Isolation, Culture And In Vitro Multipotential Of Goat Marrow-Derived Mesenchymal Stem Cells

The mesenchymal stem cells (MSCs) were one of the two kinds of important stem cells derived from bone marrow. There are many events showed the differentiation multipotentials of the bone marrow MSCs in vitro and in vivo. The data suggested that the MSCs would be one of the main seed cells source for cell/gene therapy and tissue engineering in future. However, There are no a systemic study on goat MSCs, especially adult goat, in the isolation, culture, differentiation, and so on. The aim of this paper is to illuminate the biological property and multipotentials of the goat bone marrow MSCs, so as to apply MSCs to diseases therapy and bioactive compounds production in future, and subsequently to establish the goat bone marrow MSCs line. For above reasons, the isolation, culture, proliferation and growth characteristics, surface markers, and in vitro differentiation of adult Guanzhong dairy goat marrow-derived MSCs were assayed. The results are as follows: 1. The MSCs can be derived from goat bone marrow by density gradient centrifuge separation or cell attachment to tissue culture plates. And the MSCs can be sub-cultured and expanded stably. In our test, the MSCs were successfully derived from adult Guanzhong dairy goat iliac crest bone marrow and sub-cultured to the 21st passage. The goat MSCs have been expanded to 2.8×108 cells. 2. In morphology, the goat MSCs are polygonal, triangal, or long fusiform cells, similar to human MSCs. 3. The scanning electron microscope showed that many protuberances, microspikes, and filopodia were found on the goat MSCs'coarseness surface. And the microspikes of goat MSCs formed a kind of interlaced net when many cells co-existed. The transmission electron microscope showed many rough endoplasmic reticulums (RER) and mitochondrions are found in the cells, which suggested the puissant secreted function of the cells. The typical characteristics of the MSCs were different from other cells, such as the nuclei of the MSCs were infantile, the nucleus-plasma ratio was high and the nucleoli were in quiescent stage; there were a few organelles in the cells. 4. The goat MSCs expressed the cellular markers for the MSCs, such as CD29, CD44, CD105, CD106, CD166 etc. (positive), but did not express CD14, CD34, CD45, and c-kit (negative), the markers of hematopoietic stem cells. It is help for identification of the MSCs to assay those markers. The result also showed the goat MSCs expressed TGF-α. The reason should be further studied. 5. The 5-azacytosine could induce goat MSCs to differentiate into the cardiomyocytes phenotype, which expressed α-actin (positive) and PAS (periodic acid Schiff's) reaction positive and secreted hepatin. That provided a scientific foundation for establishing animal cellular model of cardiopathy therapy. The potential of cardiaomyocytes differentiation was an important criterion for distinguishing the bone marrow-derived mesenchymal stem cells from hematopoietic stem cells. 6. The results showed that goat MSCs gradually changed their configuration from fibroblast-like to typical β-like cells and formed mass colonies after being induced by HGF and nicotinamide. The β-like cells were DTZ (dithizone) stained positive and expressed PDX-1 (pancreatic duodenal homeobox-1) and Nestin (positive), and secreted the insulin. Therefore, the results could provide a scientific foundation for diabetes cellular therapy with non-pancreatic derived cells or be as a cellular bioreactor to produce the insulin in vitro. 7. The goat MSCs can be induced to differentiate into neuron phenotype by the β-mercaptoethanol. After the treatment of the β-mercaptoethanol, the form of the cells changed and expressed the neuron markers, such as Nestin, NF-200 (neurofilament-200), GFAP (glial fibrillary acidic protein), and NSE (neuron specific enolase). But it was necessary for appraising their function to further study. 8. Under induction of dexamethasone (DEX), ascorbate-phosphate (Vita min C), and β-glycerophosphate (β-gly), the goat MSCs gradually changed their configuration from polygonal, triangal, or long fusiform cells, to cubiform, then elliptic, and even rotundity. That meant the MSCs differentiated into osteoblast, which expressed alkaline phosphatase (ALP+) and alizarin red stain positive. 9. The results also showed that the goat MSCs expressed some surface markers of the embryo stem cells, such as Oct-4, SSEA-1, SSEA-4 (positive). That suggested the possible reason of the MSCs maintained multipotential. It need to further study for elucidating the exact reason. Above all, we obtained goat bone marrow mesenchymal stem cells successfully, whichwere capable of differentiating into the cellular phenotypes of all the three germ layers. In accordance with the morphology, ultrastructure, surface markers and differentiation potentials, the cells derived from the adult Guangzhong dairy goat marrow should be identified as the MSCs. Although it is no doubt that the MSCs reveal a vastly potential applied foreground, many works should be done before the MSCs could be applied in production and clinical practice.