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1.
New Egyptian Journal of Medicine [The]. 2009; 41 (3): 232-239
in English | IMEMR | ID: emr-111429

ABSTRACT

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


Subject(s)
Animals, Laboratory , Cells, Cultured , Hepatocyte Growth Factor , Cell Differentiation , Mice
2.
New Egyptian Journal of Medicine [The]. 2009; 41 (4): 318-328
in English | IMEMR | ID: emr-111490

ABSTRACT

As a source of hematopoietic stem cells [HSCs], umbilical cord blood [UCB] has the advantages of speed of availability, tolerance of more than one HLA mismatch, and a low incidence of severe graft-versus-host disease [GVHD]. Hence, it represents a promising, alternative non-costly and non-invasive source for prospective stem cell based therapy. In this study we investigated the angiogenic potential of ex vivo expanded human umbilical cord blood CD 133* stem cells transplanted into mice with chronic hepatic fibrosis induced by Schistosomiasis infection. Histopathological, ultrastructural and immunohistochemical analysis of mice liver sections were done to detect specific human angiogenic markers. Umbilical cord blood was obtained from healthy pregnant females after delivery and mononuclear cells were collected by density gradient using Ficoll Hypaque. Enrichment for the CD 133* stem cells was done by positive selection using the Magnetic Activated Cell Sorting system and magnetic microbeads. Cells were cultured in prirnaly ex vivo expansion medium for three weeks. Flowcytomeric analysis of the cultured cells was done in each step to identify the CD 133* cells. Schistosomiasis was induced in Swiss Albino mice by intradermal injection of schistosoma cercariae. Twenty two weeks post schistosoma infection a total of 0.3 x 106 human CD 133* stem cells were injected intrahepatically in mice. Accordingly, mice were divided into three groups: Group 1 [infected, transplanted]; Group 2 [infected controls] and Group 3 [healthy, transplanted]. All mice were sacrificed 3 wks after cell transplantation was done in groups I and 3. Histopathology and Electron microscopy showed an obvious increase in the capillary network and the small blood vessels and a decrease in the fibrosis known for this stage of the disease. By immunohistochemical analysis the cellular constituents of these newly formed blood vessels showed positive expression of the human-specific angiogenic markers CD31, CD34 and von Willebrand Factor [vWF]. Few hepatocyte like polygonal cells showed positive expression of human Vascular Endothelial Growth Factor [VEGF] and inducible Nitric Oxide Synthase [iNOS]. Ex vivo expanded CD 133* human stem cells incorporate into the liver of schistosoma infected mice enhancing local angiogenesis and hepatic neovascularization. These preliminary results obtained suggest a dual benefit of CD 133* cells in cell therapy in hepatic diseases based on its capability of hematopoietic and endothelial differentiation. We suggest that the CD 133* cells contribute to repair in a paracrine manner by creating a permissive environment that enables rapid proliferation and survival of damaged cells rather than through direct differentiation to hepatocytes


Subject(s)
Schistosomiasis/complications , Liver/pathology , Fetal Blood/cytology , Antigens, CD/blood , Stem Cells , Angiogenesis Inducing Agents , Flow Cytometry/methods , Liver/ultrastructure , Microscopy, Electron , Immunohistochemistry/methods
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