David G. Kent

Long-Term Propagation of Distinct Hematopoietic Differentiation Programs In Vivo

Heterogeneity in the differentiation behavior of hematopoietic stem cells is well documented but poorly understood. To investigate this question at a clonal level, we isolated a subpopulation of adult mouse bone marrow that is highly enriched for multilineage in vivo repopulating cells and transplanted these as single cells, or their short-term clonal progeny generated in vitro, into 352 recipients. Of the mice, 93 showed a donor-derived contribution to the circulating white blood cells for at least 4 months in one of four distinct patterns. Serial transplantation experiments indicated that two of the patterns were associated with extensive self-renewal of the original cell transplanted. However, within 4 days in vitro, the repopulation patterns subsequently obtained in vivo shifted in a clone-specific fashion to those with less myeloid contribution. Thus, primitive hematopoietic cells can maintain distinct repopulation properties upon serial transplantation in vivo, although these properties can also alter rapidly in vitro.

Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect

The regulation of HSC proliferation and engraftment of the BM is an important but poorly understood process, particularly during ontogeny. Here we show that in mice, all HSCs are cycling until 3 weeks after birth. Then, within 1 week, most became quiescent. Prior to 4 weeks of age, the proliferating HSCs with long-term multilineage repopulating activity displayed an engraftment defect when transiting S/G2/M. During these cell cycle phases, their expression of CXC chemokine ligand 12 (CXCL12; also referred to as stromal cell-derived factor 1 [SDF-1]) transiently increased. The defective engrafting activity of HSCs in S/G2/M was reversed when cells were allowed to progress into G1 prior to injection or when the hosts (but not the cells) were pretreated with a CXCL12 antagonist. Interestingly, the enhancing effect of CXCL12 antagonist pretreatment was exclusive to transplants of long-term multilineage repopulating HSCs in S/G2/M. These results demonstrate what we believe to be a new HSC regulatory checkpoint during development. They also suggest an ability of HSCs to express CXCL12 in a fashion that changes with cell cycle progression and is associated with a defective engraftment that can be overcome by in vivo administration of a CXCL12 antagonist.

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays

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.

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

Hematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150(+) subset of the EPCR(+)CD48(-)CD45(+) fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150(-) subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.

Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties

Hematopoietic stem cells (HSCs) execute self-renewal divisions throughout fetal and adult life, although some of their properties do alter. Here we analyzed the magnitude and timing of changes in the self-renewal properties and differentiated cell outputs of transplanted HSCs obtained from different sources during development. We also assessed the expression of several “stem cell” genes in corresponding populations of highly purified HSCs. Fetal and adult HSCs displayed marked differences in their self-renewal, differentiated cell output, and gene expression properties, with persistence of a fetal phenotype until 3 weeks after birth. Then, 1 week later, the HSCs became functionally indistinguishable from adult HSCs. The same schedule of changes in HSC properties occurred when HSCs from fetal or 3-week-old donors were transplanted into adult recipients. These findings point to the existence of a previously unrecognized, intrinsically regulated master switch that effects a developmental change in key HSC properties.

The Lin28b–let-7–Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells

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.

Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.

Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations

Summary Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

Comprehensive microRNA expression profiling of the hematopoietic hierarchy

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.