Nguyen Van Thuan

Equivalency of Nuclear Transfer‐Derived Embryonic Stem Cells to Those Derived from Fertilized Mouse Blastocysts

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT‐ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT‐ESCs, we established more than 150 NT‐ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT‐ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocyst‐derived ESCs in terms of their expression of pluripotency markers in the presence of tissue‐dependent differentially DNA methylated regions, in DNA microarray profiles, and in high‐coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT‐ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research.

Chromatin Decondensation and Nuclear Reprogramming by Nucleoplasmin

ABSTRACT Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1β and TIF1β from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.

The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice

Since the birth of Cumulina, the first mouse clone produced by somatic cell nuclear transfer (SCNT), the success rate of cloning in mice has been extremely low compared with other species and most of the inbred mouse strains have never been cloned. Recently, our laboratory has found that treatment of SCNT mouse embryos with trichostatin A, a histone deacetylase inhibitor (HDACi), improved the full-term development of B6D2F1 mouse clones significantly. However, this was not effective for the inbred strains. Here, we show for the first time that by treating SCNT embryos with another HDACi, scriptaid, all the important inbred mouse strains can be cloned, such as C57BL/6, C3H/He, DBA/2, and 129/Sv. Moreover, the success of somatic nuclear reprogramming and cloning efficiency via nuclear transfer technique is clearly linked to the competent de novo synthesis of nascent mRNA in cloned mouse embryos.

Establishment of Male and Female Nuclear Transfer Embryonic Stem Cell Lines from Different Mouse Strains and Tissues

Abstract Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F1 mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.

Production of cloned mice by somatic cellnuclear transfer

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains <5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cells genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in ≥3 months.

Effect of Trichostatin A on Chromatin Remodeling, Histone Modifications, DNA Replication, and Transcriptional Activity in Cloned Mouse Embryos

Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5′-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.

Regulation of chromatin and chromosome morphology by histone H3 modifications in pig oocytes

Oocyte growth, maturation, and activation are complex processes that include transcription, heterochromatin formation, chromosome condensation and decondensation, two consecutive chromosome separations, and genomic imprinting. The objective of this study was to investigate changes in histone H3 modifications in relation to chromatin/chromosome morphology in pig oocytes during their growth, maturation, and activation. During the growth phase, histone H3 was acetylated at lysines 9, 14, and 18 (K9, K14, and K18), and became methylated at K9 when the follicles developed to the antral stage (oocyte diameter, 90 mum). When the fully grown oocytes (diameter, 120 mum) started their maturation, histone H3 became phosphorylated at serine 28 (S28) and then at S10, and deacetylated at K9, K14, and K18 as the chromosomes condensed. After the electroactivation of mature oocytes, histone H3 was reacetylated and dephosphorylated concomitant with the decondensation of the chromosomes. Histone H3 kinase activity increased over a similar time course to that of the phosphorylation of histone H3-S28 during oocyte maturation, and this activity decreased as histone H3-S10 and H3-S28 began to be dephosphorylated after the activation of the mature oocytes. These results suggest that the chromatin morphology of pig oocytes is regulated by the acetylation/deacetylation and the phosphorylation/dephosphorylation of histone H3, and the phosphorylation of histone H3 is the key event in meiotic chromosome condensation in oocytes. The inhibition of histone deacetylase with trichostatin A (TSA) inhibited the deacetylation and phosphorylation of histone H3, and chromosome condensation. Therefore, the deacetylation of histone H3 is thought to be required for its phosphorylation in meiosis. Although histone H3 acetylation and phosphorylation were reversible, the histone methylation that was established during the oocyte growth phase was stable throughout the course of oocyte maturation and activation.

Differentiation and Transplantation of Functional Pancreatic Beta Cells Generated from Induced Pluripotent Stem Cells Derived from a Type 1 Diabetes Mouse Model

The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.

Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail‐tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4‐expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (>90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.

Similar Time Restriction for Intracytoplasmic Sperm Injection and Round Spermatid Injection into Activated Oocytes for Efficient Offspring Production

Abstract The injection of male haploid germ cells, such as spermatozoa and round spermatids, into preactivated mouse oocytes can result in the development of viable embryos and offspring. However, it is not clear how the timing of intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) affects the production of offspring. We carried out ICSI and ROSI every 20 min for up to 4 h after the activation of mouse oocytes by Sr2+ and compared the late-stage development of ICSI- and ROSI- treated oocytes, including the formation of pronuclei, blastocyst formation, and offspring production. The rate of pronucleus formation (RPF) after carrying out ICSI started to decrease from >95% at 100 min following oocyte activation and declined to <20% by 180 min. In comparison, RPF by ROSI decreased gradually from >70% between 0 and 4 h after activation. The RPFs were closely correlated with blastocyst formation. Offspring production for both ICSI and ROSI decreased significantly when injections were conducted after 100 min, a time at which activated oocytes were in the early G1 stage of the cell cycle. These results suggest that spermatozoa and round spermatids have different potentials for inducing the formation of a male pronucleus in activated oocytes, but ICSI and ROSI are both subject to the same time constraint for the efficient production of offspring, which is determined by the cell cycle of the activated oocyte.