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The human iPS cell line VUZUZLi001-A (hVH-1) was generated from human foreskin fibroblasts to be used as a control line. Reprogramming was performed by retroviral transduction of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. Resource table., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Beneficial effects of erythropoietin (EPO) have been reported in acute kidney injury (AKI) when administered prior to induction of AKI. We studied the effects of EPO administration on renal function shortly after ischemic AKI. For this purpose, rats were subjected to renal ischemia for 30 min and EPO was administered at a concentration of 500 U/kg either i.v. as a single shot directly after ischemia or with an additional i.p. dose until 3 days after surgery. The results were compared with AKI rats without EPO application and a sham-operated group. Renal function was assessed by measurement of serum biochemical markers, histological grading, and using an isolated perfused kidney (IPK) model. Furthermore, we performed flow cytometry to analyze the concentration of endothelial progenitor cells (EPCs) in the peripheral blood and renal vessels. Following EPO application, there was only a statistically non-significant tendency of serum creatinine and urea to improve, particularly after daily EPO application. Renal vascular resistance and the renal perfusion rate were not significantly altered. In the histological analysis, acute tubular necrosis was only marginally ameliorated following EPO administration. In summary, we could not demonstrate a significant improvement in renal function when EPO was applied after AKI. Interestingly, however, EPO treatment resulted in a highly significant increase in CD133- and CD34-positive EPC both in the peripheral blood and renal vessels., (© 2015 International Federation for Cell Biology.)

Ischemic acute kidney injury (AKI) is associated with high morbidity and frequent complications. Repeated episodes of AKI may lead to end-stage renal failure. The pathobiology of regeneration in AKI is not well understood and there is no effective clinical therapy that improves regeneration. The Notch signaling pathway plays an essential role in kidney development and has been implicated in tissue repair in the adult kidney. Here, we found that kidneys after experimental AKI in mice showed increased expression of Notch receptors, specifically Notch1-3, of the Notch ligands Jagged-1 (Jag1), Jag2 and Delta-like-4 (Dll4) and of the Notch target genes Hes1, Hey2, HeyL, Sox9 and platelet-derived growth factor receptor β (Pdgfrb). Treatment of ischemic mice with the x03B3;-secretase inhibitor DBZ blocked Notch signaling and specifically downregulated the expression of Notch3 and the Notch target genes Hes1, Hey2, HeyL and Pdgfrb. After DBZ treatment, the mice developed less interstitial edema and displayed altered interstitial inflammation patterns. Furthermore, serum urea and creatinine levels were significantly decreased from 6 h onwards when compared to control mice treated with DMSO only. Our data are consistent with an amelioration of the severity of kidney injury by blocking Notch activation following AKI, and suggest an involvement of Notch-regulated Pdgfrb in AKI pathogenesis., (© 2016 S. Karger AG, Basel.)

Human adipose-derived stroma cells (ADSCs) have successfully been employed in explorative therapeutic studies. Current evidence suggests that ADSCs are unevenly distributed in subcutaneous adipose tissue; therefore, the anatomical origin of ADSCs may influence clinical outcomes. This study was designed to investigate proliferation and differentiation capacities of ADSCs from the gluteal and abdominal depot of 8 females. All had normal BMI (22.01 ± 0.39 kg/m(2) ) and waist circumference (81.13 ± 2.33 cm). Examination by physicians and analysis of 31 laboratory parameters did not reveal possibly confounding medical disorders. Gluteal and abdominal adipose tissue was sampled by en bloc resection on day 7 (±1) after the last menses. Histological examination did not reveal significant depot-specific differences. As assessed by BrdU assay, proliferation of cells from both depots was similar after 24 h and analysis of 15 cell surface markers by flow cytometry identified the isolated cells as ADSCs, again without depot-specific differences. ADSCs from both depots differentiated poorly to chondroblasts. Gluteal ADSCs displayed significantly higher adipogenic differentiation potential than abdominal cells. Osteogenic differentiation was most pronounced in gluteal cells, whereas differentiation of abdominal ADSCs was severely impaired. Our data demonstrate a depot-specific difference in ADSC differentiation potential with abdominal cells failing to meet the criteria of multipotent ADSCs. This finding should be taken into account in future explorations of ADSC-derived therapeutic strategies., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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-β1; 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-β1 served as controls. Proliferation, migration, and release of TGF-β1 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-β1. 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-β1, which was not provided by our scaffold design. The initial release of TGF-β1 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-β1 is preferred to foster type II and avoid type I collagen expression., (Copyright © 2012 Wiley Periodicals, Inc.)

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., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Mesenchymal stromal cells (MSC) have been introduced into the field of tissue-engineered airway transplantation. Since patients with extensive tracheal defects often require an open tracheotomy, this study investigated if MSC could be obtained from the adipose tissue of the neck during this procedure. Cells were isolated by plastic adherence from the adipose tissue of 8 patients. Cell isolates were analyzed for (i) proliferation, (ii) the expression of CD marker molecules and (iii) multilineage differentiation. The isolated spindle-shaped cells showed a high proliferation capacity and the flow cytometric analysis revealed a distinct population meeting the criteria for MSC. Using classical MSC cultivation protocols the characterized cells showed adipogenic, chondrogenic and osteogenic differentiation for all analyzed cell isolates. This study was able to demonstrate that sufficient amounts of stem/progenitor cells can be easily isolated from adipose tissue of the neck obtained during open tracheotomy. These cells may be a source for future tracheal replacement therapies.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

Differentiation of embryonic stem (ES) cells via embryoid bodies has been established as an appropriate model to study the development of various cell types in vitro. Here, we show that murine induced pluripotent stem (iPS) cells, reprogrammed by exogenous expression of the two transcription factors Oct4 and Klf4 (2F OK iPS), differentiate into chondrocytes in vitro characterized by the appearance of Alcian blue-stained nodules and the expression of cartilage-associated genes and proteins. Quantitatively, the chondrogenic differentiation potential of 2F OK iPS and ES cells was found to be similar. Further, we demonstrate the induction of chondrogenic iPS cell differentiation by certain members of the transforming growth factor-β family (BMP-2, TGF-β(1)). The number of Alcian blue-positive nodules and the expression of the cartilage marker molecule collagen type II increased after application of BMP-2, whereas simultaneous treatment with both BMP-2 and TGF-β(1) showed no significant effect on gene expression., (Copyright © 2012 S. Karger AG, Basel.)

Background Aims: In vitro cultured mesenchymal stromal cells (MSC) are characterized by a short proliferative lifespan, an increasing loss of proliferation capacity and progressive reduction of differentiation potential. Laminin-1, laminin-5, collagen IV and fibronectin are important constituents of the basement membrane extracellular matrix (ECM) that are involved in a variety of cellular activities, including cell attachment and motility., Methods and Results: The in vitro proliferation capacity of MSC was significantly improved when the cells were incubated in the presence of basement membrane ECM proteins. For example, a mixture of proteins improved proliferation capacity 250-fold in comparison with standard conditions after five passages. Furthermore, in colony-forming unit-fibroblast (CFU-F) assays colony numbers and size were significantly extended. Blocking specific integrin cell-surface receptors, positive effects on the proliferation capacity of MSC were inhibited. Additionally, when MSC were co-cultivated with ECM proteins, cells maintained their multipotential differentiation capacity throughout many culture passages in comparison with cells cultivated on plastic. However, expansion of MSC on laminin-5 suppressed any subsequent chondrogenic differentiation., Conclusions: Our results suggest that expansion of bone marrow-derived MSC in the presence of ECM proteins is a powerful approach for generating large numbers of MSC, showing a prolonged capacity to differentiate into mesodermal cell lineages, with the exception of the lack of chondrogenesis by using laminin-5 coating.

Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.

The aim of this study was to examine the influence of matrix elasticity on the maintenance of the chondrogenic phenotype of chondrocytes cultured in monolayer. We used a two-dimensional culturing system in which polyacrylamide gels with different concentrations of bis-acrylamide were coated with collagen type I. Matrices with a Young's modulus of 4, 10, 40, and 100 kPa were produced, as determined by atomic force microscopy. Porcine chondrocytes were cultivated on these matrices at a low density for 7 days. The proliferation of cells was analyzed by 5-Bromo-2'-deoxy-uridine incorporation. Maintenance of the chondrogenic phenotype was analyzed by measuring collagen type I, type II, and aggrecan gene expression, immunofluorescence staining for collagen type II, and phalloidin staining for actin filaments. Cellular proliferation and actin organization were decreased on matrices of 4 kPa compared with stiffer substrates. The differentiated phenotype of the chondrocytes grown on matrices of 4 kPa was stabilized, indicated by higher collagen type II and aggrecan, and lower collagen type I expression. These findings indicate that chondrocytes sense the elasticity of the matrix and might be used for the design of scaffolds with mechanical properties specifically tailored to support the chondrogenic phenotype in tissue engineering applications.

The adult bone marrow has been generally considered to be composed of hematopoietic tissue and the associated supporting stroma. Within the latter compartment, a subset of cells with multipotent differentiation capacity exists, usually referred to as mesenchymal stem cells. Mesenchymal stem cells can easily be expanded ex vivo and induced to differentiate into several cell types, including osteoblasts, adipocytes and chondrocytes. Up to now, mesenchymal stem cells have gained wide popularity. Despite the rapid growth in this field, irritations remain with respect to the defining characteristics of these cells, including their differentiation potency, self-renewal and in vivo properties. As a consequence, there is a growing tendency to challenge the term mesenchymal stem cell, especially with respect to the stem cell characteristics. Here, we revisit the experimental origins of mesenchymal stem cells, their classical differentiation capacity into mesodermal lineages and their immunophenotype in order to assess their stemness and function. Based on these essentials, it has to be revisited if the designation as a stem cell remains an appropriate term.

Background: Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues., Results: We introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR., Conclusions: We were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells.

Currently, adult stem cells are attracting significant interest in regenerative medicine and tissue engineering. These cells have been isolated from various tissue sources; however, in most cases, adult stem cells useful for tissue engineering and regeneration are present at a low frequency. High numbers of stem cells with an effective and reliable potential for differentiation are needed for clinical applications. Thus, the identification of new stem cell sources and the establishment of optimized cell culture conditions that allow for the amplification of stem cells are of utmost relevance. In addition, the isolation procedure should ideally be minimally invasive and possibly be performed under local anesthesia. We report here for the first time on the identification of adult stem cells with mesenchymal characteristics in human parotid gland tissue. Cells were isolated from freshly resected specimens of parotid glands using enzymatic digestion and plastic adhesion protocols. Following an initial proliferation period and short-term culture for four passages, immunophenotyping revealed the presence of mesenchymal stem cell markers. In the presence of tissue-specificinduction medium, stem cells could be differentiated into adipogenic, osteogenic, and chondrogenic cell types. Tissue-specific differentiation was confirmed by histochemical and immunocytochemical staining as well as by RT-PCR for defined marker genes. This study is, to the best of our knowledge, the first report on the isolation and differentiation of stem cells from adult human parotid glands. Although isolated from an endodermal tissue source, these stem cells share many characteristics with MSCs. Easy accessibility and a high differentiation potential make salivary gland-derived stem cells a promising source for future applications in regenerative medicine.

The transcription factor Sox9 plays an important role during chondrogenesis. After early conditional inactivation of Sox9 in mesenchymal limb bud cells of mice, mesenchymal condensations as well as cartilage and bone are completely absent in the developing limbs. We analyzed chondrogenic differentiation of Sox9-/- mouse embryonic stem cells in vitro, using two clones with different targeted mutations. We found that the development of mature and hypertrophic chondrocytes is completely inhibited in the absence of Sox9 confirming that Sox9 is required for the formation of cartilage. In contrast, Sox9+/- mouse embryonic stem cells showed continuous but reduced differentiation into mature chondrocytes. Interestingly, the formation of early chondrogenic condensations expressing characteristic marker genes such as scleraxis, Sox5 and Sox6 was not inhibited in the absence of Sox9 in vitro. Thus, we propose that the earliest step of chondrogenesis could be regulated by a non cell-autonomous function of Sox9.

This unit describes the protocols used for cultivation of murine embryonic stem (ES) cells and their differentiation into chondrogenic cell types in vitro. ES cells cultivated as cellular aggregates, so-called embryoid bodies (EBs), differentiate spontaneously into chondrogenic cell types recapitulating cellular events of chondro- and osteogenesis. The undifferentiated ES cells differentiate into mesenchymal prechondrogenic cells in the EB outgrowths. These progenitor cells aggregate and form mesenchymal condensations. During further cultivation, these cells form cartilage nodules, show a phenotype typical for chondroblasts, and start to express marker molecules of cartilage tissue. Later, the chondrocytes become hypertrophic, and finally, marker molecules indicating bone formation can be detected in the nodules. This unit also contains protocols for characterization of the differentiated cells by immunostaining, mRNA-in situ hybridization, electron microscopy, and RT-PCR analysis.

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.

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

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.

The model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates (embryoid bodies, EBs) can be used to analyze cell differentiation from a pluripotent stem cell via progenitor cells up to terminally differentiated cell types. ES cells are known to be pluripotent; they have the capacity to differentiate into any cell lineage of the three germ layers. Using various ES cell lines, we characterized chondrogenic and osteogenic differentiation in EBs by histochemical staining, immunostaining, mRNA-in situ hybridization, and reverse transcriptase polymerase chain reaction analysis. Here, we describe in detail our established protocols to analyze chondrogenic differentiation of ES cells. We summarize different ways to modulate ES cell-derived chondrogenic differentiation.

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.

Pluripotent mouse embryonic stem (ES) cells differentiate in vitro spontaneously into cell types of all three primary germ layers when cultivated as cell aggregates, so-called 'embryoid bodies'. Many reports have shown that this system recapitulates cellular developmental processes and gene expression patterns of early embryogenesis. During ES cell differentiation, efficient and directed differentiation into a specific cell type is influenced by many parameters, for example, the batch of the serum used or the application of growth factors and signalling molecules. Because all ES cell lines are considered to be pluripotent, one should not expect remarkable differences regarding their spontaneous differentiation efficiencies. However, here we show that different ES cell lines exhibit a variable degree of spontaneous chondrogenic differentiation indicating that lines with a specific differentiation capacity could be selected. This is an important aspect if ES cells are applied for tissue regeneration.

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.

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-beta family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity.