Laurence Lagneaux

Mesenchymal Stem Cells Derived from CD133‐Positive Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression, and Plasticity

In this study, we used a common procedure to assess the potential of mobilized peripheral blood (MPB) and umbilical cord blood (UCB) as sources of mesenchymal stem cells (MSCs) in comparison with bone marrow (BM). We tested three methods: plastic adhesion supplemented with 5% of BM‐MSC conditioned medium, unsupplemented plastic adhesion, and selection of CD133‐positive cells. MSCs derived from MPB or UCB are identified by their positive expression of mesenchymal (SH2, SH3) and negative expression of hematopoietic markers (CD14, CD34, CD45, HLA‐DR). We observed that the CD133‐positive cell fraction contains more MSCs with high proliferative potential. Placed in appropriate conditions, these cells proved their capacity to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal/glial cells. MPB‐ and UCB‐MSCs express Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity. The selection of CD133‐positive cells enabled us to obtain a homogeneous population of MSCs from UCB and MPB. These sources may have a major clinical importance thanks to their easy accessibility.

Bisphosphonates induce breast cancer cell death in vitro

Breast cancer frequently spreads to bone and is almost always associated with osteolysis. This tumor‐induced osteolysis is caused by increased osteoclastic bone resorption. Bisphosphonates are used successfully to inhibit bone resorption in tumor bone disease and may prevent development of new osteolytic lesions. The classical view is that bisphosphonates only act on bone cells. We investigated their effects on breast cancer cells using three human cell lines, namely, MCF‐7, T47D, and MDA.MB.231, and we tested four structurally different bisphosphonates: clodronate, pamidronate, ibandronate, and zoledronate. We performed time course studies for each bisphosphonate at various concentrations and found that all four compounds induced a nonreversible growth inhibition in both MCF‐7 and T47D cell lines in a time‐ and dose‐dependent manner. The MDA.MB.231 cell line was less responsive. Bisphosphonates induced apoptosis in MCF‐7 and cell necrosis in T47D cells. The inhibition of MCF‐7 cell proliferation could be reverted almost completely by the benzyloxycarbonyl‐Val‐Ala‐Asp(OMe)‐fluoromethyl ketone (z‐VAD‐fmk) inhibitor of caspases, suggesting that the apoptotic process observed in the MCF‐7 cell line is mediated, at least partly, by the caspase system. Caspase activity was little changed by bisphosphonates in T47D cells and the inhibitor of caspase did not modify bisphosphonates effects. In summary, we found that bisphosphonates inhibit breast cancer cell growth by inducing cell death in vitro. Such effects could contribute to the beneficial role of bisphosphonates in the treatment and the prevention of tumor‐induced osteolysis.

microRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic leukemia and improves disease risk stratification.

Aberrant expression of microRNAs has been recently associated with chronic lymphocytic leukemia (CLL) outcome. Although disease evolution can be predicted by several prognostic factors, a better outcome individualization in a given patient is still of utmost interest. Here, we showed that miR-29c and miR-223 expression levels decreased significantly with progression from Binet stage A to C were significantly lower in poor prognostic subgroups (defined by several prognostic factors) and could significantly predict treatment-free survival (TFS) and overall survival (OS). Furthermore, we developed a quantitative real-time polymerase chain reaction (qPCR) score combining miR-29c, miR-223, ZAP70, and LPL (from 0 to 4 poor prognostic markers) to stratify treatment and death risk in a cohort of 110 patients with a median follow-up of 72 months (range, 2-312). Patients with a score of 0/4, 1/4, 2/4, 3/4, and 4/4 had a median TFS of greater than 312, of 129, 80, 36, and 19 months, respectively (hazard ratio, HR(0/4 < 1/4 < 2/4 < 3/4 < 4/4) = 17.00, P < .001). Patients with a score of 0-1/4, 2-3/4, and 4/4 had a median OS of greater than 312, of 183 and 106 months, respectively (HR(0/4 < 1/4 < 2/4 < 3/4 < 4/4) = 13.69, P = .001). This score will help to identify, among the good and poor prognosis subgroups, patients who will need early therapy and thus will require a closer follow-up.

Mesenchymal stromal cells use PGE2 to modulate activation and proliferation of lymphocyte subsets: Combined comparison of adipose tissue, Wharton’s Jelly and bone marrow sources

Due to their immunomodulatory properties, adipose tissue (AT) and Whartons Jelly (WJ) constitute valuable alternatives to BM as sources of MSCs for managing graft-versus-host disease. To ensure the efficiency of AT- and WJ-MSCs implies the characterization of their immunomodulatory functions in comparison to those of BM. In this study, we investigated the capacity of AT- and WJ-MSCs to modulate lymphocyte reactions in response to different stimuli as well as the specificity of this immunomodulation. AT- and WJ-MSC displayed potent immunosuppressive effects on lymphocyte responses in a dose-dependent manner. These effects included the prevention of lymphocyte activation as well as the suppression of T-cell proliferation regardless of the stimuli used to activate lymphocytes. These effects were mediated through the expression of COX1/COX2 enzymes and by the production of PGE2. CD4(+) and CD8(+) T-lymphocytes were equally targeted by MSCs demonstrating that the immunomodulation was not restricted to a specific T-cell subpopulation.

Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6

BACKGROUND AIMS Mesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures. METHODS The effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis. RESULTS MSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses. CONCLUSIONS MSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.

Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential

BACKGROUND BM mesenchymal stem cells (MSC) have the capacity for renewal and the potential to differentiate into multiple tissues. In this study, we compared different enrichment methods to obtain MSC from BM. METHODS Three different methods were compared with a view to obtaining MSC more rapidly from BM: negative selection (RosetteSep and MACS) and plastic adhesion. The three cell fractions were grown in complete alpha-minimum essential medium in order to evaluate their proliferative capacity, their phenotype during culture and their potential to differentiate into adipocytes, osteocytes and chondrocytes. Identification of MSC was performed by immunofluorescence with putative mesenchymal markers SH2 and SH3 but also with hematopoietic markers. RESULTS After negative selection, only 1+/-0.2% and 2.9+/-0.8% of cells were recovered from BM with the RosetteSep and MACS methods, respectively. However, negative depletion permitted a homogeneous population of MSC, with more than 90% SH2+ and SH3+ cells, to be obtained rapidly and in large quantities after 10 days of culture. Similar homogeneity was observed after three passages if the plastic adhesion was used as selection method and after an average of 25-30 days of culture. Different levels of MSC maturity were also suggested by the variable level expression of Stro-1. DISCUSSION Depleting selection by RosetteSep may represent an easy method of obtaining MSC rapidly from BM with the aim of potential therapeutic use.

Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-MEM medium.

Abstract:  The expansion of mesenchymal stem cells (MSCs) strongly depends on the culture conditions and requires medium supplemented with 10–20% fetal calf serum (FCS) to generate relevant numbers of cells. However, the presence of FCS is a major obstacle for their clinical use. Therefore, we have evaluated the capacity of expansion of MSC in a commercial serum‐free medium (UC) supplemented with a serum substitute (ULTROSER®) in comparison with a classical medium α‐MEM containing 15% FBS. Bone marrow‐mononuclear cells collected from 12 volunteer healthy donors were expanded in two different culture media. MSCs isolated in the both media were morphologically similar and expressed identical phenotypic markers. After the primoculture (P0) and one passage, we obtained significantly more MSC and CFU‐F progenitors in UC medium than in αMEM. Their multipotentiality was preserved during culture, as well as their capacity to support haematopoiesis. In conclusion, our observations strongly suggest that UC is an optimal medium for ex vivo expansion of MSC: it allows a better cell expansion, preserves cell multipotentiality, reduces the culture period and contains low concentration of serum substitute. This medium seems suitable for clinical scale expansion of MSC.

Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are involved in tissue repair and in the regulation of immune responses. MSC express Toll-like receptors (TLR) known to link innate and adaptive immunity. We hypothesized that TLR signaling could influence human MSC (hMSC) function. Here, we show that hMSC express TLR1, TLR2, TLR3, TLR4, TLR5, and TLR6 but not TLR7, TLR8, TLR9, and TLR10. In inflammatory conditions mimicked by culturing hMSC in an inflammatory environment, TLR2, TLR3, and TLR4 are upregulated, whereas TLR6 is downregulated. Interleukin (IL)-1 beta, IL-6, IL-12p35 and transforming growth factor-beta mRNAs are constitutively expressed by hMSC. Inflammation leads to an increase in IL-1 beta, IL-6, IL-12p35, and transforming growth factor-beta transcription and is characterized by IL-23p19 and IL-27p28 transcription. In this setting, poly(I:C) further augments IL-6, IL-12p35, IL-23p19, and IL-27p28 transcription, whereas lipopolysaccharide (LPS) increases IL-23p19 and IL-27p28 transcription. By upregulating TLR3 and TLR4 transcription, inflammation increases the hMSC responsiveness to LPS and poly(I:C), leading to a proinflammatory shift in their cytokine profile. The hMSC osteogenic potential does not change after TLR triggering but stimulation with LPS and poly(I:C) results in a decrease in their immunosuppressive capabilities. In conclusion, TLR activation in hMSC may affect their function and could modify their in vivo fate, especially in an inflammatory context.

Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

BackgroundNeuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC) could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes.MethodsBM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin). By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3.ResultsUsing microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin). Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin). Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3), NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate receptor channel (AMPA3) in the presence of two agonist glutamate, AMPA or CNQX antagonist.ConclusionOur results demonstrate that BM-MSC have the potential to differentiate in neuronal cells with specific gene expression and functional properties. BM-MSC are thus promising candidates for cell-based therapy of neurodegenerative diseases

The source of human mesenchymal stromal cells influences their TLR profile as well as their functional properties

Mesenchymal stromal cells (MSC) can be expanded from different sources. We compared the influence of inflammation and TLR ligation on the phenotype and function of MSC derived from bone marrow (BM), adipose tissue (AT), and Whartons jelly (WJ). WJ-MSC were featured by a lack of TLR4 expression. While inflammation upregulated TLR3 in all three MSC types, TLR4 upregulation was observed only on BM-MSC. TLR ligation increased the production of inflammatory cytokines in BM- and AT-MSC but not in WJ-MSC and augmented anti-inflammatory cytokines in AT-MSC. Although inflammation increased in all MSC types the secretion of inflammatory cytokines, additional TLR triggering did not have further effect on WJ-MSC. The immunosuppressive potential of WJ-MSC on MLR was affected neither by inflammation nor by TLR triggering. This resistance was related to an overproduction of HGF. These data indicate that MSC source could be of importance while designing immunomodulating cell therapy in transplantation.