In this study, a customized WQI (Seoul water quality index, S-WQI) for urban rivers that can ultimately reflect their characteristics was developed by modifying and supplementing the existing Bascaron WQI calculation method through linkage with statistical methods such as factor analysis. We used the water quality data generated monthly at 17 water quality monitoring networks (WQMNs) in Seoul for 18 years, from 2002 to 2019. Result of a research, the monthly S-WQI showed an average 70 out of 100, 'good (II)' grades, whereas the average water quality grade according to the environmental standards was 'slightly good (II),' with an R-2 value of 0.8298. The annual S-WQI was found to be 39 (bad) to 97 (very good), with an average of 72 (good). Through this study, S-WQI, a customized WQI for urban rivers, was judged to be a reasonable index that can represent the characteristics of urban river water quality. This is because it is easy to apply and is a calculation method that uses relatively fewer water quality items than the WQI calculated in the past, and it is highly likely to be linked to the currently implemented water quality grade system. In addition, to extend the application of WQI to various water quality survey points, based on the calculation methodology performed to derive the indices in this study, such as modified S-WQI (MS-WQI), by adding new water quality items and changing some items, it is also possible to develop an advanced customized WQI for urban rivers considering watershed characteristics and measurement items.

We developed a classification model and a real-time prediction model for short-term dissolved oxygen (DO) at the junction of the Han River in Anyangcheon, where water quality accidents occur frequently. The classification model is an analysis model that derives the main factors affecting DO changes in the Anyangcheon mobile water quality monitoring network using decision tree, random forest, and XGBoost. The model identified the key factors affecting DO changes to be electrical conductivity, cumulative precipitation, total nitrogen, and water temperature. Random forest (sensitivity, 0.9962; accuracy, 0.9981) and XGBoost (sensitivity, 1.0000; accuracy, 0.9822) showed excellent classification performance. The real-time prediction model for short-term DO we developed adopted artificial neural network (ANN), long short-term memory (LSTM), and gated recurrent unit (GRU) algorithms. LSTM (R-2 = 0.93 - 0.97, first half; R-2 = 0.95 - 0.96, second half) and GRU (R-2 = 0.94 - 0.98, first half; R-2 = 0.96 - 0.98, second half) significantly outperformed ANN (R-2 = 0.64 - 0.86). The LSTM and GRU models we developed used real-time automatic measurement data, targeting urban rivers that are sensitive to water quality changes and are waterfront areas for citizens. They can quickly reflect and simulate short-term, real-time changes in water quality compared with existing static models.

Abstract Background Aging has detrimental effects on the ovary, such as a progressive reduction in fertility and decreased hormone production, that greatly reduce the quality of life of women. Thus, the current study was undertaken to investigate whether human placenta-derived mesenchymal stem cell (hPD-MSC) treatment can restore the decreases in folliculogenesis and ovarian function that occur with aging. Methods Acclimatized 52-week-old female SD rats were randomly divided into four groups: single hPD-MSC (5 × 105) therapy, multiple (three times, 10-day intervals) hPD-MSC therapy, control (PBS), and non-treated groups. hPD-MSC therapy was conducted by tail vein injection into aged rats. The rats were sacrificed 1, 2, 3, and 5 weeks after the last injection. hPD-MSC tracking and follicle numbers were histologically confirmed. The serum levels of sex hormones and circulating miRNAs were detected by ELISA and qRT-PCR, respectively. TGF-β superfamily proteins and SMAD proteins in the ovary were detected by Western blot analysis. Results We observed that multiple transplantations of hPD-MSCs more effectively promoted primordial follicle activation and ovarian hormone (E2 and AMH) production than a single injection. After hPD-MSC therapy, the levels of miR-21-5p, miR-132-3p, and miR-212-3p, miRNAs associated with the ovarian reserve, were increased in the serum. Moreover, miRNAs (miR-16-5p, miR-34a-5p, and miR-191-5p) with known adverse effects on folliculogenesis were markedly suppressed. Importantly, the level of miR-145-5p was reduced after single- or multiple-injection hPD-MSC therapy, and we confirmed that miR-145-5p targets Bmpr2 but not Tgfbr2. Interestingly, downregulation of miR-145-5p led to an increase in BMPR2, and activation of SMAD signaling concurrently increased primordial follicle development and the number of primary and antral follicles. Conclusions Our study verified that multiple intravenous injections of hPD-MSCs led to improved ovarian function via miR-145-5p and BMP-SMAD signaling and proposed the future therapeutic potential of hPD-MSCs to promote ovarian function in women at advanced age to improve their quality of life during climacterium.

We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening.

The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.

Human adult stem cells are a readily available multipotent cell source that can be used in regenerative medicine. Despite many advantages, including low tumorigenicity, their rapid senescence and limited plasticity have curtailed their use in cell-based therapies. In this study, we isolated CD34/CD73-double-positive (CD34(+)/CD73(+)) testicular stromal cells (HTSCs) and found that the expression of CD34 was closely related to the cells' stemness and proliferation. The CD34(+)/CD73(+) cells grew in vitro for an extended period of time, yielding a multitude of cells (5.6×10(16) cells) without forming tumors in vivo. They also differentiated into all three germ layer lineages both in vitro and in vivo, produced cartilage more efficiently compared to bone marrow stem cells and, importantly, restored erectile function in a cavernous nerve crush injury rat model. Thus, these HTSCs may represent a promising new autologous cell source for clinical use.

Human pre-ovulatory follicular fluid (FF) contains a higher concentration of melatonin than serum. The aim of this study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcomes of an in-vitro maturation (IVM) IVF-embryo transfer programme for patients with polycystic ovarian syndrome (PCOS). Melatonin concentrations in the culture media of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes after using IVM media with or without melatonin were analysed. In the culture media of GC or COC, melatonin concentrations gradually increased. When human chorionic gonadotrophin priming protocols were used, implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control group (P<0.05). Pregnancy rates were also higher, although not significantly. The findings suggest that the addition of melatonin to IVM media may improve the cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes. It is speculated that follicular melatonin may be released from luteinizing GC during late folliculogenesis and that melatonin supplementation may be used to improve the clinical outcomes of IVM IVF-embryo transfer. Melatonin is primarily produced by the pineal gland and regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. Interestingly, human pre-ovulatory follicular fluid contains a higher concentration of melatonin than serum. However, in contrast to animal studies, the direct role of melatonin on oocyte maturation in the human system has not yet been investigated. So, the aim of the study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcome of an in-vitro maturation (IVM) IVF-embryo transfer programme for PCOS patients. The melatonin concentrations in culture medium of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes of IVM IVF-embryo transfer using IVM medium alone or supplemented with melatonin were analysed. In the culture media of GC or COC, the melatonin concentration gradually increased. With human chorionic gonadotrophin priming, the pregnancy and implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control (P<0.05). Our findings suggest that follicular melatonin is released from luteinizing GC during late folliculogenesis and plays a positive role in oocyte maturation. Therefore, addition of melatonin into IVM medium may improve cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes.

Recent advances in the stem cell biology, including the development of optimized cell type specific culture systems, and the broader understandings of biochemical and molecular signals involved in cell self-renewal and differentiation have made the cell based therapy closer to the practical uses. As of now, at least 180 adult stem cell therapies are being used or tested in real life in the forms of bone marrow stem cell, mesenchymal stem cell, cord blood stem cell, fetal brain stem cell and fat stem cell transplantations. This novel technology holds such a promising therapeutic potentials that Food and Drug Administration of U. S. A. approved a clinical trial for patients of spinal cord injuries using human ES cell derived oligodendrocytes in early 2009.Stem cells have two important properties that distinguish themselves from other types of cells; they can both proliferate without changing their phenotypes indefinitely, and they also can differentiate into one or more new kind of cells depending on the culture conditions. Thus, stem cell therapy could be most effective to treat the diseases that are marked by the loss of cells. The typical examples are Parkinson's disease, Alzheimer's disease, diabetes, heart failure, blindness, spinal cord injury and stroke. Additionally, stem cell derivatives can be used in drug discovery as well. In last decade, various types of stem cells have been identified from the preimplantation stage embryos, fetus, placenta, and adult tissues. Moreover, it is now almost a common practice to make induced pluripotent stem cells (iPS) from various adult somatic cells using only a few defined factors. Thus it is feasible to generate patient specific stem cells with less controversy in near future. However, human ES cells firmly remained as "the gold standard" because of their greatest potentials to become any type of cells in the body.The vast knowledge obtained from human ES cell research in the past decade has made cell based therapy more promising than ever. Even the recent establishment of iPS cell technology is the culmination of human ES cells research. Thus, here, the current status of clinical applications using fetal, adult and embryonic stem cell based therapies will be introduced and the future directions of stem cell application in the clinical trials will be discussed.

The Second Biennial International Collaborative Symposium on Stem Cell Research, held in Seoul, Korea, on 18-19 September 2008, showcased talks by a roster of established and emerging leaders in stem cell biology, and demonstrated how far and fast the field has moved in the last 2 years.

To date, all methods to generate induced pluripotent stem cells (iPSCs) require the use of genetic materials and/or potentially mutagenic molecules. Here we report the generation of stable iPSCs from human fibroblasts by directly delivering four reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell-penetrating peptide (CPP). These protein-induced human iPSCs (p-hiPSCs) exhibited similarity to human embryonic stem cells (hESCs) in morphology, proliferation, and expression of characteristic pluripotency markers. p-hiPSC lines produced with these recombinant proteins were successfully maintained for more than 35 passages and differentiated into derivatives of all three embryonic germ layers both in vitro and in teratomas. This system eliminates the potential risks associated with the use of viruses, DNA transfection, and potentially harmful chemicals and in the future could potentially provide a safe source of patient-specific cells for regenerative medicine. Over a decade ago, Wilmut and colleagues showed that adult somatic cells could be reprogrammed back to an undifferentiated embryonic state using somatic cell nuclear transfer (SCNT) (Wilmut et al., 1997Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H. Nature. 1997; 385: 810-813Crossref PubMed Scopus (3911) Google Scholar). However, since that time, attempts to generate patient-specific cells using SCNT have proven unsuccessful (Chung et al., 2009Chung Y. Bishop C.E. Treff N.R. Walker S.J. Sandler V.M. Becker S. Klimanskaya I. Wun W.S. Dunn R. Hall R.M. et al. Cloning Stem Cells, 2009https://doi.org/10.1089/clo.2009.0004Google Scholar, French et al., 2008French A.J. Adams C.A. Anderson L.S. Kitchen J.R. Hughes M.R. Wood S.H. Stem Cells. 2008; 26: 485-493Crossref PubMed Scopus (194) Google Scholar). In 2006, a new and less controversial method of reprogramming somatic cells to pluripotency was reported by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17372) Google Scholar). This and subsequent studies confirmed that mouse and human somatic cells can be reprogrammed to the pluripotent state via viral transduction with the same or similar sets of reprogramming factors (Maherali et al., 2007Maherali N. Sridharan R. Xie W. Utikal J. Eminli S. Arnold K. Stadtfeld M. Yachechko R. Tchieu J. Jaenisch R. et al.Cell Stem Cell. 2007; 1: 55-70Abstract Full Text Full Text PDF PubMed Scopus (1355) Google Scholar, Okita et al., 2007Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3397) Google Scholar, Park et al., 2008Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2302) Google Scholar, Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13805) Google Scholar, Wernig et al., 2007Wernig M. Meissner A. Foreman R. Brambrink T. Ku M. Hochedlinger K. Bernstein B.E. Jaenisch R. Nature. 2007; 448: 318-324Crossref PubMed Scopus (2160) Google Scholar, Yu et al., 2007Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (7740) Google Scholar). Although the therapeutic potential of iPSCs has been demonstrated in animal models of sickle cell anemia and Parkinson's disease (Hanna et al., 2007Hanna J. Wernig M. Markoulaki S. Sun C.W. Meissner A. Cassady J.P. Beard C. Brambrink T. Wu L.C. Townes T.M. et al.Science. 2007; 318: 1920-1923Crossref PubMed Scopus (1207) Google Scholar, Wernig et al., 2008Wernig M. Zhao J.P. Pruszak J. Hedlund E. Fu D. Soldner F. Broccoli V. Constantine-Paton M. Isacson O. Jaenisch R. Proc. Natl. Acad. Sci. USA. 2008; 105: 5856-5861Crossref PubMed Scopus (982) Google Scholar), these cells contain multiple viral vector integrations that make them unsuitable for human clinical trials. The use of genome-integrating viruses could cause insertional mutagenesis and unpredictable genetic dysfunction (Okita et al., 2007Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3397) Google Scholar, Yamanaka, 2007Yamanaka S. Cell Stem Cell. 2007; 1: 39-49Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). To address whether it is possible to generate hiPSCs without the use of viral or DNA vectors, we attempted to deliver four reprogramming proteins—Oct4, Sox2, Klf4, and c-Myc—directly into somatic cells. A major hurdle for intracellular delivery of macromolecules such as proteins is their limited ability to cross the cellular membrane (Belting et al., 2005Belting M. Sandgren S. Wittrup A. Adv. Drug Deliv. Rev. 2005; 57: 505-527Crossref PubMed Scopus (173) Google Scholar). In 1988, Frankel and Pabo found that the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein can overcome this hurdle with a short basic segment residing at amino acids 48–60 that allows this protein to penetrate the cell membrane and activate HIV-specific genes (Frankel et al., 1988Frankel A.D. Bredt D.S. Pabo C.O. Science. 1988; 240: 70-73Crossref PubMed Scopus (329) Google Scholar, Frankel and Pabo, 1988Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2213) Google Scholar). This and other naturally occurring peptides capable of overcoming the cell membrane barrier contain a high proportion of basic amino acids (e.g., arginine or lysine) and are known as CPPs (El-Sayed et al., 2009El-Sayed A. Futaki S. Harashima H. AAPS J. 2009; 11: 13-22Crossref PubMed Scopus (347) Google Scholar, Ziegler et al., 2005Ziegler A. Nervi P. Durrenberger M. Seelig J. Biochemistry. 2005; 44: 138-148Crossref PubMed Scopus (197) Google Scholar). In order to test our hypothesis that CPP-anchored reprogramming proteins may directly reprogram human somatic cells without genetic manipulation and/or chemical treatments, we first examined whether red fluorescent protein (RFP) fused with a 9 arginine (RFP-9R) (Wender et al., 2000Wender P.A. Mitchell D.J. Pattabiraman K. Pelkey E.T. Steinman L. Rothbard J.B. Proc. Natl. Acad. Sci. USA. 2000; 97: 13003-13008Crossref PubMed Scopus (1367) Google Scholar) could penetrate into COS-7 cells and human newborn fibroblasts (HNFs). RFP-9R was efficiently delivered into both cell types within a few hours, even when in the context of whole-cell extracts (see Figure S1 available online). We then generated stable HEK293 cell lines that could express each of the four human reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) fused with 9R and the myc tag. High expression of these proteins was confirmed in HEK293 cell lines by western blotting analyses (Figure S2). When HNFs were treated with cell extracts from the HEK293 cell lines, efficient intracellular translocation of each recombinant protein was observed within 8 hr (Figure 1A). Notably, in contrast to RFP-9R, which was translocated to the cytoplasm, it appeared that most recombinant reprogramming proteins were translocated to the nucleus, while some remained in the cytoplasm (Figure 1A and Figure S1). In an initial series of experiments, 5 × 105 HNFs were treated with combined total extracts of four HEK293 cell lines for 16 hr (see “Protocol 1” in Figure S3). After washing, cells were incubated for 6 days in ES media 1 and then transferred onto mouse embryonic feeders (MEFs). The transferred cells were incubated with ES media 2 for up to 4 weeks. Despite numerous attempts, we did not observe the formation of reprogrammed colonies using this protocol. We next treated with the same total extracts for 16 hr followed by washing and incubation with ES media 1 for 8 hr/day for 6 days (“Protocol 2” in Figure S3). By day 7, most cells did not survive, and no colonies formed after further incubation on MEF. One potential reason for the lack of success is that, in contrast to virus- or other DNA-based methods, the reprogramming factors were not provided continuously and thus were in short supply. Therefore, we tested whether repeated protein treatment cycles (16 hr protein treatment followed by 6 day incubation in ES media 1) (Figure 1B) could yield hiPSCs. Using this approach, after three or four rounds of treatment, several colonies with iPSC-like morphology were observed (Figure 1C), although none of these colonies showed alkaline phosphatase (AP) activity, suggesting only rudimentary reprogramming. When this procedure was repeated for further cycles, the number of iPSC-like colonies significantly increased, and approximately half of the resulting colonies were AP positive starting from the sixth cycle (Figure 1D). In contrast, no such colonies were formed at any stage when extracts of naive HEK293 cells were used. AP-positive colonies with iPSC-like morphology were handpicked and transferred onto MEFs in the presence of ES media 2 and ES media 3 for 7 days each. Five hiPSC-like colonies were established, and two of them were maintained and characterized in this study. These two cell lines (p-hiPS01 and p-hiPS02) have been successfully maintained for more than 35 passages and exhibit morphology similar to that of hESCs, characterized by large nuclei and scant cytoplasm (Figure 1C). Overall, the establishment of these hiPSC-like colonies took about 8 weeks, approximately double that seen with viral transduction (Park et al., 2008Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2302) Google Scholar, Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13805) Google Scholar, Yu et al., 2007Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (7740) Google Scholar). At present, the efficiency of hiPSC generation is significantly lower using this protein-based protocol (about 0.001% of input cells; Figure 1D), compared to virus-based protocols (about 0.01% of input cells) (Park et al., 2008Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2302) Google Scholar, Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13805) Google Scholar, Yu et al., 2007Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (7740) Google Scholar). In order to determine whether the p-hiPSCs have hESC-like properties, we examined them for expression of markers of pluripotency. As shown in Figure 1C, both cell lines prominently expressed ESC markers, including AP, Oct4, Nanog, tumor-rejection antigen (TRA)1-60, stage-specific embryonic antigen (SSEA)-3, and SSEA-4. Quantitative reverse transcription PCR (qRT-PCR) analysis confirmed that both lines expressed endogenous mRNAs of ESC markers: Oct4, Nanog, Sox2, reduced expression 1 (Rex1), growth and differentiation factor 3 (Gdf3), and telomerase reverse transcriptase (hTERT) levels were dramatically higher than those of HNF cells (up to 100-fold, and comparable to hESCs) (Figure 2A). The expression patterns of ESC pluripotency markers were indistinguishable from hESCs (H9), strongly suggesting that appropriate epigenetic reprogramming had occurred in the p-hiPSCs. Bisulfite sequencing analyses further showed that the promoter regions of the pluripotency genes Nanog and Oct4 were significantly demethylated in both p-hiPSC lines and the hESC H9 line, whereas the same regions were densely methylated in the parental HNF cells (Figure 2C). hiPSC lines from the starting HNFs were also generated using retroviral vectors expressing the same four reprogramming factors (Park et al., 2008Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2302) Google Scholar, Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13805) Google Scholar, Yu et al., 2007Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (7740) Google Scholar). These cells displayed similar characteristics and properties as the p-hiPSCs (Figure S4), and one of them (rv-hiPS01) was used as a control. When global gene expression was compared using the Affymetrix Array U133 Plus 2.0, analyzing over 47,000 human transcripts, both p-hiPSC and rv-hiPS01 showed high similarity to hES H9, but not to HNFs (Figure 2B, Figure S5). Since hES H9 and rv-hiPS01 were used as control cell lines, it was important to rule out the possibility that the new p-hiPSCs were derived from contaminating cells. RT-PCR analyses detected all four transgene mRNAs in rv-hiPS01 cells, but not in p-hiPS01 and p-hiPS02 cell lines (Figure S6). Furthermore, DNA fingerprinting demonstrated that the patterns of both p-hiPSC lines and rv-hiPS01 cells were identical to the parental HNF cells but different from those of the hES (H9) cells and HEK293 cells (Figure S7), thus confirming that both p-hiPSC lines are derived from HNF cells. Both p-hiPSC lines exhibited the same karyotype as the starting HNF cells (Figure S8). When the p-hiPSCs were allowed to form embryoid bodies (EBs) by suspension culture, they readily differentiated into cells of all three germ layers (Figure 2D, Figure S8). After 8 days, well-formed EB structures were observed from both p-hiPSC clones. When these EB-like structures were incubated on gelatin-coated tissue culture plates in ITSFn media for 15–25 days, they differentiated to a wide range of cell types, including neural, muscle, and endodermal cells, among others. Immunocytochemical analyses demonstrated the existence of different cell types positive for hepatocyte necrosis factor 3β (HNF 3β, endoderm marker), α-fetoprotein (AFP, endoderm marker), smooth-muscle actin (SMA, mesoderm marker), desmin (mesoderm marker), Tuj1 (ectoderm marker), nestin (ectoderm marker), and tyrosine hydroxylase (TH, ectoderm marker) (Figure 2D, Figure S8). In addition, teratoma formation was observed after transplantation of p-hiPSCs under the kidney capsule of nude mice for 6–8 weeks. These teratomas contained tissues from all three germ layers including neural tissues (ectoderm), epidermal tissues (ectoderm), striated muscle (mesoderm), adipose tissue (mesoderm), cartilage (mesoderm), respiratory epithelium (endoderm), and intestinal-like epithelial tissues (endoderm) (Figure 2E, Figure S8), confirming that both p-hiPSC clones exhibit pluripotency both in vitro and in vivo. Protein-based hiPSC technology offers a new and potentially safe method for generating patient-specific stem cells that does not require the destruction of ex utero embryos. This system completely eliminates genome manipulation and DNA transfection, resulting in human iPSCs suitable for drug discovery, disease modeling, and future clinical translation. In this regard, the present study demonstrates the “proof of concept” that human iPSCs can be generated by direct protein delivery without genetic manipulation. Other studies suggest that it may be possible to replace and/or further reduce the number of factors required for reprogramming (Huangfu et al., 2008Huangfu D. Osafune K. Maehr R. Guo W. Eijkelenboom A. Chen S. Muhlestein W. Melton D.A. Nat. Biotechnol. 2008; 26: 1269-1275Crossref PubMed Scopus (1082) Google Scholar, Li et al., 2009Li W. Wei W. Zhu S. Zhu J. Shi Y. Lin T. Hao E. Hayek A. Deng H. Ding S. Cell Stem Cell. 2009; 4: 16-19Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, Shi et al., 2008Shi Y. Desponts C. Do J.T. Hahm H.S. Scholer H.R. Ding S. Cell Stem Cell. 2008; 3: 568-574Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). To minimize/avoid chromosomal disruption, adenovirus and plasmid transfection have been successfully used to generate iPSCs in the mouse system (Kaji et al., 2009Kaji K. Norrby K. Paca A. Mileikovsky M. Mohseni P. Woltjen K. Nature. 2009; 458: 771-775Crossref PubMed Scopus (1011) Google Scholar, Okita et al., 2008Okita K. Nakagawa M. Hyenjong H. Ichisaka T. Yamanaka S. Science. 2008; 322: 949-953Crossref PubMed Scopus (1549) Google Scholar). Also, Thomson and his colleagues reported generation of hiPSCs by transfection with nonintegrating episomal vectors (Yu et al., 2009Yu J. Hu K. Smuga-Otto K. Tian S. Stewart R. Slukvin I.I. Thomson J.A. Science. 2009; 324: 797-801Crossref PubMed Scopus (1679) Google Scholar). In addition, piggyBac transposon (Kaji et al., 2009Kaji K. Norrby K. Paca A. Mileikovsky M. Mohseni P. Woltjen K. Nature. 2009; 458: 771-775Crossref PubMed Scopus (1011) Google Scholar, Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hamalainen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1394) Google Scholar) and Cre-recombinase excisable viruses (Soldner et al., 2009Soldner F. Hockemeyer D. Beard C. Gao Q. Bell G.W. Cook E.G. Hargus G. Blak A. Cooper O. Mitalipova M. et al.Cell. 2009; 136: 964-977Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar) have been used to generate hiPSCs. While the transgenes can be excised by inducible gene expression once reprogramming is established (Soldner et al., 2009Soldner F. Hockemeyer D. Beard C. Gao Q. Bell G.W. Cook E.G. Hargus G. Blak A. Cooper O. Mitalipova M. et al.Cell. 2009; 136: 964-977Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar, Stadtfeld et al., 2008Stadtfeld M. Nagaya M. Utikal J. Weir G. Hochedlinger K. Science. 2008; 322: 945-949Crossref PubMed Scopus (1286) Google Scholar, Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hamalainen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1394) Google Scholar), residual sequences and chromosomal disruptions may still result in harmful alterations that could pose clinical risks. The DNA vector-free, direct protein transduction system described here eliminates limitations that may be caused by viral or any other DNA-based reprogramming methods. However, the generation of p-hiPSCs is very slow and inefficient and requires further optimization. In particular, the whole-protein extracts used in the present study limited the concentrations of factors delivered into the target cells, thus suggesting that p-hiPSCs may be more efficiently generated using purified reprogramming proteins. Recently, Ding and his colleagues reported the generation of mouse iPSCs by combining the use of recombinant reprogramming proteins and the small molecule valproic acid (Zhou et al., 2009Zhou H. Wu S. Joo J.Y. Zhu S. Han D.W. Lin T. Trauger S. Bien G. Yao S. Zhu Y. et al.Cell Stem Cell. 2009; 4: 381-384Abstract Full Text Full Text PDF PubMed Scopus (1455) Google Scholar). In this study, mouse iPSCs were not generated when only recombinant proteins were used. In contrast, the system described here generated human iPSCs with direct delivery of reprogramming proteins in the absence of any chemical treatment. One possible explanation for these differences is that we used reprogramming proteins expressed in mammalian cells, while Ding and colleagues used refolded proteins after expression in E. coli. Since chemicals such as valproic acid and/or genetic manipulation may induce mutations, it has been suggested that whole genomic sequencing would be necessary if such methods are used to generate iPSCs (Yamanaka, 2009Yamanaka S. Cell. 2009; 137: 13-17Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). In conclusion, the system described here eliminates the potential risks associated with chromosomal integrations and/or mutations and may allow the translation of hiPSC technology into the clinic. This work was supported by National Institutes of Health (NIH) grants MH48866 and DC 006501 and by International Grants from the CHA University, Korean Stem Cell Research Center, and Dongyang Corporation Co. in Korea. The authors thank Dr. V. Morgan (Harvard Partners Center for Genetics and Genomics), Dr. J. Kim (Harvard Medical School), and Dr. J. Lee (Shippensburg University) for microarray analysis and Ms. J. Johnson (Cell Line Genetics) for karyotyping analysis. R.L. is an employee and shareholder of Advanced Cell Technology and a scientific advisor for Stem Cell and Regenerative Medicine International. K.Y.C. is a shareholder of CHA Bio and Diostech Co., Ltd., and Stem Cell and Regenerative Medicine International. Microarray data can be assessed at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE16093, GSM402806, GSM402752, GSM402717, GSM402708, and GSM402707. Download .pdf (.47 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Supplemental References, Eight Figures, and Four Tables

Cell Culture Human newborn fibroblasts (HNF) were purchased from ATCC (CCL-117) and cultured in Dulbecco’s modified Minimal Essential Medium (DMEM, Invitrogen, Carlsbad, CA), supplemented with 2mM L-glutamine (Invitrogen, Grand island, NY). 1mM β-mercaptoethanol, 1x non-essential amino acids (NEAA; Invitrogen, Carlsbad, CA), 15% fetal bovine serum (FBS, Hyclone, Thermo Scientific, Logan UT), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Grand island, NY). Reprogramming experiments were started when the cultures reached 10-20% confluence. Cultures were maintained at 37°C and 5% CO2, and the media changed every other day. For mouse embryonic fibroblast (MEF) isolation, uteri were isolated from 13.5-day-pregnant CD1 mice and washed with phosphatebuffered saline (PBS). The head and visceral tissues were removed, and the remaining bodies washed with fresh PBS, transferred into a 0.1 mM trypsin/1 mM EDTA solution, and incubated for 20 min. After incubation, MEF culture medium (DMEM containing 15% defined FBS) was added and pipetted up and down to dissociate cells. MEFs were used as feeders at passages one to three.

You have accessJournal of UrologyPodium 47, Tuesday, May 22, 2007, 3:30 - 5:30 pm1 Apr 20071672: Early Phase I/II Clinical Trial Results for Human Cord Blood Stem Cell Injection Therapy for Stress Urinary Incontinence Jong No Lee, Chester J. Koh, Christopher S. Lee, Hyung Min Chung, Kwang Yul Cha, and Jin Young Paek Jong No LeeJong No Lee More articles by this author , Chester J. KohChester J. Koh More articles by this author , Christopher S. LeeChristopher S. Lee More articles by this author , Hyung Min ChungHyung Min Chung More articles by this author , Kwang Yul ChaKwang Yul Cha More articles by this author , and Jin Young PaekJin Young Paek More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)31860-3AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1672: Early Phase I/II Clinical Trial Results for Human Cord Blood Stem Cell Injection Therapy for Stress Urinary Incontinence." The Journal of Urology, 177(4S), p. 554 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 554 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Jong No Lee More articles by this author Chester J. Koh More articles by this author Christopher S. Lee More articles by this author Hyung Min Chung More articles by this author Kwang Yul Cha More articles by this author Jin Young Paek More articles by this author Expand All Advertisement Loading ...

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.

OBJECTIVE:To report the survival rate of oocytes and the rate of successful pregnancies obtained from super-rapid cooling of oocytes using slush nitrogen (SN(2)).DESIGN:Prospective clinical research.SETTING:A university-affiliated hospital.PATIENT(S):Twenty-eight infertile women who underwent 30 cycles of IVF-ET using previously vitrified oocytes.INTERVENTION(S):Oocytes were vitrified by super-rapid cooling using SN(2).MAIN OUTCOME MEASURE(S):Morphological normality of thawed oocytes and clinical outcome.RESULT(S):In 30 cycles of ovarian stimulation for IVF, 364 surplus oocytes from 28 patients were vitrified using SN(2). Three hundred two (85.1% +/- 2.9%) of the oocytes survived after warming. Fertilization and cleavage rates were 77.4% +/- 3.5% (168/218) and 94.3% +/- 2.1% (158/168), respectively. Thirteen pregnancies (43.3%) resulted from 30 uterine transfers of 120 embryos with an implantation rate of 14.2% (17/120). There were no differences between the pregnancy rate after vitrification/warming and that obtained from routine noncryopreserved oocytes.CONCLUSION(S):The present report suggests that super-rapid cooling may improve the clinical efficacy of human oocyte vitrification and may be a valuable tool for human assisted reproductive technologies.

BACKGROUND:Three typical folate metabolism enzymes-i.e. methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS) and MS reductase (MTRR) in the folate cycle-play a critical role in DNA synthesis and methylation reactions. We evaluated whether polymorphisms of these three enzymes are associated with non-obstructive male infertility.METHOD:Three hundred and sixty patients with non-obstructive infertility and 325 fertile men without any chromosomal abnormalities were included in this study. The single-nucleotide polymorphism (SNP) analysis was performed by pyrosequencing and PCR-restriction fragment length polymorphism (RFLP) analysisRESULTS:The frequencies of MTHFR 677TT and MTRR 66GG genotypes were higher in non-obstructive infertile men compared with those in fertile men. By classifying 360 infertile patients into 174 azoospermia and 186 oligoasthenoteratozoospermia (OAT) subjects, the MTHFR 677TT and MS 2756GG types were significantly associated with the azoospermia group (P = 0.0227 and 0.0063, respectively). The frequency of MTRR 66GG was significant in the OAT group (P = 0.0014 versus fertile males).CONCLUSIONS:By analysis of a large number of subjects and a more specific patient selection, we showed the first genetic evidence that MTHFR C677T, MS A2756G and MTRR A66G genotypes were independently associated with male infertility. Each SNP of the three enzymes may have a different impact on the folate cycle during spermatogenesis.