Sadafumi Suzuki

Imbalance of NKp44+NKp46− and NKp44−NKp46+ Natural Killer Cells in the Intestinal Mucosa of Patients With Crohn's Disease

BACKGROUND & AIMS Mucosal natural killer (NK) cells that produce interleukin (IL)-22 mediate intestinal homeostasis and inflammation in mice. However, their role in the pathogenesis of human inflammatory bowel diseases (IBDs) is not known. We investigated intestinal NK cells in intestinal mucosa samples of patients with Crohns disease (CD). METHODS We isolated lamina propria NK cells from intestinal mucosal samples of patients with IBD and subjects without IBD (controls) and analyzed expression patterns of cell surface molecules and cytokine production. Interactions between lamina propria NK cells and intestinal macrophages were examined. RESULTS In intestinal mucosa samples from controls, NKp44 and NKp46 were expressed differentially on CD3(-)CD56(+) NK cells, NKp44(+)NKp46(-) (NKp44(+)) NK cells expressed CD127 and the transcription factor retinoic acid-related orphan receptor C (RORC) and produced IL-22 whereas NKp44(-)NKp46(+) (NKp46(+)) NK cells did not express CD127 or RORC and produced interferon (IFN)-gamma. NKp46(+) NK cells were predominant in intestinal mucosa of patients with CD compared with controls or patients with ulcerative colitis. Upon interaction with intestinal inflammatory macrophages NKp46(+), NK cells from patients with CD were activated via IL-23 and produced IFN-gamma; this activation required cell-to-cell contact. CONCLUSIONS The balance of NKp44(+)/NKp46(+) NK cells is disrupted in intestinal mucosa of patients with CD. NKp46(+) NK cells might mediate the pathogenesis of CD by producing IFN-gamma.

Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α.

Platelet-derived growth factor receptor α (PDGFR-α) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-α+Sca-1+ (PαS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PαS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PαS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 °C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45+Ter-119+)-positive cells. Approximately 10,000 PαS MSCs may then be isolated per mouse. The total protocol takes ∼7 h to complete.

Side population of pancreatic cancer cells predominates in TGF-β-mediated epithelial to mesenchymal transition and invasion

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up‐regulated E‐cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF‐β, SP cells changed their shape into mesenchymal‐like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E‐cadherin expression level. TGF‐β induced EMT‐associated gene alteration such as reduction of E‐cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)‐2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF‐β treatment, whereas MP cells did not respond to TGF‐β‐mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro‐invasion, and in vivo metastasis, as compared to MP cells. Because micro‐invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression.

LNGFR+THY-1+VCAM-1hi+ Cells Reveal Functionally Distinct Subpopulations in Mesenchymal Stem Cells

Summary Human mesenchymal stem cells (hMSCs), which conventionally are isolated based on their adherence to plastic, are heterogeneous and have poor growth and differentiation, limiting our ability to investigate their intrinsic characteristics. We report an improved prospective clonal isolation technique and reveal that the combination of three cell-surface markers (LNGFR, THY-1, and VCAM-1) allows for the selection of highly enriched clonogenic cells (one out of three isolated cells). Clonal characterization of LNGFR+THY-1+ cells demonstrated cellular heterogeneity among the clones. Rapidly expanding clones (RECs) exhibited robust multilineage differentiation and self-renewal potency, whereas the other clones tended to acquire cellular senescence via P16INK4a and exhibited frequent genomic errors. Furthermore, RECs exhibited unique expression of VCAM-1 and higher cellular motility compared with the other clones. The combination marker LNGFR+THY-1+VCAM-1hi+ (LTV) can be used selectively to isolate the most potent and genetically stable MSCs.

Purified Mesenchymal Stem Cells Are an Efficient Source for iPS Cell Induction

Background Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the forced expression of defined transcription factors. Although most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear. Methodology/Principal Findings Here, we generated iPS cells from PDGFRα+ Sca-1+ (PαS) adult mouse mesenchymal stem cells (MSCs) and PDGFRα− Sca-1− osteo-progenitors (OP cells), and compared the induction efficiency and quality of individual iPS clones. MSCs had a higher reprogramming efficiency compared with OP cells and Tail Tip Fibroblasts (TTFs). The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency. Conclusions/Significance Our findings suggest that a purified source of undifferentiated cells from adult tissue can produce high-quality iPS cells. In this context, prospectively enriched MSCs are a promising candidate for the efficient generation of high-quality iPS cells.

Cell surface N-glycans mediated isolation of mouse neural stem cells.

The isolation of neural stem cells (NSCs) from the brain has been hampered by the lack of valid cell surface markers and the requirement for long‐term in vitro cultivation that may lead to phenotype deterioration. However, few suitable specific cell surface antigens are available on NSCs that could be used for their prospective isolation. The present study demonstrated that the expression of complex type asparagine‐linked oligosaccharide (N‐glycans) was detected on brain cells dissociated from embryonic and adult brain using Phaseolus vulgaris erythroagglutinating lectin (E‐PHA) which binds to biantennary complex type N‐glycans, and demonstrated that E‐PHA bound preferentially to purified NSCs, but not to neurons, microglia, or oligodendrocyte precursor cells. The labeling of dissociated mouse embryonic brain cells or adult brain cells with E‐PHA enabled the enrichment of NSCs by 25‐fold or 9‐fold of the number of neurosphere‐forming cells in comparison to that of unsorted cells, respectively. Furthermore, a lectin blot analysis revealed the presence of several glycoproteins which were recognized by E‐PHA in the membrane fraction of the proliferating NSCs, but not in the differentiated cells. These results indicate that complex type N‐glycans is a valuable cell surface marker for living mouse NSCs from both the embryonic and adult brain.

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

Homeodomain Transcription Factor Meis1 Is a Critical Regulator of Adult Bone Marrow Hematopoiesis

Hematopoietic stem cells in the bone marrow have the capacity to both self-renew and to generate all cells of the hematopoietic system. The balance of these two activities is controlled by hematopoietic stem cell-intrinsic regulatory mechanisms as well as extrinsic signals from the microenvironment. Here we demonstrate that Meis1, a TALE family homeodomain transcription factor involved in numerous embryonic developmental processes, is selectively expressed in hematopoietic stem/progenitor cells. Conditional Meis1 knockout in adult hematopoietic cells resulted in a significant reduction in the hematopoietic stem/progenitor cells. Suppression of hematopoiesis by Meis1 deletion appears to be caused by impaired self-renewal activity and reduced cellular quiescence of hematopoietic stem/progenitor cells in a cell autonomous manner, resulting in stem cell exhaustion and defective long-term hematopoiesis. Meis1 deficiency down-regulated a subset of Pbx1-dependent hematopoietic stem cell signature genes, suggesting a functional link between them in the maintenance of hematopoietic stem/progenitor cells. These results show the importance of Meis1 in adult hematopoiesis.

Intraindividual dynamics of transcriptome and genome-wide stability of DNA methylation

Cytosine methylation at CpG dinucleotides is an epigenetic mechanism that affects the gene expression profiles responsible for the functional differences in various cells and tissues. Although gene expression patterns are dynamically altered in response to various stimuli, the intraindividual dynamics of DNA methylation in human cells are yet to be fully understood. Here, we investigated the extent to which DNA methylation contributes to the dynamics of gene expression by collecting 24 blood samples from two individuals over a period of 3 months. Transcriptome and methylome association analyses revealed that only ~2% of dynamic changes in gene expression could be explained by the intraindividual variation of DNA methylation levels in peripheral blood mononuclear cells and purified monocytes. These results showed that DNA methylation levels remain stable for at least several months, suggesting that disease-associated DNA methylation markers are useful for estimating the risk of disease manifestation.

A Genome-Wide Expression Profile of Adrenocortical Cells in Knockout Mice Lacking Steroidogenic Acute Regulatory Protein

Steroidogenic acute regulatory protein (StAR) facilitates cholesterol transfer into the inner mitochondrial membrane in the acute phase of steroidogenesis. Mice lacking StAR (Star(-/-)) share phenotypes with human individuals having congenital lipoid adrenal hyperplasia including compromised production of steroid hormones and florid accumulation of cholesterol esters in adrenal glands and gonads. To define a specific pattern of molecular changes with StAR deficiency, we performed transcriptome analysis of adrenal cells selectively isolated by fluorescent-activated cell sorting at embryonic d 17.5 or 18.5 in seven wild-type (Star(+/+)) or four Star(-/-) mice having the transgene targeting the enhanced green fluorescent protein to cell lineages that express StAR. A gene expression profile was obtained by whole-mouse genome microarray and confirmed by quantitative real-time PCR, identifying 1206 and 767 significantly up-regulated and down-regulated genes, respectively, in Star(-/-) mice compared with Star(+/+) mice (fold difference ≥ 2 and P value < 0.05 with false discovery rate < 0.2). In Star(-/-) mice, expression levels of genes involved in cholesterol efflux and the inflammatory response were significantly up-regulated, whereas those related to steroid hormone biosynthesis or cholesterol biosynthesis and influx were not significantly changed. Immunoreactive Iba1 or F4/80 (macrophage marker) in adrenal glands of Star(-/-) mice was detected not only in an increased number of resident macrophages but also in most adrenocortical cells. These findings expand our understanding of the pathophysiology of adrenal glands with the disruption of StAR and propose a reciprocal interaction between adrenocortical cells and resident macrophages inside adrenal glands of Star(-/-) mice.