RESUMO
Recent publications showed that highly selected hone marrow derived stem cells can differentiate into hepatocyte like cells. Bone marrow, considered as a rich source of adult stem cells, contains not only hematopoietic and endothelial stem cells, but also precursors of other tissues of mesenchymal origin. The different growth factors used in the culture media are critical factors directly affecting the hepatocytic differentiation and expansion process. Differentiation of adult bone marrow stem cells [BMC] into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocklail. Here, we investigated whether the in vitro differentiation efficacy can be enhanced by sequential addition of liver-specific factors in a time-dependent order that closely resembles their secretion pattern during in vivo liver embryogenesis. Fibroblast growth factors [FGFa and b], Leukeamia inhibitory factor [L1F] Stem cell factor [SCF], Hepatocyte growth factor [HGF] and Oncostatin M [OSM] were used as differentiation factors. Mice bone marrow cells were cultured for one week in a primary culture media and the adherent cells were then subcultured in the directed differentiation media including the above mentioned growth factors added either as a cocktail or in a sequential manner for comparison. In the course of cell differentiation, cell morphology was observed, and the expression of albumin by the transdif Terentiated cells was examined by immunofluorescence and Western blot analysis. Some epithelial-like cells or polygonal hepatocyte like cells appeared in the directed differentiation medium within 12 days, and the number and size of colonies of epithehial-like cells or polygonal cells increased in the course of the cell directed differentiation. The sequential addition of growth factors setup featured relatively earlier and more intense differentiation to hepatocyte like cells. By immunofluoresence analysis albumin expressions appeared, lasted and increased throughout the later directed differentiation. Albumin was found in the cell membrane and scattered in the cytoplasm by immunofluorescent staining. On day 21, the ratio of albumin-positive cells was 85% in the sequentially exposed cells in contrast to the cocktail treatment where the ratio of albumin expressing cells was 65%. We propose that the addition of the hepatocyte growth factor to the culture media is the pivotal step in the trans-differentiation protocol. Moreover the sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult mice BMC into functional hepatocyte-like cells