Distinct human α(1,3)-fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells.

In humans, six α(1,3)-fucosyltransferases (α(1,3)-FTs: FT3/FT4/FT5/FT6/FT7/FT9) reportedly fucosylate terminal lactosaminyl glycans yielding Lewis-X (LeX; CD15) and/or sialyl Lewis-X (sLeX; CD15s), structures that play key functions in cell migration, development, and immunity. Prior studies analyzing α(1,3)-FT specificities utilized either purified and/or recombinant enzymes to modify synthetic substrates under nonphysiological reaction conditions or molecular biology approaches wherein α(1,3)-FTs were expressed in mammalian cell lines, notably excluding investigations using primary human cells. Accordingly, although significant insights into α(1,3)-FT catalytic properties have been obtained, uncertainty persists regarding their human LeX/sLeX biosynthetic range across various glycoconjugates. Here, we undertook a comprehensive evaluation of the lactosaminyl product specificities of intracellularly expressed α(1,3)-FTs using a clinically relevant primary human cell type, mesenchymal stem cells. Cells were transfected with modified mRNA encoding each human α(1,3)-FT, and the resultant α(1,3)-fucosylated lactosaminyl glycoconjugates were analyzed using a combination of flow cytometry and MS. The data show that biosynthesis of sLeX is driven by FTs-3, -5, -6, and -7, with FT6 and FT7 having highest potency. FT4 and FT9 dominantly biosynthesize LeX, and, among all FTs, FT6 holds a unique capacity in creating sLeX and LeX determinants across protein and lipid glycoconjugates. Surprisingly, FT4 does not generate sLeX on glycolipids, and neither FT4, FT6, nor FT9 synthesizes the internally fucosylated sialyllactosamine VIM-2 (CD65s). These results unveil the relevant human lactosaminyl glycans created by human α(1,3)-FTs, providing novel insights on how these isoenzymes stereoselectively shape biosynthesis of vital glycoconjugates, thereby biochemically programming human cell migration and tuning human immunologic and developmental processes.

Glycoengineering of E-Selectin Ligands by Intracellular versus Extracellular Fucosylation Differentially Affects Osteotropism of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E-selectin ligands that direct homing of blood-borne cells to bone marrow. Previously, we described a method to engineer E-selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP-Fucose, enforcing transient surface expression of the potent E-selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E-selectin ligands created via FTVI-exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI (FUT6-modRNA) into human MSCs. FTVI-exofucosylation (i.e., extracellular fucosylation) and FUT6-modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E-selectin ligand levels, and shear-based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E-selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E-selectin ligands and also induced a more sustained expression of E-selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly-fucosylated cells compared to extracellularly-fucosylated cells. This study represents the first direct analysis of E-selectin ligand expression programmed on human MSCs by FTVI-mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. Stem Cells 2016;34:2501-2511.

Platelet bioreactor: accelerated evolution of design and manufacture.

Platelets, responsible for clot formation and blood vessel repair, are produced by megakaryocytes in the bone marrow. Platelets are critical for hemostasis and wound healing, and are often provided following surgery, chemotherapy, and major trauma. Despite their importance, platelets today are derived exclusively from human volunteer donors. They have a shelf life of just five days, making platelet shortages common during long weekends, civic holidays, bad weather, and during major emergencies when platelets are needed most. Megakaryocytes in the bone marrow generate platelets by extruding long cytoplasmic extensions called proplatelets through gaps/fenestrations in blood vessels. Proplatelets serve as assembly lines for platelet production by sequentially releasing platelets and large discoid-shaped platelet intermediates called preplatelets into the circulation. Recent advances in platelet bioreactor development have aimed to mimic the key physiological characteristics of bone marrow, including extracellular matrix composition/stiffness, blood vessel architecture comprising tissue-specific microvascular endothelium, and shear stress. Nevertheless, how complex interactions within three-dimensional (3D) microenvironments regulate thrombopoiesis remains poorly understood, and the technical challenges associated with designing and manufacturing biomimetic microfluidic devices are often under-appreciated and under-reported. We have previously reviewed the major cell culture, platelet quality assessment, and regulatory roadblocks that must be overcome to make human platelet production possible for clinical use [1]. This review builds on our previous manuscript by: (1) detailing the historical evolution of platelet bioreactor design to recapitulate native platelet production ex vivo, and (2) identifying the associated challenges that still need to be addressed to further scale and validate these devices for commercial application. While platelets are among the first cells whose ex vivo production is spearheading major engineering advancements in microfluidic design, the resulting discoveries will undoubtedly extend to the production of other human tissues. This work is critical to identify the physiological characteristics of relevant 3D tissue-specific microenvironments that drive cell differentiation and elaborate upon how these are disrupted in disease. This is a burgeoning field whose future will define not only the ex vivo production of platelets and development of targeted therapies for thrombocytopenia, but the promise of regenerative medicine for the next century.

Progress and obstacles towards generating hematopoietic stem cells from pluripotent stem cells.

PURPOSE OF REVIEW: Human pluripotent stem cells (PSCs) have the potential to provide an inexhaustible source of hematopoietic stem cells (HSCs) that could be used in disease modeling and in clinical applications such as transplantation. Although the goal of deriving definitive HSCs from PSCs has not been achieved, recent studies indicate that progress is being made. This review will provide information on the current status of deriving HSCs from PSCs, and will highlight existing challenges and obstacles. RECENT FINDINGS: Recent strides in HSC generation from PSCs has included derivation of developmental intermediates, identification of transcription factors and small molecules that support hematopoietic derivation, and the development of strategies to recapitulate niche-like conditions. SUMMARY: Despite considerable progress in defining the molecular events driving derivation of hematopoietic progenitor cells from PSCs, the generation of robust transplantable HSCs from PSCs remains elusive. We propose that this goal can be facilitated by better understanding of the regulatory pathways governing HSC identity, development of HSC supportive conditions, and examining the marrow homing properties of PSC-derived HSCs.

Cellular barcoding tool for clonal analysis in the hematopoietic system.

Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important.

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.

Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) regenerated in vivo display sustained differences in their self-renewal and differentiation activities. Variations in Steel factor (SF) signaling are known to affect these functions in vitro, but the cellular and molecular mechanisms involved are not understood. To address these issues, we evaluated highly purified HSCs maintained in single-cell serum-free cultures containing 20 ng/mL IL-11 plus 1, 10, or 300 ng/mL SF. Under all conditions, more than 99% of the cells traversed a first cell cycle with similar kinetics. After 8 hours in the 10 or 300 ng/mL SF conditions, the frequency of HSCs remained unchanged. However, in the next 8 hours (ie, 6 hours before any cell divided), HSC integrity was sustained only in the 300 ng/mL SF cultures. The cells in these cultures also contained significantly higher levels of Bmi1, Lnk, and Ezh2 transcripts but not of several other regulators. Assessment of 21 first division progeny pairs further showed that only those generated in 300 ng/mL SF cultures contained HSCs and pairs of progeny with similar differentiation programs were not observed. Thus, SF signaling intensity can directly and coordinately alter the transcription factor profile and long-term repopulating ability of quiescent HSCs before their first division.

Regulation of hematopoietic stem cells by the steel factor/KIT signaling pathway.

Understanding the intrinsic pathways that regulate hematopoietic stem cell (HSC) proliferation and self-renewal responses to external signals offers a rational approach to developing improved strategies for HSC expansion for therapeutic applications. Such studies are also likely to reveal new targets for the treatment of human myeloid malignancies because perturbations of the biological processes that control normal HSC self-renewal divisions are believed to drive the propagation of many of these diseases. Here, we review recent findings that point to the importance of using stringent functional criteria to define HSCs as cells with longterm repopulating activity and evidence that activation of the KIT receptor and many downstream effectors serve as major regulators of changing HSC proliferative and self-renewal behavior during development.

Hematopoietic stem cell aging and self-renewal.

A functional decline of the immune system occurs during organismal aging that is attributable, in large part, to changes in the hematopoietic stem cell (HSC) compartment. In the mouse, several hallmark age-dependent changes in the HSC compartment have been identified, including an increase in HSC numbers, a decrease in homing efficiency, and a myeloid skewing of differentiation potential. Whether these changes are caused by gradual intrinsic changes within individual HSCs or by changes in the cellular composition of the HSC compartment remains unclear. However, of note, many of the aging properties of HSCs are highly dependent on their genetic background. In particular, the widely used C57Bl/6 strain appears to have unique HSC aging characteristics compared with those of other mouse strains. These differences can be exploited by using recombinant inbred strains to further our understanding of the genetic basis for HSC aging. The mechanism(s) responsible for HSC aging have only begun to be elucidated. Recent studies have reported co-ordinated variation in gene expression of HSCs with age, possibly as a result of epigenetic changes. In addition, an accumulation of DNA damage, in concert with an increase in intracellular reactive oxygen species, has been associated with aged HSCs. Nevertheless, whether age-related changes in HSCs are programmed to occur in a certain predictable fashion, or whether they are simply an accumulation of random changes over time remains unclear. Further, whether the genetic dysregulation observed in old HSCs is a cause or an effect of cellular aging is unknown.

Optimizing human Treg immunotherapy by Treg subset selection and E-selectin ligand expression.

While human Tregs hold immense promise for immunotherapy, their biologic variability poses challenges for clinical use. Here, we examined clinically-relevant activities of defined subsets of freshly-isolated and culture-expanded human PBMC-derived Tregs. Unlike highly suppressive but plastic memory Tregs (memTreg), naïve Tregs (nvTreg) exhibited the greatest proliferation, suppressive capacity after stimulation, and Treg lineage fidelity. Yet, unlike memTregs, nvTregs lack Fucosyltransferase VII and display low sLeX expression, with concomitant poor homing capacity. In vitro nvTreg expansion augmented their suppressive function, but did not alter the nvTreg sLeX-l°w glycome. However, exofucosylation of the nvTreg surface yielded high sLeX expression, promoting endothelial adhesion and enhanced inhibition of xenogeneic aGVHD. These data indicate that the immature Treg glycome is under unique regulation and that adult PBMCs can be an ideal source of autologous-derived therapeutic Tregs, provided that subset selection and glycan engineering are engaged to optimize both their immunomodulation and tropism for inflammatory sites.

mRNA-mediated glycoengineering ameliorates deficient homing of human stem cell-derived hematopoietic progenitors.

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.

Isolation and Assessment of Single Long-Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow.

Hematopoietic stem cells with long-term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45(+) EPCR(+) CD150(+) CD48(-) (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision. © 2016 by John Wiley & Sons, Inc.

Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics.

OBJECTIVE: The Hoechst 33342-effluxing side population (SP) of adult mouse bone marrow (BM) contains most of the hematopoietic stem cells (HSCs). Here we measured the HSC content of specific subsets of SP cells and then used a highly HSC-enriched fraction to investigate the effect of different growth factors on the initial rate of HSC proliferation in vitro and the accompanying maintenance (or loss) of HSCs in the first-division progeny. MATERIALS AND METHODS: Staining with Rhodamine-123 (Rho) was used to subfractionate lineage marker-negative (lin-) SP cells. Cells were assayed for HSCs by examining their ability to generate sustained (>4 months) multi-lineage lympho-myeloid clones in irradiated hosts. Cultures of single lin- Rho- SP cells were used to monitor growth factor effects on HSC proliferation and function. RESULTS: More than 40% of mice injected with single lin- Rho- SP cells showed long-term lympho-myeloid reconstitution. Some clones peaked within 8 weeks but others developed more slowly apparently unrelated to the pattern of lineage representation. 3/3 clones tested repopulated secondary mice. Either Steel factor+interleukin-11 (+/- flt3-ligand) or Steel factor+thrombopoietin stimulated at least 75% of single lin- Rho- SP cells to divide in vitro with the same synchronous kinetics. However, in the first cocktail, the frequency of HSCs among the first-division doublets was preserved but in the latter it was greatly diminished. CONCLUSION: Exogenous growth factors can differentially affect the ability of HSCs to execute a self-renewal division within a single cell cycle even when the kinetics of proliferation are the same.

No monkeying around: clonal tracking of stem cells and progenitors in the macaque.

Clonal tracking of hematopoietic stem and progenitor cells (HSPCs) has proven valuable for studying their behavior in murine recipients. Now in Cell Stem Cell, Kim et al. (2014) and Wu et al. (2014) extend these analyses to nonhuman primates, providing insights into dynamics of HSPC expansion and lineage commitment following autologous transplantation.

Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.

Hematopoietic stem cell (HSC) populations change with aging, but the extent to which this is caused by qualitative versus quantitative alterations in HSC subtypes is unclear. Using clonal assays, in this study we show that the aging HSC compartment undergoes both quantitative and qualitative changes. We observed a variable increase of HSC pool size with age, accompanied by the accumulation of predominantly myeloid-biased HSCs that regenerate substantially fewer mature progeny than young myeloid-biased HSCs and exhibit reduced self-renewal activity as measured by long-term secondary transplantation. Old HSCs had a twofold reduction in marrow-homing efficiency and a similar decrease in functional frequency as measured using long-term transplantation assays. Similarly, old HSCs had a twofold reduced seeding efficiency and a significantly delayed proliferative response compared with young HSCs in long-term stromal cell co-cultures but were indistinguishable in suspension cultures. We show that these functional defects are characteristics of most or all old HSCs and are not indicative of a nonfunctional subset of cells that express HSC markers. Furthermore, we demonstrate that cells with functional properties of old HSCs can be generated directly from young HSCs by extended serial transplantation, which is consistent with the possibility that they arise through a process of cellular aging.

Hematopoietic stem cell quiescence: yet another role for p53.

p53, sometimes referred to as the "guardian of the genome," helps regulate cell-cycle arrest, DNA-damage repair, apoptosis, and senescence. Adding to this list, in this issue of Cell Stem Cell, Liu et al. (2009) show that p53 also plays a role in regulating hematopoietic stem cell quiescence.

Characterization of mouse hematopoietic stem and progenitor cells.

The unit describes functional assays for the quantification of mouse hematopoietic stem cells and progenitor cells. The competitive repopulating unit (CRU) assay detects transplantable mouse hematopoietic stem cells with the capacity to regenerate all of the blood cell lineages for extended time periods in vivo. The long-term culture-initiating cell (LTC-IC) assay, founded on the bone marrow long-term culture system, measures primitive hematopoietic progenitors based on their capacity to produce myeloid progeny for at least four weeks. Colony-forming cell (CFC) assays, performed in semisolid medium cultures to assess mouse pre-B, megakaryocyte, erythroid, granulocyte-monocyte, and multipotential hematopoietic progenitors are also described. These assays are powerful tools for evaluating human stem cell (HSC) and progenitor content in various hematopoietic tissues, during development as well as in the adult animal, and in cell populations manipulated ex vivo.

Combining transcriptional profiling and genetic linkage analysis to uncover gene networks operating in hematopoietic stem cells and their progeny.

Stem cells are unique in that they possess both the capacity to self-renew and thereby maintain their original pool as well as the capacity to differentiate into mature cells. In the past number of years, transcriptional profiling of enriched stem cell populations has been extensively performed in an attempt to identify a universal stem cell gene expression signature. While stem-cell-specific transcripts were identified in each case, this approach has thus far been insufficient to identify a universal group of core "stemness" genes ultimately responsible for self-renewal and multipotency. Similarly, in the hematopoietic system, comparisons of transcriptional profiles between different hematopoietic cell stages have had limited success in revealing core genes ultimately responsible for the initiation of differentiation and lineage specification. Here, we propose that the combined use of transcriptional profiling and genetic linkage analysis, an approach called "genetical genomics", can be a valuable tool to assist in the identification of genes and gene networks that specify "stemness" and cell fate decisions. We review past studies of hematopoietic cells that utilized transcriptional profiling and/or genetic linkage analysis, and discuss several potential future applications of genetical genomics.

Isolation and assessment of long-term reconstituting hematopoietic stem cells from adult mouse bone marrow.

Suspensions of multipotent hematopoietic stem cells with long-term repopulating activity can now be routinely isolated from adult mouse bone marrow at purities of 30%. A robust method for obtaining these cells in a single step using multiparameter cell sorting to isolate the CD45(mid)lin(-)Rho(-)SP subset is described here, together with a detailed protocol for assessing their regenerative activity in mice transplanted with single cells. These procedures provide unprecedented power and precision for characterizing the molecular and biological properties of cells with hematopoietic stem cell activity at the single cell level.

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.