Staphylococcal enterotoxin C injection in combination with ascorbic acid promotes the differentiation of bone marrow-derived mesenchymal stem cells...

Staphylococcal enterotoxin C injection is established as a clinical therapy for delayed healing or disunion of bone fractures. In the present study, the effects of staphylococcal enterotoxin C injection in combination with ascorbic acid (SEC-AA) on the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) and their influences on the mineralization of osteoblasts were investigated. SEC-AA treatment induced increased levels of alkaline phosphatase activity in MSCs and increased numbers of alizarin red-stained calcified nodules, indicating enhanced differentiation of MSCs into osteoblasts. The findings demonstrated that SEC-AA promoted the differentiation of MSCs into osteoblasts and accelerated the cytopoiesis of osteoblasts. Our data provide a cytological model for bone fracture therapy aimed at shortening the time required for healing and improving the clinical outcome, and also provide a theoretical basis for inducible differentiation of MSCs, mineralization of osteoblasts and reconstruction of bone tissues.

ARTICLE

Alkaline Phosphatase-positive Colony Formation is a Sensitive, Specific and Quantitative Indicator of Undifferentiated Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP+) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows: (1) Most colonies obtained under these conditions consist of 30 AP+ cells that co-express OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. (2) Most such colonies are derived from SSEA3+ cells. (3) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multi-lineage differentiation in embryoid bodies (EBs). (4) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (5) CFC frequencies decrease when differentiation is induced, either by exposure to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP+ clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.