Search

Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

Correct reprogramming of epigenetic marks in the donor nuclei is a prerequisite for successful cloning by somatic cell transfer. In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts for the doxycycline-inducible expression of Kdm4b, a demethylase that removes histone 3 lysine 9 trimethylation (H3K9me3) and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels reduced ∼3-fold and ∼5-fold, respectively, compared to non-induced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation), further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared to non-induced, non-starved control fibroblasts. Following somatic cell transfer, Kdm4b-BEF fibroblasts reprogrammed significantly better into cloned blastocysts than non-induced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase rates of post-blastocyst development from implantation to survival into adulthood. In summary, KDM4B only improved somatic cell reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1–6 days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH, and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92% ,8 5%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 for MPC vs. MT vs. MF, respectively, P , 0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively). assisted reproductive technology, developmental biology, early development, embryo

Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus-free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling (‘2i’). Non-invasive real-time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthesis and did not expand after passaging. Following transfection, non-proliferative primary colonies expressed discriminatory markers of pluripotency, including endogenous iPSC factors, CDH1, DPPA3, NANOG, SOCS3, ZFP42, telomerase activity, Tra-1-60/81 and SSEA-3/4, but not SSEA-1. This indicates that they had initiated a self-sustaining pluripotency programme. Bovine iPSC-like cells maintained a normal karyotype and differentiated into derivatives of all three germ layers in vitro and in teratomas. Our study demonstrates that conversion into induced pluripotency can occur in quiescent cells, following a previously undescribed route of direct cell reprogramming. This identifies a major species-specific barrier for generating iPSCs and provides a chemically defined screening platform for factors that induce proliferation and maintain pluripotency of embryo-derived pluripotent stem cells in livestock.

Interspecies somatic cell NT (SCNT) can be used in the conservation of endangered animals but only when there is an abundant source of compatible oocytes and recipients. The objective of this study was to compare the efficiency of intra- and interspecies SCNT in sheep using zona-free embryo reconstruction methods. Skin fibroblasts from either an argali (Ovis ammon) or control (Ovis aries) ram were used as donor cells for SCNT between passages 2 to 5 and following culture in medium containing 0.5% FCS for 4 to 6 days. Single cells were electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes obtained from domestic ewes. In an additional experiment with argali, a double cytoplast (DC-SCNT) procedure was used whereby a second cytoplast was fused to the first reconstruct within 1 h. Reconstructs were artificially activated ~25 h after the start of maturation using ionomycin and 6-DMAP. Zona-free parthenogenote (PG) control oocytes were activated around the same time. In each treatment, 10 to 12 zona-free embryos where cultured in microwells formed in 20-μL drops of modified synthetic oviduct fluid under oil. Half the medium was replaced on Day 3, and developing embryos were transferred to individual 5-μL drops on Day 6. Development on Day 7 was expressed as a percentage of cleaved embryos. Statistical significance was determined using Fisher’s exact test for embryo development and two-tailed t-test for embryo cell numbers. Total embryo development on Day 7 was significantly greater with intraspecies sheep SCNT compared with interspecies argali SCNT (34/157 = 21.7% v. 34/363 = 9.4%, respectively; P

Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) bovine adult male fibroblasts versus non-starved, diploid G1 controls. Mechanically synchronized G1 cells were generated by manual selection or mitotic shake-off and processed within 3 h post-mitosis. Based on morphological assessment and 5-ethyl-2′-deoxyuridine (EdU) incorporation during continuous labelling, >93% of cells were captured in G1. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay (ELISA), we show that G0 fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3, and H3K27me3, but not H3K9me1. They were also significantly hypoacetylated at H3K9 and H4K5, hyperacetylated at H4K12, and unchanged at H4K16 positions. Furthermore, G0 cells significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as polycomb group proteins EED, SUZ12, PHC1, and RING2. Following NT into metaphase-arrested oocytes, G0 chromatin condensed slower than that of G1 cells, indicating a more relaxed configuration. After 7 days of in vitro culture, H3K9me3, but not H4K4me3, H3K27me3, SUZ12, and RING2, remained hypomethylated in G0- versus G1-derived NT blastocysts, both in the inner cell mass and trophectoderm (730 v. 550 nuclei from 55 v. 42 G0 v. G1 blastocysts, respectively; n = 7 NT runs). Reduced H3K9me3 levels correlated with significantly increased mRNA abundance of the H3K9me3-specific histone demethylase KDM4B (or JMJD2B) in NT blastocysts. Expression of other pluripotency-related factors (NANOG, SOX2, STELLA, and IIFITM3), imprinted genes (SNRPN), and histone demethylases (KDM4A) was not affected in G0-derived blastocysts (32 G0 v. 55 G1 blastocysts; n = 4). Following NT, G0 donors developed significantly better into cloned blastocysts (175/382 = 46% v. 122/332 = 37% for G0 v. G1, respectively; n = 7, P