Age-specific changes of mesenchymal stem cells are paralleled by upregulation of CD106 expression as a response to an inflammatory environment.

Regeneration, tissue remodeling, and organ repair after injury, which rely on the regulated activity of tissue-borne stem cells, become increasingly compromised with advancing age. Mesenchymal stroma cells were isolated from bone of differently aged healthy donors. The rare population of mesenchymal stem cells (MSCs) contained in the primary cell isolates barely declined in number, yet the stem cells displayed diminished long-term proliferation potential relative to the donor age and the expression of vascular cell adhesion molecule-1 (VCAM-1; CD106) was elevated on primary MSCs. In CD106(bright) MSCs, the abundance of a panel of stemness transcription factors remained unchanged. Because the CD106 level could be further enhanced by proinflammatory cytokines, we considered the rate of VCAM-1 expression to be a good reflection of an endogenous inflammatory milieu to which the MSCs are exposed. Treatment of MSCs with increasing doses of interferon-γ exerted no immediate influence on their self-renewal capacity. However, it impacted on the differentiation potential toward the adipogenic or osteogenic lineage. Moderately elevated levels of inflammatory stimuli supported osteoblastogenesis whereas the same treatment reduced adipogenic differentiation in MSCs from young and intermediately aged donors. In MSCs from elderly donors, however, osteoblastogenesis was greatly diminished in an inflammatory environment whereas adipogenic differentiation remained unchanged. Conclusively, moderate levels of inflammatory stimuli are being interpreted by MSCs at a young age as instructive signals for osteoblastogenesis, whereas at old age, an inflammatory milieu may effectively suppress bone remodeling and repair by tissue-borne MSCs while uninterrupted adipogenic differentiation may lead to adipose upgrowth.

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.

lin-Sca-1+ cells and age-dependent changes of their proliferation potential are reliant on mesenchymal stromal cells and are leukemia inhibitory factor dependent.

Aging as a process is paralleled by a variety of hematological alterations. Characteristic features are a diminished homeostatic control of blood cell production and a decline in immune functions. It is generally accepted that stromal cells play a basal role in hematopoiesis by providing survival and differentiation signals, by secreting cytokines, or through direct contact with hematopoietic stem cells, thereby supporting the generation and replenishment of hematopoi- etic progenitor cells (HPC). Here we demonstrated that HPC-related colony formation is positively influenced by mesenchymal stromal cells (MSCs) when grown in co-culture, in particular regarding the number of primary granulocyte/macrophage colony-forming units as well as with respect to the average size of the formed colonies. These effects were more pronounced when the MSCs originated from young donors than from old ones. Because leukemia inhibitory factor (LIF) plays an important role during hematopoiesis, properties of lin- Sca-1+ cells and MSCs derived from LIF-deficient mice (LIF-/-) were determined both ex vivo and in vitro. LIF-/- animals contain a significantly reduced number of lin- Sca-1+ cells, nevertheless the replating capacity of LIF-/- HPCs was found to be generally unchanged when compared to those from LIF+/+ animals. However, when cocultured with MSCs, LIF-/- lin- Sca-1+ cells exhibited comparable characteristics to HPCs derived from old wild-type animals.

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.

Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan.

Mesenchymal stem cells (MSC) are capable of differentiating into bone, fat, cartilage, tendon and other organ progenitor cells. Despite the abundance of MSC within the organism, little is known about their in vivo properties or about their corresponding in vivo niches. We therefore isolated MSC from spongy (cancellous) bone biopsies of healthy adults. When compared with the surrounding marrow, a fourfold higher number of colony-forming units was found within the tight meshwork of trabecular bone surface. At these sites, oxygen concentrations range from 1% to 7%. In MSC cultured at oxygen as low as 3%, rates for cell death and hypoxia-induced gene transcription remained unchanged, while in vitro proliferative lifespan was significantly increased, with about 10 additional population doublings before reaching terminal growth arrest. However, differentiation capacity into adipogenic progeny was diminished and no osteogenic differentiation was detectable at 3% oxygen. In turn, MSC that had previously been cultured at 3% oxygen could subsequently be stimulated to successfully differentiate at 20% oxygen. These data support our preliminary finding that primary MSC are enriched at the surface of spongy bone. Low oxygen levels in this location provide a milieu that extends cellular lifespan and furthermore is instructive for the stemness of MSC allowing proliferation upon stimulation while suppressing differentiation.

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.

Aging of murine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are able to differentiate into distinct lineages such as adipo-, osteo-, and chondrocytes. MSCs were isolated from three mouse strains, which are short- (SAMP6, 9.7 months), medium- (SAMR1, 16.3 months), or long-lived (C57BL/6, 28 months). We investigated primary colony-forming units with regard to bone marrow stroma and found differences that correlate with mean life expectancies of the particular genetic backgrounds. However, MSC derived from the various mouse strains behaved equivalently in vitro with respect to growth rate. By genomic means, we analyzed the cellular milieu in vivo and found considerable differences among the various mouse strains. This implies that, although individual MSCs show an equivalent differentiation potential in vitro, the primary stem cells are greatly influenced by their molecular environment.

Techniques in gerontology: cell lines as standards for telomere length and telomerase activity assessment.

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.

Strong binding of bioactive BMP-2 to nanocrystalline diamond by physisorption.

Nano-crystalline diamond (NCD)-coated surfaces were efficiently functionalized with bone morphogenetic protein-2 (BMP-2) by means of physisorption. Due to their randomly oriented texture, NCD-coated surfaces appear to bind complex molecules firmly. Applying various highly sensitive analytical methods, the interaction was found extremely stable. The strength of the experimentally measured adherence between BMP-2 and NCD was further corroborated by theoretical calculations. Oxygen treatment rendered NCD hydrophilic by the appearance of surface oxygen containing groups. This particular NCD surface exhibited even higher binding energies towards BMP-2 than the hydrophobic surface, and this surface was also favoured by cultured cells. Most importantly in this context, bound BMP-2 was found fully active. When cultured on BMP-2-treated NCD, osteosarcoma cells strongly up-regulated alkaline phosphatase, a specific marker for osteogenic differentiation. Hence, this simple method will allow generating highly versatile surfaces with complex biomimetic coatings, essentials for novel medical devices and implants as well as for innovative scaffolds in tissue engineering.

Mesenchymal stem cell aging.

Stem cells are located throughout the adult body of higher organisms, supporting a continuous renewal and repair of tissues. Unique abilities of stem cells are self-renewal and multipotential differentiation. It is, therefore, of critical importance for an organism to maintain and control quantity and quality of stem cells within a given pool. Otherwise, when something goes awry within a stem cell, it is likely to have far-reaching effects. Mesenchymal stem cells (MSC) derived from various sources such as bone marrow or fat have been expanded in culture and differentiated in vitro into several lineages such as adipocytes, osteocytes or chondrocytes. In particular, aged human MSC show a decline in differentiation potential as well as in proliferation rate. The latter most likely reflects the fact that aged MSC suffer from eroded telomeres. Besides the individual age of the cell, stem and progenitor cell functions are influenced by the cellular environment, i.e. the niche and the architecture of the tissue, they reside in. This contribution reviews current knowledge about MSC aging (in vitro or in vivo), and respective difficulties for tissue engineering and stem cell therapy.

CD25-expressing CD8+ T cells are potent memory cells in old age.

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions.

d-amino acids are present in some peptides from amphibian skin. These residues are derived from the corresponding L-amino acids present in the respective precursors. From skin secretions of Bombinae, we have isolated an enzyme that catalyzes the isomerization of an L-Ile in position 2 of a model peptide to D-allo-Ile. In the course of this reaction, which proceeds without the addition of a cofactor, radioactivity from tritiated water is incorporated into the second position of the product. The amino acid sequence of this isomerase could be deduced from cloned cDNA and genomic DNA. After expression of this cDNA in oocytes of Xenopus laevis, isomerase activity could be detected. Polypeptides related to the frog skin enzyme are present in several vertebrate species, including humans.