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Epigenetic memory gained by priming with osteogenic induction medium improves osteogenesis and other properties of mesenchymal stem cells.

Rui Y, Xu L, Chen R, Zhang T, Lin S, Hou Y, Liu Y, Meng F, Liu Z, Ni M, Tsang KS, Yang F, Wang C, Chan HC, Jiang X, Li G - Sci Rep (2015)

Bottom Line: In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2.And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters.Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Orthopaedics, Zhongda Hospital, Southeast University, 87 Ding Jia Qiao, Nanjing 210009, Jiangsu, PR China [2] Department of Orthopaedics &Traumatology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, PR China.

ABSTRACT
Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

No MeSH data available.


Related in: MedlinePlus

De-Os-rMSCs exhibited advantages in clonogenicity, proliferation, cell survival and migration.(A) Clongenic assay. The untreated rMSCs and De-Os-rMSCs were plated at 100 cells per 20 cm2 dish and cultured for 10 days, respectively. The cells were then stained with 0.5% crystal violet to view the colonies. (B) Cell proliferation assay. The rMSCs and De-Os-rMSCs were plated at 2,000 cells/well in normal medium in a 96-well plate and incubated at 37 °C with 5% CO2. At day 3, cell proliferation was assessed using the BrdU assay as described in Materials and Methods. The data are expressed as mean ± SD (n = 3), *p < 0.05. (C) De-Os-rMSCs exhibited survival advantage over untreated rMSCs. The untreated rMSCs and De-Os-rMSCs were plated in 96-well plates. At days 3, these cells were challenged with 0–500 μM H2O2 for 12 hours. Then, the cells were incubated with Alamar Blue for 2.5 hours at 37 °C. The metabolic rate of the cells was determined at 570 nm, with reference wavelength at 600 nm. Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (D&E) De-Os-rMSCs exhibited advantage in migration over untreated rMSCs. An equal number of untreated rMSCs and De-Os-rMSCs suspended in α-MEM were added into the upper layer of BD Falcon cell culture insert, respectively, and the rMSCs migrated through the membrane were detected with crystal violet staining. The number of MSCs that passed through the membrane was counted (n = 3). Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (F&G) Total RNA were extracted from rMSCs and De-Os-rMSCs. The relative expression levels of p53 and CXCR4 were checked by qRT-PCR. β-actin was used as an internal control. The data are expressed as mean ± SD (n = 3), *p < 0.05.
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f3: De-Os-rMSCs exhibited advantages in clonogenicity, proliferation, cell survival and migration.(A) Clongenic assay. The untreated rMSCs and De-Os-rMSCs were plated at 100 cells per 20 cm2 dish and cultured for 10 days, respectively. The cells were then stained with 0.5% crystal violet to view the colonies. (B) Cell proliferation assay. The rMSCs and De-Os-rMSCs were plated at 2,000 cells/well in normal medium in a 96-well plate and incubated at 37 °C with 5% CO2. At day 3, cell proliferation was assessed using the BrdU assay as described in Materials and Methods. The data are expressed as mean ± SD (n = 3), *p < 0.05. (C) De-Os-rMSCs exhibited survival advantage over untreated rMSCs. The untreated rMSCs and De-Os-rMSCs were plated in 96-well plates. At days 3, these cells were challenged with 0–500 μM H2O2 for 12 hours. Then, the cells were incubated with Alamar Blue for 2.5 hours at 37 °C. The metabolic rate of the cells was determined at 570 nm, with reference wavelength at 600 nm. Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (D&E) De-Os-rMSCs exhibited advantage in migration over untreated rMSCs. An equal number of untreated rMSCs and De-Os-rMSCs suspended in α-MEM were added into the upper layer of BD Falcon cell culture insert, respectively, and the rMSCs migrated through the membrane were detected with crystal violet staining. The number of MSCs that passed through the membrane was counted (n = 3). Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (F&G) Total RNA were extracted from rMSCs and De-Os-rMSCs. The relative expression levels of p53 and CXCR4 were checked by qRT-PCR. β-actin was used as an internal control. The data are expressed as mean ± SD (n = 3), *p < 0.05.

Mentions: To determine whether De-Os-rMSCs exhibit any difference in stem cell potency, we evaluated the colony forming ability, cell proliferation, cell survival and cell migration in naïve rMSCs and De-Os-rMSCs. Our results showed that De-Os-rMSCs formed more and larger colonies as compared to untreated rMSCs Fig. 3A). This could be related to the increase in cell proliferation of De-Os-rMSCs, as demonstrated by Brdu assay (Fig. 3B). In addition, De-Os-rMSCs exhibited a survival advantage over untreated rMSCs under the circumstance of oxidative stress. As shown in Fig. 3C, De-Os-rMSCs had higher cell survival capacity compared to untreated rMSCs when both of them were challenged by H2O2 at different concentrations for 12 hours. As revealed by transwell migration assay, the migratory capability was also significantly enhanced in De-Os-rMSCs compared to untreated rMSCs (Fig. 3D,E). In addition, we also checked the expression levels of some genes that were known to be involved in cell proliferation and migration by qRT-PCR, and the result showed that p53 and CXCR4 were significantly increased in De-Os-rMSCs (Fig. 3F,G), implying that upregulation of p53 and CXCR4 might account for the increased proliferation and migration showed by De-Os-rMSCs.


Epigenetic memory gained by priming with osteogenic induction medium improves osteogenesis and other properties of mesenchymal stem cells.

Rui Y, Xu L, Chen R, Zhang T, Lin S, Hou Y, Liu Y, Meng F, Liu Z, Ni M, Tsang KS, Yang F, Wang C, Chan HC, Jiang X, Li G - Sci Rep (2015)

De-Os-rMSCs exhibited advantages in clonogenicity, proliferation, cell survival and migration.(A) Clongenic assay. The untreated rMSCs and De-Os-rMSCs were plated at 100 cells per 20 cm2 dish and cultured for 10 days, respectively. The cells were then stained with 0.5% crystal violet to view the colonies. (B) Cell proliferation assay. The rMSCs and De-Os-rMSCs were plated at 2,000 cells/well in normal medium in a 96-well plate and incubated at 37 °C with 5% CO2. At day 3, cell proliferation was assessed using the BrdU assay as described in Materials and Methods. The data are expressed as mean ± SD (n = 3), *p < 0.05. (C) De-Os-rMSCs exhibited survival advantage over untreated rMSCs. The untreated rMSCs and De-Os-rMSCs were plated in 96-well plates. At days 3, these cells were challenged with 0–500 μM H2O2 for 12 hours. Then, the cells were incubated with Alamar Blue for 2.5 hours at 37 °C. The metabolic rate of the cells was determined at 570 nm, with reference wavelength at 600 nm. Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (D&E) De-Os-rMSCs exhibited advantage in migration over untreated rMSCs. An equal number of untreated rMSCs and De-Os-rMSCs suspended in α-MEM were added into the upper layer of BD Falcon cell culture insert, respectively, and the rMSCs migrated through the membrane were detected with crystal violet staining. The number of MSCs that passed through the membrane was counted (n = 3). Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (F&G) Total RNA were extracted from rMSCs and De-Os-rMSCs. The relative expression levels of p53 and CXCR4 were checked by qRT-PCR. β-actin was used as an internal control. The data are expressed as mean ± SD (n = 3), *p < 0.05.
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Related In: Results  -  Collection

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f3: De-Os-rMSCs exhibited advantages in clonogenicity, proliferation, cell survival and migration.(A) Clongenic assay. The untreated rMSCs and De-Os-rMSCs were plated at 100 cells per 20 cm2 dish and cultured for 10 days, respectively. The cells were then stained with 0.5% crystal violet to view the colonies. (B) Cell proliferation assay. The rMSCs and De-Os-rMSCs were plated at 2,000 cells/well in normal medium in a 96-well plate and incubated at 37 °C with 5% CO2. At day 3, cell proliferation was assessed using the BrdU assay as described in Materials and Methods. The data are expressed as mean ± SD (n = 3), *p < 0.05. (C) De-Os-rMSCs exhibited survival advantage over untreated rMSCs. The untreated rMSCs and De-Os-rMSCs were plated in 96-well plates. At days 3, these cells were challenged with 0–500 μM H2O2 for 12 hours. Then, the cells were incubated with Alamar Blue for 2.5 hours at 37 °C. The metabolic rate of the cells was determined at 570 nm, with reference wavelength at 600 nm. Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (D&E) De-Os-rMSCs exhibited advantage in migration over untreated rMSCs. An equal number of untreated rMSCs and De-Os-rMSCs suspended in α-MEM were added into the upper layer of BD Falcon cell culture insert, respectively, and the rMSCs migrated through the membrane were detected with crystal violet staining. The number of MSCs that passed through the membrane was counted (n = 3). Values are expressed as mean ± SD of three independent experiments. *p < 0.05. (F&G) Total RNA were extracted from rMSCs and De-Os-rMSCs. The relative expression levels of p53 and CXCR4 were checked by qRT-PCR. β-actin was used as an internal control. The data are expressed as mean ± SD (n = 3), *p < 0.05.
Mentions: To determine whether De-Os-rMSCs exhibit any difference in stem cell potency, we evaluated the colony forming ability, cell proliferation, cell survival and cell migration in naïve rMSCs and De-Os-rMSCs. Our results showed that De-Os-rMSCs formed more and larger colonies as compared to untreated rMSCs Fig. 3A). This could be related to the increase in cell proliferation of De-Os-rMSCs, as demonstrated by Brdu assay (Fig. 3B). In addition, De-Os-rMSCs exhibited a survival advantage over untreated rMSCs under the circumstance of oxidative stress. As shown in Fig. 3C, De-Os-rMSCs had higher cell survival capacity compared to untreated rMSCs when both of them were challenged by H2O2 at different concentrations for 12 hours. As revealed by transwell migration assay, the migratory capability was also significantly enhanced in De-Os-rMSCs compared to untreated rMSCs (Fig. 3D,E). In addition, we also checked the expression levels of some genes that were known to be involved in cell proliferation and migration by qRT-PCR, and the result showed that p53 and CXCR4 were significantly increased in De-Os-rMSCs (Fig. 3F,G), implying that upregulation of p53 and CXCR4 might account for the increased proliferation and migration showed by De-Os-rMSCs.

Bottom Line: In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2.And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters.Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Orthopaedics, Zhongda Hospital, Southeast University, 87 Ding Jia Qiao, Nanjing 210009, Jiangsu, PR China [2] Department of Orthopaedics &Traumatology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, PR China.

ABSTRACT
Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

No MeSH data available.


Related in: MedlinePlus