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Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

Kim JH, Jee MK, Lee SY, Han TH, Kim BS, Kang KS, Kang SK - PLoS ONE (2009)

Bottom Line: Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage.Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1.The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biotechnology, Seoul National University, Seoul, Republic of Korea.

ABSTRACT

Background: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages.

Methodology/principal findings and conclusions: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

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Effect of exogenic Oct4 and several stemness gene expressions on adipogenic, osteogenic, and chondrogenic differentiation potencies.(A) Oct4 knockdown actively induces adipogenic, osteogenic, and chondrogenic differentiation. Differentiation potency was evaluated by fat, bone, and chondrocyte staining and lineage specific gene expression pattern by RT-PCR or western blotting using tissue specific markers. (B) Exogenic stemness genes expression affect on mesodermal differentiation potency. After differentiation induction of Oct4/ATSCs and control ATSCs, the number of positive clones was quantified after fat (Oil Red O), bone (Van Kossa), and chondrocyte staining (Van Gieson) and data presented relative density of positive population (%). (C) Effect of stemness genes overexpression on differentiation of ATSCs into bone, fat, and chondrocyte and their lineage specific gene expression analysis after differentiation induction.
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pone-0007166-g005: Effect of exogenic Oct4 and several stemness gene expressions on adipogenic, osteogenic, and chondrogenic differentiation potencies.(A) Oct4 knockdown actively induces adipogenic, osteogenic, and chondrogenic differentiation. Differentiation potency was evaluated by fat, bone, and chondrocyte staining and lineage specific gene expression pattern by RT-PCR or western blotting using tissue specific markers. (B) Exogenic stemness genes expression affect on mesodermal differentiation potency. After differentiation induction of Oct4/ATSCs and control ATSCs, the number of positive clones was quantified after fat (Oil Red O), bone (Van Kossa), and chondrocyte staining (Van Gieson) and data presented relative density of positive population (%). (C) Effect of stemness genes overexpression on differentiation of ATSCs into bone, fat, and chondrocyte and their lineage specific gene expression analysis after differentiation induction.

Mentions: ATSCs have been identified as skeletal tissue progenitors, and differentiate into osteoblast-like cells in cultures supplemented with ascorbic acid with a glucocorticoid source. Calcium and lipid droplets begin to accumulate in ATSCs following 2–4 weeks of induction in osteogenic and adipogenic differentiation media. We studied the effect of Oct4 overexpression on mesodermal lineage differentiation into fat, bone, and chondrocyte of ATSCs. Oct4 knockdown ATSCs were shown to accumulate significant quantities of calcium and lipid droplets, and differences were apparent in the efficiency of nodule and lipid droplet formation between the naive and siOct4/ATSC. As shown in Figure 5A, up to three times as many lipid droplets and nodules were detected in siOct4/ATSCs compared to the control. After culturing siOct4/ATSCs (passage 5) in osteogenic differentiation media, cells were stained for calcium deposits. These results were generally consistent with what has been observed in conjunction with the overexpression of adipogenesis- and osteogenesis-related transcription factors, including RXR, osteonectin, AP, and PPAR-gamma, after Oct4 mediated cell reprogramming (Fig. 5A). Moreover, when mesodermal differentiation was induced of Oct/4, Sox2, and Nanog overexpressed ATSCs, the differentiation efficiencies of each gene was prominently decreased along with each lineage specific (Fig. 5A and 5B). To determine the transdifferentiation activity of the Oct4/ATSC, the neurogenic potency was evaluated through transdifferentiation into a neural lineage. After neural induction in differentiated Oct4/ATSCs, we observed an extreme upregulation of TuJ and MAP2ab and low levels of Nestin expression in Oct4/ATSCs after neural differentiation and also neurosphere formation efficiency was higher than control ATSCs (Fig. 6A). When we engrafted Oct4/ATSCs in the fetal mouse brain, the engrafted cells effectively transdifferentiated into TuJ- and NF160-positive neurons in contrast of that of control ATSCs 5 weeks after engraftment (Fig. 6D). A large population of differentiated Oct4/ATSCs showed phenotypic characteristics of astrocytes (GFAP), and neurons (MAP2ab and NF160 [approximately 45–60% of the total population]; Fig. 6D). The siOct4/ATSCs did not appear to have neurogenic morphology and neural gene expression 7–10 days after neural differentiation induction (data not showed).


Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

Kim JH, Jee MK, Lee SY, Han TH, Kim BS, Kang KS, Kang SK - PLoS ONE (2009)

Effect of exogenic Oct4 and several stemness gene expressions on adipogenic, osteogenic, and chondrogenic differentiation potencies.(A) Oct4 knockdown actively induces adipogenic, osteogenic, and chondrogenic differentiation. Differentiation potency was evaluated by fat, bone, and chondrocyte staining and lineage specific gene expression pattern by RT-PCR or western blotting using tissue specific markers. (B) Exogenic stemness genes expression affect on mesodermal differentiation potency. After differentiation induction of Oct4/ATSCs and control ATSCs, the number of positive clones was quantified after fat (Oil Red O), bone (Van Kossa), and chondrocyte staining (Van Gieson) and data presented relative density of positive population (%). (C) Effect of stemness genes overexpression on differentiation of ATSCs into bone, fat, and chondrocyte and their lineage specific gene expression analysis after differentiation induction.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747014&req=5

pone-0007166-g005: Effect of exogenic Oct4 and several stemness gene expressions on adipogenic, osteogenic, and chondrogenic differentiation potencies.(A) Oct4 knockdown actively induces adipogenic, osteogenic, and chondrogenic differentiation. Differentiation potency was evaluated by fat, bone, and chondrocyte staining and lineage specific gene expression pattern by RT-PCR or western blotting using tissue specific markers. (B) Exogenic stemness genes expression affect on mesodermal differentiation potency. After differentiation induction of Oct4/ATSCs and control ATSCs, the number of positive clones was quantified after fat (Oil Red O), bone (Van Kossa), and chondrocyte staining (Van Gieson) and data presented relative density of positive population (%). (C) Effect of stemness genes overexpression on differentiation of ATSCs into bone, fat, and chondrocyte and their lineage specific gene expression analysis after differentiation induction.
Mentions: ATSCs have been identified as skeletal tissue progenitors, and differentiate into osteoblast-like cells in cultures supplemented with ascorbic acid with a glucocorticoid source. Calcium and lipid droplets begin to accumulate in ATSCs following 2–4 weeks of induction in osteogenic and adipogenic differentiation media. We studied the effect of Oct4 overexpression on mesodermal lineage differentiation into fat, bone, and chondrocyte of ATSCs. Oct4 knockdown ATSCs were shown to accumulate significant quantities of calcium and lipid droplets, and differences were apparent in the efficiency of nodule and lipid droplet formation between the naive and siOct4/ATSC. As shown in Figure 5A, up to three times as many lipid droplets and nodules were detected in siOct4/ATSCs compared to the control. After culturing siOct4/ATSCs (passage 5) in osteogenic differentiation media, cells were stained for calcium deposits. These results were generally consistent with what has been observed in conjunction with the overexpression of adipogenesis- and osteogenesis-related transcription factors, including RXR, osteonectin, AP, and PPAR-gamma, after Oct4 mediated cell reprogramming (Fig. 5A). Moreover, when mesodermal differentiation was induced of Oct/4, Sox2, and Nanog overexpressed ATSCs, the differentiation efficiencies of each gene was prominently decreased along with each lineage specific (Fig. 5A and 5B). To determine the transdifferentiation activity of the Oct4/ATSC, the neurogenic potency was evaluated through transdifferentiation into a neural lineage. After neural induction in differentiated Oct4/ATSCs, we observed an extreme upregulation of TuJ and MAP2ab and low levels of Nestin expression in Oct4/ATSCs after neural differentiation and also neurosphere formation efficiency was higher than control ATSCs (Fig. 6A). When we engrafted Oct4/ATSCs in the fetal mouse brain, the engrafted cells effectively transdifferentiated into TuJ- and NF160-positive neurons in contrast of that of control ATSCs 5 weeks after engraftment (Fig. 6D). A large population of differentiated Oct4/ATSCs showed phenotypic characteristics of astrocytes (GFAP), and neurons (MAP2ab and NF160 [approximately 45–60% of the total population]; Fig. 6D). The siOct4/ATSCs did not appear to have neurogenic morphology and neural gene expression 7–10 days after neural differentiation induction (data not showed).

Bottom Line: Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage.Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1.The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biotechnology, Seoul National University, Seoul, Republic of Korea.

ABSTRACT

Background: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages.

Methodology/principal findings and conclusions: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

Show MeSH
Related in: MedlinePlus