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Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

BackgroundFollowing acute myocardial infarction (MI), irreversible damage to the myocardium can only be reduced by shortening the duration between symptom onset and revascularization. While systemic hypothermia has shown promising results in slowing pre-revascularization myocardial damage, it is resource intensive and not conducive to prehospital initiation. We hypothesized that topical neck cooling (NC), an easily implemented therapy for en route transfer to definitive therapy, could similarly attenuate myocardial ischemia-reperfusion injury (IRI).MethodsUsing an in vivo mouse model of myocardial IRI, moderate systemic hypothermia or NC was applied following left coronary artery (LCA) occlusion and subsequent reperfusion, at early, late, and post-reperfusion intervals. Vagotomy was performed after late NC in an additional group. Hearts were harvested to measure infarct size.ResultsBoth hypothermia treatments equally attenuated myocardial infarct size by 60% compared to control. The infarct-sparing effect of NC was temperature-dependent and timing-dependent. Vagotomy at the gastroesophageal junction abolished the infarct-sparing effect of late NC. Cardiac perfusate isolated following ischemia had significantly reduced cardiac troponin T, HMGB1, cell-free DNA, and interferon α and β levels after NC.ConclusionsTopical neck cooling attenuates myocardial IRI in a vagus nerve-dependent manner, with an effect comparable to that of systemic hypothermia. NC attenuated infarct size when applied during ischemia, with earlier initiation resulting in superior infarct sparing. This novel therapy exerts a cardioprotective effect without requiring significant change in core temperature and may be a promising practical strategy to attenuate myocardial damage while patients await definitive revascularization.

Metformin is increasingly prescribed in pregnancy, with beneficial maternal effects. However, it is not known how metformin-treatment impacts metabolism and energy production in the developing feto-placental unit. We assessed the human placental response to metformin using both in vivo and in vitro treated samples. trophoblasts were derived from placentas collected from non-laboured Caesarean deliveries at term, then treated in vitro with metformin (0.01 mM, 0.1 mM or vehicle). Metformin-concentrations were measured using liquid-chromatography mass-spectrometry. Oxygen consumption in cultured-trophoblasts was measured using a Seahorse-XF Mito Stress Test. Markers of oxidative-stress were assayed using qRT-PCR. Metformin-transporter mRNA and protein-levels were determined by quantitative RT-PCR and Western-blotting respectively. Metformin concentrations were also measured in sample trios (maternal plasma/fetal plasma/placental tissue) from pregnancies exposed to metformin on clinical-grounds. Maternal and fetal metformin concentrations in vivo were highly correlated over a range of concentrations (R2 = 0.76, p < 0.001; average fetal:maternal ratio 1.5; range 0.8–2.1). Basal respiration in trophoblasts was reduced by metformin treatment (0.01 mM metformin; p < 0.05, 0.1 mM metformin; p < 0.001). Mitochondrial-dependent ATP production and proton leak were reduced after treatment with metformin (p < 0.001). Oxidative stress markers were significantly reduced in primary-trophoblast-cultures following treatment with metformin. There is a close linear relationship between placental, fetal, and maternal metformin concentrations. Primary-trophoblast cultures exposed to clinically-relevant metformin concentrations have reduced mitochondrial-respiration, mitochondrial-dependent ATP-production, and reduced markers of oxidative-stress. Given the crucial role of placental energy-production in supporting fetal growth and well-being during pregnancy, the implications of these findings are concerning for intrauterine fetal growth and longer-term metabolic programming in metformin-exposed pregnancies.

Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration–regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.

Peter Wallbridge,1,2 Mark Hew,1,3,4 Selina M Parry,5 Louis Irving,1,2 Daniel Steinfort1,2 1Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC, Australia; 2Department of Respiratory and Sleep Medicine, Royal Melbourne Hospital, Parkville, VIC, Australia; 3Public Health and Preventive Medicine, Monash University, Clayton, VIC, Australia; 4Allergy, Asthma & Clinical Immunology, Alfred Health, Prahran, VIC, Australia; 5Department of Physiotherapy, The University of Melbourne, Parkville, VIC, AustraliaCorrespondence: Peter WallbridgeDepartment of Respiratory Medicine, Level 5 East, Royal Melbourne Hospital, Grattan St, Parkville, VIC 3050, AustraliaTel +61 3 9342 7000Fax +61 3 9342 3141Email peter.wallbridge@mh.org.auBackground and Objectives: Parasternal intercostal ultrasound morphology reflects spirometric COPD severity. Whether this relates to the systemic nature of COPD or occurs in response to hyperinflation is unknown. We aimed to assess changes in ultrasound parasternal intercostal muscle quantity and quality (echogenicity) in response to relief of hyperinflation. We hypothesised that reduction in hyperinflation following endobronchial valve (EBV) insertion would increase ultrasound parasternal thickness and decrease echogenicity.Methods: In this prospective cohort study, eight patients with severe COPD underwent evaluation of health-related quality of life, lung function, and sonographic thickness of 2nd parasternal intercostal muscles and diaphragm thickness, both before and after EBV insertion. Relationships between physiological and radiographic lung volumes, quality of life and ultrasound parameters were determined.Results: Baseline FEV1 was 1.02L (SD 0.37) and residual volume (RV) was 202% predicted (SD 41%). Median SGRQ was 63.26 (range 20– 70.6). Change in RV (− 0.51 ± 0.9L) following EBV-insertion showed a strong negative correlation with change in parasternal thickness (r = − 0.883) ipsilateral to EBV insertion, as did change in target lobe volume (− 0.89 ± 0.6L) (r = − 0.771). Parasternal muscle echogenicity, diaphragm thickness and diaphragm excursion did not significantly change.Conclusions: Dynamic changes in intercostal muscle thickness on ultrasound measurement occur in response to relief of hyperinflation. We demonstrate linear relationships between intercostal thickness and change in hyperinflation following endobronchial valve insertion. This demonstrates the deleterious effect of hyperinflation on intrinsic inspiratory muscles and provides an additional mechanism for symptomatic response to EBVs.Keywords: endobronchial valve, COPD, ultrasound, respiratory muscle, measurement

Abstract Background Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors. Methods GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7. Results Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD. Conclusions These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

The contribution of the different waves and sites of developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre. Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%-40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1-/- concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5-13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called “pre-HSCs.” Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed “11a- eKLS” (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs.

Background We previously demonstrated that ischemically injured cardiomyocytes release cell‐free DNA and HMGB1 (high mobility group box 1 protein) into circulation during reperfusion, activating proinflammatory responses and ultimately exacerbating reperfusion injury. We hypothesize that cell‐free DNA and HMGB1 mediate myocardial ischemia‐reperfusion injury by stimulating plasmacytoid dendritic cells (pDCs) to secrete type I interferon (IFN‐I). Methods and Results C57BL/6 and interferon alpha receptor‐1 knockout mice underwent 40 minutes of left coronary artery occlusion followed by 60 minutes of reperfusion (40′/60′ IR) before infarct size was evaluated by 2,3,5‐Triphenyltetrazolium chloride–Blue staining. Cardiac perfusate was acquired in ischemic hearts without reperfusion by antegrade perfusion of the isolated heart. Flow cytometry in pDC‐depleted mice treated with multiple doses of plasmacytoid dendritic cell antigen‐1 antibody via intraperitoneal injection demonstrated plasmacytoid dendritic cell antigen‐1 antibody treatment had no effect on conventional splenic dendritic cells but significantly reduced splenic pDCs by 60%. pDC‐depleted mice had significantly smaller infarct size and decreased plasma interferon‐α and interferon‐β compared with control. Blockade of the type I interferon signaling pathway with cyclic GMP‐AMP synthase inhibitor, stimulator of interferon genes antibody, or interferon regulatory factor 3 antibody upon reperfusion similarly significantly attenuated infarct size by 45%. Plasma levels of interferon‐α and interferon‐β were significantly reduced in cyclic GMP‐AMP synthase inhibitor‐treated mice. Infarct size was significantly reduced by >30% in type I interferon receptor monoclonal antibody–treated mice and interferon alpha receptor‐1 knockout mice. In splenocyte culture, 40′/0′ cardiac perfusate treatment stimulated interferon‐α and interferon‐β production; however, this effect disappeared in the presence of cyclic GMP‐AMP synthase inhibitor. Conclusions Type I interferon production is stimulated following myocardial ischemia by cardiogenic cell‐free DNA/HMGB1 in a pDC‐dependent manner, and subsequently activates type I interferon receptors to exacerbate reperfusion injury. These results identify new potential therapeutic targets to attenuate myocardial ischemia‐reperfusion injury.

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

Small numbers of hematopoietic stem cells (HSCs) generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through in vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as Lin-KIT(+)SCA-1(+)CD150(+)CD34-) are CD11A(+) and lack long-term self-renewal potential. We propose that CD11A(+)Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are multipotent progenitors and CD11A-Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are true HSCs.