Studies on Improvement of Bone Marrow-derived Cell Establishment and Manipulation in Chicken

Abstract

Bone marrow-derived cells have an enormous value for the research on mesenchymal stem cell and cell transformation. And also Mesenchymal-derived, multipotent cells have become a valuable resource for cell-to-tissue regeneration and experimental modelling for differentiation and reprogramming. In the chicken, bone marrow-derived cells can induce somatic chimerism and if their pluripotency is confirmed, they could be used as resources to expand the applications of chicken pluripotent cells for various purposes. Bone marrow cells are considered as the basic material for clinical application to injuries and degenerative diseases. For clinical implementation of bone marrow cells, their stable maintenance is important for optimizing manipulation protocol of chicken bone marrow cells and the large-scale expanded culture system has been used to high reproducibility of bone marrow cells. And also no information on cell culture properties have been reported because of their heterogeneous phenotype and mixed cell population. In this study, optimized and improved manipulation system of chicken bone marrow derived cells was established and by evaluating the effect of addition of growth factors on the growth and maintenance of chicken bone marrow derived cells.

First, I observed two different populations were maintained: a spindle shape dominant population consisting mainly of fibroblast-like cells and a cuboidal shape dominant population. So, I investigated A significant correlation affecting cell retrieval was detected between the parameters of body weight and leg length in the spindle shape cell-dominant and cuboidal shape cell-dominant populations. Additionally, analysis of cell kinetics, protein marker expression of the chicken bone marrow cells at passage 5. As the results, based on these observations, we conclude that chicken bone marrow cells possess different cellular characteristics compared with the bone marrow cells of mice and humans, and that a different approach and culture regimen may be necessary for manipulating chicken bone marrow-derived multifunctional cells.

Next, I investigated the necessity of growth factors and different basic media to modify the culture system of chicken bone marrow cells and the change of cellular properties in chicken bone marrow cells through a suspended culture. Four-day-old white leghorn chicks were employed for experimental animal and the isolated bone marrow derived cells were cultured in different four types media(High glucose DMEM, Low glucose DMEM, F-12/DMEM, αMEM) to which 5 ng/ml bFGF and 500 unit/ml LIF were supplemented or not. To monitor of the effect of growth factors on the capacity of cell maintenance in vitro, primary cell attachment, CFU-F colony number, proliferative activity was evaluated as experimental parameters. As a results, the number of CFU-F-positive colonies was higher (p<0.05) after the bFGF and LIF addition than after no addition, which resulted improved primary cell attachment and also better proliferative activity was detected in the growth factor supplementation group.

Additionally, analysis of pluripotency- or differentiation-related gene expression and protein marker expression in the chicken bone marrow derived cells were detected. Different expression profile in the expression of several genes was detected after the supplementation or not, which differed from that of primordial germ cells and embryonic fibroblasts. And also, flow cytometry analysis data shows more expression rate detected such as CD44, CD105, MCAM in growth factors supplementation group.

In conclusion, the combined addition of bFGF and LIF to culture medium improved the culture efficiency of chicken BMCs of mixed population, which may contain various reprogrammable cells.