Large-scale secretome analyses unveil the superior immunosuppressive phenotype of umbilical cord stromal cells as compared to other adult mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs), given their regenerative potential, are being investigated as a potential therapeutic tool for cartilage lesions. MSCs express several bioactive molecules which act in a paracrine fashion to modulate the tissue microenvironment. Yet, little is known about the divergence of these signalling molecules in different MSC populations. The present study investigated secretomes of stromal cells harvested from Hoffa's fat pad (HFPSCs), synovial membrane (SMSCs), umbilical cord (UCSCs) and cartilage (ACs) by quantitative liquid chromatography-mass spectrometry (LC-MS/MS) proteomics. Also, multiplex protein arrays and functional assays were performed to compare the constitutive immunomodulatory capabilities of different MSCs. Proteins involved in extracellular matrix degradation and inflammation, such as matrix metalloproteinases (MMPs), interleukin (IL)-17 and complement factors, were downregulated in UCSCs as compared to adult cell sources. Additionally, secretion of transforming growth factor (TGF)-ß1 and prostaglandin E2 (PGE2) was enhanced in UCSC supernatants. UCSCs were superior in inhibiting peripheral blood mononuclear cell (PBMC) proliferation, migration and cytokine secretion as compared to adult stromal cells. SMSCs significantly suppressed the proliferation of PBMCs only if they were primed with pro-inflammatory cytokines. Although all cell types repressed human leukocyte antigen-DR isotype (HLA-DR) surface expression and cytokine release by activated macrophages, only UCSCs significantly blocked IL-6 and IL-12 production. Furthermore, UCSCs supernatants increased aggrecan gene expression in two-dimensional chondrocyte cultures. The data demonstrated that UCSCs displayed superior anti-inflammatory and immunosuppressive properties than stromal cells from adult tissues. This allogeneic cell source could potentially be considered as an adjuvant therapy for articular cartilage repair.

Non-linear optical microscopy as a novel quantitative and label-free imaging modality to improve the assessment of tissue-engineered cartilage.

OBJECTIVE: Current systems to evaluate outcomes from tissue-engineered cartilage (TEC) are sub-optimal. The main purpose of our study was to demonstrate the use of second harmonic generation (SHG) microscopy as a novel quantitative approach to assess collagen deposition in laboratory made cartilage constructs. METHODS: Scaffold-free cartilage constructs were obtained by condensation of in vitro expanded Hoffa's fat pad derived stromal cells (HFPSCs), incubated in the presence or absence of chondrogenic growth factors (GF) during a period of 21 d. Cartilage-like features in constructs were assessed by Alcian blue staining, transmission electron microscopy (TEM), SHG and two-photon excited fluorescence microscopy. A new scoring system, using second harmonic generation microscopy (SHGM) index for collagen density and distribution, was adapted to the existing "Bern score" in order to evaluate in vitro TEC. RESULTS: Spheroids with GF gave a relative high Bern score value due to appropriate cell morphology, cell density, tissue-like features and proteoglycan content, whereas spheroids without GF did not. However, both TEM and SHGM revealed striking differences between the collagen framework in the spheroids and native cartilage. Spheroids required a four-fold increase in laser power to visualize the collagen matrix by SHGM compared to native cartilage. Additionally, collagen distribution, determined as the area of tissue generating SHG signal, was higher in spheroids with GF than without GF, but lower than in native cartilage. CONCLUSION: SHG represents a reliable quantitative approach to assess collagen deposition in laboratory engineered cartilage, and may be applied to improve currently established scoring systems.

Mesenchymal stromal cells from human umbilical cords display poor chondrogenic potential in scaffold-free three dimensional cultures.

Many researchers world over are currently investigating the suitability of stromal cells harvested from foetal tissues for allogeneic cell transplantation therapies or for tissue engineering purposes. In this study, we have investigated the chondrogenic potential of mesenchymal stromal cells (MSCs) isolated from whole sections of human umbilical cord or mixed cord (UCSCs-MC), and compared them with cells isolated from synovial membrane (SMSCs), Hoffa's fat pad (HFPSCs) and cartilage. All MSCs were positive for surface markers including CD73, CD90, CD105, CD44, CD146 and CD166, but negative for CD11b, CD19, CD34, CD45 and HLA-DR in addition to CD106 and CD271. Chondrogenic potential of all cell sources was studied using 3D pellet cultures incubated in the presence of different combinations of anabolic substances such as dexamethasone, IGF-1, TGF-ß1, TGF-ß3, BMP-2 and BMP-7. BMP-2 and dexamethasone in combination with TGF-ß1 or TGF-ß3 excelled at inducing chondrogenesis on SMSCs, HFPSCs and chondrocytes, as measured by glycosaminoglycans and collagen type II staining of pellets, quantitative glycosaminoglycan expression, quantitative PCR of cartilage signature genes and electron microscopy. In contrast, none of the tested growth factor combinations was sufficient to induce chondrogenesis on UCSCs-MC. Moreover, incubation of UCSCs-MC spheroids in the presence of cartilage pieces or synovial cells in co-cultures did not aid chondrogenic induction. In summary, we show that in comparison with MSCs harvested from adult joint tissues, UCSCs-MC display poor chondrogenic abilities. This observation should alert researchers at the time of considering UCSCs-MC as cartilage forming cells in tissue engineering or repair strategies.

Dynamics of circulating tumor cells in early breast cancer under neoadjuvant therapy.

At present, the majority of patients with breast cancer are diagnosed at early stages of disease development. However, a considerable number of such cases develop secondary malignancies after a relatively short period of time. The presence of circulating tumor cells (CTCs) has been proposed as a strong biomarker to predict disease recurrence in metastatic breast cancer. However, the prognostic significance is not clear in early breast cancer. We present results on CTC determination in peripheral blood in non-metastatic breast cancer patients in the context of neoadjuvant treatment. Twenty-six breast cancer patients, scheduled for neoadjuvant therapy, were enrolled in a prospective study, of which 24 were able to complete therapy. CTC assessment was performed by sorting out cytokeratin-positive cells from 10 ml of peripheral blood using immunomagnetic separation, followed by immunocytochemical characterization of cells. Seventeen blood samples out of 24 patients were CTC-positive when collected prior to neoadjuvant chemotherapy. No significant correlations were found between the presence of CTCs and lymph node status (p=0.1), histological type (p=0.802), stage (p=0.43) or overall survival (OS) (p=0.599). Thirteen CTC-positive samples were observed in blood samples collected after treatment. Univariate analyses revealed that the presence of CTCs was related to OS when the detection was positive both before and after treatment (p=0.023). CTCs can be a strong prognostic marker in early breast cancer. The persistence of CTCs before and after treatment can identify a subpopulation of patients with an increased risk of recurrence.

Clinical relevance associated to the analysis of circulating tumour cells in patients with solid tumours

The distant growth of tumour cells escaping from primary tumours, a process termed metastasis, represents the leading cause of death among patients affected by malignant neoplasias from breast and colon. During the metastasis process, cancer cells liberated from primary tumour tissue, also termed circulating tumour cells (CTCs), travel through the circulatory and/or lymphatic systems to reach distant organs. The early detection and the genotypic and phenotypic characterisation of such CTCs could represent a powerful diagnostic tool of the disease, and could also be considered an important predictive and prognostic marker of disease progression and treatment response. In this article we discuss the potential relevance in the clinic of monitoring CTCs from patients suffering from solid epithelial tumours, with emphasis on the impact of such analyses as a predictive marker for treatment response (AU)

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