Adipose tissue serves as a source of adipokines and cytokines with both local and systemic actions in health and disease. In this study, we examine the hypothesis that multipotent human adipose-derived stem cells (ASCs), capable of differentiating along the adipocyte, chondrocyte, and osteoblast pathways, contribute to adipose tissue-derived cytokine secretion. Following exposure to basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), the ASCs significantly increase their secretion of hepatocyte growth factor (HGF), a cytokine implicated in hematopoiesis, vasculogenesis, and mammary epithelial duct formation. Ascorbic acid synergizes with these inductive factors, further increasing HGF levels. Following exposure to lipopolysaccharide, ASCs increase their secretion of both hematopoietic (granulocyte/monocyte, granulocyte, and macrophage colony stimulating factors, interleukin 7) and proinflammatory (interleukins 6, 8, and 11, tumor necrosis factor alpha) cytokines based on ELISA and RT-PCR. In co-cultures established with umbilical cord blood-derived CD34(+) cells, the ASCs support long-term hematopoiesis in vitro. Furthermore, in short-term 12-day co-cultures, the ASC maintain and expand the numbers of both myeloid and lymphoid progenitors. These observations are consistent with the functionality of the secreted cytokines and confirm recent reports by other laboratories concerning the hematopoietic supportive capability of ASCs. We conclude that the ASCs display cytokine secretory properties similar to those reported for bone marrow-derived mesenchymal stem cells (MSCs).

No abstract

Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP ؊/؊ cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP ؊/؊ embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP ؊/؊ cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP ؊/؊ cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP ؊/؊ phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development.The CpG dinucleotide is an important regulatory component of mammalian genomes. The cytosine of this dinucleotide serves as the target for methylation by DNA methyltransferase (Dnmt) enzymes, which functions as a critical epigenetic modification of DNA. Methylated DNA is correlated with heterochromatin and transcriptionally inactive genes, while actively expressed genes are generally hypomethylated (58). Cytosine methylation may also represent a defense mechanism to silence parasitic repetitive DNA elements present in mammalian genomes (72,78). In addition, cytosine methylation is involved in the processes of genomic imprinting, in which paternal and maternal alleles of a gene exhibit distinct patterns of cytosine methylation and expression (65), and X chromosome inactivation, in which one X chromosome in each cell of a female becomes irreversibly inactivated during early development (52). The CpG dinucleotide is underrepresented in mammalian genomes (5 to 10% of the expected frequency), presumably due to the propensity of 5-methylcytosine to undergo spontaneous deamination to form thymine (8). Approximately 50% of human and mouse genes reside near unmethylated CpG islands, which contain the statistically expected frequency of CpG dinucleotides.Global cytosine methylation patterns inherited from gametes are erased during early embryogenesis (morula), followed by a wave of de novo DNA methylation in the blastocyst upon implantation (44). Dnmt3a and Dnmt3b are de novo methyltransferases that preferentially recognize unmethylated CpG motifs (49), while Dnmt1 is a maintenance methyltransferase that recognizes hemimethylated DNA (5), the imme...

Ubiquitin-mediated degradation targets cell cycle regulators for proteolysis. Much of the ubiquitin pathway's substrate specificity is conferred by E3 ubiquitin ligases, and cullins are core components of some E3s. CUL-4A encodes one of six mammalian cullins and is amplified and/or overexpressed in breast cancer, which suggests a role in regulating cell cycle progression. To examine CUL-4A's physiologic function, we generated a CUL-4A deletion mutation in mice. No viable CUL-4A ؊/؊ pups and no homozygous mutant embryos as early as 7.5 days postcoitum (dpc) were recovered. However, CUL-4A ؊/؊ blastocysts are viable, hatch, form an inner cell mass and trophectoderm, and implant (roughly 4.5 dpc), indicating that CUL-4A ؊/؊ embryos die between 4.5 and 7.5 dpc. Despite 87% similarity between the Cul-4A and Cul-4B cullins, the CUL-4A ؊/؊ lethal phenotype indicates that CUL-4A has one or more distinct function(s). Surprisingly, 44% fewer heterozygous pups were recovered than expected by Mendelian genetics, indicating that many heterozygous embryos also die during gestation due to haploinsufficiency. Taken together, our findings indicate that appropriate CUL-4A expression is critical for early embryonic development.Ubiquitin-mediated degradation plays a critical role in controlling the turnover of cell cycle regulators (reviewed in references 11 and 66). The ATP-dependent attachment of ubiquitin to a ubiquitin-activating enzyme (E1) activates ubiquitin for transfer to a ubiquitin-conjugating enzyme (E2) and then to a ubiquitin ligase (E3), which transfers ubiquitin to a substrate protein (reviewed in references 17 and 46). Repetition of this ubiquitin transferase reaction results in the attachment of a polyubiquitin chain to the substrate, which is then recognized by the 26S proteasome and degraded. Much of the ubiquitin pathway's substrate specificity derives from E3 ligases, so regulating E3 activity is likely to be important for controlling ubiquitin-mediated degradation. Cullins are a core component of a subset of E3 ligases (described below). In this report, we describe in vivo studies that examine the physiologic function of the CUL-4A cullin.

We have designed cosmid vectors for rapid genomic "walking" and restriction mapping. These vectors contain the transcription promoters from either bacteriophage SP6, T7, or T3 flanking a unique BamHI cloning site. Mammalian expression modules encoding the dominant marker neomycin phosphotransferase or the amplifiable dihydrofolate reductase gene expressed from SV40 promoters were inserted for use in gene transfer studies. Restriction sites for the enzymes Not I and Sfi I, which cut mammalian DNA very infrequently, have been engineered near the transcriptional promoters to enable the excision of most inserts as single, full-length fragments. Genomic libraries representative of mouse, human, and hamster genomes were constructed by inserting 33-to 44-kilobase-pair (kbp) DNA fragments, generated by partial cleavage of genomic DNA with Mbo I or Sau3A, into the unique BamHI site. Digestion of recombinant cosmids with restriction enzymes that cleave frequently but do not disrupt the transcriptional promoters generates two small DNA templates for the synthesis of end-specific RNA probes to facilitate directional "walking." Cosmid restriction maps can be determined rapidly by one of several methods. The cosmids and methods we describe should have wide utility in determining the functional and structural organization of complex eukaryotic genomes and for physically linking distant genetic loci.