Biological Characteristics Of Rat Placenta Derived Mesenchymal Stem Cells

Background and purposeThe mesenchyma stem cells (MSCs) belongs to one kind of the adult stem cells, it originates from the mesoderm. The source of MSCs are wide, including different species, like mouse,rat, rabbit, sheep, pig, monkey, human; As well as from wide range of tissues, such as bone marrow, adipose, muscle, liver, pancreas, amniotic fluid, umbilical cord blood, umbilical cord, placenta, and so on. MSCs except has stem cell's general characteristics: Self-renewal and multi-direction differentiation potential. With the progress of research on MSCs, we discovered that MSCs still has some unique charicteristics of supporting hemopoiesis stem cell transplant, the immunomodulation and paracrine processes. As a result, these characteristics of MSCs enables it to become a focus in the fields of cell-based therapy and tissue engineering, researchers put it into treating many kinds of human diseases. These diseases including the multiple sclerosis, GVHD, acute myocardial infarction, heart failure, last stage of liver disease, Crohn's disease, diabetes, and so on. During the past decade, by the numerous researchers' endeavor around the world, it has no doutely that tremendous advancements have been made from significant in vitro and in vivo preclinical studies and clinical trials. Most of them got optimism outcomes and had no obvious side effects. Currently, it seems that there is no universal source for MSCs suitable for all conceivable clinical applications. Moreover, there is still exists dispute about put MSCs into treating some human diseases, for instance the identification of MSCs is lacks of unique molecular markers, its long-term effects and security, and so on. Therefore regarding comprehensively use MSCs in clinical, we still need work hard for it. We try to establish a method of separates MSCs from the rat placenta through this research, and to observes its biology characteristics in vitro.Materials and Methods1 AnimalOne Sprague Dawley (SD) rat, it was about 13-15d pregnant and was bought from animal center of the PLA's Academy of Military Medical Sciences.2 MethodsRat placenta derived MSCs (r-pl-MSCs) were separated and obtained by collagenase-digested and adherent methods. Morphology of r-pl-MSCs were observed with inverted microscope every day. Growth kinetics was measured with MTT assay and growth curve was drawn.Their surface antigen and cell cycle were detected by Flow Cytometer. Adipogenic differentiation and osteogenic differentiation were tested by immunohistochemistry.3 Data analysisAll data were dealed with GraphPad Prism software package.Results1 Morphological characteristics of r-pl-MSCsThe primary cultured cells adhered after 8 hours, and adherent cells displayed spindle or triangle shape. These cells formed fibroblast colony-forming units within 24 hours and gradually displayed the whirlpool-liked growth pattern. From the first generation cells to the third generation cells showed different shape and size. The size and morphology of 4th generation cells were almost same, the mainly cells were shaped like spindle.2 Growth characteristics of r-pl-MSCsThe third generation cells'growth curve displayed that these cells grew very slowly in the first day(it's called latent period). But they entered into rapid multiplication period from the second day and transfered into plateau after 5 days. The double times of these cells was 42 hours.3 Cell cycle testing of r-pl-MSCsCell cycle measure showed the proportion of cells in G0/G1 phase, S phase, G2 phase was respectively 81.07%,11.49%,7.44%. CV was 4.38%.4 Immunophenotype of r-pl-MSCsImmunophenotype analysis of both 4th and 7th generation cells were showed they expressed CD29 and CD90, but no CD45.5 Adipogenic and osteogenic differentiation of r-pl-MSCsAfter adipogenic differentiation and stained by oil red O, there are red oil drops in the cells; after osteogenic differentiation and Von-Kossa stain, there are black particle in the cells.Conclusion1. We can saperate MSCs from rat placenta.2. The morphology of r-pl-MSCs was similar with bone marrow derived MSCs and umbilical cord derived MSCs. All of them mainly displayed spindle-shape and whirlpool-liked growth pattern.3. r-pl-MSCs expressed CD29 and CD90, but no CD454. r-pl-MSCs have potentials of adipogenic and osteogenic differentiation in special cultrue conditions in vitro.5. r-pl-MSCs can serve as one new source of MSCs for further research.