Alain Delforge

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.

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

BACKGROUNDnBM 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.nnnMETHODSnThree 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.nnnRESULTSnAfter 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.nnnDISCUSSIONnDepleting 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:u2002 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.

Ultrasonic low-energy treatment: A novel approach to induce apoptosis in human leukemic cells

OBJECTIVEnWe evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity.nnnMATERIALS AND METHODSnHuman leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose.nnnRESULTSnThe technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism.nnnCONCLUSIONnThe cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.

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

In vitro study of matrix metalloproteinase/tissue inhibitor of metalloproteinase production by mesenchymal stromal cells in response to inflammatory cytokines: the role of their migration in injured tissues

BACKGROUND AIMSnThe transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production.nnnMETHODSnThe migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel in the presence and absence of stromal cell-derived (SDF)-1alpha, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels.nnnRESULTSnWe have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli.nnnCONCLUSIONSnOur observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.

Heterogenous response of B lymphocytes to transforming growth factor-beta in B-cell chronic lymphocytic leukaemia: correlation with the expression of TGF-beta receptors

We investigated the potential role of transforming growth factor‐beta (TGF‐β) on spontaneous and cytokine‐induced proliferation of B‐cell chronic lymphocytic leukaemia (B‐CLL) cells in vitro. Purified B lymphocytes from 21 B‐CLL patients were cultured for 5u2003d in the presence of medium alone, IL‐2 and/or IL‐10, in the presence or absence of TGF‐β, and proliferation was measured by 3H‐thymidine incorporation. TGF‐β inhibited B‐cell proliferation in the majority of patients (15/21) but no inhibition was detected in 6/21 patients whatever the type of stimulant used. Addition of neutralizing antibodies to TGF‐β increased spontaneous and cytokine‐induced proliferation; this effect was dose dependent and specific because addition of an irrelevant chicken IgG had no effect on B‐CLL proliferation. In resistant patients, neutralizing antibodies to TGF‐β did not increase the proliferation. The expression of TGF‐β receptors on B‐CLL cells was significantly lower than the one observed on normal CD5+ B lymphocytes for which the sensitivity to TGF‐β inhibition was more marked than in CLL. In addition, we found a strong correlation between the response of leukaemic B cells to TGF‐β inhibitory action and the expression of TGF‐β receptors on these cells. In summary, TGF‐β appears to function in CLL as a negative regulator of B lymphocytes but loss of responsiveness to this factor accompanied by a decrease of TGF‐β receptor expression, might provide a selective advantage to B‐CLL lymphocytes.

Early induction of apoptosis in B-chronic lymphocytic leukaemia cells by hydroxychloroquine: activation of caspase-3 and no protection by survival factors

We have investigated the effect of hydroxychloroquine (HCQ), an anti‐rheumatic drug, on malignant B cells from 20 patients with B‐chronic lymphocytic leukaemia (B‐CLL). HCQ induced a decrease in cell viability in a dose‐ and time‐dependent manner. The mean IC50 was 32u2003±u20037 μg/ml (range, 10‐75 μg/ml) for 24 h of exposure. This cytotoxic effect was owing to apoptosis, as demonstrated by morphological changes, annexin V binding capacity and DNA fragmentation (28u2003±u20034% of apoptotic cells as early as 5 h post incubation, increasing to 82u2003±u20034% at 18 h post treatment). The apoptosis was associated with caspase‐3 activation because the cleavage and activity of caspase‐3 were increased by HCQ. The amount of bcl‐2 protein was reduced during apoptosis, evidenced using quantitative flow cytometry. As early as 1 h post‐HCQ treatment, a reduction of the mitochondrial transmembrane potential was measured by 3,3’‐dihexyloxacarbocyanine iodide. Interestingly, the HCQ effect was not affected by exposure to interleukin‐4 or co‐culture with bone marrow stromal cells. Our observations suggest that HCQ may offer a new therapeutic tool in the treatment of B‐CLL patients.

TGF-beta activity and expression of its receptors in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in Western countries and results from the accumulation of B-lymphocytes which are functionally abnormal and predominantly non-cycling in vivo. Consequently, it is important to understand why B-CLL cells accumulate in GO phase. Since TGF-beta is an important negative regulator of the immune system, a loss of responsiveness to this factor might provide a selective advantage to B-CLL cells. Here we review data on the role of TGF-beta in B-CLL. We show that the B-CLL cell response to TGF-beta signals is abnormal in vitro (inhibition of proliferation and induction of apoptosis). This lack of response of B-CLL cells to TGF-beta inhibition appears to be accompanied by a decrease or a loss of TGF-beta receptor expression.

Comparative analysis of cytokines released by bone marrow stromal cells from normal donors and B-cell chronic lymphocytic leukemic patients.

We studied the production of cytokines (G-CSF, GM-CSF, IL-6, LIF and IL-10) by bone marrow stromal cells of five untreated patients with B-CLL, in Rai stage 0, I and II, and of 8 healthy subjects. The production of G-CSF, GM-CSF, LIF and IL-10 did not differ significantly between controls and B-CLL patients. However, the ability of stromal cells to release IL-6 in response to LPS was decreased in all patients: 36 +/- 5 ng/ml versus 123 +/- 47 ng/ml for normal controls (p < 0.004). Moreover, a soluble activity that inhibited hematopoietic colony formation was detected in B-CLL stromal cell conditioned media. Some potential inhibitors were envisaged and the results indicated an increased production of TGF-beta by B-CLL stromal cells compared to normal stromal cells (respectively 53 +/- 10 versus 15 +/- 4 ng/ml, p < 0.03). The reduced capacity of B-CLL stromal cells to produce IL-6 was associated with this excessive release of TGF-beta; indeed, addition of anti-TGF-beta neutralizing antibody to B-CLL stromal cells, before LPS stimulation, totally normalized the production of IL-6. TGF-beta and IL-6 were also measured in serum samples from normal subjects and B-CLL patients. No significant difference was seen in the production of total TGF-beta (bioactive and latent forms) between normal and B-CLL sera but the mean level of bioactive protein in B-CLL sera was increased in comparison with normal sera (1.74 +/- 0.44 versus 0.67 +/- 0.2 ng/ml, p < 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)