Mutations in mitochondrial DNA (mtDNA) are associated with serious human diseases and inherited from mother's eggs. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%). However, a significant portion of ST zygotes (52%) displayed abnormal fertilization as determined by irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell (ESC) isolation (38%) rates were comparable to controls. All ESC lines derived from ST zygotes displayed normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing ESCs similar to controls.

SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

We wish to correct a number of figure-related and typographical errors that appeared in the article above. None of these errors affect the conclusions of the paper.In Figures 2F and S5 (upper-right), we presented two phase-contrast photos of fields of cells, correctly labeled as SCNT-derived hESO-NT1 and IVF-derived hESO-7, respectively. These images are the same fields of cells shown in the top two images of Figure 6D; however, in Figure 6D, we inadvertently switched the labels on the images. This re-use of the images was intentional, but we should have indicated this in the original legend for Figure 6. We have corrected the labeling error in Figure 6D.In Figure S6, the scatterplot presenting a comparison between biological HDF-f replicates #2 and #3 is an inadvertent duplication of the scatterplot presenting the comparison of HDF-f replicates #1 and #3. This plot has been replaced in the figure online and is shown below.In Figure 1, the number of SCNT embryos for I/DMAP group (n = 51) has been corrected to 53.In Figure 5D, the numbers of plated blastocysts for agonist and antagonist were reversed and have been corrected to agonist (n = 4) and antagonist (n = 17).In the Experimental Procedures, the age range of oocyte donors in the paper was listed as 23-31; however, the range has been corrected to 23-33.In Table S2, percentages for fused oocytes in the 10 nM TSA for 24 hr group (95.4) and for compact morula (CM) in the 5 nM TSA for 12 hr group (26.0) have been corrected to 96.3 and 28.0, respectively.In Table S3, we incorrectly reported several figures due to errors that occurred in converting the raw patient data, from which these values are calculated, from a file created with Mac-based software to a file in the analogous Windows-based software. The following corrections have been made: number of oocytes in the antagonist group 11.7 ± 5.6 has been changed to 10.2 ± 4.9; number of oocytes in the agonist group, 20.5 ± 11.9 to 16.3 ± 5.2; AMH level in the antagonist group, 2.8 ± 0.5 to 2.5 ± 0.5; AFC in the antagonist group, 23.1 ± 7.2 to 23.2 ± 7.2; FSH dosage in antagonist group, 958.3 ± 241.7 to 966.7 ± 247.3; number of hMG ampoules in antagonist group, 8.5 ± 1.6 to 10.2 ± 4.2; number of hMG ampoules in agonist group, 8.8 ± 0.9 to 8.8 ± 1.0; stimulation days in antagonist group, 8.7 ± 1.6 to 8.7 ± 0.8; and stimulation days in agonist group, 9 ± 0.8 to 9.8 ± 1.0. We have confirmed that these differences do not affect any of the statistical conclusions originally reported.In Table S4, short tandem repeats (STR) readings for egg donor A in D6S291 and D6S276 loci were reversed and have been corrected to 199/209 for D6S291 and 245/249 for D6S276.

High-mobility and low-voltage-operated organic field-effect transistors (OFETs) are demonstrated by the design of a new fluorinated benzothiadiazole-based conjugated polymer with fluorinated high-k polymer dielectrics. A record-breaking high hole mobility of 9.0 cm(2) V(-1) s(-1) for benzothiadiazole-based semiconducting polymers is achieved by the excellent planarity of the semiconducting polymer.

SUMMARY The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.

Summary Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the 4-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.

No abstract

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E 90 K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E 90 K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.protein trafficking | protein misrouting | intracellular trafficking