Mouse Fetal Fibroblasts Research Articles

OR11-04 Activatable Glucocorticoid Receptor Agonists As Treatments For The Consequences Of Preterm Birth

Abstract Disclosure: R.S. Dhavan: None. K.L. Short: None. J. Ng: None. M.J. Wallace: None. P. Monaghan-Nichols: None. D. DeFranco: None. T.J. Cole: None. Glucocorticoid (GC) signaling is essential for normal fetal organ development. During late gestation a surge of endogenous GCs contributes to organ maturation, particularly important for the lung where thinning the mesenchymal tissue cause an increased alveolar-gas exchange surface area and decreased gas-exchange diffusion distance that is critical for lung function after birth. Currently potent synthetic GCs such as betamethasone (Bet) or dexamethasone (Dex) are administered antenatally to accelerate fetal lung maturation and reduce the risk of respiratory distress with preterm birth. There are however growing concerns that systemic exposure to powerful synthetic GCs such as Dex is associated with detrimental side effects, particularly in the developing fetal brain. We are currently assessing novel activatable and potentially partially selective agonists of the glucocorticoid receptor (GR) as new antenatal steroid treatments of preterm birth. One such GR agonist is a steroid prodrug called ciclesonide (Cic) that is activated in vivo to the GR agonist Des-Cic by a family of intracellular serine esterase enzymes, called carboxylesterases (Ces). We have previously demonstrated that postnatal administration of Cic and Dex drive similar stimulation of important preterm lung-specific biomarkers but in contrast to Dex, Cic produced no postnatal growth retardation, no reduction in brain weight, and no alteration in levels of neural myelination. In this current study we show by western blot analysis and immunofluorescence that the CES enzymes are expressed strongly in epithelial cells and the mesenchyme of peripheral organs such as the lung and kidney but are expressed at very low levels in the fetal and adult mouse brain, providing a basis for the preferential activation of Cic to Des-Cic only in peripheral organs. To examine the transcriptome changes induced by Cic vs Dex in the fetal mouse lung we have assessed cultured primary mouse fetal fibroblasts (the primary cell target for GCs during lung development) stimulated by 1μM Dex and Cic, for 6 hours. Changes in the fibroblast transcriptome was assessed by microarray analysis and relevant targets validated by real-time-qPCR. Analysis of the top 30 induced and repressed gene targets, including Fkbp5, Crispld2, Tgm2 and Zbtb16, demonstrated that Dex and Cic have almost identical activity profiles. Induction of transcriptional changes was completely lost in GR-null- derived fetal lung fibroblasts demonstrating specificity via the GR. The data suggests that Cic is able to regulate a respiratory genomic signature similar to Dex. Further studies will examine the effects of Cic vs Beta and Dex in mouse models of preterm birth, particularly in the context of unwanted side-effects in neural development. Presentation: Friday, June 16, 2023

Quantitative analysis of mitochondrial DNA in porcine-mouse cloned embryos.

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture

BackgroundThe liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of hepatocytes is used for the understanding of liver physiology as well as for the drug development. Hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. Alternatively, systems using animal models and hepatocellular carcinoma cells have been established, but interspecies differences, variation between human cell sources and limited hepatic functions are among the challenges faced when using these models. Therefore, there is still a need for a highly stable method to purify human hepatocytes with functional sufficiency. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply.MethodsWe first established a growth culture system for hepatocytes derived from patients with drug-induced liver injury using fetal mouse fibroblasts and EMUKK-05 medium. We then evaluated the morphology, proliferative capacity, chromosome stability, gene and protein expression profiles, and drug metabolic capacity of hepatocytes in early, middle and late passages with and without puromycin. In addition, hepatic maturation in 3D culture was evaluated from morphological and functional aspects.ResultsIn our culture system, the stable proliferation of human hepatocytes was achieved by co-culturing with mouse fetal fibroblasts, resulting in dedifferentiation into hepatic progenitor-like cells. We purified human hepatocytes by selection with cytocidal puromycin and cultured them for more than 60 population doublings over a span of more than 350 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytocidal antibiotics because of enhanced drug-metabolizing activity.ConclusionsThese results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

Interaction of fibroblasts and induced pluripotent stem cells with poly(vinyl alcohol)-based hydrogel substrates.

Induced pluripotent stem cells (iPSCs) provide a promising means of creating custom-tailored cell lines for cellular therapies. Their application in regenerative medicine, however, depends on the possibility that the maintenance and differentiation of cells and organs occur under defined conditions. One major component of stem cell culture systems is the substrate, where the cells must attach and proliferate. The present study aimed to investigate the putative cytotoxic effects of poly(vinyl alcohol) (PVA)-based matrices on the in vitro culture of mouse fetal fibroblasts. In addition, the PVA-based hydrogels were used to determine the capacity of bovine induced pluripotent stem cells (biPSCs) to adhere and proliferate on synthetic substrates. Our results show that both cell types interacted with the substrate and presented proliferation during culture. The biPSCs formed new colonies when cell suspensions were placed onto the hydrogel surface for culture. These results may represent a new characterized xeno-free clinical grade culture system to be widely applied in cell-based therapies.

3212 Development of a Contractile Fontan Circuit to Decrease Central Venous Pressures in Single Ventricle Patients

OBJECTIVES/SPECIFIC AIMS: Children born with a single ventricle congenital heart defect requires three invasive open-heart surgeries in the first three years of life. The third operation, the Fontan procedure, includes connection of the vena cava (VC) to the pulmonary artery (PA) using a bio-inert conduit to reduce work required by the right ventricle (RV). While this operation greatly extends the lives of HLHS patients, the Fontan circuit eventually fails, and the only solution is a scarcely available donor heart. This failed circuit is explained by the “Fontan paradox” where central venous pressures build up over time, causing increased systemic resistance and congestion. The absence of the sub-pulmonary ventricle leads to abnormal hemodynamics associated with life-threatening complications. We believe that decreasing central venous pressures through the use of a tissue engineered contractile, patient specific conduit will decrease the amount and severity of complications caused by the “Fontan paradox.” We will use amniotic fluid derived induced pluripotent stem cells (AF-iPSCs) differentiated into cardiomyocytes (CMs) to generate flow within a biodegradable conduit. Additionally, AF-iPSC will be differentiated into structural support cells (SSCs), including cardiac fibroblasts and epicardium. Several studies suggest advanced contraction and structure of CMs in specific ratios with SSCs, particularly mouse and human fetal fibroblasts. In combination, these cells have shown advanced tissue organization and function through mechanically and electrically aligned junctions. This allows them to have a magnitude higher contractile force than CMs alone, making them ideal for increasing pressure within a tissue engineered construct. This poster focuses on the differentiation and selection of SSCs. METHODS/STUDY POPULATION: AF-iPSCs differentiation began at roughly 80% confluency. Mesoderm formation occurred via WNT pathway modulation by supplementing RPMI+insulin media with 0.5 ng/mL BMP4 at day 0, followed by 3 ng/mL BMP4, 2 ng/mL Activin A, and 5 ng/mL BFGF for four days. Then, RPMI+insulin media was supplemented with 10 ng/mL of BMP4 until day fifteen for epicardial formation. Cells were lifted to induce epithelial-to-mesenchymal transition (EMT) and RPMI-insulin media was supplemented with 10 ng/mL BFGF for cardiac fibroblasts. They were then harvested and characterized using immunofluorescence. Planned experiments include RT-qPCR for further characterization of cardiac fibroblasts. Additionally, a fibroblast isolation plating technique will be utilized to obtain cardiac fibroblast from AF-iPSC CMs and AF-iPSC epicardium. Commercially obtained human cardiac fibroblasts will be utilized as a control for all studies. RESULTS/ANTICIPATED RESULTS: Immunofluorescence (IF) revealed positive expression of vimentin and α-SMA indicating a fibroblast and vascular smooth muscle phenotype after supplementation with 10 ng/mL of BMP4 after EMT induction. It is expected that IF of epicardial formation at day 15 will show positive expression of WT1, a well-known epicardial marker. We also suspect RT-qPCR will reveal high expression of cardiac fibroblast specific markers COL1A1, PDGFA, TCF21, and THSB1. We expect to yield a higher number of cardiac fibroblast from the small molecule AF-iPSC differentiation compared to a timed plating technique of AF-iPSC CMs and AF-iPSC epicardium (separately plated). Results will be quantified and compared using the aforementioned techniques. DISCUSSION/SIGNIFICANCE OF IMPACT: Discussion/significance of impact: Although fibroblasts make up a large portion of cells in the heart and greatly enhance CM function, they are poorly characterized in the literature and not easily obtained. This study will provide an efficiency comparison on the best method for acquiring cardiac fibroblast for cardiac tissue engineering applications as we move forward with translational cardiac pediatric research.

Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency

Mouse somatic cells can be reprogrammed to pluripotency by the ectopic expression of four pluripotency transcription factors, Oct4, Sox2, cmyc, and Klf4. Usually, silencing of the exogenous reprogramming factors is considered to be essential for complete reprogramming and differentiation. In the vast majority of studies, murine pluripotency transcription factor sequences have been used for the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSC). The effectiveness of xenogeneic transcription factors in miPSC generation has not yet been investigated in detail. Here, we evaluated transposon-based vectors with four porcine pluripotency factors for their ability to reprogram mouse fetal fibroblasts (MEFs) harboring an Oct4-EGFP reporter construct to pluripotency. Additionally, we examined the effects of the non-silenced heterologous transgenes on the expression levels of key endogenous pluripotency markers and the differentiation capacities of the miPSC. Within 8 days of transfection with porcine reprogramming transcription factors the MEFs acquired typical compact miPSC morphology and upregulated expression of endogenous Oct4 and other critical pluripotency genes. Consequently, the transgenes under the control of the TetO promoter became silenced, while the CAG-controlled constructs were expressed throughout the period of culture. Despite the continuous transgene expression, the CAG-miPSC showed normal morphology and were capable of differentiation into the three primary germ layers in vitro and in vivo. However, the expression levels of important endogenous pluripotency markers, Klf4, c-myc, Rex1, and Utf1, were significantly lower in CAG-miPSC compared with TetO-miPSC with silenced reprogramming cassettes. Surprisingly, the endogenous Oct4 and Sox2 expression levels were not affected by the residual transgene expression. Our results suggest that porcine reprogramming transcription factors are suitable for production of miPSC, but silencing of the heterologous transgenes may be necessary for complete reprogramming to pluripotency.

Chronic exposure to simulated space conditions predominantly affects cytoskeleton remodeling and oxidative stress response in mouse fetal fibroblasts.

Microgravity and cosmic rays as found in space are difficult to recreate on earth. However, ground-based models exist to simulate space flight experiments. In the present study, an experimental model was utilized to monitor gene expression changes in fetal skin fibroblasts of murine origin. Cells were continuously subjected for 65 h to a low dose (55 mSv) of ionizing radiation (IR), comprising a mixture of high‑linear energy transfer (LET) neutrons and low-LET gamma-rays, and/or simulated microgravity using the random positioning machine (RPM), after which microarrays were performed. The data were analyzed both by gene set enrichment analysis (GSEA) and single gene analysis (SGA). Simulated microgravity affected fetal murine fibroblasts by inducing oxidative stress responsive genes. Three of these genes are targets of the nuclear factor‑erythroid 2 p45-related factor 2 (Nrf2), which may play a role in the cell response to simulated microgravity. In addition, simulated gravity decreased the expression of genes involved in cytoskeleton remodeling, which may have been caused by the downregulation of the serum response factor (SRF), possibly through the Rho signaling pathway. Similarly, chronic exposure to low-dose IR caused the downregulation of genes involved in cytoskeleton remodeling, as well as in cell cycle regulation and DNA damage response pathways. Many of the genes or gene sets that were altered in the individual treatments (RPM or IR) were not altered in the combined treatment (RPM and IR), indicating a complex interaction between RPM and IR.

Transplantation of Fetal Instead of Adult Fibroblasts Reduces the Probability of Ectopic Ossification During Tendon Repair

Although cell transplantation therapy can effectively promote functional tendon repair, occasional ectopic ossification during tendon regeneration undermines its efficacy. The effect of transplanted cell types on ectopic ossification has not yet been systematically evaluated. This study compared the rate of ectopic ossification during tendon repair upon transplantation with mouse fetal fibroblasts (FFs) and their adult counterparts (adult fibroblasts [AFs]). Alkaline phosphatase (ALP) staining, immunofluorescence, and gene expression analysis were used to compare the spontaneous osteogenic differentiation of FFs and AFs in vitro. X-ray, histology, and gene expression analysis were used to investigate the ectopic ossification in a mouse Achilles tendon repair model in vivo. ALP staining and immunofluorescence data in vitro showed that FFs had less spontaneous osteogenic differentiation capacity, and lower expression of runt-related transcription factor 2 (runx2). For the in vivo study, the FFs transplant group displayed reduced ectopic ossification (2/7 vs. 7/7, Mann-Whitney test p<0.01) at 14 weeks post-transplantation and enhanced tendon repair (general histological score at week 6, 7.53 vs. 10.56, p<0.05). More chondrocytes formed at 6 weeks, and all mice developed bone marrow at 14 weeks post-transplantation in the AFs transplant group. Gene expression analysis of the regenerated tissue showed significantly higher expression levels of transforming growth factor beta1 (TGF-β1) and transforming growth factor beta3 (TGF-β3) in the AFs group during the early stages of tendon repair. Our study demonstrates that transplantation of fetal instead of AFs is more promising for tendon repair, underscoring the importance of the origin of seed cells for tendon repair.

86 EFFECT OF HDAC2 INTERFERENCES ON HISTONE MODIFICATIONS IN MOUSE EARLY EMBRYOS

One of the histone deacetylase members, HDAC2 plays an important role in chromatin remodeling and transcriptional repression. The present study was designed to improve histone acetylations by knocking down the expression of histone deacetylase with RNA interferences. According to the published mouse HDAC2 mRNA sequence (NM_008229.2, GI:87162463), as well as shRNA design principles, 3 interference fragments of 107 to 126 bps, 879 to 898 bps, and 1240 to 1259 bps and a negative control sequence were designed and synthesized, respectively. Four interference vectors expressing a red fluorescent protein were successfully constructed, which were named pCDsRed2-shRNA107, pCDsRed2-shRNA879, pCDsRed2-shRNA1240, and pCDsRed2-shRNAcontrol, respectively. These vectors were then transfected respectively to mouse fetal fibroblast cells. Real-time, quantitative PCR of the transgenic cells showed that the vectors mentioned above resulted in 0.81, 0.73, 0.16, and 0.80 times knockdown of hdac2 expression when compared with the nontransfected cells, which suggested that the piece of 1240 to 1259 bp was the most effective RNAi region. Immunofluorescent staining of the transgenic cells showed that the histone acetylations and methylations of H4K5ac, H3K9ac, H3K9me2, H3K27me3, and H3K4me3 were significantly higher in all of the interference vectors than they were in the negative control. Vector pCDsRed2-shRNA1240 was the most effective RNAi. After injection of pCDsRed2-shRNA1240 into zygote pronuclei, embryos developed to 2-cell, 8-cell embryos and blastocysts decreased to 96.6, 85.3 (P &lt; 0.05), and 35.6 (P &lt; 0.01) of the control group, respectively. High levels of H3K9ac, H4K5ac, H3K4me3, and H3K27me3 were observed in the RNAi embryos when compared with the controls. These results indicated that the knockdown of hdac2 by RNAi decreased the expression of HDAC2 and induced high expression of histone acetylations in both somatic cells and early embryos. This work was supported by the national basic research program of china (No. 2012CB22306).

Simulated microgravity decreases apoptosis in fetal fibroblasts

Space travel is a major challenge for human beings. Especially, the mechanisms through which space conditions might alter animal development have been questioned for a long time. The two major physical stress factors that are of relevance in this context are space radiation and weightlessness. While it has been extensively shown that high doses of ionizing radiation induce deleterious effects on embryonic development, so far, little is known about the potential harmful effects of radiation in combination with microgravity on the developing organism. In the present study, we investigated the effects of simulated microgravity on irradiated STO mouse fetal fibroblast cells using a random positioning machine (RPM). Radiation-induced cell cycle changes were not affected when cells were subjected to simulated microgravity for 24 h. Moreover, no morphological differences were observed in irradiated samples exposed to simulated microgravity compared to cells that were exclusively irradiated. However, microgravity simulation significantly decreased the level of apoptosis at all doses as measured by caspase-3 activity and it prevented cells from undergoing radiation-induced size increase up to 1 Gy.

ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3 –)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

X-irradiation induces cell death in fetal fibroblasts

The impact of ionizing radiation on developing organisms has been widely studied for risk assessment purposes. Even though efforts have been made to decrease received doses to as low as reasonably achievable, the possibility of accidental exposure has to be considered as well. Mammalian gestation is usually divided into three periods. Radiation exposure during the 'pre-implantation period' may essentially result in embryonic mortality while exposure during the 'organogenesis period' may characteristically lead to malformations. In humans, the 'fetal period' is one of particular sensitivity to radiation induction of mental retardation, especially if the exposure occurs between weeks 8-15 of gestation. It is also admitted that prenatal irradiation may increase the risk of leukemia and childhood cancer, with an equal risk over the whole pregnancy. The aim of this study was to investigate the effects of moderate to high doses of X-irradiation on mouse skin fetal fibroblasts, one of the cell types subjected to the highest dose of radiation. Exposure of the cells to X-rays led to a rapid and significant increase in γ-H2AX foci, indicative of high levels of DNA double strand breaks. High doses (>2 Gy) also led to a pronounced G2-arrest and a decrease in the number of cells in the S phase, which was followed after 24 h by a decrease in cell survival and an increase in the level of apoptosis and necrosis. This study shows that mouse fetal skin fibroblasts are sensitive to high doses of X-irradiation. Furthermore, we report a better repair for higher doses than lower, which seems to indicate that little DNA damage is not necessarily repaired immediately. However, more sensitive approaches are necessary to identify the risk associated with low doses of radiation.

The Effect of Berberine Treatment on the Reversibility of the Development of Mouse Zygotes and Gametes, and on the Fertilization and Subsequent Development

As reported previously, berberine, the main component extracted from Coptis rhizome and Phellodendron, has potential as a contraceptive for animals since berberine has a strong inhibitory effect on the in vitro development of mouse zygotes and on fetal development in vivo. The present study was undertaken to examine the effect of berberine treatment on the reversibility of the development of zygotes and gametes, and on the fertilization and subsequent development in the mouse. The reversibility of the berberine-induced inhibition of the development of mouse zygotes was dependent on the concentration used and treatment period. Berberine treatment did not inhibit the fertilizing capacity of epididymal spermatozoa and the fertilizability of oocytes at the second metaphase stage. The present study demonstrated that in vitro development of mouse zygotes is about 100 times more sensitive to berberine than the in vitro growth of mouse fetal fibroblast cells. The high stability of berberine at low temperatures for at least for 12 months and the high sensitivity of preimplantation embryos to berberine is useful information when considering the administration of berberine to females as a contraceptive.

PROGRESS TOWARDS THE DERIVATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS

Porcine induced pluripotent cells (iPSC) are considered an important large animal model for developing personalized stem cell therapies. Since the derivation of the first mouse and human iPSC, there have been relatively few reports regarding the reprogramming of pig somatic cells into pluripotency, exclusively with the use of transcription factors from human and mouse origin. To investigate whether using species-specific transcription factors would allow for an efficient reprogramming of porcine somatic cells, we have developed a Sleeping Beauty (SB) transposon system based on the porcine OCT4, SOX2, C-MYC, and KLF4 sequences and have tested it in the reprogramming of mouse and porcine fetal fibroblasts and pig EGC-like cells. Transfection of mouse embryonic fibroblasts with a multicistronic SB-tetO-pOCT4-F2A-pSOX2-T2A-pC-MYC-E2A-pKLF4-ires-Tomato vector resulted in the formation of iPSC colonies, which inactivated the exogenous transcription factors and upregulated their endogenous pluripotency genes. These cells maintained mESC-like morphology, formed embryoid bodies, and differentiated into different cell types in culture, including rhythmically contracting cardiac myocytes. In contrast, porcine fibroblasts and EGC-like cells transfected with the same transposon vector did not proliferate in culture and did not form any iPSC colonies. We then transfected these cells with multiple bi-cistronic vectors SB-Ef1a-pOCT4-ires-Tomato, SB-Ef1a-pSOX2-ires-Neo, SB-Ef1a-pC-MYC-ires-Puro, and SB-Ef1a-pKLF4-ires-Puro. As a result, both cell types formed multiple colonies with mouse ESC-like morphology. Clones established from individually picked colonies from transfected fetal fibroblasts maintained this morphology for 5-6 passages, after which they became flat and epithelial-like. They expressed endogenous SOX2, C-MYC, KLF4, and E-Cadherin, but not OCT4. At the same time, clones derived from EGC-like cells proliferated at accelerated rate and maintained their morphology for over 10 passages at the time of this writing. While the exogenous genes were expressed continuously during this period, the cells expressed also endogenous OCT4, SOX2, TERT, STELLA, TDH, and CHD1; however, C-MYC, KLF4, NANOG, and E-Cadherin expression was relatively low. These cells are currently being characterized for pluripotency. Despite the use of porcine transcription factors, the overall reprogramming of porcine cells appears to be still less efficient compared with mouse fibroblasts. Our results suggest that the species and tissue origin of the somatic cells may play a more important role in the reprogramming to pluripotency than the origin of the transcription factors used. In this respect, optimization of culture conditions may be necessary in order to allow for efficient and complete reprogramming of porcine somatic cells. This work is supported by a research grant by the Deutsche Forschungsgemeinschaft.

Differential Expression of MMP-2 and -9 and their Inhibitors in Fetal Lung Cells Exposed to Mechanical Stretch: Regulation by IL-10

Abnormal remodeling of the extracellular matrix (ECM) has been implicated in the pathogenesis of bronchopulmonary dysplasia. However, the contribution of lung parenchymal cells to ECM remodeling after mechanical injury is not well defined. The objective of these studies was to investigate in vitro the release of MMP-2 and -9 and their respective inhibitors TIMP-2 and -1, and to explore potential regulation by IL-10. Mouse fetal epithelial cells and fibroblasts isolated on E18-19 of gestation were exposed to 20% cyclic stretch to simulate lung injury. MMP-2 and MMP-9 activity were investigated by zymography and ELISA. TIMP-1 and TIMP-2 abundance were analyzed by Western blot. We found that mechanical stretch increased MMP-2 and decreased TIMP-2 in fibroblasts, indicating that excessive stretch promotes MMP-2 activation, expressed as the MMP-2/TIMP-2 ratio. Incubation with IL-10 did not change MMP-2 activity. In contrast, mechanical stretch of epithelial cells decreased MMP-9 activity and the MMP-9/TIMP-1 ratio by 60-70%. When IL-10 was added, mechanical stretch increased the MMP-9/TIMP-1 ratio by 50%. We conclude that mechanical stretch differentially affects MMP-2/9 and their inhibitors in fetal lung cells. IL-10 modulates MMP-9 activity through a combination of effects on MMP-9 and TIMP-1 levels.

Generation of a Slc39a8 hypomorph mouse: Markedly decreased ZIP8 Zn2+/[formula omitted] transporter expression

Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn2+/(HCO3-)2 symporter, moving both ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac – in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.

Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT-EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N(1),N(11)-diethylnorspermine), CPENSpm (N(1)-ethyl-N(11)-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N(1)-ethyl-N(11)-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT-EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT-PCR (reverse transcription-PCR) results suggested that the analogue-affected regulation of SSAT-EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.

A New Mechanism for Aging: Chemical “Age Spots” in Immortal DNA Strands in Distributed Stem Cells

The existence of immortal DNA strands (IDSs) in distributed stem cells (DSCs) of adult human tissues was first inferred by Cairns. Cairns deduced the existence of IDSs by connecting two seemingly disparate observations - one his own and the other belonging to Lark. Cairns noted a mathematical discrepancy between predicted human tissue cell mutation rates and human cancer incidence. He integrated this insight with Lark's earlier discovery of non-random mitotic chromosome segregation in both plant root tip cells and mouse fetal fibroblast cultures to predict the existence of IDSs as the essential elements of a mutation-defense mechanism in DSCs. Since Cairns' seminal prediction, several laboratories have identified IDSs in diverse mammalian cells with DSC properties. Past studies focused on the potential roles of IDSs as originally envisioned in DSC genetic fidelity or in the maintenance of the DSC phenotype. Another possible consequence of IDSs, aging, has received little attention. Herein, the potential for cumulative chemical modifications and decompositions (i.e., "age spots") of IDSs in DSCs to act as a major determinant of human aging is considered. If accrued chemical alterations of IDSs prove to be essential determinants of aging, then a means to restore IDSs may yield new strategies for tissue rejuvenation.

Slc39a14 Gene Encodes ZIP14, A Metal/Bicarbonate Symporter: Similarities to the ZIP8 Transporter

The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd(2+) (K(m) = 0.14 versus 1.1 microM) and Mn(2+) uptake (K(m) = 4.4 versus 18.2 microM); 3) ZIP14A- and ZIP14B-mediated Cd(2+) uptake is most inhibited by Zn(2+), and next by Mn(2+) and Cu(2+); 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd(2+) uptake is dependent on extracellular HCO(3)(-); 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd(2+) is likely to act as a rogue hitchhiker-displacing Zn(2+) or Mn(2+) and entering the body to cause unwanted cell damage and disease.

Retinoic acid induces differential expression of MMP-1&-9 in fetal and adult fibroblasts

Antony, Anuja K. MD; Bari, Sina BS; Longaker, Michael MD; Lorenz, Peter H. MD Author Information