Differentiation Of Sca-1～+ Cells From Mouse Fetal Liver And CD34～+ Cells From Human Into Neural Cells In Vivo And In Vitro

Objectives:To observe whether fetal liver stem cells from mouse or human possess potentials of differentiation into neural cells in vivo and in vitro, to study its plasticity, to probe its rules of differentiation, and to establish the model of neural cell differentiation from fetal liver.The study includes three parts: mouse fetal liver Sca-l+ cells differentiate into neural cells in vivo, mouse fetal liver Sca-1+ cells differentiate into neuronal cells in vitro, human fetal liver CD34+ cells differentiate into neuronal cells in vitro.Methods:1. mouse fetal liver Sca-l+ cells differentiate into neural cells in vivoSex of 14.5-day-old C57BL/6J mouse fetuses was determined by rapid PCR analysis of sry gene, which total time is less than 3.5 hours. Sca-l+ cells from male fetal liver were isolated with a magnetic cell sorting kit for mouse Sca-l+cells, and transplanted into lethally irradiated 8-10 week-old female mice (10.0Gy) via tail veins (2.0+103 cells/mouse). Mice were bred in the sterilizedanimal facility. Reconstitution of hematopoietic system in lethally irradiated and Sca-l+ cell- transplanted mice was observed by numbering mononuclear cells in peripheral blood, and was analysed by the PCR assay of mouse sry gene. The donor cells and their characteristics in recipient brains were detectedand identified by FISH and immunohistochemistry double-staining analysis at 60, 120, 180 days after transplantation.2. mouse fetal liver Sca-l+ cells differentiate into neuronal cells in vitroSca-l+ cells from 14.5-day-old mouse fetal livers were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS), and passaged at a ratio of 1:3 when cells reached more than 80% confluence. The 5passage cells were induced by 10-3 M b-mercaptoethanol (b-ME) and 5+10-7M all-trans-retinoic acid (RA) for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days. Culture plates and coverslips were coated with10g/ml fibronectin. The morphology was observed under contrast phasemicroscope. The characteristics of treated cells were assayed by immunocytochemistry staining analysis and semi-quantitative RT-PCR method (p-actin as internal control) at 5 hours, or 5 days.3. human fetal liver CD34+ cells differentiate into neuronal cells in vitro1) cell culture:CD34+ cells from naturally aborted 5-month-old human fetal liver were isolated with a magnetic cell sorting kit for human CD34, and cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS), and passaged at a ratio of 1:3 when cells reached more than 80% confluence.2) b-ME and RA induced neural differentiation:The 5 passage cells were induced by 10-3 M b-mercaptoethanol (b-ME) and 5 +10-7 M all-trans-retinoic acid (RA) for 24 hours, respectively, and then incubated in serum-free medium for 5 hours to 5 days. Culture plates and coverslips were coated with 10g/ml fibronectin. The morphology was observed under contrast phase microscope. The characteristics of treated cellswere assayed by immunocytochemistry staining analysis and semi-quantitative RT-PCR method (p-actin as internal control) at 5 hours or 5 days.3) b-ME and BHA induced neural differentiation:The 5 passage cells were pretreated in DMEM/F12 + 10% FCS + 1mM P-ME for 24 hours, and washed with PBS, and then induced by serum-free medium containing 2% DMSO + 0.2 mM BHA + 2 mM b-ME for 5 days.Culture plates and coverslips were coated with 10g/ml fibronectin. Themorphology was observed under contrast phase microscope. The characteristics of treated cells were assayed by immunocytochemistry staining analysis and semi-quantitative RT-PCR method (P-actin as internal control) at 5 hours or 5 days.Results:1. mouse fetal liver Sca-l+ cells differentiate into neural cells in vivoMice transplanted with Sca-l+ cells all survived more than 6 months, mice non- transplanted with Sca-l+ ce...