scholarly journals Double sperm cloning (DSC) is a promising strategy in mammalian genetic engineering and stem cell research

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhi-ping Zhang ◽  
Jun-tao Zhang ◽  
Shu-cheng Huang ◽  
Xiu-yuan He ◽  
Li-xin Deng
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Immunological Response ◽  
Epigenetic Memory ◽  
Donor Cells ◽  
Induced Pluripotent ◽  
The Many ◽  
Artificial Activation

Abstract Embryonic stem cells (ESCs) derived from somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) are promising tools for meeting the personalized requirements of regenerative medicine. However, some obstacles need to be overcome before clinical trials can be undertaken. First, donor cells vary, and the reprogramming procedures are diverse, so standardization is a great obstacle regarding SCNT and iPSCs. Second, somatic cells derived from a patient may carry mitochondrial DNA mutations and exhibit telomere instability with aging or disease, and SCNT-ESCs and iPSCs retain the epigenetic memory or epigenetic modification errors. Third, reprogramming efficiency has remained low. Therefore, in addition to improving their success rate, other alternatives for producing ESCs should be explored. Producing androgenetic diploid embryos could be an outstanding strategy; androgenic diploid embryos are produced through double sperm cloning (DSC), in which two capacitated sperms (XY or XX, sorted by flow cytometer) are injected into a denucleated oocyte by intracytoplasmic sperm injection (ICSI) to reconstruct embryo and derive DSC-ESCs. This process could avoid some potential issues, such as mitochondrial interference, telomere shortening, and somatic epigenetic memory, all of which accompany somatic donor cells. Oocytes are naturally activated by sperm, which is unlike the artificial activation that occurs in SCNT. The procedure is simple and practical and can be easily standardized. In addition, DSC-ESCs can overcome ethical concerns and resolve immunological response matching with sperm providers. Certainly, some challenges must be faced regarding imprinted genes, epigenetics, X chromosome inactivation, and dosage compensation. In mice, DSC-ESCs have been produced and have shown excellent differentiation ability. Therefore, the many advantages of DSC make the study of this process worthwhile for regenerative medicine and animal breeding.

scholarly journals Human therapeutic cloning, pitfalls and lack luster because of rapid developments in induced pluripotent stem cell technology

2014 ◽  
Vol 8 (1) ◽  
pp. 5-10
Author(s):  
Song Hua ◽  
Henry Chung ◽  
Kuldip Sidhu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Pluripotent Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Therapeutic Cloning ◽  
Induced Pluripotent

AbstractBackground: Therapeutic cloning is the combination of somatic cell nuclear transfer (SCNT) and embryonic stem cell (ES) techniques to create specific ES cells that match those of a patient. Because ES cells derived by nuclear transfer (SCNT ES cells) are genetically identical to the donor, it will not generate rejection by the host’s immune system and thus therapeutically may be more acceptable. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cell artificially derived from an adult somatic cell by inducing a forced expression of a set of specific pluripotent genes. In the past few years, rapid progress in reprogramming and iPS technology has been made, and it seems to shadow any progress made in SCNT programs.Objective: This review compares the application perspective of SCNT with that of iPS in regenerative medicine.Methods:We conducted a literature search using the MEDLINE (PubMed), Wiley InterScience, Springer, EBSCO, and Annual Reviews databases using the keywords “iPS”, “ES”, “SCNT” “induced pluripotent stem cells”, “embryonic stem cells”, “therapeutic cloning”, “regenerative medicine”, and “somatic cell nuclear transfer”. Only articles published in English were included in this review.Results: These two methods both have advantages and disadvantages. Nevertheless, by using SCNT to generate patient-specific cell lines, it eliminates complications by avoiding the use of viral vectors during iPS generation. Success in in vitro matured eggs from aged women and even differentiation of oocytes from germ stem cells will further enhance the application of SCNT in regenerative medicine.Conclusion: Human SCNT may be an appropriate mean of generating patient stem cell lines for clinical therapy in the near future.

Epigenetic Memory Enables the Dominant Generation of Adult-Type Erythrocytes From Human Induced Pluripotent Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 648-648 ◽  
Author(s):  
Naoya Takayama ◽  
Shiya Sano ◽  
Takafumi Shimizu ◽  
Ryoichiro Kawahata ◽  
Hiroshi Endo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Epigenetic Modification ◽  
Globin Gene ◽  
Globin Genes ◽  
In Vitro Differentiation ◽  
Epigenetic Memory ◽  
Adult Type ◽  
Induced Pluripotent

Abstract Abstract 648 It is well known that “globin switching” during erythropoiesis is associated with the pathophysiology of sickle cell anemia, as well as an approach to ameliorating some hemoglobinopathies. Furthermore, the process of globin switching represents an important paradigm for developmental gene regulation. Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are attractive tools for studying the ontogeny of human erythropoiesis because they exhibit in vitro differentiation toward various erythrocytes with embryonic (e), fetal (g) or adult (b) globin genes. However, earlier studies mainly showed that erythroid differentiation from human ESCs/iPSCs stopped at fetal erythrocytes and yielded few erythrocytes expressing b-globin (Chang, Blood 2006, 2010; Lapillonne, Haematologica 2010). In addition, successful differential reprogramming through somatic cell nuclear transfer and the use of iPSCs raised to the possibility that the poor yield of b-globin-expressing erythrocytes may result from incomplete genomic methylation or deregulated epigenetic modification. Recent studies using mouse iPSCs suggest the presence of “epigenetic memory” reflecting the tissue of origin. In that context, we attempted to establish an in vitro differentiation culture system that would preferentially yield adult-type erythrocytes derived from human iPSCs and would enable the study of b-globin gene switching during erythrocyte ontogeny. We initially established iPSCs from human dermal fibroblasts (HDFs) (f-iPSCs) or cord blood-derived CD34+/CD45+ cells (b-iPSCs) using retroviral vectors harboring human reprogramming factors (OCT3/4, SOX2, KLF4 and/or c-MYC). Gene expression analyses showed comparable patterns of gene clustering in f-iPSCs and b-iPSCs, and the b-globin gene was undetectable in either undifferentiated iPSC type. We then compared the hematopoietic colony forming capacities of 6 f-iPSC clones and 10 b-iPSC clones using a previously established culture system in which an “iPS-Sac” structure manifests an “in vitro hematopoietic niche” that generates multipotential hematopoietic progenitors. The b-iPSCs produced a higher number of iPS-sac structures and mixed-lineage or BFU-E colonies than f-iPSCs (Mixed colonies: 261±39.4 vs. 36±13.8, p<0.01; BFU-E: 192±35.4 vs. 11.8±6.7 per 1×105 iPSCs, p<0.01). Moreover, as a result of terminal differentiation, erythrocytes were generated much more efficiently from b-iPSCs than f-iPSCs under erythrocyte-specific culture conditions. Finally, we selected two clones from each group to further analyze erythroid maturation and globin switching in erythrocytes generated from b-iPSCs and f-iPSCs. RT-PCR and immunochemical assays revealed limited differentiation by f-iPSC-derived erythrocytes (i.e., most were at the fetal stage), which is consistent with previous reports (Chang, Blood 2010), but b-iPSCs efficiently generated adult-type erythrocytes expressing b-globin (f-iPSCs, 21.0±4.7% vs. b-iPSCs, 54.7±4.2% b-globin+ p<0.01). In addition, the number of enucleated erythrocytes was higher from b-iPSCs than f-iPSCs. These data strongly suggest that genes regulating g- to b-globin switching are suppressed in f-iPSCs, possibly by epigenetic modification. Studies of the mechanisms underlying b-globin gene expression are clinically important, as they can provide the basis for potential gene therapeutic and reactivation strategies employing fetal globin genes to treat various hemoglobinopathies. Thus, b-iPSCs could be an abundant source of adult-type erythrocytes for use in clinical applications. Disclosures: No relevant conflicts of interest to declare.

scholarly journals “Epigenetic Memory” Phenomenon in Induced Pluripotent Stem Cells

Acta Naturae ◽  
2013 ◽  
Vol 5 (4) ◽  
pp. 15-21 ◽  
Author(s):  
E. A. Vaskova ◽  
A. E. Stekleneva ◽  
S. P. Medvedev ◽  
S. M. Zakian
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Somatic Tissue ◽  
Epigenetic Memory ◽  
High Throughput Analysis ◽  
Tissue Of Origin ◽  
Induced Pluripotent ◽  
Covalent Histone Modifications

To date biomedicine and pharmacology have required generating new and more consummate models. One of the most perspective trends in this field is using induced pluripotent stem cells (iPSCs). iPSC application requires careful high-throughput analysis at the molecular, epigenetic, and functional levels. The methods used have revealed that the expression pattern of genes and microRNA, DNA methylation, as well as the set and pattern of covalent histone modifications in iPSCs, are very similar to those in embryonic stem cells. Nevertheless, iPSCs have been shown to possess some specific features that can be acquired during the reprogramming process or are remnants of epigenomes and transcriptomes of the donor tissue. These residual signatures of epigenomes and transcriptomes of the somatic tissue of origin were termed epigenetic memory. In this review, we discuss the epigenetic memory phenomenon in the context of the reprogramming process, its influence on iPSC properties, and the possibilities of its application in cell technologies.

scholarly journals iPSC Preparation and Epigenetic Memory: Does the Tissue Origin Matter?

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1470
Author(s):  
Giuseppe Scesa ◽  
Raffaella Adami ◽  
Daniele Bottai
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Epigenetic Memory ◽  
Differentiated Cells ◽  
Ipsc Generation ◽  
Tissue Of Origin ◽  
Induced Pluripotent ◽  
The Impact

The production of induced pluripotent stem cells (iPSCs) represent a breakthrough in regenerative medicine, providing new opportunities for understanding basic molecular mechanisms of human development and molecular aspects of degenerative diseases. In contrast to human embryonic stem cells (ESCs), iPSCs do not raise any ethical concerns regarding the onset of human personhood. Still, they present some technical issues related to immune rejection after transplantation and potential tumorigenicity, indicating that more steps forward must be completed to use iPSCs as a viable tool for in vivo tissue regeneration. On the other hand, cell source origin may be pivotal to iPSC generation since residual epigenetic memory could influence the iPSC phenotype and transplantation outcome. In this paper, we first review the impact of reprogramming methods and the choice of the tissue of origin on the epigenetic memory of the iPSCs or their differentiated cells. Next, we describe the importance of induction methods to determine the reprogramming efficiency and avoid integration in the host genome that could alter gene expression. Finally, we compare the significance of the tissue of origin and the inter-individual genetic variation modification that has been lightly evaluated so far, but which significantly impacts reprogramming.

scholarly journals Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms

2016 ◽  
Vol 113 (43) ◽  
pp. 12202-12207 ◽  
Author(s):  
Timothy Alexander Hore ◽  
Ferdinand von Meyenn ◽  
Mirunalini Ravichandran ◽  
Martin Bachman ◽  
Gabriella Ficz ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Retinoic Acid ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Epigenetic Memory ◽  
Mechanistic Insight ◽  
Induced Pluripotent ◽  
Tet Enzymes

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.

scholarly journals Proteasomes in Protein Homeostasis of Pluripotent Stem Cells

Acta Naturae ◽  
2017 ◽  
Vol 9 (3) ◽  
pp. 39-47 ◽  
Author(s):  
А. V. Selenina ◽  
А. S. Tsimokha ◽  
А. N. Tomilin
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Basic Research ◽  
Ubiquitin Proteasome System ◽  
Protein Homeostasis ◽  
Cellular Processes ◽  
Induced Pluripotent

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are subjects of high interest not only in basic research, but also in various applied fields, particularly, in regenerative medicine. Despite the tremendous interest to these cells, the molecular mechanisms that control protein homeostasis in these cells remain largely unknown. The ubiquitin-proteasome system (UPS) acts via post-translational protein modifications and protein degradation and, therefore, is involved in the control of virtually all cellular processes: cell cycle, self-renewal, signal transduction, transcription, translation, oxidative stress, immune response, apoptosis, etc. Therefore, studying the biological role and action mechanisms of the UPS in pluripotent cells will help to better understand the biology of cells, as well as to develop novel approaches for regenerative medicine.

scholarly journals Current status and future perspectives of HLA-edited induced pluripotent stem cells

2020 ◽  
Vol 40 (1) ◽  
Author(s):  
Keiko Koga ◽  
Bo Wang ◽  
Shin Kaneko
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Es Cells ◽  
Embryonic Stem ◽  
The Body ◽  
Current Status ◽  
Induced Pluripotent

Abstract In 2007, Human-induced pluripotent stem cells (iPSCs) were generated by transducing four genes (Oct3/4, Sox2, Klf4, c-Myc). Because iPSCs can differentiate into any types of cells in the body and have fewer ethical issues compared to embryonic stem (ES) cells, application of iPSCs for regenerative medicine has been actively examined. In fact, iPSCs have already been used for clinical applications, but at present, only autologous iPSC-derived grafts or HLA homozygous iPSC-derived grafts are being transplanted into patients following HLA matching. HLA is an important molecule that enables the immune system differentiates between self and non-self-components; thus, HLA mismatch is a major hurdle in the transplantation of iPSCs. To deliver inexpensive off-the-shelf iPSC-derived regenerative medicine products to more patients, it is necessary to generate universal iPSCs that can be transplanted regardless of the HLA haplotypes. The current strategy to generate universal iPSCs has two broad aims: deleting HLA expression and avoiding attacks from NK cells, which are caused by HLA deletion. Deletion of B2M and CIITA genes using the CRISPR/Cas9 system has been reported to suppress the expression of HLA class I and class II, respectively. Transduction of NK inhibitory ligands, such as HLA-E and CD47, has been used to avoid NK cell attacks. Most recently, the HLA-C retaining method has been used to generate semi-universal iPSCs. Twelve haplotypes of HLA-C retaining iPSCs can cover 95% of the global population. In future, studying which types of universal iPSCs are most effective for engraftment in various physiological conditions is necessary.

scholarly journals Non-integrating Methods to Produce Induced Pluripotent Stem Cells for Regenerative Medicine: An Overview

2020 ◽  
Author(s):  
Immacolata Belviso ◽  
Veronica Romano ◽  
Daria Nurzynska ◽  
Clotilde Castaldo ◽  
Franca Di Meglio
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Safety Issues ◽  
Advantages And Disadvantages ◽  
Induced Pluripotent

Induced Pluripotent Stem cells (iPSC) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Due to their autologous origin from adult somatic cells, iPSCs are considered a tremendously valuable tool for regenerative medicine, disease modeling, drug discovery and testing. iPSCs were first obtained by introducing specific transcription factors through retroviral transfection. However, cell reprogramming obtained by integrating methods prevent clinical application of iPSC because of potential risk for infection, teratomas and genomic instability. Therefore, several integration-free alternate methods have been developed and tested thus far to overcome safety issues. The present chapter provides an overview and a critical analysis of advantages and disadvantages of non-integrating methods used to generate iPSCs.

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