Mesenchymal stem cell proliferation and mineralization but not osteogenic differentiation are strongly affected by extracellular pH.

UNLABELLED: Osteomyelitis is a serious complication in oral and maxillofacial surgery affecting bone healing. Bone remodeling is not only controlled by cellular components but also by ionic and molecular composition of the extracellular fluids in which calcium phosphate salts are precipitated in a pH dependent manner. OBJECTIVE: To determine the effect of pH on self-renewal, osteogenic differentiation and matrix mineralization of mesenchymal stem cells (MSCs). METHODS: We selected three different pH values; acidic (6.3, 6.7), physiological (7.0-8.0) and severe alkaline (8.5). MSCs were cultured at different pH ranges, cell viability measured by WST-1, apoptosis detected by JC-1, senescence was analyzed by ß-galactosidase whereas mineralization was detected by Alizarin Red and osteogenic differentiation analyzed by Real-time PCR. RESULTS: Self-renewal was affected by pH as well as matrix mineralization in which pH other than physiologic inhibited the deposition of extracellular matrix but did not affect MSCs differentiation as osteoblast markers were upregulated. The expression of osteocalcin and alkaline phosphatase activity was upregulated whereas osteopontin was downregulated under acidic pH. CONCLUSION: pH affected MSCs self-renewal and mineralization without influencing osteogenic differentiation. Thus, future therapies, based on shifting acid-base balance toward the alkaline direction might be beneficial for prevention or treatment of osteomyelitis.

Decoding Cytoskeleton-Anchored and Non-Anchored Receptors from Single-Cell Adhesion Force Data.

Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking ß1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that ß1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions.

Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.

BACKGROUND: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies. RESULTS: Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC. CONCLUSIONS: Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

Loss of tenomodulin results in reduced self-renewal and augmented senescence of tendon stem/progenitor cells.

Tenomodulin (Tnmd) is a well-known gene marker for the tendon and ligament lineage, but its exact functions in these tissues still remain elusive. In this study, we investigated Tnmd loss of function in mouse tendon stem/progenitor cells (mTSPC) by implicating a previously established Tnmd knockout (KO) mouse model. mTSPC were isolated from control and Tnmd KO tail tendons and their stemness features, such as gene marker profile, multipotential, and self-renewal, were compared. Immunofluorescence and reverse transcriptase-polymerase chain reaction analyses for stem cell-, tenogenic-, osteogenic-, and chondrogenic-related genes confirmed their stemness and lineage specificity and demonstrated no profound differences between the two genotypes. Multipotential was not significantly affected since both cell types differentiated successfully into adipogenic, osteogenic, and chondrogenic lineages. In contrast, self-renewal assays validated that Tnmd KO TSPC exhibit significantly reduced proliferative potential, which was also reflected in lower Cyclin D1 levels. When analyzing possible cellular mechanisms behind the observed decreased self-renewability of Tnmd KO TSPC, we found that cellular senescence plays a major role, starting earlier and cumulating more in Tnmd KO compared with control TSPC. This was accompanied with augmented expression of the cell cycle inhibitor p53. Finally, the proliferative effect of Tnmd in TSPC was confirmed with transient transfection of Tnmd cDNA into Tnmd KO TSPC, which rescued their proliferative deficit. Taken together, we can report that loss of Tnmd affects significantly the self-renewal and senescence properties, but not the multipotential of TSPC.

Activation of EphA4 and EphB2 Reverse Signaling Restores the Age-Associated Reduction of Self-Renewal, Migration, and Actin Turnover in Human Tendon Stem/Progenitor Cells.

Tendon tissues, due to their composition and function, are prone to suffer age-related degeneration and diseases as well as to respond poorly to current repair strategies. It has been suggested that local stem cells, named tendon stem/progenitor cells (TSPCs), play essential roles in tendon maintenance and healing. Recently, we have shown that TSPC exhibit a distinct age-related phenotype involving transcriptomal shift, poor self-renewal, and elevated senescence coupled with reduced cell migration and actin dynamics. Here, we report for the first time the significant downregulation of the ephrin receptors EphA4, EphB2 and B4 and ligands EFNB1 in aged-TSPC (A-TSPC). Rescue experiments, by delivery of target-specific clustered proteins, revealed that activation of EphA4- or EphB2-dependent reverse signaling could restore the migratory ability and normalize the actin turnover of A-TSPC. However, only EphA4-Fc stimulation improved A-TSPC cell proliferation to levels comparable to young-TSPC (Y-TSPC). Hence, our novel data suggests that decreased expression of ephrin receptors during tendon aging and degeneration limits the establishment of appropriate cell-cell interactions between TSPC and significantly diminished their proliferation, motility, and actin turnover. Taken together, we could propose that this mechanism might be contributing to the inferior and delayed tendon healing common for aged individuals.

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7.

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression. HMSC were isolated from 18 healthy or osteoporotic donors. Migration was assessed using a collagen IV coated micro-slide linear gradient chamber and time-lapse microscopy. Invasion was analyzed utilizing an ECM coated transmembrane invasion assay. Quantitative real-time RT PCR was performed for the ECM binding integrins α1, α2, α3, α4, α5, α11, αv and ß1. HMSC from osteoporotic patients showed a significant increase of migration upon BMP-2 or FCS stimulation, as well as a significant increase of invasion upon BMP-2, BMP-7 or FCS stimulation. Nevertheless, the migration and invasion capacity was significantly decreased compared to healthy controls. Out of all integrins analyzed, collagen binding integrin α2 was significantly downregulated in hMSC from osteoporotic patients. In conclusion, we here demonstrate for the first time osteoporosis associated alterations in BMP mediated hMSC recruitment. These findings may underlie the reduced healing of osteoporotic fractures. Nevertheless, the maintained migration and invasion response upon BMP stimulation illustrates the therapeutic potential of these clinically approved substances in the treatment of osteoporotic fractures. Another therapeutic target may be the downregulation of the collagen binding integrin α2 in hMSC from osteoporotic patients.

Integrin signaling in skeletal development and function.

Integrins are cell surface receptors that connect extracellular matrix (ECM) components to the actin cytoskeleton and transmit chemical and mechanical signals into the cells through adhesion complexes. Integrin-activated downstream pathways have been implicated in the regulation of various cellular functions, including proliferation, survival, migration, and differentiation. Integrin-based attachment to the matrix plays a central role in development, tissue morphogenesis, adult tissue homeostasis, remodeling and repair, and disturbance of the ECM-integrin-cytoskeleton signaling axis often results in diseases and tissue dysfunction. Increasing amount of in vitro and in vivo evidences suggest that integrins are pivotal for proper development, function, and regeneration of skeletal tissues. In this paper, we will summarize and discuss the role of integrins in skeletogenesis and their influence on the physiology and pathophysiology of cartilage, bone, and tendon.

Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration.

Although the link between altered stem cell properties and tissue aging has been recognized, the molecular and cellular processes of tendon aging have not been elucidated. As tendons contain stem/progenitor cells (TSPC), we investigated whether the molecular and cellular attributes of TSPC alter during tendon aging and degeneration. Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained. Senescence analysis showed a premature entry into senescence of the A-TSPC, a finding accompanied by an upregulation of p16(INK4A). To identify age-related molecular factors, we performed microarray and gene ontology analyses. These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton. Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments. Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell-matrix interactions and the ROCK kinase pathway might be key players in TSPC aging. Taken together, we propose that during tendon aging and degeneration, the TSPC pool is becoming exhausted in terms of size and functional fitness. Thus, our study provides the first fundamental basis for further exploration into the molecular mechanisms behind tendon aging and degeneration as well as for the selection of novel tendon-specific therapeutical targets.

Probing the interaction forces of prostate cancer cells with collagen I and bone marrow derived stem cells on the single cell level.

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1ß1 and α2ß1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.

Conversion of human bone marrow-derived mesenchymal stem cells into tendon progenitor cells by ectopic expression of scleraxis.

Tendons and ligaments (T/L) are dense connective tissues of mesodermal origin. During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, we investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Gene expression analysis demonstrated that collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. From these results, we conclude that Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research.

IKK-2 is required for TNF-alpha-induced invasion and proliferation of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-kappaB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-alpha (TNF-alpha), which is known to be a key regulator of the NF-kappaB pathway. Therefore, we hypothesized that TNF-alpha may lead to MSC invasion and proliferation by activation of the NF-kappaB pathway. TNF-receptor 1 and 2, NF-kappaB (p65), and IkappaB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-alpha caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-alpha strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-kappaB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-alpha stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-kappaB pathway caused a significant reduction in the basal proliferation rate. TNF-alpha stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-alpha led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-kappaB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-alpha-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-kappaB signal transduction pathway.

Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy.

Within the bone lie several different cell types, including osteoblasts (OBs) and mesenchymal stem cells (MSCs). The MSCs are ideal targets for regenerative medicine of bone due to their differentiation potential towards OBs. Human MSCs exhibit two distinct morphologies: rapidly self-renewing cells (RS) and flat cells (FC) with very low proliferation rates. Another cell type found in pathological bone conditions is osteosarcoma. In this study, we compared the topographic and morphometric features of RS and FC cells, human OBs and MG63 osteosarcoma cells by atomic force microscopy (AFM). The results demonstrated clear differences: FC and hOB cells showed similar ruffled topography, whereas RS and MG63 cells exhibited smoother surfaces. Furthermore, we investigated how selected substrates influence cell morphometry. We found that RS and MG63 cells were flatter on fibrous substrates such as polystyrene and collagen I, but much more rounded on glass, the smoothest surface. In contrast, cells with large area, namely FC and hOB cells, did not exhibit pronounced changes in flatness with regards to the different substrates. They were, however, remarkably flatter in comparison to RS and MG63 cells. We could explain the differences in flatness by the extent of adhesion. Indeed, FC and hOB cells showed much higher content of focal adhesions. Finally, we used the AFM to determine the cellular Young's modulus. RS, FC and hOB cells showed comparable stiffness on the three different substrates, while MG63 cells demonstrated the unique feature of increased elasticity on collagen I. In summary, our results show, for the first time, a direct comparison between the morphometric and biophysical features of different human cell types derived from normal and pathological bone. Our study manifests the opinion that along with RNA, proteomic and functional research, morphological and biomechanical characterization of cells also reveals novel cell features and interrelationships.

Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system.

The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The discussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies.