SOX9 Gene Expression Research Articles

Static Magnetic Field Effect on Cell Alignment, Growth, and Differentiation in Human Cord-Derived Mesenchymal Stem Cells.

This investigation is performed to evaluate the impact of static magnetic field on the Cell growth alignment, and differentiation potential in Human Mesenchymal Stem cells derived from human newborn cords. In vitro-cultured mesenchymal stem cells derived from human newborn cords were exposed to SMF up to 24mT and compared with the control (unexposed) cultures. Viability was assessed via Trypan Blue staining and MTT assay. Cell cycle progression was studied after flow cytometry data analysis. Sox-2, Nanong, and Oct-4 Primers used for RT-PCR experiment. Morphological studies showed that the exposed cells were significantly aligned in parallel bundles in a correlation with the magnetic field lines. Viability measurements showed a significant reduction in cell viability which was noted after exposure to static magnetic field and initiated 36h after the end of exposure time. Flow cytometric data analysis confirmed a decrease in G1 phase cell population within the treated and cultured groups compared with the corresponding control samples. However, the induced changes were recovered in the cell cultures after the post-exposure culture recovery time which may be attributed to the cellular repair mechanisms. Furthermore, the proliferation rate and Oct-4 gene expression were reduced due to the 18mT static magnetic field exposure. The significant proliferation rate decrease accompanied by the Sox-2, Nanong, and Oct-4 gene expression decline, suggested the differentiation inducing effects of SMF exposure. Exposure to Static Magnetic fields up to 24mT affects mesenchymal stem cell alignment and proliferation rate as well as mRNA expression of Sox-2, Nanong, and Oct-4 genes, therefore can be considered as a new differentiation inducer in addition to the other stimulators.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia.

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation.

Scleraxis Is Essential for Tendon Differentiation by Equine Embryonic Stem Cells and in Equine Fetal Tenocytes.

The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes. Strikingly, differentiating ESCs and fetal tenocytes without scleraxis failed to reorganize a three-dimensional (3D) matrix and generate artificial tendons. This was associated with a significantly reduced survival. In contrast, there was no effect on the survival and remodeling capacity of adult tenocytes following scleraxis knockdown. Overexpression of scleraxis in fetal tenocytes rescued gene expression, cell survival in 3D, and subsequent matrix contraction. Together, these results demonstrate that scleraxis is not only essential for ESC differentiation into tenocytes but that it also has an active role in maintaining fetal tenocytes, which is then redundant in adult tenocytes.

Expression of Sox5, Sox8 and Sox9 Genes in Golden Pompano Trachinotus ovatus (Linnaeus 1758) Larvae During Ontogeny and in Response to Water Temperature

Sox5, Sox8, and Sox9 genes in golden pompano larvae were cloned and analyzed in this study. In the first trial, the expression of Sox genes during ontogeny of fish larvae in the first 18 days was examined, after which the expression of Sox genes in tissues was evaluated on 18 days post hatching (DPH). Subsequently, the response of Sox genes to water temperature (23, 26, and 29oC) on day 12 DPH and 18 DPH were compared. The expression levels of Sox8, Sox9a, and Sox9b peaked around 2 DPH before first feeding during fish ontogeny, and remained at low levels from 3-18 DPH. The expressions of Sox5 and Sox9a were affected by water temperature on 12 DPH and 18 DPH, while the expression of Sox8 was not affected by water temperature on 12 DPH and 18 DPH. The expression of Sox9b was affected by water temperature on 12 DPH. This study detected gene expression of Sox5, Sox8, and Sox9 at the early stage of development of golden pompano larvae. The time dependent expression of Sox genes in fish larvae is important for the understanding of bone ontogeny of fish larvae in early life.

Expression of Sox5, Sox8 and Sox9 Genes in Golden Pompano Trachinotus ovatus (Linnaeus 1758) Larvae During Ontogeny and in Response to Water Temperature

Sox5, Sox8, and Sox9 genes in golden pompano larvae were cloned and analyzed in this study. In the first trial, the expression of Sox genes during ontogeny of fish larvae in the first 18 days was examined, after which the expression of Sox genes in tissues was evaluated on 18 days post hatching (DPH). Subsequently, the response of Sox genes to water temperature (23, 26, and 29oC) on day 12 DPH and 18 DPH were compared. The expression levels of Sox8, Sox9a, and Sox9b peaked around 2 DPH before first feeding during fish ontogeny, and remained at low levels from 3-18 DPH. The expressions of Sox5 and Sox9a were affected by water temperature on 12 DPH and 18 DPH, while the expression of Sox8 was not affected by water temperature on 12 DPH and 18 DPH. The expression of Sox9b was affected by water temperature on 12 DPH. This study detected gene expression of Sox5, Sox8, and Sox9 at the early stage of development of golden pompano larvae. The time dependent expression of Sox genes in fish larvae is important for the understanding of bone ontogeny of fish larvae in early life.

Di-(2-ethylhexyl) Phthalate-Induced Hippocampus-Derived Neural Stem Cells Proliferation.

The brain and spinal cord have a limited capacity for self-repair under damaged conditions. One of the best options to overcome these limitations involves the use of phytochemicals as potential therapeutic agents. In this study, we have aimed to investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) on hippocampus-derived neural stem cells (NSCs) proliferation to search phytochemical candidates for possible treatment of neurological diseases using endogenous capacity. In this experimental study, neonatal rat hippocampus-derived NSCs were cultured and treated with various concentrations of DEHP (0, 100, 200, 400 and 600 µM) and Cirsium vulgare (C. vulgare) hydroethanolic extract (0, 200, 400, 600, 800 and 1000 µg/ml) for 48 hours under in vitro conditions. Cell proliferation rates and quantitative Sox2 gene expression were evaluated using MTT assay and real-time reverse transcription polymerase chain reaction (RT-PCR). We observed the highest average growth rate in the 400 µM DEHP and 800 µg/ml C. vulgare extract treated groups. Sox2 expression in the DEHP-treated NSCs significantly increased compared to the control group. Gas chromatography/mass spectrometry (GC/ MS) results demonstrated that the active ingredients that naturally occurred in the C. vulgare hydroethanolic extract were 2-ethyl-1-hexanamine, n-heptacosane, 1-cyclopentanecarboxylic acid, 1-heptadecanamine, 2,6-octadien-1-ol,2,6,10,14,18,22-tetracosahexaene, and DEHP. DEHP profoundly stimulated NSCs proliferation through Sox2 gene overexpression. These results provide and opportunity for further use of the C. vulgure phytochemicals for prevention and/or treatment of neurological diseases via phytochemical mediated-proliferation of endogenous adult NSCs.

The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers.

Low back pain is a major public health issue in the Western world, one main cause is believed to be intervertebral disc (IVD) degeneration. To halt/diminish IVD degeneration, cell therapy using different biomaterials e.g. hydrogels as cell carriers has been suggested. In this study, two different hydrogels were examined (in vitro) as potential cell carriers for human mesenchymal stem cells (hMSCs) intended for IVD transplantation. The aim was to investigate cell-survival and chondrogenic differentiation of hMSCs when cultured in hydrogels Puramatrix® or Hydromatrix® and potential effects of stimulation with growth hormone (GH). hMSCs/hydrogel cultures were investigated for cell-viability, attachment, gene expression of chondrogenic markers SOX9, COL2A1, ACAN and accumulation of extracellular matrix (ECM). In both hydrogel types, hMSCs were viable for 28days, expressed integrin β1 which indicates adhesion of hMSCs. Differentiation was observed into chondrocyte-like cells, in a higher extent in hMSCs/Hydromatrix® cultures when compared to hMSCs/Puramatrix® hydrogel cultures. Gene expression analyses of chondrogenic markers verified results. hMSCs/hydrogel cultures stimulated with GH displayed no significant effects on chondrogenesis. In conclusion, both hydrogels, especially Hydromatrix® was demonstrated as a promising cell carrier in vitro for hMSCs, when directed into chondrogenesis. This knowledge could be useful in biological approaches for regeneration of degenerated human IVDs.

Repair of cartilage defects in osteoarthritis rats with induced pluripotent stem cell derived chondrocytes.

BackgroundThe incapacity of articular cartilage (AC) for self-repair after damage ultimately leads to the development of osteoarthritis. Stem cell-based therapy has been proposed for the treatment of osteoarthritis (OA) and induced pluripotent stem cells (iPSCs) are becoming a promising stem cell source.ResultsThree steps were developed to differentiate human iPSCs into chondrocytes which were transplanted into rat OA models induced by monosodium iodoacetate (MIA). After 6 days embryonic body (EB) formation and 2 weeks differentiation, the gene and protein expression of Col2A1, GAG and Sox9 has significantly increased compare to undifferentiated hiPSCs. After 15 weeks transplantation, no immune responses were observed, micro-CT showed gradual engraftment and the improvement of subchondrol plate integrity, and histological examinations demonstrated articular cartilage matrix production.ConclusionshiPSC could be an efficient and clinically translatable approach for cartilage tissue regeneration in OA cartilages.

Increased Chondrogenic Potential of Mesenchymal Cells From Adipose Tissue Versus Bone Marrow‐Derived Cells in Osteoarthritic In Vitro Models

Primarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment. 3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1β, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition, and organization. Both hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1β by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs. hADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. J. Cell. Physiol. 232: 1478-1488, 2017. © 2016 Wiley Periodicals, Inc.

Sodium Tungstate for Promoting Mesenchymal Stem Cell Chondrogenesis.

Articular cartilage has a limited ability to heal. Mesenchymal stem cells (MSCs) derived from the bone marrow have shown promise as a cell type for cartilage regeneration strategies. In this study, sodium tungstate (Na2WO4), which is an insulin mimetic, was evaluated for the first time as an inductive factor to enhance human MSC chondrogenesis. MSCs were seeded onto three-dimensional electrospun scaffolds in growth medium (GM), complete chondrogenic induction medium (CCM) containing insulin, and CCM without insulin. Na2WO4 was added to the media leading to final concentrations of 0, 0.01, 0.1, and 1 mM. Chondrogenic differentiation was assessed by biochemical analyses, immunostaining, and gene expression. Cytotoxicity using human peripheral blood mononuclear cells (PBMCS) was also investigated. The chondrogenic differentiation of MSCs was enhanced in the presence of low concentrations of Na2WO4 compared to control, without Na2WO4. In the induction medium containing insulin, cells in 0.01 mM Na2WO4 produced significantly higher sulfated glycosaminoglycans, collagen type II, and chondrogenic gene expression than all other groups at day 28. Cells in 0.1 mM Na2WO4 had significantly higher collagen II production and significantly higher sox-9 and aggrecan gene expression compared to control at day 28. Cells in GM and induction medium without insulin containing low concentrations of Na2WO4 also expressed chondrogenic markers. Na2WO4 did not stimulate PBMC proliferation or apoptosis. The results demonstrate that Na2WO4 enhances chondrogenic differentiation of MSCs, does not have a toxic effect, and may be useful for MSC-based approaches for cartilage repair.

Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.

Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.

MiR-145 modulates lncRNA-ROR and Sox2 expression to maintain human amniotic epithelial stem cell pluripotency and β islet-like cell differentiation efficiency

In this study, we observed a great reduction in the expression of the endogenous long noncoding RNA ROR (lncRNA-ROR) and the stem cell transcription factor Sox2, in contrast to a marked increase in miR-145 expression, during the course of in vitro induced differentiation of human amniotic epithelial stem cells (HuAECs). Bioinformatics analysis and the luciferase reporter assay revealed binding of miR-145 to specific sites in lncRNA-ROR and Sox2, silencing their expression. Overexpression of a lncRNA-ROR-specific siRNA effectively downregulated the expression levels of Sox2 and other stem cell markers in HuAECs while weakening the efficiency of HuAEC differentiation into β islet-like cells. Moreover, the in vitro response of HuAEC-derived β islet-like cells to extracellular stimuli and C-peptide release by these cells were markedly weakened in the siRNA-ROR transfection group. Furthermore, the in vivo expression of β islet-like cell biomarkers was substantially reduced in HuAECs in the siRNA-ROR transfection group, and their in vivo β islet-like cell differentiation and insulin release capacities were reduced in a streptozocin-induced diabetic rat model. The experimental results indicate that lncRNA-ROR effectively maintains Sox2 gene expression through competitive binding to miR-145, achieving pluripotency maintenance in HuAECs and regulation of their directed β islet-like cell differentiation efficiency.

Dimorphic DNA methylation during temperature-dependent sex determination in the sea turtle Lepidochelys olivacea

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination.

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

We previously reported that perlecan, a heparan-sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage-synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2-/- -Tg (Hspg2-/- ;Col2a1-Hspg2Tg/- ) mice, in which the perlecan-knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic-, osteogenic-, and adipogenic-potentials were examined in the Hspg2-/- -Tg SMCs compared to the control SMCs prepared from wild-type Hspg2+/+ -Tg (Hspg2+/+ ;Col2a1-Hspg2Tg/- ) littermates. In a culture condition permitting proliferation, both control and Hspg2-/- -Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2-/- -Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2-/- -Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837-846, 2017.

Tissue- and time-directed electroporation of CAS9 protein-gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration.

A rapid method for temporally and spatially controlled CRISPR-mediated gene knockout in vertebrates will be an important tool to screen for genes involved in complex biological phenomena like regeneration. Here we show that in vivo injection of CAS9 protein–guide RNA (gRNA) complexes into the spinal cord lumen of the axolotl and subsequent electroporation leads to comprehensive knockout of Sox2 gene expression in SOX2+ neural stem cells with corresponding functional phenotypes from the gene knockout. This is particularly surprising considering the known prevalence of RNase activity in cerebral spinal fluid, which apparently the CAS9 protein protects against. The penetrance/efficiency of gene knockout in the protein-based system is far higher than corresponding electroporation of plasmid-based CRISPR systems. We further show that simultaneous delivery of CAS9–gRNA complexes directed against Sox2 and GFP yields efficient knockout of both genes in GFP-reporter animals. Finally, we show that this method can also be applied to other tissues such as skin and limb mesenchyme. This efficient delivery method opens up the possibility for rapid in vivo genetic screens during axolotl regeneration and can in principle be applied to other vertebrate tissue systems.

Porous microcarrier-enabled three-dimensional culture of chondrocytes for cartilage engineering: A feasibility study.

Cartilage repair is substantially intractable due to poor self-healing ability. Porous microspheres can be a fascinating three-dimensional matrix for cell culture and injectable carrier in cartilage engineering. In this study, we assessed the feasible use of porous biopolymer microspheres for chondrocyte carriers. When seeded onto the blended biopolymer microspheres and followed by a dynamic spinner flask culture, the chondrocytes showed robust growth behaviors during the culture period. The gene expressions of SOX9, type II collagen, and aggrecan were significantly upregulated after 2-week of culture. Furthermore, immunolocalization of type II collagen and secretion of glycosaminolglycan became prominent. The results suggest the feasible usefulness of the porous microspheres as the cell culture matrix and the subsequent delivery into cartilage defects.

REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence.

Hyaluronic acid (HA) plays a fundamental role in cell polarity and hydrodynamic processes, affording significant modulation of proliferation, migration, morphogenesis and senescence, with deep implication in the ability of stem cells to execute their differentiating plans. The Radio Electric Asymmetric Conveyer (REAC) technology is aimed to optimize the ions fluxes at the molecular level in order to optimize the molecular mechanisms driving cellular asymmetry and polarization. Here, we show that treatment with 4-methylumbelliferone (4-MU), a potent repressor of type 2 HA synthase and endogenous HA synthesis, dramatically antagonized the ability of REAC to recover the gene and protein expression of Bmi1, Oct4, Sox2, and Nanog in ADhMSCs that had been made senescent by prolonged culture up to the 30th passage. In senescent ADhMSCs, 4-MU also counteracted the REAC ability to rescue the gene expression of TERT, and the associated resumption of telomerase activity. Hence, the anti-senescence action of REAC is largely dependent upon the availability of endogenous HA synthesis. Endogenous HA and HA-binding proteins with REAC technology create an interesting network that acts on the modulation of cell polarity and intracellular environment. This suggests that REAC technology is effective on an intracellular niche level of stem cell regulation.

Role of Progenitor Cells in Liver Regeneration after Subtotal Resection.

In the liver of rats subjected to subtotal liver resection (80% organ weight), the expression of sox9 gene and SOX9 protein content increased and cells with hepatocyte morphology expressing SOX9 appeared; the proportion of cells expressing cytokeratin-19 also increased. Based on these data, we cannot completely exclude the involvement of resident progenitor cells and hepatocyte reprogramming in liver regeneration after subtotal resection, however, the contribution of these processes seems to be insignificant. The leading mechanism of liver mass recovery after subtotal resection is proliferation of hepatocytes.

Estimating the Survival Probability of Nuclear-Transfer Embryos before Embryo Transfer by a Novel Biopsy: Oct4 and Sox2 Gene Expression Patterns of a Monozygotic Twin Blastocyst Separated at the 2-Cell Stage of Nuclear-Transfer Embryos

The survival probability of mouse blastocysts developed from nuclear transfer (NT) was estimated prior to embryo transfer using a novel biopsy method. NT embryos were separated into two at the 2-cell stage to produce monozygotic twin blastocysts from a single NT oocyte. We first examined efficient culture methods to produce monozygotic twin blastocysts. Then one of the two blastocysts was used for gene expression analysis, and the other was transferred to a recipient mouse. Co-culture of monozygotic twin blastomeres with fertilized embryos or culture in a small well in vitro improved the developmental potential to the blastocyst stage and increased the blastocyst cell number. Although 21.9% of twin somatic cell NT embryos had the same expression levels of Oct4 and Sox2 genes as fertilized embryos, no fetuses were obtained after transfer to recipients.

Estimating the Survival Probability of Nuclear-Transfer Embryos before Embryo Transfer by a Novel Biopsy: Oct4 and Sox2 Gene Expression Patterns of a Monozygotic Twin Blastocyst Separated at the 2-Cell Stage of Nuclear-Transfer Embryos

Abstract: The survival probability of mouse blastocysts developed from nuclear transfer (NT) was estimated prior to embryo transfer using a novel biopsy method. NT embryos were separated into two at the 2-cell stage to produce monozygotic twin blastocysts from a single NT oocyte. We first examined efficient culture methods to produce monozygotic twin blastocysts. Then one of the two blastocysts was used for gene expression analysis, and the other was transferred to a recipient mouse. Co-culture of monozygotic twin blastomeres with fertilized embryos or culture in a small well in vitro improved the developmental potential to the blastocyst stage and increased the blastocyst cell number. Although 21.9% of twin somatic cell NT embryos had the same expression levels of Oct4 and Sox2 genes as fertilized embryos, no fetuses were obtained after transfer to recipients.