Pluripotency Marker Genes Research Articles

Digital PCR-Based Gene Expression Analysis Using a Highly Multiplexed Assay with Universal Detection Probes to Study Induced Pluripotent Stem Cell Differentiation into Cranial Neural Crest Cells.

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type, offering a valuable tool for research in developmental biology, regenerative medicine, and disease modeling. In this study, iPSCs were differentiated into cranial neural crest cells (CNCCs) over a 14-day period. RNA was extracted from these cells at day 0 (iPSCs), day 7, and day 14 to evaluate successful differentiation through the expression analysis of pluripotency and CNCC marker genes.A key focus was the conversion of existing qPCR assays into multiplexed RT-dPCR assays utilizing universal detection probes for precise gene expression analysis during the differentiation of induced pluripotent stem cells (iPSCs) into cranial neural crest cells (CNCCs). We aimed to leverage the superior precision, sensitivity, and multiplexing-degree of dPCR, particularly in quantifying low-abundance targets. We conducted a comparative analysis of the temporal expression patterns of crucial marker genes using both qPCR and dPCR.Our experiments revealed that the four five-plex dPCR assays could successfully detect and quantify the pluripotency and CNCC marker genes and evaluate CNCC differentiation. We observed the expected downregulation of pluripotency genes during differentiation. Conversely, the upregulation of CNCC markers validates the successful differentiation process. In conclusion, SYBR Green I gene expression qPCR assays can be readily converted into multiplex dPCR assays using universal detection probes.Overall, this work underscores the potential of dPCR as a valuable tool for molecular profiling in stem cell research, offering robust, precise, and efficient gene expression analysis. The findings suggest that while qPCR remains a reliable method for routine applications, dPCR provides particular advantages for high-precision, low-sample input studies, expanding the analytical toolbox for stem cell differentiation and gene expression research.

Analysis of the pluripotent and germline marker gene expression, and the state of X chromosome reactivation of primordial germ cells in pig gonads

The gonadal primordial germ cells (PGCs) possess a unique state of pluripotency and X chromosome activity. However, extensive evidence indicates developmental variability in PGCs across different species. This study aims to evaluate the pluripotency status, specific gene expression patterns, and X chromosome reactivation (XCR) of pig gonadal PGCs. Single-cell RNA-seq revealed significant heterogeneity within the population of gonadal PGCs. Notably, these PGCs expressed high levels of pluripotency markers OCT4, PRDM14, and NANOG, while lacking SOX2 expression. Through the screening of marker genes and subsequent protein expression validation, we identified growth differentiation factor 3 (GDF3) as a specific surface marker for pig gonadal PGCs, facilitating their efficient purification for further study. Furthermore, analysis of gonadal PGCs demonstrated complete XCR. This was evidenced by the absence of repressive histone modifications (H3K27me3, H3K9me3, and H2AK119ub), the lack of X inactive specific transcript (XIST) RNA FISH signal, and the doubled expression of X-linked genes. Additionally, these PGCs expressed high levels of genes associated with epigenetic modification, chromatin remodeling, and XIST-associated RNA-binding. These factors likely play a crucial role in regulating pluripotency and X chromosome activity. In summary, this study reveals the heterogeneity in pig gonadal PGCs and identifies GDF3 as a specific surface marker. It also elucidates the expression patterns of pluripotency transcription factors and the events involved in XCR.

Generation of ID1/3 knockout human embryonic stem cell lines (WAe009-A-2A and WAe009-A-2B) derived from H9 using CRISPR/Cas9

ID1 and ID3 are the members of the Inhibitor of DNA Binding (ID) protein family, which negatively regulates the basic helix-loop-helix (bHLH) transcription factors by forming heterodimers, are involved in neurodevelopment, cardiovascular development, and tumor metastasis. We created twoID1/3knockout cell lines from a human embryonic stem cell (hESC) line (H9) by CRISPR/Cas9-mediated gene targeting. These cell lines maintain stem cell morphology, a normal karyotype, and the expression of pluripotent marker genes. Additionally, they retain their in vivo differentiation potential. Thecell lines are valuable tools for studying the roles of ID1/3 in neurodevelopment and cardiovascular diseases.

Induction of Pluripotent Stem Cells from Muscle Cells of Large Yellow Croaker (Larimichthys Crocea) Via Electrotransfection.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

BCL6B-dependent suppression of ETV2 hampers endothelial cell differentiation

BackgroundB-cell CLL/lymphoma 6 member B (BCL6B) operates as a sequence-specific transcriptional repressor within the nucleus, playing crucial roles in various biological functions, including tumor suppression, immune response, stem cell self-renew, and vascular angiogenesis. However, whether BCL6B is involved in endothelial cell (EC) development has remained largely unknown. ETS variant transcription factor 2 (ETV2) is well known to facilitate EC differentiation. This study aims to determine the important role of BCL6B in EC differentiation and its potential mechanisms.MethodsDoxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown were established and subjected to differentiate into ECs and vessel organoids (VOs). RNA sequencing analysis was performed to identify potential signal pathways regulated by BCL6B during EC differentiation from hiPSCs. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pluripotency and vascular-specific marker genes expression. EC differentiation efficiency was determined by Flow cytometry analysis. The performance of EC was evaluated by in vitro Tube formation assay. The protein expression and the vessel-like structures were assessed using immunofluorescence analysis or western blot. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP)-PCR analysis were used to determine the regulatory relationship between BCL6B and ETV2.ResultsFunctional ECs and VOs were successfully generated from hiPSCs. Notably, overexpression of BCL6B suppressed while knockdown of BCL6B improved EC differentiation from hiPSCs. Additionally, the overexpression of BCL6B attenuated the capacity of derived hiPSC-ECs to form a tubular structure. Furthermore, compared to the control VOs, BCL6B overexpression repressed the growth of VOs, whereas BCL6B knockdown had little effect on the size of VOs. RNA sequencing analysis confirmed that our differentiation protocol induced landscape changes for cell/tissue/system developmental process, particularly vascular development and tube morphogenesis, which were significantly modulated by BCL6B. Subsequent experiments confirmed the inhibitory effect of BCL6B is facilitated by the binding of BCL6B to the promoter region of ETV2, led to the suppression of ETV2's transcriptional activity. Importantly, the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression.ConclusionsBCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.

Comparative study of PGCs cultivation systems HiS and FAcs: a transcriptomic and cellular biology perspective

In chicken, primordial germ cells (PGC) are crucial for the preservation and manipulation of genetic resources in poultry production. The HiS and FAcs culture systems are two important methods for the in vitro cultivation of chicken PGCs. The purpose of this study was to compare and analyze the two cultivation systems for PGCs (His and FAcs culture systems) to assess their efficacy and applicability in supporting PGC growth, maintaining PGC characteristics, and lineage transmission ability. The study found that both HiS and FAcs culture systems could maintain the basic biological characteristics of chicken PGCs, including the simultaneous expression of pluripotency and reproductive marker genes, as well as the presence of abundant glycogen granules. Subsequently, we identified 2,145 differentially expressed genes (DEG) through RNA sequencing. GO and KEGG analysis revealed a large number of DEGs enriched in the cell adhesion and calcium ion binding pathways, and the analysis found that these genes maintained a higher level in HiS-PGCs. Further personalized analysis found that the regulatory genes for maintaining PGC pluripotency were highly expressed in HiS-PGCs, while germ cell-related genes showed similar expression in both systems. Additionally, through RNA sequencing data and cell proliferation ability, it was found that PGCs in the FAcs system had a higher proliferation rate and a faster cell cycle. Finally, it was discovered that the expression of cell migration-related genes was maintained at a higher level in HiS-PGCs, but the migration efficiency of HiS-PGCs did not show a significant difference compared to FAcs-PGCs. These results suggest that both HiS and FAcs culture systems can maintain the proliferation and basic characteristics of chicken PGCs, but differences exist in cell proliferation, pluripotency regulation, and cell adhesion. These findings provide new information for optimizing PGC cultivation systems and are important for the preservation and genetic improvement of chicken PGCs.

Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Generation of FOXJ1-EGFP knock-in reporter human embryonic stem cell line, WAe001-A-2D, using CRISPR/Cas9-based gene targeting

Forkhead box protein J1 (FOXJ1), a member of the forkhead family, is an important transcription factor regulating multiciliated cell differentiation and motile ciliogenic program. Here, we established a FOXJ1- EGFP knock-in human embryonic stem cell (hESC) line by inserting a P2A-EGFP gene cassette of FOXJ1 using CRISPR/Cas9 system. The reporter cell line retained a normal karyotype, expressed comparable pluripotent marker genes, and maintained differentiation potential. This reporter cell line enables live identification of multiciliated cells during the general lung differentiation and will be a valuable tool for studying the multiciliated cell differentiation, ciliogenesis and mechanism of related pulmonary diseases.

DNMT1 Y495C mutation interferes with maintenance methylation of imprinting control regions

Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) is a rare autosomal dominant neurological disorder due to missense mutations in DNA methyltransferase 1 (DNMT1). To investigate the nature of the dominant effect, we compared methylomes of transgenic R1wtDnmt1 and R1Dnmt1Y495C mouse embryonic stem cells (mESCs) overexpressing WT and the mutant mouse proteins respectively, with the R1 (wild-type) cells. In case of R1Dnmt1Y495C, 15 out of the 20 imprinting control regions were hypomethylated with transcript level dysregulation of multiple imprinted genes in ESCs and neurons. Non-imprinted regions, minor satellites, major satellites, LINE1 and IAP repeats were unaffected. These data mirror the specific imprinting defects associated with transient removal of DNMT1 in mESCs, deletion of the maternal-effect DNMT1o variant in preimplantation mouse embryos, and in part, reprogramming to naïve human iPSCs. This is the first DNMT1 mutation demonstrated to specifically affect Imprinting Control Regions (ICRs), and reinforces the differences in maintenance methylation of ICRs over non-imprinted regions. Consistent with nervous system abnormalities in the HSAN1E disorder and involvement of imprinted genes in normal development and neurogenesis, R1Dnmt1Y495C cells showed dysregulated pluripotency and neuron marker genes, and yielded more slender, shorter, and extensively branched neurons. We speculate that R1Dnmt1Y495C cells produce predominantly dimers containing mutant proteins, leading to a gradual and specific loss of ICR methylation during early human development.

Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.

Derivation of embryonic stem cells from wild-derived mouse strains by nuclear transfer using peripheral blood cells

Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging to Mus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.

Generation of CD16A gene knockout human embryonic stem cell line using CRISPR/Cas9

CD16A is a receptor for the Fc portion of immunoglobulin G, and is involved in the antibody dependent cellular cytotoxicity (ADCC) of nature killer cells(Zhu et al., 2020) and antibody dependent enhancement (ADE) of virus infections(Wan et al., 2020). However, the role of CD16A in human embryonic stem cell modeled development has been merely documented. Hence, to illustrate the role of CD16A in the human cell development, we reported a CD16A knockout human embryonic stem(hESC) cell line via CRISPR/Cas9 mediated gene knockout. The CD16A mutated cell line displayed normal karyotype, pluripotent stem cell marker gene expression and differentiation potential.

Identifying Transient Cells During Reprogramming via Persistent Homology.

Single-cell RNA sequencing is a powerful method that helps delineate the regulatory mechanisms shaping the diverse cellular populations. Heterogeneous cell populations consist of individual cells, and the expression of distinct sets of genes can differentiate one sub-population of cells from another, as they are responsible for the emergence of distinct cellular phenotypes. Of particular importance are cells at transition states that bridge these different cellular phenotypes. In this study, we develop a method to identify the cells at transition states bridging different cellular phenotypes. Our approach is based on persistent homology, which enabled us to identify the group of cells located on the boundaries between different sub-populations of cells. We applied this method to study the reprogramming of human fibroblasts toward induced pluripotent stem cells using single-cell time-course data. Even though only the data that is representative of the early stages of the reprogramming process are analyzed, we are able to uncover transient cells bridging different cell sub-populations. The most prominent group of transient cells are found to be enriched for NANOG, which is a known stem cell transcription factor that takes part in the maintenance of pluripotency and other stem cell marker genes. Overall, our method can identify cells in transient states bridging major cellular phenotypes, even though they are only a small fraction of the overall cell population. We also discuss how this approach can link the topology of the surface of cellular transcripts and bring order to the transition between cellular states and how it automatically uncovers the underlying time process.

Species-Specific Enhancer Activity of OCT4 in Porcine Pluripotency: The Porcine OCT4 Reporter System Could Monitor Pluripotency in Porcine Embryo Development and Embryonic Stem Cells.

The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Generation of a human induced pluripotent stem cell (iPSC) line from skin fibroblasts of a patient carrying an E363Q mutation in PSEN1 gene

Alzheimer’s disease (AD) is a common neurodegenerative disease characterized by cognitive decline even leading to incapacity, which prevalence estimates that about 5% of AD cases are caused by mutations in genes such as Presenilin-1 (PSEN1). Here we report the generation and characterization of an iPSC line derived from a patient carrying an E363Q mutation in PSEN1 gene. The iPSC line we generated presented a typical morphology, normal karyotype, free from Sendai viral vectors and exogenous factors, expressed endogenous pluripotency marker genes and proteins, which could form embryoid bodies in vitro and form teratoma in vivo as well, demonstrating its pluripotency.

Defining the Pluripotent Marker Genes for Identification of Teleost Fish Cell Pluripotency During Reprogramming.

Pluripotency is a transient state in early embryos, which is regulated by an interconnected network of pluripotency-related genes. The pluripotent state itself seems to be highly dynamic, which leads to significant differences in the description of induced pluripotent stem cells from different species at the molecular level. With the application of cell reprogramming technology in fish, the establishment of a set of molecular standards for defining pluripotency will be important for the research and potential application of induced pluripotent stem cells in fish. In this study, by BLAST search and expression pattern analysis, we screen out four pluripotent genes (Oct4, Nanog, Tdgf1, and Gdf3) in zebrafish (Danio rerio) and crucian carp (Carassius). These genes were highly expressed in the short period of early embryonic development, but significantly down-regulated after differentiation. Moreover, three genes (Oct4, Nanog and Tdgf1) have been verified that are suitable for identifying the pluripotency of induced pluripotent stem cells in zebrafish and crucian carp. Our study expands the understanding of the pluripotent markers of induced pluripotent stem cells in fish.

Fish Pluripotent Stem-Like Cell Line Induced by Small-Molecule Compounds From Caudal Fin and its Developmental Potentiality.

The technique of induced pluripotent stem cells has significant application value in breeding and preserving the genetic integrity of fish species. However, it is still unclear whether the chemically induced pluripotent stem cells can be induced from non-mammalian cells or not. In this article, we first verify that fibroblasts of fish can be chemically reprogrammed into pluripotent stem cells. These induced pluripotent stem-like cells possess features of colony morphology, expression of pluripotent marker genes, formation of embryoid bodies, teratoma formation, and the potential to differentiate into germ cell-like cells in vitro. Our findings will offer a new way to generate induced pluripotent stem cells in teleost fish and a unique opportunity to breed commercial fish and even save endangered fish species.

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Derivation of Ringed Seal (Phoca hispida) Induced Multipotent Stem Cells.

Induced pluripotent stem (iPS) cells have been produced just for a few species among order Carnivora: snow leopard, Bengal tiger, serval, jaguar, cat, dog, ferret, and American mink. We applied the iPS cell derivation protocol to the ringed seal (Phoca hispida) fibroblasts. The resulting cell line had the expression of pluripotency marker gene Rex1. Differentiation in embryoid body-like structures allowed us to register expression of AFP, endoderm marker, and Cdx2, trophectoderm marker, but not neuronal (ectoderm) markers. The cells readily differentiated into adipocytes and osteocytes, mesoderm cell types of origin. Transcriptome analysis allowed us to conclude that the cell line does not resemble human pluripotent cells, and, therefore, most probably is not pluripotent. Thus, we produced ringed seal multipotent stem cell line capable of differentiation into adipocytes and osteocytes.