| Objectives:1. To observe and investigate the changes of in vitro cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) in the process of morphology, phenotype, growth dynamics and differentiation capacity;2. To explore the different gene expression profiles of young and old hUC-MSCs and discuss the possible mechanisms.Methods:1. Cell culture:hUC-MSCs were isolated from human umbilical cord by attachment method. Cells at passage3were served as the control group and those at passage15were considered as the aged group. Morphology of those two kinds of cells was observed under normal microscope and scanning electron microscope.2. Growth and proliferation:both control and aged group were cultured in96-hole plates, each of them had8repeated holes and was cultivated for6days. OD values were measured at450nm wavelength everyday in order to draw cell growth curves.3. Phenotype detection:cells of control and aged groups were digested into single cell liquid. Phenotypes of CD34, CD45, CD44and CD105were detected by flow cytometry.4. Differentiation capacity:cells of control and aged groups were respectively cultured in osteogenic and adipogenic induction medium. Cells were fixed and dyed after14days in order to observe the difference between the two groups.5. Gene expression profile analysis:cells were washed and disposed with Trizol in order to extract total RNA. The differences of gene expression were revealed with the whole human genome oligo microarray and data was detected with standardization analysis. Several differential genes were selected for further confirmation by Gene Ontology and KEGG pathway.6. PCR test:According to the results of gene expression profiles, several differently expressed genes of the two groups were selected for further confirmation by quantitative reverse transcription-polymerase chain reaction. Those genes included BAX, FADD, JAK2, IGF3BP5, PDK1, Col-Ⅲ, Col-Ⅳ, SMAD2, SAMD4, BMPR2, CCND2, etc.Results1. Cell morphology:the third passage cells were fibroid and slender and there were about500cells per square millimeter while the aged cells grew bigger and wider in size and150could be found per square millimeter. When observed under scanning electron microscope, the third passage cells were plump and had a lot of microvilli distributed uniformly, but aged cells were more flat and had many pseudopods.2.Growth and proliferation:cells of both control group and aged group had a stagnant period for about12to24hours after inoculation, then got into the proliferation stage. Cells of the control group grew more quickly and on the fifth day they entered the platform period with a larger amount of cells. Aged cells had a longer stagnant period and they got to platform period quickly and the total amount was also less.3. Phenotype detection:by testing cell phenotypes of both control and aged groups, results showed that expressions of CD34and CD45were negative, CD105and CD44were obviously positive. There was no difference between the two groups.4. Differentiation capacity:after being cultured in osteogenic and adipogenic induction medium for14days, cells of both control and aged groups could be induced into osteoblasts and adipocytes. There was calcific extracellular matrix among the osteogenic induced cells. By using the alizarin red S and alkaline phosphatase (ALP) staining, most cells had positive signals. When cultured in adipogenic induction medium for14days, there were about50percent cells formed lipochondrion which showed positive signals by oil red O staining. Results showed that both control and aged cells had the characteristics of MSC, but the aged cells had a decreased ability to differentiate.5. Gene expression profiling:according to KEGG pathway analysis, compared with control group there were20up-regulated and49down-regulated pathways (p<0.05) in aged cells. Those pathways correlate to synthetic biology, metabolism, autoimmune diseases, degenerative diseases, extracellular matrix and cell proliferation, etc. There were more than8thousand genes expressed differently between cells of two groups among the tested41thousand genes.2000genes were selected for Gene Ontology analysis, those genes were found to be associated with DNA transcription, RNA translation, synthetic biology, metabolism, signal transduction, cell proliferation, migration and connection, etc.7. PCR test:differently expressed genes were checked by quantitative reverse transcription-polymerase chain reaction. Compared with control group, genes such as BAX, FADD, JAK2, IGF3BP5, PDK1and MGMT were up-regulated in aged cells and Col-Ⅲ, Col-Ⅳ, SMAD2, SAMD4, BMPR2, CCND2and PAK2were down-regulated.Conclusions1.Cells of both passage3and passage15had the ability to proliferation, osteogenic and adipogenic differentiation and had the same phenotypes, but compared with cells of control group, the ability of aged cells were decreased..2. Functional categories showed that the down-regulated genes and pathways in aged cells concentrate upon those related to cell proliferation, migration, metabolism, synthesis, connection and extracellular matrix meanwhile those up-regulated genes in aged cells relate to autoimmune diseases and degenerative diseases3. After hUC-MSCs were cultured in vitro for several passages, their morphologies became larger and more flat. Cells became senescent due to the declines in metabolism, proliferation and differentiation activities. |
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