Feeder-free growth of undifferentiated human embryonic stem cells.

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

goosecoid expression represses Brachyury in embryonic stem cells and affects craniofacial development in chimeric mice.

The homeobox gene goosecoid, originally identified in Xenopus, is expressed in the organizer or its equivalent during gastrulation in the frog, chick, zebrafish and mouse. To investigate the role of goosecoid in mouse development, we have generated embryonic stem cells that stably overexpress the murine homolog of goosecoid. These cells show a repression of the gastrulation-associated gene Brachyury. Interestingly, repression of Brachyury is conserved between Xenopus and mouse despite the lack of conservation of the Brachyury promoter. Further characterization of the goosecoid-overexpressing ES cells revealed that they maintain the expression of stage-specific embryonic antigen-1, and teratomas derived from goosecoid-overexpressing cells show the presence of cell types derived from all three germ layers. Some highly chimeric mice derived from goosecoid-overexpressing cells displayed skull defects. These observations suggest that goosecoid may play a role in specification of anterior mesendodermal fates and specifically in mouse craniofacial development.

The diagnostic value of BAC for identifying alcohol-related problems among DWI offenders: another look.

The present study examines the association between breath alcohol concentration (BAC) at arrest and problem drinking for a sample of 1,283 male DWI offenders in the US Army. The results indicated a moderate but statistically significant association between BAC at arrest and DSM-III diagnosis. BAC's ability to indicate problem drinking was also compared with the diagnostic ability of three well-known, paper-and-pencil instruments designed for that purpose. BAC performed as well in identifying problems with alcohol as did the MAST, the MacAndrew Scale of the MMPI, and the Vaillant.

Mechanisms of genomic imprinting in mammals.

This chapter can be summarized by the following main points: Genomic imprinting results in the functional nonequivalence of the maternal and paternal genomes, thereby preventing the development of viable parthenogenotes and androgenotes in eutherian mammals. Imprinting may have arisen as a result of the specialized evolutionary requirements of the parental genomes or may have been an obligatory step in the development of placentation. A substantial proportion of transgenes and a smaller number of endogenous genes demonstrate imprinted pattern of expression in mice and humans. An analysis of DNA methylation in somatic tissues and germ cells during embryonic and postnatal development reveals dynamic changes, particularly during gametogenesis and early embryogenesis. The nature and timing of these changes suggest that DNA methylation may be involved in genomic imprinting. Imprinted genes display complex methylation patterns. Many aspects of these patterns are consistent with a role for methylation in the imprinted phenotype, although it is currently unclear whether methylation functions in the establishment of imprinting or plays a secondary role in the maintenance of the imprinted pattern of expression. Studies underway to identify new imprinted genes may help elucidate both the function and mechanism of genomic imprinting.

The prevalence of antisocial behavior among U.S. Army DWI offenders.

This study compares arrest records for three groups of male soldiers. The first group of 76 had been arrested for DWI, completed a 5-day, in-patient evaluation/education program and were subsequently re-arrested, all within the period from January 1985 through December 1987. The second group of 76 was composed of a random sample, matched by age and ethnicity who had completed the 5-day program following a DWI but had not been re-arrested. The third group was a control group of 76, matched by age and ethnicity, but with no record of DWI, who were randomly selected from the same military units as the initial two groups. Soldiers with one DWI had significantly more arrests than did soldiers in the control group; soldiers with two DWI arrests had significantly more arrests than either of the other groups. The data indicate that soldiers apprehended for DWI are more likely than non-arrestees to be arrested for a wide variety of antisocial behaviors.

The late retinoic acid induction of laminin B1 gene transcription involves RAR binding to the responsive element.

Transcription of the murine laminin B1 (LB1) gene is induced by retinoic acid (RA), but responds only 24-28 h after RA treatment in F9 EC cells. Here we have shown by gel retardation assay that all three retinoic acid receptors (RARs) alpha, beta and gamma expressed in Cos cells can bind directly to the previously characterized retinoic acid response element (RARE) of the LB1 promoter, albeit with a weaker affinity than to the RAR-beta gene RARE. Three stereo-aligned TGACC-like motifs are crucial for this binding. Interestingly, the capacity of RAR-alpha, -beta and -gamma to bind the LB1 RARE appears to be differentially modulated by factor(s) present in HeLa cells infected with RAR-expressing vaccinia virus vectors. Analyses of LB1 RARE mutants provide a strong correlation between RA-inducibility in vivo and efficiency of RAR binding in vitro. Thus, RARs can participate directly in transcriptional induction of the LB1 gene, even though this induction is cycloheximide sensitive and RARs are present in F9 cells prior to RA addition.

A retinoic acid-responsive element is present in the 5' flanking region of the laminin B1 gene.

The retinoic acid (RA)-associated differentiation of murine F9 teratocarcinoma stem cells results in dramatic changes in gene expression. The cellular gene encoding the B1 subunit of the extracellular matrix protein laminin is transcriptionally activated by RA, and its transcription is further enhanced by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) during the differentiation of F9 stem cells into extraembryonic parietal endoderm cells. We now report that expression vectors encoding the human RA receptors RAR-alpha, RAR-beta, and RAR-gamma can activate chloramphenicol acetyltransferase (CAT) expression from laminin B1 promoter/CAT expression vectors (e.g., p1.6LAMCAT) in RA-treated F9 cells, as measured in a transient transfection assay. Bt2cAMP does not further enhance the RA-associated increase in CAT activity. Through the use of deletion and mutation analyses, the RA-responsive element (RARE) of the murine laminin B1 gene has been defined as a 46-base-pair element between -477 and -432 of the laminin B1 5' flanking region. Insertion of a region of DNA containing this RARE in either orientation into a thymidine kinase promoter/CAT expression vector causes CAT expression to be activated 5- to 9-fold by the cotransfected human RAR-alpha or RAR-beta constructs in RA-treated F9 cells, and this RARE also functions in human HeLa cells. In contrast, this RARE in the p1.6LAMCAT vector does not activate CAT expression when cotransfected into F9 stem cells with the c-erbA gene in the presence of thyroid hormone. This suggests that the laminin B1 gene is activated by RA but not by thyroid hormone in vivo.

An evaluation and education program for driving while intoxicated offenders.

This report addresses the development of a mandatory, inpatient, evaluation and education program aimed at reducing Driving While Intoxicated (DWI) at a major Army installation. The program was designed to provide extensive evaluation to all soldiers within a week of the DWI. The majority of soldiers are young males, a population known for heavy drinking and a disproportionate amount of both motor-vehicle accidents and fatalities. Among the initial 490 soldiers admitted to the program, 88% were found to meet DSM-III criteria for alcohol abuse or alcohol dependence. Factors associated with a diagnosis of abuse or dependence were age; scores on the Vaillant alcohol questionnaire, the MacAndrews and F scales of the MMPI, the Mortimer-Filkins questionnaire; BAC at time of arrest; a prior history of alcohol-related problems; and certain blood hematology and chemistry values. Results indicate that DWI is a marker for serious alcohol problems.