Modulation of Cell Fate Using Nuclear and Cytoplasmic Extracts

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

It has been speculated that incomplete epigenetic reprogramming of the somatic cell genome is the primary reason behind the developmental inefficiencies and postnatal abnormalities observed after nuclear transplantation in domestic animal clones. One chromosome structure that is altered in dividing somatic cells is telomere length-the terminal ends of linear chromosomes capped by repetitive sequences of G-rich noncoding DNA, (TTAGGG)", and specific binding proteins. Telomeres are critical structures that function in maintaining chromosome stability and ensure the full replication of coding DNA by acting as a buffer to terminal DNA attrition due to the end replication problem. Telomere shortening limits cellular proliferation through a DNA damage signal activating permanent cell cycle arrest at a critical telomere length or through structural telomere alterations that prevents effective chromosome capping. Telomere-mediated signaling of cellular senescence has been established for many somatic cell types in vitro, except for germ cells, cancer lines, and regenerative tissues in which telomere length is maintained primarily by the ribonucleoprotein telomerase, a reverse transcriptase that synthesizes TTAGGG repeats de novo onto the chromosome ends. Telomere length discrepancies have been reported in animal clones as being shorter, no different, and even longer than in age-matched control animals, but the etiology is not yet understood. Possible explanations include differences in donor cell type and the efficiency of telomerase reprogramming. This chapter summarizes the conventional protocols and recent advances in telomere length and telomerase activity measurement that will help elucidate the mechanism(s) behind telomere length deregulation in somatic cell clones and its role in chromosomal instability, cellular senescence, and organismal aging in vivo.

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

Does in vivo aging of mouse oocytes affect mitochondrial function? Mitochondrial function was impaired in post-ovulatory in vivo-aged mouse oocytes and microinjection of somatic cell mitochondria did not rescue poor fertilization and embryonic development rates. The mechanisms underlying the decline in oocyte quality associated with oocyte aging remain unknown, although studies have suggested that the decline is regulated by mitochondrial dysfunction. However, only a limited number of studies have provided direct evidence implicating mitochondrial dysfunction in oocyte quality during the aging of oocytes. We used post-ovulatory, in vivo-aged mouse oocytes as a model for studying low-quality oocytes in oocyte aging. Superovulated oocytes released from the oviduct at 14 h and 20-24 h post-hCG injection were designated as 'fresh' and 'aged' oocytes, respectively. Membrane potentials and oxygen consumption in single oocytes were evaluated as measures of mitochondrial function in fresh and aged oocytes. Mitochondrial transcriptional factor A (TFAM) expression levels were examined by western blotting, and colocalization of mitochondria and TFAM was analyzed by measuring immunofluorescence in fresh and aged oocytes. IVF and blastocyst formation rates were calculated after oocyte microinjection with mitochondria derived from liver cells. The average mitochondrial membrane potential in fresh oocytes was significantly higher than that in aged oocytes (P<0.05). The average oxygen consumption rate in aged oocytes was significantly lower than that in fresh oocytes (P<0.05). Although total TFAM expression was unchanged, its colocalization with mitochondria decreased in aged oocytes. IVF and blastocyst formation rates for mitochondrion-injected aged oocytes were not significantly different from those for buffer-injected aged oocytes. Not applicable. A limitation of this study is that we did not examine the effects of microinjecting mitochondria from other somatic cell types into aged oocytes on their fertilization and embryonic development rates. The results from the present study showed that poor embryonic development was associated with impairment of mitochondrial functions in in vivo-aged oocytes. However, the microinjection of mitochondria from liver cells did not improve the low fertilization and embryonic development rates of aged oocytes. It remains to be demonstrated whether oocyte quality can be rescued by the transfer of cytosolic factors or cellular organelles, such as the endoplasmic reticulum or mitochondria, from specific cell types. This study was supported by Grants-in-Aid for General Science Research to Toshifumi Takahashi (No. 25462550) and Hideki Igarashi (No. 26462474). The funding source played no role in study design in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The authors have no conflict of interest to disclose.

A single regulatory pathway coordinates the number of germ cells and somatic cells in the ovary, according to a study in Drosophila [1]. The Hippo pathway regulates the number of stem cell niches and the proliferation of germ cells within those niches, while assuring the proper balance of somatic and germ cells. Hippo is known to regulate organ size and stem cell proliferation in numerous tissues. Tissue-specific knock-outs of this kinase, for instance, result in oversized organs. But little is known about how the different cell types within an organ communicate with each other to assure that their proliferation is coordinated and proportionate. In the new study, Didem Sarikaya and Cassandra Extavour show that Hippo can act as such a coordinator—regulating proliferation of different cell types using distinct mediators, and mediating communication between cell types. There are two major somatic cell types in the Drosophila ovary. The terminal filament cells determine the number of germ cell niches, and the intermingled cells are thought to help determine how many germ cells populate each niche. The researchers overor under-expressed various Hippo family members, in either somatic or germ cells, to understand how cell number is established and coordinated. They found that the canonical Hippo pathway cellautonomously regulated the number of terminal filament cells, thereby controlling the number of germ cell niches. The Hippo pathway also cell-autonomously regulated the number of intermingled cells via a distinct set of mediators. In germ cells, proliferation was cell-autonomously regulated not by Hippo but by Yorkie, a transcriptional co-activator that in other tissues operates downstream of Hippo. Hippo was also found to coordinate communication between germ cells and intermingled cells to generate roughly proportional numbers of these cell types. Future studies should help unlock how the Hippo pathway works with other regulators that affect cell proliferation in the ovary, such as insulin. Meanwhile, the new study neatly lays out a blueprint for how the number of germ cells is established in the ovary in coordination with supporting somatic cells.

Genetically based diseases constitute a major human health burden, and de novo germline mutations represent a source of heritable genetic alterations that can cause such disorders in offspring. The availability of transgenic rodent systems with recoverable, mutation reporter genes has been used to assess the occurrence of spontaneous point mutations in germline cells. Previous studies using the lacI mutation reporter transgenic mouse system showed that the frequency of spontaneous mutations is significantly lower in advanced male germ cells than in somatic cell types from the same individuals. Here we used this same mutation reporter transgene system to show that female germ cells also display a mutation frequency that is lower than that in corresponding somatic cells and similar to that seen in male germ cells, indicating this is a common feature of germ cells in both sexes. In addition, we showed that statistically significant differences in mutation frequencies are evident between germ cells and somatic cells in both sexes as early as mid-fetal stages in the mouse. Finally, a comparison of the mutation frequency in a general population of early type A spermatogonia with that in a population enriched for Thy-1-positive spermatogonia suggests there is heterogeneity among the early spermatogonial population such that a subset of these cells are predestined to form true spermatogonial stem cells. Taken together, these results support the disposable soma theory, which posits that genetic integrity is normally maintained more stringently in the germ line than in the soma and suggests that this is achieved by minimizing the initial occurrence of mutations in early germline cells and their subsequent gametogenic progeny relative to that in somatic cells.

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (DNMT1o) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as in cloned embryos. The objectives of this study were to identify the alternative splicing variants of DNMT1 in porcine oocytes and determine the gene expression pattern of the different DNMT1 isoforms during embryo development. Total RNA was isolated from a pool of 50 zona free mature porcine oocytes using the Trizol method. A RACE System (Invitrogen, Carlsbad, CA) was used to amplify the 5′ cDNA end of DNMT1. Polymerase chain reaction products were separated by electrophoresis, recovered from the agarose gel, subcloned into a cloning vector, and sequenced. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa DNMT1 genomic sequence located on chromosome 2 (CU462940). RNA levels of the DNMT1 isoforms were analyzed in porcine oocytes and in vivo embryos, as well and in cells from ovary, lung, spleen, liver, heart, and skin by quantitative PCR using the AACT method. DNMT1 protein levels of porcine oocytes and different somatic cells types were analyzed by Western blot. Two new DNMT1o RNA isoforms were identified (EU908730 and EU908731). The previously reported DNMT1s isoform (DQ060156) was expressed at low but constant levels in oocytes, as well as in embryos from the 2-cell to the blastocyst stage. Abundant RNA levels of EU908730 and EU908731 were detected in oocytes and embryos from the 2-cell to the 8- to 16-cell stage. Levels of these DNMT1o transcripts were low at the morula and blastocyst stage. Although DQ060156 was present in all the somatic cell types analyzed, EU908730 and EU908731 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by Western blot analysis, EU908730 and EU908731 RNAs translate one DNMT1o enzyme (ˆ170 KDa). Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, while somatic cells produce only DNMT1s protein. In conclusion, this study demonstrates that porcine oocytes express a DNMT1o RNA in addition to the DNMT1s somatic transcript. The 2 newly identified RNA isoforms are produced by alternative splicing of the DNMT1 gene and translate a DNMT1o protein that is unique to oocytes and early embryos. Analysis of the oocyte and somatic DNMT1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.

BackgroundThe Monopterus albus serves as a unique model for studying sex reversal, transitioning naturally from female to male. However, the origins of male germline stem cells (GSCs) and the roles of somatic cells during sex reversal remain poorly understood.MethodsWe performed single-cell RNA sequencing (scRNA-seq) on ovarian, ovotestis, and testicular tissues to construct a gonadal cell atlas. RNA fluorescence in situ hybridization (RNA-FISH) validated cell subpopulations.ResultsCell types of germ cells and somatic cells in gonads were identified, and their differentiation trajectories during sex reversal were depicted. Our results show that GSCswdr17+tep1− in the ovaries possess bipotential differentiation capacity and can transform into GSCswdr17+ tep1+ with the capability to differentiating into sperm during sex reversal. Two theca cell subpopulations cooperate to synthesize steroid precursors in ovaries, with Leydig cells in testes likely originating from theca cell transitions. Proliferative gdf9+ follicle cells promoted folliculogenesis and persisted in male gonads with reduced hsd17b1 expression. Stromal heterogen-eity analysis revealed bmp4+ mesenchymal stem cells (MSCsbmp4+) as potential precursors for Sertoli cells in testes.ConclusionsThis study provides a comprehensive cellular roadmap of gonadal cell dynamics during sex reversal in Monopterus albus. Our findings unveil the molecular mechanisms underlying germline and somatic cell fate determination, offering novel insights into vertebrate sex reversal and potential therapeutic strategies for disorders of sex development (DSD).Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04810-8.

Somatic cell nuclear transfer (SCNT) is a powerful technique, although challenging, to study reprograming into the totipotent state of differentiated nuclei in mammals. This procedure was initially applied in farm animals, then rodents, and more recently in primates. Nuclear transfer of embryonic stem cells is known to be more efficient, but many types of somatic cells have now been successfully reprogramed with this procedure. Moreover, SCNT reprograming is more effective on a per cell basis than induced Pluripotent Stem Cells (iPSC) and provides interesting clues regarding the underlying processes. In this chapter, we describe the protocol of nuclear transfer in mouse that combines cell cycle synchronization of the donor cells, enucleation of metaphase II oocyte and Piezo-driven injection of a donor cell nucleus followed by activation of the reconstructed embryos and nonsurgical transfer into pseudo-pregnant mice. Moreover, this protocol includes two facultative steps to erase the epigenetic "memory" of the donor cells and improve chromatin remodeling by histones modifications targeting.

Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.

Development of bovine embryos in chemically defined medium at different levels of polyvinyl alcohol

Chapter 22 - Induced Pluripotent Stem Cells as Human Disease Models

Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

Embryonic stem cells (ESCs) first derived from the inner cell mass of blastocyst-stage embryos have the unique capacity of indefinite self-renewal and potential to differentiate into all somatic cell types. Similar developmental potency can be achieved by reprogramming differentiated somatic cells into induced pluripotent stem cells (iPSCs). Both types of pluripotent stem cells provide great potential for fundamental studies of tissue differentiation, and hold promise for disease modeling, drug development, and regenerative medicine. Although much has been learned about the molecular mechanisms that underlie pluripotency in such cells, our understanding remains incomplete. A comprehensive understanding of ESCs and iPSCs requires the deconstruction of complex transcription regulatory networks, epigenetic mechanisms, and biochemical interactions critical for the maintenance of self-renewal and pluripotency. In this review, we will discuss recent advances gleaned from application of global "omics" techniques to dissect the molecular mechanisms that define the pluripotent state.