Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),

Abstract Abstract 3415 Introduction: The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal. Methods: As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC). Results: There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1. Conclusion: SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing. Acknowledgments: This project was supported by NIAID U191A168021-06 and NIA R21AG034254. Disclosures: No relevant conflicts of interest to declare.

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Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro

Blood ◽

10.1182/blood.v120.21.3468.3468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3468-3468

Author(s):

Xiaoyang Ling ◽

Ye Chen ◽

Peter P. Ruvolo ◽

Vivian Ruvolo ◽

Zhiqiang Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Biology ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Bone Marrow Niche ◽

In Vivo Experiments ◽

Cell Gene Expression

Abstract Abstract 3468 Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology. Disclosures: No relevant conflicts of interest to declare.

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Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Differences Underlying Variations in Pathogenicity

mBio ◽

10.1128/mbio.00969-14 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 113

Author(s):

Christophe Bécavin ◽

Christiane Bouchier ◽

Pierre Lechat ◽

Cristel Archambaud ◽

Sophie Creno ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Cell Biology ◽

Virulence Genes ◽

Constitutive Expression ◽

Nucleotide Polymorphisms ◽

Pathogenic Potential ◽

Bacterial Gene ◽

Content Type

ABSTRACTFor nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogenListeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strainin vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infectionin vivo, highlighting the fact thatin vitroinvasion assays are not sufficient for evaluating the pathogenic potential ofL. monocytogenesstrains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using differentL. monocytogenesstrains.IMPORTANCEOver the past 3 decades,Listeriahas become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studyingListeriause primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator ofListeriavirulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may useListeriaas a tool to study the pathophysiology of listeriosis and immune responses.

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Gene Expression Studies Reveal a Potential Mechanism of Glivec Resistance in Patients with Ph+ ALL Treated.

Blood ◽

10.1182/blood.v106.11.4374.4374 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4374-4374

Author(s):

To Ha Loi ◽

David D.F. Ma

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

B Cell ◽

Cell Lines ◽

Unexpected Finding ◽

B Cell Differentiation ◽

Down Regulation ◽

Blast Cells

Abstract In Ph+ ALL, patients respond to Glivec but nearly always relapse due to acquired resistance. The biological processes associated with Glivec resistance in Ph+ ALL are yet to be fully elucidated. The aim of this study is to use microarray and Q-PCR technology to dissect changes in signaling pathways of blast cells in Ph+ ALL patients treated with Glivec. Peripheral blood (PB) samples were taken before treatment and on consecutive days after administration of Glivec from two Ph+, c-ALLA+ and CD19+ ALL patients. Blast cells were isolated and their gene expression assayed using 19K cDNA microarrays. Over 400 differentially expressed genes were identified with at least a 1.5-fold up- or down-regulation in treated cells compared to cells collected pre-treatment. Based on these gene expression results, three main gene ontology groups were further evaluated: Apoptosis, Proliferation and B cell differentiation. The up-regulation of Bim and Bcl-6, and down regulation of Cyclin D2, confirms the induction of apoptosis via the FOXO3a pathway in cells treated with Glivec in vivo. Interestingly, both the proliferation genes, Tcl1-A and PKCe, and B cell differentiation associated genes, including CD79a, ETS1 and a cohort of IGH and Igl and k genes, were up-regulated during therapy. These gene expression changes observed in vivo were confirmed by Q-PCR in the Ph+ cell lines K562 (derived from CML blast crisis) and SUP-B15 (derived from ALL) treated with Glivec. The unexpected finding of increased expression of pro-proliferative genes and B cell differentiation genes by microarray revealed potential links with early B cell development and B cell receptor (BCR) signaling. Evidence for apoptosis and proliferation of Ph+ cell lines treated with Glivec were then examined by FACs. After 5 days of treatment with Glivec, 90% of K562 and 50% SUP-B15 cells underwent apoptosis. Furthermore, cell cycle analysis revealed the existence of a population of cells in G2 phase even after 6 days of Glivec treatment in SUP-B15 but not K562 cells, thus providing evidence of a population of cells undergoing proliferation during Glivec treatment in vitro. In summary, our in vivo observations supported by in vitro experiments suggest that Glivec induces the majority of Ph+ ALL blasts to undergo apoptosis. However, as treatment is prolonged, a population of ALL cells escapes death and undergoes proliferation and differentiation. We hypothesise that Glivec induced differentiation and proliferation of Ph+ cells may result in the clonal enrichment of cells resistant to Glivec.

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Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2566 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2566-2575 ◽

Cited By ~ 48

Author(s):

T E Blank ◽

M P Woods ◽

C M Lebo ◽

P Xin ◽

J E Hopper

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Strong Support ◽

Constitutive Expression ◽

Mutant Proteins ◽

Binding Characteristics ◽

Isolation And Characterization ◽

In Cells

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

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A Comparison of Gene Expression Signatures from Breast Tumors and Breast Tissue Derived Cell Lines

Disease Markers ◽

10.1155/2001/850531 ◽

2001 ◽

Vol 17 (2) ◽

pp. 99-109 ◽

Cited By ~ 83

Author(s):

Douglas T. Ross ◽

Charles M. Perou

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Cell Biology ◽

Expression Patterns ◽

Tumor Biology ◽

Clinical Samples ◽

Analysis Tool

Cell lines derived from human tumors have historically served as the primary experimental model system for exploration of tumor cell biology and pharmacology. Cell line studies, however, must be interpreted in the context of artifacts introduced by selection and establishment of cell linesin vitro. This complication has led to difficulty in the extrapolation of biology observed in cell lines to tumor biologyin vivo. Modern genomic analysis tool like DNA microarrays and gene expression profiling now provide a platform for the systematic characterization and classification of both cell lines and tumor samples. Studies using clinical samples have begun to identify classes of tumors that appear both biologically and clinically unique as inferred from their distinctive patterns of expressed genes. In this review, we explore the relationships between patterns of gene expression in breast tumor derived cell lines to those from clinical tumor specimens. This analysis demonstrates that cell lines and tumor samples have distinctive gene expression patterns in common and underscores the need for careful assessment of the appropriateness of any given cell line as a model for a given tumor subtype.

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Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture

10.1101/2021.03.07.434268 ◽

2021 ◽

Author(s):

Anne E.C. Nichols ◽

Sarah E. Miller ◽

Luke J. Green ◽

Michael S. Richards ◽

Alayna E. Loiselle

Keyword(s):

Cell Biology ◽

Tendon Healing ◽

Marker Genes ◽

Cell Models ◽

Isolation Method ◽

Tendon Cell ◽

Tendon Cells ◽

Tissue Systems

AbstractTendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell sub-populations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation method preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior.Statement of clinical significanceThe development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.

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Species-specific differences in coumarin-induced hepatotoxicity as an example toxicogenomics-based approach to assessing risk of toxicity to humans

Human & Experimental Toxicology ◽

10.1177/0960327107087910 ◽

2008 ◽

Vol 27 (1) ◽

pp. 23-35 ◽

Cited By ~ 35

Author(s):

T Uehara ◽

N Kiyosawa ◽

T Shimizu ◽

K Omura ◽

M Hirode ◽

...

Keyword(s):

Gene Expression ◽

Diclofenac Sodium ◽

Species Difference ◽

Human Hepatocytes ◽

Differentially Expressed ◽

Glutathione Metabolism ◽

Marker Genes ◽

Species Specific

One expected result from toxicogenomics technology is to overcome the barrier because of species-specific differences in prediction of clinical toxicity using animals. The present study serves as a model case to test if the well-known species-specific difference in the toxicity of coumarin could be elucidated using comprehensive gene expression data from rat in-vivo, rat in-vitro, and human in-vitro systems. Coumarin 150 mg/kg produced obvious pathological changes in the liver of rats after repeated administration for 7 days or more. Moreover, 24 h after a single dose, we observed minor and transient morphological changes, suggesting that some early events leading to hepatic injury occur soon after coumarin is administered to rats. Comprehensive gene expression changes were analyzed using an Affymetrix GeneChip® approach, and differentially expressed probe sets were statistically extracted. The changes in expression of the selected probe sets were further examined in primary cultured rat hepatocytes exposed to coumarin, and differentially expressed probe sets common to the in-vivo and in-vitro datasets were selected for further study. These contained many genes related to glutathione metabolism and the oxidative stress response. To incorporate human data, human hepatocyte cultured cells were exposed to coumarin and changes in expression of the bridging gene set were examined. In total, we identified 14 up-regulated and 11 down-regulated probe sets representing rat–human bridging genes. The overall responsiveness of these genes to coumarin was much higher in rats than humans, consistent with the reported species difference in coumarin toxicity. Next, we examined changes in expression of the rat–human bridging genes in cultured rat and human hepatocytes treated with another hepatotoxicant, diclofenac sodium, for which hepatotoxicity does not differ between the species. Both rat and human hepatocytes responded to the marker genes to the same extent when the same concentrations of diclofenac sodium were exposed. We conclude that toxicogenomics-based approaches show promise for overcoming species-specific differences that create a bottleneck in analysis of the toxicity of potential therapeutic treatments.

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Atacicept (TACI-Ig) Inhibits Growth of TACIhigh Primary Myeloma Cells in SCID-Hu Mice.

Blood ◽

10.1182/blood.v108.11.842.842 ◽

2006 ◽

Vol 108 (11) ◽

pp. 842-842

Author(s):

Shmuel Yaccoby ◽

Stacey R. Dillon ◽

Wen Ling ◽

Paul Perkins ◽

John R. Tisdale ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Biology ◽

B Lymphocyte ◽

Predictive Marker ◽

Cell Types ◽

Tumor Burden ◽

Delayed Growth

Abstract Two TNF family members known to play key roles in normal B cell biology, BLyS/BAFF (B-lymphocyte stimulator/B cell activating factor) and APRIL (A PRoliferation-Inducing Ligand), also promote the survival of various malignant B cell types, including multiple myeloma (MM). BLyS binds to 3 TNF-R-related receptors, BCMA (B-cell maturation antigen, TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF-receptor), whereas APRIL binds to TACI and BCMA and to heparan sulfate proteoglycans (HSPG) such as CD138. All MM cells express HSPG and one or more of these 3 receptors, and incubation of MM cells with BLyS and/or APRIL leads to enhanced survival of these malignant cells in vitro. Stromal cells and osteoclasts residing in the bone marrow (BM) produce BLyS and APRIL and thus provide a paracrine source of these survival factors MM cells. Inhibition of BLyS and APRIL in vitro using a soluble receptor, TACI-Ig, causes cultured MM cells to die rapidly. It has been shown (Moreaux et al, Blood 106:1021) that differences in TACI gene expression can distinguish tumors with a BM microenvironment dependence signature (TACIhigh) from those with a plasmablastic signature (TACIlow), suggesting that TACIhigh MM cells may be more sensitive to growth factor withdrawal. We tested the ability of atacicept (TACI-Ig) to inhibit MM growth in the SCID-hu model of MM (Yaccoby et al, Blood 92:2908). Myelomatous SCID-hu mice were constructed by implanting a human fetal bone into which primary MM cells were directly injected. Changes in levels of tumor burden were monitored by weekly measurements of serum human monotypic immunoglobulins (hIg), and confirmed by histology. We compared the ability of atacicept and BAFFR-Ig (which binds BLyS, but not APRIL) to inhibit MM cell growth in this model. Upon establishment of myeloma growth, mice were injected intraperitoneally with atacicept (5 or 10 mg/kg, 3 times a wk; n=7), BAFFR-Ig (10 mg/kg, 3 times a wk; n=5) or with vehicle (PBS; n=7) for 6 wks. Compared to controls, in which tumor burden (hIg) increased by 1007±260% (avg±SEM) from pre-treatment levels, atacicept treatment markedly reduced tumor burden in 5 experiments in which TACIhigh MM cells were used, and delayed growth or had no effect in 2 experiments using TACIlow MM cells (avg increase in tumor burden of 302±184% for all 7 experiments; p<0.01 vs. control). Inhibition of tumor infiltration was confirmed by histology. Preliminary data also indicate that atacicept can inhibit growth of bortezomib-resistant MM cells in vivo. The level of TACI gene expression by MM cells generally correlated with response to atacicept. In vitro MM cell/osteoclast co-culture experiments confirmed these results, as atacicept inhibited growth of TACIhigh MM cells, but had a minimal effect on growth of TACIlow MM cells. In contrast, BAFFR-Ig treatment in the SCID-hu model resulted in tumor reduction in 1 experiment, delayed growth in 2 experiments and no effect in 2 experiments (avg increase in hIg of 762±298 and 208±46% in control and BAFFR-Ig groups, respectively; p<0.078), suggesting that APRIL plays a non-redundant role in survival of tumor cells from certain MM patients. Our results suggest that atacicept might represent an important new treatment for MM, and that TACI gene expression may prove to be a useful predictive marker for this therapeutic intervention.

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

Biochemical Journal ◽

10.1042/bj20120172 ◽

2012 ◽

Vol 447 (2) ◽

pp. 239-248 ◽

Cited By ~ 17

Author(s):

Yoshinao Oki ◽

Hiromasa Ono ◽

Takeharu Motohashi ◽

Nobuki Sugiura ◽

Hiroyuki Nobusue ◽

...

Keyword(s):

Granulosa Cells ◽

Cell Biology ◽

Cell Lineage ◽

Marker Genes ◽

Specific Cell ◽

Specific Marker ◽

Matrix Mineralization ◽

Differentiated Cells

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.

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