ABSTRACT
BACKGROUND & OBJECTIVES: Next generation transplantation medicine aims to develop stimulating cocktail for increased ex vivo expansion of primitive hematopoietic stem and progenitor cells (HSPC). The present study was done to evaluate the cocktail GF (Thrombopoietin + Stem Cell factor + Flt3-ligand) and homing-defining molecule Stromal cell-derived factor 1 (SDF1) for HSPC ex vivo expansion. METHODS: Peripheral blood stem cell (n=74) harvests were analysed for CD34hiCD45lo HSPC. Immunomagnetically enriched HSPC were cultured for eight days and assessed for increase in HSPC, colony forming potential in vitro and in vivo engrafting potential by analyzing human CD45+ cells. Expression profile of genes for homing and stemness were studied using microarray analysis. Expression of adhesion/homing markers were validated by flow cytometry/ confocal microscopy. RESULTS: CD34hiCD45lo HSPC expansion cultures with GF+SDF1 demonstrated increased nucleated cells (n=28, P+ cells (n=8, P=0.021) and increased colony forming units (cfu) compared to unstimulated and GF-stimulated HSPC. NOD-SCID mice transplanted with GF+SDF1-HSPC exhibited successful homing/engraftment (n=24, PInterpretation & conclusions: Cocktail of cytokines and SDF1 showed good potential to successfully expand HSPC which exhibited enhanced ability to generate multilineage cells in short-term and long-term repopulation assay. This cocktail-mediated stem cell expansion has potential to obviate the need for longer and large volume apheresis procedure making it convenient for donors.
Subject(s)
Cell Proliferation/drug effects , Hematopoietic Stem Cells/cytology , Stem Cells/cytology , Animals , Antigens, CD34/genetics , Cell Proliferation/genetics , Cell Self Renewal/drug effects , Chemokine CXCL12/administration & dosage , Chemokine CXCL12/metabolism , Gene Expression Regulation, Developmental/drug effects , Hematopoietic Stem Cells/drug effects , Humans , Leukocyte Common Antigens/administration & dosage , Leukocyte Common Antigens/metabolism , Membrane Proteins/administration & dosage , Membrane Proteins/metabolism , Mice , Stem Cell Factor/administration & dosage , Stem Cell Factor/metabolism , Stem Cells/drug effects , Thrombopoietin/administration & dosage , Thrombopoietin/metabolismABSTRACT
The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of 'oxygenase' and 'reductase' components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol 'ortho' ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3-5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30-40 µM and V max = 34-48 µmol min(-1) mg(-1). However, reductase from benzoate grown cells showed K m = 49 µM and V max = 10 µmol min(-1) mg(-1). Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4.
