Martine Massy

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

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.

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 5 d 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.