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 ng͞ml Steel factor, 20 ng͞ml IL-11, and 1 ng͞ml 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 HSCcontaining 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.video microscopy ͉ time-lapse imaging ͉ cell-cycle kinetics ͉ cell behavior ͉ lineage tracking T ime-lapse video imaging offers unique opportunities to determine how specific physical properties of individual living cells change with respect to one another over time and under different conditions. Time-lapse micrography has been used for more than half a century (1-4) to study cell morphology during attachment and migration (5, 6), cell lifetimes (7, 8), growth (9), death (2, 10), contact inhibition (11), clonal heterogeneity (12), and mitosis (13). Software for extracting and analyzing cell lineage (14) Here, we asked whether time-lapse video imaging could be used to identify previously unidentified behavioral traits of hematopoietic stem cells (HSCs) with functionally validated long-term multilineage repopulating activity in vivo. A number of groups have reported methods for obtaining highly purified (Ͼ20% pure) populations of HSCs from normal adult mouse bone marrow (23-28). One of these methods involves isolating cells lacking surface markers characteristic of mature blood cells (i.e., lineage marker-negative, or lin Ϫ cells) and able to efflux the fluorescent dyes, Rhodamine-123 (Rho Ϫ cells) and Hoechst 33342 (25). Efflux of Hoechst 33342 results in the appearance of a side population of cells (SP cells) in two-dimensional plots of fluorescent events (29). In mouse bone marrow (BM), the subset of lin Ϫ Rho Ϫ SP cells represents Ϸ0.004...
Transgene variegation is caused by epigenetic switching between expressing and silent states. gamma-retrovirus vectors can be variegated in stem cells, but the dynamics of epigenetic remodeling during transgene variegation are unknown. Here, we measured variegated enhanced green fluorescent protein gamma-retrovirus expression over 4 days in individual embryonic stem cells while tracking cells in order to create expression lineage trees: 56 colony founder cells and their progeny were tracked over seven generations. Nineteen lineages produced synchronized inheritable trajectories of transgene silencing or reactivation, indicative of epigenetic remodeling with long-term stable inheritance. Short-term fluctuations in fluorescence intensity were also observed, which contributed low-amplitude variation to transgene expression level. These two processes have different frequencies and inheritability, but together contribute to variegated transgene expression. Inhibition of DNA methylation with 5-azacytidine eliminated long-term transgene silencing over 4 days, but short-term fluctuations continued. Our approach applies real-time imaging technology to track the long-term dynamics of transgene expression to investigate the timing and expression patterns leading to variegation.
Background: There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo‐like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates “lineage informatics,” a lineage‐centric approach to cytomics. Methods: We cultured cells in a confined monolayer using a novel “gap chamber” that produces images with confocal‐like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics. Results: Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo‐like than traditional culture systems. Conclusions: The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long‐term cell imaging. © 2006 International Society for Analytical Cytology
Recently described strategies for isolating nearly pure populations of hematopoietic stem cells (HSCs) from normal adult mouse bone marrow (BM) offer new opportunities to identify previously unrecognized properties of HSCs Unfortunately, most phenotypic markers thus far found to characterize HSCs are not tightly linked to the functional potential of these cells but, instead, vary independently when their activation status is altered. Here we describe the results of experiments in which single CD45+Lin-Rho-SP adult mouse BM cells were micromanipulated into microwells of a specially designed silicone array and were monitored in real time (every 3 min) for a total of 4 days by high resolution digital time lapse photography to track the behavior of each cell and its clonal progeny. During this time, the cells were maintained at 37°C in a feeder-free, serum-free medium supplemented with 300 ng/mL SF, 20 ng/mL IL-11, and 1ng/mL Flt3-L. Under these conditions, 64 of 66 input cells divided at least once, and 63 divided at least twice. Only 2/715 cells tracked died. Final clones varied in size from 1-92 cells, corresponding to 0–7 cell cycles. The initial division occurred after 41±12 hr, and the 2nd and 3rd divisions another 18±5 and 16±4 hr later. Synchrony of sister cell divisions within clones and the presence of cells displaying uropodia (lagging protrusions) or lamellipodia (leading protrusions) were common. HSC activity in 83 initial CD45+Lin-Rho-SP cells or the 66 derived clones was assessed by transplanting these individually into sublethally irradiated Ly5-congenic W41/W41 recipients, which were then analyzed for longterm, multilineage, donor-derived WBCs (>1% @ 16 wk). 33% (27/83), of the initial cells were HSCs (producing 1–84% of WBCs @ 16 wk) and 27% (18/66) of the 4-day clones had HSC activity (2–83% of WBCs @ 16 wk), showing that many input HSCs had executed self-renewal divisions. Cells in clones containing HSCs had longer 1st, 2nd, and 3rd cycle times compared to cells within non-repopulating clones (5.4±2.3 hr, 3.7±1.0 hr, and 2.8±0.6 hr longer, respectively, p<.02 for each) and the cumulative time to the 3rd division was on average, 12±2 hr longer (p<.001) for cells in clones with HSCs. Similarly, clones containing HSCs underwent fewer divisions overall (3.2±0.2 vs 4.2±0.2, p=.003) and were therefore smaller (10±1 vs 26±3 cells, p<.001). All 24 clones in which >50% of cells had a cumulative time to their 3rd division of ≤ 65.3 hr (ie, < mean - 0.5 SD) lacked HSC activity. However, neither of the 2 cells that did not divide in the 4-day period were HSCs. The 22 clones in which all cells displayed uropodia within the final 12 hr of culture also lacked HSCs. Combined, these parameters allowed the 18 HSC-containing clones to be identified in a subset of 30 out of the original 66; ie, 2x more frequently. To test the validity of these parameters for predicting HSC-containing clones, a 2nd experiment was performed and similarly analyzed. In this case, these parameters identified 5 HSC-containing clones in a subset of 34 out of an original total of 76, again a 2-fold increase. These studies illustrate the potential of this novel monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal events.
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