Treatment Of Donor Cells Research Articles

Development to term of cloned cattle derived from donor cells treated with valproic acid.

Cloning of mammals by somatic cell nuclear transfer (SCNT) is still plagued by low efficiency. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this regard, most factors that promote chromatin decondensation, including histone deacetylase inhibitors (HDACis), have been found to increase nuclear reprogramming efficiency, making their use common to improve SCNT rates. Herein we used valproic acid (VPA) in SCNT to test whether the treatment of nuclear donor cells with this HDACi improves pre- and post-implantation development of cloned cattle. We found that the treatment of fibroblasts with VPA increased histone acetylation without affecting DNA methylation. Moreover, the treatment with VPA resulted in increased expression of IGF2R and PPARGC1A, but not of POU5F1. However, when treated cells were used as nuclear donors no difference of histone acetylation was found after oocyte reconstruction compared to the use of untreated cells. Moreover, shortly after artificial activation the histone acetylation levels were decreased in the embryos produced with VPA-treated cells. With respect to developmental rates, the use of treated cells as donors resulted in no difference during pre- and post-implantation development. In total, five clones developed to term; three produced with untreated cells and two with VPA-treated cells. Among the calves from treated group, one stillborn calf was delivered at day 270 of gestation whereas the other one was delivered at term but died shortly after birth. Among the calves from the control group, one died seven days after birth whereas the other two are still alive and healthy. Altogether, these results show that in spite of the alterations in fibroblasts resulting from the treatment with VPA, their use as donor cells in SCNT did not improve pre- and post-implantation development of cloned cattle.

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibro- blast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficul- ties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular repro- gramming.

Posttranslational Modification Of The Thrombopoietin Receptor Regulates Cytokine Signal Transduction and Hematopoietic Stem Cell Engraftment

BackgroundThrombopoietin (TPO) is a key cytokine regulating hematopoietic stem cell (HSC) quiescence and self-renewal, and has been shown to augment ex vivo expansion and maintenance of HSCs in culture. Impaired TPO signaling is associated with bone marrow failure and thrombocytopenia. TPO mediates intracellular signal transduction by binding its cell surface receptor, cMpl, and transgenic mice deficient in cMpl expression have an age-progressive reduction in HSC content. An improved understanding of the mechanisms regulating HSC proliferation, self-renewal, and quiescence is essential to optimize transplantation, expansion, and other therapeutic applications of HSCs.Posttranslational modification of proteins in the Golgi results in conjugation of complex-type N-glycans on glycoproteins destined for the cell surface. A complete deficiency of glycosylation results in failure of transport of proteins to the plasma membrane, while glycosylation with complex branched carbohydrates regulates a wide variety of functions at the cell surface including receptor clustering, cross-linking and trafficking. N-glycosylation of tyrosine kinase cytokine receptors has been shown to enhance their signaling potential by sparing receptor internalization via endocytosis, thus promoting receptor retention at the cell surface (Partridge EA et al, Science 2004). cMpl is known to undergo N-linked glycosylation, with defects in posttranslational processing associated with polycythemia vera. However, little is known about the functional significance of glycosylation of cMpl on TPO signaling and HSC function.Here we show that glycosylation of cMpl regulates TPO signal transduction in an endocytosis-dependent manner, resulting in enhanced short-term engraftment in a mouse model of in utero hematopoietic cell transplantation (IUHCT). Methods293T cells stably overexpressing the TPO receptor cMpl were treated with either the α-mannosidase II inhibitor swainsonine (1 μg/ml) to inhibit complex N-glycan branching or the hexosamine biosynthetic pathway substrate UDP-GlcNAc (12.5 – 50 mM) to increase N-glycan production. Activation of signaling proteins was measured by immunoblotting and immunofluorescence studies. Clathrin- and caveolin-dependent endocytosis was inhibited by potassium depletion and nystatin treatment, respectively. The effect of posttranslational cMpl modification on donor cell engraftment was assessed in a murine model of IUHCT. Fetal liver from gestational day 14 Balb/c recipient fetuses were assessed for donor cell chimerism 48 hours after transplantation with 10e6 B6-GFP bone marrow cells with or without UDP-GlcNAc treatment. ResultsInhibition of N-linked glycosylation by swainsonine treatment resulted in reduced downstream signal transduction following TPO stimulation, with significantly decreased levels of phosphorylation of MAPK (Figure 1A). Pharmacologic disruption of endocytosis was found to ablate the effects of swainsonine treatment, with equivalent and robust activation of MAPK in swainsonine-treated and untreated cells. Upregulation of N-linked glycosylation by supplementation of the glycosylation pathway with UDP-GlcNAc treatment resulted in a dose-dependent increase in signaling following TPO stimulation, with increased levels of phosphorylation of MAPK (Figure 1B). Finally, UDP-GlcNAc treatment of donor cells resulted in significantly higher engraftment levels at 48 hours post IUHCT compared to controls (20.18 +/- 2.77 versus 30.27 +/- 1.985, p=0.0417). [Display omitted] ConclusionsThese results support a role for cMpl glycosylation in modulating TPO signal transduction in an endocytosis-dependent manner, and suggest that enhanced cMpl glycosylation can optimize HSC function resulting in increased levels of engraftment following IUHCT. Ongoing studies of receptor trafficking, including internalization by clathrin-dependent and –independent endocytosis, are needed to further elucidate the functional significance of complex N-glycans on cMpl fate. These studies will ultimately improve our understanding of how TPO signaling may be manipulated to control HSC self-renewal and quiescence to optimize in vitro expansion and HSC transplantation. Disclosures:No relevant conflicts of interest to declare.

Effect of Histone Deacetylase Inhibitor Valproic Acid Treatment on Donor Cell Growth Characteristics, Cell Cycle Arrest, Apoptosis, and Handmade Cloned Bovine Embryo Production Efficiency

In this study, we tested the effects of valproic acid (VPA), a known histone deacetylase inhibitor (HDACi), on the growth characteristics, apoptosis, and cell cycle stages distribution of donor cells, as well as cloning efficiency, embryo development, and histone methylation. Our results showed that treatment of donor cells with VPA (2.5 mM, 5.0 mM, 7.5 mM, or 10 mM) for 24 h resulted in altered cell proliferation, extent of apoptosis and necrosis, and cell cycle stage distribution, whereas no changes in cell viability and chromosomal complements were observed. Measurement of relative gene expression using real-time PCR of a few developmentally important genes in treated donor cells showed decreased expression of HDAC1 and increased expression of BAX (p<0.05). No change in relative expression of HDAC2 and Bcl2 was noticed. Treatment of donor cells with VPA for 24 h before electrofusion significantly (p<0.05) increased the blastocyst formation rate of somatic cell nuclear transfer (SCNT) embryos compared to the control embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in SCNT blastocysts derived from VPA-treated donor cells were significantly decreased compared to the control blastocysts (p<0.05). Immunolocalization studies revealed that the levels of histone H3 at lysine 9 (H3K9me3) were lower in VPA-treated donor cells derived cloned blastocysts than nontreated cloned embryos, and was at the level of in vitro fertilization (IVF) counterparts, although no effects of treatments were found in donor cells. Our study demonstrates that the use of VPA in SCNT has been beneficial for efficient reprogramming of donor cells. Its effect on histone methylation in cloned embryos correlates with their developmental potential and may be a useful epigenetic marker to predict the efficiency of SCNT.

15-OR

Solid phase immunoassays enable us to identify unacceptable donor HLA specific antigens (DSA) which allows for the prediction of compatible donor/recipient combinations. This process is referred to as the virtual crossmatch (vXM). The aim of the study was to evaluate the performance of the vXM against the final flow cytometric crossmatch (FCXM). All FCXM performed in the past three years were included in this study. Flow cytometry antibody screening and Luminex single antigen bead assay (LSA) were performed to determine the presence of HLA antibodies and their specificities. MFI = 1000 was used as cutoff for LSA. A final FCXM either prospective or retrospective for the transplant recipients was performed with potential donors selected upon the vXM. Donor cells used for final crossmatch were treated with pronase. Total 2070 FCMX were recorded and analyzed in the study. Among these crossmatches, 1891 were vXM negative, 768 crossmatches were performed for HLA sensitized recipients with 179 positive vXM due to presence of DSA. There were total 297 positive final FCXM, of them 123 were expected and 174 (9%) were unexpected compared with the vXM results. The overall prediction rate of the final negative FCXM was 91%. The overall unexpected positive final FCXM was 8%. The majority of the unexpected final T cell positive and B cell negative FCXM became negative when the FCXM repeated with non-pronase treated donor cells. Our vXM practice provided a >90% correct predication of negative FCXM. We could expected that the unexpected positive final FCXM mainly caused by non-HLA reactivity, for example, the pronase treatment and/or presence of antibodies not directed to HLA because the current LSA could finely define the specificities of HLA antibodies. However, it could not be excluded that the presence of anti-DP and/or DQA1 antibodies could trigger a positive B cell FCXM in cases where donors’ DP and DQA1 were not typed. This will be further investigated.

Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.

Treatment of fetal fibroblasts with DNA methylation inhibitors and/or histone deacetylase inhibitors improves the development of porcine nuclear transfer-derived embryos

This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50nM) for 1h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5nM 5-aza-dC for 1h and subsequently treated with 50nM TSA for another 1h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were significantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5nM 5-aza-dC and 50nM TSA for 1h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos.

Effects of trichostatinAon histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos

The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.

45 EFFECT OF DEACETYLASE INHIBITOR VALPROIC ACID ON BOVINE CULTURED SOMATIC CELLS

Normal development depends on a precise sequence of changes in the configuration of chromatin. Epigenetic modifications, such as histone acetylation and methylation and DNA methylation, control the precise tissue-specific gene expressions. Valproic acid (VPA) is an aliphatic acid compound and a deacetylase inhibitor that influences cellular proliferation, differentiation, apoptosis, and migration. Several studies have shown that treatment of fused, cloned embryos with VPA improved embryo development. However, whether the treatment of donor cells with VPA affected cloned embryo development was not clear. In the present study, bovine fibroblast cells were treated with VPA at 0, 0.25, 0.5, 1.0, 2.0, and 4.0 mM concentrations for 24 and 48 h, respectively. The cell growth, cell cycle, diploid composition, and histone modifications were examined. The results showed that (1) when the cells were treated with VPA at and over 2.0 mM concentrations for 24 or 48 h, cell growth was significantly inhibited; (2) VPA treatment resulted in a decrease in cell diploid composition; (3) VPA induced the arrestment of cells at the G0/G1 stages; (4) VPA treatment increased early cell apoptosis; (5) VPA at a 4.0 mM concentration and treatment of cells for 24 h improved H3K9 acetylation; however, treatment of cells with VPA decreased H3K9 methylation in all experimental groups; and (6) when the cells treated with VPA at 0, 0.25, 0.5, 1.0, and 2.0 mM concentrations for 24 h were used as donor cells for NT, the cleavage rates were 83.2, 80.0, 82.1, 80.5, and 65.5%, respectively, and the percentages of blastocyst development were 30.5, 29.6, 29.2, 25.0, and 15.5%, respectively. When the cells were treated with VPA at 4.0 mM concentration, the cleavage (65.5%, 76/116) and blastocyst development (15.5%, 18/116) significantly decreased as compared with the control [83.2% (158/190) and 30.5% (58/190), respectively]. The cell numbers of the blastocysts derived from VPA treatment at 0, 0.25, 0.5, 1.0, and 2.0 mM concentrations were 89.6, 88.6, 87.6, 75.0, and 71.3, respectively, which decreased with the increase in VPA concentration. In conclusion, VPA affected cell growth and histone modifications in a concentration-dependent manner. The VPA-treated cells did not improve the cloned embryo development and even decreased blastocyst when VPA was at a high concentration. This work was supported by the National Basic Research Program of China (no. 2012CB22306).

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals.

The Immunization of Recipient with Donor Sodium Chloride Mummified Non-Dividing Splenocytes Significantly Prolongs Allogeneic Heart Survival

Prior contact of recipient with donor transplantation antigens evokes humoral and cellular rejection reaction. Blood transfusions, pregnancy and previous grafts may cross-reactively immunize recipient against donor antigens. In all the listed situations donor antigen is mainly provided in “passenger cells” These cells colonize recipient lymphoid tissues and produce progenies. This enhances the host-versus-graft immune response and subsequently rejection ensues. The question arises whether metabolically immobilized non-dividing (mummified) donor cells, mimicking passenger cells, administered to recipient prior to transplantation may mitigate rejection reaction, presumably by evoking formation of enhancing antibodies. Immobilization can be achieved by treatment of donor cells with pulverized NaCl. These cells retain their molecular structure but do not divide. Immunohistopathology of these cells shown normal expression of MHC I and II antigens. Aim: to immunize recipient with donor NaCl-treated splenocytes prior to heart transplantation for prolongation of graft survival. Methods: BN rat spleen fragments were placed in pulverized NaCl and 7 d later implanted intraperitoneally into LEW rat on d 0 and 7. Seven days later heart tx from BN was performed. Non-immunized rats served as controls. No immunosuppression was given. Results: the immunized LEW did not reject BN heart until d 20 (20±2), whereas control rats rejected the graft within 6±1 days (p< 0.001). On histology, rejected hearts from immunized recipients revealed hypertrophied muscle with few infiltrates, in contrast to controls with dense infiltrates and necrotic areas. The MLR revealed decreased reaction to donor lymphocytes. Deposition of IgG in graft vessels could be seen on immunohistochemical pictures. Conclusions: immunization of recipient with mummified splenocytes retaining their molecular structure may be responsible for prolongation of allogeneic heart survival.

Treatment of porcine donor cells and reconstructed embryos with the melatonin enhances cloning efficiency

Treatment of porcine donor cells and reconstructed embryos with the melatonin enhances cloning efficiency

179-P

This study was to discuss flow cytometry crossmatch (FCXM) results of a kidney transplant patient who consistently showed unexpected T positive and B negative FCXM results in the absence of unacceptable antigens (UA). Possible CREG reactivity were considered as a possible cause. Flow PRA and Luminex single antigen bead assay (LSA) were performed to determine the presence of HLA antibodies. Results of 12 FCXMs with pronase treated donor cells were retrospectively analyzed. Additional crossmatches with absorbed and non-absorbed sera were performed to clarify CREG reactivity. Patient had previous transplant from a living donor in 2003 and developed a weak anti-HLA-A68, and strong anti-DR13, -DR52 DSAs in addtion to a few non-DSA antibodies. Among the donors of 12 FCXMs, one had both class I and class II UAs and 6 had only class II UAs. All T cell FCXMs were positive or borderline positive regardless of having UAs, while all B cell FCXMs were negative with donors without class II UAs. When donors HLA typing were scrutinized, 8/12 donors had CREG2 antigens. Therefore, we speculated that the CREG2 antigens might have crossreacted with patient’s anti-HLA-A68 antibody giving T positive, B negative (shifted) FCXM results. To confirm our speculation, the patient serum was absorbed with HLA-A68 cells and crossmatched with CREG2 and non-CREG2 donors. Absorbed serum showed nearly 20 MCS reduction in T cell FCXM and 50 MCS reduction in B cell FCXM with the CREG2 donor compared with non-absorbed serum, which correlated with several hundred MFI reduction of anti-HLA-A68 antibody strength detected by LSA. In contrast, no difference was observed between the reactivities of absorbed versus non-absorbed sera when crossmatched with non-CREG2 donor. Based on our findings we suggest that unexpected T cell positive, B cell negative (shifted) FCXM could be partially caused by CREG2 reactivity in addition to non-HLA interference.

CD26 protease inhibition improves functional response of unfractionated cord blood, bone marrow, and mobilized peripheral blood cells to CXCL12/SDF-1

Hematopoietic stem cell transplantation (HSCT) is an important treatment option for patients with malignant and nonmalignant hematologic diseases. Methods to improve transplant efficiency are being explored with the intent to improve engraftment and immune reconstitution post-HSCT. A current approach under investigation involves treatment of donor cells with inhibitors that target the protease CD26, a negative regulator of the chemokine CXCL12/stromal cell-derived factor-1. CD26 inhibitor treatment has been shown to improve the functional response of CD34(+) cord blood (CB) cells, but not CD34(+) granulocyte colony-stimulating factor-mobilized peripheral blood stem cells, to CXCL12/stromal cell-derived factor-1. The effect of CD26 inhibitors on unfractionated CB, bone marrow, or granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells has not been evaluated previously. We observed that although CB had greater CD26 expression than bone marrow or mobilized peripheral blood, treatment with a CD26 inhibitor (Diprotin A) resulted in increased responsiveness to stromal cell-derived factor-1 for all three mononuclear cell sources tested. This suggests that clinical therapeutic benefit might be gained by using CD26 inhibitors as a strategy to improve engraftment of unfractionated mobilized peripheral blood cells as well as CB cells.

Epigenetic reprogramming of Yak iSCNT embryos after donor cell pre-treatment with oocyte extracts

The treatment of donor cells with oocyte extracts before inter-species somatic cell nuclear transfer (iSCNT) is a novel method for cellular reprogramming. This study aims to evaluate the effect of pre-treatment donor cell with oocyte extracts on the early developmental competence of yak iSCNT embryos. Yak fibroblasts were reversibly permeabilized with streptolysin O, and then treated with yak oocyte extracts (YOE) or bovine oocyte extracts (BOE) prior to iSCNT. The 8-cell and blastocyst formation increased significantly compared with the control group (P<0.05) when donor cells pre-treated with YOE or BOE. The relative expression level of embryo-specific genes TBP1 and Mash2 were also up-regulated both in the blastocysts of the YOE and BOE groups. In addition, the methylation level of pluripotency-specific genes (Oct4 and Nanog) in the blastocysts of the YOE and BOE groups were similar to that of its IVF counterpart (53.1%, 48.8% vs. 40.1%; 24.8%, 26.5% vs. 35.9%). Our results suggested that pre-treatment of donor cells with oocyte extracts can improve nuclear–cytoplasmic reprogramming; thus representing a novel way to improve the efficiency of yak iSCNT.

Treatment of Nuclear-Donor Cells or Cloned Zygotes with Chromatin-Modifying Agents Increases Histone Acetylation But Does Not Improve Full-Term Development of Cloned Cattle

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

XenopusEgg Extract Treatment Reduced Global DNA Methylation of Donor Cells and Enhanced Somatic Cell Nuclear Transfer Embryo Development in Pigs

The efficiency to produce offspring by somatic cell nuclear transfer (SCNT) is low. It has been showed that treatment of donor cells with Xenopus oocyte extract increased live births in ovine and handmade cloned embryo development in pigs. Scriptaid treatment after oocyte activation is another approach to improve SCNT efficiency. The present study was carried out to investigate (a) the effects of treatment of donor cells with Xenopus egg extract on donor cell DNA methylation at days 0 and 4 with two digitonin permeabilization concentrations (10 and 15 μg/mL), (b) the effects of treatment of donor cells with Xenopus egg extract on early development of cloned embryos, and (c) the effects of combined treatments, treating donor cells with extract before nuclear transfer and treatment of cloned embryos with scriptaid after oocyte activation, on embryo development. Compared to the control, a decrease of DNA methylation in donor cells was observed at 2.5 h after extract treatment. However, this effect was not observed after the cells were cultured for four more days. More embryos developed into blastocysts in the Xenopus egg extract-treated group than in the control (13.4±1.9% vs. 9.1±1.9%, p=0.01). Furthermore, scriptaid treatment of cloned embryos further increased the frequency of development to blastocyst, compared to the control reconstructed with the same extract-treated cells (22.5±0.9% vs. 15.3±0.9%, p<0.01). In addition, egg extract treatments increased the cell number in the blastocysts. This study demonstrated that Xenopus egg extract treatment reduced donor cell DNA methylation and enhanced the SCNT embryo development. Moreover, the combined treatments of donor cells with egg extract before nuclear transfer and of cloned embryos with scriptaid could improve cloned embryo development additively.

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2′-deoxycytidine

Summary Reprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2'-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P &lt; 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.