Expression of inflammatory cytokines in mesenchymal stromal cells is sensitive to culture conditions and simple cell manipulations.

BACKGROUND: Mesenchymal stromal cells (MSCs) can be used in several clinical applications. While MSCs are frequently cultured in fetal bovine serum for in vitro experimentation, human serum supplements are required for cells to be used in patients. Here we show how different human serum supplements and in vitro manipulations used during the cell culture impact on MSC proliferation rate and expression of inflammatory molecules. METHODS: MSCs were cultured in medium supplemented with human plasma or serum combined with human platelet lysate (PL) and/or basic fibroblast growth factor (FGF2). Real time RT-PCR and western blot were used to assess expression of inflammatory cytokines. RESULTS: Serum with addition of FGF2 gave the fastest proliferation rate. However, serum with FGF2 also increased expression of genes encoding inflammatory cytokines. The most favorable expansion condition for chondrogenic differentiation and inhibition of cartilage matrix degrading enzymes was plasma supplemented with PL and FGF2. Detachment of cells using trypsin gave considerable upregulation of inflammatory cytokine mRNAs which lasted for up to 24 h, with concomitant increase in protein levels. Even the gentle act of changing medium led to upregulation of cytokine mRNA, caused by addition of fresh serum. DISCUSSION: Different culture conditions and simple cell manipulation influence proliferation rate and expression of inflammatory genes. Supplementing culture medium with allogeneic AB serum and FGF2 during monolayer expansion supported cell expansion better than other supplements, but also induced the highest levels of inflammatory cytokines and gave inferior results for chondrogenic differentiation. The importance of the composition of the culture medium and even gentle in vitro manipulation of the cells should be taken into account in the planning of procedures using in vitro expanded MSCs.

Liposome delivery of microRNA-145 to mesenchymal stem cells leads to immunological off-target effects mediated by RIG-I.

Synthetic microRNAs regulate gene expression when transfected into cells, and may be used in strategies for molecular therapy both in vitro and in vivo. Liposomal transfection reagents are frequently used as delivery vehicles in both settings. Here, we report on the immunological off-target effects observed following liposome transfection of synthetic microRNA-145 into human mesenchymal stem cells and human articular chondrocytes (hAC). The immune response was independent on endosome delivery and toll-like receptors (TLRs) but was mediated by retinoic acid inducible-gene 1 (RIG-I). Upregulation of immune genes required liposomal delivery, as no immune response was observed after electroporation of smiR-145 directly in to the cytosol, suggesting a new role of RIG-I. Immune response was seen both with blunt ended and 2-nucleotide 3' overhang versions of synthetic miR-145, and occurred in the absence of a 5'ppp cap. Mutations in a centrally placed poly (UUUU) sequence reduced, but did not abolish the immune response. Interestingly, exposure to liposomes alone led to upregulation of several immune genes, including RIG-I mRNA. However, this process was not mediated by RIG-I. This insight is important for researchers to avoid unexpected results from gene transfer experiments in vitro and unwanted immune responses following the use of lipid-based transfection reagents in vivo.

Comparison between articular chondrocytes and mesenchymal stromal cells for the production of articular cartilage implants.

Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs. Articular chondrocytes produced more extracellular matrix per seeded cell than mesenchymal stromal cells. Quantitative proteomics analysis showed that articular chondrocyte discs contained more articular cartilage proteins, while mesenchymal stromal cell discs had more proteins associated with cartilage hypertrophy and bone formation. Sequencing analysis revealed more microRNAs associated with normal cartilage in articular chondrocyte discs, and large-scale target predictions, performed for the first time for in vitro chondrogenesis, suggested that differential expression of microRNAs in the two disc types were important mechanisms behind differential synthesis of proteins. We conclude that articular chondrocytes should be preferred over mesenchymal stromal cells for tissue engineering of articular cartilage.

Effect of three-dimensional culture and incubator gas concentration on phenotype and differentiation capability of human mesenchymal stem cells.

To obtain sufficient numbers of cells for tissue engineering applications, human bone marrow-derived mesenchymal stem cells (hBM-MSC) are commonly cultured as monolayers in incubators containing room air. In this study, we investigated whether three-dimensional (3D) culture conditions and incubator gas concentrations more similar to those observed in vivo impacted on cell expansion, differentiation capability, or phenotype of hBM-MSC. We found that 3D culture alone increased the expression of some molecules involved in osteogenic and adipogenic differentiation. In contrast, 3D culture did not induce chondrogenic differentiation, but enhanced the response to the chondrogenic differentiation medium. Changing the oxygen concentration to 6% and the carbon dioxide concentration to 7.5% did not impact on the results of any of our assays, showing that the hyperoxia of room air is not detrimental to hBM-MSC proliferation, differentiation, or phenotype.

Single-Cell RNA Sequencing of In Vitro Expanded Chondrocytes: MSC-Like Cells With No Evidence of Distinct Subsets.

OBJECTIVE: To investigate the heterogeneity of in vitro expanded chondrocytes used for autologous chondrocyte implantation. METHODS: Human articular chondrocytes were expanded in vitro for 14 days, sorted into 86 single cells using fluorescence-activated cell sorting and subjected to single-cell RNA sequencing. Principal component, Cross R2 hierarchical clustering, and differential gene expression analyses were used for data evaluation. Flow cytometry and single-cell RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) was used to validate the results of the RNA sequencing data Polyclonal chondrocyte populations from the same donor were differentiated in vitro toward the osteogenic and adipogenic lineages. RESULTS: There was considerable variation in gene expression between individual cells, but we found no evidence for separate cell subpopulations based on principal component, hierarchical clustering, and differential gene expression analysis. Most of the cells expressed all the markers defining mesenchymal stem cells, and as polyclonal chondrocyte populations from the same donor were shown to differentiate into osteocytes and adipocytes in vitro, these cells formally qualify as mesenchymal stem cells. CONCLUSIONS: In vitro expanded chondrocytes consist of one single population of cells with heterogeneity in gene expression between the cells. Dedifferentiated chondrocytes qualify as mesenchymal stem cells as they fulfill all the criteria suggested by the International Society for Cellular Therapy.

Extensive downregulation of immune gene expression by microRNA-140-3p 5' isomiR in an in vitro model of osteoarthritis.

Objective: MicroRNA-140-3p is the most prevalent form of canonical miR-140 in native chondrocytes. IsomiRs are sequence variants of microRNAs with potentially distinct functionalities. Here we present functional studies of canonical microRNA-140-3p and two of its most prevalent isomiRs, a 5' isomiR and a 3' isomiR, in an inflammation-induced model of osteoarthritis (OA). Method: Canonical miR-140-3p, the 5' isomiR and the 3' isomiR were overexpressed separately in chondrocytes from three donors and subsequently subjected to an inflammatory milieu mediated by interleukin 1 beta and tumor necrosis factor alpha. RNA sequencing was performed on the cells to investigate the altered transcriptomes, RT-qPCR was performed to validate important observations, and western blot analysis was carried out to further study key inflammatory molecules. Results: The three microRNAs downregulated many of the same genes. However, the 5' isomiR showed a much greater target spectrum compared to the other two miRNAs, and downregulated cascades of genes downstream of interferon beta, interferon gamma and interleukin 1 beta as well as genes involved in several other inflammatory and antiviral pathways. In addition the 5' isomiR downregulated practically all HLA class II and class I genes. Conclusion: Introduction of the 5' isomiR led to downregulation of genes essential for some of the most important inflammation cascades and virtual silencing of genes responsible for antigen presentation. These observations may indicate a very promising therapeutic potential for the 5' isomiR for OA and several inflammatory conditions, particularly HLA associated immune conditions including many arthritic diseases.

MicroRNAs mediated regulation of glutathione peroxidase 7 expression and its changes during adipogenesis.

Glutathione peroxidase 7 (GPx7) acts as an intracellular stress sensor/transmitter and plays an important role in adipocyte differentiation and the prevention of obesity related pathologies. For this reason, finding the regulatory mechanisms that control GPx7 expression is of great importance. As microRNAs (miRNAs) could participate in the regulation of GPx7 expression, we studied the inhibition of GPx7 expression by four selected miRNAs with relation to obesity and adipogenesis. The effect of the transfection of selected miRNAs mimics on GPx7 expression was tested in three cell models (HEK293, SW480, AT-MSC). The interaction of selected miRNAs with the 3'UTR of GPx7 was followed up on using a luciferase gene reporter assay. In addition, the levels of GPx7 and selected miRNAs in adipose tissue mesenchymal stem cells (AT-MSC) and mature adipocytes from four human donors were compared, with the changes in these levels during adipogenesis analyzed. Our results show for the first time that miR-137 and miR-29b bind to the 3'UTR region of GPx7 and inhibit the expression of this enzyme at the mRNA and protein level in all the human cells tested. However, no negative correlation between miR-137 nor miR-29b level and GPx7 was observed during adipogenesis. Despite the confirmed inhibition of GPx7 expression by miR-137 and miR-29b, the action of these two molecules in adipogenesis and mature adipocytes must be accompanied by other regulators.

Scaffold-Free Engineering of Human Cartilage Implants.

OBJECTIVE: Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs. DESIGN: Articular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young's modulus. RESULTS: Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young's modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface. CONCLUSION: Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.

Robust profiling of microRNAs and isomiRs in human plasma exosomes across 46 individuals.

microRNAs (miRNAs) are small double stranded RNA molecules consisting of two complementary strands called the 5p and 3p arms. Following imprecise processing and/or addition of nucleotides at the ends, miRNA biogenesis can give rise to variants called isomiRs. Exosomes are small vesicles released by cells. They have attracted attention due to their potential use in biomarker development because of their content of biomolecules, including miRNAs and isomiRs. Exosomes are found in body fluids such as plasma. In this study we used next generation sequencing to investigate the distribution of 5p and 3p arms of both miRNAs and isomiRs in plasma exosomes from 46 individuals. Among the canonical miRNAs there was similar prevalence between 5p and 3p miRNAs. Most of the miRNAs had isomiRs, and in approximately half of the cases an isomiR was more abundant than the corresponding canonical miRNA. Most of the isomiRs were generated from 5p miRNAs. There were very small differences in the concentration of canonical miRNA and isomiR sequences between donors, suggesting tight control of isomiR generation and sorting into exosomes. IsomiRs are abundant in plasma exosomes and should be included in analysis when plasma exosomal miRNAs are investigated as potential biomarkers for disease development.

Multi-pathway Protective Effects of MicroRNAs on Human Chondrocytes in an In Vitro Model of Osteoarthritis.

Osteoarthritis (OA) is the most common degenerative joint disease. One of the main pathogenic factors of OA is thought to be inflammation. Other factors associated with OA are dysregulation of microRNAs, reduced autophagic activity, oxidative stress, and altered metabolism. microRNAs are small non-coding RNAs that are powerful regulators of gene expression. miR-140-5p is considered a cartilage-specific microRNA, is necessary for in vitro chondrogenesis, has anti-inflammatory properties, and is downregulated in osteoarthritic cartilage. Its passenger strand, miR-140-3p, is the most highly expressed microRNA in healthy cartilage and increases during in vitro chondrogenesis. miR-146a is a well-known anti-inflammatory microRNA. Several studies have illustrated its role in OA and autoimmune diseases. We show that, when human chondrocytes were transfected individually with miR-140-5p, miR-140-3p, or miR-146a prior to stimulation with interleukin-1 beta and tumor factor necrosis-alpha as an inflammatory model of OA, each of these microRNAs exhibited similar protective effects. Mass spectrometry analysis provided an insight to the altered proteome. All three microRNAs downregulated important inflammatory mediators. In addition, they affected different proteins belonging to the same biological processes, suggesting an overall inhibition of inflammation and oxidative stress, enhancement of autophagy, and restoration of other homeostatic cellular mechanisms, including metabolism.

Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.

Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5 ng/ml FGF-2 [ACK(AB + FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5 ng/ml FGF2 [Lympho(AB + FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGFlow) or 10% AB +10 ng/ml FGF2 [Direct(AB + FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1α and IL-1ß expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

microRNA-140 Inhibits Inflammation and Stimulates Chondrogenesis in a Model of Interleukin 1ß-induced Osteoarthritis.

Osteoarthritis is a serious disease of articular cartilage. The pathogenic factors contributing to this disorder are inflammation, extracellular matrix degradation and failure to rebuild the articular cartilage. Preclinical studies suggest that microRNA-140 may play a protective role in osteoarthritis development, but little is known about the mechanism by which this occurs. Here we present the results of forced expression of microRNA-140 in an in vitro model of osteoarthritis, evaluated by global proteomics analysis. We show that inflammation was reduced through the altered levels of multiple proteins involved in the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 pathway. microRNA-140 upregulated many of the components involved in the synthesis of hyaline extracellular matrix and reduced the levels of aggrecanases and syndecan 4, thus potentially both increasing cartilage repair and reducing cartilage breakdown. These results show how forced expression of microRNA-140 is likely to counteract all three pathogenic processes, and support the idea that intra-articular injection of microRNA-140 may benefit patients suffering from early osteoarthritis.

microRNA-140 targets RALA and regulates chondrogenic differentiation of human mesenchymal stem cells by translational enhancement of SOX9 and ACAN.

Lesions of articular cartilage do not heal spontaneously. One treatment strategy would be to make cartilage in the laboratory by directed chondrogenic differentiation of mesenchymal stem cells (MSCs). To promote our understanding of the molecular control of chondrogenesis, we have compared the changes in microRNAs (miRNAs) during in vitro chondrogenesis of MSCs with those observed in uncultured and dedifferentiated articular chondrocytes (ACs). Several miRNAs showed a reciprocal relationship during the differentiation of MSCs and dedifferentiation of ACs. miR-140-5p and miR-140-3p changed the most during in vitro chondrogenesis, they were the miRNAs most highly expressed in tissue-engineered chondrocytes, and they were also among the miRNAs most highly expressed in uncultured ACs. There was a 57% overlap for the 100 most highly expressed miRNAs in differentiated MSCs and uncultured ACs, but for other miRNAs, the expression pattern was quite different. We transiently and stably inhibited and overexpressed miR-140-5p and miR-140-3p in differentiating MSCs and dedifferentiating ACs, respectively, to describe global effects and identify and validate new targets. Surprisingly, SOX9 and aggrecan proteins were found to be downregulated in anti-miR-140 transduced differentiating MSCs despite unchanged mRNA levels. This suggests that miR-140 stimulates in vitro chondrogenesis by the upregulation of these molecules at the protein level. RALA, a small GTPase, was identified as a miR-140 target and knockdown experiments showed that RALA regulated SOX9 at the protein level. These observations shed new light on the effect of miR-140 for chondrogenesis in vitro and in vivo.

Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage.

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage.

Human primary articular chondrocytes, chondroblasts-like cells, and dedifferentiated chondrocytes: differences in gene, microRNA, and protein expression and phenotype.

In this study we have isolated human primary uncultured articular chondrocytes. When these cells are allowed to proliferate within their own extracellular matrix (ECM), they begin to produce hyaline ECM molecules similar to embryological chondroblasts. These cells are called chondroblast-like cells. Upon continued culture these cells spread onto the plastic surface and dedifferentiate. We have characterized these three stages of chondral cells by gene expression and expression of microRNAs (miRNAs) and proteins. Gene expression was quantified by real-time reverse transcriptase (RT) polymerase chain reaction, miRNA expression by miRNA arrays, and protein synthesis by extra- and intracellular flow cytometry. Many of the genes, miRNAs, and proteins were differentially expressed in the different stages of chondral cells. In the context of cellular therapy, expression of some genes is a cause for concern. The best source of cells for treatment of lesions of hyaline cartilage has not yet been identified. Adult chondroblast-like cells may be strong candidates. Profound understanding of how expression of genes and synthesis of proteins are regulated in these cells, for instance, by miRNAs, may reveal new strategies for improving their synthesis of hyaline ECM. This insight is important to be able to use these cells in the clinic.