Bernhard Schewe

Novel Markers for the Prospective Isolation of Human MSC

Abstract:  The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)‐derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271+ population. The recognized CD271+ populations were fractionated by fluorescence‐activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU‐F. The results showed that only the CD271bright but not the CD271dim population contained CFU‐F. Two‐color flow cytometry analysis revealed that only the CD271bright population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271bright population but no other BM cells. The new MSC‐specific molecules included the platelet‐derived growth factor receptor‐β (CD140b), HER‐2/erbB2 (CD340), frizzled‐9 (CD349), the recently described W8B2 antigen, as well as cell‐surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9‐3C2F1, and HEK‐3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM‐MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1

Mesenchymal stem cells are self-renewing cells with the ability to differentiate into osteocytes, chondrocytes and adipocytes. This article describes a subset of mesenchymal stem cells with distinct phenotypic and functional properties. Background Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells. Design and Methods Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining. Results Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90-fold enriched in the MSCA-1+CD56− fraction and ~180-fold in the MSCA-1+CD56+ fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1+CD56− mesenchymal stem cells subset and CD166 to MSCA-1+CD56± mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1+CD56± cells whereas adipocytes emerged exclusively from MSCA-1+CD56− cells. The culture of single sorted MSCA-1+CD56+ cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities. Conclusions Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1+CD56+ subset is an attractive starting population for autologous chondrocyte transplantation.

Chondrogenic Potential of Human Adult Mesenchymal Stem Cells Is Independent of Age or Osteoarthritis Etiology

Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age‐ or OA etiology‐dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA‐etiology groups (age‐related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase‐polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor‐site morbidity, typically containing >107 mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible.

Remodeling of Articular Cartilage and Subchondral Bone After Bone Grafting and Matrix-Associated Autologous Chondrocyte Implantation for Osteochondritis Dissecans of the Knee

Background: Osteochondritis dissecans (OCD) of the knee is a challenging problem. Previously, the authors implemented a novel 1-step surgical procedure for OCD treatment consisting of matrix-associated autologous chondrocyte implantation (ACI) and simultaneous bone reconstruction including the subchondral lamina. Purpose: This study presents the 2-to 5-year results after this technique, assessing correlations of clinical function and cartilage and bone remodeling processes. Study Design: Case series; Level of evidence, 4. Methods: Twenty-six patients with symptomatic condylar knee OCD (International Cartilage Repair Society OCD III/IV) were treated with matrix-associated ACI and monocortical cancellous cylinders for defect filling and subchondral bone plate reconstruction using cortical graft layers as novel subchondral lamina. Evaluations were performed with clinical rating scales and 1.5-T magnetic resonance imaging using the magnetic resonance observation of cartilage repair tissue (MOCART) score and a newly implemented subchondral lamina remodeling grade. Results: The defect size was 5.3 ± 2.3 cm2. The defect depth was 8.7 ± 2.4 mm. After a follow-up of 39.8 ± 12.0 months, all scores improved significantly. Nineteen patients (73%) reached good/excellent results in the Lysholm-Gillquist score (preoperatively: 53.2 ± 18.0 points; latest follow-up: 88.5 ± 9.5 points) and the Cincinnati knee rating score (preoperatively: 51.7 ± 13.0 points; latest follow-up: 84.6 ± 11.7 points) and significant improvements in the subjective International Knee Documentation Committee (IKDC) score by 27.9% (preoperatively: 50.5% ± 16.1%; latest follow-up: 78.4% ± 13.4%). The MOCART score reached 62.4 ± 18.9 points. The clinical improvement and tissue remodeling occurred simultaneously and timed; thus, the cartilage defect filling and the lamina remodeling grades correlated significantly with each other, the follow-up time, and almost all clinical scores. Conclusion: The simultaneous reconstruction of deep osteochondral defects of the knee OCD with monocortical cancellous cylinders and matrix-associated ACI is a biological, 1-step alternative to osteochondral cylinder transfer or conventional ACI that leads to good clinical and magnetic resonance imaging results after an intermediate follow-up period. The present study demonstrated simultaneous remodeling processes of articular cartilage repair tissue and subchondral lamina; this synchronization is not yet understood and deserves further investigation.

Articular cartilage defects in the knee-basics, therapies and results

Full-thickness defects of the articular cartilage in the knee joint have lower regenerative properties than chondral lesions of the ankle. In order to avoid early osteoarthritis, symptomatic articular cartilage defects in younger patients should undergo biological reconstruction as soon as possible. Various surgical procedures are available to biologically resurface the articular joint line. Numerous animal experiments and clinical studies have shown that early biological reconstruction of circumscribed cartilage defects in the knee is superior to conservative or delayed surgical treatment. This superiority refers not only to defect healing but also to the elimination of changes following secondary osteoarthritis. The various surgical procedures can be differentiated by the range of indications and the final outcome. Additional malalignment, meniscus tears and/or ligament instabilities should be treated simultaneously with the cartilage resurfacing. The mid- and long-term results of the various current techniques are promising, but further modifications and improvements are needed.

Human mesenchymal stromal cells express CD14 cross‐reactive epitopes

Mesenchymal stromal cells (MSCs) do not express a unique definite epitope or marker gene. As such, minimal criteria were recently established for defining multipotent MSC. These criteria include expression of CD73, CD90, CD105, and a lack of hematopoietic marker expression. However, we detected binding of a CD14 antibody on bone marrow‐ and placenta‐derived MSC and investigated the staining of CD14 antibodies on these MSC in more detail. The MSC were isolated from human bone marrow and placenta tissue, expanded, characterized by quantitative RT‐PCR, flow cytometry, and immunocytochemistry and differentiated to generate osteoblasts, chondrocytes, and adipocytes. The CD14‐cross‐reactive MSCs were enriched by cell sorting. Human peripheral blood mononuclear cells, fibroblasts, and hematopoietic cell lines served as controls. Utilizing four different clones of CD14 monoclonal antibodies, we found that three CD14 reagents stained the MSC. Two CD14 antibodies (HCD14 and M5E2) clearly marked the CD90+ MSC population with distinct intensities, clone 134 620 generated a shift in flow cytometry histograms, but clone MΦP9 did not stain MSC. Transcripts encoding CD14 or the CD14 protein were not detected in MSC. We confirm that bone marrow‐ and placenta‐derived MSC do not express CD14 and that the CD14 antibody MΦP9 discriminates between monocytes and MSC more efficiently than the other antibodies employed here. This investigation does not contradict previous work but provides a more accurate characterization of MSC.

Stand und Trends in der biologischen Rekonstruktion von Knorpelschäden des Kniegelenks

Der hyaline Gelenkknorpel wird nicht durchblutet und hat keinen Anschluss an das Lymphoder Nervensystem Seine Ernährung erfolgt ausschließlich durch Diffusion. Der synovialen Flüssigkeit, die auch als Gelenkschmiere wirkt, kommt dabei eine wichtige Funktion zu. Beim Gehen oder unter normaler Belastung wird das Knorpelgewebe komprimiert, dabei tritt Flüssigkeit aus dem Knorpel aus und Stoffwechselendprodukte werden ausgeschwemmt. Nach Entlastung saugt das Knorpelgewebe Flüssigkeit aus dem Gelenkspalt zurück. Durch diesen Vorgang werden die Knorpelzellen (Chondrozyten) mit Zuckern, Aminosäuren, Fetten und anderen Nährstoffen versorgt.