Mouse Embryonic Fibroblast Feeder Layers Research Articles

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves­tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 μm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments.

231 Novel protocol for the invitro maintenance and expansion of adult epidermal LGR5+ stem cells

Currently, the standard for treatment of full-thickness skin wounds is skin grafts or bioengineered skin substitutes; however, this method is limited by the amount of intact donor skin and lack of follicles and architecture. Thus, a protocol is needed for the expansion and differentiation of adult epidermal and hair follicle stem cells for use in scaffold mediated tissue engineering. Recently, we developed a transgenic porcine model in which H2B-GFP is under the control of the LGR5 promoter. LGR5 is an established marker of stem cells, meaning this model can be used to track the development and behaviour of these cells. The focus of this project was to create a novel culture method for the maintenance and expansion of LGR5+ epidermal adult stem cells utilising the green fluorescent protein (GFP) tag. Single cell epidermal stem cells were isolated from porcine skin using dispase II (10mgmL−1; Sigma) and trypsin (0.05%; Corning). Porcine fetal fibroblasts (PFF) or mouse embryonic fibroblasts (MEF) were grown to 95% confluence in a 6-well plate. Feeder layer cells were mitotically inactivated by incubation with mitomycin C (Sigma Aldrich, 10μgmL−1). Three different media were tested: basal medium [Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, Corning; Ham's F12, ThermoFisher; fetal bovine serum, Gemini Bio-Products], basal media + 5-azacytidine (Sigma Aldrich) and CHIR99021 (Tocris), or basal media + keratinocyte growth supplements (transferrin, hydrocortisone, T3, adenine, insulin, cholera toxin; Sigma Aldrich, epidermal growth factor; R&D Systems). Epidermal cells were plated in each medium for both PFF and MEF feeder layers. Experiments were performed in technical duplicates and replicated 3 times. On Day 9, total numbers of colonies in each well were counted and number of GFP-positive cells were quantified using ImageJ (National Institutes of Health). Results in Table 1 show that overall, the MEF feeder layer was able to support a higher rate of growth (P<0.05) and maintain the LGR5+ lineage at a higher proportion under all of the experimental conditions (P<0.05). In the growth-supplemented media, MEFs had fewer colonies than PFFs, but MEF colonies were, on average, 2.5 times larger (P<0.05). Conditions containing 5-aza and CHIR were the only conditions to maintain the LGR5+ lineage on the feeder layer. Statistically significant differences (P<0.05) were determined using two-way ANOVA, followed by Tukey's HSD test. Next, LGR5+ cells will be plated in media containing additional growth factors to stimulate expansion, while using CHIR and 5-aza to maintain the LGR5+ lineage. This protocol could be used in scaffolds to create three-dimensional growth of skin invitro and lead to better grafts for burn victims. Table 1.Growth of LGR5+ cells in different media including 5-azacytidine (5-aza), CHIR 99021 (CHIR), and keratinocyte growth supplements Group1 Basal medium (BM) BM + 5-aza+ CHIR BM + growth supplements No. of colonies/well MEF 127.7±40.8AB 189.3±16.9A 87.3±14.6B PFF 65.0±14.1A 83.3±17.0AB 148±33.7B Average no. of GFP+ cells per frame MEF 0.5±0.8B 65.7±18.4A 1.8±1.7B PFF 0.9±1.0B 22.6±4.5A 0.3±0.6B A,BValues within rows with different superscripts differ (P ≤ 0.05). 1MEF=mouse embryonic fibroblasts; PFF=porcine fetal fibroblasts.

Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

Abstract 209: iPSC Derived Cardiomyocytes Reproduce Divergent Phenotypes Caused by a LQTS Type-1 Likely Pathogenic Mutation

The purpose of this work was to investigate if iPSC derived cardiomyocytes would reproduce the divergent phenotypes observed in a proband and mother that carried the same likely-pathogenic variant in KCNQ1 gene. A 15 years old proband had syncope and cardiac arrest while swimming being diagnosed with LQTS based on a 12 lead ECG. The mother’s ECG showed no abnormalities. Genomic DNA from blood of daughter and mother was extracted using standard procedures. All coding exons and flanking intronic sequences of KCNQ1 (NM_000218) genes were amplified by polymerase chain reaction. Direct sequencing was performed on a 3500XL Genetic Analyzer. Data was analyzed with Geneious software for identification of mutations. For iPSC generation CD71+ CD36+ erythroblasts were reprogrammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit on irradiated mouse embryonic fibroblast feeder layers. Differentiation into cardiomyocytes was accomplished using the protocol described by Lian et al. (2012). Electrophysiological recordings were obtained from cardiomyocytes after 30–45 days of differentiation. Cells were transferred to acrylic plates pretreated with Matrigel and kept in culture for 3 days before microelectrode action potential (AP) were registered. The ECG recordings showed a corrected QT interval (QTc) of 516 ms for the daughter and 436 ms for the mother. Sequencing showed that both daughter and mother had a c.1763C>T mutation resulting in p.Ile588Thr substitution. iPSC generated from daughter, mother and an unrelated control displayed normal karyotype and expressed pluripotent genes, besides spontaneously differentiating into cells of the three germ layers. Differentiation into cardiomyocytes was ascertained by immunofluorescence and flow cytometry for troponin T. In all differentiations more than 70% of cells were cardiomyocytes. Action potentials recordings showed that ventricular cardiomyocytes from the daughter exhibited significantly longer action potential durations at 90% repolarization than the mother’s or the unrelated control’s cardiomyocytes. Cardiomyocytes derived from iPS cells reproduced the clinical phenotypes exhibited by daughter and mother.

Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions.

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells.

To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.

Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion

Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However, medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers, primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4, Sox2 Klf4, and c-Myc (OSKM) transcription factor genes, with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore, the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion, especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions

Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, β-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

Single‐cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.

Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line

PurposeMouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application.MethodsA novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells.ResultsESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation.ConclusionsHuman feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.

283 GENERATION AND CHARACTERIZATION OF BOVINE INDUCED PLURIPOTENT STEM CELLS

Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production. This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.

285 GENERATION OF CANINE INDUCED PLURIPOTENT STEM CELLS FROM CANINE FETAL FIBROBLAST AND ADULT FIBROBLAST OF CLONED DOG

Induced pluripotent stem cells (iPSC) derived from a patient’s fibroblasts have been used as fine resources for studying disease mechanisms and therapeutic strategies. The dog is considered invaluable in human disease research because its genetic diseases are strikingly similar to those of human. Therefore, we generated cloned dogs and transgenic cloned dogs via somatic cell nuclear transfer. In this study, we tried to derive canine iPSCs from canine fibroblasts to establish a way to make iPSC from skin fibroblasts of transgenic cloned dogs. We isolated canine fetal fibroblast (FF) from normal beagles and adult skin fibroblast (ASF) from cloned beagles. Both ASF and FF were infected with all-in-one retroviral vector that delivers human reprogramming factors (Oct4, Sox2, Klf4, c-Myc). Ten to twenty-one days after infection, the colony-shaped structure was picked and plated on a mouse embryonic fibroblast (MEF) feeder layer, pretreated with mitomycin C. Then, all cells were cultured with DMEM/F12 supplemented with 20% fetal bovine serum, 5 ng mL–1 basic fibroblast growth factor (bFGF), 5 ng mL–1 LIF, 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin. Alkaline phosphatase (AP) activity and expression of Oct4, Sox2, SSEA1, and SSEA4, were observed in the cells to characterise the iPS cell colonies. In vitro differentiation of 10th-passage canine iPSC was performed through embryonic body formation. About 50 canine iPS-like colonies were formed on a 100-mm dish. As a result, the canine iPSC from FF (iPSC-FF) and canine iPSC from ASF (iPSC-ASF) showed typical colony morphology, and both stained positively for AP. The expression of pluripotency-associated transcription factors Oct4 and Sox2 was positively displayed in iPSC-FF colonies. The stem cell markers SSEA1 and SSEA4 were negative in canine iPSC-FF. The canine iPS-FF spontaneously differentiated into all 3 germ layers in vitro, showing positive expressions of βIII-tubulin (ectoderm), α-SMA (mesoderm), and GATA6 (endoderm). As for iPS-ASF, characterisation and in vitro differentiation experiment are in progress. These results show that canine iPS-FF are similar to embryonic stem cells in terms of morphology and the ability to differentiate into 3 germ layers. Although we did not demonstrate complete verification of canine iPS-ASF of the cloned dog, their morphology, AP expression, and iPS-FF generation should indicate the possibility of iPSC production in the cloned dog. In conclusion, retroviral transduction of 4 human reprogramming factors can reprogram canine fetal fibroblasts into canine iPSC. The technique of producing canine iPSC will stimulate the utilisation of transgenic cloned dogs and expand the range of human diseases or therapeutic application. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311011-05-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.

Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors

Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

A high G418-resistant neoR transgenic mouse and mouse embryonic fibroblast (MEF) feeder layers for cytotoxicity and gene targeting in vivo and in vitro

Aminoglycoside antibiotics have been in use since 1944 with the discovery of streptomycin. The aim of this study was to derive a new, highly resistant multicopy neoR transgenic mouse strain, named TgN3Ems, by random insertion of the plasmid, pPGKneobpA, and compare the level of drug resistance of wild-type and transgenic mice in vivo and corresponding primary mouse embryonic fibroblasts (MEFs) in vitro to a model neomycin analog, G418. The expression neoR in transgenic animals caused a 5-fold increase in the approximate lethal dose of G418, compared to wild type. No adverse pathological changes were found for the transgenic mice treated with G418, as they all died within minutes after injection. In contrast, the G418 treatment of wild-type mice resulted in a marked liver and kidney toxicity detected microscopically and via increases of serum biomarkers for liver and kidney damage. In addition, there was a mild bone marrow and lymphoid depletion. In in vitro studies, the transgenic MEFs survived 20-fold higher G418 levels, compared to the wild-type MEF cells. Therefore, TgN3Ems transgenic mice could be used as a source of G418-resistant feeder cells for gene targeting. Since the expression of drug-resistance genes in transgenic animals confers resistance to toxicity, the TgN3Ems mice might serve as a tool applicable in drug design.

2013 INTRODUCTION OF VASA, DAZL, DAZ3, AND BOULE IN THE DIRECT REPROGRAMMING OF GERM CELLS FROM FIBROBLASTS DERIVED FROM ADULT TESTIS TISSUE

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20112013 INTRODUCTION OF VASA, DAZL, DAZ3, AND BOULE IN THE DIRECT REPROGRAMMING OF GERM CELLS FROM FIBROBLASTS DERIVED FROM ADULT TESTIS TISSUE Hideyuki Kobayashi, Koichi Nagao, Yusuke Oka, Masato Nagata, Fumito Yamabe, keiichiro Takasugi, Shuichi Kamimura, Norie Tanaka, Kuri Suzuki, and Koichi Nakajima Hideyuki KobayashiHideyuki Kobayashi Tokyo, Japan More articles by this author , Koichi NagaoKoichi Nagao Tokyo, Japan More articles by this author , Yusuke OkaYusuke Oka Tokyo, Japan More articles by this author , Masato NagataMasato Nagata Tokyo, Japan More articles by this author , Fumito YamabeFumito Yamabe Tokyo, Japan More articles by this author , keiichiro Takasugikeiichiro Takasugi Tokyo, Japan More articles by this author , Shuichi KamimuraShuichi Kamimura Tokyo, Japan More articles by this author , Norie TanakaNorie Tanaka Tokyo, Japan More articles by this author , Kuri SuzukiKuri Suzuki Tokyo, Japan More articles by this author , and Koichi NakajimaKoichi Nakajima Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2241AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The forced expression of 4 transcription factors–Oct4, Sox2, Klf4, and c-Myc–permits reprogramming of fibroblasts into pluripotent stem cells. These reprogrammed cells are referred to as induced pluripotent stem (iPS) cells. It has been shown that human adult germline stem cells from testicular tissue (haGSCs) have pluripotent properties similar to those of human embryonic stem cells (hESCs). However, recent research has indicated that haGSCs and fibroblasts have a similar gene expression profile and differ in character from hESCs. The pluripotency of haGSCs has thus been questioned. Notably, it was reported that the DAZL, DAZ, and BOULE genes modulate primordial germ cell and haploid gamete formation. Therefore, we examined whether the introduction of VASA, DAZL, DAZ3, and BOULE would permit direct reprogramming of germ cells derived from human testicular fibroblasts. METHODS After obtaining informed consent, testis tissue was collected from infertile men. The tissues were mechanically dissected and dissociated by enzymatic treatment. The cells were then incubated with 7% FBS and DMEM for 14 days. We established fibroblasts from human testicular cultures. VASA, DAZL, DAZ3, and BOULE were delivered to fibroblasts by using a lentiviral vector system. The fibroblasts were then cultured with 7% FBS and DMEM for 4 days, after which they were grown on either a mouse embryonic fibroblast feeder layer, a 0.1% gelatin dish, a HEMA/MMA-coated dish, or an uncoated dish; all dishes were incubated with ES medium and basic fibroblast growth factor. After 20–25 days, cells were collected and their characteristics were investigated by RT-PCR. RESULTS There were no morphological changes in iPS cells generated from testicular fibroblasts with VASA, DAZL, DAZ3, and BOULE; however, RT-PCR confirmed that the cells expressed the germ cell markers VASA, DAZL, and DAZ. CONCLUSIONS We attempted direct reprogramming of germ cells derived from human testicular fibroblasts; however, the introduction of VASA, DAZL, DAZ3, and BOULE did not result in generation of complete germ cells. Nevertheless, we maintain that direct reprogramming of germ cells is possible under suitable conditions. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e805 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hideyuki Kobayashi Tokyo, Japan More articles by this author Koichi Nagao Tokyo, Japan More articles by this author Yusuke Oka Tokyo, Japan More articles by this author Masato Nagata Tokyo, Japan More articles by this author Fumito Yamabe Tokyo, Japan More articles by this author keiichiro Takasugi Tokyo, Japan More articles by this author Shuichi Kamimura Tokyo, Japan More articles by this author Norie Tanaka Tokyo, Japan More articles by this author Kuri Suzuki Tokyo, Japan More articles by this author Koichi Nakajima Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium

Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).

A New Feeder-Free Technique to Expand Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

The optimal maintenance of human embryonic stem cells (hESC) in vitro is generally observed in the presence of a feeder-layer of mouse embryonic fibroblasts in a serum-containing medium. Various approaches are now available to remove the feeder requirement. Today, the best feeder-free system for the maintenance of hESC and induced pluripotent stem (iPS) cells is based on a serum-replacement medium on a matrice of Matrigel. Some reports have also shown feeder- free maintenance of hESC in the presence of laminin, fibronectin, vitronectin or collagen IV. However these combinations are expensive. Here we describe an alternative to the current feeder-free short-term amplification of hESC and iPS cells using culture grade plastic dishes pre-coated with 10-20% fetal calf serum. hESC retain expression of various stem cell markers and also retain the ability to differentiate in vitro. Similar results were observed with human iPS cells. This feeder-free culture system is reliable, convenient and allows for the rapid amplification of hESC and iPS cells to numbers suitable for many cell biology techniques.

Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice

Objective To establish a long-term culture system for mouse spermatogonial stem cells(SSCs). Methods Testis cells from 4-6 days postpartum male transgenic BALB/C mice were collected by a modified two-step enzymatic digestion method.After three differential adherence selections,the enriched germ cells were finally suspended in StemPro-34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast(MEF)feeder layer.20 ng/ml Glial cell line-derived neurotrophic factor,10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation.Aduh male BALB/C mice,4-5 weeks old,underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice.Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control.Testes of recipient mice were observed by a fluorescence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method,the vitality of isolated testis cells was more than 98%and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3-4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP.HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis.Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months.The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility. Key words: Spermatogonia; Stem cells; Cell culture techniques; Transplantation; Mice

101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P &lt; 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.