Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis.

BACKGROUND: Hematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry. RESULTS: With fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling. CONCLUSIONS: These comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

Modes and Mechanisms of Salivary Gland Epithelial Cell Death in Sjogren's Syndrome.

Sjogren's syndrome is an autoimmune disease in middle and old-aged women with a dry mucosal surface, which is caused by the dysfunction of secretory glands, such as the oral cavity, eyeballs, and pharynx. Pathologically, Sjogren's syndrome are characterized by lymphocyte infiltration into the exocrine glands and epithelial cell destruction caused by autoantibodies Ro/SSA and La/SSB. At present, the exact pathogenesis of Sjogren's syndrome is unclear. Evidence suggests epithelial cell death and the subsequent dysfunction of salivary glands as the main causes of xerostomia. This review summarizes the modes of salivary gland epithelial cell death and their role in Sjogren's syndrome progression. The molecular mechanisms involved in salivary gland epithelial cell death during Sjogren's syndrome as potential leads to treating the disease are also discussed.

Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis.

Upon migrating into the tissues, hematopoietic stem cell (HSC)-derived monocytes differentiate into macrophages, playing a crucial role in determining innate immune responses towards external pathogens and internal stimuli. However, the regulatory mechanisms underlying monocyte-to-macrophage differentiation remain largely unexplored. Here we divulge a previously uncharacterized but essential role for an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), in monocyte-to-macrophage maturation. FLRT2 is almost undetectable in human monocytic cell lines, human peripheral blood mononuclear cells (PBMCs), and mouse primary monocytes but significantly increases in fully differentiated macrophages. Myeloid-specific deletion of FLRT2 (Flrt2ΔMyel ) contributes to decreased peritoneal monocyte-to-macrophage generation in mice in vivo, accompanied by impaired macrophage functions. Gain- and loss-of-function studies support the promoting effect of FLRT2 on THP-1 cell and human PBMC differentiation into macrophages. Mechanistically, FLRT2 directly interacts with Unc-5 netrin receptor B (UNC5B) via its extracellular domain (ECD) and activates Akt/mTOR signaling. In vivo administration of mTOR agonist MYH1485 reverses the impaired phenotypes observed in Flrt2ΔMyel mice. Together, these results identify FLRT2 as a novel pivotal endogenous regulator of monocyte differentiation into macrophages. Targeting the FLRT2/UNC5B-Akt/mTOR axis may provide potential therapeutic strategies directly relevant to human diseases associated with aberrant monocyte/macrophage differentiation.

Preclinical transfusion-dependent humanized mouse model of beta thalassemia major.

A preclinical humanized mouse model of beta thalassemia major or Cooley anemia (CA) was generated by targeted gene replacement of the mouse adult globin genes in embryonic stem cells. The mouse adult alpha and beta globin genes were replaced with adult human alpha globin genes (alpha2alpha1) and a human fetal to adult hemoglobin (Hb)-switching cassette (gamma(HPFH)deltabeta(0)), respectively. Similar to human infants with CA, fully humanized mice survived postnatally by synthesizing predominantly human fetal Hb, HbF (alpha(2)gamma(2)), with a small amount of human minor adult Hb, HbA2 (alpha(2)delta(2)). Completion of the human fetal to adult Hb switch after birth resulted in severe anemia marked by erythroid hyperplasia, ineffective erythropoiesis, hemolysis, and death. Similar to human patients, CA mice were rescued from lethal anemia by regular blood transfusion. Transfusion corrected the anemia and effectively suppressed the ineffective erythropoiesis, but led to iron overload. This preclinical humanized animal model of CA will be useful for the development of new transfusion and iron chelation regimens, the study of iron homeostasis in disease, and testing of cellular and genetic therapies for the correction of thalassemia.

Loading of metal isotope-containing intercalators for mass cytometry-based high-throughput quantitation of exosome uptake at the single-cell level.

Nanometer-sized exosomes are being widely studied as cell-to-cell communicators and versatile drug vehicles. Characterizations of the biodistribution of these exosomes are essential for the evaluation of their biological functions and drug delivery efficacy. However, current technologies for exosome tracking rely on fluorescence and have the disadvantages of being low throughput due to the limited number of available channels and spectral spillover. Here, we reported the development of an engineering approach that involves loading of metal isotope-containing intercalators into exosomes to quantify exosome uptake at the single-cell level. We demonstrate that mass cytometry in conjunction with highly multivariate cellular phenotyping enables high-throughput identification of the in vivo fate of exosomes. Inspired by these insights into cellular distribution, we optimized the administration methods for exosome-based drug delivery, verifying the anticancer efficacy of these exosomes in a mouse model of breast cancer. The evaluation of exosome's fate in vivo at the single-cell level provides valuable insights into the functions of exosomes in vivo and facilitates the improvement of exosome-based therapy.

High-throughput single-cell analysis of exosome mediated dual drug delivery, in vivo fate and synergistic tumor therapy.

Exosomes could serve as delivery platforms, owing to their good biocompatibility, stability, and long blood circulation time. Tracking the biological fate of exosomes in vivo is essential for evaluating their functions, delivery efficacy, and biosafety, and it is invaluable for guiding exosome-based therapy. Here, we merged a single-cell technique, mass cytometry, with in vivo uptake analysis to comprehensively reveal the fate of exosomes at the single-cell level. In tandem with multivariate cellular phenotyping, in vivo uptake of exosomes labeled with heavy metal-containing tags was quantified in a high-throughput manner. Interestingly, an organ-dependent uptake landscape of exosomes by diverse cell types was distinctly demonstrated, which implied that cancer cells seemed to preferably take up more released drugs from the exosomes. Using these cellular insights, the administration method of drug-loaded exosomes was optimized to elevate their accumulation in tumor sites and minimize their spread into healthy organs. Dual drug-loaded exosomes were locally administered and superior synergistic tumor treatment effects were achieved in a solid tumor model. The disclosure of exosome cellular distribution, together with the successful engineering of exosomes with multiple anticancer capacities, provides a new level of insight into optimizing and enhancing exosome-based drug delivery and synergistic tumor therapy.

Allogeneic bone marrow transplant in the absence of cytoreductive conditioning rescues mice with ß-thalassemia major.

ß-thalassemia is a group of inherited blood disorders that result in defects in ß-globin chain production. Cooley anemia (CA), or ß-thalassemia major, is the most severe form of the disease and occurs when an individual has mutations in both copies of the adult ß-globin gene. Patients with CA fail to make adult hemoglobin, exhibit ineffective erythropoiesis, experience severe anemia, and are transfusion dependent for life. Currently, allogeneic bone marrow transplantation (BMT) is the only cure; however, few patients have suitable donors for this procedure, which has significant morbidity and mortality. In this study, a novel humanized murine model of CA is rescued from lethal anemia by allogeneic BMT in the absence of cytoreductive conditioning. A single intravenous postnatal injection of allogeneic bone marrow results in stable, mixed hematopoietic chimerism. Five months after transplantation, donor cells accounted for approximately 90% of circulating erythrocytes and up to 15% of hematopoietic stem and progenitor cells. Transplanted mice are transfusion independent, have marked improvement of hematological indices, exhibit no growth retardation or signs of graft-versus-host disease, and are fertile. This study describes a method for the consistent engraftment of allogeneic donor hematopoietic cells that rescues a humanized mouse model of CA from lethal anemia, all in the absence of toxic cytoreductive conditioning.

An In Vivo Gain-of-Function Screen Identifies the Williams-Beuren Syndrome Gene GTF2IRD1 as a Mammary Tumor Promoter.

The broad implementation of precision medicine in cancer is impeded by the lack of a complete inventory of the genes involved in tumorigenesis. We performed in vivo screening of ∼1,000 genes that are associated with signaling for positive roles in breast cancer, using lentiviral expression vectors in primary MMTV-ErbB2 mammary tissue. Gain of function of five genes, including RET, GTF2IRD1, ADORA1, LARS2, and DPP8, significantly promoted mammary tumor growth. We further studied one tumor-promoting gene, the transcription factor GTF2IRD1. The mis-regulation of genes downstream of GTF2IRD1, including TßR2 and BMPR1b, also individually promoted mammary cancer development, and silencing of TßR2 suppressed GTF2IRD1-driven tumor promotion. In addition, GTF2IRD1 is highly expressed in human breast tumors, correlating with high tumor grades and poor prognosis. Our in vivo approach is readily expandable to whole-genome annotation of tumor-promoting genes.

The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance.

The Par polarity proteins play key roles in asymmetric division of Drosophila melanogaster stem cells; however, whether the same mechanisms control stem cells in mammals is controversial. Although necessary for mammary gland morphogenesis, Par3 is not essential for mammary stem cell function. We discovered that, instead, a previously uncharacterized protein, Par3-like (Par3L), is vital for mammary gland stem cell maintenance. Par3L function has been mysterious because, unlike Par3, it does not interact with atypical protein kinase C or the Par6 polarity protein. We found that Par3L is expressed by multipotent stem cells in the terminal end buds of murine mammary glands. Ablation of Par3L resulted in rapid and profound stem cell loss. Unexpectedly, Par3L, but not Par3, binds to the tumour suppressor protein Lkb1 and inhibits its kinase activity. This interaction is key for the function of Par3L in mammary stem cell maintenance. Our data reveal insights into a link between cell polarity proteins and stem cell survival, and uncover a biological function for Par3L.

Epithelial homeostasis.

Epithelia form intelligent, dynamic barriers between the external environment and an organism's interior. Intercellular cadherin-based adhesions adapt and respond to mechanical forces and cell density, while tight junctions flexibly control diffusion both within the plasma membrane and between adjacent cells. Epithelial integrity and homeostasis are of central importance to survival, and mechanisms have evolved to ensure these processes are maintained during growth and in response to damage. For instance, cell competition surveys the fitness of cells within epithelia and removes the less fit; extrusion or delamination can remove apoptotic or defective cells from the epithelial sheet and can restore homeostasis when an epithelial layer becomes too crowded; spindle orientation ensures two-dimensional growth in simple epithelia and controls stratification in complex epithelia; and transition to a mesenchymal phenotype enables active escape from an epithelial layer. This review will discuss these various mechanisms and consider how they are subverted in disease.

Human globin knock-in mice complete fetal-to-adult hemoglobin switching in postnatal development.

Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult ß-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human ß-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and ß-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-ß-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.

Humanized mouse models of Cooley's anemia: correct fetal-to-adult hemoglobin switching, disease onset, and disease pathology.

beta thalassemia major or Cooley's Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This summary describes novel preclinical humanized mouse models of CA that survive on human fetal hemoglobin at birth and are blood-transfusion dependent for life upon completion of their human fetal-to-adult hemoglobin switch after birth. These CA models are the first to recapitulate the temporal onset of the disease in human patients. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; examining the onset of disease pathology; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Humanized Mouse Model of Cooley's Anemia.

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia.

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFbeta-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFbeta-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFbeta-SMMHC/FLT3-ITD-expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFbeta-SMMHC-expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFbeta-SMMHC/FLT3-ITD-reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFbeta-SMMHC in an animal model of inv(16)-associated AML.