Germ Layer Derivatives Research Articles

Ferroptosis: The functions of Nrf2 in human embryonic stem cells

Human embryonic stem cells (hESCs) have the capacity of self-renewal as well as differentiation towards three germ layer derivatives which makes them as a source of therapeutic application. hESCs are tremendously prone to cell death after dissociation into single cells. Therefore, it technically hinders their applications. Our recent study has revealed that hESCs can be prone to ferroptosis which differs from those in earlier explorations reporting that cellular detachment results in a process cited as anoikis. Ferroptosis occurs via increasing intracellular iron. Therefore, this form of programmed cell death is distinct from other cell deaths in terms of biochemistry, morphology, and genetics. Ferroptosis is found by excessive iron which plays an important part role in reactive oxygen species (ROS) generation through the Fenton reaction as a cofactor. Many genes are related to ferroptosis under the control of nuclear factor erythroid 2-related factor 2 (Nrf2) which is a transcription factor regulating the expression of genes to protect cells from oxidative stress. Nrf2 was demonstrated to take a perilous role in the suppression of ferroptosis by regulating the iron, antioxidant defense enzymes, usage, and restoration of glutathione, thioredoxin, and NADPH. Mitochondrial function is another target of Nrf2 to control cell homeostasis through the modulation of ROS production. In this review, we will give a succinct overview of lipid peroxidation and discuss the major players in the ferroptotic cascade. Additionally, we discussed the important role of the Nrf2 signaling pathway in mediating lipid peroxidation and ferroptosis, with a focus on known Nrf2 target genes that inhibit these processes and their possible role in hESCs.

Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

Generation of three human induced pluripotent stem cell lines, LUMCi024-A, LUMCi025-A, and LUMCi026-A, from two patients with combined oxidative phosphorylation deficiency 8 and a related control

Combined Oxidative Phosphorylation Deficiency 8 (COXPD8) is an autosomal recessive disorder causing lethal childhood-onset hypertrophic cardiomyopathy. Homozygous or compound heterozygous mutations in the nuclear-encoded mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene underly the pathology. We generated induced pluripotent stem cells (hiPSCs) from two patients carrying the heterozygous compound c.1774 C>T, c.2188 G>A and c.2872 C>T AARS2 mutations, as well as a related healthy control carrying the c.2872 C>T AARS2 mutation. All hiPSC-lines expressed pluripotency markers, maintained a normal karyotype, and differentiated towards the three germ layer derivatives in vitro. These lines can be used to model COXPD8 or mitochondrial dysfunction.

Generation of Pluripotent Stem Cells Using Somatic Cell Nuclear Transfer and Induced Pluripotent Somatic Cells from African Green Monkeys.

Patient-specific stem cells derived from somatic cell nuclear transfer (SCNT) embryos or from induced pluripotent stem cells (iPSCs) could be used to treat various diseases with minimal immune rejection. Many studies using these cells have been conducted in rats and mice; however, there exist numerous dissimilarities between the rodents and humans limiting the clinical predictive power and experimental utility of rodent experiments alone. Nonhuman primates (NHPs) share greater homology to human than rodents in all respects, including genomics, physiology, biochemistry, and the immune system. Thus, experimental data obtained from monkey studies would be more predictive for designing an effective cell replacement therapy in humans. Unfortunately, there are few iPSC lines and even fewer SCNT lines that have been derived in NHPs, hampering broader studies in regenerative medicine. One promising potential therapy would be the replacement of dopamine neurons that are lost in Parkinson's disease. After dopamine depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the African green monkey (Chlorocebus sabaeus) shows the most complete model of Parkinsonism compared with other species and brain pathology and behavioral changes are almost identical to those in humans after accidental exposure to MPTP. Therefore, we have developed a SCNT procedure to generate multiple pluripotent stem cell lines in this species for studies of possible treatment of Parkinsonism and for comparing with cells derived from iPSCs. Using 24 female monkeys as egg donors and 7 somatic cell donor monkeys, we have derived 11 SCNT embryonic stem cell lines that expressed typical stemness genes and formed all three germ layer derivatives. We also derived two iPSC lines using an episome-mediated reprogramming factor delivery system. This report describes the process for deriving these cell lines and proving their pluripotency for differentiation into various potentially therapeutic cells.

Origins of Intraindividual Genetic Variation in Human Fetuses

BackgroundIntraindividual copy number variation (CNV) origin is largely unknown. They might be due to aging and/or common genome instability at the preimplantation stage while contribution of preimplantation in human intraindividual CNVs occurrence is unknown. To address this question, we investigated mosaicism and its origin in the fetuses of natural conception.MethodsWe studied normal fetuses following therapeutic abortion due to maternal indications. We analyzed the genome of 22 tissues of each fetus by array comparative genomic hybridization for intraindividual CNVs. Each tissue was studied in 2 microarray experiments; the reciprocal aberrations larger than 40 Kb, identified by comparing tissues of each fetus, were subsequently validated using quantitative polymerase chain reaction.ResultsThrough intraindividual comparison, frequency of reciprocal events varied from 2 to 9. According to the distribution pattern of the frequent CNV in derivatives of different germ layers, we found that its origin is early development including preimplantation, whereas CNVs with low frequency have occurred in later stages. Shared CNVs in both fetuses were belonged to thymus and related to the functional role of genes located in these CNVs.ConclusionsThe origin of some of fetal CNVs is preimplantation stage. Each organ might inherit CNVs with an unpredictable pattern due to the extensive cell mixing/migration in embryonic development.

Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation

BackgroundTo maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes.MethodsElectrospun nanofibrous substrates with different reduced Young’s modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively.ResultsThe results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner.ConclusionsLineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications.

Principles for the regulation of multiple developmental pathways by a versatile transcriptional factor, BLIMP1.

Single transcription factors (TFs) regulate multiple developmental pathways, but the underlying mechanisms remain unclear. Here, we quantitatively characterized the genome-wide occupancy profiles of BLIMP1, a key transcriptional regulator for diverse developmental processes, during the development of three germ-layer derivatives (photoreceptor precursors, embryonic intestinal epithelium and plasmablasts) and the germ cell lineage (primordial germ cells). We identified BLIMP1-binding sites shared among multiple developmental processes, and such sites were highly occupied by BLIMP1 with a stringent recognition motif and were located predominantly in promoter proximities. A subset of bindings common to all the lineages exhibited a new, strong recognition sequence, a GGGAAA repeat. Paradoxically, however, the shared/common bindings had only a slight impact on the associated gene expression. In contrast, BLIMP1 occupied more distal sites in a cell type-specific manner; despite lower occupancy and flexible sequence recognitions, such bindings contributed effectively to the repression of the associated genes. Recognition motifs of other key TFs in BLIMP1-binding sites had little impact on the expression-level changes. These findings suggest that the shared/common sites might serve as potential reservoirs of BLIMP1 that functions at the specific sites, providing the foundation for a unified understanding of the genome regulation by BLIMP1, and, possibly, TFs in general.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

Characterization of embryonic stem cell model of polycystic ovary syndrome.

The aims of this study are to establish an embryonic stem (ES) cell model of polycystic ovary syndrome and to characterize this ES cell line. ES cells were isolated and cultured from 322 wasted fertilized embryos from polycystic ovary syndrome (PCOS) patients in vitro. They were also characterized by development and differential markers. ES cells from PCOS subject present normal development profile with ES-specific markers such as OCT-4 and SSEA-4. These ES cells can also differentiate into three germ layer derivatives and form teratomas in vivo. ES cells from PCOS patients pose development and differentiation potentials as you would expect of cells from non-PCOS patients; therefore, they can be used as a cellular model to study the pathology of PCOS.

Human Stem Cells Can Differentiate in Post-implantation Mouse Embryos

The potency of human pluripotent stem cells (hPSCs) to differentiate into germ layer derivatives is conventionally assessed by teratoma induction and in vitro differentiation. In this issue of Cell Stem Cell, Mascetti and Pedersen (2016) demonstrate that the human-mouse post-implantation chimera offers an efficient avenue to test the germ layer differentiation potential of hPSCs in mouse embryos ex vivo.

A comparative analysis of mesenchymal stem-cell lines derived from bone marrow and limb muscle of early human embryos

The properties of mesenchymal stem-cell (MSC) lines derived from bone marrow (FetMSC) and limb muscle (M-FetMSC) of 5- to 6-week human embryos were assayed. The main cell-line characteristics were obtained at the sixth passage. The average cell-population doubling time was 33.0 ± 1.4 h for FetMSC and 25.0 ± 0.1 h for M-FetMSC. Growth curves indicated active cell proliferation. Numerical and structural karyotypic analysis showed that these lines have normal karyotype, 46, XY. Cell surface markers were analyzed by flow cytometry. It was found that the cells express CD44, CD73, CD90, CD105, and HLA-ABC surface antigens and vimentin, which are common for human MSC beings, and lack CD34 and HLA-DR. The cells were capable of osteogenic, chondrogenic, and adipogenic differentiation. Immunofluorescence and flow-cytometry assays reveled a lack of surface antigen TRA-1-60, high expression of surface antigen SSEA-4, and low expression of transcription factor Oct-4 attributed to human embryonic stem cells. Immunofluorescence analysis showed the presence of early differentiation markers, three germ-layer derivatives for human embryonic stem cells. This makes MSCs useful for repair of damaged tissue in the corresponding microenvironment. Despite their shared MSC status, the FetMSC and M-FetMSC lines displayed some interlinear differences related to growth characteristics and differentiation potential. The MFetMSC line exhibited reduced potential for adipogenic differentiation compared to the FetMSC line. Immunofluorescence analysis revealed Z-disks in M-FetMSC, but not in FetMSC, during skeletal-muscle differentiation. These findings suggest that the different microenvironment has an influence when cells are in an organism before their transplantation in vitro.

Induced Pluripotent Stem Cells from Human Placental Chorion for Perinatal Tissue Engineering Applications

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly, we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4, SOX2, KLF4, and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology, immunocytochemistry, and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives, including neural stem cells, beating cardiomyocytes, and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications.

Novel animal pole‐enriched maternal mRNAs are preferentially expressed in neural ectoderm

Many animals utilize maternal mRNAs to pre-pattern the embryo before the onset of zygotic transcription. In Xenopus laevis, vegetal factors specify the germ line, endoderm, and dorsal axis, but there are few studies demonstrating roles for animal-enriched maternal mRNAs. Therefore, we carried out a microarray analysis to identify novel maternal transcripts enriched in 8-cell-stage animal blastomeres. We identified 39 mRNAs isolated from 8-cell animal blastomeres that are >4-fold enriched compared to vegetal pole mRNAs. We characterized 14 of these that are of unknown function. We validated the microarray results for 8/14 genes by qRT-PCR and for 14/14 genes by in situ hybridization assays. Because no developmental functions are reported yet, we provide the expression patterns for each of the 14 genes. Each is expressed in the animal hemisphere of unfertilized eggs, 8-cell animal blastomeres, and diffusely in blastula animal cap ectoderm, gastrula ectoderm and neural ectoderm, neural crest (and derivatives) and cranial placodes (and derivatives). They have varying later expression in some mesodermal and endodermal tissues in tail bud through larval stages. Novel animal-enriched maternal mRNAs are preferentially expressed in ectodermal derivatives, particularly neural ectoderm. However, they are later expressed in derivatives of other germ layers.

The Transcriptional and Functional Properties of Mouse Epiblast Stem Cells Resemble the Anterior Primitive Streak

Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.

Mouse Tenm4 is required for mesoderm induction

BackgroundTenm4 is a mouse homolog of the Drosophila gene Tenascin-m (Ten-m (Odd oz)), which functions in motor neuron routing. Recently, a genome-wide association analysis for bipolar disorder identified a new susceptibility locus at TENM4 increasing the importance of understanding Tenm4. A series of Tenm4 mouse alleles showing a broad range of phenotypes were isolated after ENU mutagenesis. Here, we examine the timing and features of gastrulation failure in a loss of function allele.ResultsEmbryonic mesoderm did not form in loss of function Tenm4m1/m1 mutant embryos. Genes normally expressed in embryonic mesoderm were not expressed in the mutant, the primitive streak did not form, and markers of the anteroposterior axis were not expressed or were mislocalized. The lack of embryonic mesoderm could not be attributed to poor proliferation of the epiblast, as normal numbers of dividing cells were observed. Epiblast cells maintained expression of Pou5f1 suggesting that they remain pluripotent, but they did not have the capacity to form any germ layer derivatives in teratomas, showing that the inability to induce mesoderm is cell autonomous. Misexpression of E-cadherin and N-cadherin suggest that the embryos did not undergo an epithelial-to-mesenchymal transition. In addition, Wnt signaling did not occur in the mutants, as assessed by the TOPGAL reporter assay, while a GSK3β inhibitor partially rescued the mutant embryos, and rescued TOPGAL reporter expression.ConclusionsThese data demonstrate that Tenm4 mutants fail to form a primitive streak and to induce embryonic mesoderm. Markers of anterior posterior patterning fail to be expressed or are mislocalized. Further, Tenm4 mutants lack the ability to differentiate in a cell autonomous manner. Together, our data suggest that embryos become impaired prior to E6.5 and as a result, Wnt signaling fails to occur; however, the involvement of other signaling pathways remains to be examined.

Basal Expression of Pluripotency-Associated Genes Can Contribute to Stemness Property and Differentiation Potential

Pluripotency and stemness is believed to be associated with high Oct-3/4, Nanog, and Sox-2 (ONS) expression. Similar to embryonic stem cells (ESCs), high ONS expression eventually became the measure of pluripotency in any cell. The threshold expression of ONS genes that underscores pluripotency, stemness, and differentiation potential is still unclear. Therefore, we raised a question as to whether pluripotency and stemness is a function of basal ONS gene expression. To prove this, we carried out a comparative study between basal ONS expressing NIH3T3 cells with pluripotent mouse bone marrow mesenchymal stem cells (mBMSC) and mouse ESC. Our studies on cellular, molecular, and immunological biomarkers between NIH3T3 and mBMSC demonstrated stemness property of undifferentiated NIH3T3 cells that was similar to mBMSC and somewhat close to ESC as well. In vivo teratoma formation with all three germ layer derivatives strengthen the fact that these cells in spite of basal ONS gene expression can differentiate into cells of multiple lineages without any genetic modification. Conclusively, our novel findings suggested that the phenomenon of pluripotency which imparts ability for multilineage cell differentiation is not necessarily a function of high ONS gene expression.

Adult somatic stem cells in the human parasite Schistosoma mansoni

SummarySchistosomiasis is among the most prevalent human parasitic diseases, affecting more than 200 million people worldwide1. The etiological agents of this disease are trematode flatworms (Schistosoma) that live and lay eggs within the vasculature of the host. These eggs lodge in host tissues, causing inflammatory responses that are the primary cause of morbidity. Because these parasites can live and reproduce within human hosts for decades2, elucidating the mechanisms that promote their longevity is of fundamental importance. Although adult pluripotent stem cells, called neoblasts, drive long-term homeostatic tissue maintenance in long-lived free-living flatworms3,4 (e.g., planarians), and neoblast-like cells have been described in some parasitic tapeworms5, little is known about whether similar cell types exist in any trematode species. Here, we describe a population of neoblast-like cells in the trematode Schistosoma mansoni. These cells resemble planarian neoblasts morphologically and share their ability to proliferate and differentiate into derivatives of multiple germ layers. Capitalizing on available genomic resources6,7 and RNAseq-based gene expression profiling, we find that these schistosome neoblast-like cells express a fibroblast growth factor receptor ortholog. Using RNA interference we demonstrate that this gene is required for the maintenance of these neoblast-like cells. Our observations suggest that adaptation of developmental strategies shared by free-living ancestors to modern-day schistosomes likely contributed to the success of these animals as long-lived obligate parasites. We expect that future studies deciphering the function of these neoblast-like cells will have important implications for understanding the biology of these devastating parasites.

The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar toESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon invitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

Rejuvenation through stem cells

The discovery that schistosomes possess adult stem cells could explain the long-term persistence of these parasitic worms in humans. Targeting a protein produced by the cells might damage schistosome defences. See Letter p.476 Adult stem cells (or neoblasts) are found in free-living planarians and parasitic tapeworms and can support impressive feats of tissue regeneration. Phillip Newmark and colleagues now report the identification of adult stem cells in the human parasite Schistosoma mansoni. This trematode flatworm, also known as the blood fluke, infects millions of people worldwide. The schistosomal stem cells proliferate and differentiate into derivatives of multiple germ layers, and express a fibroblast growth factor receptor orthologue. Using RNA interference, the authors showed that this gene is required for the maintenance of the neoblast-like cells. These findings might help to elucidate the mechanisms that promote the parasite's longevity and so could be relevant for medical treatment.