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The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O -glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O -glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus. [Display omitted] • The MUC2 mucin CysD2 core structure forms a stable stalk resistant to intestinal proteases • Three flexible surface loops are exposed outside of glycosylated domains • CysD2 forms transient pH-dependent non-covalent dimers • The dimers are stabilized by transglutaminase-catalyzed covalent cross-links in colon mucus Recktenwald et al. characterize the structure of the second CysD domain of the MUC2 mucin using NMR spectroscopy. The domain shows weak homotypic interactions at physiological pH that can be stabilized by transglutaminase 3 forming isopeptide-bond stabilized dimers in the mucus of the large intestine. [ABSTRACT FROM AUTHOR]

It is challenging to measure dietary exposure with techniques that are both accurate and applicable to free-living individuals. We performed a cross-over intervention, with 24 healthy individuals, to capture the acute metabolic response of a cereal breakfast (CB) and an egg and ham breakfast (EHB). Fasting and postprandial urine samples were analyzed using 1 H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Metabolic profiles were distinguished in relation to ingestion of either CB or EHB. Phosphocreatine/creatine and citrate were identified at higher concentrations after consumption of EHB. Beverage consumption (i.e., tea or coffee) could clearly be seen in the data. 2-furoylglycine and 5-hydroxymethyl-2-furoic acid – potential biomarkers for coffee consumption were identified at higher concentrations in coffee drinkers. Thus 1 H NMR urine metabolomics is applicable in the characterization of acute metabolic fingerprints from meal consumption and in the identification of metabolites that may serve as potential biomarkers. [ABSTRACT FROM AUTHOR]

Summary During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34 + cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context. [ABSTRACT FROM AUTHOR]

Aging negatively affects hematopoiesis, with consequences for immunity and acquired blood cell disorders. Although impairments in hematopoietic stem cell (HSC) function contribute to this, the in vivo dynamics of such changes remain obscure. Here, we integrate extensive longitudinal functional assessments of HSC-specific lineage tracing with single-cell transcriptome and epitope profiling. In contrast to recent suggestions from single-cell RNA sequencing alone, our data favor a defined structure of HSC/progenitor differentiation that deviates substantially from HSC-derived hematopoiesis following transplantation. Native age-dependent attrition in HSC differentiation manifests as drastically reduced lymphoid output through an early lymphoid-primed progenitor (MPP Ly-I). While in vitro activation fails to rescue lymphoid differentiation from most aged HSCs, robust lymphopoiesis can be achieved by culturing elevated numbers of candidate HSCs. Therefore, our data position rare chronologically aged HSC clones, fully competent at producing lymphoid offspring, as a prime target for approaches aimed to improve lymphopoiesis in the elderly. [Display omitted] • Hematopoietic differentiation proceeds through obligatory intermediate cell stages • MPP Ly-I represents the earliest stage toward lymphoid-lineage commitment • Most aged HSCs fail to differentiate into MPP Ly-I and Ly-II subsets • Rare multilineage/lymphoid-competent aged HSCs can be maintained in vitro Konturek-Ciesla et al. integrate in vivo lineage tracing and single-cell profiling to native HSC-derived hematopoiesis during aging. They reveal age-dependent reductions in HSC contribution to multilineage hematopoiesis, with progressive loss of lymphoid-oriented MPP Ly-I cells. Lymphoid-competent aged HSC clones, although rare, can be expanded in vitro. [ABSTRACT FROM AUTHOR]

Summary Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1 , Tal1 , Lmo2 , and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development. [ABSTRACT FROM AUTHOR]

Summary: Prospective isolation is critical for understanding the cellular and molecular aspects of stem cell heterogeneity. Here, we identify the cell surface antigen CD9 as a positive marker that provides a simple alternative for hematopoietic stem cell isolation at high purity. Crucially, CD9 affords the capture of all hematopoietic stem cells in murine bone marrow in the absence of contaminating populations that lack authentic stem cell function. Using CD9 as a tool to subdivide hematopoietic stem-cell-containing populations, we provide evidence for heterogeneity at the cellular, functional, and molecular levels. [Copyright &y& Elsevier]

The emerging notion of hematopoietic stem and progenitor cells (HSPCs) as a low-primed cloud without sharply demarcated gene expression programs raises the question on how cellular-fate options emerge and at which stem-like stage lineage priming is initiated. Here, we investigate single-cell chromatin accessibility of Lineage−, cKit+, and Sca1+ (LSK) HSPCs spanning the early differentiation landscape. Application of a signal-processing algorithm to detect transition points corresponding to massive alterations in accessibility of 571 transcription factor motifs reveals a population of LSK FMS-like tyrosine kinase 3 (Flt3)intCD9high cells that concurrently display stem-like and lineage-affiliated chromatin signatures, pointing to a simultaneous gain of both lympho-myeloid and megakaryocyte-erythroid programs. Molecularly and functionally, these cells position between stem cells and committed progenitors and display multi-lineage capacity in vitro and in vivo but lack self-renewal activity. This integrative molecular analysis resolves the heterogeneity of cells along hematopoietic differentiation and permits investigation of chromatin-mediated transition between multipotency and lineage restriction. [Display omitted] • Distinct chromatin landscapes separate hematopoietic stem and progenitor populations • LSKFlt3intCD9high cells display concurrent stem- and multi-lineage chromatin • LSKFlt3intCD9high cells bridge multipotency and lineage restriction Safi et al. reveal a unique hematopoietic transition state, displaying concurrent stem- and multi-lineage chromatin-accessibility signatures captured by LSKFlt3intCD9high immunophenotype. LSKFlt3intCD9high cells represent extensively primed progenitors lacking self-renewal but bridging multipotency and lineage restriction. These findings permit analysis of how self-renewal and multipotency are lost in favor of lineage commitment. [ABSTRACT FROM AUTHOR]

Abstract: Optically clear dispersions of dioctadecyldimethylammonium bromide and chloride (DODAX, X=Br−, Cl−) in water can be obtained by simply mixing the amphiphiles at low concentrations (1mM) and at a temperature safely above the gel to liquid crystalline phase transition temperature (T m ≈45–48°C) of DODAX in water. Under these conditions, dynamic light scattering shows that, at room temperature, the dispersions contain two well-defined populations of large vesicles with average hydrodynamic radii (R H) of 80 and 337nm for DODAB and of 69 and 247nm for DODAC. Cryo-transmission electron microscopy (cryo-TEM) micrographs show that DODAX vesicles are unilamellar and polydisperse with apparent radius up to 800nm. The vesicles are stable for at least 1 month according to the ageing time-dependence of the turbidity and molar absorption coefficient. [Copyright &y& Elsevier]

Background & Aims: Biallelic mutations in the base-excision repair gene MYH have recently been associated with recessive inheritance of multiple colorectal adenomas. An investigation and characterization of MYH mutations in Swedish patients were therefore carried out. Methods: A set of 15 unrelated adenomatous polyposis coli (APC)–mutation negative patients from the Swedish Polyposis Registry was screened for germline mutations in the MYH gene. The patients were clinically characterized and compared with 43 APC-mutation positive probands diagnosed during the same period. Results: Disease-causing biallelic MYH mutations were identified in 6 patients (40%). The mean age at diagnosis was 47.8 years versus 34.1 years in APC-mutation positive patients (P = .015). Colorectal cancer at diagnosis of polyposis was present in 67% (4/6) of the patients, and all were right-sided, compared with only 19% versus 12.5% right-sided cancer in APC-mutation positive patients. Upper gastrointestinal manifestations were diagnosed in 1 of 5 compared with 23 of 27 in APC-mutation positive patients (odds ratio, 23; 95% confidence interval, 2–263; P = .0086). One family exhibited apparent dominant inheritance of colorectal adenomatous polyposis. Two new pathogenic mutations, MYH p.G175E and p.P391L, were identified. The mutations are argued to introduce profound changes in substrate-recognizing domains of the protein. Conclusions: Biallelic MYH mutations, including 2 novel mutations, were found in a substantial number of the patients with multiple colorectal adenomas who were negative for APC-mutation. The examined MYH-mutation positive patients were found to have higher risks of colorectal cancer at diagnosis, right-sided location of cancers, and a significantly lower incidence of upper gastrointestinal manifestations, compared with APC-mutation positive patients. [Copyright &y& Elsevier]

Analyzes the cost-effectiveness and capital costs of economic evaluations. Assumptions on constant returns to scale; Role of cost-effectiveness in decision-making; Estimation of cost function.

Quinoa is a pseudocereal grown in the Andean region of South America that is of increasing interest worldwide as an alternative staple food. We have detected a complex mixture of both odd- and even-alkyl chain alkylresorcinols (AR), branched-chain alkylresorcinols (bcAR) and methylalkylresorcinols (mAR) in ethyl acetate extracts of quinoa. We quantified the content of AR in 17 commercial samples of quinoa, and found that the mean ± SD content of AR was 58 ± 16 μg/g, bcAR was 182 ± 52 μg/g, and mAR was 136 ± 40 μg/g. AR from quinoa could also be detected in plasma after eating quinoa, indicating that some of these unique AR could be used as biomarkers of quinoa intake in humans. Further work is required to understand the role of these ARs in the quinoa plant and whether any of the novel ARs may be of particular interest in human nutrition. [ABSTRACT FROM AUTHOR]

Abstract: N,N,N-Trialkylammonioundecahydrododecaborates (1-), a novel class of compounds of interest for use as anions in ionic liquids, interact with DPPC liposomes. Increasing compound concentration causes an increasing negative ζ potential. Dissociation constants demonstrate that the binding capacity increases strongly with longer chain length. N,N,N-Trialkylammonioundecahydrododecaborates with longer alkyl chains show a detergent-like behavior: the compounds incorporate into the liposome membrane and differential scanning calorimetric experiment show already low concentrations cause a complete disappearance of the peak representing the gel-to-liquid crystalline phase transition. In contrast, compounds with shorter alkyl chains only interact with the headgroups of the lipids. Investigations by means of cryo-TEM reveal that all derivatives induce significant morphological changes of the liposomes. N,N,N-Trialkylammonioundecahydrododecaborates with short alkyl chains produce large bilayer sheets, whereas those with longer alkyl chains tend to induce the formation of open or multi-layered liposomes. We propose that the binding of N,N,N-trialkylammonioundecahydrododecaborates is mainly due to electrostatic interactions between the doubly negatively charged cluster unit and the positively charged choline headgroup; the positively charged ammonium group might be in contact with the deeper-lying negatively charged phosphate. For N,N,N-trialkylammonioundecahydrododecaborates with longer alkyl chains hydrophobic interactions with the non-polar hydrocarbon part of the membrane constitute an additional important driving force for the association of the compounds to the lipid bilayer. [Copyright &y& Elsevier]

Abstract: Thrombin was prepared from human blood plasma (batch size 1200 L). First, prothrombin was isolated by the following separation techniques: cryoprecipitation, ion-exchange chromatography (diethyl aminoethyl, DEAE-IEX), heparin affinity chromatography, a second DEAE-IEX step, and immobilized metal-affinity chromatography (IMAC). Prothrombin was then activated to thrombin, which was purified by hydrophobic interaction chromatography (HIC) and concentrated by ultrafiltration. This process is cost-effective because a waste fraction can be used from one of the steps (heparin affinity chromatography) in the commercial production of plasma-derived Factor IX (FIX). The final thrombin preparation had a purity of approximately 75% (specific activity approximately 2400 IU/mg protein), which is sufficient for its intended purpose in a fibrin glue. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Factor X (FX) activity analysis, and analytical HIC were also used to characterize the thrombin. Three substantially different techniques were used to reduce any viral activity, namely: solvent/detergent (S/D) treatment, pasteurization, and virus filtration (nanofiltration). The manufacturing process presented here would be suitable for large-scale production of thrombin with a high degree of virus safety. [Copyright &y& Elsevier]

Abstract: A conventional, cholesterol-containing liposome formulation of cisplatin has demonstrated insignificant activity in clinical trials, due in part, to insufficient release of encapsulated content following localization within solid tumors. For this reason, the development of a triggered release liposome formulation is desirable. In this report, cisplatin was encapsulated into lysolipid-containing thermosensitive liposomes (LTSL) using a novel technique, which relies on the equilibration of cisplatin across the liposomal membrane at temperatures above the gel-to-liquid crystalline phase transition temperature (T C) of the bulk phospholipid. Mild heating and drug loading into LTSL did not induce morphological changes of the liposomes. In vitro data demonstrated that >95% of encapsulated cisplatin was released from LTSL within 5min following mild heating at 42°C, while <5% was released at 37°C. Under similar conditions, lysolipid-free thermosensitive liposomes exhibited 70% release of cisplatin at 42°C, and cholesterol-containing liposomes exhibited negligible drug release at 42°C. The pharmacokinetic profiles of LTSL- and TSL-cisplatin indicated that these formulations were rapidly eliminated from circulation (terminal t 1/2 of 1.09 and 2.83h, respectively). The therapeutic utility of LTSL-cisplatin formulation will be based on strategies where hyperthermia is applied prior to the administration of the liposomal drug—a strategy similar to that used in the clinical assessment of LTSL-doxorubicin formulation. [Copyright &y& Elsevier]

Abstract: Cryo-transmission electron microscopy (cryo-TEM) was used to image the microstructure in dilute sonicated dispersions of monoolein and oleic acid. The aim of the study was to explore how different experimental parameters, such as sample composition, total lipid concentration, pH, and ageing affect the phase behaviour and aggregate structure. Our investigations show that a rich variety of lamellar and non-lamellar structures, including liposomes and particles of cubic and inverted hexagonal phase, may form depending on the experimental conditions. The results are analyzed and discussed in relation to existing phase diagrams and earlier investigations concerning phase- and structural behaviour in monoolein/oleic acid/water systems. [Copyright &y& Elsevier]

Abstract: Previous investigations have revealed that the boron cluster compound Na2B12H11SH (BSH) is very potent in causing major structural rearrangements of and leakage from phosphatidylcholine liposomes. This somewhat unexpected finding is interesting from a fundamental point of view and may also constitute the basis of future important pharmaceutical/medical applications of BSH. In order to further explore the BSH-lipid interaction, we have studied the effects caused by BSH on dimyristoyl phosphatidylcholine (DMPC) liposomes. Cryo-transmission electron microscopy showed that BSH induces aggregation, membrane rupture and increasing wall thickness of the liposomes. Differential scanning calorimetry revealed a BSH dependent shift of the gel to liquid crystalline phase transition temperature of DMPC. The zeta potential of the liposomes decreases with increasing BSH concentrations, and an apparent dissociation constant of 0.23mM was found. BSH caused leakage of liposome-encapsulated carboxyfluorescein; leakage was higher at 23°C (near the phase transition temperature) than at 15°C and 37°C. It induced lipid mixing only at very high concentrations. [Copyright &y& Elsevier]

Abstract: The hydrodynamic properties of dextrans hydrophobically modified by sterically-hindered phenols in aqueous solution were characterized by a combination of Cryogenic Transmission Electron Microscopy (Cryo-TEM), fluorescent spectroscopy, and Dynamic Light Scattering (DLS). All solutions investigated contained aggregates the structure, shape, and critical aggregation concentration of which are influenced by the amount of the inserted phenol groups in the polymer matrix. We found that increasing of temperature enhances aggregation. It was established that the critical aggregation concentration could be well approximated by a logarithmic function of the substitution degree of the glycoside units. The results suggest that the proportion of aggregates is nevertheless negligible in comparison with the proportion of single molecules. [Copyright &y& Elsevier]

In order to investigate major histocompatibility complex (MHC) class I chain-related gene A (MICA), tumor necrosis factor (TNFa), −308TNFA, and human leukocyte antigen (HLA-DR/DQ) polymorphisms in mixed connective tissue disease (MCTD), we analyzed 24 patients and 229 healthy controls from Sweden. MICA and TNFa typing was performed by polymerase chain reaction (PCR) and genotyping. HLA-DR and -DQ were genotyped using PCR–sequence specific primers (PCR-SSP) and PCR–sequence-specific oligonucleotide probe (PCR-SSOP), respectively. For analysis of −308TNFA polymorphisms we performed PCR with restriction endonuclease enzymes. We found that the MICA5.1-5.1 genotype was positively associated with MCTD. Shared epitope genes (DRB1*01 and DRB1*04) were also significantly positively associated with MCTD. Polymorphism of −308TNFA was not differently distributed in MCTD patients compared with controls. Furthermore, we demonstrated that frequencies of three estimated haplotypes were increased in MCTD patients compared with controls. Interestingly, the haplotype with MICA allele 4 together with DRB1*04 and TNF1 alleles gives the most specific pattern for MCTD patients compared with controls. Our study demonstrates a clear contribution of HLA loci in susceptibility to MCTD in the Swedish population. Susceptibility to MCTD may be linked to the MICA4/HLA-DRB1*04/TNF1 haplotype and MICA 5.1-5.1 genotype. Mixed connective tissue disease was also associated with shared epitope genes, which in RA has been associated with a more severe disease. Whether these genotypes affect the clinical phenotype of MCTD needs to be determined. [Copyright &y& Elsevier]

Phytochrome proteins guide the red/far-red photoresponse of plants, fungi, and bacteria. Crystal structures suggest that the mechanism of signal transduction from the chromophore to the output domains involves refolding of the so-called PHY tongue. It is currently not clear how the two other notable structural features of the phytochrome superfamily, the so-called helical spine and a knot in the peptide chain, are involved in photoconversion. Here, we present solution NMR data of the complete photosensory core module from Deinococcus radiodurans. Photoswitching between the resting and the active states induces changes in amide chemical shifts, residual dipolar couplings, and relaxation dynamics. All observables indicate a photoinduced structural change in the knot region and lower part of the helical spine. This implies that a conformational signal is transduced from the chromophore to the helical spine through the PAS and GAF domains. The discovered pathway underpins functional studies of plant phytochromes and may explain photosensing by phytochromes under biological conditions. • Solution NMR is used to study the changes when phytochrome is photoactivated • An unrecognized phytochrome signaling pathway via the knot and spine helix is detected • Available crystal structures have not captured these changes Animals have eyes to see their environment, but bacteria, plants, and fungi use photoreceptors proteins to sense light. How is this achieved at the molecular level? Isaksson et al. found a previously unrecognized signaling pathway in phytochrome proteins. The mechanism explains how light information is processed by the enzyme for controlling light-dependent behavior of plants, fungi, and bacteria. [ABSTRACT FROM AUTHOR]

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a sub population is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent LSC-subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). By comparing LSCs from patients with different therapeutic outcome we describe how LSC heterogeneity at diagnosis correlate with long-term therapy response. We furthermore evaluate the potential for humanized ossicle-niche xenotransplantation (Reinisch et al., 2016) as a model system for functional characterization of LSC subpopulations in vivo. [ABSTRACT FROM AUTHOR]

Recent advances in single-cell RNA-sequencing (ScRNA-seq) has accelerated the investigation of hematopoiesis. However, characterization of leukemic cells using scRNA-seq remains challenging as conventional approaches for analysis fail to consider that the transcriptomes of leukemic cells often systematically diverge from normal cells. This results in irrelevant separation of leukemic subpopulations from their healthy counterparts. We have developed a computational approach - Nabo - which compares otherwise unalignable cells. The accuracy of Nabo was validated by mapping multiple scRNA-Seq datasets from purified subpopulations to well-defined reference samples. Nabo predicted the identity of human hematopoietic stem and progenitor populations to the same accuracy as can be achieved by established cell-surface markers. We applied Nabo to uncover the heterogeneity of hematopoietic cells from different stages of acute myeloid leukemia (AML). This revealed that AML cells lacked the heterogeneity of normal CD34+ cells and were devoid of cells with a hematopoietic stem cell gene signature. Furthermore, leukemic cells mapped to distinct stages of myeloid progenitors in a patient-dependent manner. To ask whether the observed mapping of leukemic to healthy progenitor cells indicates the identity of leukemia-initiating cells (LICs), we induced the human fusion oncogene MLL-ENL in murine granulocyte-monocyte-lymphoid progenitors (GMLPs). Induced leukemic cells mapped to a distinct Flt3+ subpopulation within the healthy GMLPs. Functional assays demonstrated that Flt3+ GMLPs contained 3-4 fold more LICs than Flt3- GMLPs, indicating that LICs within GMLPs express Flt3. In summary, Nabo represents a novel tool for delineation of leukemia heterogeneity, with the potential to identify LIC populations in single-cell data. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) have the capacity to differentiate into all hematopoietic lineages, and at the same time self-renew to maintain the HSC pool. HSCs have been thoroughly investigated using immunophenotypic-, molecular- and functional-analysis resulting in the development of protocols for high-purity prospective isolation of human HSCs. However, within the current state-of-the-art HSC populations, 90% of the cells are not bona fide HSCs, confounding molecular analysis of HSCs. Thus, identification of novel immunophenotypic markers to delineate the HSC population would improve our understanding of HSC biology. To identify cell-surface markers with the potential to discriminate between functionally different cells within the HSC population, we performed antibody screens measuring the expression of 340 markers on human cord blood (CB) and bone marrow (BM). Candidate markers that divide the HSC population were included in CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of single-cell immunophenotype-/RNA sequencing analysis. This allowed us to correlate the molecular signature of each single-cell with expression of 40 cell-surface proteins, in CD34+ and CD34+CD38- populations of CB and BM. Following sequencing the cells were clustered based on molecular signature. Distinct groups with HSC, progenitor and early committed progenitor profiles were identified. Interestingly, comparison between CB and BM showed differences in progenitor proportions while the fraction of cells with HSC signature remained intact. Additionally, three novel HSC marker-candidates were discovered which are being validated. Together, using CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify three novel cell-surface marker candidates for prospective isolation of HSCs. [ABSTRACT FROM AUTHOR]