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Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24 h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P

Process of somatic cell reprogramming into induced pluripotent stem (iPS) cells can broadly be divided into an early stochastic phase and a later, hierarchical and more deterministic phase of gene activation. Between the stochastic onset and the deterministic progression while approaching pluripotency, cells upon reprogramming undergo a transient activation or repression of developmental regulators. In these intermediate cells yet acquiring pluripotency, many somatic gene expressions are lost, while various lineage-specific factors are activated. In the present study, we produced pancreatic β cells by exposing human dermal fibroblasts (HDF) at the intermediate phase of reprogramming to pancreas-specific culture conditions. To obtain the intermediate cells, reprogramming factor Oct4, Sox2, Klf4, and c-Myc were transduced into HDF using retroviruses. By Day 9 after transduction, the expression of fibroblast-associated Thy1 was lost, while the early reprogramming marker CD49d was activated, suggesting that the cells are at the early stage of intermediate phase. These cells were capable of differentiating into 3 germ layers when cultured in tri-differentiation medium (including 100 ng mL–1 Activin A, 0.1 ng mL–1 Wnt3 for endodermal differentiation; 20 ng mL–1 BMP4, 20 ng mL–1 Activin A for mesodermal differentiation; 10 μM SB431542, 600 ng mL–1 Dorsomorphin, and 35 ng mL–1 Noggin for ectodermal differentiation) and expressing lineage-specific markers Sox17 (endoderm), Brachyury (mesoderm), and Otx2 (ectoderm). Further, the endodermal cells derived from HDF at intermediate phase of reprogramming were cultured in pancreas-induction medium (KO DMEM with 1% insulin-transferrin-selenium (ITS), 10 ng mL–1 BMP4, 10 mM nicotinamide, 10 μM forskolin, 10 μM dexamethasone, and 50 ng mL–1 exendin-4). After 5 days in culture, cells expressed pancreatic progenitor marker Pdx1, as examined by immunostaining and fluorescence-activated cell sorting (FACS) analysis. After culture for additional 10 days, the cells formed spherical clusters and expressed pancreatic islet-specific genes including insulin, somatostatin, glucagon, Nkx6.1, and MafA, as determined by RT-PCR. Moreover, the production of insulin and C-peptide from the derived β cells was confirmed by immunocytochemistry and FACS analysis. In response to glucose stimulation, these cells secreted insulin and C-peptide, as measured by ELISA. The present study demonstrates that fully differentiated functional cells could be converted from somatic cells at intermediate phase of reprogramming by exposing cells to specific culture conditions. The results shown in this study may provide a faster and potentially safer way to produce functional pancreatic cells without isolation and differentiation of iPS cells.

Pre-term infants born before the end of the 37th week of pregnancy have not been exposed to their mother's womb full-term. Because they are born too soon, many of their biological systems, such as those involving the lungs and liver, are not developed enough to work property on their own. The lungs are not functional in the fetus, and the blood passes directly from the right atrium to the left atrium or from the right ventricle to the aorta. In this study, we analyzed protein profiles in the umbilical cord of term and pre-term infants by 2DE and MALDI-TOF analysis. Protein patterns of the umbilical cord were established using the pH range 4–7, isoelectric focusing (IEF) cell system, and 7.5–17.5% gradient 2-dimensional polyacrylamide gel electrophoresis (2DE). Image analysis was performed following Coomassie brilliant blue G staining to reveal spots, and spot intensities were compared using a PDQuest analysis system. Peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and a SWISS-PROT database search were utilized to identify proteins. Around 1000 protein spots were recognized by image analysis for umbilical cords from each treatment. Analysis of umbilical cords from pre-term infants revealed 35 different spots compared with those from full-term infants. Levels of 12 proteins increased, such as serum albumin precursor and tropomyosin, whereas levels of 11 proteins decreased, including 78 kDa glucose-regulated protein and protein disulfide isomerase A3. Another 12 proteins had levels that were different between 35 and 36 weeks of pregnancy. In general, proteins that increased represented precursor and cell mobility or structure-related proteins, whereas proteins that decreased included chaperon-related proteins. Expression levels of this protein were confirmed by Western blot analysis and real-time PCR. The results of this experiment suggest differential protein profiles between umbilical cords of pre-term and full-term infants that may be related to fetal circulation and early birth of infant.

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.

The proper post-transcriptional modification of recombinant human erythropoietin (rhEPO) is critical to retain its biological functions, either in vivo or in vitro. The major glycosyltransferases for the determinant of glycosylation patterns of rhEPO are N-acetylglycosaminyltransferase (GnT) and α-1-3/4 fucosyltransferase (Fut). GnT-III expression (388 ± 19.09) in the mouse mammary gland has been shown to be dramatically different from that in CHO cells, although FuT-VIII expression in CHO cells (1970 ± 255.9) is comparable to mouse mammary gland (272 ± 14.8), suggesting that the mammary gland may proceed with the proper glycosylation of rhEPO as shown in CHO cells. To identify this hypothesis and establish the rhEPO bioreactor system for mass production of protein in transgenic animals, we have generated two transgenic mouse lines that express rhEPO in milk. Both lines of transgenic mouse express only rhEPO in the lactating mammary gland, and the protein yield of rhEPO in lactating milk is comparable to that in CHO cells. After determining the protein expression in lactating milk, using three different methods – enzymatic release of oligosaccharide analysis, two-dimensional electrophoresis, and 2-aminobenzamide-labeled analysis – we report that the rhEPO produced by the animal bioreactor system has the proper glycosylation patterns as shown in CHO cell-derived Epoietin α, and has more tetra-acidic oligosaccharide structures than Epoietin α, which is the widely used rhEPO for therapeutic purposes. The in vitro biological property of transgenic mouse milk-derived rhEPO has been tested by measuring luciferase activity in MCF-7 cells, indicating that rhEPO from mammary gland up-regulates the EPO-receptor-mediated STAT5 gene expression in a dose-dependent manner the same as Epoietin α does. In addition, in vivo biological activity demonstrated that direct injection of rhEPO into a mouse vein increases blood components such as RBC and HCT. In light of these findings, we suggest that high levels of tetra-acidic structures observed in transgenic mouse milk-derived rhEPO may be related to the high level of expression of glycosiltransferases (GnT-III and FuT-VIII) in mammary gland; thus the bioreactor system using the mammary gland of a transgenic animal could be a good candidate for production of rhEPO for pharmaceutical purposes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea. D.N. Kwon is the recipient of a scholarship from the BK21 program, granted by the Ministry of Education, Korea.