An efficient approach to isolation and characterization of pre- and postnatal umbilical cord lining stem cells for clinical applications.

There have been various forms of mesenchymal stem cell-like (MSC-like) cells isolated from umbilical cords (UCs). The isolation of umbilical cord lining stem cells (ULSCs) may be of great value for those interested in a possible treatment to several disease/disorders. Unlike umbilical cord blood cells, these cells are unique because they can be expanded to therapeutically relevant numbers and cryopreserved for several different uses. Here we efficiently isolate stem cells from a small segment of pre- and postnatal UCs, and obtain therapeutically relevant amounts of ULSCs within 3 weeks. We demonstrate their growth potential and characterize them using immunocytochemistry, flow cytometry, and RT-PCR. In addition, we differentiate ULSCs into multiple lineages. Pre- and postnatal ULSCs are morphologically similar to mesenchymal stem cells (MSCs) and easily expand to greater than 70 population doublings. They express pluripotent markers Oct4 and nanog at the protein and RNA level. Flow cytometry demonstrates that they express markers indicative of MSCs in addition to high SSEA-4 expression. ULSCs are easily differentiated into osteogenic, adipogenic, chondrogenic, cardiogenic, and neurogenic cells. Pre- and postnatal ULSCs are characteristically similar in respect to their growth, marker expression, and plasticity, demonstrating they are highly conserved throughout development. ULSCs have phenotypic and genotypic properties of MSCs. These studies demonstrate the therapeutic potential of an otherwise discarded tissue. They are a perfect HLA match for the donor and an excellent match for immediate family members; therefore, they may serve as a therapeutic cell source.

A putative mesenchymal stem cells population isolated from adult human testes.

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Dielectric relaxation of DNA aqueous solutions.

We report on a detailed characterization of complex dielectric response of Na-DNA aqueous solutions by means of low-frequency dielectric spectroscopy (40 Hz-110 MHz). Results reveal two broad relaxation modes of strength 20

Screening and fundamental length scales in semidilute Na-DNA aqueous solutions.

The fundamental length scales in semidilute Na-DNA aqueous solutions have been investigated by dielectric spectroscopy. The low- and the high-frequency relaxation modes are studied in detail. The length scale of the high-frequency relaxation mode at high DNA concentrations can be identified with the de Gennes-Pfeuty-Dobrynin correlation length of polyelectrolytes in semidilute solution, whereas at low DNA concentrations and in the low added salt limit the length scale shows an unusual exponent reminiscent of semidilute polyelectrolyte chains with hydrophobic backbone. The length scale of the low-frequency relaxation mode corresponds to a Gaussian chain composed of correlation blobs in the low added salt limit, and to the Odijk-Skolnick-Fixman value of the single chain persistence length in the high added salt limit.

Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells.

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

The many shapes of mitochondrial membranes.

The roles of mitochondria in cell death and in aging have generated much excitement in recent years. At the same time, however, a quiet revolution in our thinking about mitochondrial ultrastructure has begun. This revolution started with the use of vital dyes and of green fluorescent protein fusion proteins, showing that mitochondria are very dynamic structures that constantly move, divide and fuse throughout the life of a cell. More recently, some of the first proteins contributing to these various processes have been discovered. Our view of the internal structures of mitochondria has also changed. Three-dimensional reconstructions obtained with high voltage electron microscopy show that cristae are often connected to the mitochondrial inner membrane by thin tubules. These new insights are brought to bear on the wealth of data collected by conventional electron microscopic analysis.

Differential usage of two 5' splice sites in a complex exon generates additional protein sequence complexity in chicken CLIP-170 isoforms.

Reverse transcription-polymerase chain reaction amplification and cloning of cDNA encoding the chicken CLIP-170(11) isoform of the Cytoplasmic Linker Protein 170 gene revealed an unusual source of protein sequence variation. In addition to differential combinatorial splicing of two cassette exons to yield four CLIP-170 protein isoforms, we found differential usage of alternative 5'-splice junctions in a single exon. Splicing at the downstream site yields message containing 18 bp of nucleotide sequence that is missing from message spliced at the more 5' site. This 18 bp sequence encodes a segment of 6 amino acids that fills a gap in the alignment of chicken and human CLIP-170 homologue sequences. Differential usage of the 5'-splice junctions in this complex exon appears to be tissue- rather than isoform-specific.

Identification and expression of two novel CLIP-170/Restin isoforms expressed predominantly in muscle.

CLIP-170 and Restin, microtubule-binding proteins originally cloned from human cells, are identical except for a stretch of 35 amino acids present in Restin, but missing from CLIP-170. Here we present the discovery of two novel isoforms of the CLIP-170/Restin gene in both chickens and humans. One of the new isoforms, named CLIP-170(11), contains an 11 amino acid insert instead of the 35 amino acid insert found in Restin. Eight of these 11 amino acids, including a helix-breaking proline residue, are perfectly conserved between chickens and humans. The second new isoform, named CLIP-170(11+35), contains both the 11 and 35 amino acid inserts in tandem. PCR analysis of chicken genomic DNA revealed that all four isoforms result from differential splicing of two exons in a region of the CLIP-170 gene that contains approximately 8.6 kb of intervening sequence. We found that the CLIP-170(11) and CLIP-170(11+35) are expressed preferentially in muscle tissues. Chicken and human skeletal muscle express predominantly CLIP-170(11) and to a lesser extent CLIP-170 and CLIP-170(11+35). Adult chicken cardiac and smooth muscles also express CLIP-170(11) and CLIP-170(11+35), but CLIP-170 is the predominant isoform in these muscles as it is in all other tissues except brain. The ratios of CLIP-170 isoform expression found in embryonic and adult chicken cardiac muscles reveal that isoform expression is regulated differentially in different developmental stages as well as in different tissues.

Cloning and expression of chicken CLIP-170 and restin isoforms.

We have cloned cDNA for the chicken homologues of human CLIP-170 and Restin and characterized expression of chicken CLIP-170 and Restin messages in a variety of chicken tissues. Chicken CLIP-170 and Restin, like the human homologues, differ only in a stretch of 35 amino acids present in Restin but missing from CLIP-170. This Restin-specific insert is perfectly conserved between the chicken and human sequences at both the protein and nucleotide level and contributes an additional five heptads to one of the heptad repeat regions in the central alpha-helical coiled-coil rod domain. Other highly conserved chicken and human CLIP-170/Restin regions confirm the importance of certain protein domains as crucial for protein function, including two CAP-Gly microtubule-binding motifs in the N-terminal globular head domain and two CCHC metal-binding motifs in the C-terminal globular tail domain. We have used Southern DNA blot analysis and PCR amplification of exon-intron junctions of chicken genomic DNA to confirm that CLIP-170 and Restin are isoforms encoded by the same gene. Semiquantitative RT-PCR analysis of CLIP-170 and Restin mRNA expression revealed expression of both isoforms in a variety of chicken tissues but in different ratios. In the tissues tested, except brain, the message for CLIP-170 was more abundant than that for Restin. Comparison of the levels of CLIP-170 and Restin messages in RNA from chicken and human intestinal epithelial cells revealed remarkably similar ratios in the two species. Our data suggest that expression of CLIP-170 and Restin is differentially regulated and that the two isoforms have distinct functions in a wide variety of cells.