The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

Mouse haematopoietic stem cells (HSCs) undergo a postnatal transition in several properties, including a marked reduction in their self-renewal activity. We now show that the developmentally timed change in this key function of HSCs is associated with their decreased expression of Lin28b and an accompanying increase in their let-7 microRNA levels. Lentivirus-mediated overexpression of Lin28 in adult HSCs elevates their self-renewal activity in transplanted irradiated hosts, as does overexpression of Hmga2, a well-established let-7 target that is upregulated in fetal HSCs. Conversely, HSCs from fetal Hmga2(-/-) mice do not exhibit the heightened self-renewal activity that is characteristic of wild-type fetal HSCs. Interestingly, overexpression of Hmga2 in adult HSCs does not mimic the ability of elevated Lin28 to activate a fetal lymphoid differentiation program. Thus, Lin28b may act as a master regulator of developmentally timed changes in HSC programs with Hmga2 serving as its specific downstream modulator of HSC self-renewal potential.

Summary Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in the adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.

i HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV's structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies. HIV-1 is notorious for its genetic diversity and its ability to adapt to selection pressures (44,87,116). Despite this, within-host HIV-1 evolution in response to antiretroviral (61, 72), host cellular immune (15,50,71,94,95), antibody (46), and vaccine-induced (103) selection pressures occurs along generally predictable mutational pathways (3, 84). Studying these evolutionary pathways can offer insight into the immunopathogenesis of HIV-1 and may help inform the design of immune-based interventions and vaccines.Substantial progress has been made in our understanding of HIV-1's ability to evade human leukocyte antigen (HLA) class I-restricted CD8 ϩ cytotoxic T-lymphocytes (CTL). In particular, application of novel statistical methods (13,25,84) to large population-based data sets of linked host and viral genetic information has facilitated the systematic identification of HLA-associated immune escape and covarying mutations in 20,60,81,99,104), revealing important insights into HIV-1 adaptation to its host. We now appreciate that immune selection represents a major force shaping HIV-1 diversity (3,80,...

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 normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors.

SummaryIncreasing evidence of functional and transcriptional heterogeneity in phenotypically similar cells examined individually has prompted interest in obtaining parallel methylome data. We describe the development and application of such a protocol to index-sorted murine and human hematopoietic cells that are highly enriched in their content of functionally defined stem cells. Utilizing an optimized single-cell bisulfite sequencing protocol, we obtained quantitative DNA methylation measurements of up to 5.7 million CpGs in single hematopoietic cells. In parallel, we developed an analytical strategy (PDclust) to define single-cell DNA methylation states through pairwise comparisons of single-CpG methylation measurements. PDclust revealed that a single-cell epigenetic state can be described by a small (<1%) stochastically sampled fraction of CpGs and that these states are reflective of cell identity and state. Using relationships revealed by PDclust, we derive near complete methylomes for epigenetically distinct subpopulations of hematopoietic cells enriched for functional stem cell content.

By comparing fetal and adult B-lymphopoiesis, the authors identify a prepro–B-cell subset in humans that marks the origin of B-cell lineage commitment in utero.