Adipose-tissue-derived therapeutic cells in their natural environment as an autologous cell therapy strategy: the microtissue-stromal vascular fraction.

The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31+, smooth muscle actin+). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix.

Molecular pathways of early CD105-positive erythroid cells as compared with CD34-positive common precursor cells by flow cytometric cell-sorting and gene expression profiling.

Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34(+)) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34(+) progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-ß signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository.