Derivation and characterization of putative embryonic stem cells from cloned rabbit embryos.

The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investigated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthenogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17, 17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell-specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription-polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibroblasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo.

Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors.

We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent.

The J-domain of heat shock protein 40 can enhance the transduction efficiency of arginine-rich cell-penetrating peptides.

Sense and antisense oligonucleotide pairs encoding cell-penetrating peptides PTD (Tat47-57), DPV3A, E162, pVEC, R11, and TP13 were used to construct two sets of pET22b-CPP-DsRed and pET22b-CPP-J-DsRed vectors for CPP-DsRed and CPP-J-DsRed recombinant proteins expression. PTD-DsRed, DPV3A-DsRed, PTD-J-DsRed, and DPV3A-J-DsRed recombinant proteins were expressed in a soluble form. PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins were able to escape from E. coli host cells into the culture medium. The membrane-penetrating activity of PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins to mammalian cells was more effective than that of PTD-DsRed and DPV3A-DsRed. The route of the cellular membrane translocation of these recombinant proteins is suggested via macropinocytosis followed by an endosomal escape pathway.

Leukemia inhibitory factor and fibroblast growth factor 2 critically and mutually sustain pluripotency of rabbit embryonic stem cells.

Effects of leukemia inhibitory factor (LIF) and fibroblast growth factor 2 (FGF2) on establishment and maintenance of rabbit embryonic stem cell (rESC) lines were assessed. When grown on MEF feeders, rESC lines derived from fertilized embryos were established and maintained in medium containing paracrine factors LIF (via STAT3) and/or FGF2 (via MEK-ERK1/2 and PI3K-AKT). However, high levels of ERK1/2 and AKT activities in rESCs were crucial for maintaining their undifferentiated proliferation. Although rESCs under the influence of either LIF (500, 1,000, and 2,000 U/ml) or FGF2 (5, 10, and 20 ng/ml) alone had enhanced expression of pluripotency markers, peak expression occurred when both LIF (1,000 U/ml) and FGF2 (10 ng/ml) were applied. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors limited growth of rESCs and caused remarkable losses of self-renewal capacity; therefore, we inferred that STAT3, ERK, and AKT had essential roles in maintaining rESC proliferation and self-renewal. We concluded that LIF and FGF2 jointly maintained the undifferentiated state and self-renewal of rESCs through an integrative signaling module.

Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated viable, healthy, and fertile chimeric mice with 100 % coat color chimerism.Both vial coculture and hypertonic microinjection methods are useful and effective alternatives for producing germline chimeric or F0 mice efficiently and reliably. Furthermore, both novel methods are also good for induced pluripotent stem cells (iPSCs) to generate chimeric embryos.

Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury.

High concentrations of oxygen aggravate the severity of lung injury in patients requiring mechanical ventilation. Although mesenchymal stem cells have been shown to effectively attenuate various injured tissues, there is limited information regarding a role for amniotic fluid stem cells (AFSCs) in treating acute lung injury. We hypothesized that intravenous delivery of AFSCs would attenuate lung injury in an experimental model of hyperoxia-induced lung injury. AFSCs were isolated from EGFP transgenic mice. The in vitro differentiation, surface markers, and migration of the AFSCs were assessed by specific staining, flow cytometry, and a co-culture system, respectively. The in vivo therapeutic potential of AFSCs was evaluated in a model of acute hyperoxia-induced lung injury in mice. The administration of AFSCs significantly reduced the hyperoxia-induced pulmonary inflammation, as reflected by significant reductions in lung wet/dry ratio, neutrophil counts, and the level of apoptosis, as well as reducing the levels of inflammatory cytokine (IL-1ß, IL-6, and TNF-α) and early-stage fibrosis in lung tissues. Moreover, EGFP-expressing AFSCs were detected and engrafted into a peripheral lung epithelial cell lineage by fluorescence microscopy and DAPI stain. Intravenous administration of AFSCs may offer a new therapeutic strategy for acute lung injury (ALI), for which efficient treatments are currently unavailable.

Proteomic profiling of rabbit embryonic stem cells derived from parthenotes and fertilized embryos.

Rabbit embryonic stem (rES) cells can be derived from various sources of embryos. However, understanding of the gene expression profile, which distincts embryonic stem (ES) cells from other cell types, is still extremely limited. In this study, we compared the protein profiles of three independent lines of rabbit cells, i.e., fibroblasts, fertilized embryo-derived stem (f-rES) cells, and parthenote-derived ES (p-rES) cells. Proteomic analyses were performed using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Collectively, the expression levels of 100 out of 284 protein spots differed significantly among these three cell types (p<0.05). Of those differentially expressed spots, 91% were identified in the protein database and represented 63 distinct proteins. Proteins with known identities are mainly localized in the cytoplasmic compartments (48%), nucleus (14%), and cytoskeletal machineries (13%). These proteins were majorly involved in biological functions of energy and metabolic pathways (25%), cell growth and maintenance (25%), signal transduction (14%), and protein metabolisms (10%). When protein expression levels among cell types were compared, six proteins associated with a variety of cellular activities, including structural constituents of the cytoskeleton (tubulins), structural molecule (KRT8), catalytic molecules (α-enolase), receptor complex scaffold (14-3-3 protein sigma), microfilament motor proteins (Myosin-9), and heat shock protein (HSP60), were found highly expressed in p-rES cells. Two proteins related to HSP activity and structural constituent of cytoskeleton in f-rES cells, and one structural molecule activity protein in fibroblasts showed significantly higher expression levels (p<0.05). Marker protein expressions in f-rES and p-rES cells were further confirmed by Western blotting and immunocytochemical staining. This study demonstrated unique proteomic profiles of the three rabbit cell types and revealed some novel proteins differentially expressed between f-rES and p-rES cells. These analyses provide insights into rES cell biology and would invite more in-depth studies toward rES cell applications.

An efficient and mass reproducible method for vitrifying mouse embryos on a paper in cryotubes.

To date, the most successful and popular vitrification method is based on the minimum volume cooling (MVC) concept, in which embryos are vitrified in a very small volume of vitrification solution (VS) and then stored in cryotubes in liquid nitrogen (LN2). Unfortunately, these methods need special devices and may not be suitable for vitrifying a large number of embryos. Theoretically, more embryos in VS on a paper (MVC concept) in cryotubes can be vitrified effectively. Therefore, this study directly vitrifies mouse embryos on a Kimwipes tissue in an 1.8 mL cryotube. The ICR 2-celled to blastocyst embryos were used for testing this procedure. In Treatment 1, embryos transferred with 1-2 µL of VS into a cryotube. Treatment 2 was similar to Treatment 1 except that the cryotube was filled with LN2. Treatment 3 was identical to Treatment 1 except that a small piece (5 mm²) of a sterilized Kimwipes tissue was placed on the top of VS. Treatment 4 was identical to Treatment 3 except for the cryotube being filled with LN2. After each treatment, the cryotubes were capped and transferred to a LN2 tank. After warming, the recovered embryos were cultured in KSOM+AA for 1-3 days. There were no differences in the recovery rate, overnight survival rate, blastocyst rate, and birth rate after embryo transfer among all treatment groups. Our results demonstrated an alternative simple, efficient, and mass reproducible method for vitrifying mouse embryos using papers as a vehicle and cryotubes as a container.

Simple and efficient derivation of mouse embryonic stem cell lines using differentiation inhibitors or proliferation stimulators.

The inhibition of endogenous differentiation-inducing signaling or the enhancement of growth capacity and viability of preimplantation embryos, via 2i (PD0325901 and CHIR99021), dramatically improves the establishment of mouse embryonic stem cells (mESCs). Using adrenocorticotropic hormone fragments 1-24 (ACTH 1-24), which enhances survival and/or proliferation of mESCs, also increases the derivation of mESCs from single blastomeres significantly. The CHIR99021 pathway and the proposed ACTH pathway are likely different. Therefore, this study aimed to assess the synergetic effects of 2i and ACTH 1-24 on derivation of mESCs. Results in the present study demonstrate that germline-transmitted mESCs could be efficiently derived from ICR and C57BL/6J at 0.5-4.5 days postcoitum denuded zygotes to blastocysts or isolated blastomeres of 2-8-cell embryos and cultured in 10 µL droplets with human foreskin fibroblast (Hs68) or STO (a mouse embryonic fibroblast line) feeders and in knockout serum replacement (KSR) ESC medium containing 2i or ACTH 1-24. The overall success rates for C57BL/6J and ICR were 56.2% when cultured in 2i+ACTH 1-24, 26.6% in 2i, 6.7% in ACTH 1-24, and 4.8% in KSR ESC medium. These results imply that CHIR99021 and ACTH 1-24 are synergistically enhancing the establishment of mESCs. The proposed protocol also demonstrates a highly efficient and reproducible method, has a simple layout, is easy to apply, and could be used as an alternative method for routinely establishing mESC lines.

Docosahexaenoic acid promotes dopaminergic differentiation in induced pluripotent stem cells and inhibits teratoma formation in rats with Parkinson-like pathology.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic (DA) neurons in the midbrain. Induced pluripotent stem (iPS) cells have shown potential for differentiation and may become a resource of functional neurons for the treatment of PD. However, teratoma formation is a major concern for transplantation-based therapies. This study examined whether functional neurons could be efficiently generated from iPS cells using a five-step induction procedure combined with docosahexaenoic acid (DHA) treatment. We demonstrated that DHA, a ligand for the RXR/Nurr1 heterodimer, significantly activated expression of the Nurr1 gene and the Nurr1-related pathway in iPS cells. DHA treatment facilitated iPS differentiation into tyrosine hydroxylase (TH)-positive neurons in vitro and in vivo and functionally increased dopamine release in transplanted grafts in PD-like animals. Furthermore, DHA dramatically upregulated the endogenous expression levels of neuroprotective genes (Bcl-2, Bcl-xl, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor) and protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis in iPS-derived neuronal precursor cells. DHA-treated iPS cells significantly improved the behavior of 6-hydroxydopamine (6-OHDA)-treated PD-like rats compared to control or eicosapentaenoic acid-treated group. Importantly, the in vivo experiment suggests that DHA induces the differentiation of functional dopaminergic precursors and improves the abnormal behavior of 6-OHDA-treated PD-like rats by 4 months after transplantation. Furthermore, we found that DHA treatment in iPS cell-grafted rats significantly downregulated the mRNA expression of embryonic stem cell-specific genes (Oct-4 and c-Myc) in the graft and effectively blocked teratoma formation. Importantly, 3 Tesla-magnetic resonance imaging and ex vivo green fluorescence protein imaging revealed that no teratomas were present in transplanted grafts of DHA-treated iPS-derived DA neurons 4 months after implantation. Therefore, our data suggest that DHA plays a crucial role in iPS differentiation into functional DA neurons and that this approach could provide a novel therapeutic approach for PD treatment.

LIF and FGF cooperatively support stemness of rabbit embryonic stem cells derived from parthenogenetically activated embryos.

We investigated the individual and combined effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor 2 (bFGF2) on the derivation and maintenance of rabbit embryonic stem cell lines isolated from parthenogenetic activated embryos (p-rES). First, we demonstrated that p-rES cell lines can be prevented from differentiation via LIF (STAT3) and bFGF2 (MEK-ERK1/2 and PI3K-AKT) signaling on MEF feeders. High levels of ERK1/2 and AKT activities were crucial for maintaining p-rES cells in an undifferentiated state. Although the p-rES cells under the influence of LIF (500, 1000, and 2000 U/mL) or bFGF2 (5, 10, and 20 ng/mL) alone showed enhanced expression in the pluripotency markers, the highest levels of marker expressions coincided with the simultaneous presence of LIF (1000 U/mL) and bFGF2 (10 ng/mL). The phosphorylation status of LIF and bFGF2 downstream signaling molecules including STAT3, ERK, and AKT was also intensively involved in the maintenance of p-rES cell proliferation and self-renewal. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors caused remarkable losses of self-renewal capacity of p-rES cells. We conclude that bFGF2 and LIF by itself are self-sufficient in maintaining the state of undifferentiation and self-renewal of rabbit p-ES cells, yet are most effective when acting concomitantly.

Collagen IV significantly enhances migration and transplantation of embryonic stem cells: involvement of α2ß1 integrin-mediated actin remodeling.

Embryonic stem (ES) cell transplantation represents a potential means for the treatment of degenerative diseases and injuries. As appropriate distribution of transplanted ES cells in the host tissue is critical for successful transplantation, the exploration of efficient strategies to enhance ES cell migration is warranted. In this study we investigated ES cell migration under the influence of various extracellular matrix (ECM) proteins, which have been shown to stimulate cell migration in various cell models with unclear effects on ES cells. Using two mouse ES (mES) cell lines, ESC 26GJ9012-8-2 and ES-D3 GL, to generate embryoid bodies (EBs), we examined the migration of differentiating cells from EBs that were delivered onto culture surfaces coated with or without collagen I, collagen IV, Matrigel, fibronectin, and laminin. Among these ECM proteins, collagen IV exhibited maximal migration enhancing effect. mES cells expressed α2 and ß1 integrin subunits and the migration enhancing effect of collagen IV was prevented by RGD peptides as well as antibodies against α2 and ß1 integrins, indicating that the enhancing effect of collagen IV on cell migration was mediated by α2ß1 integrin. Furthermore, staining of actin cytoskeleton that links to integrins revealed well-developed stress fibers and long filopodia in mES cells cultured on collagen IV, and the actin-disrupting cytochalasin D abolished the collagen IV-enhanced cell migration. In addition, pretreatment of undifferentiated or differentiated mES cells with collagen IV resulted in improved engraftment and growth after transplantation into the subcutaneous tissue of nude mice. Finally, collagen IV pretreatment of osteogenically differentiated mES cells increased osteogenic differentiation-like tissue and decreased undifferentiation-like tissue in the grafts grown after transplantation. Our results demonstrated that collagen IV significantly enhanced the migration of differentiating ES cells through α2ß1 integrin-mediated actin remodeling and could promote ES cell transplantation efficiency, which may be imperative to stem cell therapy.

The signals of FGFs on the neurogenesis of embryonic stem cells.

BACKGROUND: Neural induction is a complex process and the detailed mechanism of FGF-induced neurogenesis remains unclear. METHODS: By using a serum-free neural induction method, we showed that FGF1 dose-dependently promoted the induction of Sox1/N-cadherin/nestin triple positive cells, which represent primitive neuroblasts, from mouse embryonic stem (ES) cells. RESULTS: We demonstrated that FGF1, FGF2, and FGF4, but not FGF8b, enhanced this neurogenesis. Especially, FGF-enhanced neurogenesis is not mediated through the rescue of the apoptosis or the enhancement of the proliferation of Sox1+ cells. We further indicated that the inactivation of c-Jun N-terminal kinase-1 (JNK-1) and extracellular signal-related kinase-2 (ERK-2), but not p38 mitogen-activated protein kinase (MAPK), inhibited the neural formation through the inhibition of ES differentiation, but not through the formation of endomesodermal cells. CONCLUSIONS: These lines of evidence delineated the roles of FGF downstream signals in the early neural differentiation of ES cells.

Ginkgolide B induces apoptosis via activation of JNK and p21-activated protein kinase 2 in mouse embryonic stem cells.

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signaling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards the proliferation and development of mouse embryonic stem cells and blastocysts in vitro, and causes developmental injury in vivo. However, the precise molecular mechanisms underlying its actions are currently unknown. Here, our study further revealed that GKB induced apoptotic biochemical changes, including activation of JNK, caspase-3, and p21-activated protein kinase 2 (PAK2), in ESC-B5 mouse embryonic stem cells. Treatment of ESC-B5 cells with a JNK-specific inhibitor (SP600125) reduced GKB-induced activation of both JNK and caspase-3, indicating that JNK activity is required for GKB-induced caspase activation. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 showed that caspase-3 activation is required for PAK2 activation and both of these activations are required for GKB-induced apoptosis in ESC-B5 cells.

Characterization of embryonic stem cell lines derived from New Zealand white rabbit embryos.

The purposes of this study were to examine technical details in deriving and maintaining rabbit embryonic stem (rES) cell lines and to analyze their characteristics. When STO cells were used as feeder cells, no rES cell lines were established using either intact blastocysts or inner cell masses (ICMs). On the mouse embryonic fibroblasts (MEF) feeder, rES cell lines were efficiently (24%) derived. Addition of leukemia inhibitory factor (LIF) to the cells cultured on the MEF feeders further increased the derivation efficiency (57%) of rES cells. The fact that LIF induced serine-phosphorylation of STAT3 suggested LIF-dependent maintenance of rES cells. Most of the rES cell lines expressed AP, SSEA-4, Oct4, TRA-1-60, and TRA-1-81. Western blot or RT-PCR analysis also confirmed the expression of Oct4, Nanog, and Sox2. When induced to form EBs in vitro or injected to the severe combined immunodeficiency (SCID) mice, the rES cells generated embryoid bodies (EBs) and teratomas with three germ layers expressing the marker genes including MAP2, Desmin, and GATA4, respectively. In conclusion, rabbit ES cell lines can be efficiently established using our current protocols with LIF supplement. These ES cells express pluripotent stem cell markers and retain their capability to differentiate into different tissue cells. Furthermore, rES cells depend on LIF for self-renewal, likely via the JAK-STAT pathway.

Porcine type III RNA polymerase III promoters for short hairpin RNA expression.

Based on the highly conserved sequences of small nuclear RNA and small cytoplasmic RNA between vertebrate species, three porcine type III RNA polymerase III promoters, pY1, pY3 and pU6, were identified by using genomic DNA walking. To test the functional relationship of these sequences, the human H1 promoter of pSUPER-EGFP-l-neo vector was substituted with these three promoters to create the ppPol III-MCS vectors. The strength of each promoter was measured by its ability to derive expression of shRNA to repress expression of luciferase via RNA interference in the pig kidney epithelial cell line LLC-PK1. We determine that the ranking of promoter strength in descending order is pU6 > pY1 > pY3.

LUZP deficiency affects neural tube closure during brain development.

LUZP is a leucine zipper-containing protein predominantly expressed in the brain. The functional significance of LUZP remains unknown. To explore the role of LUZP in brain development, a knockout mouse strain with a lacZ knock-in (Luzp-KO/lacZ-KI) has been established. LacZ reporter expression driven by the endogenous Luzp promoter was detected in the neuroepithelium and the cardiac tissue. Luzp(-/-) mice exhibited perinatal death, presumably due to the accompanied complex cardiovascular defects. Luzp(-/-) embryos displayed a cranial neural tube closure defect (NTD), with exposed brain tissues. Ectopic expression of Sonic-hedgehog, which is a protein known to be involved in neural tube closure, and elevated apoptosis were observed in the dorsal lateral neuroepithelium of the NTD Luzp(-/-) hindbrain. These findings assign a novel function of LUZP in the embryonic development of brain.

FSH-sensitive murine sertoli cell lines immortalized by human telomerase gene hTERT express the androgen receptor in response to TNF-alpha stimulation.

Sertoli cells are regulated by follicular stimulating hormone (FSH) and testosterone secreted by the pituitary gland and Leydig cells, respectively. However, the expression of the FSH receptor and androgen receptor were undetectable in both primary cultured Sertoli cells and Sertoli cell lines immortalized by SV40 large T antigen. Two Sertoli cell lines, B6Sc-2 and B6Sc-3, were established from the testis of 19-day-old C57BL/6 mice testis by immortalization with human telomere reverse transcriptase. These Sertoli cell lines expressed FSH receptors and the total phosphoprotein patterns were converted after FSH treatment. Additionally, immunological methods demonstrated that these cell lines expressed characteristic Sertoli cell proteins, such as tyrosine-tubulin, vimentin and stem cell factor (SCF). Reverse transcription-polymerase chain reaction (RT-PCR) also indicates that they express Sertoli specific mRNAs, such as Amh, claudin11 and ZO-1. The expression of the androgen receptor in both B6Sc-2 and B6Sc-3 cells could be induced by TNF-alpha treatment. The present results indicate that these Sertoli cell lines are more native than others and may thus provide useful tools for in vitro studies.

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method.

AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of Nucleofector electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectin-coated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination. The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.

An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials.

The generation of germline competent chimeric mice via embryonic stem (ES) cells is a crucial step in developing gene-manipulated mouse models. To date, techniques for generating chimeric mice include direct microinjection of ES cells into the cavity of 3.5-d post-coitum (dpc) blastocysts and aggregating or coculturing 2.5 dpc zona pellucida-free (denuded) embryos with ES cells. We present here a procedure that is simple and reproducible for mass producing (10-150 embryos/vial/time) chimeric embryos by coculturing denuded 8-cell embryos and morula in 0.8 mL KSOM-AA medium containing 5 x 10(5)mL-1 purified green fluorescence protein-expressing ES cells (either fresh or thawed) in an 1.7 mL Eppendorf vial for 3h. The resulting chimeras had substantial levels of chimerism and high germline transmission rates. Therefore, the method developed in this study can provide a simple and mass reproducible alternative method (to germline transmitter chimeric mice), without technological and instrumental difficulties, for generating chimeric embryos.