Donor cell differentiation,reprogramming, and cloning efficiency: Elusive or illusive correlation?

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages.     

Fish stem cell cultures

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.     

Stemness,fusion and renewal of hematopoietic and embryonic stem cells

Development of replacement cell therapies awaits the identification of factors that regulate nuclear reprogramming and the mechanisms that control stem cell renewal and differentiation. Once such factors and signals will begin to be elucidated, new technologies will have to be envisaged where uniform differentiation of adult or embryonic stem cells along one differentiation pathway can be induced. Controlled differentiation of stem cells will require the engineering of niches and extracellular signal combinations that would amplify a particular signaling network and allow uniform and selective differentiation. Three recent advances in stem cell research open the possibility to approach engineering studies for cell replacement therapies. Fusion events between stem cells and adult cells or between adult and embryonic stem cells have been shown to result in altered fates and nuclear reprogramming of cell hybrids. Hematopoietic stem cells were shown to require Wnt signaling in order to renew. The purification of Wnt proteins would allow their use as exogenous purified cytokines in attempts to amplify stem cells before bone marrow transplantation. The homeodomain protein Nanog has been shown to be crucial for the embryonic stem cell renewal and pluripotency. However, the cardinal question of how stemness is preserved in the early embryo and adult stem cells remains opened.     

Nuclear transfer in cats and its application

Nuclear transfer (NT) technology is typically used for generating identical individuals, but it is also a powerful resource for understanding the cellular and molecular aspects of nuclear reprogramming. Most recently, the procedure has been used in humans for producing patient-specific embryonic stem cells. The successful application of NT in cats was demonstrated by the birth of domestic and non-domestic cloned kittens at a similar level of efficiency to that reported for other mammalian species. In cats, it has been demonstrated that either in vivo or in vitro matured oocytes can be used as donor cytoplasts. The length of in vitro oocyte maturation affects in vitro development of reconstructed embryos, and oocytes matured in vitro for shorter periods of time are the preferred source of donor cytoplasts. For NT, cat somatic cells can be synchronized into the G0/G1 phase of the cell cycle by using different methods of cell synchronization without affecting the frequency of in vitro development of cloned embryos. Also, embryo development to the blastocyst stage in vitro is not influenced by cell type, but the effect of cell type on the percentage of normal offspring produced requires evaluation. Inter-species NT has potential application for preserving endangered felids, as live offspring of male and female African wildcats (AWC, Felis silvestris lybica) have been born and pregnancies have been produced after transferring black-footed cat (Felis nigripes) cloned embryos into domestic cat (Felis silvestris catus) recipients. Also, successful in vitro embryo development to the blastocyst stage has been achieved after inter-generic NT of somatic cells of non-domestic felids into domestic cat oocytes, but no viable progeny have been obtained. Thus, while cat cytoplasm induces early nuclear remodeling of cell nuclei from a different genus, the high incidence of early embryo developmental arrest may be caused by abnormal nuclear reprogramming. Fetal resorption and abortions were frequently observed at various stages of pregnancy after transfer of AWC cloned embryos into domestic cat recipients. Abnormalities, such as abdominal organ exteriorization and respiratory failure and septicemia were the main causes of death in neonatal cloned kittens. Nonetheless, several live domestic and AWC cloned kittens have been born that are seemingly normal and healthy. It is important to continue evaluating these animals throughout their lives and to examine their capability for natural reproduction.     

Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.     

Nuclear transfer into mouse oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).     

Somatic cell nuclear transfer: Past, present and future perspectives

It is now over a decade since the birth, in 1996, of Dolly the first animal to be produced by nuclear transfer using an adult derived somatic cell as nuclear donor. Since this time similar techniques have been successfully applied to a range of species producing live offspring and allowing the development of transgenic technologies for agricultural, biotechnological and medical uses. However, though applicable to a range of species, overall, the efficiencies of development of healthy offspring remain low. The low frequency of successful development has been attributed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Many studies have demonstrated that such reprogramming occurs by epigenetic mechanisms not involving alterations in DNA sequence, however, at present the molecular mechanisms underlying reprogramming are poorly defined. Since the birth of Dolly many studies have attempted to improve the frequency of development, this review will discuss the process of animal production by nuclear transfer and in particular changes in the methodology which have increased development and survival, simplified or increased robustness of the technique. Although much of the discussion is applicable across species, for simplicity we will concentrate primarily on published data for cattle, sheep, pigs and mice.     

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.     

转基因红鲤体细胞的核移植

以F4代转hGH基因红鲤体细胞（肾脏和尾鳍）及培养18代的F4代转hGH基因红鲤尾鳍细胞为核供体,泥鳅或黄河鲤成熟卵为受体,进行了核移植,以探讨外源F4代转基因鱼体外源基因的分布与存在形式,稳定性和克隆转基因鱼的可能性。F4代红鲁肾脏细胞核与泥鳅卵配合的核移植胚胎有12．4％发育到囊胚,0．33％发育到神经胚;F4代尾鳍细胞核移入泥鳅卵后的重组胚发育到囊胚,神经胚、肌节期和肌肉效应期的胚胎分别为24．5％、0．3％、0．2％和0．1％;对照卵无发育。F4代红鲤尾鳍培养细胞与黄河鲤卵子配合的重组胚胎有50．53％发育到囊胚,5．69％发育到原肠胚,0．53％发育到神经胚,0．4％发育到肌节期。说明由于同种细胞核与卵细胞的相容性高于异种核卵的相容性,早期发育率高;而由于培养细胞的异倍化,后期的发育率降低。用PCR技术对供体鱼不同个体及同一体不同组织外源基因检测,结果100％个体为阳性鱼,而且不同组织的阳性率也是100％,说明外源基因均匀分布在不同组织中。无论F4代转基因鱼的肾脏细胞、尾鳍细胞还是培养的尾鳍细胞作核移植供体,核移植胚胎中hGH基因的检出率为100％。说明F4代转基因红鲤个体不同细胞都存在hGH基因,而且经长期培养不会丢失。表明F4代转基因红鲤中的外源hGH基因已基本稳定,体细胞核移植可以作为获得同质化转基因鱼的有效手段,但核移植效率还很低。另外还讨论了核质的相容性、细胞周期的协调、染色体的变异等因素对核移植的影响。     

哺乳动物体细胞克隆技术的研究进展

尽管与已有十几年发展史的哺乳动物胚胎细胞克隆技术相比,其体细胞克隆技术的研究仅有不到三年的时间;然而,鉴于哺乳动物体细胞克隆技术在供核效率等方面所具备的明显优势,其应用价值远高于胚胎细胞克隆技术。随着对哺乳动物体细胞克隆技术基本机理的进一步广泛深入的研究,它将在大量克隆优良种畜、扩大同基因型实验动物种群、培育转基因动物和保护濒危野生动物遗传资源等方面发挥巨大作用。     

Epigenetic reprogramming in mammalian nuclear transfer

With the exception of lymphocytes, the various cell types in a higher multicellular organism have basically an identical genotype but are functionally and morphologically different. This is due to tissue-specific, temporal, and spatial gene expression patterns which are controlled by genetic and epigenetic mechanisms. Successful cloning of mammals by transfer of nuclei from differentiated tissues into enucleated oocytes demonstrates that these genetic and epigenetic programs can be largely reversed and that cellular totipotency can be restored. Although these experiments indicate an enormous plasticity of nuclei from differentiated tissues, somatic cloning is a rather inefficient and unpredictable process, and a plethora of anomalies have been described in cloned embryos, fetuses, and offspring. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. In this review, we discuss the roles of various epigenetic mechanisms, including DNA methylation, chromatin remodeling, imprinting, X chromosome inactivation, telomere maintenance, and epigenetic inheritance in normal embryonic development and in the observed abnormalities in clones from different species. Nuclear transfer represents an invaluable tool to experimentally address fundamental questions related to epigenetic reprogramming. Understanding the dynamics and mechanisms underlying epigenetic control will help us solve problems inherent in nuclear transfer technology and enable many applications, including the modulation of cellular plasticity for human cell therapies.     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.     

How to make insulin-producing pancreatic β cells for diabetes treatment

Around 400 million people worldwide suffer from diabetes mellitus.The major pathological event for Type 1 diabetes and advanced Type 2 diabetes is loss or impairment of insulin-secreting β cells of the pancreas.For the past 100 years,daily insulin injection has served as a life-saving treatment for these patients.However,insulin injection often cannot achieve full glucose control,and over time poor glucose control leads to severe complications and mortality.As an alternative treatment,islet transplantation has been demonstrated to effectively maintain glucose homeostasis in diabetic patients,but its wide application is limited by the scarcity of donated islets.Therefore,it is important to define new strategies to obtain functional human β cells for transplantation therapies.Here,we summarize recent progress towards the production of β cells in vitro from pluripotent stem cells or somatic cell types including a cells,pancreatic exocrine cells,gastrointestinal stem cells,fibroblasts and hepatocytes.We also discuss novel methods for optimizing β cell transplantation and maintenance in vivo.From our perspective,the future of βcell replacement therapy is very promising although it is still challenging to control differentiation of β cells in vitro and to protect these cells from autoimmune attack in Type 1 diabetic patients.Overall,tremendous progress has been made in understanding βcell differentiation and producing functional β cells with different methods.In the coming years,we believe more clinical trials will be launched to move these technologies towards treatments to benefit diabetic patients.     

Nuclear reprogramming in mammalian somatic cell nuclear cloning

Nuclear cloning is still a developing technique used to create genetically identical animals by somatic cell nuclear transfer into unfertilized eggs. Despite an intensive effort in a number of laboratories, the success rate of obtaining viable offspring from this technique remains less than 5%. In the past few years many investigators reported the reprogramming of specific nuclear activities in cloned animals, such as genome-wide gene expression patterns, DNA methylation, genetic imprinting, histone modifications and telomere length regulation. The results highlight the tremendous difficulty the clones face to reprogram the original differentiation status of the donor nuclei. Nevertheless, nuclei prepared from terminally differentiated lymphocytes can overcome this barrier and produce apparently normal mice. Study of this striking nuclear reprogramming activity should significantly contribute to our understanding of cell differentiation in more physiological settings.