| Objective: To explore the isolation, purification and expansion method of human umbilical cord derived mesenchymal stem cells and to investigate there biological characteristics.Method: The umbilical cords (UCs) were obtained from department of obstetrics and gynecology of our hospital after infermed consent. UC (gestational age, 9-40weeks) were collected and processed within 2 hours after normal deliveries. The UCs were internally washed with phosphate-buffered saline (PBS) containing penicillin and streptomycin, then filled with 1% collagenaseⅣin PBS; the extremities were clamped and incubated for 20 minutes at 37℃. The collagenase solution with the detached cell was harvested, and the vein was washed twice again to gather the rest of the cells. Pellets was resuspended in DMEM-LG medium containing 10% fatal calf sera,20mmol/L HEPES,2 mmol/L L-glutamin, and 1mmol/L sodium pyravate. The cell was seeded at a concentration of 108cells/L in 25ml calture flask. After incubation for 48 hrs, the suspernatant was removed and the medium was changed, every 3 days with fresh medium. Cells were isolated by treatment with 2.5g/L trypsin for passage. The UCs were diced into small fragment and the resulting small piece of UC was cultured in the medium mentioned above.The cells were cultured consecutively in vitro to test their abilty in proliferation and passage. Immunofluorescence and flow cytometry were employed to determine the phenotype of those cells.Results: Our studies demonstrated that MSC like cells could be isolated from UCs both of two methods with similar characteristics in morphology. 1 week after UC fragments were seeded into culture plate, some mesenchymal stem cell, which appearance likes fibroblast cells, migrated from the diced UC and reached 80% confluence in abort 3 weeks in vitro. By using another method, adherent cells with same appearance in morphology could be found after incubation in 24 hours in vitro. They can form colonies after cultured in 10 to 14 days. Most of the colonies were fibroblast like cells and like paving stone type cell. MSCs isolated from UCs had the ability to potential to proliferate and passage and maintained their biological characteristic after 20 passages in vitro. The phenotype analysis showed that MSC derived from UCs expressed highly CD29, CD44, and CD449e. They also expressed low level of CD106 and CD11b. but MSCs didn't express the marker such as CD34 which belong to hematopoietic stem cells. The doubling time of P4 or P10 of MSCs was 33.1 and 35.2 hours respectively as determined by MTT assays. Cell cycle analysis showed that some of 60-70% MSCs were at G0-G1 phase. Conclusion Fibroblast like cells could be harvested by both of tissue fragment adherent method and collagenase digesting method. UCs derived fibrolast-like cells have strong ability to proliferationan and have the similar phenotype with bone marrow mesenchymal shem cell, indicating that thay are new member of MSCs family.Part two: Neural induction of human umbilical cord derived mesenchymal stem cellsObjective To discuss the condition of differentiate human umbilical cord derived mesenchymal stem cells into neurons in vitro by basic fibroblast growth factor (bFGF) and N2 suplentment.Method The passage 4th MSCs were seeded in flask to preparation coverslip. After 3-4 days, when cells reached 60% confluence, 30ng/mlbFGF and N2 supplement were added to induce the UCMSCs , and contrasted with the cells that had not been induced. A phase contrast microscope was used to observethe differentiation of MSCs everyday. Immunohistochemistry technique was employed to identify the differentiated MSCs. Such as Nestin, neuron specific enolase (NSE) and NF, The differential rates of neuron-like cells were calculated every day.Result UCMSCs bagan to contract and became irregularly shaped at 24 hours. After 3 days, the cells morphology changed more obviously, the cell become triangular or cone-shaped with multipolar ling processes. The processes continued to elaborate and to display many branches, some branch of different cells connected with each other, just like dendrite, forming a net. After 5 days, most of cells occurred to neuron-like cell change.then the partial cells fall off, dead .the most cells can survive above 10 day. Nestin occurred first after 24 hours of bFGF treatment, reached the 35.03%, for days later the positive cells exhibited value peak.(70.88%), the NF and NSE markers expressed except 47.51%, 50.1% at 7th day.Conclusion MSCs from human umbilical cord do have a potential to differentiate into neural like cells. Combination of N2 culture medium with bFGF synergistically promotes the MSC to differentiate into neuron like cells. As a kind of stem cell isolate from off-fall of delivery, UCMSCs eliminate the ethical or social concerns and are easily obtainable. Our research paves the way for the means therapeutic and biotechnological study of stem cells. |
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