Stem Cell Traits In Long-term Co-culture Revealed By Time-lapse Imaging

Background:Physiological stem cells in vivo are able to undergo different fate choices includingmigration, self-renewal, differentiation, apoptosis and quiescence under specificconditions in which the stem cell niche is believed to serve as one of the central elements.Advances in the isolation of highly purified populations of rare hematopoietic stem cells(HSC) and hematopoietic progenitor cells (HPC) from bone marrow provideopportunities to better understand stem cell traits and to seek improvements for their useas therapeutic agents. But once these valued cells are isolated, relevant in vitro modelsare needed for testing features and determinants of normal stem cell function. A good invitro model should support and permit a quantitative analysis for the different stem cellbehaviors and outcomes at single cell level. Such a model would be useful for evaluatingvariables that control these functions and for assisting the development of techniques forexpansion of HSCs with specific therapeutic purposes.Mixtures of currently known hematopoietic cytokines do not maintain the long-term invivo repopulating ability of HSCs in liquid culture for longer than days. In contrast, as anearly demonstration of the importance of the microenvironment for HSC preservation,co-cultures of hematopoietic cells and primary bone marrow stromal cells without addedcytokines have been shown to preserve repopulating HSCs for periods of up to severalweeks in vitro. Thus, the co-culture system appears to at least partially recreate thehypothesized "stem cell niche" that constitutes a specialized micro-environmentinvolving cell-cell interactions that control stem cell numbers and function in vivo.Crucial cell-cell interactions are only poorly understood, so the co-culture system canprovide a useful model for investigating stem cell-stromal cell interactions, and it mayalso serve as a platform upon which to build a better understanding of essentialcomponents of the stem cell niche, particularly when combined with time-lapse imagingand tracking at single cell resolution. The cobblestone areas (CAs) are regions of hematopoietic cell clusters that proliferate beneath the stromal cell mantle and have beendemonstrated to be associated with the activities of both HSC and HPC, dependent uponthe specific time point of the readout. Their long-term (＞4weeks) presence of CA in coculturesforms the basis of a classical in vitro surrogate for HSCs. CAs are oftensurrounded by colony cells on top of the stroma (COS), which are thought to be thedifferentiating progeny of CA, although direct evidence for this association is still lacking.Therefore, kinetics of CA and COS in the long-term co-culture system in partrecapitulates the hematopoietic regeneration in vivo.For high content analysis of small numbers of rare cell types, time lapse imaging hasbecome an important tool that continues to come of age with improvements in hardwareautomation, software, data storage/retrieval, and computer speed and power. Target cellscan be continuously tracked at single cell level and multiple parameters can be monitoredat high resolution over time while cells retain the capacity for further functional ormolecular analysis. Time-lapse analysis of HSCs in long-term co-culture has not beenwell documented in detail, due to many problems such as photo-toxicity by long-termfrequent fluorescence imaging and difficulty of tracking the desired input cells in thepresence of overwhelming numbers of stromal cells.Objective: In this study, we attempted to overcome the problems associated with timelapseanalysis of HSCs in long-term co-culture by improving the imaging and trackingstrategy and software using the well-defined HSC and HPC subsets from GFP-transgenicmice. We sought to determine whether traits such as quiescence, transmigration,division and differentiation occurred differently between hematopoietic precursors withdifferent phenotypes and we hypothesized that such traits would be more evident usingthe stromal co-culture as opposed to the studies using liquid culture. A deeperunderstanding of stem cell-niche engagement and subsequent behaviors would beenhanced by technologies enabling the tracking of individual stem cells at the clonal levelin the classical long-term co-culture (LTC) which mimics the complexity of the bonemarrow microenvironment in vivo and may support self-renew potential of stem cells.Materials and Methods: 1. Well characterized subsets of murine HSCs were sorted using a MoFlo High-SpeedCell Sorter and multi-color flow cytometric analysis based upon positive and negativeCD34 expression among the lineage~-, c-Kit~+ and Sca-1~+ (LKS) bone marrow cells. Thelineage~-, c-Kit~+ and Sca-1~- (LKS~-) progenitor populations were also included forcomparing to the two primitive HSC types. Prior to performing time-lapse analysis, wevalidated the subsets using standard in vivo and in vitro functional assays. Multi-lineagedifferentiation potential was determined as the percentage of single cells (n=180 for eachsubset) that gave rise to all four morphological phenotypes: neutrophils,monocyte/macrophage, erythroid, and megakaryoctye after approximately two weeks inliquid culture. In vivo competitive repopulation was compared between CD34~-LKS (50cells/mouse) and CD34~+LKS (300 cells/mouse) hematopoietic stem cells sorted fromB6/SJL (CD45.1) mice and injected through tail vein with bone marrow fromCD45.1/CD45.2 mice as competitors into lethally irradiated C57BL/6 (CD45.2) mice asrecipients. Peripheral blood was analyzed as % chimerism by flow cytometry at 3 weeks,12 weeks, and 5 months after transplantation (n=5 recipient mice).2. Bone marrow nucleated cells with limiting dilution assay were cultured onto primarystromal cells (PS), AFT-024 (AFT) or osteoblastic cell line (OB) for 5 weeks. All thestromal cells were plated at 100% confluence and irradiated with 15Gy before use.CAFCs were scored at 5 weeks to compare the yield on different sources of stromal cells.3. To document the relation between the current state-of-the-art immunophenotypes ofhematopoietic stern/progenitor cells and LTC, the classical in vitro surrogate assay,sorted hematopoietic subsets were serially diluted and seeded onto primary stromallayers. Two cell types with different morphologies, cobblestone area (CA) and colony onstroma (COS), were scored every week for up to 6 weeks.4. The sorted hematopoietic subsets from green fluorescent protein (GFP) transgenicmice were cultured onto the prepared primary stromal layers in 24-well plate and imagedreal-time for more than 8 weeks. Multiple target cells in the plate were memorized in thesystem and revisited every 10 minutes. Exposure of cells and associated photo-toxicityfrom fluorescence excitation were minimized by automating and synchronizing lamp andcamera shutters and further reduced while at the same time sufficient images producedfor confirming and re-establishing tracks over extended time periods by acquiring onlyone fluorescent image set (at 100 minute intervals) for every 10 bright-field images taken at 10-minute intervals. Composites of adjacent images in rectangular mosaics were usedfor global viewing or precise tracking of individual cells moving across viewfields.Results:1. In the single cell culture and differentiation assay, more than 30% of CD34~-LKS werecapable of differentiation into 4 myeloid lineages compared with less than 10% ofCD34~+LKS and almost none of LKS~- cells. A chi-square test was used to determine thestatistical significance among the three groups (~*p＜0.001). Competitive repopulation inlethally irradiated mice demonstrated sustained long-term engraftment by 50 CD34~-LKS~+at over 15% peripheral blood chimerism after 21 weeks and even higher in the earlystage, whereas engraftment by 300 CD34~+LKS~+ resulted in declining chimerism toapproximately 1% after 21 weeks.2. In long-term co-culture system, the CAFC yield on PS was the highest (1/1.2×10~5)compared with AFF024 (1/2.7×10~5) and osteoblastic cell line (1/2×10~6).3. Dynamics of cobblestone area formation in the LTC system with primary bone marrowstromal cells were highly correlated to the primitiveness of the subsets. The long-term (＞4weeks) presence of CA in co-cultures forms the basis of a classical in vitro surrogatefor LT-HSCs and likewise, CAs appearing at early stage (week 1 to 2) and middle stage(week 3 to 4) reflect the activities of myeloid HPCs and ST-HSCs.4. (1) The transition from CA under the stromal cell mantle into dispersed migrating cellson top of the stroma (COS) were directly observed using time-lapse imaging. The HSCswere able to initiate multiple waves, rather than a durable single wave as observed in thetraditional LTC, of CA formation and COS expansion beyond 2 and 4 weeks,respectively, but HPC expansion was not evident beyond the second week, despiteindistinguishable morphology of CA between the cell subtypes. CAs can merge, splitand flow into adjacent areas in a fluid-like manner, exhibiting an intercellular attractionthat keeps them clustered despite their migratory activity. (2) On average the proportionof time spent beneath the stroma was greatest for the LT-HSC cell type and intermediatebetween LT-HSC and HPC for the ST-HSC cell type (p＜0.05). (3) Retrospectivetracking of individual CAFCs revealed a longer interval (37.8 hours) on average beforefirst division for LT-HSC compared with CAFC from ST-HSC and HPC populations (31.9 and 24 hours respectively). But by the 2nd generation, there were no significantdifferences in inter-division times between cell types.(4) We did not find evidence for greater heterogeneity between sisters among the LT-HSCcells in comparison with ST-HSC or HPC subsets (standard deviations for sisterpairs were 0.76 hours, 0.65 hours, and 0.75 hours, respectively).Conclusions:1. On the basis of these functional validations, we refer to the CD34~-LKS subset as "long-term"repopulating HSC (LT-HSC), the CD34~+LKS subset as "short-term" repopulatingHSC (ST-HSC) and the LKS~- subset as HPC throughout the remainder of thismanuscript.2. In long-term co-culture system, CAFC yield was dependent on the source of stromalcells.3. All three populations on primary stroma in LTC formed unimodal curves of CA, butthe time of apprearing and peak were different. COS had a similar kinetics to CA.4. Our study demonstrates for the first time that the long-term hematopoietic co-culturesystem can be monitored for more than 5 weeks in high spatial and temporal resolutionwith the time-lapse imaging system. We compared multiple parameters of the behaviorsof individual GFP+ cells from the different subsets of HSCs and HPCs. We analyzeddivision times and tracked transmigration behavior of individual cells that gave rise toCAs, and then followed the kinetics of CA formation through the stage of transition fromCA to COS. Moreover, we were able to monitor the CA activity for more than 5 weeksin order to correlate it with the immuno-phenotypes of HSC and HPC as defined with thein vivo model.5. Real-time imaging provided visualization of the process from single cell to colony.The kinetics of late CA expansion, which correlates with in vivo self-renewal potential ofHSC, as well as multiple variables such as transmigration, mitotic quiescence, anddifferentiation can be semi-quantitatively measured with our in vitro system. Our studyrepresents an important starting point from which the long-term co-culture system can beaugmented to provide a better in vitro model for bone marrow stem cell niches.