Differences of cell surface marker expression between bone marrow- and kidney-derived murine mesenchymal stromal cells and fibroblasts.

Mesenchymal stromal cells (MSC) are undifferentiated, multipotent adult cells with regenerative properties. They are particularly relevant for therapeutic approaches due to the simplicity of their isolation and cultivation. Since MSC show an expression pattern of cell surface marker, which is almost identical to fibroblasts, many attempts have been made to address the similarities and differences between MSC and fibroblasts. In this study we aimed to isolate murine MSC from bone marrow (BM) and kidney to characterize them in comparison to fibroblasts. Cells were isolated from murine kidney, BM and abdominal skin by plastic adherence and subsequently characterized by analysing their capability to build colony-forming unit-fibroblasts (CFU-F), their morphology, their proliferation, expression of telomerase activity and cell surface antigens as well as their differentiation capacity. Plastic adherent cells from the 3 mouse tissues showed similar morphology, proliferation profiles and CFU-F building capacities. However, while MSC from BM and kidney differentiated into the adipogenic, chondrogenic and osteogenic direction, fibroblasts were not able to do so efficiently. In addition, a tendency for lower expression of telomerase was found in the fibroblast population. Proliferating cells from kidney and BM expressed the MSC-specific cell surface markers CD105 and Sca-1 on a significantly higher and CD117 on a significantly lower level compared to fibroblasts and were thereby distinguishable from fibroblasts. Furthermore, we found that certain CD markers were specifically expressed on a higher level, either in BM-derived cells or fibroblasts. This study demonstrates that murine MSC isolated from different organs express certain specific markers, which enable their discrimination.

Minor cartilage collagens type IX and XI are expressed during embryonic stem cell-derived in vitro chondrogenesis.

Cartilage development is a complex process that can be analyzed using numerous model systems. We have previously shown that in vitro differentiation of murine embryonic stem (ES) cells via embryoid bodies (EBs) recapitulates the cellular differentiation steps of chondrogenesis. However, differentiated chondrocytes lose their characteristic phenotype when they are kept in monolayer culture. This dedifferentiation process is one of the main obstacles of cartilage tissue engineering and could not be analyzed using the EB model system. The aim of this study was to further characterize the chondrogenic nodules derived by in vitro-differentiation of murine ES cells for the distribution of collagen types II, IX and XI in comparison to in vitro dedifferentiating primary chondrocytes from murine embryonic ribs. Expression of cartilage collagens and other extracellular matrix proteins was analyzed using immunostaining, cytochemical stainings and quantitative RT-PCR. We show that ES cell-derived chondrocyte differentiation starts with mesenchymal condensations synthesizing high amounts of fibronectin. Later, the matrix of the mature cartilage nodules consists of type II collagen, proteoglycans and the minor collagens type IX and XI. The nodules show a three-dimensional structure with multiple layers of collagen type II-positive cells. At late differentiation stages these chondrocytes were located at lateral regions of the nodules. Similar to the distribution pattern of collagen type II positive cells, the cells staining positive for collagen type IX and XI were present in the surface regions, but not in the central areas of the chondrogenic nodules. During cultivation of the primary murine rib chondrocytes expression of chondrogenic marker genes such as collagen type II and aggrecan declined and many chondrocytes lost characteristic cartilage matrix proteins and converted to an elongated, fibroblastoid shape with prominent actin stress fibers. Chondrogenic differentiation of murine ES cells combined with monolayer culture of embryonic rib chondrocytes is a valuable tool to study changes in the expression pattern during differentiation and dedifferentiation of chondrocytes.

Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-ε-caprolactone scaffolds.

The objective of this study was to develop a scaffold for mesenchymal stromal cell (MSC) recruitment, proliferation, and chondrogenic differentiation. The concept behind the design is to mimic the cartilage matrix and contain stimulatory agents that make continuous supply of inductive factors redundant. Nanofibrous (N: ~400 nm) and microfibrous (M: ~10 µm) poly-ε-caprolactone (PCL) scaffolds were combined with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan (HA) and 200 ng transforming growth factor-beta 1 (TGF-ß1; NTGF/MTGF), or 0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone marrow and cultured without growth factors in vitro. Cultures with chondrogenic medium supplemented with TGF-ß1 served as controls. Proliferation, migration, and release of TGF-ß1 were investigated. Cell differentiation was evaluated by polymerase chain reaction (PCR) and real-time PCR. NTGF and MTGF exhibited primarily an initial release of TGF-ß1. None of the factors released by the scaffolds recruited MSCs. The expression of aggrecan was dependent on the scaffold ultrastructure with nanofibers promoting increasing and microfibers decreasing expression levels. Composites containing HA demonstrated elevated seeding efficiency and lower type I collagen expression. Expression of type II collagen was dependent on continuous or late supply of TGF-ß1, which was not provided by our scaffold design. The initial release of TGF-ß1 induced an expression of type I collagen and osteogenic marker genes. In conclusion, nanofibrous PCL scaffolds with or without augmentation are suitable for chondrogenic initiation of MSCs. Initial release of HA is sufficient in terms of directing the implanted MSCs toward a chondrogenic end, whereas a late release of TGF-ß1 is preferred to foster type II and avoid type I collagen expression.

Simvastatin modulates mouse embryonic stem cell-derived chondrogenesis in vitro.

It has been studied in detail that cellular differentiation during chondrogenesis can be recapitulated in vitro by differentiation of embryonic stem (ES) cells as embryoid bodies (EBs). We here used this model system of cartilage development to analyze the effect of simvastatin, a potentially embryotoxic substance. Statins are a group of drugs used to treat hypercholesterolaemia. We found that simvastatin activated cartilage nodule formation during EB differentiation. Extended application of simvastatin resulted in enhanced expression of cartilage marker molecules and prolonged persistence of cartilage nodules. Expression of collagen type II was upregulated during simvastatin-induced chondrogenic ES cell differentiation as demonstrated by quantitative real time PCR. However, immunostaining for cartilage marker molecules revealed that cartilage nodules within simvastatin-treated EBs were defective, bearing cavities of cell loss. Furthermore, caspase activity was reduced in comparison to untreated controls indicating reduced apoptosis. Taken together, we may speculate that simvastatin prolongs survival of chondrocytes and disrupts cellular integrity of cartilage nodules during EB development by affecting apoptotic mechanisms. The study underlines that ES cell-derived EBs are a useful in vitro model to screen substances for their embryotoxic and teratogenic potential.

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation.

Notch signaling is involved in several cell lineage determination processes during embryonic development. Recently, we have shown that Sox9 is most likely a primary target gene of Notch1 signaling in embryonic stem cells (ESCs). By using our in vitro differentiation protocol for chondrogenesis from ESCs through embryoid bodies (EBs) together with our tamoxifen-inducible system to activate Notch1, we analyzed the function of Notch signaling and its induction of Sox9 during EB differentiation towards the chondrogenic lineage. Temporary activation of Notch1 during early stages of EB, when lineage determination occurs, was accompanied by rapid and transient Sox9 upregulation and resulted in induction of chondrogenic differentiation during later stages of EB cultivation. Using siRNA targeting Sox9, we knocked down and adjusted this early Notch1-induced Sox9 expression peak to non-induced levels, which led to reversion of Notch1-induced chondrogenic differentiation. In contrast, continuous Notch1 activation during EB cultivation resulted in complete inhibition of chondrogenic differentiation. Furthermore, a reduction and delay of cardiac differentiation observed in EBs after early Notch1 activation was not reversed by siRNA-mediated Sox9 knockdown. Our data indicate that Notch1 signaling has an important role during early stages of chondrogenic lineage determination by regulation of Sox9 expression.

Stem cell-derived chondrocytes for regenerative medicine.

The regenerative capacity of cartilage is limited. Transplantation methods used to treat cartilage lesions are based mainly on primary cultures of chondrocytes, which dedifferentiate during cultivation in vitro and lose their functional properties. Stem cells are considered as an alternative source to generate cells for two reasons: first, they can almost indefinitely divide in culture, and second, they are able to differentiate into various mature cell types. Herein, we asked the question whether chondrocytes could be differentiated from mouse embryonic stem (ES) cells to a state suitable for regenerative use. When cultivated as embryoid bodies (EBs), murine ES cells differentiate into mesenchymal progenitor cells, which progressively develop into mature, hypertrophic chondrocytes and transdifferentiate into calcifying cells recapitulating all of the cellular processes of chondrogenesis. Chondrocytes isolated from EBs exhibit a high regenerative capacity. They dedifferentiate initially in culture, but later reexpress stable characteristics of mature chondrocytes. However, in cultures of chondrocytes isolated from EBs, additional mesenchymal cell types can be observed. Mesenchymal stem (MS) cells from bone marrow have already been used in tissue engineering settings. We compared the chondrogenic differentiation of MS and ES cells.

In vivo matrix-guided human mesenchymal stem cells.

Microfracture of subchondral bone results in intrinsic repair of cartilage defects. Stem or progenitor cells from bone marrow have been proposed to be involved in this regenerative process. Here, we demonstrate for the first time that mesenchymal stem (MS) cells can in fact be recovered from matrix material saturated with cells from bone marrow after microfracture. This also introduces a new technique for MS cell isolation during arthroscopic treatment. MS cells were phenotyped using specific cell surface antibodies. Differentiation of the MS cells into the adipogenic, chondrogenic and osteogenic lineage could be demonstrated by cultivation of MS cells as a monolayer, as micromass bodies or mesenchymal microspheres. This study demonstrates that MS cells can be attracted to a cartilage defect by guidance of a collagenous matrix after perforating subchondral bone. Protocols for application of MS cells in restoration of cartilage tissue include an initial invasive biopsy to obtain the MS cells and time-wasting in vitro proliferation and possibly differentiation of the cells before implantation. The new technique already includes attraction of MS cells to sites of cartilage defects and therefore may overcome the necessity of in vitro proliferation and differentiation of MS cells prior to transplantation.

Evidence for posttranscriptional regulation of the multi K homology domain protein vigilin by a small peptide encoded in the 5' leader sequence.

Vigilin, a K homology (KH) protein has been found in all eukaryotic species studied. It has a unique structure of 14-15 consecutively arranged KH domains which apparently mediate RNA-protein binding. Cloning and sequencing of the mouse vigilin cDNA confirmed that the amino acid sequences of vertebrate vigilins are highly conserved and contain conserved sequence motifs of nuclear import and export sequences. The human and murine vigilin mRNAs carry two alternatively spliced 5' exons. In the 5' leader region of one of the splice variants, variant 1A, we found an upstream open reading frame (uORF) highly conserved between mouse and human. Here we present for the first time evidence that a 13 amino acid long peptide encoded by this uORF is an inhibitor of vigilin expression operating on a posttranscriptional level. We propose that the two structurally different 5' leader sequences of the human vigilin mRNA are involved in the regulation of vigilin biosynthesis.

Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects.

Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.

Estradiol has a direct impact on the exocrine pancreas as demonstrated by enzyme and vigilin expression.

BACKGROUND: Estrogen receptors have been found in the exocrine pancreas; however, the exact role of estrogen in pancreatic enzyme synthesis and secretion remains to be elucidated. Vigilin, a multi-KH domain protein, is part of a tRNA-containing ribonucleoprotein complex and may be a suitable marker for stimulation of the translational machinery. In the present study, we investigated the influence of estradiol and compared it to CCK on the expression of vigilin, trypsin and amylase in rat pancreatic acini. METHODS: Acini were isolated and incubated with CCK or estradiol. The change in amylase and trypsin levels in the medium and in cell extracts were determined using a photometric method. The change in vigilin mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. RESULTS: Treatment of isolated exocrine pancreatic cells with estradiol caused stimulation of amylase and trypsin production and inhibition of secretion, while treatment with CCK showed only a minor effect on enzyme production and resulted mainly in a stimulation of secretion. Further we found an increase in vigilin mRNA and protein expression in acini stimulated with both CCK-8 and estradiol. CONCLUSION: Our data suggest that estradiol may play a role in inducing exocrine enzyme production but not secretion, and that vigilin, as a marker for translational activity, is stimulated in parallel to the pancreatic enzymes: amylase and trypsin.