Adipose tissue is an easily accessible and abundant source of stem cells. Adipose stem cells (ASCs) are currently being researched as treatment options for repair and regeneration of damaged tissues. The standard culture conditions used for expansion of ASCs contain fetal bovine serum (FBS) which is undefined, could transmit known and unknown adventitious agents, and may cause adverse immune reactions. We have described a novel culture condition which excludes the use of FBS and characterised the resulting culture. Human ASCs were cultured in the novel culture medium, which included complement protein C3. These cultures, called C-ASCs, were compared with ASCs cultured in medium supplemented with FBS. Analysis of ASCs for surface marker profile, proliferation characteristics and differentiation potential indicated that the C-ASCs were similar to ASCs cultured in medium containing FBS. Using a specific inhibitor, we show that C3 is required for the survival of C-ASCs. This novel composition lends itself to being developed into a defined condition for the routine culture of ASCs for basic and clinical applications.

ObjectiveBone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations.Materials and MethodsRat MSC were plated at 20, 200, and 2000 cells/cm2 and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations.ResultsWe found optimal cell growth at a plating density of 200 cells/cm2. Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential.ConclusionOur findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.

OBJECTIVE:The aim of this study was to examine the effects of the route of administration [intrabone marrow (IBM) vs intravenous (IV)] and the role of conditioning with irradiation in optimizing mesenchymal stem cell (MSC) transplantation. MATERIALS AND METHODS:To determine if irradiation resulted in depletion of colony-forming unit fibroblasts (CFU-F), which might favor the engraftment of donor MSC, the number of CFU-Fs was assayed from animals receiving either hemibody irradiation (HBI) or total body irradiation (TBI). RESULTS:TBI resulted in a marked reduction of CFU-F numbers that spontaneously resolved, whereas animals receiving HBI did not experience depletion of CFU-F. Animals receiving MSC grafts by the IV route had higher numbers of marrow CFU-F. MSC were transduced using retroviral vectors encoding the neomycin resistance gene (Neo(R)) and a second gene encoding either the human soluble tumor necrosis factor receptor (hsTNFRII) or beta-galactosidase (beta-Gal). MSCs were administered by either the IV or IBM route to animals receiving HBI. The Neo(R) transgene was detectable in hematopoietic tissues of all animals and nonhematopoietic tissues in a single animal. Evidence of transgene expression was documented by detection of beta-Gal(+) cells in BM smears and transiently elevated serum levels of hsTNFRII. CONCLUSION:These studies indicate that 1) MSC possess the ability to engraft and persist in an unrelated mismatched allogeneic hosts; 2) 250-cGy HBI did not favor engraftment of MSC; 3) the IBM route was not more effective than the IV route in delivering MSC grafts; and 4) transplanted MSC preferentially localized to the marrow rather than nonhematopoietic tissues.

Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron-like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [2002] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time-lapse studies. Similar neurite-like processes were induced by treating MSC with DMSO only or with actin filament-depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages. (C) 2004 Wiley-Liss, Inc.

. The inner centromere protein (INCENP), which has previously been described in chicken, frog and mouse, is required for correct chromosome segregation and cytokinesis. We have identified the human INCENP gene by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized it to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. Recently we showed that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Here we demonstrate that, consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and we show here that HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines.

Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

This review is concerned with the role of chromosomal nonhistone proteins in three important aspects of mitotic events: chromosome condensation, sister chromatid separation at the metaphase:anaphase transition, and interactions between the chromosomes and cytoskeleton that occur during construction of the mitotic spindle and cleavage furrow. Emphasis will be given to the potential roles of topoisomerase II and the chromosome passenger proteins in these events. Other important aspects of mitotic events such as the regulation of the G2-->M transition, the structural changes that affect the nuclear envelope and other organelles during mitosis, and the mechanism of chromosome movement will not be considered here. Despite long histories of often elegant experimentation, all three of our chosen subjects remain areas of lively, ongoing controversy. Thus, although recent advances appear to have taken us many steps closer to an understanding of the underlying mechanisms, we suspect that final answers will be some time in coming.

This review is concerned with the role of chromosomal nonhistone proteins in three important aspects of mitotic events: chromosome condensation, sister chromatid separation at the metaphase:anaphase transition, and interactions between the chromosomes and cytoskeleton that occur during construction of the mitotic spindle and cleavage furrow. Emphasis will be given to the potential roles of topoisomerase II and the chromosome passenger proteins in these events. Other important aspects of mitotic events such as the regulation of the G(2)-->M transition, the structural changes that affect the nuclear envelope and other organelles during mitosis, and the mechanism of chromosome movement will not be considered here. Despite long histories of often elegant experimentation, all three of our chosen subjects remain areas of lively, ongoing controversy. Thus, although recent advances appear to have taken us many steps closer to an understanding of the underlying mechanisms, we suspect that final answers will be some time in coming.

The INCENPs (INnerCENtromereProteins - Mr133 and 145 kDa) are tightly bound to chromatin until early metaphase, and arrive at the metaphase spindle plate as integral components of the chromosomes (Cooke et al., 1987; Earnshaw & Cooke, 1991). It is thus surprising that the INCENPs leave the chromosomes during the latter part of metaphase to become associated with fibrous structures traversing the metaphase plate (presumably the overlapping polar microtubules of the spindle). Following the onset of anaphase, the INCENPs remain closely associated with the stem body material in the central spindle and also become associated with the inner surface of the cell membrane at the position where the cleavage furrow subsequently forms.By combining library screening with reverse-transcriptase PCR, we have recovered cDNA clones that contain the complete open reading frames (ORFs) of the two chicken INCENPs. Class 1 INCENP cDNA contains an ORF of 839 codons, which includes a predicted central coiled-coil domain of about 200 residues.

AbstractWe have used six different monoclonal and polyclonal antibodies, four different antigen preparations and two different detection systems to compare Western blotting with Coomassie Blue prestained gels (Jackson and Thompson, Electrophoresis, 1984, 5, 35–42) to blotting of unstained gels with Amido Black or Fast Green post‐transfer staining of the nitrocellulose. Contrary to a recent report (Harper et al., Anal. Biochem., 1986, 157, 270–274), in which post‐staining with Coomassie Blue was determined to severely inhibit immunoreactivity, we find using prestained Coomassie Blue gels to be extremely useful in most cases, although, rarely, inhibition of the immune reaction may take place. Post‐staining with Amido Black or Fast Green may also prove useful, but is not recommended since similar inhibition occurs, and there are the added disadvantages of not being able to “pre‐view” the gel pattern and not being able to use stored gels. Further studies indicated that the rare loss of immunoreactivity seen with Coomassie Blue prestained gels is most likely due to the necessary fixation step, and not the stain itself (as has been suggested), since the protein‐dye complex is dissociated during transfer.

The pathobiology of malignant mesothelioma (MM) in man is obscure, although the neoplasm usually occurs in individuals who have experienced heavy exposure to amphibole asbestos. In an effort to better understand the pathogenesis of MM in man, we initiated studies using the rat model developed by earlier workers. 1 Wagner JC Berry G Mesotheliomas in rats following inoculation with asbestos. Br J Cancer. 1969; 23: 567 Crossref PubMed Scopus (138) Google Scholar , 2 Wagner JC Berry G Timbrell V Mesotheliomata in rats after inoculation with asbestos and other minerals. Br J Cancer. 1973; 28: 173 Crossref PubMed Scopus (225) Google Scholar , 3 Davis JMG Histogenesis and fine structure of peritoneal tumors produced in animals by injections of asbestos. Natl Cancer Inst. 1974; 52: 1823 Crossref Scopus (36) Google Scholar , 4 Davis JMG The histopathology and ultrastructure of pleural mesotheliomas produced in the rat by injections of crocidolite asbestos. Br J Exp Pathol. 1979; 60: 642 PubMed Google Scholar , 5 Bolton RE Davis JMG Donaldson K Wright A Variations in the carcinogenicity of mineral fibres. Ann Occup Hyg. 1982; 26: 569 Crossref PubMed Scopus (38) Google Scholar Weanling Fischer-344 female rats were inoculated intraperitoneally with 25 mg of either UICC Rhodesian chrysotile or UICC crocidolite suspended in Hanks' buffered salt solution. The animals were evaluated at weekly intervals and sacrificed when and if ascites developed. The majority of the animals developed tumors from 6 to 24 months after inoculation. They involved the viscera and implanted as small foci over the peritoneal surface and the gut. The lesions exhibited a remarkable resemblance histologically to MM occurring in man. A number of studies have been initiated with tumor tissue obtained at the time of autopsy. Many but not all tumors can be cultured in vitro using Eagle's basal medium supplemented with insulin, hydrocortisone, selenium and transferrin and containing 10 or 20% fresh fetal bovine serum. In part, variability in tumor growth reflects the histologic features of the lesion inasmuch as the cells of epithelial tumors can be dissociated and implanted with relative ease, whereas fibrosarcomatous lesions rarely yield cells that grow in vitro. The chromosomal makeup of individual tumor cells was evaluated by flow cytometric analysis of cell DNA and by classic karyotype studies. Because the tumors in vivo and cell cultures in vitro exhibited a low degree of mitotic activity, it was difficult to prepare adequate numbers of metaphases for detailed analysis. Thus, flow cytometric studies provided exceptionally useful information on the chromosomal makeup of the tumors.

After long latency periods, DMM develop in rat inoculated into the pleural or peritoneal cavity with either chrysotile or crocidolite asbestos. Histologically, the tumors resemble the human lesion being either fibrosarcomatous or epithelial (or mixtures of the two cell types). Tumor tissue from most, but not all, lesions grow in serum containing medium in vitro. These tumor cells consistently are tetroploid or aneuploid; occasionally marker chromosomes are found. After a series of passages chemically defined serum-free medium maintains the growth of cells from many tumors in vitro. Cells in culture usually grow in monolayers but nodular masses of proliferating tumor cells develop from the cell sheet and readily float free in the medium. These seemingly spherical balls of cells can be used to establish fresh cultures, allowing the initial monolayers to grow indefinitely. The fine structural features of the nodular tumor masses have now been studied in detail. They consist of vacuolated epithelial cells which are replete with vellumentous villi. Experimentally-induced DMM in animals have characteristics similar to their human counterparts; implantation of metastases may develop from foci similar to those observed to form in cultures.