Emma Muiños-López

Characterization of microRNA expression profiles in normal and osteoarthritic human chondrocytes.

BackgroundOsteoarthritis (OA) is a multifactorial disease characterized by destruction of the articular cartilage due to environmental, mechanical and genetic components. The genetics of OA is complex and is not completely understood. Recent works have demonstrated the importance of microRNAs (miRNAs) in cartilage function. MiRNAs are a class of small noncoding RNAs that regulate gene expression and are involved in different cellular process: apoptosis, proliferation, development, glucose and lipid metabolism. The aim of this study was to identify and characterize the expression profile of miRNAs in normal and OA chondrocytes and to determine their role in the OA.MethodsChondrocytes were moved to aggregate culture and evaluated using histological and qPCR techniques. miRNAs were isolated and analyzed using the Agilent Human miRNA Microarray.ResultsOf the 723 miRNAs analyzed, 7 miRNAs showed a statistically significant differential expression. Amongst these 7 human miRNAs, 1 was up-regulated in OA chondrocytes (hsa-miR-483-5p) and 6 were up-regulated in normal chondrocytes (hsa-miR-149*, hsa-miR-582-3p, hsa-miR-1227, hsa-miR-634, hsa-miR-576-5p and hsa-miR-641). These profiling results were validated by the detection of some selected miRNAs by qPCR. In silico analyses predicted that key molecular pathways potentially altered by the miRNAs differentially expressed in normal and OA chondrocytes include TGF-beta, Wnt, Erb and mTOR signalling; all of them implicated in the development, maintenance and destruction of articular cartilage.ConclusionsWe have identified 7 miRNAs differentially expressed in OA and normal chondrocytes. Our potential miRNA target predictions and the signalling cascades altered by the differentially expressed miRNAs supports the potential involvement of the detected miRNAs in OA pathology. Due to the importance of miRNA in mediating the translation of target mRNA into protein, the identification of these miRNAs differentially expressed in normal and OA chondrocyte micropellets could have important diagnostic and therapeutic potential. Further studies are needed to know the function of these miRNAs, including the search of their target mRNA genes, which could lead to the development of novel therapeutic strategies for the OA treatment.

Multilineage differentiation potential of cells isolated from the human amniotic membrane

The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM‐derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion‐derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real‐time reverse transcription‐polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well‐defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem‐cell markers SSEA‐4 and STRO‐1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy. J. Cell. Biochem. 111: 846–857, 2010.

Human amniotic membrane as an alternative source of stem cells for regenerative medicine

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.

Quantification of Cells Expressing Mesenchymal Stem Cell Markers in Healthy and Osteoarthritic Synovial Membranes

Objective. To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints. Methods. Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence. Results. Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis. Conclusion. Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

Effects of Severe Hypoxia on Bone Marrow Mesenchymal Stem Cells Differentiation Potential

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed.

Molecular profile and cellular characterization of human bone marrow mesenchymal stem cells: donor influence on chondrogenesis.

BACKGROUND The use of autologous or allogenic stem cells has recently been suggested as an alternative therapeutic approach for treatment of cartilage defects. Bone marrow mesenchymal stem cells (BM-MSCs) are well-characterized multipotent cells that can differentiate into different cell types. Understanding the potential of these cells and the molecular mechanisms underlying their differentiation should lead to innovative protocols for clinical applications. The aim of this study was to evaluate the usefulness of surface antigen selection of BM-MSCs and to understand the mechanisms underlying their differentiation. METHODS MSCs were isolated from BM stroma and expanded. CD105+ subpopulation was isolated using a magnetic separator. We compared culture-expanded selected cells with non-selected cells. We analyzed the phenotypic profiles, the expression of the stem cell marker genes Nanog, Oct3/4, and Sox2 and the multi-lineage differentiation potential (adipogenic, osteogenic, and chondrogenic). The multi-lineage differentiation was confirmed using histochemistry, immunohistochemistry and/or real-time polymerase chain reaction (qPCR) techniques. RESULTS The selected and non-selected cells displayed similar phenotypes and multi-lineage differentiation potentials. Analyzing each cell source individually, we could divide the six donors into two groups: one with a high percentage of CD29 (β1-integrin) expression (HL); one with a low percentage of CD29 (LL). These two groups had different chondrogenic capacities and different expression levels of the stem cell marker genes. CONCLUSIONS This study showed that phenotypic profiles of donors were related to the chondrogenic potential of human BM-MSCs. The chondrogenic potential of donors was related to CD29 expression levels. The high expression of CD29 antigen seemed necessary for chondrogenic differentiation. Further investigation into the mechanisms responsible for these differences in BM-MSCs chondrogenesis is therefore warranted. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous human BM-MSCs for articular cartilage repair.

Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.

Combination of Intra-Articular and Intraosseous Injections of Platelet Rich Plasma for Severe Knee Osteoarthritis: A Pilot Study

The aim of this study was to assess a novel approach to treating severe knee osteoarthritis by targeting synovial membrane, superficial articular cartilage, synovial fluid, and subchondral bone by combining intra-articular injections and intraosseous infiltrations of platelet rich plasma. We explored a new strategy consisting of intraosseous infiltrations of platelet rich plasma into the subchondral bone in combination with the conventional intra-articular injection in order to tackle several knee joint tissues simultaneously. We assessed the clinical outcomes through osteoarthritis outcome score (KOOS) and the inflammatory response by quantifying mesenchymal stem cells in synovial fluid. There was a significant pain reduction in the KOOS from baseline (61.55 ± 14.11) to week 24 (74.60 ± 19.19), after treatment (p = 0.008), in the secondary outcomes (symptoms, p = 0.004; ADL, p = 0.022; sport/rec., p = 0.017; QOL, p = 0.012), as well as VAS score (p < 0.001) and Lequesne Index (p = 0.008). The presence of mesenchymal stem cells in synovial fluid and colony-forming cells one week after treatment decreased substantially from 7.98 ± 8.21 MSC/μL to 4.04 ± 5.36 MSC/μL (p = 0.019) and from 601.75 ± 312.30 to 139.19 ± 123.61  (p = 0.012), respectively. Intra-articular injections combined with intraosseous infiltrations of platelet rich plasma reduce pain and mesenchymal stem cells in synovial fluid, besides significantly improving knee joint function in patients with severe knee osteoarthritis. This trial is registered on EudraCT with the number 2013-003982-32.

Modulation of Synovial Fluid-Derived Mesenchymal Stem Cells by Intra-Articular and Intraosseous Platelet Rich Plasma Administration

The aim of this study was to evaluate the effect of intra-articular (IA) or a combination of intra-articular and intraosseous (IO) infiltration of Platelet Rich Plasma (PRP) on the cellular content of synovial fluid (SF) of osteoarthritic patients. Thirty-one patients received a single infiltration of PRP either in the IA space (n = 14) or in the IA space together with two IO infiltrations, one in the medial femoral condyle and one in the tibial plateau (n = 17). SF was collected before and after one week of the infiltration. The presence in the SF of mesenchymal stem cells (MSCs), monocytes, and lymphocytes was determined and quantified by flow cytometry. The number and identity of the MSCs were further confirmed by colony-forming and differentiation assays. PRP infiltration into the subchondral bone (SB) and the IA space induced a reduction in the population of MSCs in the SF. This reduction in MSCs was further confirmed by colony-forming (CFU-F) assay. On the contrary, IA infiltration alone did not cause variations in any of the cellular populations by flow cytometry or CFU-F assay. The SF of osteoarthritic patients contains a population of MSCs that can be modulated by PRP infiltration of the SB compartment.

Human Amniotic Membrane: A Potential Tissue and Cell Source for Cell Therapy and Regenerative Medicine

The human amniotic membrane (HAM) is the innermost membrane surrounding the fetus. HAM is a highly abundant and readily available tissue that is becoming appreciated as an alternative to adult bone marrow mesenchymal stem cells (BM-MSCs) useful for cell therapy and regenerative medicine. This tissue provides high efficiency in noninvasive and safe MSC recovery with no intrusive procedures. HAM contains two cell types from different embryological origins: human amnion epithelial cells (hAECs), derived from the embryonic ectoderm, and human amnion mesenchymal stromal cells (hAMSCs), derived from the embryonic mesoderm. hAMSCs and hAECs are immune-privileged cells that can be isolated without the sacrifice of human embryos, avoiding immunological rejection problems and the ethical conflict of using human embryonic stem cells (hESCs). Regarding their immunophenotype, both cell types demonstrate the expression of the common well-defined human mesenchymal and embryonic stem cell markers and the absence of hematopoietic markers. Moreover, both cell populations have similar multipotential for in vitro differentiation into all three germ layers: ectoderm, mesoderm, and endoderm lineages. Indeed, the potential application of amnion-derived cells in a variety of diseases, in particular those associated with degenerative processes, is under clinical or preclinical investigation. The HAM has other biological properties important for tissue engineering, including anti-fibrosis, anti-inflammatory, anti-scarring, antimicrobial, as well as adequate mechanical properties and low immunogenicity. Therefore, amnion allografts are widely applied in ophthalmology, plastic surgery, dermatology, and gynecology. In this chapter, the localization, isolation, characterization, and differentiation potential of amnion-derived cells are discussed. Moreover, the potential clinical applications of either amnion-derived cells or the whole HAM are also reviewed.