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Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts.

ABSTRACT: Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (fetal bovine serum) proliferated and dedifferentiated into fibroblast-like cells. We referred to these cells as DFOG (dedifferentiated follicular granulosa) cells. Microarray analysis showed that DFOG cells lost expression of GC-specific marker genes, but gained the expression of osteogenic marker genes during dedifferentiation. After osteogenic induction, DFOG cells underwent terminal osteoblast differentiation and matrix mineralization in vitro. Furthermore, when DFOG cells were transplanted subcutaneously into SCID mice, these cells formed ectopic osteoid tissue. These results indicate that DFOG cells derived from GCs can differentiate into osteoblasts in vitro and in vivo. We suggest that GCs provide a useful model for studying the mechanisms of transdifferentiation into other cell lineages in functionally differentiated cells.

SUBMITTER: Oki Y

PROVIDER: S-EPMC3459222 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts.

Oki Yoshinao Y Ono Hiromasa H Motohashi Takeharu T Sugiura Nobuki N Nobusue Hiroyuki H Kano Koichiro K

The Biochemical journal 20121001 2

Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium s ...[more]

PMID: 22839299

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Project description:The aim of this study is the identification of genes and gene networks involved in pig ovarian follicular development. cDNA nylon micro-arrays (2849 sequenced clones) were designed from different libraries : four subtractive suppressive hybridization libraries (generated from small versus large and small versus medium follicles) and a pig multi-tissue cDNA library. Granulosa cells were isolated from healthy follicles (small, medium or large), 24 h or 96 h after the end of a progestagen treatment. The RNA isolated from these cells was used to hybridize the micro-arrays and hybridisations were performed in duplicate. The images were quantified using Bzscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Keywords: cell type comparison Granulosa cells were isolated from healthy follicles (small, medium or large). The RNA isolated from these cells was used to hybridize nylon micro-arrays. The arrays contained PCR products from 2848 pig cDNA clones (GPL3978), spotted in duplicate on two separate fields of the same membrane (last number 1 of the local ID corresponds to the left field and 2 corresponds to right field). The data have been generated from 14 RNA follicle pools (five Large, five Medium and four Small follicles pools). For each follicle pool, 2 radioactive labellings were performed (before last number of the local ID corresponds to the duplicate number). Each membrane was exposed 6, 12 or 24 hours (depending on the saturation signal) to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The imaging plates were scanned thereafter with a phosphor imaging system at a 25 µm resolution (BAS-5000, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The images were quantified using semi-automated software, BZScan (Lopez F, Rougemont J, Loriod B, Bourgeois A, Loi L, Bertucci F, Hingamp P, Houlgatte R, and Granjeaud S. Feature extraction and signal processing for nylon DNA microarrays. BMC Genomics 5: 38, 2004). Fixed circle segmentation, i.e. a grid process with a fixed spot diameter was applied. The hybridization images were quantified by the extraction of the intensity for each spot. A new version of BZsan (AGscan solfware) is available at : http://mulcyber.toulouse.inra.fr/projects/agscan/ The experimental data were managed using the free BASE software (Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 3: SOFTWARE0003, 2002.) modified by Sigenae team to accept radioactive experiments. Finally, data consisted in (14 RNA x 2 labellings x 2 fields) 14 probes, 28 hybridisations, 42 images (14 images were filtred out for background too hight).

Project description:These vector oligonucleotide hybridizations were performed to determine the quality of the spotting process. EST with low signal values of the vector array were excluded from the analysis, since they probably were mis-amplified or mis-spotted Keywords: spotting control cDNA arrays (GPL3978) were first hybridized with a vector oligonucleotide labeled (T7 probe) with 33P-ATP. After washing, the arrays were exposed 6 hours to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The imaging plates were scanned thereafter with a phosphor imaging system at a 25 µm resolution (BAS-5000, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The images were quantified using semi-automated software, BZScan (Lopez F, Rougemont J, Loriod B, Bourgeois A, Loi L, Bertucci F, Hingamp P, Houlgatte R, and Granjeaud S. Feature extraction and signal processing for nylon DNA microarrays. BMC Genomics 5: 38, 2004). Fixed circle segmentation, i.e. a grid process with a fixed spot diameter was applied. The hybridization images were quantified by the extraction of the intensity for each spot. A new version of BZsan (AGscan solfware) is available at : http://mulcyber.toulouse.inra.fr/projects/agscan/ The experimental data were managed using the free BASE software (Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 3: SOFTWARE0003, 2002.) modified by Sigenae team to accept radioactive experiments. After stripping, the arrays were hybridized with a complex target. The data correspond to the hybridization of the first membrane DEV-1 and the last one DEV-20. The arrays contained PCR products from 2848 pig cDNA clones (GPL3978), spotted in duplicate on two separate fields of the same membrane (last number 1 of the local ID corresponds to the left field and 2 corresponds to right field). Finally, data consisted in four data files and were used to delete clones with a bad quality of spotting.

Dataset Information

Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts.

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Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts.

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