Jörg Fiedler

Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells

Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types, and may also replace or repair diseased brain tissue. NSCs can be directly isolated from fetal or adult nervous tissue, or derived from embryonic stem cells. Here, we describe the efficient conversion of human adult bone marrow stromal cells (hMSC) into a neural stem cell-like population (hmNSC, for human marrow-derived NSC-like cells). These cells grow in neurosphere-like structures, express high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, MSl1 as well as otx1 and nestin, but lose the characteristics of mesodermal stromal cells. In the presence of selected growth factors, hmNSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Clonal analysis demonstrates that individual hmNSCs are multipotent and retain the capacity to generate both glia and neurons. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases.

BMP‐2, BMP‐4, and PDGF‐bb stimulate chemotactic migration of primary human mesenchymal progenitor cells

For bone development, remodeling, and repair; the recruitment of mesenchymal progenitor cells (MPC) and their differentiation to osteoblasts is mandatory. The process of migration is believed to be regulated in part by growth factors stored within the bone matrix and released by bone resorption. In this study, primary human MPCs and to osteoblasts differentiated progenitor cells were examined for chemotaxis in response to human basic fibroblast growth factor (rhbFGF), human transforming growth factor beta 1 (rhTGF‐β1), human platelet derived growth factor bb (rhPDGF‐bb), human bone morphogenetic protein‐2 (rhBMP‐2), and recombinant bone morphogenetic protein‐4 of Xenopus laevis (rxBMP‐4) from 0.001 to 1.0 ng/ml each. The results of migration were expressed as a chemotactic index (CI). Migration of primary human progenitor cells was stimulated by rhBMP‐2, rxBMP‐4, and rhPDGF‐bb in a dose‐dependent manner. The increase of CI was up to 3.5‐fold for rhBMP‐2, 3.6‐fold for rxBMP‐4, and up to 22‐fold for rhPDGF‐bb, whereas rhTGF‐β1 and rhbFGF did not stimulate cell migration in the concentration range tested. In contrast differentiated progenitor cells behave similar to primary human osteoblasts. RhBMP‐2, rhPDGF‐bb, and rhTGF‐β1 stimulated the migration from 2.2 to 2.4‐fold each, while rxBMP‐4 and rhbFGF reached only a CI of 1.7–1.6. The effect of rhBMP‐2, rxBMP‐4, and rhPDGF‐bb as chemoattractive proteins for primary human MPC, including the change in response to growth factors after differentiation suggests a functional role for recruitment of MPCs during bone development and remodeling, as well as fracture healing. J. Cell. Biochem. 87:305‐312, 2002.

TNFα promotes osteogenic differentiation of human mesenchymal stem cells by triggering the NF-κB signaling pathway

Mesenchymal stem cells are multipotent cells able to differentiate into different mesenchymal lineages. Studies in the past had suggested that two of these mesenchymal differentiation directions, the chondrogenic and the myogenic differentiation, are negatively regulated by the transcription factor NF-kappaB. Although osteogenic differentiation has been extensively studied, the influence of NF-kappaB on this differentiation lineage was not subject of detailed analyses in the past. We have analyzed the consequences of TNF-alpha treatment and genetic manipulation of the NF-kappaB pathway for osteogenic differentiation of hMSCs. Treatment of hMSCs during differentiation with TNF-alpha activates NF-kappaB and this results in enhanced expression of osteogenetic proteins like bone morphogenetic protein2 (BMP-2) and alkaline phosphatase (ALP). In addition, enhanced matrix mineralization was observed. The direct contribution of the NF-kappaB pathway was confirmed in cells that express a constitutively active version of the NF-kappaB-inducing kinase IKK2 (CA-IKK2). The IKK2/NF-kappaB-induced BMP-2 up-regulation results in the enhancement of RUNX2 and Osterix expression, two critical regulators of the osteogenic differentiation program. Interestingly, a genetic block of the NF-kappaB pathway did not interfere with osteogenic differentiation. We conclude that TNFalpha mediated NF-kappaB activation, although not absolutely required for BMP-2 expression and matrix mineralization nevertheless supports osteogenic differentiation and matrix mineralization by increasing BMP-2 expression. Our results therefore suggest that NF-kappaB activation may function in lineage selection during differentiation of hMSCs by fostering osteogenic differentiation at the expense of other differentiation lineages.

Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers

We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2–12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints.

Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting.

OBJECTIVE Identification, quantification and isolation of subpopulations with characteristics of mesenchymal progenitor cells (MPC) from the synovial membrane (SM) from patients with osteoarthritis (OA). METHOD Cells from the SM of patients with end stage OA who underwent total knee joint replacement were enzymatically isolated. One aliquot was directly analyzed by fluorescence automated cell sorting (FACS) using various combinations of surface markers of bone marrow MPC (CD9, CD44, CD54, CD90, and CD166). Remaining cells were cultivated on plastic, expanded over several passages, analyzed by FACS again and tested for their osteo- and chondrogenic potential. The differentiation was analyzed by immuno-/histochemistry and by RT-PCR for the expression of lineage related marker genes. RESULTS Using FACS analysis we could show that the relative proportion of subpopulations expressing triplicate combinations of CD9, CD44, CD54, CD90 and CD166 in the SM from OA patients varies between 3 and 10%. Upon cultivation their relative amount markedly increased to values between 24 and 48%. Within the heterogeneous cell populations it was possible to induce osteogenic and chondrogenic differentiation. Initial sorting for CD9/CD90/CD166 triplicate positive cells proved that this subpopulation contains cells with multipotency for mesenchymal differentiation and thus characteristics of MPC. CONCLUSION Our results show that SM from OA patients contains cells that express typical combinations of MPC surface markers and have the potency of osteogenic and chondrogenic differentiation. Their relative enrichment during in vitro cultivation and the possibility of cell sorting to get more homogenous populations offer interesting perspectives for possible future therapeutic applications.

To go or not to go: Migration of human mesenchymal progenitor cells stimulated by isoforms of PDGF

The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet‐derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet‐derived growth factor AA, ‐BB, and heterodimeric PDGF‐AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF‐AA, rhPDGF‐BB, and rhPDGF‐AB. The effect was highest in MPCs and for rhPDGF‐BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP‐2 as well as rxBMP‐4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF‐β1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF‐AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone.

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols.

Human adult bone marrow‐derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT‐PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, β‐tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell‐like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs.

The effect of substrate surface nanotopography on the behavior of multipotnent mesenchymal stromal cells and osteoblasts.

Hexagonally arranged Gold nanoparticles with controllable diameters and inter-particle distances were deposited on thick SiO2 layers on top of Si wafers and used as masks during subsequent reactive ion etching. In this way, arrays of nanopillars are obtained with well-defined diameters (10/30 nm), inter-pillar distances (50-120 nm) and heights (20-35 nm), all on the nanoscale. Such nanotopographies served as substrate for multipotent mesenchymal stromal cells (MSC) and human osteoblasts (OB) allowing to study cellular responses to purely topographically patterned interfaces. Focus was put on adhesion, proliferation and differentiation of the cells. It turned out experimentally that adhesion is comparable for both cell types practically independent of topographical details at the substrate surface. Topography induced proliferation enhancement, however, is again independent of geometrical details in case of MSC, but significantly sensitive to pillar height in case of OB with a clear preference towards short nanopillars (20 nm). A high sensitivity to topographic details is also observed for osteogenic differentiation of MSC, in that case with a preference towards higher nanopillars (50 nm). The present experimental data also allow the important conclusion that cell proliferation and differentiation can be optimized simultaneously by fine-tuning nanoscaled topographical parameters.

CYR61/CCN1 and WISP3/CCN6 are chemoattractive ligands for human multipotent mesenchymal stroma cells

Background:CCN-proteins are known to be involved in development, homeostasis and repair of mesenchymal tissues. Since these processes implicate recruitment of cells with the potential to be committed to various phenotypes, we studied the effect of CYR61/CCN1 and WISP3/CCN6 on migration of human bone marrow derived mesenchymal stroma cells (MSCs) in comparison to in vitro osteogenic differentiated MSCs using a modified Boyden chamber assay.Results:CYR61 and WISP3 were purified as fusion proteins with a C-terminal Fc-tag from baculovirus infected SF21 cells using protein G sepharose columns. CYR61 and WISP3 stimulated cell migration of undifferentiated MSCs in a dose-dependent manner. CYR61 and WISP3 had similar effects on committed osteogenic precursor cells. Checkerboard analysis revealed that CYR61 and WISP3 stimulated true directed cell migration (chemotaxis) of MSCs and committed osteogenic precursors. In MSCs the chemotactic activity of WISP3 but not CYR61 was mediated through integrin ανß 5.Conclusion:Our results indicate that CYR61 and WISP3 can function as soluble ligands transmitting chemotactic signals to human MSCs but differ in the involvement of integrin ανß 5. This may be relevant for their possible role in connective tissue repair.

Copper and silver ion implantation of aluminium oxide-blasted titanium surfaces: proliferative response of osteoblasts and antibacterial effects.

Implant infection still represents a major clinical problem in orthopedic surgery. We therefore tested the in vitro biocompatibility and antibacterial effects of copper (Cu)- and silver (Ag)-ion implantation. Discs of a commonly used titanium alloy (Ti6AlV4) with an aluminium oxide-blasted surface were treated by Cu- or Ag-ion implantation with different dosage regimen (ranging from 1e15–17 ions cm−2 at energies of 2–20 keV). The samples were seeded with primary human osteoblasts and cell attachment and proliferation was analyzed by an MTT-assay. In comparison to the reference titanium alloy there was no difference in the number of attached viable cells after two days. After seven days the number of viable cells was increased for Cu with 1e17 ions cm−2 at 2 and 5 keV, and for Ag with 1e16 ions cm−2 at 5 keV while it was reduced for the highest amount of Ag deposition (1e17 ions cm−2 at 20 keV). Antibacterial effects on S.aureus and E.coli were marginal for the studied dosages of Cu but clearly present for Ag with 1e16 ions cm−2 at 2 and 5 keV and 1e17 ions cm−2 at 20 keV. These results indicate that Ag-ion implantation may be a promising methodological approach for antibacterial functionalization of titanium implants.