Elena Jones

Markers for Characterization of Bone Marrow Multipotential Stromal Cells

Given the observed efficacy of culture-expanded multipotential stromal cells, also termed mesenchymal stem cells (MSCs), in the treatment of graft-versus host and cardiac disease, it remains surprising that purity and potency characterization of manufactured cell batches remains rather basic. In this paper, we will initially discuss surface and molecular markers that were proposed to serve as the indicators of the MSC potency, in terms of their proliferative potential or the ability to differentiate into desired lineages. The second part of this paper will be dedicated to a critical discussion of surface markers of uncultured (i.e., native) bone marrow (BM) MSCs. Although no formal consensus has yet been reached on which markers may be best suited for prospective BM MSC isolation, markers that cross-react with MSCs of animal models (such as CD271 and W8-B2/MSCA-1) may have the strongest translational value. Whereas small animal models are needed to discover the in vivo function on these markers, large animal models are required for safety and efficacy testing of isolated MSCs, particularly in the field of bone and cartilage tissue engineering.

Synovial fluid mesenchymal stem cells in health and early osteoarthritis: Detection and functional evaluation at the single‐cell level

OBJECTIVE Arthritic synovial fluid (SF) contains mesenchymal stem cells (MSCs), which could simply reflect their shedding from diseased joint structures. This study used the bovine model to explore SF MSCs in health and enumerated them at the earliest stages of human osteoarthritis (OA) in radiographically normal joints. METHODS Clonogenicity and multipotentiality of normal bovine SF MSCs were compared with donor-matched bone marrow (BM) MSCs at the single-cell level. The colony-forming unit-fibroblastic assay was used for MSC enumeration. The XTT assay was employed to assess cell proliferation, and flow cytometry was used to investigate the marker phenotype of bovine and human SF MSCs. RESULTS Single MSCs were present in normal bovine SF, and 96% of them were able to expand at least 1 million-fold. These cells were CD271-, multipotential, considerably more clonogenic, and less adipogenic than matched BM MSCs. In both pellet assays and on polyglycolic acid scaffolds, SF clones displayed consistent chondrogenic differentiation, while BM clones were variable. MSCs were present in arthroscopically normal human joints and were increased 7-fold in early OA (P = 0.034). Their numbers correlated with numbers of free microscopic synovial tissue fragments (r = 0.826, P < 0.0001). OA SF had a growth-promoting effect on synovial MSCs. CONCLUSION This study confirms the presence of MSCs in normal SF and shows their numerical increase in early human OA. SF MSCs are likely to originate from synovium. These findings provide a platform for the exploration of the potential role of SF MSCs in joint homeostasis and for investigation of their utility in novel joint regeneration strategies.

Optimization of a Flow Cytometry-Based Protocol for Detection and Phenotypic Characterization of Multipotent Mesenchymal Stromal Cells from Human Bone Marrow

To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony‐forming unit‐fibroblast (CFU‐F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry‐based methods for their evaluation.

A biomarker-based mathematical model to predict bone-forming potency of human synovial and periosteal mesenchymal stem cells.

OBJECTIVE To develop a biomarker-based model to predict osteogenic potency of human mesenchymal stem cells (MSCs) from synovial membrane and periosteum. METHODS MSC populations were derived from adult synovium and periosteum. Phenotype analysis was performed by fluorescence-activated cell sorting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Telomere lengths were determined by Southern blot analysis. In vitro osteogenesis was assessed quantitatively by measurements of alkaline phosphatase activity and calcium deposits. To investigate bone formation in vivo, MSCs were seeded onto osteoinductive scaffolds and implanted subcutaneously in nude mice. Bone was assessed by histology, and the human origin investigated by in situ hybridization for human Alu genomic repeats. Quantitation was achieved by histomorphometry and real-time RT-PCR for human osteocalcin. Analysis at the single-cell level was performed with clonal populations obtained by limiting dilution. Multiple regressions were used to explore the incremental predictive value of the markers. RESULTS Periosteal MSCs had significantly greater osteogenic potency than did synovial MSCs inherent to the single cell. Bone was largely of human origin in vivo. Within the same tissue type, there was variability between different donors. To identify predictors of osteogenic potency, we measured the expression levels of osteoblast lineage genes in synovial and periosteal clonal MSCs prior to osteogenic treatment. We identified biomarkers that correlated with osteogenic outcome and developed a mathematical model based on type I collagen and osteoprotegerin expression that predicts the bone-forming potency of MSC preparations, independent of donor-related variables and tissue source. CONCLUSION Our findings indicate that our quality-control mathematical model estimates the bone-forming potency of MSC preparations for bone repair.

The Effect of Bone Morphogenetic Protein-2, Bone Morphogenetic Protein-7, Parathyroid Hormone, and Platelet-Derived Growth Factor on the Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Osteoporotic Bone

Introduction: It has been previously shown that in patients with osteoporosis, mesenchymal stem cell (MSC) growth rate and osteogenic potential is decreased contributing to inferior fracture consolidation. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2), BMP-7, parathyroid hormone (PTH), and platelet-derived growth factor (PDGF) on proliferation and osteogenic differentiation of MSCs derived from patients with osteoporosis. Materials and Methods: Trabecular bone was obtained from 10 patients (four males, mean age 76 years) with lower extremity osteoporotic fractures. MSCs were isolated by enzymatic digestion. Functional assays of proliferation and osteogenic differentiation were performed under the influence of a wide range of concentrations of BMP-2, BMP-7, PTH, and PDGF-BB. Proliferation was assessed using CFU-F and XTT assays. Osteogenic differentiation was assessed by alkaline phosphatase activity and total calcium production. Results: MSC proliferation was found to be stimulated by supplementation with BMP-7 and PDGF-BB, whereas BMP-2 and PTH had little effect. The largest increase in proliferation rate was observed after administration 100 ng/mL of BMP-7. All four molecules induced alkaline phosphatase activity and calcium production in growing osteoblasts with a dose-dependent effect noted. BMP-2 and BMP-7 at their highest studied concentration (100 ng/mL) produced a threefold increase in the osteogenic potential of MSCs. Conclusion: BMP-7, BMP-2, PTH, and PDGF-BB were observed to have a positive effect on osteogenic differentiation of MSCs. BMP-7 and PDGF-BB (in high doses) could be considered most potentially advantageous because they enhance both proliferation and osteogenic differentiation of MSCs derived from elderly osteoporotic bone.

Mesenchymal stem cells in rheumatoid synovium: enumeration and functional assessment in relation to synovial inflammation level

Objective: Achieving joint regeneration in rheumatoid arthritis (RA) represents a future challenge. Autologous synovial mesenchymal stem cells (MSCs) could be therapeutically exploited. However, the inflammatory milieu in the RA synovium could adversely affect endogenous MSC function. To test this hypothesis, the frequency and multipotency of RA synovial MSCs was evaluated in relation to existing synovial inflammation. Methods: Synovial inflammation was measured using the arthroscopic visual analogue score (VAS) and further validated using immunohistochemistry and flow cytometry. Highly proliferative clonogenic in vivo MSCs were enumerated following fluorescence-activated cell sorting and expansion for 20 population doublings. MSC multipotency was quantified following standard in vitro culture expansion and trilineage differentiation assays. Real-time PCR, flow cytometry and ELISA were used to evaluate pro- and anti-chondrogenic molecules in standard polyclonal synovial MSCs. Results: The arthroscopic VAS significantly correlated with synovial macrophage infiltration. In RA, synovial MSC chondrogenesis was inhibited in direct relation to VAS (r = −0.777, p<0.05) and reduced compared with control osteoarthritis (OA)-MSCs (p<0.05). In vivo, MSCs resided in the synovial fibroblastic/stromal fraction (CD45−CD31−) and were reduced in frequency in relation to VAS (r = −0.695, p<0.05). In RA-MSCs, CD44 levels correlated negatively with inflammation and positively with chondrogenesis (r = −0.830 and r = 0.865, respectively). Cytokine production and Sox9 expression was similar in RA-MSCs and OA-MSCs. Conclusions: There is a negative relationship between synovial MSC chondrogenic and clonogenic capacities and the magnitude of synovitis in RA. Effective suppression of joint inflammation is therefore necessary for the development of autologous MSC treatments aimed at cartilage regeneration in RA.

Functional properties of cartilaginous tissues engineered from infrapatellar fat pad-derived mesenchymal stem cells

Articular cartilage has a poor intrinsic capacity for self-repair. The advent of autologous chondrocyte implantation has provided a feasible method to treat cartilage defects. However, the associated drawbacks with the isolation and expansion of chondrocytes from autologous tissue has prompted research into alternative cell sources such as mesenchymal stem cells (MSCs) which have been found to exist in the bone marrow as well as other joint tissues such as the infrapatellar fat pad (IFP), synovium and within the synovial fluid itself. In this work we assessed the chondrogenic potential of IFP-derived porcine cells over a 6 week period in agarose hydrogel culture in terms of mechanical properties, biochemical content and histology. It was found that IFP cells underwent robust chondrogenesis as assessed by glycosaminoglycan (1.47+/-0.22% w/w) and collagen (1.44+/-0.22% w/w) accumulation after 42 days of culture. The 1Hz dynamic modulus of the engineered tissue at this time point was 272.8 kPa (+/-46.8). The removal of TGF-beta3 from culture after 21 days was shown to have a significant effect on both the mechanical properties and biochemical content of IFP constructs after 42 days, with minimal increases occurring from day 21 to day 42 without continued supplementation of TGF-beta3. These findings further strengthen the case that the IFP may be a promising cell source for putative cartilage repair strategies.

Distinct mesenchymal progenitor cell subsets in the adult human synovium

OBJECTIVE To analyse the heterogeneity at the single-cell level of human mesenchymal progenitor cells from SM. METHODS Cell populations were enzymatically released from the knee joint synovium of adult human individuals. Single cell-derived clonal populations were obtained by limiting dilution and serially passaged to determine growth rates. Phenotypic analysis was carried out by flow cytometry. Replicative senescence was assessed by the senescence-associated beta-galactosidase staining. Telomere lengths were determined semiquantitatively by Southern blotting. Telomerase activity was measured using a real-time quantitative telomerase repeat amplification procedure. Culture-expanded clonal populations were subjected to in vitro differentiation assays to investigate their mesenchymal multipotency. RESULTS The 50 clones analysed displayed wide variations in the proliferation rates, even within the same donor sample. The time taken to reach 20 population doublings ranged from 44 to 130 days. The phenotype of the clones tested was compatible with that of mesenchymal stem cells. Mean telomere lengths ranged from 5.2 to 10.9 kb with positive linear trend with telomerase activity, but no correlation with proliferative rates or cell senescence. All clones tested were capable of chondrogenic and osteogenic differentiation, though with large variability in potency. In contrast, only 30% of the clones were adipogenic. CONCLUSIONS We report for the first time the co-existence, within the synovium, of progenitor cell subsets with distinct mesenchymal differentiation potency. Our findings further emphasize the need for strategies to purify cell populations with the clinically desired tissue formation potentials.

Growing bone and cartilage: THE ROLE OF MESENCHYMAL STEM CELLS

The management of large bony defects and cartilage loss continues to be a challenge to trauma and orthopaedic surgeons. The treatment of these conditions has evolved over the years and has comprised of many techniques, such as autologous bone grafting, distraction osteogenesis and free fibular

Transcriptional profile of native CD271+ multipotential stromal cells: Evidence for multiple fates, with prominent osteogenic and Wnt pathway signaling activity

OBJECTIVE Controversy surrounds the identity and functionality of rare bone marrow-derived multipotential stromal cells (BM-MSCs), including their differentiation capabilities, their relationship to pericytes and hematopoiesis-supporting stromal cells, and the relevance of their culture-expanded progeny in studies of skeletal biology and development of cell-based therapies. The aim of this study was to clarify the nature of candidate BM-MSCs by profiling transcripts that reflect different aspects of their putative functions in vivo. METHODS Rare, sorted BM-derived CD45(-/low) CD271(bright) (CD271) cells were analyzed using 96-gene expression arrays focused on transcripts relevant to mesenchymal-lineage differentiation (toward bone, cartilage, fat, or muscle), hematopoietic and stromal support, and molecules critical to skeletal homeostasis. These cells were compared to matched CD45+ CD271- hematopoietic-lineage cells, culture-expanded MSCs, and skin fibroblasts. When feasible, transcription was validated using flow cytometry. RESULTS CD271 cells had a transcriptional profile consistent with the multiple fates of in vivo MSCs, evident from the observed simultaneous expression of osteogenic, adipogenic, pericytic, and hematopoiesis-supporting genes (e.g., SP7 [osterix], FABP4 [fatty acid binding protein 4], ANGPT1 [angiopoietin 1], and CXCL12 [stromal cell-derived factor 1], respectively). Compared to culture-expanded MSCs and fibroblasts, CD271 cells exhibited greater transcriptional activity, particularly with respect to Wnt-related genes (>1,000-fold increased expression of FRZB [secreted frizzled-related protein 3] and WIF1 [Wnt inhibitory factor 1]). A number of transcripts were identified as novel markers of MSCs. CONCLUSION The native, BM-derived in vivo MSC population is endowed with a gene signature that is compatible with multiple functions, reflecting the topographic bone niche of these cells, and their signature is significantly different from that of culture-expanded MSCs. This indicates that studies of the biologic functions of MSCs in musculoskeletal diseases, including osteoporosis and osteoarthritis, should focus on in vivo MSCs, rather than their culture-adapted progeny.