ABSTRACT
BACKGROUND:Human umbilical cord mesenchymal stem cells (hUC-MSCs) may be mutated duringin vitro culture based on the spontaneous malignant transformation of adult stem cells and tumor stem cell theory, and there may be a risk of tumorigenesis after in vivo transplantation. Therefore, to establish and perfect the in vitro safety testing procedures will actively promote the clinical application of stem cells. OBJECTIVE:To investigate the tumorigenic mechanism of hUC-MSCs and the expression level of DNA methyltransferase (DNMTs) in hUC-MSCs. METHODS:Primary hUC-MSCs were isolated and expanded by tissue adherent culture. 3-Methycholanthrene was used to cause the malignant transformation in hUC-MSCs (experimental group), followed by morphological observation and tumorigenesis experiment in nude mice. Then, the tumor tissues were obtained and identified by pathological examination and primary cell culture, and the levels of DNMTs mRNA in hUC-MSCs treated with 3-methycholanthrene and dimethyl sulfoxide (control group) were detected by real-time RT-PCR and compared. RESULTS AND CONCLUSION:hUC-MSCs treated with 3-methycholanthrene led to malignant transformation, which showed malignant growth and non-integer ploidy changes in the cell nuclei, and formed a malignant tumor in immune-deficient mice after injection. Compared with the control group, the cells in the experimental group showed higher expression of DNMTs mRNA as detected by real-time RT-PCR. To conclude, hUC-MSCs can trigger malignant transformation in the morphology and the epigenetics under certain conditions. DNMTs can be a candidate for prevention against malignant transformation of transplanted stem cells.
ABSTRACT
BACKGROUND: Human umbilical cord mesenchymal stem cells (HUMSCs) can differentiate into insulin-producing cellsafter induced by chemical drugs or co-culture methods, but insulin secretion is extremely low. Therefore, to inducemature pancreatic beta cell differentiation from stem cells by adenovirus transfection of specific genes involved in thedevelopment of pancreas is a research hotspot in recent years.OBJECTIVE: To study the differentiation potential of HUMSCs into insulin-producing cells after transfection withmusculoaponeurotic fibrosarcoma oncogene homologue A (MafA).METHODS: Ad-MafA-EGFP was transferred into passage 3 HUMSCs. After 7 days of induction, changes of cellmorphology were observed by inverted phase contrast microscope. Expression of pancreatic cell-specific genes(glucagon, PDX1, Nkx2.2) was detected by PCR technique.RESULTS AND CONCLUSION: After Ad-MafA-EGFP transfection, no significant morphological changes were observedin the HUMSCs under inverted phase contrast microscope. It was confirmed by fluorescence microscope thatAd-MafA-EGFP was transferred into the HUMSCs. After induction, the expression of human pancreatic precursorcell-related genes, including glucagon, PDX1 and Nkx2.2, was increased as detected by PCR. To conclude, thesefindings could provide experimental evidence for further differentiation and maturation of pancreatic β cells fromHUMSCs.
ABSTRACT
Objective To establish and identify the induced pluripotent stem cell(iPSC) line reprogrammed from human umbilical cord mesenchymal cells(HuMSCs).Methods HuMSCs were cultured by adhesion method,and OCT4,SOX2,KLF4,c-Myc,NANOG,LIN-28 were transfected into HuMSCs with lentiviral victor to reprogramme HuMSCs into iPSC.Morphological observation,pluripotency genes (SOX2,TDGF1,THY-1,OCT4,REX1 and TERF1) expression,alkaline phosphatase detection,karyotype analysis,embryonic stem cells (ESC) specific proteins (NANOG,OCT4,SSEA-4,TRA-1-81) immunofluorescence staining,differentiated into teratomas in vivo(inject the iPSC into SCID mice) and embryniod bodies in vitro were performed to exam the pluripotency of the iPSC.Results Four days after being infected by lentivirus,the HuMSCs became round-shape; 10 days after infection,some embryonic stem(ESC)-like colonies appeared.Fourteen days after infection,picked up the regularly shaped colonies and cultured several passages.About 1.25％ HuMSCs were reprogrammed into iPSC.The iPSC presented clone-like growth like ESC.All the cells were positive to alkaline phosphatase staining and expressed the pluripotency genes.The iPSC also expressed the ESC specific proteins,and karyotype analysis showed normal chromosome caryotype (46,XY).Furthermore,the iPSC could form embryoid bodies in vitro,expressed alpha fetoprotein(AFP),smooth muscle actin(SMA) and β-tubulin.The iPSC could alsoform teratomas in vivo.Conclusion OCT4,SOX2,KLF4,c-Myc,NANOG,LIN-28 can reprogram HuMSCs into iPSC efficiently.