate the vast amounts of data generated by such studies [9,11,47,48]. The success of two-dimensional biomaterials approaches to study stem cell regulation in culture is contingent on the availability of markers and/or behaviors that accurately predict stem cell fate in vivo [49]. "
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