Effects Of Different Dimensions Of Culture On The Stemness Of Mesenchymal Stem Cells And Its Mechanism

Part 1 Investigation of the different effects of 2D and 3D culture on the stemness of mesenchymal stem cells and the mechanismObjective: Mesenchymal stem cells(MSCs)are a class of adult stem cells with self-renewal,multidirectional differentiation potential,extensive proliferation and paracrine functions.Due to the advantages of non-ethical controversy,low tumorigenicity and low immunogenicity,adipose mesenchymal stem cells(ADSCs),placental mesenchymal stem cells(PD-MSCs),etc.have been used as an important source of seed cells for cell therapy and tissue engineering.Related studies have shown that traditional two-dimensional(2D)cell culture cannot replicate the real in vivo microenvironment,leading to a decrease in stemness of stem cells in long-term culture;whereas,the use of 3D culture can lead to stronger stemness of some MSCs than 2D culture,but the mechanism is not clear.It was shown that MEK/ERK signaling pathway activation and inhibition can regulate the expression of pluripotency transcription factors in stem cells and that cytochalasins relax the cytoskeleton to regulate the expression of transcription factors.The aim of this study is to investigate the different effects of 3D and 2D culture on the stemness of human ADSCs and PD-MSCs,and to make a preliminary exploration of the possible mechanisms from different signaling pathways.Methods:(1)Characterization of ADSCs and PD-MSCs under 3D culture,cell spheres were prepared using 96 U low adsorption plates,the sphere-forming effect of human adipose stem cell spheres(ADSCs)and human placental stem cell spheres(PD-MSCs)cells under different days was observed by scanning electron microscopy,and the internal survival of cell spheres was observed by dead or alive staining.(2)The expression of positive markers CD44 and CD90 and negative markers CD34 and CD45 on the surface of human adipose stem cells(ADSCs)and human placental stem cells(PD-MSCs)were detected by flow cytometry.For both human adipose stem cells(ADSCs)and human placental stem cells(PD-MSCs),1,000 cells each were grown in 6-well plates for 14 days.Crystalline violet staining was performed to detect the clonogenic ability of ADSCs and PD-MSCs after 2D and 3D culture.(3)Differences in the expression of pluripotency transcription factors between ADSCs and PD-MSCs were compared under 2D and 3D culture conditions,and the expression of transcription factors Nanog,Oct4 and Sox2 was detected by immunofluorescence,RT-q PCR and Western blot in both modalities.(4)Differential expression of MEK/ERK signaling pathway in ADSCs and PD-MSCs were compared under 2D and 3D culture conditions,and the expression of transcription factors Nanog,Oct4,and Sox2 were detected by Western blot.The expression of the three stem cell transcription factors Nanog,Oct4,and Sox2 was detected by Western blot after the addition of MEK/ERK signaling pathway activator FGF4 and inhibitor PD 98059 under different dimensional culture conditions.To observe whether MEK/ERK signaling pathway is involved in the mechanism by which culture dimension affects the stemness of ADSCs and PD-MSCs.(5)Different concentrations of cytochalasin(CYD)were added and the expression of stem cell transcription factors Nanog,Oct4,and Sox2 was detected by Western blot.To observe whether the cytoskeleton is involved in the different dimensions on the stemness of ADSCs.Results:(1)Scanning electron microscopy results showed that human adipose stem cell spheres(ADSCs)and human placental stem cell spheres(PD-MSCs)formed spheres best at 5 to 7 days,with more regular and firm spheres.The dead and live staining results showed that both cell spheres had live cells on the outside of the spheres after 5 days of culture,with a few dead cells on the inside of the spheres.(2)The immunophenotypic as well as proliferation differences between ADSCs and PD-MSCs were compared under 2D and 3D culture conditions.The crystalline violet staining results showed that the 3D group had stronger clonogenic ability than the 2D group.Flow cytometric results showed that positive stem cell markers CD44 and CD90 decreased and negative markers CD34 and CD45 increased in the 3D group of ADSCs and PD-MSCs compared with the 2D group.(3)Immunofluorescence results showed that the fluorescence intensity of ADSCs and PD-MSCs 3D cytosolic stemness factors(Nanog,Oct4,Sox2)were significantly stronger than 2D culture.RT-q PCR showed that the expression of ADSCs cytosolic stemness factors Nanog and Oct4 m RNA were higher than 2D group(P<0.01).PD-MSCs cytosolic stemness factors Nanog,Oct4,Sox2 m RNA expression were significantly higher than 2D group(P<0.01).The expression of stemness factors Nanog,Oct4,and Sox2 m RNA were significantly higher in ADSCs than in 2D group(P<0.01).western blot examined the effect of 2D and 3D culture methods on the three stemness transcription factors,and the expression of Nanog and Sox2 in ADSCs was higher in 3D than in 2D(P<0.01),and the expression of Nanog,Sox2 in PD-MSCs was higher in 3D than in 2D(P<0.01).Sox2 expression was higher in 3D than in 2D(P<0.05).(4)The expression differences of MEK/ERK signaling pathway in ADSCs and PD-MSCs were compared under 2D and 3D culture conditions.western blot detected the expression of transcription factors Nanog,Oct4,Sox2.there was no difference in the expression of MEK,phosphorylated(P-MEK)expression,3D group was lower than 2D group(P<0.05).expression of ERK The results showed no significant difference between 3D and 2D groups,phosphorylated(P-ERK)expression,ADSCs3 D group was lower than 2D group(P<0.05)PD-MSCs 3D group was lower than 2D group(P<0.01).However,after activation or inhibition of MEK/ERK signaling pathway in 2D and 3D culture conditions,Western blot assays showed that the expression of three stem cell transcription factors Nanog,Oct4 and Sox2 in ADSCs and PD-MSCs did not differ significantly in different dimensional culture situations.(5)After administration of different doses of cytoleptin,Western Blotting results showed that the expression of the stem cell transcription factor Nanog,Oct4,Sox2 was downregulated relative to the normal group(P<0.05).Conclusion:(1)Human adipose stem cells and placental stem cells 3D cell spheres showed a decrease in stem cell positive indicators CD44 and CD90 compared to 2D culture.(2)Human adipose stem cells and placental stem cells cultured in 3D cell spheres showed significantly enhanced expression of the stemness transcription factors Nanog,Oct4,Sox2 genes and proteins compared to 2D culture,and this change was independent of the cytoskeleton as well as the MEK/ERK signaling pathway.Part 2 Effect of culture dimension switch on stemness of human umbilical cord stem cellsObjective: A previous study found that culture dimensional shift could affect the change of stemness transcription factors in rat adipose stem cells and subsequently alter stemness.In this study,primary human umbilical cord mesenchymal stem cells(UC-MSCs)were used to observe the alteration of stemness transcription factor expression under the change of culture dimension switch.Methods:(1)Stem cell identification of UC-MSCs.Flow cytometry was performed to detect the expression of positive markers CD44 and CD90 and negative markers CD34 and CD45 on the surface of primary human umbilical cord MSCs.(2)Effect of conversion of different dimensional cultures on the stemness of UC-MSCs.Human primary umbilical cord mesenchymal stem cells(UC-MSC)were extracted and digested and directly primaryed into two stages of culture;the first stage of 2D and 3D cell sphere culture for 6 days,the second stage was divided into 2D culture for 6 days after digestion and continued with 2D culture for 6 days(2D-2D group),2D culture for 6 days after digestion and converted to 3D cell sphere culture for 6 days(2D-3D group),3D cell sphere culture for 6 days after digestion and Continued with 3D cell sphere culture for 6 days(3D-3D group),3D cell sphere culture for 6 days after digestion converted to 2D culture for 6 days(2D-3D group).Immunofluorescence and RT-q PCR were performed to detect the expression of first and second stage transcription factors Nanog,Oct4 and Sox2,respectively.Results:(1)Primary cultured umbilical cord MSCs showed positive stem cell markers(CD44 and CD90 >90% and negative markers CD34 and CD45 below 5%)by flow analysis,consistent with stem cell characteristics.(2)Immunofluorescence experiments of primary 2D and 3D groups revealed that the 3D group was stronger than the 2D group.m RNA expression of stem transcription factors(Nanog,Oct4,Sox2)was detected by RT-q PCR,and the 3D group was stronger than the 2D group(P<0.01).After the conversion of different culture dimensions,the fluorescence intensity and m RNA expression of the four groups were compared,and the expression of three transcription factors(Nanog,Sox2,Oct4)was significantly higher in the 2D-3D group than in the 2D-2D,3D-2D and 3D-3D groups by RT-q PCR(P<0.01),and the expression of three transcription factors was stronger in the 3D-3D group than in the 3D-2D group(P< 0.01).Conclusion:(1)Stemness of umbilical cord primary stem cell culture was significantly enhanced after conversion from 2D culture to 3D cell sphere culture.(2)Stemness of umbilical cord primary stem cell culture decreased after conversion from 3D cell sphere culture to 2D culture.