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.
Full-textDOI: · Available from: David G Kent, May 19, 2015
SourceAvailable from: David G Kent[Show abstract] [Hide abstract]
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.
[Show abstract] [Hide abstract]
ABSTRACT: Cellular heterogeneity represents an increasingly appreciated aspect for research in life science. To address this issue, we have developed a nano-volume well array chip that allows larger-scale isolation and propagation of single cells. Notably, the chip enables single-cell analysis of freshly isolated primary cells at a high-resolution. With an average height of 130 ± 10 μm and an average diameter of 80 ±10 μm, each nano-volume well can hold up to 0.4 nL of volume, and is compatible with both adherent as well as 3D suspension cultures. Simultaneous time-lapse imaging of thousands of nano-volume wells allows to monitor cell division, as well as tracking of cell fate, and/or alterations in the microscopic cellular morphology and/or markers expression. To demonstrate its application, we employed the system for propagating and tracking of cancer stem cells (CSCs). CSCs could be monitored over three consecutive days by time-lapse high-resolution imaging at the single-cell level. We could demonstrate that non-CSCs do not dedifferentiate into CSCs, while CSCs were able to give rise to both CSCs and non-CSCs by undergoing symmetric and asymmetric division, respectively. Altogether, we have developed a novel nano-volume well array chip that significantly ameliorates clonal propagation and high-resolution image analysis of rare cells.Nanomedicine Nanotechnology Biology and Medicine 03/2014; 5(2):191. DOI:10.4172/2157-7439.1000191 · 5.98 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Tissue homeostasis requires the presence of multipotent adult stem cells that are capable of efficient self-renewal and differentiation; some of these have been shown to exist in a dormant, or quiescent, cell cycle state. Such quiescence has been proposed as a fundamental property of hematopoietic stem cells (HSCs) in the adult bone marrow, acting to protect HSCs from functional exhaustion and cellular insults to enable lifelong hematopoietic cell production. Recent studies have demonstrated that HSC quiescence is regulated by a complex network of cell-intrinsic and -extrinsic factors. In addition, detailed single-cell analyses and novel imaging techniques have identified functional heterogeneity within quiescent HSC populations and have begun to delineate the topological organization of quiescent HSCs. Here, we review the current methods available to measure quiescence in HSCs and discuss the roles of HSC quiescence and the various mechanisms by which HSC quiescence is maintained. © 2014. Published by The Company of Biologists Ltd.Development 12/2014; 141(24):4656-4666. DOI:10.1242/dev.106575 · 6.27 Impact Factor