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Proteins can be modified by lipids in various ways, for example by myristoylation, palmitoylation, farnesylation, and geranylgeranylation—these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable toin vivosystems. Here, we introduce a proteomic methodology for global analyses of endogenous lipidation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore lipidation sites in HeLa cells, and identified a total of 90 lipidation sites, including 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence lipidated proteins identified by myristic acid analog-based chemical proteomics. Isolation of lipidated peptides from digests prepared with different proteases enabled the identification of different lipidated sites, extending the coverage. Moreover, our peptide-centric approach successfully identified dually modified peptides having myristoylation and palmitoylation. Finally, we analyzedin vivomyristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of various protein lipidations.

BACKGROUND: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. METHODS: The Actin CreER /R26 VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions. RESULTS: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105 high Sca1 low , which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105 low Sca1 high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. CONCLUSIONS: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system. An analysis of skeletal stem cells in mice reveals that bone ageing occurs at the level of local niches affecting skeletal and haematopoietic lineage output, which may influence systemic aspects of multi-organ physiological ageing.

Atopic dermatitis (AD) is a skin disease heterogeneous both in terms of clinical manifestations and molecular profiles. It is increasingly recognized that AD is a systemic rather than a local disease and should be assessed in the context of whole-body level biology. In this study, we integrated RNA-seq data of both skin and PBMC along with clinical data from 115 AD patients and matched 14 healthy controls aiming to comprehensively capture the molecular signature associated with specific clinical presentation. Analysis of cross-tissue ligand-receptor coupling suggested increase of skin-PBMC interactions in AD patients compared to healthy controls. We built a regression model that predicts clinical phenotypes of AD using transcriptome modules identified from weighted gene co-expression network analysis of RNA-seq data in skin tissue and PBMC. We identified differential immunological signatures associated with two qualitatively differential skin manifestations of AD, erythema and papulation. Furthermore, we applied the regression model established in the cross-sectional dataset to a longitudinal dataset collected monthly from 30 AD patients for up to a year for personalized monitoring of disease trajectory and examined longitudinal heterogeneity in association with clinical presentation. Three patient clusters identified on the basis of longitudinal features of blood tests and PBMC transcriptome modules were found to be associated with longitudinal features in clinical severity as well as in the medication history. Our approach serves as a framework for effective clinical investigation to gain a holistic view of pathophysiology of complex human diseases by highlighting inter- and intra- patient heterogeneity.

Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles1–9, it has been difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution. Expression profiling on 75,000 single cells creates a comprehensive cell atlas of the human lung that includes 41 out of 45 previously known cell types and 14 new ones.

SummaryThe stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

Fragility fractures have a limited capacity to regenerate, and impaired fracture healing is a leading cause of morbidity in the elderly. The recent identification of a highly purified bona fide human skeletal stem cell (hSSC) and its committed downstream progenitor cell populations provides an opportunity for understanding the mechanism of age‐related compromised fracture healing from the stem cell perspective. In this study, we tested whether hSSCs isolated from geriatric fractures demonstrate intrinsic functional defects that drive impaired healing. Using flow cytometry, we analyzed and isolated hSSCs from callus tissue of five different skeletal sites (n = 61) of patients ranging from 13 to 94 years of age for functional and molecular studies. We observed that fracture‐activated amplification of hSSC populations was comparable at all ages. However, functional analysis of isolated stem cells revealed that advanced age significantly correlated with reduced osteochondrogenic potential but was not associated with decreased in vitro clonogenicity. hSSCs derived from women displayed an exacerbated functional decline with age relative to those of aged men. Transcriptomic comparisons revealed downregulation of skeletogenic pathways such as WNT and upregulation of senescence‐related pathways in young versus older hSSCs. Strikingly, loss of Sirtuin1 expression played a major role in hSSC dysfunction but re‐activation by trans‐resveratrol or a small molecule compound restored in vitro differentiation potential. These are the first findings that characterize age‐related defects in purified hSSCs from geriatric fractures. Our results provide a foundation for future investigations into the mechanism and reversibility of skeletal stem cell aging in humans., Human skeletal stem cells (hSSCs) are present and accumulate at fracture sites independent of age. Impaired healing in the elderly might underlie functional defects in hSSCs which are exacerbated in women. Re‐activation of Sirt1 rejuvenates geriatric hSSCs independent of sex.

Objective The purpose of this research was to develop a deep-learning model to assess radiographic finger joint destruction in RA. Methods The model comprises two steps: a joint-detection step and a joint-evaluation step. Among 216 radiographs of 108 patients with RA, 186 radiographs were assigned to the training/validation dataset and 30 to the test dataset. In the training/validation dataset, images of PIP joints, the IP joint of the thumb or MCP joints were manually clipped and scored for joint space narrowing (JSN) and bone erosion by clinicians, and then these images were augmented. As a result, 11 160 images were used to train and validate a deep convolutional neural network for joint evaluation. Three thousand seven hundred and twenty selected images were used to train machine learning for joint detection. These steps were combined as the assessment model for radiographic finger joint destruction. Performance of the model was examined using the test dataset, which was not included in the training/validation process, by comparing the scores assigned by the model and clinicians. Results The model detected PIP joints, the IP joint of the thumb and MCP joints with a sensitivity of 95.3% and assigned scores for JSN and erosion. Accuracy (percentage of exact agreement) reached 49.3–65.4% for JSN and 70.6–74.1% for erosion. The correlation coefficient between scores by the model and clinicians per image was 0.72–0.88 for JSN and 0.54–0.75 for erosion. Conclusion Image processing with the trained convolutional neural network model is promising to assess radiographs in RA.

Although single cell RNA sequencing studies have begun providing compendia of cell expression profiles, it has proven more difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here we describe droplet- and plate-based single cell RNA sequencing applied to ∼75,000 human lung and blood cells, combined with a multi-pronged cell annotation approach, which have allowed us to define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 of 45 previously known cell types or subtypes and 14 new ones. This comprehensive molecular atlas elucidates the biochemical functions of lung cell types and the cell-selective transcription factors and optimal markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signaling interactions including sources and targets of chemokines in immune cell trafficking and expression changes on lung homing; and identifies the cell types directly affected by lung disease genes and respiratory viruses. Comparison to mouse identified 17 molecular types that appear to have been gained or lost during lung evolution and others whose expression profiles have been substantially altered, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions, and interactions are achieved in development and tissue engineering and altered in disease and evolution.

Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

The hematopoietic stem cell (HSC) is arguably the most extensively characterized tissue stem cell. Since its identification by prospective isolation1, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology including, self-renewal2–4, differentiation, aging, niche5, and diversity6–8. Here we demonstrate by unbiased multi-step screening, identification of a single gene, Hoxb5 (homeobox B5 also known as Hox-2.1), whose expression in the bone marrow (BM) is limited to the long-term HSC (LT-HSC) in mice. Utilizing a single-color tri-mCherry reporter mouse driven by endogenous Hoxb5 regulation, only the Hoxb5-positive HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients, and critically, in secondary recipients. Only 7–35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse BM, we show that >94% of LT-HSC (Hoxb5+) are directly attached to VE-cadherin-positive cells, implicating a perivascular space as a near homogenous localization of the LT-HSC.

Accurately assessing the changes in cellular differentiation status in response to drug treatments or genetic manipulations is crucial for understanding the tumorigenesis process and developing novel therapeutics for human cancer. Here we have developed a novel systems biology approach, called the Lineage Maturation Index (LMI), to define the changes in differentiation state of cancer cells based on their gene expression profiles. We have been focusing on hematopoietic malignancies, but this approach is applicable to many human cancer types. We define the expression profile of N genes within a cell to be a point in an N-dimensional space. A lineage vector is derived from a progenitor cell (such as a hematopoietic stem cell) to a mature cell (such as a granulocyte) based on the expression of these N genes. By projecting the expression profile of an unknown cell onto the lineage vector, an LMI can be computed for the differentiation state of this specific cell. Upon drug treatments or genetic manipulations, a shift in LMI from stem cells towards mature cell types indicates differentiation. We have confirmed that the LMI approach can track the differentiation of normal hematopoietic cells. We have further shown that differentiation is a major consequence of various targeted therapies against leukemia irrespective of the driving oncogenes. Most importantly, we have used this approach to analyze the Connectivity Map HL60 leukemia data set for drugs that can induce leukemia cells to differentiate. In addition to drugs known to have differentiation activities (such as all-trans retinoic acid and etoposide), we discovered that several novel drugs (such as dihydroergotamine, mebendazole, and clonidine) have potent differentiation activities in leukemia cells. Currently, we are expanding our study into multiple types of solid tumors, such as breast, prostate, and colon cancers. Our LMI approach is highly robust in characterizing differentiation of the cancer cells and discovering novel differentiation therapies for human cancer. Citation Format: Yulin Li, Jun Seita, Dean Felsher, David Dill. Discovery of differentiation therapeutics using a systems biology approach. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-35.

Accurately assessing the changes in cellular differentiation status in response to drug treatments or genetic manipulations is crucial for understanding the tumorigenesis process and developing novel therapeutics for human cancer. Here we have developed a novel systems biology approach, called the Lineage Maturation Index (LMI), to define the changes in differentiation state of cancer cells based on their gene expression profiles. We have been focusing on hematopoietic malignancies, but this approach is applicable to many human cancer types. We define the expression profile of N genes within a cell to be a point in an N-dimensional space. A lineage vector is derived from a progenitor cell (such as a hematopoietic stem cell) to a mature cell (such as a granulocyte) based on the expression of these N genes. By projecting the expression profile of an unknown cell onto the lineage vector, an LMI can be computed for the differentiation state of this specific cell. Upon drug treatments or genetic manipulations, a shift in LMI from stem cells towards mature cell types indicates differentiation. We have confirmed that the LMI approach can track the differentiation of normal hematopoietic cells. We have further shown that differentiation is a major consequence of various targeted therapies against leukemia irrespective of the driving oncogenes. Most importantly, we have used this approach to analyze the Connectivity Map HL60 leukemia data set for drugs that can induce leukemia cells to differentiate. In addition to drugs known to have differentiation activities (such as all-trans retinoic acid and etoposide), we discovered that several novel drugs (such as dihydroergotamine, mebendazole, and clonidine) have potent differentiation activities in leukemia cells. Currently, we are expanding our study into multiple types of solid tumors, such as breast, prostate, and colon cancers. Our LMI approach is highly robust in characterizing differentiation of the cancer cells and discovering novel differentiation therapies for human cancer. Citation Format: Yulin Li, Jun Seita, Dean Felsher, David L. Dill. A systems biology approach for the discovery of differentiation therapeutics. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-12.

Precursors of hematopoietic stem cells, termed pre-HSCs, are intermediate precursors during the maturation process from hemogenic endothelial cells (HECs) to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at E10.5-11.5. Pre-HSCs acquire an efficient adult-repopulating ability only after ex vivo co-culture, which precludes understanding of their native hematopoietic capacity. Our group recently identified the native repopulating ability of the pre-HSC population using immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that a freshly isolated pre-HSC population exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multi-lineage repopulating capacity. In addition, out lineage tracing mouse model labeling cdh5+ ECs by tamoxifen injection at various embryonic time points suggests that HECs at E9.5-10.5 produce HSCs and B-1 lineage cells simultaneously, while later HECs at E11.5 predominantly produce HSC and minimum B-1 lineage. The HSC-labeling using Fgd5CreERT2 mice also shows the minimum contribution of B-1a cell production of fetal liver HSCs at E14.5. To understand this unexpected B-1 lymphocyte bias of the pre-HSC population and acquisition of multi-lineage potential during commitment to the HSC fate, we performed single-cell RNA-sequencing (scRNA-seq) using E11.5 pre-HSCs, and E12.5 and E14.5 Fetal liver HSCs. The velocity analysis of the scRNA-seq data indicates the presence of a divergent point into two directions: features of HSPC and B-lymphoid progenitors. Our results display the cellular landscape of HSC and B-1 cell development from HECs and heterogeneity of the pre-HEC population.

Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray platform gene expression commons and individually manipulated in human hematopoietic stem and progenitor cells (HSPCs). Deletion of some of the candidate genes (the hit genes) by CRISPR/Cas9 led to decreased MkP generation during HSPC differentiation, while more MkPs were produced when some hit genes were overexpressed in HSPCs. We then demonstrated that overexpression of these genes can increase the frequency of mature megakaryocytic colonies by functional colony forming unit-megakaryocyte (CFU-Mk) assay and the release of platelets after in vitro maturation. Finally, we showed that the histone deacetylase inhibitors could also increase MkP differentiation, possibly by regulating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro.

While hematopoietic stem cells (HSCs) have been extensively studied at the population level, little is known about the lineage commitment of individual clones. Here, we provide comprehensive maps ofin vivoHSC clonal development in mice under homeostasis and after depletion of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been depleted by irradiation or by an anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiates while dominantly expanding and exhibiting lineage bias. We identified the cellular origins of clonal dominance and lineage bias, and uncovered the lineage commitment pathways that lead HSC clones to differential blood production. This study reveals surprising alterations in HSC regulation by irradiation, and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) sustain daily blood production through a complex step-wise lineage commitment process. In this work, we present the first comprehensive study of HSC lineage commitment at the clonal level and identify new HSC regulatory mechanisms that are undetectable by conventional population level studies. First, we uncover distinct HSC clonal pathways that lead to differential blood production and imbalances. Second, we reveal that HSC regulation under physiological conditions is strikingly different from that after injury. Third, we present a comprehensive map of HSC activities in vivo at the clonal level.

SummaryHematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

SummaryHow 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.

Summary: Age-associated thymic involution results in diminished T cell output and function in aged individuals. However, molecular mediators contributing to the decline in thymic function during early thymic involution remain largely unknown. Here, we present transcriptional profiling of purified thymic stromal subsets from mice 1, 3, and 6 months of age spanning early thymic involution. The data implicate unanticipated biological functions for a subset of thymic epithelial cells. The predominant transcriptional signature of early thymic involution is decreased expression of cell-cycle-associated genes and E2F3 transcriptional targets in thymic epithelial subsets. Also, expression of proinflammatory genes increases with age in thymic dendritic cells. Many genes previously implicated in late involution are already deregulated by 3–6 months of age. We provide these thymic stromal data sets, along with thymocyte data sets, in a readily searchable web-based platform, as a resource for investigations into thymocyte:stromal interactions and mechanisms of thymic involution. : Heterogeneous thymic stromal cells regulate proper T cell development. Age-associated thymic involution results in diminished T cell output and immunity. Ki et al. now present transcriptional profiling of six thymic stromal subsets during early thymic involution. They conclude that diminished E2F3 activity in thymic epithelial cells and an augmented inflammatory signature in dendritic cells are early hallmarks of involution. Data are provided on a web-based platform to facilitate discovery of molecular mediators of thymocyte:stromal cell crosstalk and early thymic involution.

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRPα axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.

Summary Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot find VSELs in mouse BM with any of the reported stem cell potentials, specifically for hematopoiesis. We found that: (1) most events within the “VSEL” flow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to confirm the existence of pluripotent VSELs., Highlights • We rigorously evaluated the mouse VSEL hypothesis • Most CD45−/intLin−SCA-1+ events were neither “very small” nor diploid cells • No pluripotency was detected in CD45−/intLin−SCA-1+ cells, regardless of their size • Existence of adult mouse VSELs in the bone marrow remains dubious

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody–mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody–mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8 + )] but decreased priming of OT-II T cells (CD4 + ). The CD4 + T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3 + ) regulatory T cells. Macrophages following anti-CD47–mediated phagocytosis primed CD8 + T cells to exhibit cytotoxic function in vivo . This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.

Summary Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. Video Abstract

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment., National Institutes of Health (U.S.) (NIH grant RO1-DK70055), National Institutes of Health (U.S.) (NIH Grant RO1-DK59279), National Institutes of Health (U.S.) (American Recovery and Reinvestment Act (RC2-HL102815)), National Institutes of Health (U.S.) (NIH (K99HL093212-01)), Cooley’s Anemia Foundation, National Institutes of Health (U.S.) (NIH LLS (3567-07)), National Institutes of Health (U.S.) (NIH grant R37CA054358), National Institutes of Health (U.S.) (NIH grant P50HG003233), National Institutes of Health (U.S.) (NIH grant R01AI047457), National Institutes of Health (U.S.) (NIH Grant R01AI047458), National Institutes of Health (U.S.) (CA86065), National Institutes of Health (U.S.) (HL099999), Thomas and Stacey Siebel Foundation, California Institute for Regenerative Medicine (Fellowship T1-00001)