Lineage-specific Markers Research Articles

Label-free isolation and cultivation of patient-matched human mammary epithelial and stromal cells from normal breast tissue.

Breast cancer is primarily derived from mammary epithelial cells, the main cell type in human mammary glands. The majority of knowledge gained thus far around breast cancer has come from research using immortalized epithelial cell lines. The use of primary cells derived from breast tissue can be used in research to provide more biological relevance representative of the heterogeneous nature of breast cancer development and metastasis in its natural microenvironment. However, the successful isolation and propagation of human primary mammary gland cells can be costly and difficult due to their complex in vivo microenvironment and sensitivity when isolated. Here, we present a gentle isolation method for viable human mammary epithelial cells (hMECs) and donor-matched human mammary fibroblasts (hMFbs) from human mammary gland tissue. We isolated, expanded and passaged the hMECs and hMFbs in vitro and characterized cultures using cell-specific markers. A total of four primary cell lines were isolated and established from normal breast tissue and characterized through various markers, including pan cytokeratin (panCK), CK14, CD44, CD31, fibronectin and vimentin by immunofluorescence. To determine functional potential for subsequent studies, epithelial cells were examined via Matrigel® assays to assess spheroid development. Both cell type cultures expressed lineage specific markers with hMECs but not hMFbs forming spheroid structures in 3D Matrigel® assays. Our analyses confirm the successful isolation of two different cell phenotypes from normal breast tissues. This robust technique provides an inexpensive and accessible approach for mammary cell isolation.

Up-regulation of Aquaporin 5 Defines Spasmolytic Polypeptide-Expressing Metaplasia and Progression to Incomplete Intestinal Metaplasia.

Background & AimsMetaplasia in the stomach is highly associated with development of intestinal-type gastric cancer. Two types of metaplasias, spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia (IM), are considered precancerous lesions. However, it remains unclear how SPEM and IM are related. Here we investigated a new lineage-specific marker for SPEM cells, aquaporin 5 (AQP5), to assist in the identification of these 2 metaplasias.MethodsDrug- or Helicobacter felis (H felis) infection-induced mouse models were used to identify the expression pattern of AQP5 in acute or chronic SPEM. Gene-manipulated mice treated with or without drug were used to investigate how AQP5 expression is regulated in metaplastic lesions. Metaplastic samples from transgenic mice and human gastric cancer patients were evaluated for AQP5 expression. Immunostaining with lineage-specific markers was used to differentiate metaplastic gland characteristics.ResultsOur results revealed that AQP5 is a novel lineage-specific marker for SPEM cells that are localized at the base of metaplastic glands initially and expand to dominate glands after chronic H felis infection. In addition, AQP5 expression was up-regulated early in chief cell reprogramming and was promoted by interleukin 13. In humans, metaplastic corpus showed highly branched structures with AQP5-positive SPEM. Human SPEM cells strongly expressing AQP5 were present at the bases of incomplete IM glands marked by TROP2 but were absent from complete IM glands.ConclusionsAQP5-expressing SPEM cells are present in pyloric metaplasia and TROP2-positive incomplete IM and may be an important component of metaplasia that can predict a higher risk for gastric cancer development.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even>64N) and extremely large cell (40-60 µm). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

A Review on Molecular Factors Affecting Stomach Development

Stomach as a highly important portion of the GIT which is responsible for storage, digestion of food, immune defense, and hormonal organization of metabolic homeostasis; it originates from posterior foregut. The transcription factors such as Forkhead Box A (Foxa1/2/3), Gata Binding Protein (Gata4/6), Hnf1 Homeobox B (Hnf1b), and Sry- Box Transcription Factor 2 (Sox2) have a crucial role in gastric specification. Endodermal foregut patterning and regionalization, mesenchymal differentiation and cell lineage specification of stomach are affected by many essential factors such as Retinoic acid (RA) signalling, Wingless-related integration site (Wnt) signals, fibroblast growth factor (FGF) signals, Bone morphogenetic protein (BMP) signalling, sonic hedgehog (Shh) signalling and GATA family .There is a lack for understanding the organ lineage relationships and lineage-specific markers during stomach development and differentiation. The objectives of this review are to focus on the normal development and the mesenchymal differentiation of the stomach and to highlight the relatively limited information we have about genes and transcription factors associated with stomach specification, regionalization, and differentiation of component cell lineages. Keywords: Stomach, Specification, Regionalization, Transcription Factor, Lineage-specific markers, Genes, Cell lineage specification. Read more →

Abstract 1018: SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid proliferation of leukemic blasts and high rate of acquired resistance to drugs. Differentiating agent ATRA has been established as a backbone of APL treatment, however its activity in other leukemias is limited. Knowing that Cyclin Dependent Kinase 8 (CDK8) can maintain tumor dedifferentiation and embryonic stem cell pluripotency, we investigated whether CDK8 inhibitor SEL120 could effectively target leukemia by induction of lineage commitment. SEL120 is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human phase Ib clinical trial with SEL120 in AML or HR-MDS patients is currently ongoing. To better understand the mechanism of action of SEL120 in AML, we studied effects of SEL120 on differentiation as one of the important anti-leukemic activities of the compound. Methods Global transcriptional changes were analyzed by RNAseq at different time points to capture early and long-term effects of SEL120. Genome-wide profiling of DNA-binding was performed by CHIPseq. Cell cycle, proliferation and lineage specific markers were studied by flow cytometry. Differentiation potential of AML cells was studied in semi-solid methylcellulose-based media to asses colony formation of SEL120 treated blasts. Results SEL120 treatment leads to decreased CDK8 occupancy and increased RNA Pol II occupancy as well as changes in the peak distribution among promoter and enhancer regions. SEL120 could repress many “stemness” genes and induce the expression of genes involved in lineage commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1 and GATA1/2. Moreover, for the first time, we showed that prolonged exposure of AML CD34+ cells to SEL120 could lead to colony formation in semi-solid media. Detailed analysis by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, characterizing differentiation and the presence of cells of myeloid or erythroid/megakaryocytic origin. Conclusion In addition to established role of CDK8 in regulation of tumor suppressor genes we present evidence for an essential role of CDK8 in lineage controlling-functions. Previously we reported specific cytotoxicity of SEL120 on cells positive for stemness markers such as CD34+. We further expanded on these studies, showing profound morphological changes in AML blasts during prolonged exposure to SEL120, correlating with increased expression of myeloid and erythroid/megakaryocytic markers. Based on these findings further studies are warranted to investigate the efficacy of SEL120 in anemia associated with bone marrow failures in AML and MDS. Combination of direct effects of SEL120 on viability of cells, with a strong differentiation potential, represents a promising profile for a drug in successful leukemia treatment. Citation Format: Urszula Pakulska, Elzbieta Adamczyk, Katarzyna Dziedzic, Katarzyna Wiklik, Michal Combik, Michal Mikula, Aniela Golas, Marta Obacz, Magdalena Masiejczyk, Przemyslaw Juszczynski, Magdalena Cybulska, Milena Mazan, Krzysztof Brzozka, Tomasz Rzymski. SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1018.

Th1/17 Cells Are Expanded in Bronchial Alveolar Lavage (BAL) Fluid from Leukemia Patients with Checkpoint Inhibitor-Induced Pneumonitis

A number of clinical trials are evaluating immune checkpoint inhibitors in patients with AML/MDS with encouraging results (Daver N et al, EHA 2017; Garcia-Manero et al, EHA 2017). Approximately 5-10% of patients with leukemia treated with checkpoint inhibitors had immune related adverse event (irAE) of pneumonitis, one of the life-threatening irAEs. Increased awareness, distinction from infectious pneumonia, and early intervention are critical in the management of pneumonitis-irAEs in patients with leukemia. Especially in the setting of acute leukemia the ability to distinguish immune mediated pneumonitis from infectious pneumonia is critical to determine whether or not the addition of high-dose steroids is warranted. The mechanisms of pneumonitis-irAE, the most important step for risk stratification and early detection remain elusive.MethodsWe collected bronchial alveolar lavage (BAL) fluid from seven leukemia patients (6 AML/MDs and 1 Richter transformation) who received checkpoint inhibitor based therapy on clinical trials, and subsequently developed respiratory symptoms and had a bronchoscopy. As a control, we collected BAL fluid from two AML/MDS patients who had never received checkpoint inhibitor therapy. We stained BAL CD4 T cell with lineage specific surface markers using the following antibodies; anti-CD4 BUV396, anti-CXCR3 BV 421, anti-CD45RA BV786, anti-CD3 PerCP, anti-CCR6 FITC, anti-CD25 PE, anti-PD-1 PE-Dazzle, anti-CXCR5 APC, and anti-CD127 APC 700. BAL CD4 T cell subsets, including Th1, 2, 17, follicular helper T, and regulatory T cells, were enumerated and the proportion and profile of the CD4 T cell subsets within the total lymphocytes were compared between pneumonitis group and control group.ResultPatient demographics are described in Figure 1A. Mean age of the patients with pneumonitis-irAE was 56. Most of them were on nivolumab in combination with chemotherapy/hypomethylator therapy. The patients developed pneumonitis 52.7 ± 77.8 days (mean ± DS) after the first exposure to a checkpoint inhibitor. Compared to the control group, lymphocytes were expanded in pneumonitis group (% lymphocytes, disease versus control: 32.3% vs. 19.0%). This observation led us to further characterize the lymphocytes in available BAL samples, especially CD4 T cells given their central role in immune efficacy and toxicity. Interestingly, Th1/17 hybrid cells (CXCR3hi CCR6hi), known to be pathogenic in autoimmune diseases, were expanded in the pneumonitis group compared to the control (8.24 % vs 3.19 %) (Figure 1B and C) These Th1/17 hybrid cells express high level of PD-1, suggesting their hyperactive status. Although premature, there was a trend that regulatory T cells were decreased in pneumonitis group (Data not shown). Surface staining of CD4 T cells in BAL fluid revealed that both disease and control had no follicular helper T cells, the CD4 T cell subset governing humoral immunity. Consistently, B cells were barely detected in BAL in both groups (Data not shown).ConclusionThese results suggest the possibility of increased Th1/17 hybrid cells with decreased Tregs as a biomarker of pneumonitis. The high PD-1 expression on Th1/17 hybrid cells suggests activation (or exhaustion) in the lungs in patients with irAEs. Further investigation of Th1/17 hybrid cells and correlation with peripheral blood may provide an insight into the mechanisms of pneumonitis-irAEs and biomarkers for early detection. In parallel, decreased Treg in the BAL may indicate an increased susceptibility to immune activation and inflammation seen in irAEs. [Display omitted] DisclosuresKantarjian:Novartis: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding. Sharma:Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Evelo: Consultancy, Other: stock; Astellas: Consultancy; EMD Serono: Consultancy; Consetellation: Other: stock; Amgen: Consultancy; Kite Pharma: Consultancy, Other: stock; Neon: Consultancy, Other: stock; BMS: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy. Daver:Otsuka America Pharmaceutical, Inc.: Consultancy; Karyopharm: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Kiromic: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Jazz: Consultancy; Incyte Corporation: Honoraria, Research Funding.

Comprehensive Immunophenotyping Panel in Japan Enables to Detect Rare Subtypes in Childhood Leukemia

[Background] Although the development of genome wide screening for risk stratification in childhood leukemia, immunophenotyping is still essential to decide initial treatment because of quick duration to make diagnosis. Immunophenotyping is also very useful to detect rare subtypes in leukemia. From 2011, Japanese pediatric leukemia/lymphoma study group (JPLSG) started nation-wide immunophenotyping with comprehensive panel including more than 50 antigens for childhood hematological malignancy. This panel led easier diagnosis not only for typical leukemia subtypes such as acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML), but for rare subtypes including mixed phenotype acute leukemia (MPAL), acute undifferentiated leukemia (AUL), and blastic plasmacytoid dendritic cell neoplasms (BPDCN). We report here the significance of comprehensive immunophenotyping panel, then clinical manifestations and immunophenotypic peculiarity of rare subtypes in childhood leukemia.[Methods] Immunophenotyping was done with comprehensive panel (Table 1) and diagnosis was done according to WHO 2008 classification. Briefly, determination of lineage was usually decided by major lineage specific markers such as myeloperoxidase (MPO), CD19, and cytoplasmic CD3. Then, more detailed categorization was done by analysis with other lineage, differentiation, or activation markers.[Result] In 4,033 case from January 2012 to December 2016, major immunophenotypes were as follows; B-precursor/pre-B (BCP-) ALL was 2,141, T cell (T-) ALL 348, AML 842, B-ALL 74, Lymphomas 171, transient myeloproliferative disorder (TAM) 244, chornic myelogenous leukemia (CML) 72. Out of other 141 cases, we also found 32 MPAL, 16 AUL, and 7 BPDCN. According to detailed immunophenotyping, MPAL cases could be divided into 3 distinct clusters, T-ALL/ AML-M1 biphenotypic subtype (8 cases), BCP-ALL/AML-M5 bilineal subtype (11), and BCP-ALL with MPO expression subtype (13). Furthermore, distinct cluster, ‘early T cell progenitor ALL (ETP)-like subtype’ was also distinguished from AUL.[Discussion] Acute leukemia of ambiguous lineage and BPDCN are both rare subtypes of acute leukemia. According to WHO 2008 classification, acute leukemia of ambiguous lineage is further classified as MPAL, AUL and natural killer lymphoblastic leukemia. In this study, we evaluate 32 MPAL, 16 AUL and 7 BPDCN. It is of note that we could not identify no natural killer lymphoblastic leukemia in this childhood leukemia cohort. We also evaluate 32 MPAL cases, which showed 3 distinct subgroups. T-ALL oriented cases indicate only one subtype ‘T-ALL/AML-M1 biphenotypic’, which indicated relatively older age, usually diagnosed as AML-M1 with morphologic FAB classification, and received mostly myeloid oriented therapy. BCP-ALL related cases were also divided into two groups, BCP-ALL/AML-M5 bilineal, and BCP-ALL with MPO expression. Bilineal leukemia often found in infantile ALL with MLL gene rearrangement (4 out of 11 cases), but still found 7 older cases without MLL gene rearrangemnt. Bilineal leukemia occasionally cause lineage-switch relapse, so diagnosis of bilineal leukemia seems to be very significant. Recent studies showed leukemia with ZNF384 fusion indicate CD10 negative or low BCP-ALL, and tended to express MPO. Our cases also tended to indicate negative or low CD10 expression. In addition, ETP-ALL like cases were positive for T cell antigens such as CD2 and/or CD7 and also some myeloid antigens but lacked cytoplasmic CD3 expression.In conclusion, we identified a spectrum of rare subtypes in childhood leukemia, including MPAL, AUL, and BPDCN by adoption of comprehensive immunophnotyping panel. In most of childhood MPAL cases were divided into 3 distinct clusters. It must be useful information for detection of genetic abnormality. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HIV-induced neuroinflammation inhibits oligodendrocyte maturation via glutamate-dependent activation of the PERK arm of the integrated stress response.

Despite combined antiretroviral therapy (cART), HIV-associated neurocognitive disorder (HAND) affects 30-50% of HIV-positive patients. Importantly, persistent white matter pathologies, specifically corpus callosum thinning and disruption of white matter microstructures observed in patients with HAND despite viral control through cART, raise the possibility that HIV infection in the setting of suboptimal cART may perturb oligodendrocyte (OL) maturation, function and/or survival, influencing HAND persistence in the cART era. To examine the effect of HIV infection on OL maturation, we used supernatants of primary human monocyte-derived macrophages infected with HIV (HIV/MDMs) to treat primary cultures of rat oligodendrocyte precursor cells (OPCs) during their differentiation to mature OLs. Using immunostaining for lineage-specific markers, we found that HIV/MDMs significantly inhibited OPC maturation. Based on our previous studies, we examined the potential role of several signaling pathways, including ionotropic glutamate receptors and the integrated stress response (ISR), and found that AMPA receptors (AMPAR)/kainic acid (KA) receptors (KARs) mediated the HIV/MDMs-induced defect in OL maturation. We also found that the treatment of OPC cultures with glutamate or AMPAR/KAR agonists phenocopied this effect. Blocking ISR activation, specifically the PERK arm of the ISR, protected OPCs from HIV/MDMs-mediated inhibition of OL maturation. Further, while glutamate, AMPA, and KA activated the ISR, inhibition of AMPAR/KAR activation prevented ISR induction in OPCs and rescued OL maturation. Collectively, these data identify glutamate signaling via ISR activation as a potential therapeutic pathway to ameliorate white matter pathologies in HAND and highlight the need for further investigation of their contribution to cognitive impairment.

Alveolar Bone Marrow Gli1+ Stem Cells Support Implant Osseointegration

Osseointegration is the key issue for implant success. The in vivo properties of cell populations driving the osseointegration process have remained largely unknown. In the current study, using tissue clearing–based 3-dimensional imaging and transgenic mouse model-based lineage tracing methods, we identified Gli1+ cells within alveolar bone marrow and their progeny as the cell population participating in extraction socket healing and implant osseointegration. These Gli1+ cells are surrounding blood vessels and do not express lineage differentiation markers. After tooth extraction and delayed placement of a dental implant, Gli1+ cells were activated into proliferation, and their descendants contributed significantly to new bone formation. Ablation of Gli1+ cells severely compromised the healing and osseointegration processes. Blockage of canonical Wnt signaling resulted in impaired recruitment of Gli1+ cells and compromised bone healing surrounding implants. Collectively, these findings demonstrate that Gli1+ cells surrounding alveolar bone marrow vasculature are stem cells supporting dental implant osseointegration. Canonical Wnt signal plays critical roles in regulating Gli1+ stem cells.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Gender-specific characteristics of hypertrophic response in cardiomyocytes derived from human embryonic stem cells.

Introduction: Gender-specific phenotypes of the heart were reported with respect to both physiology and pathology. While most differences were associated with the sex hormones, differential expression of genes received special attention, particularly X-Y chromosomes’ genes.Methods: Here, we compared cardiogenesis by gene expression analysis of lineage specific markers and X-Y chromosomes’ genes, during in vitro differentiation of XY and XX human embryonic stem cells (hESC), in a hormone-free setup. Results: Downregulation of pluripotency marker (NANOG) and upregulation of cardiac mesoderm and progenitor markers (GATA4, TBX5, NKX2.5, ISL1) was remained temporally similar in differentiating XY and XX hESCs. Isoproterenol treatment of XY and XX hESC-derived cardiomyocytes (hESCCM) induced hypertrophy in a sex-specific manner, with female cardiomyocytes showing response at higher isoproterenol concentration and a later time point of differentiation. Interestingly, KDM5C as an X-linked gene, was markedly upregulated in both hypertrophied male and female cardiomyocytes. Conclusion: Collectively, our results indicated a temporally identical cardiogenesis, but more susceptibility of XY hESC-CM to hypertrophic stimulus in a hormone-free condition.

The diversity of adult lung epithelial stem cells and their niche in homeostasis and regeneration.

The adult lung, a workhorse for gas exchange, is continually subjected to a barrage of assaults from the inhaled particles and pathogens. Hence, homeostatic maintenance is of paramount importance. Epithelial stem cells interact with their particular niche in the adult lung to orchestrate both natural tissue rejuvenation and robust post-injury regeneration. Advances in single-cell sequencing, lineage tracing, and living tissue imaging have deepened our understanding about stem cell heterogeneities, transition states, and specific cell lineage markers. In this review, we provided an overview of the known stem/progenitor cells and their subpopulations in different regions of the adult lung, and explored the regulatory networks in stem cells and their respective niche which collectively coordinated stem cell quiescence and regeneration states. We finally discussed relationships between dysregulated stem cell function and lung disease.

Tissue Engineering of Cartilage Using Collagen Scaffold Enriched with Plant Polysaccharides.

Degenerative diseases associated with articular cartilage pose a huge burden on health care economics. The nature of the tissue involved and the changes therein do not allow self-healing; and most of these problems are progressive. Tissue engineering offers some solutions provided we focus on the right kind of cells and the appropriate surrounding niches created for a particular tissue. The present study deals with the formation of polysaccharide rich stable scaffold of collagen after cross-linking with oxidized gum arabic. The scaffold was tested for its biocompatibility and ability to support cells. The in vitro cytotoxicity of the scaffolds toward induced pluripotent stem cells and chondrocytes was evaluated. Evaluation of expression of lineage specific markers indicates differentiation of induced pluripotent stem cells to chondrogenic lineage and maintenance of chondrocytes per se when grown in the scaffold. Animal studies were carried out to study the efficacy of the scaffold to repair the knee injuries. Cells along with the scaffold appeared to be the best filling, in repair of injured cartilage. These studies show that these scaffolds are potential candidates in applications such as tissue engineering of cartilage.

The rapid, mass invasion of New Zealand by North American Daphnia "pulex".

Nonindigenous members of the Daphnia pulex complex have been found in many lakes in New Zealand (NZ) in the past 20 years, suggesting a recent invasion. However, very little is known about the precise phylogenetic origin of invasive Daphnia, whether each lake is invaded by a single clone or multiple clones, the lineage of the invasive clones, and whether they are obligately asexual clones. Furthermore, the source and time of arrival of the invasive genotype(s) are unclear. We address these questions by genomic sequencing of Daphnia populations from 13 lakes in the South Island and 1 lake in the North Island, NZ. All biallelic sites in these NZ populations have similar numbers of reads for the two parental alleles, suggesting each NZ population originates from a single asexual clone. Based on 25,643 monomorphic lineage-specific markers, the invasive Daphnia in the South Island were found to be Daphnia pulicaria Forbes, while those in the North Island are hybrids of D. pulicaria Forbes and D. cf. pulex sensu Hebert. Both the South and North Island Daphnia are phylogenetically clustered with North American Daphnia, thereby suggesting their North American origins. We found also that all South Island clones contain identical mitochondrial genomes, suggesting the origin and proliferation from a single founder clone, which we experimentally verified to be an obligate asexual. Estimates from molecular data imply a colonization time for the South Island clones of ~60 years ago, with a likely invasion route associated with the introduction of salmonids from North America.

Differential controls of MAIT cell effector polarization by mTORC1/mTORC2 via integrating cytokine and costimulatory signals

Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor β chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1β induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1β to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1βR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.

Adhesion GPCR Latrophilin-2 Specifies Cardiac Lineage Commitment through CDK5, Src, and P38MAPK.

SummaryIdentifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we investigated the functioning of a cardiomyogenic cell-surface marker, latrophilin-2 (LPHN2), an adhesion G-protein-coupled receptor, in cardiac differentiation. LPHN2 was selectively expressed in cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs) during mouse and human pluripotent stem cell (PSC) differentiation; cell sorting with an anti-LPHN2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knockdown or knockout PSCs did not express cardiac genes. We used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways, to assess molecular mechanisms underlying LPHN2-induced cardiac differentiation. LPHN2-dependent phosphorylation was the strongest for cyclin-dependent kinase 5 (CDK5) at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of LPHN2 signaling. These findings provide a valuable strategy for isolating CPCs and CMCs from PSCs and insights into the still-unknown cardiac differentiation mechanisms.

Evaluation of Ranzoni etal.: Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

This article shows an example of the peer review process for "Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis" (Ranzoni etal., 2021).

Lin28 Inhibits the Differentiation from Mouse Embryonic Stem Cells to Glial Lineage Cells through Upregulation of Yap1.

The RNA-binding protein Lin28 regulates neurogliogenesis in mammals, independently of the let-7 microRNA. However, the detailed regulatory mechanism remains obscured. Here, we established Lin28a or Lin28b overexpression mouse embryonic stem cells (ESCs) and found that these cells expressed similar levels of the core pluripotent factors, such as Oct4 and Sox2, and increased Yap1 but decreased lineage-specific markers compared to the control ESCs. Further differentiation of these ESCs to neuronal and glial lineage cells revealed that Lin28a/b overexpression did not affect the expression of neuronal marker βIII-tubulin, but dramatically inhibited the glial lineage markers, such as Gfap and Mbp. Interestingly, overexpression of Yap1 in mouse ESCs phenocopied Lin28a/b overexpression ESCs by showing defect in glial cell differentiation. Inhibition of Yap1/Tead-mediated transcription with verteporfin partially rescued the differentiation defect of Lin28a/b overexpression ESCs. Mechanistically, we demonstrated that Lin28 can directly bind to Yap1 mRNA, and the induction of Yap1 by Lin28a in mESCs is independent of Let7. Taken together, our results unravel a novel Lin28-Yap1 regulatory axis during mESC to glial lineage cell differentiation, which may shed light on glial cell generation in vitro.

English

BACKGROUND Differentiating prostate carcinoma (PCa) arising in the neck of urinary bladder from high grade urothelial cancer (UCa) with prostatic extension can be a difficult task for histopathologist due to similar morphologic characteristics and overlapping clinical manifestations in the two diseases. These two tumours often occur in association with one another but have different potential therapeutic strategies and prognostic implications. We have investigated p63 immunohistochemical (IHC) marker as simple first line marker adjuvant to histopathological examination. METHODS In this prospective study, total 50 cases including 25 cases of urothelial carcinoma and 25 cases of prostatic carcinoma were taken. Tumour grade was determined according to standard H&E staining and scoring system. p63 expressions were determined by immunohistochemical staining of all the cases. The obtained results were analysed and evaluated using chi-square statistical test to determine whether p63 IHC can be used as simple first line marker tool with a high sensitivity and specificity. RESULTS p63 was not expressed in any of the 25 cases of prostatic carcinoma cases while in urothelial carcinoma it was expressed in 23 of 25 (92 %) cases. p63 IHC staining expression is positive in all histological grades of urothelial carcinomas. 2 out of 25 cases of urothelial carcinomas were negative for p63 IHC expression. None of the prostatic adenocarcinomas expressed p63 staining. Sensitivity of p63 stain in differentiating UCa with PCa was 92 % in our study, specificity of p63 stain in differentiating UCa with PCa was found to be 100 %. CONCLUSIONS p63 can be used as a screening first line IHC marker to distinguish urothelial carcinomas from prostatic adenocarcinomas. For challenging and unresolved cases both of these have limited sensitivity; thus, authors recommend two lineagespecific markers one each for UCa (GATA3, S100P) and PCa (NKX3.1, P501S, PSMA) should be used for definitive diagnosis. KEYWORDS p63 Immunohistochemistry, Urothelial Carcinoma, Prostatic Adenocarcinoma

Hepatitis B Virus DNA Integration and Clonal Expansion of Hepatocytes in the Chronically Infected Liver.

Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.