Leptin receptor/CD295 is upregulated on primary human mesenchymal stem cells of advancing biological age and distinctly marks the subpopulation of dying cells.

During the lifetime of an adult organism, stem cells face extrinsic and intrinsic aging. Mesenchymal stem cells (MSC) can be expanded in culture, and the proliferation potential of individual cell isolates before growing senescent appear to be dependent on fitness and age of the donor, respectively. To date no molecular markers are available, which specifically reflect the degree of cellular aging in a population of MSC. Employing a genomic approach, we noticed that the gene encoding leptin receptor (also termed OB-R) is differentially regulated in MSC derived from aged donors as well as in MSC that had been stressed due to cultivation under hyperoxic conditions. We further observed that the leptin receptor transcript levels in primary MSC isolates are inversely correlated with the prospective number of generations that are ahead of these cells in culture, i.e., the number of population doublings that will occur in long term culture prior to cessation of growth due to replicative senescence. The MSC subpopulation, which exhibited distinctly elevated levels of leptin receptor or CD295 at the cell surface, is indistinguishable from dying cells. Considered together with the observation that primary MSC derived from healthy individuals showed proliferation capacities that declined at differentially increasing rates, we concluded that attenuation of MSC proliferation potential during aging greatly relies on the strictly increasing withdrawal of cells due to cell death.

High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris.

Hyaluronidase from honey bee was recombinantly expressed as a secreted glycoprotein in Pichia pastoris. The active enzyme was produced in milligram quantities per liter of primary culture. When changing the codons of the original transcript to triplet sequences preferred by P. pastoris, no further increase of protein product could be achieved. After expression of a fusion protein by linking hyaluronidase and human serum albumin together with the recognition sequence for the protease, factorXa, fragmented protein products were obtained in the culture supernatant. Only after replacement of the hinge region with a serine-glycine-rich linker, stable full-length fusion protein could be generated. The protein products were purified by cation exchange chromatography at pH 5.0 and pure enzyme fractions were further characterized in detail. The biochemical properties of the product matched those of crude hyaluronidase within bee venom: the native and the recombinant enzyme exhibited activity over a pH range from 3 to 8 (maximum: 3.8), at temperatures as low as 4 degrees C and up to 90 degrees C (maximum 62 degrees C), and at ionic strength as high as 2 M salt. Recombinant bee hyaluronidase efficiently degrades 6-S-chondroitin sulfate (chondroitin sulfate C) as well as 4-S-chondroitin sulfate (chondroitin sulfate A), the latter to a lesser extent. Only very little hydrolase activity towards chondroitin sulfate B (dermatan sulfate) was detectable.

Changes of the Functional Capacity of Mesenchymal Stem Cells due to Aging or Age-Associated Disease - Implications for Clinical Applications and Donor Recruitment.

SUMMARY: In contrast to stem cells of embryonic origin, autologous tissue-specific stem cells are easier to introduce into the clinical practice. In this context, molecular and cellular changes, which alter tissue-specific stem cell properties with age, are of particular interest since elderly patients represent the main target group for cell-based therapies. The clinical use of mesenchymal stem cells is an emerging field, especially because this stem cell type appears to be amenable for the treatment of a large number of diseases, such as non-healing bone defects and fractures, inflammatory relief during arthritis, and the repair of suspensory ligament tears. More than that, mesenchymal stem cells provoke effective immune suppression in the context of graft-versus-host disease. Here, we present a comprehensive overview of the recent findings with special attention to age-related changes of mesenchymal stem cell properties and the consequential impact on tissue regeneration and repair, together with the current perception concerning their therapeutic application potential as well as the challenges associated with their clinical use.

Mouse testicular hyaluronidase-like proteins SPAM1 and HYAL5 but not HYALP1 degrade hyaluronan.

Besides SPAM1 (sperm adhesion molecule 1; formerly named PH-20), further hyaluronidase-like proteins, HYAL5 (hyaluronoglucosaminidase 5) and HYALP1 (hyaluronoglucosaminidase pseudogene 1) are also expressed in murine testicular tissue. As they share a high degree of sequence similarity with known hyaluronidases, all three polypeptides could potentially exhibit hyaluronidase activity, a function that is beneficial for spermatozoa in order to penetrate the hyaluronan-rich cumulus, which surrounds the oocyte. Recently, it was reported that SPAM1-deficient mice are fertile and spermatozoa derived from mutant mice still exhibit hyaluronidase activity [Baba, Kashiwabara, Honda, Yamagata, Wu, Ikawa, Okabe and Baba (2002) J. Biol. Chem. 277, 30310-30314]. We have now recombinantly expressed mouse SPAM1, HYAL5 and HYALP1 in Xenopus laevis oocytes and determined their respective expression pattern in testis. Transcripts of all three genes are expressed in seminiferous tubules in regions where maturing spermatogenic cells reside. SPAM1 and HYAL5 but not HYALP1 proteins exhibit hyaluronidase activity at neutral pH. The two active hyaluronidases are both bound to the cell surface via a glycosylphosphatidylinositol anchor. Furthermore, structural characteristics are discussed that are necessary for hyaluronidases in order to exhibit hyaluronan cleavage.