Stem cells isolated from adult rat muscle differentiate across all three dermal lineages

Abstract

Adult stem cells capable of differentiating into phenotypes from all three dermal layers were isolated from adult rat muscle. Stem cells were obtained by enzymatic digestion, followed by primary culture in Eagle's minimum essential medium +10% preselected horse serum. When the cells reached confluence, they were released by trypsin, filtered to remove differentiated myotubes, and then slow frozen in 7.5% dimethylsulfoxide to -80 degrees C. Thawed cells were the stem cells and were induced to differentiate with the nonspecific differentiating agent dexamethasone at concentrations of 10(-10)-10(-6) M. After a 6-week treatment with dexamethasone, the cells were assayed by immunohistochemistry for phenotypes of the mesodermal, ectodermal, and endodermal lineages. Examples of mesodermal phenotypes identified were as follows: bone, cartilage, and skeletal, smooth, and cardiac muscle. Ectodermal phenotypes identified were as follows: neurons and oligodendrocytes. Hepatocyte phenotypes identified represented the endodermal lineage. All the phenotypes were observed only with treatment with dexamethasone. However, nestin was observed in the absence of dexamethasone and may be a marker for uncommitted pluripotent stem cells. The results show that adult muscle contains pluripotent stem cells capable of differentiating across all three dermal lineages. Such cells could be used in the context of tissue engineering.

Osteosarcoma Cells Differentiate into Phenotypes from all Three Dermal Layers

“…First, when MASCs are treated with dexamethasone, each phenotype has a characteristic range of dexamethasone concentrations at which phenotypic expression is most marked [31,32,50,58]. Both osteosarcoma cell lines exhibited the same pattern of differentiation in response to dexamethasone as MASCs.…”

“…Third, cells positive for markers for several phenotypes were present at any given dexamethasone concentration, which is expected as dexamethasone is a nonspecific inducer and thus will induce multiple phenotypes. When isolating MASCs from normal tissue, we selected for stem cells by the freeze-thaw method which kills committed cells present in the tissue while maintaining undifferentiated cell viability [31,32,50,58]. Both osteosarcoma cell lines were cultured in fetal calf serum by ATCC or Dr. Rodan.…”

“…We checked the karyotype of both cell lines when they were received and then before the experiments. Both cell lines were obtained frozen and then were grown and tested as described for mammalian MASCs [32,50]. They were thawed and plated at a density of 100,000 cells per 100-mm culture dish (Corning Glass Works, Corning, NY, USA) that previously had been coated with 1% gelatin (Fisher Scientific, Pittsburgh, PA, USA) in Eagle's minimal essential medium (EMEM) + 10% preselected horse serum.…”

“…Cryopreservation was performed in freezing chambers (Fisher Scientific, Norcross, GA, USA) to slow freeze the cells to À80°C [32,50]. After being frozen for at least 24 hours, aliquots of the frozen cells were thawed and plated at a density of 5000 cells per 16-mm well in all 24 wells of five 24-well gelatin-coated culture plates (Corning) in EMEM + 10% horse serum to start the experiments.…”

“…Stem cells are defined by two parameters: extended proliferation potential and the ability to differentiate into one or more phenotypes [3,4,6,56]. Normal adult tissue contains multipotent adult stem cells that retain the ability to differentiate into several mesodermal phenotypes from all three dermal lineages [32,50] and progenitor stem cells that have the limited differentiation capability of forming only the cell types in that tissue [51]. Tumors are thought to arise from clonal expansion of a progenitor cell to a group of cells consisting of partially or wholly differentiated cells that no longer divide and a small percentage of cells that continue to divide -cancer stem cells [11,15,16,42,51,53,59].…”