Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.RT-qPCR | stem cell | hematopoiesis | microfluidic | single cell

Objective-Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect.Methods-To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10-to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay).Results-These experiments revealed remarkable expansions of Nucleoporin98-Homeoboxtransduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion.Conclusion-These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.The self-renewal function of hematopoietic stem cells (HSCs) is essential to their ability to sustain lifelong hematopoiesis and to regenerate the hematopoietic system after myeloablative treatments. This property is the basis of an increasing range of applications of HSC transplants to treat various malignant and genetic disorders [1,2]. Further improvements in the safety and therapeutic utility of HSC transplants can be readily envisaged if robust methods for largescale ex vivo expansion of HSCs were available. For example, the low absolute numbers of HSCs in most cord blood samples [3] [4] restrict the clinical utility of these products. A method for significantly expanding HSCs could not only address these insufficiencies but also broaden the exploitation of reduced conditioning regimens and associated therapeutic benefits.One approach that has allowed some HSC expansion in vitro to be achieved has focused on the identification of optimized combinations and concentrations of externally acting growth factors and related molecules [5][6][7][8][9][10][11][12]. A complementary approach has been to identify intrinsic regulators such as transcription factors [13] and key mediators of signaling pathways [14,15] that can be manipulated to activate or promote HSC self-renewal divisions. A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numb...

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