Human Cell Populations Research Articles

Abstract 3020: Human leukocyte antigen (HLA) typing of a broad panel of cancer patient-derived xenograft (PDX) models for immune therapies

Abstract Background In immuno-oncology research, an appropriate microenvironment for both human tumor cells and human immune cells is an important but challenging factor for successful development of relevant preclinical models. Humanized patient-derived xenograft (PDX) models can support establishment of such an environment based on matching human leukocyte antigen (HLA) profiles of PDX and compatible human immune cell populations. In this study, we determined the individual HLA types of a broad panel of 291 established PDX models from 18 different tumor entities. Furthermore, we performed comprehensive HLA matching analyses of all PDX models and 13 peripheral blood mononuclear cell (PBMC) donors to enable personalized studies with HLA-matched PDX. Methods The HLA profile of each PDX was determined comprising HLA class I, II and non-class types in 4-digit resolution. Matching analyses were performed according to donor-recipient HLA matching criteria recommended by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN). For comparative HLA profile analyses, allele frequencies of 8862 German stem cell donors provided by The Allele Frequency Net Database were downloaded. Results The individual HLA profiles of all PDX models were generated comprising HLA-A, -B and -C (class I), HLA-DQA1, -DQB1, -DRB1, -DPA1 and -DPB1 (class II) as well as HLA-H (non-class) types. Comparative analyses of HLA profiles revealed that obtained frequencies of PDX HLA alleles are comparable with frequencies of a representative population of 8862 German stem cell donors. HLA profile matching analyses performed for all PDX models and PBMC donors resulted in 15 matches regarding PDX from head and neck, lung, mammary and pancreatic carcinoma as well as from endometrial cancer, glioma, lymphoma, melanoma and sarcoma. Dependence of immune therapy efficacy on matched HLA profiles was shown exemplarily for a head and neck squamous cell cancer (HNSCC) PDX. The HNSCC PDX was humanized with PBMC from 5 donors representing different degrees of HLA matching (33 - 100 %). Treatment with the checkpoint inhibitor Nivolumab caused a more powerful tumor growth inhibition in matched HLA profiles compared to non-matched profiles. This indicated a relation between antitumoral immune therapy effect and HLA matching in preclinical models. Conclusions We generated a comprehensive HLA profile portfolio containing information of a broad panel of PDX models. Matched HLA profiles of PDX models and PBMC donors enable personalized, preclinical immune-oncology studies to support the development of novel therapeutic strategies. Citation Format: Theresia Conrad, Annika Wulf-Goldenberg, Maria Stecklum, Michael Becker, Konrad Klinghammer, Jens Hoffmann. Human leukocyte antigen (HLA) typing of a broad panel of cancer patient-derived xenograft (PDX) models for immune therapies [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 3020.

A Novel Model of Human Hemopoiesis Based on Single Cell Functional and Transcriptional Analysis of Lympho-Myeloid Progenitors

Introduction: Human hemopoiesis produces 10 billion new, terminally mature blood cells daily; a production that is also rapidly responsive to external stimuli. Dysregulation of this complex process can lead to hemopoietic and immune deficiencies and blood cancers. In humans, the hemopoietic progenitor hierarchy producing lymphoid and myeloid lineages is unclear. Multiple progenitor populations give rise to lymphoid and myeloid cells but they remain incompletely characterized at the immunophenotypic, transcriptional and functional level. Here, we aim to understand the clonal functional output and transcriptional programs of primary human lympho-myeloid progenitor populations - the lymphoid-primed multipotent progenitor (LMPP) (Goardon et al., Cancer Cell, 2011), the multi-lymphoid progenitor (MLP) (Doulatov et al., Nature Immunology, 2010) and the granulocyte-macrophage progenitor (GMP).Methods: We devised a FACS strategy to prospectively purify eight human hemopoietic stem and progenitor cell populations. We functionally compared the LMPP, MLP and GMP in vitro by quantitative CFU assays, three different single cell liquid cultures or limit dilution analysis and in vivo by transplantation into a humanized ossicle mouse model (Reinisch et al., Nature Medicine, 2016). We performed population and single cell RNA sequencing and Fluidigm qRT-PCR to understand the relationship between the functional and transcriptional heterogeneity within human lympho-myeloid progenitor populations.Results: Our study comprehensively characterized the LMPP, MLP and GMP lympho-myeloid progenitor populations. Both LMPP and MLP are very rare within the mononuclear fraction (1 in 104 and 1 in 105 cells in CB and BM, respectively). We cultured 4357 single LMPP, GMP and MLP cells (isolated from 25 cord blood units and equivalent to ~1010 mononuclear cells) under three different culture conditions, one of which is described for the first time. We observed marked functional heterogeneity in the three lympho-myeloid progenitor populations. Focusing on the wells that gave single cell-initiated cultures, the majority of cells from LMPP, MLP and GMP gave uni-lineage output (60-90%). Bi-lineage output was less common (up to 35%), while multi-lineage output was the least frequent and only observed from LMPP and GMP cells (up to 15%). In vivo transplantation using the humanized ossicle mouse model increased the engraftment of lympho-myeloid progenitors 10-fold compared to previous reports. In vivo, the LMPP and GMP gave robust engraftment but the MLP substantially less engraftment. The LMPP gave rise to both myeloid and B cell engraftment, the GMP to myeloid engraftment and the MLP mainly to B cell output. Population RNA sequencing revealed distinct transcriptional signatures of these populations: MLP signature was enriched for lymphoid-affiliated genes, GMP for myeloid-affiliated genes and LMPP had a hybrid lympho-myeloid signature. Single cell RNA sequencing of 320 LMPP, MLP and GMP cells showed that the 3 progenitor populations form a transcriptional continuum. Linking the single cell gene expression to FACS indexing allowed exclusive purification of lymphoid- and myeloid-only progenitors from the current LMPP and GMP populations.Conclusions: These data change our understanding of human hematopoiesis and offer a radically new model of human lympho-myeloid progenitor specification. This model may have important implications for human immune deficiencies and hemopoietic malignancies.BS and DK contributed equally to this work. FH and AR contributed equally to this work. DisclosuresQuek:Celgene Corporation: Research Funding. Vyas:Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

Differential Effect of Non-Thermal Plasma RONS on Two Human Leukemic Cell Populations.

Simple SummaryAs the number of investigations into the use of non-thermal plasma (NTP) for cancer treatment expands, it is becoming apparent that susceptibility of different cancer cells to NTP varies. We hypothesized that such differences could be attributed to the cell type-dependent interactions between NTP-generated reactive oxygen and nitrogen species (RONS) and the target cells. To test this hypothesis, we examined how two different human leukemic cell lines—Jurkat T lymphocytes and THP-1 monocytes—influence hydrogen peroxide and nitrite content in media after NTP exposure. We also assessed the potential of NTP to enhance immunogenicity in these cells and assayed phagocytosis of NTP-exposed leukemic cells by macrophages. Our results highlight the significance of target-mediated modulation of plasma chemical species in the development and clinical use of protocols involving plasma sources for use in cancer therapeutic application.Non-thermal plasma application to cancer cells is known to induce oxidative stress, cytotoxicity and indirect immunostimulatory effects on antigen presenting cells (APCs). The purpose of this study was to evaluate the responses of two leukemic cell lines—Jurkat T lymphocytes and THP-1 monocytes—to NTP-generated reactive oxygen and nitrogen species (RONS). Both cell types depleted hydrogen peroxide, but THP-1 cells neutralized it almost immediately. Jurkat cells transiently blunted the frequency-dependent increase in nitrite concentrations in contrast to THP-1 cells, which exhibited no immediate effect. A direct relationship between frequency-dependent cytotoxicity and mitochondrial superoxide was observed only in Jurkat cells. Jurkat cells were very responsive to NTP in their display of calreticulin and heat shock proteins 70 and 90. In contrast, THP-1 cells were minimally responsive or unresponsive. Despite no NTP-dependent decrease in cell surface display of CD47 in either cell line, both cell types induced migration of and phagocytosis by APCs. Our results demonstrate that cells modulate the RONS-mediated changes in liquid chemistry, and, importantly, the resultant immunomodulatory effects of NTP can be independent of NTP-induced cytotoxicity.

Single-cell lipidomics with high structural specificity by mass spectrometry

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.

Aspartame induces cancer stem cell enrichment through p21, NICD and GLI1 in human PANC-1 pancreas adenocarcinoma cells

This study aimed to investigate the molecular effects of the common natural sugar glucose and artificial sweetener aspartame on cancer stem cell (CSC) population and cancer aggressiveness of PANC-1 human pancreas adenocarcinoma cells. According to our findings while aspartame exposure significantly increased the CSC population, high glucose had no effect on it. The epithelial-mesenchymal transition marker N-cadherin increased only in the aspartame group. The findings indicate that a high level of glucose exposure does not effect the invasion and migration of PANC-1 cells, while aspartame increases both of these aggressiveness criteria. The findings also suggest that a high concentration of glucose maintains CSC population through induction of nuclear Oct3/4 and differentiation to parental cells via increasing cytoplasmic c-myc. Aspartame exposure to PANC-1 cells activated AKT and deactivated GSK3β by increasing levels of ROS and cytoplasmic Ca+2, respectively, through T1R2/T1R3 stimulation. Then p-GSK3β(Ser9) boosted the CSC population by increasing pluripotency factors Oct3/4 and c-myc via NICD, GLI1 and p21. In the aspartame group, T1R1 silencing further increased the CSC population but decreased cell viability and suppressed the p21, NICD and GLI activation. The presence and amount of T1R subunits in the membrane fraction of PANC-1 cells are demonstrated for the first time in this study, as is the regulatory effect of T1R1's on CSC population. In conclusion, the present study demonstrated that long-term aspartame exposure increases CSC population and tumor cell aggressiveness through p21, NICD, GLI1. Moreover, while aspartame had no tumorigenic effect, it could potentially advance an existing tumor.

Performance based clone selection and automated media change to improve the reproducability of human mesenchymal stromal cell populations

Performance based clone selection and automated media change to improve the reproducability of human mesenchymal stromal cell populations

Mixed species live biotherapeutic product improves immunological tolerance in food allergy

Abstract The human gut microbiota is central to early-life immune system education, including the establishment of immune tolerance, and thus represents an important target for the development of preventative interventions against food allergy (FA). The composition of the gut microbiota, and specific bacterial members within the microbiota, have been shown to impact allergic disease development in animal models by limiting basophil hematopoiesis, influencing macrophage polarization, and enhancing regulatory T cell activity. With a growing body of evidence showing the gut microbiota is an important environmental factor contributing to FA development comes an opportunity to translate this knowledge into novel approaches for treating and preventing FA. Using a proprietary human gut microbiota consortium, we have developed a mixed species live biotherapeutic product (LBP) to alleviate FA development and severity. We show that this LBP can directly interact with human immune cell populations modulating immune signaling pathways including TGF-beta, FFARs, chemotaxis, and NFkB inhibition. We also demonstrate that supplementation with this LBP can reduce OVA induced oral anaphylactic severity in mice with humanized gut microbiota. This protective effect may be attributed to increased fecal antigen-specific IgA levels and enhanced intestinal TGFb levels. These data describe immunological mechanism of a mixed species LBP that is protective in mouse FA model.

Novel Primary Human Cancer Stem-Like Cell Populations from Non-Small Cell Lung Cancer: Inhibition of Cell Survival by Targeting NF-κB and MYC Signaling.

There is growing evidence that cancer stem cells (CSCs), a small subpopulation of self-renewal cancer cells, are responsible for tumor growth, treatment resistance, and cancer relapse and are thus of enormous clinical interest. Here, we aimed to isolate new CSC-like cells derived from human primary non-small cell lung cancer (NSCLC) specimens and to analyze the influence of different inhibitors of NF-κB and MYC signaling on cell survival. CSC-like cells were established from three squamous cell carcinomas (SCC) and three adenocarcinomas (AC) of the lung and were shown to express common CSC markers such as Prominin-1, CD44-antigen, and Nestin. Further, cells gave rise to spherical cancer organoids. Inhibition of MYC and NF-κB signaling using KJ-Pyr-9, dexamethasone, and pyrrolidinedithiocarbamate resulted in significant reductions in cell survival for SCC- and AC-derived cells. However, inhibition of the protein–protein interaction of MYC/NMYC proto-oncogenes with Myc-associated factor X (MAX) using KJ-Pyr-9 revealed the most promising survival-decreasing effects. Next to the establishment of six novel in vitro models for studying NSCLC-derived CSC-like populations, the presented investigations might provide new insights into potential novel therapies targeting NF-κB/MYC to improve clinical outcomes in NSCLC patients. Nevertheless, the full picture of downstream signaling still remains elusive.

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.

Chromatin coordinates stemness

Stem Cells Blood cells are produced from stem cells in the bone marrow and thymus and transition between quiescence and activation depending on the body's needs. Although generally quiescent, long-term hematopoietic stem cells (LT-HSCs) self-renew and can transform into short-term HSCs (ST-HSCs) to produce multilineage blood cells. Takayama et al. used single-cell analysis and a sequencing approach called ATAC-seq on various human hematopoietic cell populations. The method identified chromatin accessibility signatures that distinguish LT-HSCs and activated hematopoietic stem and progenitor cells (HSPCs). The activated HSPC signature displays binding to sites on the transcription factor CTCF, which is a mediator of three-dimensional (3D) genome organization. As HSCs differentiate, CTCF interacts with chromatin to silence loops that enclose genes. Thus, CTCF acts as a gatekeeper for coordinating the 3D chromatin interactions that regulate hematopoietic cell dynamics. Cell Stem Cell 28 , P488 (2021).

Field size effects on DNA damage and proliferation in normal human cell populations irradiated with X-ray microbeams

To clarify the health risks of internal radiation exposure, it is important to investigate the radiological effects of local exposure at cell levels from radioactive materials taken up by organs. Focusing on the response of cell populations post-irradiation, X-ray microbeams are very effective at reproducing the effects of local exposure within an internal exposure in vitro. The present study aims to clarify the effects of local exposure by investigating the response of normal human cell (MRC-5) populations irradiated with X-ray microbeams of different beam sizes to DNA damage. The populations of MRC-5 were locally irradiated with X-ray microbeams of 1 Gy at 0.02–1.89 mm2 field sizes, and analyzed whether the number of 53BP1 foci as DSB (DNA double strand break) per cell changed with the field size. We found that even at the same dose, the number of DSB per cell increased depending on the X-irradiated field size on the cell population. This result indicated that DNA damage repair of X-irradiated cells might be enhanced in small size fields surrounded by non-irradiated cells. This study suggests that X-irradiated cells received some signal (a rescue signal) from surrounding non-irradiated cells may be involved in the response of cell populations post-irradiation.

The age of adult pilocytic astrocytoma cells

Adult pilocytic astrocytomas (PAs) have been regarded as indistinguishable from pediatric PAs in terms of genome-wide expression and methylation patterns. It has been unclear whether adult PAs arise early in life and remain asymptomatic until adulthood, or whether they develop during adulthood. We sought to determine the age and origin of adult human PAs using two types of “marks” in the genomic DNA. First, we analyzed the DNA methylation patterns of adult and pediatric PAs to distinguish between PAs of different anatomic locations (n = 257 PA and control brain tissues). Second, we measured the concentration of nuclear bomb test-derived 14C in genomic DNA (n = 14 cases), which indicates the time point of the formation of human cell populations. Our data suggest that adult and pediatric PAs developing in the infratentorial brain are closely related and potentially develop from precursor cells early in life, whereas supratentorial PAs might show age and location-specific differences.

Nanopore Sequencing Reveals Global Transcriptome Signatures of Mitochondrial and Ribosomal Gene Expressions in Various Human Cancer Stem-like Cell Populations.

Cancer stem cells (CSCs) are crucial mediators of tumor growth, metastasis, therapy resistance, and recurrence in a broad variety of human cancers. Although their biology is increasingly investigated within the distinct types of cancer, direct comparisons of CSCs from different tumor types allowing comprehensive mechanistic insights are rarely assessed. In the present study, we isolated CSCs from endometrioid carcinomas, glioblastoma multiforme as well as adenocarcinomas of lung and prostate and assessed their global transcriptomes using full-length cDNA nanopore sequencing. Despite the expression of common CSC markers, principal component analysis showed a distinct separation of the CSC populations into three clusters independent of the specific type of tumor. However, GO-term and KEGG pathway enrichment analysis revealed upregulated genes related to ribosomal biosynthesis, the mitochondrion, oxidative phosphorylation, and glycolytic pathways, as well as the proteasome, suggesting a great extent of metabolic flexibility in CSCs. Interestingly, the GO term "NF-kB binding" was likewise found to be elevated in all investigated CSC populations. In summary, we here provide evidence for high global transcriptional similarities between CSCs from various tumors, which particularly share upregulated gene expression associated with mitochondrial and ribosomal activity. Our findings may build the basis for identifying novel therapeutic strategies targeting CSCs.

Multiparameter Flow Cytometry Analysis of the Human Spleen Applied to Studies of Plasma-Derived EVs From Plasmodium vivax Patients.

The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45+ cells included B (30%), CD4+ T (16%), CD8+ T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria.

Comparison of DNA repair and radiosensitivity of different blood cell populations

Despite the frequent use of ionising radiation (IR) in therapy and diagnostics and the unavoidable exposure to external radiation sources, our knowledge regarding the radiosensitivity of human blood cell populations is limited and published data, obtained under different experimental conditions, are heterogeneous. To compare the radiosensitivity of different hematopoietic cell populations, we set out to determine the responses of cells obtained from peripheral blood of healthy volunteers under identical conditions (resting, non-stimulated cells). First, we measured the radiation response of T cells (Treg, Th, CTL), B cells, NK cells, CD34+ progenitor cells and monocytes obtained from peripheral blood and monocyte-derived macrophages (Mph) and immature dendritic cells (iDC) ex vivo and show that T and B cells are highly sensitive, starting to undergo apoptosis following IR with a dose as low as 0.125 Gy. Importantly, there was no clear threshold dose and cell death/apoptosis increased up to a saturation level with a dose of 2 Gy. The sensitivity decreased in the order of T cells > NK and B cells > monocytes > macrophages and iDC. The data confirm a previous report that Mph and iDC are radiation-resistant compared to their progenitor monocytes. Although non-stimulated T and B cells were highly radiation-sensitive compared to monocytes and macrophages, they were competent in the repair of DNA double-strand breaks, as shown by a decline in γH2AX foci in the post-exposure period. CD34+ cells obtained from peripheral blood also showed γH2AX decline post-exposure, indicating they are repair competent. Granulocytes (CD15+) did not display any γH2AX staining following IR. Although peripheral blood lymphocytes, the main fraction are T cells, were significantly more radiation-sensitive than monocytes, they displayed the expression of the repair proteins XRCC1, ligase III and PARP-1, which were nearly non-expressed in monocytes. To assess whether monocytes are depleted in vivo following IR, we measured the amount of T cells and monocytes in cancer patients who received total-body radiation (TBR, 6 × 2 Gy). We observed that the number of T cells in the peripheral blood significantly declined already after the first day of TBR and remained at a low level, which was accompanied by an increase in the number of γH2AX foci in the surviving CD3+ T cell fraction. In contrast, the number of monocytes did not decline extensively, reflecting their radiation resistance compared to T cells. Monocytes also showed an accumulation of γH2AX foci in vivo, but the levels were significantly lower than in T cells. CD56+ NK cells displayed a response similar to T cells. The data support the notion that unstimulated T cell subfractions are nearly equally radiation sensitive. There are, however, remarkable differences in the radiation sensitivity between the lymphoid and the myeloid lineage, with lymphoid cells being significantly more sensitive than cells of the myeloid lineage. In the myeloid lineage, macrophages and iDCs were the most radio-resistant cell types.

Mitochondrial gene expression in single cells shape pancreatic beta cells' sub-populations and explain variation in insulin pathway

Mitochondrial gene expression is pivotal to cell metabolism. Nevertheless, it is unknown whether it diverges within a given cell type. Here, we analysed single-cell RNA-seq experiments from human pancreatic alpha (N = 3471) and beta cells (N = 1989), as well as mouse beta cells (N = 1094). Cluster analysis revealed two distinct human beta cells populations, which diverged by mitochondrial (mtDNA) and nuclear DNA (nDNA)-encoded oxidative phosphorylation (OXPHOS) gene expression in healthy and diabetic individuals, and in newborn but not in adult mice. Insulin gene expression was elevated in beta cells with higher mtDNA gene expression in humans and in young mice. Such human beta cell populations also diverged in mitochondrial RNA mutational repertoire, and in their selective signature, thus implying the existence of two previously overlooked distinct and conserved beta cell populations. While applying our approach to human alpha cells, two sub-populations of cells were identified which diverged in mtDNA gene expression, yet these cellular populations did not consistently diverge in nDNA OXPHOS genes expression, nor did they correlate with the expression of glucagon, the hallmark of alpha cells. Thus, pancreatic beta cells within an individual are divided into distinct groups with unique metabolic-mitochondrial signature.

YAP Orchestrates Heterotypic Endothelial Cell Communication via HGF/c-MET Signaling in Liver Tumorigenesis.

The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET-expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. SIGNIFICANCE: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5502/F1.large.jpg.

Cubic Membranes Formation in Synchronized Human Hepatocellular Carcinoma Cells Reveals a Possible Role as a Structural Antioxidant Defense System in Cell Cycle Progression.

Cubic membranes (CMs) represent unique biological membrane structures with highly curved three-dimensional periodic minimal surfaces, which have been observed in a wide range of cell types and organelles under various stress conditions (e. g., starvation, virus-infection, and oxidation). However, there are few reports on the biological roles of CMs, especially their roles in cell cycle. Hence, we established a stable cell population of human hepatocellular carcinoma cells (HepG2) of 100% S phase by thymidine treatment, and determined certain parameters in G2 phase released from S phase. Then we found a close relationship between CMs formation and cell cycle, and an increase in reactive oxygen species (ROS) and mitochondrial function. After the synchronization of HepG2 cells were induced, CMs were observed through transmission electron microscope in G2 phase but not in G1, S and M phase. Moreover, the increased ATP production, mitochondrial and intracellular ROS levels were also present in G2 phase, which demonstrated a positive correlation with CMs formation by Pearson correlation analysis. This study suggests that CMs may act as an antioxidant structure in response to mitochondria-derived ROS during G2 phase and thus participate in cell cycle progression.

Stem cell and niche regulation in human short bowel syndrome.

Loss of functional small bowel surface area following surgical resection for disorders such as Crohn’s disease, intestinal ischemic injury, radiation enteritis, and in children, necrotizing enterocolitis, atresia, and gastroschisis, may result in short bowel syndrome, with attendant high morbidity, mortality, and health care costs in the United States. Following resection, the remaining small bowel epithelium mounts an adaptive response, resulting in increased crypt cell proliferation, increased villus height, increased crypt depth, and enhanced nutrient and electrolyte absorption. Although these morphologic and functional changes are well described in animal models, the adaptive response in humans is less well understood. Clinically the response is unpredictable and often inadequate. Here we address the hypotheses that human intestinal stem cell populations are expanded and that the stem cell niche is regulated following massive gut resection in short bowel syndrome (SBS). We use intestinal enteroid cultures from patients with SBS to show that the magnitude and phenotype of the adaptive stem cell response are both regulated by stromal niche cells, including intestinal subepithelial myofibroblasts, which are activated by intestinal resection to enhance epithelial stem and proliferative cell responses. Our data suggest that myofibroblast regulation of bone morphogenetic protein signaling pathways plays a role in the gut adaptive response after resection.

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells.

Simple SummaryThe regeneration of nearly all organs of the human body mainly depends on the functionality of adult stem cell populations that reside in their respective niches and can be activated upon injuries or other damages. These stem cell populations greatly differ in their expression profile of molecular markers, which greatly influences their potential use in regenerative medicine. Neural crest-derived stem cells are a prominent subpopulation of adult stem cells and are known for their high regenerative potential. Within this study, we compared two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells from the nasal cavity and human cardiac stem cells from the heart, using global gene expression profiling. Here, we found differences that correspond to the tissue sources of origin but also similarities in the expression of markers that are associated with the neural crest. Further classifying nasal stem cells and cardiac stem cells in a broader context, we identified clear similarities between both populations and other adherent stem cell populations compared to non-adherent progenitor cells of the blood system. The analyses provided here might help to understand the differences and similarities between different adult human stem cell populations.For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations.