Generating pluripotent stem cells: Differential epigenetic changes during cellular reprogramming

Pluripotent stem cells hold enomous potential for therapuetic applications in tissue replacement therapy. Reprogramming somatic cells from a patient donor to generate pluripotent stem cells involves both ethical concerns inherent in the use of embryonic and oocyte-derived stem cells, as well as issues of histocompatibility. Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)-derived by ectopic expression of four reprogramming factors in donor somatic cells-are superior in terms of ethical use, histocompatibility, and derivation method. However, iPSC also show genetic and epigenetic differences that limit their differentiation potential, functionality, safety, and potential clinical utility. Here, we discuss the unique characteristics of iPSC and approaches that are being taken to overcome these limitations.     

Current state and prospects for gene therapy of Duchenne muscular dystrophy in the world and in Russia

Failure of drug therapy of Duchenne muscular dystrophy (DMD) stimulated intense search for adequate methods of gene therapy (GT) which would ensure effective delivery of the dystrophin (D) gene, its long-term persistence in transfected cells, and its expression in muscle fibers. The main results of the experimental GT of DMD with the use of viral and nonviral delivery of the D gene into muscles of biological models are discussed. Delivery of a mini-gene of D with a specific muscle promoter using a modified adenoassociated virus is currently the most promising method, which will soon be available for clinical trials. The main results of the studies on the DMD GT in Russia are summarized. The results of experiments on genetic transfection of mdx mice with marker genes and various constructions with the D gene are outlined. The genes are delivered into muscles by means of gene gun, electroporation, viral oligopeptides, liposomes, microspheres, lactoferine, and other nonviral vehicles. It is emphasized that consolidation of funds and efforts of all Russian laboratories dealing with gene and cell therapy of DMD are necessary to complete the experiments and start clinical trials.     

The Current State and Prospects of the Gene Therapy of Duchenne Muscular Dystrophy Worldwide and in Russia

Failure of drug therapy of Duchenne muscular dystrophy (DMD) stimulated intense search for adequate methods of gene therapy (GT) which would ensure effective delivery of the dystrophin (D) gene, its long-term persistence in transfected cells, and its expression in muscle fibers. The main results of the experimental GT of DMD with the use of viral and nonviral delivery of the D gene into muscles of biological models are discussed. Delivery of a mini-gene of D with a specific muscle promoter using a modified adenoassociated virus is currently the most promising method, which will soon be available for clinical trials. The main results of the studies on the DMD GT in Russia are summarized. The results of experiments on genetic transfection of mdx mice with marker genes and various constructions with the D gene are outlined. The genes are delivered into muscles by means of gene gun, electroporation, viral oligopeptides, liposomes, microspheres, lactoferine, and other nonviral vehicles. It is emphasized that consolidation of funds and efforts of all Russian laboratories dealing with gene and cell therapy of DMD are necessary to complete the experiments and start clinical trials.     

Somatic genome variations in health and disease

It is hard to imagine that all the cells of the human organism (about 10(14)) share identical genome. Moreover, the number of mitoses (about 10(16)) required for the organism's development and maturation during ontogeny suggests that at least a proportion of them could be abnormal leading, thereby, to large-scale genomic alterations in somatic cells. Experimental data do demonstrate such genomic variations to exist and to be involved in human development and interindividual genetic variability in health and disease. However, since current genomic technologies are mainly based on methods, which analyze genomes from a large pool of cells, intercellular or somatic genome variations are significantly less appreciated in modern bioscience. Here, a review of somatic genome variations occurring at all levels of genome organization (i.e. DNA sequence, subchromosomal and chromosomal) in health and disease is presented. Looking through the available literature, it was possible to show that the somatic cell genome is extremely variable. Additionally, being mainly associated with chromosome or genome instability (most commonly manifesting as aneuploidy), somatic genome variations are involved in pathogenesis of numerous human diseases. The latter mainly concerns diseases of the brain (i.e. autism, schizophrenia, Alzheimer's disease) and immune system (autoimmune diseases), chromosomal and some monogenic syndromes, cancers, infertility and prenatal mortality. Taking into account data on somatic genome variations and chromosome instability, it becomes possible to show that related processes can underlie non-malignant pathology such as (neuro)degeneration or other local tissue dysfunctions. Together, we suggest that detection and characterization of somatic genome behavior and variations can provide new opportunities for human genome research and genetics.     

Pluripotency and nuclear reprogramming

Embryonic stem cells are promising donor cell sources for cell transplantation therapy, which may in the future be used to treat various diseases and injuries. However, as is the case for organ transplantation, immune rejection after transplantation is a potential problem with this type of therapy. Moreover, the use of human embryos presents serious ethical difficulties. These issues may be overcome if pluripotent stem cells are generated from patients' somatic cells. Here, we review the molecular mechanisms underlying pluripotency and the currently known methods of inducing pluripotency in somatic cells.     

Rise of the planet

Despite early failures, somatic gene therapy has recently shown renewed promise. Howy suggests that the day may come when germline gene therapy needs also to be reconsidered.EMBO reports (2013) 14, 1; doi:10.1038/embor.2012.194Human gene therapy has a short but chequered history. The recognition, in the 1980s, that many human diseases were caused by recessive single gene mutations, led inevitably to the idea that such defects could be corrected by the same technology that facilitates the creation of transgenic animals.At an early stage, scientists and clinicians explicitly eschewed the idea of making genetic modifications to the germline, even in the case of fatal diseases. This was partly to assuage public concern that such technology could be misused for eugenic purposes widely considered unethical and tainted by association with the Nazi genocide. More prosaically, the techniques for engineering changes in the genome were so novel that their safety could not be assured. Even if such hurdles could be overcome, the unpredictable effects upon subsequent generations were considered a sufficient reason to observe a self-imposed moratorium on any such ‘playing with evolution''. Germline manipulation of the human genome remains largely a taboo subject, of greater interest to Hollywood than Bethesda [1]. Indeed, editing even plant and animal genomes remains highly controversial and tightly restricted, if not completely prohibited, in many jurisdictions.Three decades later, it is time to reassess the issue in the light of our greatly expanded knowledge of genetics, and the rather limited success of non-germline approaches to genetic therapy to date.With the route blocked to germline transgenesis, the era of somatic gene therapy was born. In outline, the method seeks to replace a defective gene in those cells whose function is compromised, thus overcoming the deficiency. Numerous variations on this theme seek to target the gene or its expression to specified cell types, control its integration into the genome, incorporate molecular ‘safety triggers'' or limit the immune response to the modified cells. The experimental nature of somatic gene therapy dictated that initial trials were conducted only in cases of severely debilitating and inevitably fatal diseases. The inherent difficulties of targeting tissues within the body also restricted it, at first, to cell types where an ex vivo approach could be employed, such as blood. But even this approach to treat diseases such as severe combined immunodeficiency met with only limited success. Therapeutic benefits were seen, but were typically temporary, and some patients succumbed to serious or even fatal side-effects, for example, through the oncogenic effects of random insertions in the genome.These tragic outcomes cast a long shadow over subsequent trials. For a long time, almost the only diseases for which such an approach was contemplated were end-stage cancers, where the risk of novel neoplasms can be considered a side issue, as for radiotherapy or treatment with genotoxic drugs. Unfortunately, gene therapy for cancer, even when cleverly targeted, suffers the same methodological flaw as these older, cruder therapies, namely the practical impossibility of zapping every single tumour cell, including quiescent progenitor-type cells that may be the primary reservoir of disease. Some remissions have, however, been reported.A few brave attempts to develop the field are now under way, but fundamental safety and efficacy problems remain. Even when a non-immunogenic delivery system can be employed, a replacement gene typically elicits an immune reaction against what is, to the patient''s immune system, a foreign gene product. To be permanently effective, any such therapy requires at least a partial disabling of the recipient''s immune system, thus replacing one disease with another. The oncogenic risk associated with random insertions, as well as the common problem of transgene silencing can, in theory, be overcome by the use of targeted insertion systems based on site-specific recombinases. This has proven to be a powerful tool for creating transgenic animals in research. However, to be effective, it requires a specific landing pad in the recipient genome, and thus implies the prior use of germline genetic manipulation. In the future, it should be possible to use customized recombinases with enhanced specificity to target only one or a few ‘benign'' insertion sites in the human genome, permissive for transgene expression. But this has not yet been achieved. In weighing the ethical objections against germline gene therapy, we need to take account of the persisting problems with its somatic cousin.In the case of recessive disorders, preimplantation diagnosis offers a simple and safe alternative approach, although many people have ethical or religious objections to this procedure as well. But what if it were to be ascertained that making a specific alteration to the human genome could protect against Alzheimer disease or malaria? What if adding just a few additional copies of a tumour suppressor gene such as p53 could provide lifetime resistance to most common cancers? Preimplantation diagnosis is clearly useless for diseases acquired through somatic mutation or via epigenetic errors during development. Would it be ethical to withhold prophylactic germline ‘therapy'' if it could ensure the alleviation of suffering on a massive scale? Germline manipulation is already marching towards approval in the UK for mitochondrial DNA disorders [2]. To some this is the thin end of the wedge: to others it is a chink of light in a dark landscape.At some point in the future, humanity will have to face such questions. I believe that the continuing rapid progress in elucidating the underlying basis of disease must lead to feasible preventative strategies based on genetic technologies that essentially exist already. We need to be ready with answers.     

Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a ‘protein delivery approach’ offers a new tool for developing novel strategies for genome modification and gene therapy applications.     

Convergence of human pluripotent stem cell,organoid, and genome editing technologies

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.     

猪异种器官移植的人源化修饰

利用猪的器官来解决当前人源器官严重短缺,为解决移植器官短缺的可行的途径。用定向基因转移(gene targeting)手段,直接并准确地对α-1,3半乳糖苷转移酶（α-1,3GT）基因进行同源重组,使α-1,3GT失活,再结合猪体细胞克隆技术,对其进行人源化改造,减弱或消除排异反应。除对2-1.3GT进行基因定向修饰外,阻断由异种器官移植而激活的人类补体的串联反应是猪异种器官人源化修饰的另一途径。然而,猪内源性逆转录病毒（porcine endogenous retrovirus,PERV）造成的公共卫生问题,给异种器官移植的前景投下了阴影。因此,即要剔除导致人类排异反应的猪细胞表面的α-1,3GT及其相关的分子, 又要确保猪器官异种移植的安全性, 是尚待研究的重大课题。 Abstract:Xenotransplantation (XP) from pig into human has been considered as means to overcome the great lack of donor organ available in transplantation surgery.In order to weaken rejection between human and pig,approaches of gene targeting have been proposed to eliminate “ rejection gene”α-1,3GT from porcine cells directly and accurately.α-1,3GT knockout pigs can be produced by nuclear transfer cloning with the porcine cells(knocking out α-1,3GT).Besides the genetic modification of α-1,3GT in porcine cells,there is another technical way to interdict activity of complement in series for human by XP.However,porcine endogenous retroviruses (PERV) during XP has been thought to not be negligible in being transmitted with the xenograft to the human recipient.Therefore,it is importance task that we should not only knockout α-1,3GT and relative molecules from pigs,but also ensure safety in public health of XP from PERV.     

Somatic cell reprogramming for regenerative medicine: SCNT vs. iPS cells

Reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. However, a debate over which method is better, somatic cell nuclear transfer (SCNT) or induced pluripotent stem (iPS) cells, still persists. Both approaches have the potential to generate patient-specific pluripotent stem cells for replacement therapy. Yet, although SCNT has been successfully applied in various vertebrates, no human pluripotent stem cells have been generated by SCNT due to technical, legal and ethical difficulties. On the other hand, human iPS cell lines have been reported from both healthy and diseased individuals. A recent study reported the generation of triploid human pluripotent stem cells by transferring somatic nuclei into oocytes, a variant form of SCNT. In this essay, we discuss this progress and the potentials of these two reprogramming approaches for regenerative medicine.     

Direct mutation analysis by high-throughput sequencing: from germline to low-abundant, somatic variants

DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.     

Societal problems in human and medical genetics

The applications of human and medical genetics raise many societal and ethical problems. This paper deals with a variety of such issues posed by current and future developments in genetic counseling, genetic screening, prenatal and predictive diagnosis, and gene therapy. The promise and problems of behavioral genetics are discussed. Problems of privacy, decision making, societal pressures, stigmatization, and informed consent to genetic study are raised. Use of genetic data by insurance companies or other public groups is discussed. The rapid unfolding of genetic information affecting human health and disease is producing difficult dilemmas. New problems are likely to surface, but human ingenuity and rationality is likely to find just and compassionate solutions in most settings.     

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.     

Communication between parental and developing genomes during tetrahymena nuclear differentiation is likely mediated by homologous RNAs

Approximately 6000 DNA elements, totaling nearly 15 Mb, are coordinately excised from the developing somatic genome of Tetrahymena thermophila. An RNA interference (RNAi)-related mechanism has been implicated in the targeting of these germline-limited sequences for chromatin modification and subsequent DNA rearrangement. The excision of individual DNA segments can be inhibited if the homologous sequence is placed within the parental somatic nucleus, indicating that communication occurs between the parental and developing genomes. To determine how the DNA content of one nucleus is communicated to the other, we assessed DNA rearrangement occurring in wild-type cells that were mated to cells that contained the normally germline-limited M element within their somatic nuclei. M-element rearrangement was blocked in the wild-type cell even when no genetic exchange occurred between mating partners, a finding that is inconsistent with any genetic imprinting models. This inhibition by the parental somatic nucleus was rapidly established between 5 and 6 hr of conjugation, near or shortly after the time that zygotic nuclei are formed. M-element small RNAs (sRNAs) that are believed to direct its rearrangement were found to rapidly accumulate during the first few hours of conjugation before stabilizing to a low, steady-state level. The period between 5 and 6 hr during which sRNA levels stabilize correlates with the time after which the parental genome can block DNA rearrangement. These data lead us to suggest that homologous sRNAs serve as mediators to communicate sequence-specific information between the parental and developing genomes, thereby regulating genome-wide DNA rearrangement, and that these sRNAs can be effectively compared to the somatic genome of both parents.     

Cell Churches and Stem Cell Marketing in South Korea and the United States

The commercial provision of putative stem cell‐based medical interventions in the absence of conclusive evidence of safety and efficacy has formed the basis of an unregulated industry for more than a decade. Many clinics offering such supposed stem cell treatments include statements about the ‘ethical’ nature of somatic (often colloquially referred to as ‘adult’ stem cells) stem cells, in specific contrast to human embryonic stem cells (hESCs), which have been the subject of intensive political, legal, and religious controversy since their first derivation in 1998 1 . Christian groups—both Roman Catholic and evangelical Protestant—in many countries have explicitly promoted the medical potential and current‐day successes in the clinical application of somatic stem cells, lending indirect support to the activities of businesses marketing stem cells ahead of evidence 2 . In this article, I make a preliminary examination of how the structures and belief systems of certain churches in South Korea and the United States, both of which are home to significant stem cell marketing industries, has complemented other factors, including national biomedical funding initiatives, international economic rivalries, permissive legal structures, which have lent impetus to a problematic and often exploitative sector of biomedical commerce 3 .     

Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane

We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.