Reprogramming into pancreatic endocrine cells based on developmental cues.

Due to the increasing prevalence of type 1 diabetes and the complications arising from actual therapies, alternative treatments need to be established. In order to compensate the beta-cell deficiency associated with type 1 diabetes, current research focuses on new strategies to generate insulin-producing beta-cells for transplantation purpose, including the differentiation of stem or progenitor cells, as well as the transdifferentiation of dispensable mature cell types. However, to successfully force specific cells to adopt a functional beta-cell fate or phenotype, a better understanding of the molecular mechanisms underlying beta-cell genesis is required. The present short review summarizes the hitherto known functions and interplays of several key factors involved in the development of the different endocrine cell lineages during pancreas morphogenesis, as well as their potential to direct the generation of beta-cells. Furthermore, an emphasis is made on beta-cell regeneration and the determinants implicated

Generation of animals allowing the conditional inactivation of the Pax4 gene.

Pax4 belongs to the paired-box family of transcription factors. The analysis of loss- and gain-of-function mutant animals revealed that this factor plays a crucial role in the endocrine pancreas. Indeed, Pax4 is required for the genesis of insulin-producing beta-cells. Remarkably, the sole misexpression of Pax4 in glucagon-expressing cells is able to induce their regeneration, endow these with beta-cell features, and thereby counter chemically induced diabetes. However, the function of Pax4 in adult endocrine cells remains unclear. Herein, we report the generation of Pax4 conditional knockout mice that will allow the analysis of Pax4 function in mature beta-cells, as well as in the adult central nervous system

Characterization of human MSC propagated in animal serum-free cultures and clinical application of MSC in paediatric patients.

Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes

Characterization of human MSC propagated in animal serum-free cultures and clinical application of MSC in paediatric patients.

Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes

Isolation and expansion of functional differentiating mesenchymal stem cells with a serum deprived medium.

The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy. We evaluated a novel serum deprived medium (Quantum 333R) for the isolation and expansion of MSC as compared to the standard medium (SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 105 bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 105 BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/\u2013 1,3%) and 10% (+/\u2013 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSCSFM were smaller in the side scatter than MSC SM. Further immunophenotypical characterization showed, in the MSCSFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the "classical" MSCSM. CD73 and CD105 were upregulated in MSCSFM in comparison to MSCSM. Still, MSCSFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSCSM. In addition, to test the differentiation potential of MSCSFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications

Isolation and expansion of functional differentiating mesenchymal stem cells with a serum deprived medium.

The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy. We evaluated a novel serum deprived medium (Quantum 333R) for the isolation and expansion of MSC as compared to the standard medium (SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 105 bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 105 BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/– 1,3%) and 10% (+/– 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSCSFM were smaller in the side scatter than MSC SM. Further immunophenotypical characterization showed, in the MSCSFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the "classical" MSCSM. CD73 and CD105 were upregulated in MSCSFM in comparison to MSCSM. Still, MSCSFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSCSM. In addition, to test the differentiation potential of MSCSFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications

SMALL VOLUMES OF SUBCUTANEOUS ADIPOSE TISSUE AS SOURCE OF ADULT MESENCHIMAL STEM CELLS FOR REGENERATIVE MEDICINE AND CANCER CELL-THERAPIES

THE POSSIBILITY OF GENERATING MESENCHIMAL STEM CELLS FROM SMALL VOLUMES OF ADIPOSE TISSUE OPENS NEW PERSPECTIVES TO CLINICAL APPLICATIONS OF REGENERATIVE MEDICINE AND CANCER CELL-THERAPIE

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

Background Multipotent mesenchymal stromal cells ( MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSCFCS). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. Methods As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. Results We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSCFFPP). Moreover, MSCFFPP showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSCFFPP morphology was equivalent to MSC FCS, and MSCFFPP expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSCFFPP could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSCFFPP were indistinguishable from MSC FCS. Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSCFFPP. Discussion Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes