Differentiation Of Somatic Cells Research Articles

Influence of Growth Characteristics of Induced Pluripotent Stem Cells on Their Uptake Efficiency for Layer-by-Layer Microcarriers.

Induced pluripotent stem cells (iPSCs) have the ability to differentiate into any specialized somatic cell type, which makes them an attractive tool for a wide variety of scientific approaches, including regenerative medicine. However, their pluripotent state and their growth in compact colonies render them difficult to access and, therefore, restrict delivery of specific agents for cell manipulation. Thus, our investigation focus was set on the evaluation of the capability of layer-by-layer (LbL) designed microcarriers to serve as a potential drug delivery system to iPSCs, as they offer several appealing advantages. Most notably, these carriers allow for the transport of active agents in a protected environment and for a rather specific delivery through surface modifications. As we could show, charge and mode of LbL carrier application as well as the size of the iPSC colonies determine the interaction with and the uptake rate by iPSCs. None of the examined conditions had an influence on iPSC colony properties such as colony morphology and size or maintenance of pluripotent properties. An overall interaction rate of LbL carriers with iPSCs of up to 20% was achieved. Those data emphasize the applicability of LbL carriers for stem cell research. Additionally, the potential use of LbL carriers as a promising delivery tool for iPSCs was contrasted to viral particles and liposomes. The identified differences among those delivery tools have substantiated our major conclusion that LbL carrier uptake rate is influenced by characteristic features of the iPSC colonies (most notably colony size) in addition to their surface charges.

Nr0b1 (DAX1) mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation

Sex determinations are diverse in vertebrates. Although many sex-determining genes and pathways are conserved, the mechanistic roles of these genes and pathways in the genetic sex determination are not well understood. DAX1 (encoded by the NR0B1 gene) is a vertebrate specific orphan nuclear receptor that regulates gonadal development and sexual determination. In human, duplication of the NR0B1 gene leads to male-to-female sex reversal. In mice, Nr0b1 shows both pro-testis and anti-testis functions. We generated inheritable nr0b1 mutation in the zebrafish and found the nr0b1 mutation caused homozygous mutants to develop as fertile males due to female-to-male sex reversal. The nr0b1 mutation did not increase Caspase-3 labeling nor tp53 expression in the developing gonads. Introduction of a tp53 mutation into the nr0b1 mutant did not rescue the sex-reversal phenotype. Further examination revealed reduction in cell proliferation and abnormal somatic cell differentiation in the nr0b1 mutant gonads at the undifferentiated and bi-potential ovary stages. Together, our results suggest nr0b1 regulates somatic cell differentiation and cell proliferation to ensure normal sex development in the zebrafish.

The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing.

In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed “progressively partial endoreplication” (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical “horseshoe” pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.

525. Directed Molecular Evolution of Transcription Factors in Mammalian Cells for Enhanced Directed Cell Differentiation

The advent of cellular reprogramming has introduced new possibilities for regenerative medicine, drug discovery, and controlling cellular behavior. Recent studies have identified many natural transcription factors that are capable of directing lineage-specific differentiation in adult somatic cells. However, genetic reprogramming is often inefficient and highly variable, making it difficult to obtain sufficient quantities of completely reprogrammed cells, and thus unrealistic as a reliable source for cellular therapies and regenerative medicine applications. Directed evolution presents a well-established and currently unexplored approach for improving the intrinsic properties of reprogramming transcription factors. MyoD is the master transcription factor that defines the myogenic lineage. Overexpression of MyoD in certain cell types upregulates the myogenic gene network and induces differentiation to a myogenic phenotype. This process is well characterized and therefore provides an ideal model for developing a directed evolution scheme for mammalian transcription factors. Here we present a mammalian-based directed evolution system that enhances transcription factor-mediated genetic reprogramming in mammalian cells. A quantitative and high-throughput selection system was engineered using a cell reporter assay to isolate enhanced MyoD variants from a library of mutated gene sequences. During skeletal myoblast differentiation, MyoD directly activates expression of the myogenin transcription factor. Myogenin is one of the first markers of muscle maturation and marks commitment to myocyte differentiation. It is also important for cell fusion into functional multinucleated myotubes. Therefore, we hypothesized that engineering MyoD variants with an enhanced ability to induce expression of myogenin would lead to more effective and efficient genetic reprogramming. For this purpose, we engineered an isogenic myogenin-eGFP reporter cell line. The library of MyoD mutants were packaged into a lentiviral vector and delivered to the reporter cell line for selection. Cells with high GFP expression are indicative of hyperactive mutants with an enhanced ability to activate the target promoter and were isolated using fluorescence activated cell sorting. The variants were recovered from the genomic DNA of these sorted cells by PCR. This new library of mutants was recloned into the lentiviral vector for subsequent rounds of selection. Following ten rounds of selection, the resulting libraries were evaluated for enrichment of amino acid substitutions through next generation sequencing. Variants of interests were isolated and tested individually for their ability to induce myogenic differentiation in human cells. In this study, we demonstrate that directed evolution can be used to identify single amino acid substitutions within the master transcription factor MyoD that improve the conversion of human fibroblasts to a skeletal myocyte phenotype. This study presents a generalizable approach for enhancing the activity of mammalian transcription factors.

Tissue-specific DNA demethylation is required for proper B-cell differentiation and function

There is ample evidence that somatic cell differentiation during development is accompanied by extensive DNA demethylation of specific sites that vary between cell types. Although the mechanism of this process has not yet been elucidated, it is likely to involve the conversion of 5mC to 5hmC by Tet enzymes. We show that a Tet2/Tet3 conditional knockout at early stages of B-cell development largely prevents lineage-specific programmed demethylation events. This lack of demethylation affects the expression of nearby B-cell lineage genes by impairing enhancer activity, thus causing defects in B-cell differentiation and function. Thus, tissue-specific DNA demethylation appears to be necessary for proper somatic cell development in vivo.

Epigenetic regulation of the histone-to-protamine transition during spermiogenesis

In mammals, male germ cells differentiate from haploid round spermatids to flagella-containing motile sperm in a process called spermiogenesis. This process is distinct from somatic cell differentiation in that the majority of the core histones are replaced sequentially, first by transition proteins and then by protamines, facilitating chromatin hyper-compaction. This histone-to-protamine transition process represents an excellent model for the investigation of how epigenetic regulators interact with each other to remodel chromatin architecture. Although early work in the field highlighted the critical roles of testis-specific transcription factors in controlling the haploid-specific developmental program, recent studies underscore the essential functions of epigenetic players involved in the dramatic genome remodeling that takes place during wholesale histone replacement. In this review, we discuss recent advances in our understanding of how epigenetic players, such as histone variants and histone writers/readers/erasers, rewire the haploid spermatid genome to facilitate histone substitution by protamines in mammals.

Argonaute and Argonaute-Bound Small RNAs in Stem Cells.

Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are indispensable in small RNA-mediated regulatory pathways. AGO proteins have been identified in various types of stem cells in diverse species from plants and animals. This review article highlights recent progress on how AGO proteins and AGO-bound small RNAs regulate the self-renewal and differentiation of distinct stem cell types, including pluripotent, germline, somatic, and cancer stem cells.

Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues.

Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on loss of access to these signals. Here, we demonstrate that the differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot differentiate properly. We find that an insulin peptide produced by somatic cells immediately outside of the stem cell niche acts locally to promote somatic differentiation through Insulin-like receptor (InR) activation. These results indicate that there is a local 'differentiation' niche that upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, we demonstrate that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, we show that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that the local production of positive and negative InR signals regulates the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation are actively induced by signaling.

Wt1 directs the lineage specification of sertoli and granulosa cells by repressing Sf1 expression.

Supporting cells (Sertoli and granulosa) and steroidogenic cells (Leydig and theca-interstitium) are two major somatic cell types in mammalian gonads, but the mechanisms that control their differentiation during gonad development remain elusive. In this study, we found that deletion of Wt1 in the ovary after sex determination caused ectopic development of steroidogenic cells at the embryonic stage. Furthermore, differentiation of both Sertoli and granulosa cells was blocked when Wt1 was deleted before sex determination and most genital ridge somatic cells differentiated into steroidogenic cells in both male and female gonads. Further studies revealed that WT1 repressed Sf1 expression by directly binding to the Sf1 promoter region, and the repressive function was completely abolished when WT1 binding sites were mutated. This study demonstrates that Wt1 is required for the lineage specification of both Sertoli and granulosa cells by repressing Sf1 expression. Without Wt1, the expression of Sf1 was upregulated and the somatic cells differentiated into steroidogenic cells instead of supporting cells. Our study uncovers a novel mechanism of somatic cell differentiation during gonad development.

From Stress to Embryos: Some of the Problems for Induction and Maturation of Somatic Embryos.

Although somatic embryogenesis has been successfully achieved in numerous plant species, little is known about the mechanism(s) underlying this process. Changes in the balance of growth regulators of the culture medium, osmolarity, or amino acids as well as the genotype and developmental stage of the tissue used as initial explant may have a pivotal influence on the induction of somatic embryogenic cultures. Moreover, different stress agents (ethylene, activated charcoal, cold or heat or electrical shocks), as well as abscisic acid, can also foster the induction or further development of somatic embryos. In the process, cells first return to a stem cell-like status and then either enter their new program or dye when the stress level exceeds cell tolerance. Recalcitrance to differentiation of somatic cells into embryos is frequently observed, and problems such as secondary or recurrent embryogenesis, embryo growth arrest (at the globular stage or during the transition from torpedo to cotyledonary stage), and development of only the aerial part of somatic embryos can appear, interfering with normal germination and conversion of embryos to plants. Some solutions to solve these problems associated to embryogenesis are proposed and two very efficient somatic embryogenesis protocols for two model plant species are detailed.

Non-coding RNA in Spermatogenesis and Epididymal Maturation.

Testicular germ and somatic cells express many classes of small ncRNAs, including Dicer-independent PIWI-interacting RNAs, Dicer-dependent miRNAs, and endogenous small interfering RNA. Several studies have identified ncRNAs that are highly, exclusively, or preferentially expressed in the testis and epididymis in specific germ and somatic cell types. Temporal and spatial expression of proteins is a key requirement of successful spermatogenesis and large-scale gene transcription occurs in two key stages, just prior to transcriptional quiescence in meiosis and then during spermiogenesis just prior to nuclear silencing in elongating spermatids. More than 60 % of these transcripts are then stockpiled for subsequent translation. In this capacity ncRNAs may act to interpret and transduce cellular signals to either maintain the undifferentiated stem cell population and/or drive cell differentiation during spermatogenesis and epididymal maturation. The assignation of specific roles to the majority of ncRNA species implicated as having a role in spermatogenesis and epididymal function will underpin fundamental understanding of normal and disease states in humans such as infertility and the development of germ cell tumours.

Precocious Differentiation of Somatic and Pluripotent Stem Cells Bearing Pre-Arranged TCR

Adoptive cell transfer (ACT) of mutation-specific T lymphocytes may represent the future of immunotherapy for cancer. Current strategies utilize mature T cells isolated from tumors, but this approach may be hampered by limited persistence, advanced differentiation status, and exhaustion of transferred cells. Cellular rejuvenation is needed, and this could be accomplished through induced pluripotent stem cell (iPS) technology. IPS cells can be generated from various types of somatic cells using the four Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), and we have shown that iPS generated from T cells (T-iPS) retain their original TCR specificity upon re-differentiation into T cell lineage. With existing technology, tumor antigen-specific progenitor cells can be derived from T-iPS. These cells could have superior potential to engraft, proliferate, and persist for long periods of time. To explore this concept, we have generated a murine iPS cell line from our Pmel transgenic mouse model, in which T cells bear a pre-rearranged TCR specific for the melanoma antigen GP100. Differentiation in vitro employs the OP9-DL1 co-culture system. We characterize the DN1-DN4 stages by flow cytometry of T cell lineage differentiation markers, such as c-kit, CD3, CD4, CD8, CD25, TCRvb, and TCRvb13 (present in the Pmel transgene).To study somatic stem cell differentiation, we used lineage negative bone marrow from femurs of wild type C57BL/6 (WT) and Pmel TCR transgenic (Pmel) mice. After 10 days of culture, cells were harvested and analyzed. Frequencies of cells in the DN1-DN4 stages for WT were 2%, 41%, 55%, and 2%; for Pmel they were 5%, 31%, 39%, and 25%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 2%. In contrast, CD3 expression in Pmel cells was 85% for cells in DN1/DN2 and 95% for cells in DN3/DN4.To study pluripotent stem cell differentiation, we used WT embryonic stem cells (ES) and Pmel iPS. After 21 days of culture, cells were harvested and analyzed. The double positive compartment was 9% in WT ES and 30% in Pmel iPS; the double negative compartment was 60% and 37%. Frequencies of cells in DN1-DN4 stages for WT ES were 4%, 6%, 45%, and 45%; for Pmel they were 1%, 7%, 69%, and 23%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 13%. In contrast, CD3 expression in Pmel cells was 76% for cells in DN1/DN2 and 91% for cells in DN3/DN4.These data show for the first time that in vitro differentiation of pluripotent stem-derived progenitor cells bearing a pre-rearranged TCR will undergo precocious expression of the CD3 molecule and enhanced progression to DP stage as compared to WT controls. The pattern is consistent with that previously seen during in vitro differentiation of somatic stem cells. It appears that the presence of a pre-rearranged TCR is driving precocious development of T cells in vitro, regardless of whether the source of progenitors is from somatic (bone marrow) or pluripotent (ES/iPS) stem cells. Our ultimate goal is to intricately characterize the nature of iPS-derived T cells, and study their antitumor efficiency in vitro and in vivo, in order to evaluate their suitability for clinical use. DisclosuresNo relevant conflicts of interest to declare.

Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments.

BackgroundPrevious studies of higher order chromatin organization in nuclei of mammalian species revealed both structural consistency and species-specific differences between cell lines and during early embryonic development. Here, we extended our studies to nuclear landscapes in the human myelopoietic lineage representing a somatic cell differentiation system. Our longterm goal is a search for structural features of nuclei, which are restricted to certain cell types/species, as compared to features, which are evolutionary highly conserved, arguing for their basic functional roles in nuclear organization.ResultsCommon human hematopoietic progenitors, myeloid precursor cells, differentiated monocytes and granulocytes analyzed by super-resolution fluorescence microscopy and electron microscopy revealed profound differences with respect to global chromatin arrangements, the nuclear space occupied by the interchromatin compartment and the distribution of nuclear pores. In contrast, we noted a consistent organization in all cell types with regard to two co-aligned networks, an active (ANC) and an inactive (INC) nuclear compartment delineated by functionally relevant hallmarks. The ANC is enriched in active RNA polymerase II, splicing speckles and histone signatures for transcriptionally competent chromatin (H3K4me3), whereas the INC carries marks for repressed chromatin (H3K9me3).ConclusionsOur findings substantiate the conservation of the recently published ANC-INC network model of mammalian nuclear organization during human myelopoiesis irrespective of profound changes of the global nuclear architecture observed during this differentiation process. According to this model, two spatially co-aligned and functionally interacting active and inactive nuclear compartments (ANC and INC) pervade the nuclear space.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0038-0) contains supplementary material, which is available to authorized users.

Relationship of Oct-4 to malignant stage: a meta-analysis based on 502 positive/high Oct-4 cases and 522 negative/low case-free controls.

Octamer 4 (Oct-4), an important member of the POU domain transcription factor family, has been suggested to function as a master switch during differentiation of human somatic cells and more recently has come to be linked with neoplastic properties. The aim of this study was to evaluate the relationship between Oct-4 and cancer stage using a meta-analysis approach. Relevant articles published as of May 2015 were retrieved from the following databases: PubMed, ISI Web of Knowledge, Embase, and Chinese National Knowledge Infrastructure (CNKI). The strengths of relationship for outcomes of interest were estimated based on odds ratios (ORs) and 95% confidence intervals (CIs). A total of 11 articles on Oct-4 and cancer staging that collectively included 502 positive/high Oct-4 cases and 522 negative/low case-free controls were chosen. Positive/high Oct-4 was significantly associated with cancer stage in several kinds of cancer. Specifically, positive/high Oct-4 was associated with cancer stage III/IV (fixed effects: OR = 1.53, 95% CI = 1.12-2.10), primary tumor (T3-4) (random effects: OR = 1.93, 95% CI = 0.99-3.77), and cancer grade of differentiation (intermediate-poor) (random effects: OR = 3.45, 95% CI = 1.5-7.61). These findings suggest that positive/high Oct-4 is more strongly linked to stage III/IV cancer and cancer grade of differentiation, and is correlated with malignant characteristics that lead to poor prognosis in different types of cancer, especially in Asian. Given variability related to ethnicity and differences in cancer types, additional studies are warranted to establish the generalizability of our findings.

Identification of the specific epigenetic alterations associated with chemo-resistance via reprogramming of cancer cells

BackgroundChemo-resistance is the main obstacle in cancer therapy, limiting the effectiveness of drug treatment. Epigenetics-mediated changes are suggested as a critical factor paying the chemo-resistance phenotype. Since epigenetic modulations are a reversible phenomenon, reversion of epigenetic changes represents a promising therapeutic approach for cancer. However, heterogeneity in epigenetic marks in tumor cells makes it difficult to identify the specific epigenetic aberrations contributing to chemo-resistance. Our hypothesis aimed to explore this issue to add therapeutic options for cancer. Presentation of the hypothesisEpigenetic alterations, the main mediator of cellular reprogramming, occur rapidly upon exposure to chemotherapy. Recent studies have demonstrated that reprogramming resets/erases the epigenetic marks established during differentiation to specific somatic cell types. To overcome the heterogeneous nature of cancer cells, we will attempt to make homogenous cancer cell colonies by reprogramming. Comparison of the drug-resistant cancer cells obtained from these colonies to parent cancer cells and reprogrammed cancer cells is an effective way to determine the precise epigenetic alterations underlying specific chemo-resistance. Testing the hypothesisCellular reprogramming of cancer cells led to generation of homogenous colonies. Following lineage specification and long term drug treatment, the obtained drug resistance cells will be compared with parent cancer cells for whole genome epigenetic signature. Implications of the hypothesisA key implication of this hypothesis is that determination of the usefulness of cellular reprogramming of cancer cells enabling the identification of specific epigenetic modulation associated with particular drug resistance will enable exploration of new research avenues for cancer treatment.

MicroRNA 302/367 Cluster Effectively Facilitates Direct Reprogramming from Human Fibroblasts into Functional Neurons.

Recent studies suggest that mature somatic cells can be reprogrammed to become induced pluripotent stem cells by overexpressing specific transcription factors or microRNAs (miRNAs). Theoretically, this technique could provide a wide array of cells for therapeutics. However, the process of redifferentiation after cell reprogramming to pluripotency is inefficient and time restricted. We proposed that the differentiation of somatic cells into specific cells of another germ layer can be induced and accelerated with appropriate miRNAs and culture conditions. In human fibroblasts, we found that overexpression of pluripotency stem cell-specific miRNA-302/367 cluster, together with two other neuron-specific miRNAs (miRNA-9/9* and miRNA-124) induced fibroblasts conversion into neurons. The cells assumed neuron morphology, were positive for several neuron markers, and exhibited neuronal membrane potential feature. Moreover, concentrated expression of synaptic markers were observed in these cells in vitro and in vivo in nude mice brain, suggesting possible connectivity. To achieve efficient reprogramming, miRNA-302/367 cluster, miRNA-9/9*, and miRNA-124 were all required. The combination of the proved pluripotency-inducing miRNA-302/367 cluster and cell-specific miRNAs provides a unique strategy for one-step cellular conversion that could have important implications for studies of neuron development and neurological disease therapy.

Differentiation of Fetal Male Germline and Gonadal Progenitor Cells Is Disrupted in Organ Cultures Containing Knockout Serum Replacement.

Germ cells occupy a unique place in development through their requirement to maintain underlying totipotency while producing highly differentiated gametes. This is reflected in the expression of key regulators of pluripotency in the fetal germline and the ability of these cells to form pluripotent stem cells and germ cell tumors. Culture of whole fetal testes, including germ cells, provides a key model for studying gonad and germline development, but it is critical that such models mimic physiological development as closely as possible. We aimed to determine the effects of differing culture conditions, including serum-free and serum-containing conditions, on fetal germ cell and testis development. We tested a commonly used model that employs knockout serum replacement (KSR) to provide more defined culture conditions than media containing fetal bovine serum (FBS). In FBS conditions, cell cycle parameters in germ and Sertoli cells closely resembled normal development. In contrast, KSR significantly inhibited male germ cell entry into mitotic arrest, a key milestone in male germline development. Moreover, KSR disrupted molecular control of cell cycle and inhibited the transcription of a range of male germ cell differentiation markers. In the somatic compartment, KSR stimulated proliferation and inhibited differentiation in Sertoli cells. These data demonstrate that KSR substantially alters germ and somatic cell differentiation in fetal testis culture and should not be used to replicate normal gonadal development. In contrast, basal media with or without serum support germ and supporting cell differentiation and cell cycle dynamics that are in line with in vivo characteristics.

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.

Enhanced Rejuvenation in Induced Pluripotent Stem Cell-Derived Neurons Compared with Directly Converted Neurons from an Aged Mouse.

Most neurodegenerative diseases are late-onset and aging-related, and no effective treatments have been developed. The successful generation of induced pluripotent stem cells (iPSCs) and the direct conversion of neurons from patients' specific somatic cells have offered cell resources for disease modeling and potential cell transplantation therapy. However, to date, no systematic studies have investigated which approach is more suitable for future cell therapy. In this study, using the two approaches mentioned above in parallel we successfully obtained functional neurons from tail-tip fibroblasts (TTFs) of a 1-year-old mouse, which were characterized by specialized morphologies, neuronal marker expressions, and electrophysiological properties. Genome-wide expression analysis revealed that a set of genes related to the stress response and DNA damage were expressed at a much higher level in iNs than in diNs derived from 1-year-old TTFs. Subsequently, significant decreases in mitochondrial dysfunction and DNA damage were observed in diNs compared with iNs derived from aged TTFs. Moreover, the levels of epigenetic markers such as 5hmC, H3K4me3, H3K9me3, and H3K27me3 in iNs were more similar to those in the old TTFs compared with those in diNs, indicating that the iNs converted directly from TTFs may retain some residual epigenetic memories. By contrast, reprogramming to iPSCs not only rejuvenated the cell stages, but also erased such epigenetic memories obtained along the aging process. Taken together, the results of our study are instructive and meaningful for future clinical applications.

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.