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Cell-based therapies hold tremendous potential to treat cancer, inflammatory conditions, and degenerative diseases. However, despite a few notable successes, cell-based therapies face key clinical barriers, including cell source heterogeneity, inefficient directed migration (homing) to target tissues, cell dosing or controllability, inability to affect tissue microenvironments, and patient safety. Bioengineering may potentially enhance cell capabilities, but raises safety concerns regarding the control of genetically engineered cells in vivo. Therefore, bioengineering methods to improve cell-based therapy efficacy must prioritize predictable behavior and safety. To address these issues, the Klemke Laboratory developed a platform technology that couples cell biongineering with cell enucleation (mass removal of nuclei) to enhance cellular function and controllability. By adapting methods developed in the 1970s, mesenchymal stromal cells (MSCs) were enucleated by ultracentrifugation in discontinuous Ficoll gradients containing cytochalasin B. Combining cell bioengineering with enucleation both decreases the risk of engraftment and oncogenesis, and provides opportunities for customized pre- and/or post-enucleation bioengineering. We named these bioengineered, enucleated cells “CargocytesTM”.Cargocytes were characterized by absent nuclei, viability for 2-3 days, functional protein translational machinery, retention of receptor expression, and directional migration, and then further investigated via bioengineering with therapeutically-relevant properties. For in vitro studies, Cargocytes were bioengineered and then examined for secretion of functional cytokines and expression of functional chemokine receptors. Next, Cargocytes in vivo abilities were examined in two independent mouse models. First, bioengineered Cargocytes injected intratumorally in a mouse breast cancer model produced functional antitumor cytokine that slowed tumor progression and increased animal survival. Second, bioengineered Cargocytes injected intravenously in a mouse ear inflammation model both homed to the site of inflammation and produced functional anti-inflammatory cytokine that reduced inflammation. This suggests that bioengineered, enucleated cells are more efficacious than non-engineered cells, and lacking nuclei uniquely contributes to safety and controllability in vivo.To my knowledge, therapeutic bioengineering of enucleated cells has not been previously described, and Cargocytes represent a unique entity in the cell-based therapy field as either a stand-alone or adjuvant therapy. By retaining biologically important cell-like functions yet with advantages like small size and defined lifespan, Cargocytes potentially resolve key barriers currently limiting cell-based therapies.

Purpose Assisted reproductive technology (ART) is a widely applied fertility treatment. However, the developmental competence of aged oocytes from women of a late reproductive age is seriously reduced and the aged oocytes often fail in fertilization even when ART is used. To resolve this problem, we examined usefulness of a new method “the metaphase II spindle transfer (MESI)” as ART using mouse oocytes. Methods This work was composed of two experiments. First, 24 hours after collection, embryos from oocytes (1‐day‐old oocytes, called postovulatory‐aged oocytes), were observed, after intracytoplasmic sperm injection (ICSI), and it was found that they were not able to reach the blastocyst stage. Next, the metaphase II chromosome‐spindle complexes from 1‐day‐old oocytes were injected into cytoplasts from oocytes just collected, using piezo pulses to generate reconstructed oocytes. This procedure was named metaphase II spindle injection (MESI). Results After ICSI, embryos from the reconstructed oocytes (32/105), which contained the genes of 1‐day‐old oocytes, were able to develop into the blastocyst stage. The fragmentation rate after ICSI was 28.6%. Thus, the developmental competence of 1‐day‐old oocytes was improved by MESI. Conclusions The MESI method has the potential to improve the success rate of infertility treatments for women of a late reproductive age., The metaphase II chromosome‐spindle complexes from 1‐day‐old oocytes (postovulatory‐aged oocytes 24 hours after collection) were injected into cytoplasts from freshly collected oocytes using piezo pulses to generate reconstructed oocytes (MESI). After ICSI, the embryos from reconstructed oocytes (32/105) could develop into the blastocyst stage. However, the developmental competence of 1‐day‐old oocytes was improved by MESI.

// Lu Li 1, 4 , Lu Wang 4 , Kevin M. Prise 2 , K.N. Yu 3 , Guodong Chen 1 , Lianyun Chen 6 , Yide Mei 7 , Wei Han 1, 5 1 Anhui Province Key Laboratory of Medical Physics and Technology/Center of Medical Physics and Technology, Hefei Institutes of Physical Sciences, Chinese Academy of Sciences, Hefei, Anhui, China 2 Centre for Cancer Research & Cell Biology, Queen’s University, Belfast, UK 3 Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong 4 Clinical College of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, Anhui, China 5 Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions and School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, Suzhou, Jiangsu, China 6 Institute of Technical Biological & Agriculture Engineering, Hefei Institutes of Physical Sciences, Chinese Academy of Sciences, Hefei, Anhui, China 7 School of Life Science, University of Science and Technology of China, Hefei, Anhui, China Correspondence to: Wei Han, email: hanw@hfcas.ac.cn Keywords: radiation-induced bystander effect, cytoplasm, Akt, mTOR, cytoplast Received: September 20, 2016 Accepted: December 27, 2016 Published: February 01, 2017 ABSTRACT Cytoplasm is an important target for the radiation-induced bystander effect (RIBE). In the present work, the critical role of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in the generation of RIBE signaling after X-ray irradiation and the rapid phosphorylation of Akt and mTOR was observed in the cytoplasm of irradiated human lung adenocarcinoma epithelial (A549) cells. Targeting A549 cytoplasts with individual protons from a microbeam showed that RIBE signal(s) mediated by the Akt/mTOR pathway were generated even in the absence of a cell nucleus. These results provide a new insight into the mechanisms driving the cytoplasmic response to irradiation and their impact on the production of RIBE signal(s).

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

The process of synchronization and micro-nuclei induction in a suspension culture of Beta vulgaris, was induced by the sequential treatment with the DNA-synthesis inhibitor aphidicolin (30 mu M, 24 h) and the spindle-toxin amiprophos-methyl (32 mu M, 24 h). Mitotic arrest of divisions, spreading of G2-metaphase chromosomes, re-grouping of chromosomes, formation of a nuclear cell wall around single and re-grouped chromosomes and restitution of nuclei with a doubled DNA content was observed. The process of micro-nucleation was induced much less efficiently in Beta vulgaris than in Nicotiana plumbaginifolia. Cytological observations and monitoring of the process with flow cytometry and confocal laser scanning microscopy, was essential to follow up the course of events and to monitor the development of an efficient procedure for micro-protoplast isolation. Micro-nucleated protoplasts were fractioned by iso-osmotic Percoll gradient centrifugation to obtain heterogeneous micro-protoplast populations with cytoplasts and micro-protoplasts of different size. An enriched fraction with small sub-diploid micro-protoplasts was obtained with the equivalent DNA content of 1-4 chromosomes, as revealed by confocal laser scanning microscopy and flow cytometry. Sub-diploid micro-protoplasts with DNA amounts equivalent to 1-4 chromosomes were predominantly observed in the size classes of 1.8-10.2 mu m at a frequency of 34.2-34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micro-nucleation followed the same course of cellular events as observed in N. plumbaginifolia. The correlation between DNA content and size of micro-nuclei and micro-protoplasts was not linear and affected by the degree of DNA condensation, total amount of DNA, and the presence of cytoplasm.

Made available in DSpace on 2018-11-26T15:28:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos. Embrapa Amazonia Oriental, BR-66095100 Belem, Para, Brazil Univ Estadual Paulista, Jaboticabal, Brazil Embrapa Dairy Cattle, Valenca, Brazil Univ Sao Paulo, Fac Zootecnia & Engn Alimentos, Pirassununga, Brazil Inst Zootecnia, Sertaozinho, Brazil Embrapa Southeast Livestock, Sao Carlos, SP, Brazil Univ Estadual Paulista, Jaboticabal, Brazil

In this paper, we present a fully automated method for cell nuclei detection in Pap smear images. The locations of the candidate nuclei centroids in the image are detected with morphological analysis and they are refined in a second step, which incorporates a priori knowledge about the circumference of each nucleus. The elimination of the undesirable artifacts is achieved in two steps: the application of a distance-dependent rule on the resulted centroids; and the application of classification algorithms. In our method, we have examined the performance of an unsupervised (fuzzy C-means) and a supervised (support vector machines) classification technique. In both classification techniques, the effect of the refinement step improves the performance of the clustering algorithm. The proposed method was evaluated using 38 cytological images of conventional Pap smears containing 5617 recognized squamous epithelial cells. The results are very promising, even in the case of images with high degree of cell overlapping. Ieee Transactions on Information Technology in Biomedicine

Background The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT. Results We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog). Conclusion Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

The effects of reciprocal transplantation of meiosis-II chromosomes between senescent and young mouse oocytes were evaluated based on pre- and post-implantation development ability of resultant embryos.Karyoplasts including meiosis-II chromosomes of oocytes from senescent Rockefeller mouse/Ms-Rb(6, 15) females (10 to 12 months, age-related infertile mice) were transferred into cytoplasts of oocytes from young F(1) females (3 to 5 months). Reconstructed oocytes were fertilized in vitro, and then the resultant embryos were cultured in vitro and transferred to recipient mice.The reconstructed oocytes that consisted of aged-karyoplasts and young-cytoplasts showed significantly improved embryonic development (from 23.2% to 30.0%) and development to term (from 6.3% to 27.1%, P0.05) as compared with the oocytes reconstructed from young-karyoplasts and aged-cytoplasts.The present study showed successful rejuvenation for age-related infertility using transplantation of meiosis-II chromosomes in animal experimental models.

SummaryThe low efficiency of somatic cell nuclear transfer may be related to the ultrastructural deviations of reconstructed embryos. The present study investigated ultrastructural differences between in vivo-produced and cloned goat embryos, including intra- and interspecies embryos. Goat ear fibroblast cells were used as donors, while the enucleated bovine and goat oocytes matured in vitro as recipients. Goat–goat (GG), goat–cattle (GC) and goat in vivo-produced embryos at the 2-cell, 4-cell, 8-cell and 16-cell stages were compared using transmission electron microscopy. These results showed that the three types of embryos had a similar tendency for mitochondrial change. Nevertheless, changes in GG embryos were more similar to changes in in vivo-produced embryos than were GC embryos, which had more extreme mitochondrial deviation. The results indicate the effects of the cytoplast on mitochondria development. The zona pellucida (ZP) in all three types of embryos became thinner and ZP pores in both GC and GG embryos showed an increased rate of development, especially for GC embryos, while in vivo-produced embryos had smooth ZP. The Golgi apparatus (Gi) and rough endoplasmic reticulum (RER) of the two reconstructed embryos became apparent at the 8-cell stage, as was found for in vivo embryos. The results showed that the excretion of reconstructed embryos was activated on time. Lipid droplets (LD) of GC and GG embryos became bigger, and congregated. In in vivo-produced embryos LD changed little in volume and dispersed gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere illuminated that the nucleus function of reconstructed embryos was partly changed. In addition, at a later stage in GC embryos the nuclear envelope displayed infoldings and the chromatin was concentrated, implying that the blastomeres had an obvious trend towards apoptosis. The gap junctions of the three types of embryos changed differently and GG and GC embryos had bigger perivitelline and intercellular spaces than did in vivo-produced embryos. These results are indicative of normal intercellular communication at an early stage, but this became weaker in later stages in reconstructed embryos. In conclusion, inter- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe destruction. These ultrastructural deviations might contribute to the compromised developmental potential of reconstructed embryos.

Cell infection with Trypanosoma cruzi , the agent of Chagas’ disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34°C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus.

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Udgivelsesdato: 2006 Winter The purpose of our work was to find an efficient and reliable chemically assisted procedure for enucleation of porcine oocytes connected to the handmade cloning (HMC) technique without the potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection. After 41-42 h in vitro maturation, porcine oocytes were incubated with 0.4 microg/mL demecolcine for 45 min. Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with extrusion cones or oocytes only with polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm with the extrusion cone or adjacent to the PB was removed with a microblade. The remaining putative cytoplasts, containing the major part of the cytoplasm, were used as recipients for reconstruction with porcine fetal fibroblasts as nuclear donors. The overall efficiency achieved with chemically assisted enucleation was higher compared to oriented bisection without demecolcine incubation (90 +/- 3% vs. 81 +/- 4%, respectively; mean +/- absolute deviation [AD]). Reconstructed and activated embryos were cultured in vitro for 7 days. Fusion, cleavage and blastocyst rates were 87 +/- 7%, 97 +/- 6%, and 28 +/- 9%, respectively. These rates are at least as good as those achieved with normal HMC (81 +/- 4%, 87 +/- 8%, and 21 +/- 9%, respectively). For traditional, micromanipulator-based cloning, fusion and blastocyst rates were similar (81 +/- 10% and 21 +/- 6%, respectively), but the cleavage rate was lower (69 +/- 9%). In conclusion, chemically assisted handmade enucleation seems to be a simpler and potentially superior alternative to more conventional methods used for somatic cell nuclear transfer in pigs.

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.

We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from nuclear transfer of an identical nuclear genetic background (mural granulosa donor cells including surrounding cytoplasm) into enucleated oocytes with either Bos indicus or B. taurus mtDNA. Here we report the first cases of coexisting mtDNAs of two closely related subspecies following nuclear transfer. Heteroplasmy (0.6-2.8%) was found in 4 out of 11 cross-subspecies cloned cattle. Quantitation was performed using “amplification refractory mutation system (ARMS) allele-specific real-time PCR.” We determined that the ratio of donor cell to recipient cytoplast mtDNA copy number was 0.9% before nuclear transfer. Therefore, we concluded that the percentage of donor cell mtDNA in the heteroplasmic intersubspecific cloned animals is in accordance with neutral transmission of donor mtDNA. We determined an amino acid sequence divergence of up to 1.3% for the two subspecies-specific mtDNA haplotypes. In addition, intrasubspecific B. indicus heteroplasmy of ∼1% (but up to 7.3 and 12.7% in muscle and follicular cells of one animal) was detected in 7 out of the 9 B. indicus intrasubspecific cloned cattle.

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 mug/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3.6 kg), while the weight for females was normal.

Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections. NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment ∼8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high levels of RNA synthesis at 1 hr and moderate levels at 3 hr after fusion, although it had ceased by 20 hr after fusion. In all NTE (activated and nonactivated cytoplasts), there was no RNA synthesis seen at the 2-cell stage. However, at the 4-cell stage, weak RNA synthesis was seen in all NTE from activated cytoplasts, whereas none was observed in those from MII nonactivated cytoplasts. At the 8-cell stage, nearly all NTE from S-phase cytoplasts showed weak to moderate levels of RNA synthesis. We conclude that the nucleus reprogramming differs between NTE reconstructed from activated and nonactivated cytoplast with the former undergoing a slower cessation of RNA synthesis after fusion and earlier resumption of RNA synthesis, occurring as early as the 4-cell stage. © 1996 Wiley-Liss, Inc.

When in vitro-matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro-matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.

Aspects of the interaction between residual bodies/cytoplasts from elongated spermatids (RB/CES) and Sertoli cells were studied in vitro. Highly enriched Sertoli cells (91%: experiment A), very highly enriched Sertoli cells (greater than 96%: experiment B), as well as peritubular cells were isolated from testes of 20-day-old rats by means of hypotonic treatment. Isolated Sertoli cells and peritubular cells were also prepared from 45-day-old rats (experiment C). RB/CES were isolated by centrifugal elutriation from testes of rats aged 90-120 days. The kinetics of adhesion of RB/CES to Sertoli cells were similar in all experiments. FSH accelerated binding of RB/CES but markedly reduced the number of RB/CES phagocytosed. Co-culture of the highly enriched Sertoli cells from experiments A and C with isolated peritubular cells did not change the kinetics of adhesion of RB/CES. However, when the contamination of Sertoli cells by peritubular cells was at a minimum (experiment B), addition of peritubular cells induced a slight but significant stimulation of the binding of RB/CES. Transmission electron microscopy revealed the following events within 24 h of co-culture: adhesion of the RB/CES to microvilli of Sertoli cells; internalization of RB/CES; lysis of the membrane of RB/CES; total digestion. Therefore, FSH and peritubular cells modulate the interaction in vitro between Sertoli cells and RB/CES, and the different steps of residual body disposal can be reproduced in co-culture. The co-culture model described in this study provides a useful system for the study of phagocytic activity by Sertoli cells.

Background Different types of bioabsorbable and nonresorbable membranes have been widely used for guided tissue regeneration (GTR) with its ultimate goal of regenerating lost periodontal structures. The purpose of the present study was to evaluate the biological effects of various bioabsorbable and nonresorbable membranes in cultures of primary human gingival fibroblasts (HGF), periodontal ligament fibroblasts (PDLF) and human osteoblast-like (HOB) cells in vitro. Methods Three commercially available collagen membranes [TutoDent® (TD), Resodont® (RD) and BioGide® (BG)] as well as three nonresorbable polytetrafluoroethylene (PTFE) membranes [ACE (AC), Cytoplast® (CT) and TefGen-FD® (TG)] were tested. Cells plated on culture dishes (CD) served as positive controls. The effect of the barrier membranes on HGF, PDLF as well as HOB cells was assessed by the Alamar Blue fluorometric proliferation assay after 1, 2.5, 4, 24 and 48 h time periods. The structural and morphological properties of the membranes were evaluated by scanning electron microscopy (SEM). Results The results showed that of the six barriers tested, TD and RD demonstrated the highest rate of HGF proliferation at both earlier (1 h) and later (48 h) time periods (P < 0.001) compared to all other tested barriers and CD. Similarly, TD, RD and BG had significantly higher numbers of cells at all time periods when compared with the positive control in PDLF culture (P ≤ 0.001). In HOB cell culture, the highest rate of cell proliferation was also calculated for TD at all time periods (P < 0.001). SEM observations demonstrated a microporous structure of all collagen membranes, with a compact top surface and a porous bottom surface, whereas the nonresorbable PTFE membranes demonstrated a homogenous structure with a symmetric dense skin layer. Conclusion Results from the present study suggested that GTR membrane materials, per se, may influence cell proliferation in the process of periodontal tissue/bone regeneration. Among the six membranes examined, the bioabsorbable membranes demonstrated to be more suitable to stimulate cellular proliferation compared to nonresorbable PTFE membranes.

FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.

Six different proteins are found to be reproducibly exposed on the cell surface of chicken embryo fibroblasts (CEF) by the criterion of lactoperoxidase-catalyzed iodination (250,000, 185,000, 130,000, 100,000, 87,000, and 75,000 daltons). We wondered whether cell enucleation might lead to a differential partition of these surface proteins with the karyoplast or cytoplast membrane. We found that there is a marked enrichment of most iodinatable cell surface proteins in the cytoplast after cytochalasin-mediated enucleation of cell monolayers. Nearly all the iodinatable fibronectin remains with the cytoplast. Of the six labeled proteins, the karyoplast membrane contains a small amount of the 130 kdalton protein as well as trace levels of the 100-, 85-, and 75-kdalton proteins. Proteolysis or selective shedding of membrane proteins were not significant factors in the relative exclusion of iodinatable membrane proteins from the karyoplast. The cytoplast could replace some exposed membrane proteins after removal by trypsinization; however, fibronectin was not detectable within 10 h. That the karyoplast was not capable of membrane protein synthesis and/or insertion was suggested by the lack of any change in the labeling pattern of karyoplasts up to 8-h incubation after enucleation. A variety of control studies indicated that the surface proteins identified in this report were cell-derived and not adsorbed serum components. That some of the iodinatable proteins are intrinsic membrane proteins was suggested by their resistance to removal by conditions thought to extract extrinsic membrane proteins (i.e., low salt, high salt, and NaOH washes). lack of effect of cytoskeletal disrupting agents (preliminary evidence) suggests the nonrandom partition of membrane proteins may depend on anchoring of membrane proteins by a system(s) in the cytoplast other than intact microtubules and microfilaments.

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross-bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.

Cytoplasts and karyoplasts were obtained by ultracentrifugation of Hepatoma D23 cells on a Ficoll gradient containing cytochalasin B. Their nuclear and protein content and their metabolic activity were determined. Three i.p. injections of 2.3 x 10(7) cytoplasts were unable to protect syngeneic WAB/Not rats against an s.c. challenge of 10(4) D23 cells, whereas a similar amount of karyoplasts, or 3 injections of 10(6) irradiated D23 cells, were fully protective. Ability of cytoplasts to act as primary or secondary immunogen were also studied, and compared to that of 0.01% glutaraldehyde-treated cells, 43 degrees C heat-treated cells and 3M KCl-soluble extracts, these preparations also being of weak immunogenicity. Only heat-treated cells behaved as a primary immunogen, whereas none of the preparations provided a secondary stimulation. Moreover, when these preparations were fed in vitro to peritoneal-exudate cells before their injection into rats, cytoplasts and glutaraldehyde-treated cells showed no immunogenicity, whereas heat-treated cells induced full protection against tumour challenge. Therefore, in this tumour model, the in vivo persistence of immunogen and the presence of a nucleus are likely to be crucial in inducing transplantation resistance to tumour.

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.

The cytoplasmic ground substance of cultured cells prepared for high voltage transmission electron microscopy (glutaraldehyde/osmium fixed, alcohol or acetone dehydrated, critical-point dried) consists of slender (3-6 nm Diam) strands--the microtrabeculae (55)--that form an irregular three-dimensional lattice (the microtrabecular lattice). The microtrabeculae interconnect the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The lattice is found in all portions of the cytoplast of all cultured cells examined. The possibility that the lattice structure is an artifact of specimen preparation has been tested by (a) subjecting whole cultured cells (WI-38, NRK, chick embryo fibroblasts) to various chemical (aldehydes, osmium tetroxide) and nonchemical (freezing) fixation schedules, (b) examination of model systems (erythrocytes, protein solutions), (c) substantiating the relaibility of critical-point drying, and (d) comparing images of whole cells with conventionally prepared (plastic-embedded) cells. The lattice structure is preserved by chemical and nonchemical fixation, though alterations in ultrastructure can occur especially after prolonged exposure to osmium tetroxide. The critical-point method for drying specimens appears to be reliable as is the freeze-drying method. The discrepancies between images of plastic-embedded and sectioned cells, and images of whole, critical-point dried cells appear to be related, in part, to the electron-scattering properties of the embedding resin. The described observations indicate that the microtrabecular lattice seen in electron micrographs closely represents the nonrandom structure of the cytoplasmic ground substance of living cultured cells.

Hybrids constructed by fusion of wild-type Chinese hamster ovary cells (CHO-K1) to peroxisome-deficient CHO mutants (ZR-78.1) contain normal peroxisomes, demonstrating that the mutation(s) are recessive. "Nuclear hybrids" prepared by fusion of CHO-K1 karyoplasts to mutant ZR-78.1 occasionally fail to regain intact peroxisomes (approximately 1/300 cells). These peroxisome-deficient nuclear hybrids closely resemble the original mutant cells by biochemical criteria, but their modal chromosome number is 36-38, the same as that of CHO hybrids generated from intact cells. When the peroxisome-deficient nuclear hybrids are fused to wild-type cytoplasts, a fraction of the fusion products (at least 70%) continue to propagate normal peroxisomes indefinitely. Peroxisome biogenesis cannot be reinitiated in cells of mutant ZR-78.1 by fusion to wild-type cytoplasts. Our results suggest that a wild-type nucleus by itself is necessary but not sufficient for restoration of normal peroxisome biogenesis and that a cytoplasmic component of wild-type cells, possibly a normal peroxisome, is also required.

Neutrophilic granulocytes contain an oxidase system in their plasma membrane that can be activated to generate superoxide radicals and hydrogen peroxide. Cytochrome b, flavoprotein, and ubiquinone-50 have been proposed as components of this oxidase system. These components have been quantitated, but the results are obscured by different isolation procedures for plasma membranes from resting and activated neutrophils. This problem has now been avoided by the use of enucleated neutrophils (polymorphonuclear leukocyte cytoplasts), which are almost completely devoid of intracellular structures but contain an intact, activatable oxidase system (Roos, D., Voetman, A.A., and Meerhof, L.J. (1983) J. Cell Biol. 97, 368-377). Membranes of resting and phorbol myristate acetate-stimulated cytoplasts contain equal amounts of cytochrome b (4 pmol/milliunit of alkaline phosphatase) and also equal amounts of noncovalently bound FAD (2 pmol/milliunit of alkaline phosphatase). These findings refute the hypothesis that incorporation of cytochrome b and/or a flavoprotein into the plasma membrane constitutes the mechanism of activation of the oxidase system. Ubiquinone-50 is present neither in intact neutrophils nor in cytoplasts, excluding a role for this compound in the generation of bactericidal oxygen species by neutrophils.

Friend mouse erythroleukemia cells do not synthesize detectable levels of phenylalanine hydroxylase [phenylalanine 4-monooxygenase; L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] and hence are unable to grow in medium totally lacking tyrosine. These cells were fused with the cytoplasts of rat hepatoma cells that synthesize phenylalanine hydroxylase constitutively. Cytoplasmic hybrids [cybrids, Bunn, C. L., Douglas, C. W. & Eisenstadt, J. M. (1974) Proc. Natl. Acad. Sci. USA 71, 1681--1685] were selecte in medium without tyrosine. Cybrid clones expressed phenylalanine hydroxylase enzyme, which was of mouse type as determined by immunotitration and isoelectric focusing. This phenotype has been mainta ined even in the absence of any selective pressure. In contrast, in whole cell hybrids derived between the same parents, the expression of the phenylalanine hydroxylase gene was totally extinguished. One interpretation of these results is that the cytoplasm of rat hepatoma cells contain a positively acting factor(s) for the phenylalanine hydroxylase gene that brings about the activation of this gene in erythroleukemia cells.