Cosimo De Bari

Multipotent mesenchymal stem cells from adult human synovial membrane

OBJECTIVE To characterize mesenchymal stem cells (MSCs) from human synovial membrane (SM). METHODS Cell populations were enzymatically released from the SM obtained from knee joints of adult human donors and were expanded in monolayer with serial passages at confluence. Cell clones were obtained by limiting dilution. At different passages, SM-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatments, phenotypes of cell cultures were analyzed by histo- and immunohistochemistry and by semiquantitative reverse transcription-polymerase chain reaction for the expression of lineage-retated marker genes. RESULTS SM-derived cells could be expanded extensively in monolayer, with limited senescence. Under appropriate culture conditions, SM-derived cells were induced to differentiate to the chondrocyte, osteocyte, and adipocyte lineages. Sporadic myogenesis was also observed. Five independent cell clones displayed multilineage potential. Interestingly, only 1 clone was myogenic. Donor age, cell passaging, and cryopreservation did not affect the multilineage potential of SM-derived cells. In contrast, normal dermal fibroblasts under the same culture conditions did not display this potential. CONCLUSION Our study demonstrates that human multipotent MSCs can be isolated from the SM of knee joints. These cells have the ability to proliferate extensively in culture, and they maintain their multilineage differentiation potential in vitro, establishing their progenitor cell nature. SM-derived MSCs may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.

Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age

OBJECTIVE To assess the in vitro chondrogenic potential of adult human periosteum-derived cells (PDCs) with regard to the number of cell passages and the age of the donor. METHODS Cells were enzymatically released from the periosteum of the proximal tibia obtained from adult human donors and expanded in monolayer. PDCs were harvested at multiple passages for total RNA extraction and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) gene expression analysis. For the chondrogenesis assay, cells were plated in micromass and treated with transforming growth factor beta1 (TGFbeta1) in a chemically defined medium. At different time points, micromasses were either harvested for RT-PCR analysis for cartilage and bone markers or fixed, paraffin-embedded, and stained for cartilage matrix, and immunostained for type II collagen. RESULTS At the first 2 passages, human PDCs from young donors formed chondrogenic nodules. This spontaneous chondrogenic activity was lost upon passaging, and it was not observed in donors older than 30 years. Using a panel of marker genes, PDCs were shown to be phenotypically stable during cell expansion. Regardless of donor age or cell passage, chondrogenesis could be induced consistently by combining micromass culture and TGFbeta1 treatment. Histochemical and immunohistochemical analyses demonstrated the hyaline-like cartilage phenotype of the tissue generated in vitro. Other TGFbeta superfamily members, such as growth differentiation factor 5/cartilage-derived morphogenetic protein 1, and bone morphogenetic proteins 2, 4, and 7, were poorly chondrogenic under the same culture conditions. CONCLUSION Adult human PDCs have the potential to differentiate toward the chondrocytic lineage in vitro, retaining this property even after extensive subculture. Human PDCs are easily accessible, expandable, and maintain their chondrogenic potential, and are therefore promising progenitor cells for use in the repair of joint surface defects.

Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo

OBJECTIVE To establish a model and associated molecular markers for monitoring the capacity of in vitro-expanded chondrocytes to generate stable cartilage in vivo. METHODS Adult human articular chondrocytes (AHAC) were prepared by collagenase digestion of samples obtained postmortem and were expanded in monolayer. Upon passaging, aliquots of chondrocyte suspensions were either injected intramuscularly into nude mice, cultured in agarose, or used for gene expression analysis. Cartilage formation in vivo was documented by histology, histochemistry, immunofluorescence for type II collagen, and proteoglycan analysis by 35S-sulfate incorporation and molecular sieve chromatography of the radiolabeled macromolecules. In situ hybridization for species-specific genomic repeats was used to discriminate human-derived from mouse-derived cells. Gene expression dynamics were analyzed by semiquantitative reverse transcription-polymerase chain reaction. RESULTS Intramuscular injection of freshly isolated AHAC into nude mice resulted in stable cartilage implants that were resistant to mineralization, vascular invasion, and replacement by bone. In vitro expansion of AHAC resulted in the loss of in vivo cartilage formation. This capacity was positively associated with the expression of fibroblast growth factor receptor 3, bone morphogenetic protein 2, and alpha1(II) collagen (COL2A1), and its loss was marked by the up-regulation of activin receptor-like kinase 1 messenger RNA. Anchorage-independent growth and the reexpression of COL2A1 in agarose culture were insufficient to predict cartilage formation in vivo. CONCLUSION AHAC have a finite capacity to form stable cartilage in vivo; this capacity is lost throughout passaging and can be monitored using a nude mouse model and associated molecular markers. This cartilage-forming ability in vivo may be pivotal for successful cell-based joint surface defect repair protocols.

Dual origin of mesenchymal stem cells contributing to organ growth and repair

In many adult tissues, mesenchymal stem cells (MSCs) are closely associated with perivascular niches and coexpress many markers in common with pericytes. The ability of pericytes to act as MSCs, however, remains controversial. By using genetic lineage tracing, we show that some pericytes differentiate into specialized tooth mesenchyme-derived cells—odontoblasts—during tooth growth and in response to damage in vivo. As the pericyte-derived mesenchymal cell contribution to odontoblast differentiation does not account for all cell differentiation, we identify an additional source of cells with MSC-like properties that are stimulated to migrate toward areas of tissue damage and differentiate into odontoblasts. Thus, although pericytes are capable of acting as a source of MSCs and differentiating into cells of mesenchymal origin, they do so alongside other MSCs of a nonpericyte origin. This study identifies a dual origin of MSCs in a single tissue and suggests that the pericyte contribution to MSC-derived mesenchymal cells in any given tissue is variable and possibly dependent on the extent of the vascularity.

The regulation of differentiation in mesenchymal stem cells

Mesenchymal stromal/stem cells (MSCs) are a population of stromal cells present in the bone marrow and most connective tissues, capable of differentiation into mesenchymal tissues such as bone and cartilage. MSCs are attractive candidates for biological cell-based tissue repair approaches because of their extensive proliferative ability in culture while retaining their mesenchymal multilineage differentiation potential. In addition to its undoubted scientific interest, the prospect of monitoring and controlling MSC differentiation is a crucial regulatory and clinical requirement. Hence, the molecular regulation of MSC differentiation has been extensively studied. Most of the studies are in vitro, because the identity of MSCs in their tissues of origin in vivo remains undefined. This review addresses the current knowledge of the molecular basis of differentiation of cultured MSCs, with a particular focus on chondrogenesis and osteogenesis. Building on the information coming from developmental biology studies of embryonic skeletogenesis, several signaling pathways and transcription factors have been investigated and shown to play critical roles in MSC differentiation. In particular, the Wnt and transforming growth factor-β/bone morphogenetic protein signaling pathways are well known to modulate in MSCs the molecular differentiation into cartilage and bone. Relevant to the emerging concept of stem cell niches is the demonstration that physical factors can also participate in the regulation of MSC differentiation. Knowledge of the regulation of MSC differentiation will be critical in the design of three-dimensional culture systems and bioreactors for automated bioprocessing through mathematical models applied to systems biology and network science.

Expanded phenotypically stable chondrocytes persist in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation

Autologous chondrocyte implantation (ACI) is an established technique to repair joint surface defects. Although there is some indirect evidence that the expanded chondrocytes are required to achieve proper healing, the role they play in the repair process is not clear yet. To monitor the persistence and the phenotype of the injected chondrocytes in the repair tissue (RT) we have optimized a fluorescent labeling protocol for articular chondrocytes, which allows cell tracking in vivo for up to 14 weeks, using the fluorescent dye PKH26. We have combined in vivo cell tracking, with the immune‐detection of collagen type II protein in a goat model of ACI. Our data indicate that the implanted cells can persist for at least 14 weeks in the defects, can participate in the integration with the surrounding tissues, and become structural part of the RT, rich in collagen type II and sulfated proteoglycans. Albeit with a small number of samples, our data provide proof of principal that the implanted chondrocytes can contribute to structural cartilage repair in a goat model of ACI.

Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005

Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines – an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.

Identification of the molecular response of articular cartilage to injury, by microarray screening: Wnt‐16 expression and signaling after injury and in osteoarthritis

OBJECTIVE To characterize the molecular response of adult human articular cartilage to acute mechanical injury. METHODS An established ex vivo model was used to compare gene expression of adult human articular cartilage explants 24 hours after mechanical injury with that of uninjured controls by microarray analysis of gene expression. Confirmation for selected genes was obtained by real-time polymerase chain reaction and immunohistochemical analysis. Expression of selected genes was also investigated in preserved and osteoarthritic (OA) cartilage. RESULTS Six hundred ninety genes were significantly regulated at least 2-fold following mechanical injury. They included genes previously reported to be differentially expressed in OA versus normal cartilage or having allelic variants genetically linked to OA. Significant functional clusters included genes associated with wound healing, developmental processes, and skeletal development. The transforming growth factor beta, fibroblast growth factor, and Wnt pathways were modulated. A systematic analysis of the Wnt signaling pathway revealed up-regulation of Wnt-16, down-regulation of FRZB, up-regulation of Wnt target genes, and nuclear localization of beta-catenin in injured cartilage. In addition, in OA, Wnt-16 and beta-catenin were barely detectable in preserved cartilage areas, but were dramatically up-regulated in areas of the same joint with moderate to severe OA damage. CONCLUSION Our findings indicate that mechanical injury to adult human articular cartilage results in the activation of a signaling response, with reactivation of morphogenetic pathways. Therapeutic targeting of such pathways may improve current protocols of joint surface defect repair and/or prevent the evolution of such lesions into posttraumatic OA.

Functional Mesenchymal Stem Cell Niches in Adult Mouse Knee Joint Synovium In Vivo

OBJECTIVE We previously reported that human synovium contains cells that, after culture expansion, display properties of mesenchymal stem cells (MSCs). The objective of this study was to identify MSCs in native synovium in vivo. METHODS To identify stem cells in the synovium in vivo, a double nucleoside analog cell-labeling scheme was used in a mouse model of joint-surface injury. For labeling of slow-cycling cells, mice received iododeoxyuridine (IdU) for 30 days, followed by a 40-day washout period. For labeling of cells that proliferate after injury, mice underwent knee surgery to produce an articular cartilage defect and received chlorodeoxyuridine (CIdU) for 4 days, starting at multiple time points after surgery. Unoperated and sham-operated joints served as controls. Knee joint paraffin sections were analyzed by double and triple immunostaining to detect nucleoside analogs, conventional MSC markers, and chondrocyte-lineage markers. RESULTS Long-term-retaining, slow-cycling IdU-positive cells were detected in the synovium. At 4 days and 8 days after injury, there was marked proliferation of IdU-positive cells, which costained for CIdU. IdU-positive cells were nonhematopoietic, nonendothelial stromal cells, were distinct from pericytes, and stained positive for MSC markers. MSCs were phenotypically heterogeneous and located in topographically distinct niches in the lining layer and the subsynovial tissue. Twelve days after injury, double nucleoside-labeled cells within synovium were embedded in cartilage-specific metachromatic extracellular matrix and costained positive for the chondrocyte-lineage markers Sox9 and type II collagen. CONCLUSION Our findings provide the first evidence of the existence of resident MSCs in the knee joint synovium that undergo proliferation and chondrogenic differentiation following injury in vivo.

Activation of WNT and BMP signaling in adult human articular cartilage following mechanical injury

Acute full thickness joint surface defects can undergo repair, which involves tissue patterning and endochondral bone formation. Molecular signals regulating this process may contribute to the repair outcome, chronic evolution and, eventually, the onset of osteoarthritis. We tested the hypothesis that mechanical injury modulates morphogenetic pathways in adult human articular cartilage explants. Adjacent articular cartilage explants were obtained from preserved areas of the femoral condyles of patients undergoing arthroplasty for osteoarthritis, or from a normal joint of a patient undergoing lower limb amputation. Paired explants were individually maintained in explant culture. From each pair, one explant was mechanically injured and the other left uninjured as a control. Cultures were terminated at different time points for histochemistry, immunohistochemistry and gene expression analysis by reverse transcription real time PCR. Bone morphogenetic protein 2 (BMP-2) mRNA was upregulated in the injured explants. We detected phosphorylation of SMAD-1 and SMAD-5, consistent with activation of the bone morphogenetic protein (BMP) pathway. FRZB-1 mRNA was downregulated in the injured explants, suggesting de-repression of WNT signaling. Accordingly, expression of the canonical WNT target genes Axin-2 and c-JUN was upregulated in the injured explants. Activation of the canonical WNT signaling pathway by LiCl treatment induced upregulation of COL2A1 and Aggrecan mRNA, suggesting an anabolic effect. Phosphorylation of SMAD-1/-5 and downregulation of FRZB were confirmed in vivo in a mouse model of joint surface injury. Taken together, these data show modulation of the BMP and WNT pathways following mechanical injury in vitro and in vivo, which may play a role in the reparative response of the joint surface. These pathways may, therefore, represent potential targets in protocols of biological joint surface defect repair.