[Construction and optimization of an in vitro induction system for bone marrow-derived mesenchymal stem cell...

Objective To construct the hematopoietic microenvironment system simulating different hematopoietic sites during the embryonic stages for in vitro inducing the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hematopoietic cells, and to initially evaluate the efficiency of differentiation. Methods Stereomicroscopy and HE staining were used to observe the anatomical parts and morphology of yolk sac (YS), placenta (PL) and fetal liver (FL) of E11.5 mice. The embryonic YS, PL and FL tissues from the stages of E7.5-E9.5, E10.5-E12.5 and E13.5-E15.5 were collected for stromal cell culture, and conditioned culture media were prepared, namely, yolk sac stromal cell conditioned medium (YSSC-CM), placental SC-CM (PLSC-CM) and fetal liver SC-CM (FLSC-CM), and co-cultured with in vitro expanded SD rat BMSCs. The experimental cells were divided into control group, interleukin 6(IL-6) combined with stem cell factor (SCF) treatment group, YSSC-CM treatment group, PLSC-CM treatment group and FLSC-CM treatment group. After co-culture for 7-9 days, the floating cells in culture medium were collected. Giemsa staining was used to exam cell morphology. Direct immunofluorescence was conducted to detect CD34 and CD45 expression. Colony formation assay was performed to detect granulocyte/macrophage colony formation unit (GM-CFU) to identify differentiated cells. Results Under a stereomicroscope, PL and FL of mouse embryos had the same position as human embryos, while YS was wrapped on the outer side of embryo body and amniotic membrane. HE staining showed that PL vessel labyrinth, FL sinusoids and YS blood islands were rich in blood cells. The inverted phase-contrast microscope and cell counting results indicated that the floating cells in the culture medium of the YSSC-CM group, the PLSC-CM group and the FLSC-CM group significantly increased compared with the control group and the IL-6 combined with SCF group, especially the FLSC-CM group had the most. The floating cells in YSSC-CM group, PLSC-CM group and FLSC-CM group were similar in morphology to lymphoid or mononucleoid cells as showed by Giemsa staining, and expressed hematopoietic cell-specific surface markers CD34 and CD45. The number of hematopoietic cell colonies formed was the most in the FLSC-CM group, followed by the PLSC-CM group, and the least in the YSSC-CM group. The control group and IL-6 combined with SCF treatment group had no changes. Conclusion By collecting YS, PL, and FL according to the stage and time sequence, YSSC-CM, PLSC-CM, and FLSC-CM prepared can induce the differentiation of SD rat's BMSCs into hematopoietic cells, and FLSC-CM-treated cells have better differentiation efficiency.