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Revisiting hematopoiesis: applications of the bulk and single-cell transcriptomics dissecting transcriptional heterogeneity in hematopoietic stem cells
Abstract
Hematopoietic system (HS) is one of the most unique, adaptive and comprehensive developmental systems on which various other body systems relies on. It consists of a central pool of multipotent hematopoietic stem cells (HSCs) differentiating into lymphoid and myeloid lineage by series of gradual loss of stemness potential. Thus, this highly coordinated phenomenon of blood cell renewal ensures robust immunity and limits autoimmunity. Any disease, chronic infection or stress interrupts HS homeostasis and breaks HSCs' dormancy, thereby activating HSCs to meet the peripheral demand for different immune cells via their expansion and differentiation into more lineage-restricted progenitors, primarily within the bone marrow (BM) in adult life. Therefore, a greater understanding of the overall regulatory landscape of HSC homeostasis and their perturbations is critical for dissecting protective immunity versus autoimmunity. Recent advancements in next-generation sequencing (NGS) viz genomic, transcriptomic, epigenomic and proteogenomic methods at bulk as well as single-cell levels have increased our apprehension for HSC working model. In this review, we discussed the recent findings and computational methods used to unravel the new HSC model revised over the classical model.
... A large number of studies have revealed the significant activation of innate immune response and adaptive immune response in KD, which support the autoimmunity hypothesis. However, its low recurrence rate and the absence of a family history of autoimmune diseases in KD are inconsistent with the typical presentation of autoimmune disorders (1,30,31). In short, none of these theories of KD have been fully validated and they only partially account for the characteristics of KD. ...
... The pseudo-time analyses of KD before treatment in our and GSE168732 datasets has an impaired cell development trajectory compared with febrile and KD after treatment. Canonical cell markers show that febrile patients have four clearly differentiated states, which are myeloid lineage (state 1), lymphoid lineage 1 (state 2), erythro-megakaryocytic lineage (state 4), and lymphoid lineage 2 (state 5), but KD patients before treatment have mixed myeloid and erythro-megakaryocytic lineage and mixed lymphoid lineage 1 and lineage 2 (30). It suggests that B-cell development dysregulation in KD actually has a deep root in the very early cell lineage differentiation, which affect not only lymphoid lineage but also myeloid lineage. ...
... It further confirms that the SPI1 expression is repressed in KD before treatment, because it controls hematopoietic cells to differentiate into myeloid and lymphoid lineages. In hematopoiesis, HSPCs first give the birth to common myeloid progenitor cells and common lymphoid progenitor cells (30). The formers further split into myeloid lineage and erythro-megakaryocytic lineage, and the latter further split into T lymphoid lineage (lymphoid lineage 1) and B lymphoid lineage (lymphoid lineage 2) (30). ...
Background
Kawasaki disease (KD) is an acute systemic vasculitis that can lead to acquired heart disease in children mostly from in developed countries. The previous research showed that B cells in KD patients underwent a profound change in both the cell numbers and types after intravenous immunoglobulin (IVIG) therapy.
Methods
We performed the single-cell RNA-sequencing for the peripheral blood mononuclear cells (PBMCs) from three febrile patients and three KD patients to investigate the possible mechanism underlying B cell developmental dysfunction in KD. The pseudo-time analysis was employed to study the developmental trajectories of the PBMCs in febrile control and KD patients.
Results
Overall single-cell expression profiles show that the biological processes of immunity, B cell activation pathway and their related biological entities are repressed in KD patients before IVIG treatment compared to febrile patient and KD patients after IVIG treatment. The differentially expressed gene analyses further demonstrate that B cell signaling pathway is downregulated in B cells and plasma blast cells of KD patients before treatment while cell cycle genes and MYC gene are upregulated in dendritic cells (DCs) and hematopoietic stem and progenitor cells (HSPCs) of KD patients before treatment. The biological process of immune response is upregulated in the HSPCs of KD patients before treatment in our dataset while the biological process of inflammatory response is upregulated in the HSPCs of KD patients before treatment in GSE168732 dataset. Single-cell trajectory analyses demonstrate that KD patients before treatment have a shortened developmental path in which B cells and T cells are failed to differentiate into separate lineages. HSPD1 and HSPE1 genes show an elevated expression level in the early cell development stage of KD patients before treatment accompanied with the repression of MYC, SPI1, MT2A and UBE2C genes. Our analyses of all B cells from KD patients before treatment show most of B cells are arrested in a transitional state with an ill developmental path compared with febrile patients and KD patients after treatment.
Conclusion
Our results indicate that the immune premature HSPCs accompanied with the abnormal expression dynamics of cell cycle and SPI1 genes are the mechanism underlying B cell developmental dysfunction in KD patients.
... White blood cells, also called leucocytes are the cells of the immune system that are involved in protecting the body against both infectious diseases and foreign invaders [4]. All white blood cells are produced and derived from multipotent cells in the bone marrow known as haematopoietic stem cells and their levels of production are regulated by organs such as the spleen, liver, and kidneys [4]. ...
... White blood cells, also called leucocytes are the cells of the immune system that are involved in protecting the body against both infectious diseases and foreign invaders [4]. All white blood cells are produced and derived from multipotent cells in the bone marrow known as haematopoietic stem cells and their levels of production are regulated by organs such as the spleen, liver, and kidneys [4]. Leucocytes are found throughout the body, including the blood and lymphatic system [5]. ...
Exposure to wood dust may result in external and internal health problems which may be immediate, short-term or long-term. This study was carried out to evaluate the leucocytes and CD4 counts of individuals exposed to wood dust in Ekpoma, Edo State, Nigeria. A total of fifty individuals exposed to wood dust aged 16-60 years and of both sexes were recruited for this study. Fifty apparently healthy subjects who were not exposed to wood dust served as control. The leucocyte counts were carried out using Sysmex KX-21N Autoanalyzer and the CD4 count was determined using Flow Cytometry. In this study, the results obtained showed that the CD4 count (cells/µL) of the test subjects and control subjects were 912.60 ± 298.05 and 891.14 ± 304.61 respectively. Similarly, total leucocyte counts (x10³/μl) of both test and control subjects were 5.44 ± 1.34 and 5.34 ± 1.74 respectively. Furthermore, LYM % of the test subjects and control subjects was 41.76 ± 10.87 and 49.48 ± 8.67, NEUT % was 45.58 ± 10.19 and 37.74 ± 8.39, while MXD % was 12.38 ± 4.96 and 11.65 ± 3.69 respectively. Neutrophil % was significantly higher while lymphocyte % was significantly lower. There was a statistically significant increase in CD4 count of female subjects compared to males. Age did not affect any of the parameters studied except total leucocyte count. Duration of exposure to wood dust did not affect any of the parameters studied. In conclusion, neutrophil %, lymphocyte % and CD4 count were variably affected by wood dust. Similarly, the WBC total count of subjects in the age bracket of 51 years and above was significantly higher compared to other age groups. However, duration of exposure to wood dust did not affect any of the parameters studied. We hereby recommend that individuals exposed to wood dust should be encouraged to use Personal Protective Equipment (PPE) such as face mask while working to reduce exposure.
... It is characterized by dysregulation of both adaptive and innate immune pathways. Monocytes, which are essential components of the innate immune system, originate from hematopoietic stem and progenitor cells (HSPC) in the bone marrow and express CD14 and CD16 (1). The role of proinflammatory monocytes in autoimmune diseases is welldocumented (2,3). ...
Background
SLE is a complex autoimmune disease with deleterious effects on various organs. Accumulating evidence has shown abnormal vitamin B12 and one-carbon flux contribute to immune dysfunction. Transcobalamin II (TCN2) belongs to the vitamin B12-binding protein family responsible for the cellular uptake of vitamin B12. The role of TCN2 in SLE is still unclear.
Methods
We collected clinical information and blood from 51 patients with SLE and 28 healthy controls. RNA sequencing analysis, qPCR, and western blot confirmed the alteration of TCN2 in disease monocytes. The correlation between TCN2 expression and clinical features and serological abnormalities was analyzed. TCN2 heterozygous knockout THP1 cells were used to explore the effects of TCN2 dysfunction on monocytes. CCK-8 assay and EdU staining were used to detect cell proliferation. ELISA was conducted to assess vitamin B12, glutathione, and cytokines changes. UHPLC-MRM-MS/MS was used to detect changes in the intermediates of the one-carbon cycle. Flow cytometry is used to detect cell cycle, ROS, mitoROS, and CD14 changes.
Results
Elevated TCN2 in monocytes was correlated positively with disease progression and specific tissue injuries. Using CD14+ monocytes and TCN2 genetically modified THP1 cell lines, we found that the TCN2 was induced by LPS in serum from SLE patients. TCN2 heterozygous knockout inhibited cellular vitamin B12 uptake and one-carbon metabolism, leading to cell proliferation arrest and decreased Toll-like receptor 4 (TLR4)-mediated CCL2 release. Methionine cycle metabolites, s-adenosylmethionine and homocysteine, rescued these effects, whereas folate treatment proved to be ineffective. Folate deficiency also failed to replicate the impact of TCN2 downregulation on THP1 inflammatory response.
Conclusion
Our study elucidated the unique involvement of TCN2-driven one-carbon flux on SLE-associated monocyte behavior. Increased TCN2 may promote disease progression and tissue damage by enhancing one-carbon flux, fostering monocyte proliferation, and exacerbating TLR4 mediated inflammatory responses. The inhibition of TCN2 may be a promising therapeutic approach to ameliorate SLE.
... Single-cell RNA sequencing (scRNA-seq) characterizes the transcriptome of each individual cell in large populations. This high-throughput approach is the ideal choice to reveal the heterogeneous landscape of normal and aberrant hematopoiesis 1,2 , composed of cells characterized by differing self-renewal capacity, multipotent potential and high plasticity, and involved in infections and other diseases controlling immune responses [3][4][5] . ...
Single-cell technologies offer a unique opportunity to explore cellular heterogeneity in hematopoiesis, reveal malignant hematopoietic cells with clinically significant features and measure gene signatures linked to pathological pathways. However, reliable identification of cell types is a crucial bottleneck in single-cell analysis. Available databases contain dissimilar nomenclature and non-concurrent marker sets, leading to inconsistent annotations and poor interpretability. Furthermore, current tools focus mostly on physiological cell types, lacking extensive applicability in disease.
We developed the Cell Marker Accordion, a user-friendly platform for the automatic annotation and biological interpretation of single-cell populations based on consistency weighted markers. We validated our approach on peripheral blood and bone marrow single-cell datasets, using surface markers and expert-based annotation as the ground truth. In all cases, we significantly improved the accuracy in identifying cell types with respect to any single source database.
Moreover, the Cell Marker Accordion can identify disease-critical cells and pathological processes, extracting potential biomarkers in a wide variety of contexts in human and murine single-cell datasets. It characterizes leukemia stem cell subtypes, including therapy-resistant cells in acute myeloid leukemia patients; it identifies malignant plasma cells in multiple myeloma samples; it dissects cell type alterations in splicing factor-mutant cells from myelodysplastic syndrome patients; it discovers activation of innate immunity pathways in bone marrow from mice treated with METTL3 inhibitors.
The breadth of these applications elevates the Cell Marker Accordion as a flexible, faithful and standardized tool to annotate and interpret hematopoietic populations in single-cell datasets focused on the study of hematopoietic development and disease.
... Количество видов этих специфических рецепторов достаточно разнообразно. На сегодня на мембранах DCs наиболее часто среди других экспрессируются рецепторы CD1c, CD141, CD303 [38]. ...
Based on their own research and a review of the literature, the authors analyze the possible cellular mechanisms of the development of an inflammatory reaction after the obliteration of varicose veins with cyanoarylate adhesive compounds (CAO), which received the name phlebitis- Like abnormal Reaction (PLAR) in foreign sources. Despite the existing opinion about the “abnormal” nature of the inflammatory reaction, it is noted that the main stages of its development are fully consistent with the currently known molecular and cellular mechanisms of the response of biological tissues to contact with a foreign antigenic substance and are of a natural nature. The cause of the development of acute alterative inflammation in the vein wall is the direct contact of the endothelium with an aggressive environment, which is cyanoacrylate. A specific feature of the development of chronic inflammation in the vein wall is its productive interdaily character, which is replaced by proliferative processes. The main role in the development of successive stages of PLAR development is played by monocytic, mast and giant cells of foreign bodies, as well as the mechanisms underlying the regulation of the functional activity of these cells. During the period of cyanoacrylate biodegradation, its cellular environment corresponds to all morphological features of a phagocytoma, whose activity decreases with the biodegradation of cyanoacrylate with simultaneous connective tissue proliferation. The development of possible chronic granulomatous inflammation is based on a local autoimmune process associated with the formation of giant multinucleated epithelioid cells (Langerhans cells). In conclusion, it is emphasized that today, when using various cyanoacrylate compounds for the purpose of adhesive obliteration of veins, taking into account the accumulated clinical data and morphological studies, the final answers to the existing reasonable objections about the complete safety of the use of cyanoacrylates in clinical practice should be given by fundamental immunohistochemical and genetic studies.
... In the medical field, "infiltration" refers to the phenomenon where certain cell types invade regions outside their usual locations, resulting in an abnormal accumulation of these cells. Hematopoietic stem cells provide a notable example as they are released from the bone marrow into the bloodstream, where they mature into lymphocytes-a specific type of white blood cell in the immune system [20]. In the context of cancer, these lymphocytes infiltrate cancerous tumors, leading to their accumulation within the tumor tissue [21,22]. ...
Rotator cuff (RC) tears affect many individuals around the globe. Ambiguity of rotator cuff repair surgical outcomes is currently a limitation that is associated with fat accumulation and atrophy in the rotator cuff muscles. To improve the efficacy of rotator cuff repairs, a deeper understanding of the root causes is required. Traditionally, the term “fat infiltration” has been used to described fatty changes in muscle after rotator cuff tears. This paper introduces the concept of fat expansion as a more appropriate description for the appearance of fatty rotator cuff tear pathological changes. Furthermore, the contribution of fibroadipogenic progenitor (FAP) cells to pathological changes associated with rotator cuff injuries is presented to characterize the molecular basis of impairment. Lastly, the field of regenerative engineering is discussed as a promising solution to the pathological changes associated with rotator cuff tears.
The connection between fatty infiltration, fat expansion, fat accumulation, fibroadipogenic cells, and regenerative engineering in the context of rotator cuff tears was explored using the databases PubMed and Google Scholar.
Numerous articles have supported the role of muscle resident fibroadipogenic cells as a contributor to rotator cuff tear pathological changes. In addition, regenerative engineering solutions prove to improve the pathological changes associated with rotator cuff tears.
The term fat expansion is more appropriate to describe fat accumulation associated with rotator cuff tears, and the employment of regenerative engineering treatment strategies improve the pathological changes associated with rotator cuff tears.
Fat accumulation after rotator cuff tears has been associated with post-operative complications. Infiltration or entering of adipocytes from the external muscle environment has historically been the reported cause of the rapid increase in fat and muscle atrophy observed after rotor cuff tears. This review will dismiss the use of the term fat infiltration and acknowledge the implications of muscle resident stem cells, known as fibroadipogenic (FAP) cells, to rotator cuff tear pathological changes. Additionally, regenerative engineering, a field which seeks to regenerate various tissues using biomaterial-based scaffolds and stem cells, will be discussed as a potential solution for pathological changes.
... However, this analysis requires cells in different states during the differentiation process, including stem cells and progenitor cells, to order them along pseudo-time. Considering that tissue stem cells and progenitor cells are typically rare and difficult to identify experimentally [3,4], important processes involved in intermediate progenitor states might not be known from the analysis. To address this problem, previous studies based on bulk transcriptomes have applied phylogenetic analysis; phylogenetic analysis can infer not only tree topology-corresponding to the cell differentiation hierarchy [5]-but also ancestral states-corresponding to the states of the differentiating intermediate progenitor cells. ...
Objective
Diversification of cell types and changes in epigenetic states during cell differentiation processes are important for understanding development. Recently, phylogenetic analysis using DNA methylation and histone modification information has been shown useful for inferring these processes. The purpose of this study was to examine whether chromatin accessibility data can help infer these processes in murine hematopoiesis.
Results
Chromatin accessibility data could partially infer the hematopoietic differentiation hierarchy. Furthermore, based on the ancestral state estimation of internal nodes, the open/closed chromatin states of differentiating progenitor cells could be predicted with a specificity of 0.86–0.99 and sensitivity of 0.29–0.72. These results suggest that the phylogenetic analysis of chromatin accessibility could offer important information on cell differentiation, particularly for organisms from which progenitor cells are difficult to obtain.
... The results of the analysis for each subject are summarized in Fig. 3. The green (circle) and red (star) dots represent successful and unsuccessful treatments, respectively, such that the evaluation performed six months from the last BCG treatment (which Table 1 and the initial condition from Eq. (9). The drug administration takes place after 552 h (or 23 days) and shown by the BCG pulse (red line). ...
This work introduces the first model of immunotherapy treatment, namely the BacillusCalmette–Guerin (BCG) vaccine, for Type 1 Diabetes (T1D). The model takes into consideration the interaction network between multiple immune cell types and compartments. A set of ordinary differential equations (ODEs) is introduced to capture the connectivity between these variables and the clinical presentation of the disease. Four subsets of theT1D mice and healthy controls that exhibit normal and high-level glucose consumption are evaluated using the proposed model. Numerical results obtained for mice suggest that BCG treatment of the T1D patients that follow healthy eating habits normalizes glucose to levels observed in non-diabetic controls. Furthermore, glucose consumption profoundly influences disease progression. This outcome suggests that immunotherapy may modulate molecular and cellular manifestations of the disease but it does not eliminate T1D. Of note, our data, obtained from numerical simulations, indicate that the BCG immunotherapy treatment may benefit healthy controls on a high-glucose diet and can be used as a tool for further clinical investigation of BCG usage to control T1D.
... In humans, monocytes comprise about 2-8% of white blood cells [6]. They are produced from haematopoietic stem and progenitor cells (HSPC) in the bone marrow, circulate in the blood for about 1-3 days, and then migrate into tissues and differentiate into macrophages or dendritic cells [7]. Indeed, the influx of monocytes in response to inflammation is an important contributor to macrophage presence in tissue [8]. ...
The three subsets of human monocytes, classical, intermediate, and nonclassical, show phenotypic heterogeneity, particularly in their expression of CD14 and CD16. This has enabled researchers to delve into the functions of each subset in the steady state as well as in disease. Studies have revealed that monocyte heterogeneity is multi-dimensional. In addition, that their phenotype and function differ between subsets is well established. However, it is becoming evident that heterogeneity also exists within each subset, between health and disease (current or past) states, and even between individuals. This realisation casts long shadows, impacting how we identify and classify the subsets, the functions we assign to them, and how they are examined for alterations in disease. Perhaps the most fascinating is evidence that, even in relative health, interindividual differences in monocyte subsets exist. It is proposed that the individual’s microenvironment could cause long-lasting or irreversible changes to monocyte precursors that echo to monocytes and through to their derived macrophages. Here, we will discuss the types of heterogeneity recognised in monocytes, the implications of these for monocyte research, and most importantly, the relevance of this heterogeneity for health and disease.
... Dendritic cells are professional antigen-presenting cells, also acting as mediators between the innate and the adaptative immune systems [68]. Primary dendritic cells, namely, bone marrow-derived dendritic cells (BMDCs), represent an interesting working model, as primary BMDC cultures can be matured in a number of cell types, including dendritic cells and macrophages [69]. ...
Toll-like receptors (TLRs) are the most studied receptors among the pattern recognition receptors (PRRs). They act as microbial sensors, playing major roles in the regulation of the innate immune system. TLRs mediate their cellular functions through the activation of MyD88-dependent or MyD88-independent signaling pathways. Myd88, or myeloid differentiation primary response 88, is a cytosolic adaptor protein essential for the induction of proinflammatory cytokines by all TLRs except TLR3. While the crucial role of Myd88 is well described, the contribution of other adaptors in mediating TLR signaling and function has been underestimated. In this review, we highlight important results demonstrating that TIRAP and TRAM adaptors are also required for full signaling activity and responses induced by most TLRs.
... The human bone marrow produces about 500 billion blood cells per day, which enter the systemic circulation via permeable vasculature sinusoids within the bone marrow cavity [29]. Bone marrow consists of hematopoietic cells, BMAT, and supporting stromal cells [30]. All types of hematopoietic cells, including bone marrow and lymphoid lineages, are produced in the bone marrow, and MSCs, which can be isolated from the primary culture of the bone marrow matrix, can generate bone, adipose, and cartilage tissues [31]. ...
A variety of metabolic disorders are associated with a decrease in estradiol (E2) during natural or surgical menopause. Postmenopausal women are prone to excessive fat accumulation in skeletal muscle and adipose tissue due to the loss of E2 via abnormalities in lipid metabolism and serum lipid levels. In skeletal muscle and adipose tissue, genes related to energy metabolism and fatty acid oxidation, such as those encoding peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and estrogen-related receptor alpha (ERRα), are downregulated, leading to increased fat synthesis and lipid metabolite accumulation. The same genes regulate lipid metabolism abnormalities in the bone marrow. In this review, abnormalities in lipid metabolism caused by E2 deficiency were investigated, with a focus on genes able to simultaneously regulate not only skeletal muscle and adipose tissue but also bone metabolism (e.g., genes encoding PGC-1α and ERRα). In addition, the mechanisms through which mesenchymal stem cells lead to adipocyte differentiation in the bone marrow as well as metabolic processes related to bone marrow adiposity, bone loss, and osteoporosis were evaluated, focusing on the loss of E2 and lipid metabolic alterations. The work reviewed here suggests that genes underlying lipid metabolism and bone marrow adiposity are candidate therapeutic targets for bone loss and osteoporosis in postmenopausal women.
... Hematopoietic cells are bone-marrow derived cells that become blood-borne myeloid and lymphoid cells and migrate to various tissues, participating both in immune responses and the maintenance of specific tissues [1][2][3]. Produced in the bone marrow in the adult homeostatic state [4], hematopoietic cells consist of undifferentiated, multipotent stem and progenitor cells responsible for their self-renewal and downstream mature immune cells [5]. Hematopoietic cells are involved in the maintenance of immune homeostasis in health [6] but are also activated in disease or stress to fight infection and aid in tissue repair. ...
Hematopoietic cells play a crucial role in the adult retina in health and disease. Monocytes, macrophages, microglia and myeloid angiogenic cells (MACs) have all been implicated in retinal pathology. However, the role that hematopoietic cells play in retinal development is understudied. The temporal changes in recruitment of hematopoietic cells into the developing retina and the phenotype of the recruited cells are not well understood. In this study, we used the hematopoietic cell-specific protein Vav1 to track and investigate hematopoietic cells in the developing retina. By flow cytometry and immunohistochemistry, we show that hematopoietic cells are present in the retina as early as P0, and include microglia, monocytes and MACs. Even before the formation of retinal blood vessels, hematopoietic cells localize to the inner retina where they eventually form networks that intimately associate with the developing vasculature. Loss of Vav1 lead to a reduction in the density of medium-sized vessels and an increased inflammatory response in retinal astrocytes. When pups were subjected to oxygen-induced retinopathy, hematopoietic cells maintained a close association with the vasculature and occasionally formed ‘frameworks’ for the generation of new vessels. Our study provides further evidence for the underappreciated role of hematopoietic cells in retinal vasculogenesis and the formation of a healthy retina.
Immunotherapy utilizes immune cells to target cancer and improves treatment outcomes with few side effects. Despite the effectiveness of immunotherapy, the limited availability of monocytes, which are essential for the differentiation of antigen-presenting cells, remains a major challenge. In this study, we developed a technique for inducing monocytes from hematopoietic stem and progenitor cells by using a serum-free (SF) medium supplemented with optimal concentrations of serum substitutes and cytokines. Three key serum substitutes, namely lipids, ascorbic acid, and β-glycerophosphate, were identified through factorial design screening, with their concentrations optimized through steepest ascent path analysis. Iscove's modified Dulbecco's medium was identified as the optimal basal medium. Long-term culturing confirmed the successful induction of CD14+CD16+ and CD14+CD16- monocytes. Functional assays validated the efficacy of this technique with comparable gene expression, cytokine secretion, phagocytosis ability, and T-cell stimulating ability between SF and serum-containing cultures. Under SF conditions, high expression levels of CD16 were detected, indicating the broad range of potential applications of CD16+ monocytes. Overall, this technique represents a feasible SF alternative for monocyte generation, with potential benefits for immunotherapy.
Coronavirus disease 2019 (COVID-19) is an infectious illness caused by the SARS-CoV-2 virus. The infections can be transmitted through droplets of different sizes: when the droplet particles are >5-10 μm in diameter they are referred to as respiratory droplets, and when then are <5μm in diameter, they are referred to as droplet nuclei. Symptoms include fever, coughing, headaches, exhaustion, breathing issues, loss of smell, and loss of taste. Haematology is the study of the physiology and pathology of the cellular elements of blood. The three major cellular components of blood are red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes). Haematology laboratory is one of the essential laboratories found in hospitals that performs a wide variety of basic and advanced hematology testing on whole blood, plasma, bone marrow and other body fluids. Haematology plays a crucial role in the diagnosis of COVID-19. Blood tests, such as Full blood counts and coagulation profiles, can provide valuable information about the patient's immune response, inflammation, and potential complications associated with the virus. While these tests are not definitive for COVID-19 diagnosis on their own, they are an essential component of the overall diagnostic process when used in conjunction with other clinical and laboratory findings, including molecular tests like PCR. Haematology, therefore, contributes to a comprehensive assessment of COVID-19 patients, aiding in their management and treatment.
Sulfur quantum dots (SQDs) have emerged as an intriguing class of luminescent nanomaterial due to their exceptional physiochemical and optoelectronic properties. However, their biomedical application is still in its infancy...
Background: Bone marrow and stem cell transplantation have become conventional therapy, with the potential to cure a variety of hematologic malignancies and immunologic diseases. Severe infection is still a potentially fatal complication following transplantation, contributes significantly to morbidity, and may demand ICU admission. Objectives: The study aims to summarize current evidence on the prevalence, risk factors, and management approaches of irritable bowel syndrome in Saudi Arabia. Methodology: For article selection, the PubMed database and EBSCO Information Services were used. All relevant articles relevant to our topic and other articles were used in our review. Other articles that were not related to this field were excluded. The data was extracted in a specific format that was reviewed by the group members. Conclusion: the result of the 12 studies included, proves the high risk of infections after bone marrow transplantation and its high mortality effect. Post transplantation patient's immune system is vulnerable, opportunistic infections occur such as viruses and fungi. The highest prevalence recorded in Adenovirus infections reaching 29% of patients throat swaps, urine, and stool samples come positive affirming the diagnosis.
One of the greatest challenges in the analysis of single cells, quite often is the severe ionic background their samples exhibit. With that in mind, Asymmetrical Flow Field-Flow Fractionation (AF4),...
The cotton crop is economically important and primarily grown for its fiber. Although the genus Gossypium consists of over 50 species, only four domesticated species produce spinnable fiber. However, the genes determine the molecular phenotype of fiber, and variation in their expression primarily contributes to associated phenotypic changes. Transcriptome analyses can elucidate the similarity or variation in gene expression (GE) among organisms at a given time or a circumstance. Even though several algorithms are available for analyzing such high-throughput data generated from RNA Sequencing (RNA-Seq), a reliable pipeline that includes a combination of tools such as an aligner for read mapping, an assembler for quantitating full-length transcripts, a differential gene expression (DGE) package for identifying differences in the transcripts across the samples, a gene ontology tool for assigning function, and enrichment and pathway mapping tools for finding interrelationships between genes based on their associated functions are needed. Therefore, this chapter first introduces the cotton crop, fiber phenotype, transcriptome, then discusses the basic RNA-Seq pipeline and later emphasizes various transcriptome analyses studies focused on genes associated with fiber quality and its attributes.
Background
Single-cell RNA-sequencing (scRNA-seq) technologies and associated analysis methods have rapidly developed in recent years. This includes preprocessing methods, which assign sequencing reads to genes to create count matrices for downstream analysis. While several packaged preprocessing workflows have been developed to provide users with convenient tools for handling this process, how they compare to one another and how they influence downstream analysis have not been well studied.
Results
Here, we systematically benchmark the performance of 10 end-to-end preprocessing workflows ( Cell Ranger , Optimus , salmon alevin , alevin-fry , kallisto bustools , dropSeqPipe , scPipe , zUMIs , celseq2 , and scruff ) using datasets yielding different biological complexity levels generated by CEL-Seq2 and 10x Chromium platforms. We compare these workflows in terms of their quantification properties directly and their impact on normalization and clustering by evaluating the performance of different method combinations. While the scRNA-seq preprocessing workflows compared vary in their detection and quantification of genes across datasets, after downstream analysis with performant normalization and clustering methods, almost all combinations produce clustering results that agree well with the known cell type labels that provided the ground truth in our analysis.
Conclusions
In summary, the choice of preprocessing method was found to be less important than other steps in the scRNA-seq analysis process. Our study comprehensively compares common scRNA-seq preprocessing workflows and summarizes their characteristics to guide workflow users.
Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNA sequencing (RNA-Seq) applications have evolved in conjunction with sequence technology and bioinformatic tools advances. In most projects, bulk RNA-Seq data is used to measure gene expression patterns, isoform expression, alternative splicing and single-nucleotide polymorphisms. However, RNA-Seq holds far more hidden biological information including details of copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Recent novel and advanced bioinformatic algorithms developed the capacity to retrieve this information from bulk RNA-Seq data, thus broadening its scope. The focus of this review is to comprehend the emerging bulk RNA-Seq-based analyses, emphasizing less familiar and underused applications. In doing so, we highlight the power of bulk RNA-Seq in providing biological insights.
Sequencing technologies utilizing nucleotide conversion techniques such as cytosine-to- thymine in bisulfite-seq and thymine-to-cytosine in SLAM seq are powerful tools to explore the chemical intricacies of cellular processes. To date, no one has developed a unified methodology for aligning converted sequences and consolidating alignment of these technologies in one package. In this paper, we describe HISAT-3N (hierarchical indexing for spliced alignment of transcripts - 3 nucleotides), which can rapidly and accurately align sequences consisting of any nucleotide conversion by leveraging the powerful hierarchical index and repeat index algorithms originally developed for the HISAT software. Tests on real and simulated data sets demonstrate that HISAT-3N is faster than other modern systems, with greater alignment accuracy, higher scalability, and smaller memory requirements. HISAT-3N therefore becomes an ideal aligner when used with converted sequence technologies.
Emerging single-cell technologies profile multiple types of molecules within individual cells. A fundamental step in the analysis of the produced high-dimensional data is their visualization using dimensionality reduction techniques such as t-SNE and UMAP. We introduce j-SNE and j-UMAP as their natural generalizations to the joint visualization of multimodal omics data. Our approach automatically learns the relative contribution of each modality to a concise representation of cellular identity that promotes discriminative features but suppresses noise. On eight datasets, j-SNE and j-UMAP produce unified embeddings that better agree with known cell types and that harmonize RNA and protein velocity landscapes.
Single-cell RNA sequencing (scRNA-seq) is a high-throughput sequencing technology performed at the level of an individual cell, which can have a potential to understand cellular heterogeneity. However, scRNA-seq data are high-dimensional, noisy, and sparse data. Dimension reduction is an important step in downstream analysis of scRNA-seq. Therefore, several dimension reduction methods have been developed. We developed a strategy to evaluate the stability, accuracy, and computing cost of 10 dimensionality reduction methods using 30 simulation datasets and five real datasets. Additionally, we investigated the sensitivity of all the methods to hyperparameter tuning and gave users appropriate suggestions. We found that t-distributed stochastic neighbor embedding (t-SNE) yielded the best overall performance with the highest accuracy and computing cost. Meanwhile, uniform manifold approximation and projection (UMAP) exhibited the highest stability, as well as moderate accuracy and the second highest computing cost. UMAP well preserves the original cohesion and separation of cell populations. In addition, it is worth noting that users need to set the hyperparameters according to the specific situation before using the dimensionality reduction methods based on non-linear model and neural network.
Understanding global communications among cells requires accurate representation of cell-cell signaling links and effective systems-level analyses of those links. We construct a database of interactions among ligands, receptors and their cofactors that accurately represent known heteromeric molecular complexes. We then develop CellChat, a tool that is able to quantitatively infer and analyze intercellular communication networks from single-cell RNA-sequencing (scRNA-seq) data. CellChat predicts major signaling inputs and outputs for cells and how those cells and signals coordinate for functions using network analysis and pattern recognition approaches. Through manifold learning and quantitative contrasts, CellChat classifies signaling pathways and delineates conserved and context-specific pathways across different datasets. Applying CellChat to mouse and human skin datasets shows its ability to extract complex signaling patterns. Our versatile and easy-to-use toolkit CellChat and a web-based Explorer (http://www.cellchat.org/) will help discover novel intercellular communications and build cell-cell communication atlases in diverse tissues.
Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.
Intestinal epithelium architecture is sustained by stem cell division. In principle, stem cells can divide symmetrically to generate two identical copies of themselves or asymmetrically to sustain tissue renewal in a balanced manner. The choice between the two helps preserve stem cell and progeny pools and is crucial for tissue homeostasis. Control of spindle orientation is a prime contributor to the specification of symmetric versus asymmetric cell division. Competition for space within the niche may be another factor limiting the stem cell pool. An integrative view of the multiple links between intracellular and extracellular signals and molecular determinants at play remains a challenge. One outstanding question is the precise molecular roles of the tumour suppressor Adenomatous polyposis coli (APC) for sustaining gut homeostasis through its respective functions as a cytoskeletal hub and a down regulator in Wnt signalling. Here, we review our current understanding of APC inherent activities and partners in order to explore novel avenues by which APC may act as a gatekeeper in colorectal cancer and as a therapeutic target.
Macrophage populations in most mammalian organs consist of cells of different origin. Resident macrophages originate from erythromyeloid precursors of the yolk sac wall; maintenance of the numbers of such macrophages in postnatal ontogenesis is practically independent of bone marrow haematopoiesis. The largest populations of the resident macrophages of embryonic origin are found in the central nervous system (microglia) and liver (Kupffer cells). In contrast, skin dermis and mucous membranes become predominantly colonized by bone marrow-derived monocytes that show pronounced functional and phenotypic plasticity. In the present study, we compared Kupffer cells and monocytes using the immunophenotype, gene expression profile, proteome, and pool of microRNA. The observed differences did not consider the resident liver macrophages as purely M2 macrophages or state that monocytes have purely M1 features. Monocytes show signs of high plasticity and sensitivity to pathogen-associated molecular patterns (e.g., high levels of transcription for Tlr 2, 4, 7, and 8). In contrast, the resident liver macrophages were clearly involved in the regulation of specific organ functions (nitrogen metabolism, complement system protein synthesis).
Tooth enamel, a highly mineralized tissue covering the outermost area of teeth, is always damaged by dental caries or trauma. Tooth enamel rarely repairs or renews itself, due to the loss of ameloblasts and dental epithelial stem cells (DESCs) once the tooth erupts. Unlike human teeth, mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages. The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells; explore the regulatory mechanisms underlying enamel formation; and help answer the open question regarding the therapeutic development of enamel engineering. In the present review, we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs. The roles of DESCs during homeostasis and repair are also discussed, which should improve our knowledge regarding enamel tissue engineering.
Background
RNA sequencing (RNA-seq) is an ever increasingly popular tool for transcriptome profiling. A key point to make the best use of the available data is to provide software tools that are easy to use but still provide flexibility and transparency in the adopted methods. Despite the availability of many packages focused on detecting differential expression, a method to streamline this type of bioinformatics analysis in a comprehensive, accessible, and reproducible way is lacking.
Results
We developed the ideal software package, which serves as a web application for interactive and reproducible RNA-seq analysis, while producing a wealth of visualizations to facilitate data interpretation. ideal is implemented in R using the Shiny framework, and is fully integrated with the existing core structures of the Bioconductor project. Users can perform the essential steps of the differential expression analysis workflow in an assisted way, and generate a broad spectrum of publication-ready outputs, including diagnostic and summary visualizations in each module, all the way down to functional analysis. ideal also offers the possibility to seamlessly generate a full HTML report for storing and sharing results together with code for reproducibility.
Conclusion
ideal is distributed as an R package in the Bioconductor project (http://bioconductor.org/packages/ideal/), and provides a solution for performing interactive and reproducible analyses of summarized RNA-seq expression data, empowering researchers with many different profiles (life scientists, clinicians, but also experienced bioinformaticians) to make the ideal use of the data at hand.
Hematopoietic stem and progenitor cells (HSPC) experience a functional decline in response to chronic inflammation or aging. Haploinsufficiency of A20, or TNFAIP3, an innate immune regulator, is associated with a variety of autoimmune, inflammatory, and hematologic malignancies. Based on a prior analysis of epigenomic and transcriptomic changes during normal human aging, we find that the expression of A20 is significantly reduced in aged HSPC as compared to young HSPC. Here, we show that the partial reduction of A20 expression in young HSPC results in characteristic features of aging. Specifically, heterozygous deletion of A20 in hematopoietic cells resulted in expansion of the HSPC pool, reduced HSPC fitness, and myeloid-biased hematopoiesis. These findings suggest that altered expression of A20 in HSPC contributes to an aging-like phenotype, and that there may be a common underlying mechanism for diminished HSPC function between inflammatory states and aging.
The β-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent β-globin chain synthesis. HbE/β-thalassemia is a subtype of β-thalassemia with extremely high frequency in Asia. Studying molecular defects behind β-thalassemia is severely impeded by paucity of material from patients and lack of suitable cell lines. Approaches to derive erythroid cells from induced pluripotent stem cells (iPSCs) created from patients are confounded by poor levels of erythroid cell expansion, aberrant or incomplete erythroid differentiation and foetal/embryonic rather than adult globin expression. In this study we generate an immortalised erythroid cell line from peripheral blood stem cells of a HbE/β-thalassemia patient. Morphological analysis shows the cells are proerythroblasts with some early basophilic erythroblasts, with no change in morphology over time in culture. The line differentiates along the erythroid pathway to orthochromatic erythroblasts and reticulocytes. Importantly, unlike iPSCs, the line maintains the haemoglobin profile of the patient’s red blood cells. This is the first human cellular model for β-thalassemia providing a sustainable source of disease cells for studying underlying disease mechanisms and for use as drug screening platform, particularly for reagents designed to increase foetal haemoglobin expression as we have additionally demonstrated with hydroxyurea.
The B cell surface antigen CD19 is a target for treating B cell malignancies, such as B cell precursor acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma. The BiTE® immuno-oncology platform includes blinatumomab, which is approved for relapsed/refractory B cell precursor acute lymphoblastic leukemia and B cell precursor acute lymphoblastic leukemia with minimal residual disease. Blinatumomab is also being evaluated in combination with other agents (tyrosine kinase inhibitors, checkpoint inhibitors, and chemotherapy) in various treatment settings, including frontline protocols. An extended half-life BiTE molecule is also under investigation. Patients receiving blinatumomab may experience cytokine release syndrome and neurotoxicity; however, these events may be less frequent and severe than in patients receiving other CD19-targeted immunotherapies, such as chimeric antigen receptor T cell therapy. We review BiTE technology for treating malignancies that express CD19, analyzing the benefits and limitations of this bispecific T cell engager platform from clinical experience with blinatumomab.
Single cell transcriptomics technologies have vast potential in advancing our understanding of biology and disease. Here, Sarah Aldridge and Sarah Teichmann review the last decade of technological advancements in single-cell transcriptomics and highlight some of the recent discoveries enabled by this technology.
Significance
Bone marrow failure (BMF) syndromes are inherited life-threatening conditions characterized by low blood cell production and predisposition to cancer. In this study we report a germ line frameshift variant in the Sp1 transcription factor in a family of patients with BMF and acute leukemia. Sp1 is ubiquitously expressed in human tissues and regulates transcription for blood cell lineage specification. Dissecting the molecular function of this SP1 variant revealed a hypermorphic effect, triggering superactivation of Sp1-mediated transcription in the patients’ blood. To our knowledge, this is the first report of a naturally occurring germ line variant in SP1 that alters transcriptional networks and disrupts hematopoiesis in humans.
The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.
HOPX is a stem cell marker in hair follicles and intestines. It was shown critical for primitive hematopoiesis. We previously showed an association between higher HOPX expression and clinical characteristics related to stemness and quiescence of leukemic cells in acute myeloid leukemia (AML) patients. To further explore its physiologic functions in hematopoietic system, we generated a mouse model with hematopoietic cell-specific knockout of Hopx (Hopx−/−). In young Hopx−/− mice, the hematopoietic stem cells (HSC) showed decreased reconstitution ability after serial transplantation. Further transcriptomic study revealed decreased HSC signatures in long-term HSCs from the Hopx−/− mice. At 18 months of age, half of the Hopx−/− mice developed cytopenia and splenomegaly. Bone marrow (BM) from the sick mice showed myeloid hyperplasia with predominant mature neutrophils, and decreased progenitor cells and lymphocytes. These phenotypes suggested critical functions of Hopx in maintaining HSC quiescence. Transcriptomic study of the Hopx−/− marrow cells showed significant downregulation of the Cxcl12-Cxcr4 axis, which is critical for maintenance of HSC quiescence. We next examined the role of Hopx in AML by using the MN1 overexpression murine leukemia model. Mice transplanted with MN1-overexpressed Hopx−/− BM cells developed AML with more aggressive phenotypes compared with those transplanted with MN1-overexpressed Hopx-wild cells. Hopx−/−MN1-overexpressed leukemia cells showed higher proliferation rate and downregulation of Cxcl12 and Cxcr4. Furthermore, in human AML, BM plasma CXCL12 levels were lower in patients with lower HOPX expression. In conclusion, our study highlights the roles of Hopx in maintenance of quiescence of the hematopoietic stem cells through CXCL12 pathway in vivo and provides implication of this protein in normal and malignant hematopoiesis.
Many eukaryotic genomes harbour large numbers of duplicated sequences, of diverse biotypes, resulting from several mechanisms including recombination, whole genome duplication and retro-transposition. Such repeated sequences complicate gene/transcript quantification during RNA-seq analysis due to reads mapping to more than one locus, sometimes involving genes embedded in other genes. Genes of different biotypes have dissimilar levels of sequence duplication, with long-noncoding RNAs and messenger RNAs sharing less sequence similarity to other genes than biotypes encoding shorter RNAs. Many strategies have been elaborated to handle these multi-mapped reads, resulting in increased accuracy in gene/transcript quantification, although separate tools are typically used to estimate the abundance of short and long genes due to their dissimilar characteristics. This review discusses the mechanisms leading to sequence duplication, the biotypes affected, the computational strategies employed to deal with multi-mapped reads and the challenges that still remain to be overcome.
Circularized transcript isoforms due to back-splicing are increasingly being reported in different tissues types and pathological states including cancer. Since these circular RNAs (circRNAs) are more stable than linear messenger RNA their identification and profiling in tumor tissue could aid in stratifying patients and may serve as biomarkers. In this study, we have investigated the relationship between circRNA expression and tumor grade in a cohort of 58, mostly non-muscle-invasive bladder cancer patients. From 4571 circRNAs detected, we identified 157 that were significantly differentially expressed between tumor grades relative to the linear transcript. We demonstrated that such grade-related differences can be identified in an independent cohort, and that a large fraction of circRNAs can be, in principle, detected in urine. The differentially expressed circRNAs cluster into subgroups according to their co-expression, subgroups which are enriched for DNA repair, cell cycle and intracellular signaling genes. Since one proposed function of circRNAs is to interfere with gene-regulation by acting as microRNA “sponges,” candidates which were differentially expressed between tumor grades were investigated for potential miRNA target sites. By investigating the circRNAs from bladder cancer related pathways we demonstrated that the expression of these pathways, the circRNAs, and their parental genes are often decoupled and do not correlate, yet that some circRNAs do not follow this tendency. The present study provides the next step for the comprehensive evaluation of this novel class of RNAs in the context of non-muscle-invasive bladder cancer. Intriguingly, despite their possible function as microRNA sponges, they potentially affect host mRNA levels at the transcriptional stage, as compared to post-transcriptional control by miRNAs. Our analysis indicates differences of their activity between bladder cancer tumor stages, and their relative expression levels may provide an additional layer of information for patient stratification.
In single-cell RNA-seq (scRNA-seq) experiments, the number of individual cells has increased exponentially, and the sequencing depth of each cell has decreased significantly. As a result, analyzing scRNA-seq data requires extensive considerations of program efficiency and method selection. In order to reduce the complexity of scRNA-seq data analysis, we present scedar, a scalable Python package for scRNA-seq exploratory data analysis. The package provides a convenient and reliable interface for performing visualization, imputation of gene dropouts, detection of rare transcriptomic profiles, and clustering on large-scale scRNA-seq datasets. The analytical methods are efficient, and they also do not assume that the data follow certain statistical distributions. The package is extensible and modular, which would facilitate the further development of functionalities for future requirements with the open-source development community. The scedar package is distributed under the terms of the MIT license at https://pypi.org/project/scedar.
Background:
With the rapid development of single-cell RNA sequencing technology, it is possible to dissect cell-type composition at high resolution. A number of methods have been developed with the purpose to identify rare cell types. However, existing methods are still not scalable to large datasets, limiting their utility. To overcome this limitation, we present a new software package, called GiniClust3, which is an extension of GiniClust2 and significantly faster and memory-efficient than previous versions.
Results:
Using GiniClust3, it only takes about 7 h to identify both common and rare cell clusters from a dataset that contains more than one million cells. Cell type mapping and perturbation analyses show that GiniClust3 could robustly identify cell clusters.
Conclusions:
Taken together, these results suggest that GiniClust3 is a powerful tool to identify both common and rare cell population and can handle large dataset. GiniCluster3 is implemented in the open-source python package and available at https://github.com/rdong08/GiniClust3.
Single-cell transcriptomics offers unprecedented opportunities to infer the ligand-receptor (LR) interactions underlying cellular networks. We introduce a new, curated LR database and a novel regularized score to perform such inferences. For the first time, we try to assess the confidence in predicted LR interactions and show that our regularized score outperforms other scoring schemes while controlling false positives. SingleCellSignalR is implemented as an open-access R package accessible to entry-level users and available from https://github.com/SCA-IRCM. Analysis results come in a variety of tabular and graphical formats. For instance, we provide a unique network view integrating all the intercellular interactions, and a function relating receptors to expressed intracellular pathways. A detailed comparison of related tools is conducted. Among various examples, we demonstrate SingleCellSignalR on mouse epidermis data and discover an oriented communication structure from external to basal layers.
The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
Background:
Single-cell RNA-sequencing (scRNA-seq) is a fast emerging technology allowing global transcriptome profiling on the single cell level. Cell type identification from scRNA-seq data is a critical task in a variety of research such as developmental biology, cell reprogramming, and cancers. Typically, cell type identification relies on human inspection using a combination of prior biological knowledge (e.g. marker genes and morphology) and computational techniques (e.g. PCA and clustering). Due to the incompleteness of our current knowledge and the subjectivity involved in this process, a small amount of cells may be subject to mislabelling.
Results:
Here, we propose a semi-supervised learning framework, named scReClassify, for 'post hoc' cell type identification from scRNA-seq datasets. Starting from an initial cell type annotation with potentially mislabelled cells, scReClassify first performs dimension reduction using PCA and next applies a semi-supervised learning method to learn and subsequently reclassify cells that are likely mislabelled initially to the most probable cell types. By using both simulated and real-world experimental datasets that profiled various tissues and biological systems, we demonstrate that scReClassify is able to accurately identify and reclassify misclassified cells to their correct cell types.
Conclusions:
scReClassify can be used for scRNA-seq data as a post hoc cell type classification tool to fine-tune cell type annotations generated by any cell type classification procedure. It is implemented as an R package and is freely available from https://github.com/SydneyBioX/scReClassify.
Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73-), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38-/CD45RA-/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT).
Expression Atlas is EMBL-EBI's resource for gene and protein expression. It sources and compiles data on the abundance and localisation of RNA and proteins in various biological systems and contexts and provides open access to this data for the research community. With the increased availability of single cell RNA-Seq datasets in the public archives, we have now extended Expression Atlas with a new added-value service to display gene expression in single cells. Single Cell Expression Atlas was launched in 2018 and currently includes 123 single cell RNA-Seq studies from 12 species. The website can be searched by genes within or across species to reveal experiments, tissues and cell types where this gene is expressed or under which conditions it is a marker gene. Within each study, cells can be visualized using a pre-calculated t-SNE plot and can be coloured by different features or by cell clusters based on gene expression. Within each experiment, there are links to downloadable files, such as RNA quantification matrices, clustering results, reports on protocols and associated metadata, such as assigned cell types.
The increasingly recognized role of different types of B cells and plasma cells in protective and pathogenic immune responses combined with technological advances have generated a plethora of information regarding the heterogeneity of this human immune compartment. Unfortunately, the lack of a consistent classification of human B cells also creates significant imprecision on the adjudication of different phenotypes to well-defined populations. Additional confusion in the field stems from: the use of non-discriminatory, overlapping markers to define some populations, the extrapolation of mouse concepts to humans, and the assignation of functional significance to populations often defined by insufficient surface markers. In this review, we shall discuss the current understanding of human B cell heterogeneity and define major parental populations and associated subsets while discussing their functional significance. We shall also identify current challenges and opportunities. It stands to reason that a unified approach will not only permit comparison of separate studies but also improve our ability to define deviations from normative values and to create a clean framework for the identification, functional significance, and disease association with new populations.
Hematopoietic stem cells (HSCs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. It has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. The double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a+ runx1+) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. Transcriptome analysis revealed that gata2a+ runx1+ cells showed typical molecular signatures of HSCs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. Transplantation assays demonstrated that long-term repopulating HSCs were highly enriched within the gata2a+ runx1+ fraction. In contrast, colony-forming assays showed that gata2a− runx1+ cells abundantly contain erythroid- and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.
Transcription factor (TF)‐based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial‐like precursors that generate hematopoietic stem progenitor (HSPC)‐like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC‐like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell‐based therapeutics.
Database
Gene expression data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE130361 .
Hematopoietic stem cells (HSCs) maintain lifelong production of mature blood cells and regenerate the hematopoietic system after cytotoxic injury. Use of expanding cell surface marker panels and advanced functional analyses have revealed the presence of several immunophenotypically different HSC subsets with distinct self-renewal and repopulating capacity and bias toward selective lineage differentiation. This chapter summarizes current understanding of the phenotypic and functional heterogeneity within the HSC pool, with emphasis on the immunophenotypes and functional features of several known HSC subsets, and their roles in steady-state and emergency hematopoiesis, and in aging. The chapter also highlights some of the future research directions to elucidate further the biology and function of different HSC subsets in health and disease states.
CD3⁻CD56⁺ NK cells develop from CD34⁺ hematopoietic progenitors (HPCs) in vivo, and this process can be recapitulated in vitro. The prevailing model is that human NK cell development occurs along a continuum whereby common lymphocyte progenitors (CLPs) gradually downregulate CD34 and upregulate CD56. Acquisition of CD94 marks commitment to the CD56bright stage, and CD56bright NK cells subsequently differentiate into CD56dim NK cells that upregulate CD16 and killer immunoglobulin-like receptors (KIR). Support for this linear model comes from analyses of cell populations in secondary lymphoid tissues and in vitro studies of NK cell development from HPCs. However, several lines of evidence challenge this linear model and suggest a more branched model whereby different precursor populations may independently develop into distinct subsets of mature NK cells. A more definitive understanding of human NK cell development is needed to inform in vitro differentiation strategies designed to generate NK cells for immunotherapy. In this review, we summarize current evidence supporting the linear and branched models of human NK cell development and the challenges associated with reaching definitive conclusions.
Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.
Cell type identification is essential for single-cell RNA sequencing (scRNA-seq) studies, currently transforming the life sciences. CHETAH (CHaracterization of cEll Types Aided by Hierarchical classification) is an accurate cell type identification algorithm that is rapid and selective, including the possibility of intermediate or unassigned categories. Evidence for assignment is based on a classification tree of previously available scRNA-seq reference data and includes a confidence score based on the variance in gene expression per cell type. For cell types represented in the reference data, CHETAH's accuracy is as good as existing methods. Its specificity is superior when cells of an unknown type are encountered, such as malignant cells in tumor samples which it pinpoints as intermediate or unassigned. Although designed for tumor samples in particular, the use of unassigned and intermediate types is also valuable in other exploratory studies. This is exemplified in pancreas datasets where CHETAH highlights cell populations not well represented in the reference dataset, including cells with profiles that lie on a continuum between that of acinar and ductal cell types. Having the possibility of unassigned and intermediate cell types is pivotal for preventing misclassification and can yield important biological information for previously unexplored tissues.
Ever since hematopoietic stem cells (HSCs) were first identified half a century ago, their differentiation roadmap has been extensively studied. The classical model of hematopoiesis has long held as a dogma that HSCs reside at the top of a hierarchy in which HSCs possess self-renewal capacity and can progressively give rise to all blood lineage cells. However, over the past several years, with advances in single cell technologies, this developmental scheme has been challenged. In this review, we discuss the evidence supporting heterogeneity within HSC and progenitor populations as well as the hierarchical models revised by novel approaches mainly in mouse system. These evolving views provide further understanding of hematopoiesis and highlight the complexity of hematopoietic differentiation.
Background
Principal component analysis (PCA) is frequently used in genomics applications for quality assessment and exploratory analysis in high-dimensional data, such as RNA sequencing (RNA-seq) gene expression assays. Despite the availability of many software packages developed for this purpose, an interactive and comprehensive interface for performing these operations is lacking.
Results
We developed the pcaExplorer software package to enhance commonly performed analysis steps with an interactive and user-friendly application, which provides state saving as well as the automated creation of reproducible reports. pcaExplorer is implemented in R using the Shiny framework and exploits data structures from the open-source Bioconductor project. Users can easily generate a wide variety of publication-ready graphs, while assessing the expression data in the different modules available, including a general overview, dimension reduction on samples and genes, as well as functional interpretation of the principal components.
Conclusion
pcaExplorer is distributed as an R package in the Bioconductor project (http://bioconductor.org/packages/pcaExplorer/), and is designed to assist a broad range of researchers in the critical step of interactive data exploration.
Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease. : Ludwig et al. chart the dynamic transcriptional and chromatin landscapes as hematopoietic stem and progenitor cells differentiate into mature red blood cells. This multi-omic profiling reveals dynamic transcription factor activities and human genetic variation that modulate this process. Keywords: erythropoiesis, red blood cell, chromatin accessibility, transcriptomics, GWAS, human genetics, hematopoiesis
Maintenance of the mature blood cells requires controlled cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). While our knowledge of the gene expression changes that facilitate differentiation has made a leap forward, less is known about the cellular triggers that induce them. Biedzinski et al (2020) now uncover a new intracellular mechanism that drives myeloid differentiation: Microtubule bundles squeeze the nucleus of HSPCs and form large invaginations, thus causing changes in chromatin organization. These microtubule-induced nuclear shape changes result in gene expression profiles that favor myeloid differentiation.
CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.
Hematopoiesis has long served as a paradigm of stem cell biology and tissue homeostasis. In the past decade, the genomics revolution has ushered in powerful new methods for investigating the hematopoietic system that have provided transformative insights into its biology. As part of the advances in genomics, increasingly accurate deep sequencing and novel methods of cell tracking have revealed hematopoiesis to be more of a continuous and less of a discrete and punctuated process than originally envisioned. In part, this continuous nature of hematopoiesis is made possible by the emergent outcomes of vast, interconnected regulatory networks that influence cell fates and lineage commitment. It is also becoming clear how these mechanisms are modulated by genetic variation present throughout the population. This review describes how these recently uncovered complexities are reshaping our concept of tissue development and homeostasis while opening up a more comprehensive future understanding of hematopoiesis.
Purpose of review:
Advances in medical care and preventive measures have contributed to increasing life expectancy. Therefore, it is critical to expand our understanding of the physiological and pathophysiological adaptations of the hematological system in aging. We highlight and review the findings from recent investigations aimed at understanding the effects of aging on megakaryocytes and platelets.
Recent findings:
Biochemical and transcriptomic studies of megakaryocytes and platelets from older humans and mice have advanced our understanding of the molecular and functional characteristics of megakaryocytes and platelets during aging. These studies have led to the identification of metabolic and inflammatory pathways associated with the generation of hyperreactive platelets that may significantly contribute to the high incidence of thrombosis in aging.
Summary:
By increasing our research efforts to understand and identify the characteristics of megakaryocytes and platelets in aging, we will increase our potential to develop novel therapies aimed at decreasing the incidence of aging-associated thrombosis. These efforts will also serve as a foundation to better understand the role of megakaryocytes and platelets in other age-related hematological conditions with high thrombotic risk such as clonal hematopoiesis of indeterminate potential and myeloproliferative neoplasms.
The generation of all blood cell lineages (hematopoiesis) is sustained throughout the entire life span of adult mammals. Studies using cell transplantation identified the self-renewing, multipotent hematopoietic stem cells (HSCs) as the source of hematopoiesis in adoptive hosts and delineated a hierarchy of HSC-derived progenitors that ultimately yield mature blood cells. However, much less is known about adult hematopoiesis as it occurs in native hosts, i.e., without transplantation. Here we review recent advances in our understanding of native hematopoiesis, focusing in particular on the application of genetic lineage tracing in mice. The emerging evidence has established HSCs as the major source of native hematopoiesis, helped to define the kinetics of HSC differentiation, and begun exploring native hematopoiesis in stress conditions such as aging and inflammation. Major outstanding questions about native hematopoiesis still remain, such as its clonal composition, the nature of lineage commitment, and the dynamics of the process in humans.
Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 36 is October 6, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Purpose of review:
Although hematopoietic stem cell (HSC) function has long been studied by transplantation assays, this does not reflect what HSCs actually do in their native context. Here, we review recent technologic advances that facilitate the study of HSCs in their native context focusing on inducible HSC-specific lineage tracing and inference of hematopoietic trajectories through single-cell RNA sequencing (scRNA-Seq).
Recent findings:
Lineage tracing of HSCs at the population level using multiple systems has suggested that HSCs make a major contribution to steady-state hematopoiesis. Although several genetic systems and novel methods for lineage tracing individual hematopoietic clones have been described, the technology for tracking these cellular barcodes (in particular mutations or insertion sites) is still in its infancy. Thus, lineage tracing of HSC clones in the adult bone marrow remains elusive. Static snapshots of scRNA-Seq of hematopoietic populations have captured the heterogeneity of transcriptional profiles of HSCs and progenitors, with some cells displaying a unilineage signature as well as others with bi or multipotent lineage profiles. Kinetic analysis using HSC-specific lineage tracing combined with scRNA-Seq confirmed this heterogeneity of progenitor populations and revealed a rapid and early emergence of megakaryocytic progeny, followed by erythroid and myeloid lineages, whereas lymphoid differentiation emerged last.
Summary:
New approaches to study HSCs both in vivo through lineage tracing and at a high-resolution molecular level through scRNA-Seq are providing key insight into HSC differentiation in the absence of transplantation. Recent studies using these approaches are discussed here. These studies pave the way for integration of in-vivo clonal analysis of HSC behavior over time with single-cell sequencing data, including but not limited to transcriptomic, proteomic, and epigenomic, to establish a comprehensive molecular and cellular map of hematopoiesis.
Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.
Cell–cell communication mediated by ligand–receptor complexes is critical to coordinating diverse biological processes, such as development, differentiation and inflammation. To investigate how the context-dependent crosstalk of different cell types enables physiological processes to proceed, we developed CellPhoneDB, a novel repository of ligands, receptors and their interactions. In contrast to other repositories, our database takes into account the subunit architecture of both ligands and receptors, representing heteromeric complexes accurately. We integrated our resource with a statistical framework that predicts enriched cellular interactions between two cell types from single-cell transcriptomics data. Here, we outline the structure and content of our repository, provide procedures for inferring cell–cell communication networks from single-cell RNA sequencing data and present a practical step-by-step guide to help implement the protocol. CellPhoneDB v.2.0 is an updated version of our resource that incorporates additional functionalities to enable users to introduce new interacting molecules and reduces the time and resources needed to interrogate large datasets. CellPhoneDB v.2.0 is publicly available, both as code and as a user-friendly web interface; it can be used by both experts and researchers with little experience in computational genomics. In our protocol, we demonstrate how to evaluate meaningful biological interactions with CellPhoneDB v.2.0 using published datasets. This protocol typically takes ~2 h to complete, from installation to statistical analysis and visualization, for a dataset of ~10 GB, 10,000 cells and 19 cell types, and using five threads. CellPhoneDB combines an interactive database and a statistical framework for the exploration of ligand–receptor interactions inferred from single-cell transcriptomics measurements.
Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users.
The mechanism underlying production of various types of blood cells from hematopoietic stem and progenitor cells has been a central theme in hematology. Conventionally, hematopoietic cell populations are analyzed by cell surface markers to judge cell types and differentiation stages, and by transplantation assays to assess differentiation potential. Recently, however, next-generation sequencing technology has enabled single-cell transcriptome and epigenome analyses and cell barcoding-based lineage tracing during unperturbed hematopoiesis. These innovative assays revealed that each cell population is extensively heterogenous. Many cells within hematopoietic stem cell populations may not be multipotent, and conversely, hematopoietic progenitor cells often display self-renewal capacity. Moreover, cells tend to make their lineage choice much earlier than previously thought. Altogether, these results challenge the current hierarchical differentiation models and propose new continuous models. Single-cell analyses are expected to greatly contribute to our understanding of normal and abnormal hematopoiesis and to the development of new therapies for blood disorders.
Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function. This Review discusses advances in RNA-sequencing technologies and methods over the past decade and outlines adaptations that are enabling a fuller understanding of RNA biology, from when and where an RNA is expressed to the structures it adopts.
Unveiling the mechanisms and the cellular dynamics at the basis of human hematopoietic homeostasis has been a main focus for the scientific community since the discovery of a pool of multipotent hematopoietic stem cells (HSCs) capable of sustaining the hematopoietic output throughout life and after transplantation. Recently, new works shed light on the (1) differentiation paths, (2) size and replication rate of human HSC population at steady state, and (3) role of the distinct subpopulations comprising the hematopoietic stem and progenitor cell reservoir after transplantation. These papers exploited cutting-edge technologies, including vector integration site clonal tracking, spontaneous mutations, and deep transcriptome profiling. Here we discuss the latest updates in human hematopoietic system biology and in vivo dynamics, highlighting novel concepts and common findings deriving from different approaches and the future directions of these studies. Taken together, this information contributed to partially resolving the complexity of the in vivo HSC behavior and has major implications for HSC transplantation and gene therapy as well as for the development of future therapies.