Unique Molecular Markers Research Articles

Twist-1 Expression Represents a Novel Prognostic Factor in Chronic Myelogenous Leukemia.

Abstract 2592Poster Board II-568Chronic Myelogenous Leukemia (CML) represents one of the best characterized models for studying cancer stem cell properties and resistance to tyrosine kinase inhibitors (TKIs). A substantial proportion of CML patients develop primary or secondary resistance to such agents, and in approximately ∼30% of the cases the resistance mechanism(s) remain(s) obscure. It is known that CML leukemic stem cells could remain viable and quiescent despite the presence of growth factors that seem to protect them from apoptosis, or drugs. By performing genome-wide microarray analysis of CML sensitive and resistant cell lines, a novel oncogene Twist-1 has been shown to be deregulated in CML cells innately resistant to imatinib through, an unknown mechanism (Tipping AJ et al. Exp Hematol 2003; 31(11): 1073–1080). In this study, we have observed similar results in the same BCR-ABL+ KCL22 cell line resistant and sensitive to imatinib by quantitative RT-PCR (qRT-PCR) using specific primers and at the protein level. Twist-1 is a key player during embryogenesis that represses mesoderm differentiation and retains undifferentiated cells into a proliferative state. In adult tissues, its expression has been reported in intestinal crypts where adult gut stem cells reside. In cancer, Twist-1 overexpression has been reported in a variety of solid tumors and hematopietic malignacies (Sezary syndrome, B-Cell leukemias) and its deregulation is always associated with poor prognosis and resistance to reagents targeting microtubules (Puisieux A et al. Br J Cancer 2006; 94 (1):13–17). In addition, functional studies show that Twist-1 plays a major role in tumor promotion and progression, by inhibiting differentiation, and interfering with the p53 tumor suppressor pathway, thereby favoring cell survival (Ansieau S et al. Cancer Cell 2008; 14(1):79–89). In this context, we have analyzed Twist-1 gene expression by qRT-PCR in both CD34+ (immature cells) and CD34− (mature compartment) blood or marrow cells from CML patients at diagnosis, at different disease phases, and once TKIs resistance has occur. Results are expressed as ratio to normal CD34− cells values. We observed a 74- and 39-fold increase in CD34+ and CD34− CML cells (n= 30 patients), respectively. When Twist-1 expression could be monitored during CML progression in a same patient, a regular expression increase in the CD34+ compartment was observed [8.5 fold (n=17), 18 fold (n=4) and ≥105 fold (n=8) in chronic, accelerated and blastic phases respectively]. Twist-1 was also overexpressed in the CD34− compartment along with disease progression [between chronic and later stages (11 fold), but with no significant differences between accelerated (88 fold) and blastic (73 fold) phases]. Our results then clearly identify Twist-1 gene expression as a unique predictive molecular marker of resistance, especially in patients with unidentified mechanism of resistance as we were able at the time of diagnosis to point out 2 patients/13 that present a higher Twist-1 level relatively to the mean value and which have develop a cytogenetic resistance within 6 months (ELN criteria) without any mutation, BCR-ABL overexpression or clonal evolution. The overexpression of Twist-1 is predominant in the immature compartment during CML progression and resistance, supporting the hypothesis that resistant cells that overexpress Twist-1 might emerge from CML stem cells. In samples from patients who became resistant to imatinib within the course of the disease, Twist-1 expression follow-up at the time of cytogenetic remission, and later resistance, indicate that Twist-1 expression was transiently down regulated by TKIs. Finally, using Twist-1 specific siRNA we confirmed in CML cell lines the direct involvement of this protein in CML resistance to imatinib. Altogether, our results suggest for the first time that Twist-1 may represent a useful molecular marker to identify at diagnosis patients prone to become resistant to imatinib. The exact interactions between Twist-1 and BCR-ABL proteins pathways remain to be determined yet. Disclosures:No relevant conflicts of interest to declare.

Paediatric soft tissue tumours: from histology to molecular diagnosis

Paediatric sarcomas comprise a diverse group of relatively uncommon neoplasms, with morphological features that range from primitive round cell tumours to fasciculated spindle cell cancers. These histologies frequently overlap and cause diagnostic confusion or uncertainty, so that ancillary testing has become the standard of practice. In previous years, immunohistochemistry and electron microscopy were commonly used, but the genetic revolution has led to the discovery of unique molecular markers that can be detected using routinely fixed and embedded tissues. Detection of characteristic genetic features of paediatric sarcomas has become more readily available and offers advantages over other ancillary tests, so that general pathologists should be familiar with molecular markers and the procedures used to detect them. This brief review outlines the more common types of paediatric sarcomas and describes the morphological and genetic features that facilitate their diagnosis.

Real-time Polymerase Chain Reaction Quantification of Porphyromonas gingivalis and Tannerella forsythia in Primary Endodontic Infections

Introduction Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method The root canal levels of P. gingivalis, T. forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic ( n = 14) and asymptomatic ( n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB ( P. gingivalis), bspA ( T. forsythia), and rpoB (total bacteria) single copy genes. Results Overall, P. gingivalis, T. forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P. gingivalis and T. forsythia levels ranged from 5.65 × 10 −6 to 1.20 × 10 −2 and from 5.76 × 10 −6 to 1.35 × 10 −1. T. forsythia was highly prevalent and numerous in the study groups, whereas P. gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions The endodontic levels of P. gingivalis and T. forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin.

Searching for adult stem cells in the intestine

The epithelium of the small intestine is the most rapidly self‐renewing tissue of mammals. Vigorous proliferation of progenitor cells occurs in crypts, while their differentiated offspring leaves the crypts and travels up the flanks of the villi to suffer death by apoptosis at villus tips 3 days later (Fig 1). The crypts have long been assumed to harbour functional stem cells. » The crypts have long been assumed to harbour functional stem cells. « Yet, the absence of unique molecular markers has hampered their definitive identification. Although there has been agreement that crypts contain 4–6 independent stem cells, two hypotheses exist regarding their exact identity and location. The small intestinal crypt is pictured as a tube of proliferating cells bounded from below by Paneth cells (Fig 1A) (Bjerknes & Cheng, 2005). Since the late 1950s, the prevailing hypothesis has proposed that the stem cells reside at position +4 relative to the crypt bottom, while the first three positions are occupied by the terminally differentiated Paneth cells. Potten et al have provided experimental support for this model, by demonstrating that label‐retaining, radiation‐sensitive cells reside specifically at the +4 position (Potten, 1977; Potten et al, 1974, confirm 12 and 13). The second, more recent hypothesis is based on the identification of the crypt base columnar (CBC) cells, small cycling cells inconspicuously hidden between the Paneth cells (Cheng & Leblond, 1974a) (Fig 1A). The proposal was initially based on morphological features of these cells (Cheng & Leblond, 1974b), but clonal marking techniques have later led Leblond, Cheng and Bjerknes to propose that the CBC cells represent the true stem cells (Bjerknes & Cheng, 1981a, 1981b, 1999). Figure 1. Stem cells of the small intestine .

Defining Macropinocytosis

Macropinocytosis represents a distinct pathway of endocytosis in mammalian cells. This actin-driven endocytic process is not directly co-ordinated by the presence of cargo but can be induced upon activation of growth factor signalling pathways. The capacity to dissect the contribution of macropinocytosis to cellular processes has been hampered by a lack of unique molecular markers and defining features. While aspects of macropinosome formation and maturation are common to those shared by the other endocytic pathways, a number of key differences have recently begun to emerge and will be discussed in this study. It is now well established that macropinocytosis significantly contributes to antigen presentation by the immune system and is exploited by a range of pathogens for cellular invasion and avoidance of immune surveillance.

A split and rearranged nuclear gene encoding the iron-sulfur subunit of mitochondrial succinate dehydrogenase in Euglenozoa

BackgroundAnalyses based on phylogenetic and ultrastructural data have suggested that euglenids (such as Euglena gracilis), trypanosomatids and diplonemids are members of a monophyletic lineage termed Euglenozoa. However, many uncertainties are associated with phylogenetic reconstructions for ancient and rapidly evolving groups; thus, rare genomic characters become increasingly important in reinforcing inferred phylogenetic relationships.FindingsWe discovered that the iron-sulfur subunit (SdhB) of mitochondrial succinate dehydrogenase is encoded by a split and rearranged nuclear gene in Euglena gracilis and trypanosomatids, an example of a rare genomic character. The two subgenic modules are transcribed independently and the resulting mRNAs appear to be independently translated, with the two protein products imported into mitochondria, based on the presence of predicted mitochondrial targeting peptides. Although the inferred protein sequences are in general very divergent from those of other organisms, all of the required iron-sulfur cluster-coordinating residues are present. Moreover, the discontinuity in the euglenozoan SdhB sequence occurs between the two domains of a typical, covalently continuous SdhB, consistent with the inference that the euglenozoan 'half' proteins are functional.ConclusionThe discovery of this unique molecular marker provides evidence for the monophyly of Euglenozoa that is independent of evolutionary models. Our results pose questions about the origin and timing of this novel gene arrangement and the structure and function of euglenozoan SdhB.

Phylogenetic Diversity of Ficus Species Using HAT-RAPD Markers as a Measure of Genomic Polymorphism

To create a molecular marker characterization for twenty species of Ficus, eight decamer primers were used to randomly amplify these species genomic DNA producing a total of 172 distinct polymorphic band patterns. One band was chosen to be converted into the more robust sequence characterized amplified region (SCAR) marker format to provide a unique molecular marker characterization for the variety of Ficus hirta. This technique for species identification and char- acterization provides a morphologically independent test to verify relatedness and provide species information particularly for cases where such identification was previously untenable such as in the case of morphologically indistinguishable plant cuttings.

Immune response to Mycobacterium tuberculosis and identification of molecular markers of disease.

The complex molecular events that occur within the host during the establishment of a Mycobacterium tuberculosis infection are poorly defined, thus preventing identification of predictive markers of disease progression and state. To identify such molecular markers during M. tuberculosis infection, global changes in transcriptional response in the host were assessed using mouse whole genome arrays. Bacterial load in the lungs, the lesions associated with infection, and gene expression profiling was performed by comparing normal lung tissue to lungs from mice collected at 20, 40, and 100 days after aerosol infection with the H37Rv strain of M. tuberculosis. Quantitative, whole lung gene expression identified signature profiles defining different signaling pathways and immunological responses characteristic of disease progression. This includes genes representing members of the interferon-associated gene families, chemokines and cytokines, MHC, and NOS2, as well as an array of cell surface markers associated with the activation of T cells, macrophages, and dendritic cells that participate in immunity to M. tuberculosis infection. More importantly, several gene transcripts encoding proteins that were not previously associated with the host response to M. tuberculosis infection, and unique molecular markers associated with disease progression and state, were identified.

CD158K/KIR3DL2 Transcript Detection in Lesional Skin of Patients with Erythroderma Is a Tool for the Diagnosis of Sézary Syndrome

The distinction between Sézary syndrome (SS) and benign erythrodermic inflammatory diseases (EID) is difficult to make both clinically and on skin biopsies, since histomorphology can provide nonspecific results. New markers of circulating malignant Sézary cells have been recently described, especially CD158k/KIR3DL2 and T-plastin, but it has not been yet determined whether they could help in the diagnosis of erythroderma in skin samples. In this study, 13 frozen skin specimens from 10 SS patients and 26 from EID were analyzed for CD158k/KIR3DL2 expression using immunohistochemistry with AZ158 mAb, which also recognizes the monomeric CD158e/KIR3DL1 receptor. Although positive in all SS samples, immunohistochemistry appeared to not reliably discriminate between SS and EID. Therefore in all samples disclosing a significant staining with AZ158 mAb, CD158k/KIR3DL2, CD158e/KIR3DL1 and T-plastin mRNA expression were analyzed on the same skin specimen using conventional and/or quantitative real-time reverse transcription (RT)-PCR. Interestingly, only CD158k/KIR3DL2 transcripts were found to be significantly overexpressed in skin biopsies from patients with SS (P<0.0001), including when normalization to CD3 expression was achieved (P=0.0003). In light of these findings, CD158k/KIR3DL2 transcripts appear to be a unique molecular marker of SS in skin samples, allowing differential diagnosis with benign EID in routine practice.

Atmospheric Outflow of Anthropogenic Semivolatile Organic Compounds from East Asia in Spring 2004

To estimate the emissions of anthropogenic semivolatile organic compounds (SOCs) from East Asia and to identify unique SOC molecular markers in Asian air masses, high-volume air samples were collected on the island of Okinawa, Japan between 22 March and 2 May 2004. Contributions from different source regions (China, Japan, the Koreas, Russia, and ocean/local) were estimated by use of source region impact factors (SRIFs). Elevated concentrations of hexachlorobenzene (HCB), hexachlorcyclohexanes (HCHs), dichlorodiphenyltrichloroethanes (DDTs), and particulate-phase polycyclic aromatic hydrocarbons (PAHs) were attributed to air masses from China. A large proportion of the variation in the current-use pesticides, gas-phase PAHs, and polychlorinated biphenyl (PCB) concentrations was explained by meteorology. Chlordanes showed a technical mixture profile and similar concentrations regardless of source region. alpha/gamma HCH and trans/cis chlordane ratios did not vary significantly with different source regions and had regional averages of 2.5 +/- 1.0 and 1.2 +/- 0.3, respectively. Particulate-phase PAH concentrations were significantly correlated (p value < 0.05) with other incomplete combustion byproduct concentrations, including elemental mercury (Hg0), CO, NOx, black carbon, submicrometer aerosols, and SO2. By use of measured PAH, CO, and black carbon concentrations and estimated CO and black carbon emission inventories, the emission of six carcinogenic particulate-phase PAHs was estimated to be 1518-4179 metric tons/year for Asia and 778-1728 metric tons/year for China, respectively. These results confirm that East Asian outflow contains significant emissions of carcinogenic particulate-phase PAHs.

Detection of mutated BRAFV600E variant in circulating DNA of stage III–IV melanoma patients

BRAFV600E is the most represented somatic point mutation in cutaneous melanoma, thus providing a unique molecular marker for this disease. The development of efficient methods for its detection in free circulating DNA of patients may lead to the improvement of diagnostic and prognostic tools. With this aim, we evaluated whether BRAFV600E represents a detectable marker in the plasma/serum from melanoma patients in a pilot study. Circulating cell-free DNA was extracted from the serum or plasma of 15 healthy donors and 41 melanoma patients at different clinical stages and obtained either presurgery or after surgery during follow-up. Quantitative analysis showed higher levels of circulating free DNA in patients compared to controls, with the highest levels detected in samples obtained presurgery and at stage IV. Four different PCR methods were compared for their capacity to amplify a few copies of BRAFV600E in wild-type DNA. BRAFV600E was detectable in circulating DNA of 12 patients and in none of the controls; only 1 PCR method reproducibly amplified BRAFV600E. Positive samples were obtained from 8/13 patients at stage IV and from 4/24 patients at stage III, but not in 4 patients at stage I-II; half of the positives were obtained presurgery and half at follow-up. Correspondence between circulating DNA and related tumors were examined for 20 patients, and a correlation was found for stage IV patients. In conclusion, this method can be utilized for monitoring the disease in stage IV melanoma patients but it appears unsatisfactory for the early detection of melanoma.

Lymphatic endothelial cells, tumor lymphangiogenesis and metastasis: New insights into intratumoral and peritumoral lymphatics

Lymphatic metastasis of tumor cells represents a series of extremely complex and sequential processes that include dissemination and invasion into surrounding stromal tissues from primary tumors, penetration into lymphatic walls and implantation in regional lymph nodes, and extravasation or proliferation in parenchyma of target organs. Recent developments in lymphatic biology and research, especially the application of unique molecular markers specific for lymphatic endothelial cells (LECs), LYVE-1, Prox-1 and podoplanin have provided exciting new insights into the tumor microenvironment and LEC-tumor cell interface. To date, established factors for determining the behavior and prognosis of primary tumors have been emphasized morphologically and physiologically, i.e., lymphatic impairment and vessel density, dysfunction of lymphatic valves, interstitial fluid pressure, as well as a series of lymphangiogenic growth factors including VEGF-C/-D, and other cytokines and chemokines. Increasing knowledge of the tumor biological significance in lymphatics within the tumors (intratumoral lymphatics, ITLs) and at the tumor periphery (peritumoral lymphatics, PTLs) has greatly promoted understanding of tumor access into the lymphatic system by inducing lymphangiogenesis or by co-opting preexisting lymphatics. Therefore, the targeting PTLs and ITLs, which have been proposed as an important route for antimetastatic approach, are deemed worthy of further study in various animal tumor models and human tumors.

Spatial distribution of PM 2.5 associated organic compounds in central California

As part of the California Regional PM 2.5/PM 10 Air Quality Study, annual average concentrations of PM 2.5 associated organic compounds were measured using filter samples collected every sixth day for 24 h for 1 year. During this time many central California sites had 24-h PM 2.5 mass concentrations exceeding the National Ambient Air Quality Standard of 65 μg m −3. The highest concentration of 175 μg m −3 was recorded in Fresno on January 1, 2001. Organic speciation of 20 sites within and near the central California valley provides a measure of the spatial differences of emission sources through the use of organic molecular markers. Additionally, it provides an opportunity evaluate their utility at ambient concentrations to measure the influence of emission sources at rural and urban locations. The most abundant particulate phase organic compounds identified were polar organic compounds. Sugar anhydrates, molecular markers of wood combustion, constituted the largest weight fraction of total carbon, followed by alkanoic acids, and alkanedioic acids. Local emission sources such as residential wood combustion, gasoline and diesel vehicles were distinguished by unique molecular markers and found to vary among the annual average sites. The annual average concentrations of individual organic species within compound classes were observed to be highly correlated for many of the central California sites. The two Fresno sites were highly correlated to each other especially with respect to polycyclic aromatic hydrocarbons, but not well correlated to other sites.

CULTURAL, MORPHOLOGICAL, PHYSIOLOGICAL AND MOLECULAR STUDIES ON SOME STREPTOMYCETE ISOLATES

In this study, three isolates of Streptomyces isolated from Egyptian soil at Giza Governorate and belonging to red series were identified based on their cultural, morphological and physiological characters. According to the key proposed by Pridham and Tresner (1974), the experimental isolates ST55, ST71 and ST86 ap-peared to be related to S. lincolnensis, S. venezuelae and S. umbrinus, respectively. In addition, random amplified polymorphic DNA (RAPD)-polymerase chain reac-tion (PCR) was used to amplify the DNA genome of the three applied isolates and an identified strain to determine their fingerprints. This was carried out using 10-decamer oligonucleotides, i.e., OP-A02, OP-D01, OP-D02, OP-D05, OP-D06, OP-D07, OP-D08, OP-D11, OP-D18 and OP-D20. The PCR amplified products were detected by electrophoresing on 1.5% agarose gel and visualized by staining in eth-idium bromide and UV trasilluminator. Results showed that the same oligonucleo-tide flanking fragments differed from one isolate to another with different sizes. Monomorphic and polymorphic fragments were amplified confirming the molecular relationships as well as the genetic diversity between the four applied strains of Streptomyces. Based on the statistical analysis of RAPD-PCR polymorphisms a de-gree of similarities ranged from 33.9 to 59.2 % was found. Furthermore, frag-ment(s), whatever absent or present, were found to be unique molecular markers and could be used for characterization of some isolates. This result supported the idea for using the RAPD-PCR technique for classification of Streptomyces isolates.

The changing cellular environments of hematopoiesis in human development in utero

The hematopoietic system is indispensable from the earliest stages of development and adapts to the rapidly changing anatomy of the embryo and fetus; this takes place in such different anatomic locations as the yolk sac blood island, hepatic parenchyme, aorta-gonads-mesonephros paravascular mesenchyme, and bone marrow primary logette. We herein summarize our investigation of these serial blood-forming events in the human embryo and fetus. The access to early stages of human development, availability of a large panoply of molecular markers for human blood cell lineages, and recent development of robust assays for the earliest human hematopoietic stem cells have allowed us to gain relatively clear insight into the developmental sequence that underlies the ontogeny of human blood cells. Conversely, the control exerted by these diverse cellular environments on the emergence of human hematopoietic cells remains elusive, as is the case in animal models. We nonetheless present preliminary attempts to decipher the structure and molecular characteristics of the distinct cellular "niches" in which blood cells are produced during human gestation.

Establishment of an immortalized cell line from a precancerous lesion of lung adenocarcinoma, and genes highly expressed in the early stages of lung adenocarcinoma development

Atypical adenomatous hyperplasia (AAH) is classified as a precancerous lesion of lung adenocarcinoma. We established an immortalized AAH cell line (PL16T) and a human non-neoplastic bronchial epithelial cell line (PL16B) from the same patient by transfection with the gene for SV40 large T antigen. The expression profile of PL16T was compared with that of PL16B by the suppression subtractive hybridization method. From 704 selectively hybridized clones, we finally selected 25 fragments of mRNA that showed transcription levels more than three times higher in PL16T than in PL16B. Thirteen (52%) and eight (32%) of them encoded tumor-associated calcium signal transducer 2 (TACSTD2) and S100 calcium binding protein A2 (S100A2), respectively. The high transcription of TACSTD2 and S100A2 in PL16T was confirmed by in situ hybridization. In normal lung tissue, both TACSTD2 and S100A2 were expressed at very low levels, but seven and five of 14 AAH were positive for TACSTD2 and S100A2, respectively. The frequency of TACSTD2 positivity was increased in 16 of 22 bronchioloalveolar carcinomas (BAC) and adenocarcinoma with mixed subtype with BAC component (mixed BAC). Positivity for S100A2 occurred in four of 22 BAC and mixed BAC. The abnormal transcription of TACSTD2 and S100A2 are thought to be unique molecular markers of the preinvasive stage of lung adenocarcinoma.

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts

The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.

Polygenic analysis of ammonia-oxidizing bacteria using 16S rDNA, amoA , and amoB genes

Finding a unique molecular marker capable of quickly providing rigorous and useful phylogenetic information would facilitate assessing the diversity of ammonia-oxidizing bacteria in environmental samples. Since only one of several available markers can be used at a time in these kinds of studies, the 16S rDNA, amoA and amoB genes were evaluated individually and then compared in order to identify the one that best fits the information provided by the composite dataset. Distance-based neighbor-joining and maximum parsimony trees generated using the sequences of the three mentioned genes were analyzed with respect to the combined polygenic trees. Maximum parsimony trees were found to be more accurate than distance-based ones, and the polygenic topology was shown to best fit the information contained in the sequences. However, the taxonomic and phylogenetic information provided by the three markers separately was also valid. Therefore, either of the functional markers (amoA or amoB) can be used to trace ammonia oxidizers in environmental studies in which only one gene can be targeted.

Human Fetal Liver Derived Stem Cells Can Be Support the Maintenance of Human Embryonic Stem Cells.

Prolonged propagation of human embryonic stem (ES) cells is currently achieved by co-culture with primary or immortalized mouse embryonic fibroblast (MEF) cells. In order to replace the heterologous with homologous co-culture systems, an attempt was made using mononuclear cells derived from human fetal liver. Human fetal liver-derived mesenchymal-like stem cells (FL-MLSC) can be maintained for the prolonged period of time. They showed the characteristics of mesenchymal stem cells in various aspects. They retained a normal diploid karyotype and growth characteristics over the successive culture. Human ES cells cultured on human FL-MLSC cells up to 8 passages displayed the unique morphology and molecular markers characteristic for undifferentiated human ES cells as cultured on MEF cells. Alkaline phosphatase activity was detected in human ES cells co-cultured on human FL-MLSC. Immunocytochemical analyses showed that expressions of stage-specific embryonic antigen-3, -4 and Oct-4 were not altered on human ES cells cultured on human FLDSC. Reverse-transcriptase PCR analyses showed that similar expressions of Oct-4 and Nanog genes, markers for undifferentiated ES cells, were also observed in human ES cells cultured on both human FL-MLSC and MEF cells. Furthermore, human ES cells cultured on human FL-MLSC retained unique differentiation potentials in culture when allowed to form embryoid body. Results of this study suggest that human FL-MLSC can support the maintenance of human ES cell in vitro.

Identification of region-specific transcription factor genes in the adult mouse brain by medium-scale real-time RT-PCR

We established a medium-scale real-time RT-PCR system focusing on transcription factors and applied it to their expression profiles in the adult mouse 11 brain regions ( http://genome.gsc.riken.jp/qRT-PCR/). Almost 90% of the examined genes showed significant expression in at least one region. We successfully extracted 179 region-specific genes by clustering analysis. Interestingly, the transcription factors involved in the development of the pituitary were still expressed in the adult pituitary, suggesting that they also play important roles in maintenance of the pituitary. These results provide unique molecular markers that may account for the molecular basis of the unique functions of specific brain regions.