Enhanced efficiency of generating induced pluripotent stem （iPS） cells from human somatic cells by a combination of six transcription factors

Dear Editor, Human embryonic stem （ES） cells possess the potential to differentiate into all the cell types of the human body and provide potential applications in regenerative medicine . However, the concerns of immune rejection hamper transplantation therapies using human ES cells. To avoid the complications of immune rejection, diverse methods, such as somatic nuclear transfer （also called therapeutic cloning） and fusion of somatic ceils with human ES ceils , have been attempted to produce patient-specific pluripotent stem cells. Most of these approaches have resulted in little success. The generation of human iPS cells （induced

Therapeutic potential of periodontal ligament stem cells

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss.Due to the complexity of the periodontium,which is composed of several tissues,its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available.Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry,especially therapies using mesenchymal stem cells,as they can be isolated from a myriad of tissues.Periodontal ligament stem cells(PDLSCs)are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium.Biological insights have also highlighted PDLSCs as promising immunomodulator agents.In this review,we explore the state of knowledge regarding the properties of PDLSCs,as well as their therapeutic potential,describing current and future clinical applications based on tissue engineering techniques.

WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines

Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.

Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts

Embryonic stem （ES） cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem ceils. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-like cell lines, which expressed pluripotency markers and formed embryoid bodies （EBs） in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers. In addition, from the rat ES-like cells, we derived a rat primitive endoderm （PrE） cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and mTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.

Mesenchymal stem cells： a new strategy for immunosuppression and tissue repair

Mesenchymal stem cells （MSCs） have great potential for treating various diseases, especially those related to tissue damage involving immune reactions. Various studies have demonstrated that MSCs are strongly immunosuppressive in vitro and in vivo. Our recent studies have shown that un-stimulated MSCs are indeed incapable of immunosuppression; they become potently immunosuppressive upon stimulation with the supernatant of activated lymphocytes, or with combinations of IFN-γ, with TNF-α, IL-1α or IL-1β. This observation revealed that under certain circumstances, inflammatory cytokines can actually become immunosuppressive. We showed that there is a species variation in the mechanisms of MSC-mediated immunosuppression： immunosuppression by cytokine-primed mouse MSCs is mediated by nitric oxide （NO）, whereas immunosuppression by cytokine-primed human MSCs is executed through indoleamine 2, 3-dioxygenase （IDO）. Additionally, upon stimulation with the inflammatory cytokines, both mouse and human MSCs secrete several leukocyte chemokines that apparently serve to attract immune cells into the proximity with MSCs, where NO or IDO is predicted to be most active. Therefore, immunosuppression by inflammatory cytokine-stimulated MSCs occurs via the concerted action of chemokines and immune-inhibitory NO or IDO produced by MSCs. Thus, our results provide novel information about the mechanisms of MSC-mediated immunosuppression and for better application of MSCs in treating tissue injuries induced by immune responses.

Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.

Skeletal myogenesis by human embryonic stem cells

We have examined the myogenic potential of human embryonic stem （hES） cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

Members of the basic helix-loop-helix （bHLH） gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer （FRET） is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD 1, Mash 1, Neurogenin 1 （Ngn 1）, Neurogenin2 （Ngn2）, and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein （EYFP） and enhanced cyan fluorescence protein （ECFP）, respectively. The subcellular localization and mobility ofbHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mashl/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.

Reprogramming of germ cells into pluripotency

Primordial germ cells(PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter

Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.

Regulated expression of TATA-binding protein-related factor 3(TRF3)during early embryogenesis

RNA polymerase （Pol） Ⅱ transcription persists in TATA-box-binding protein （TBP）^-/- mutant mouse embryos, indicating TBP-independent mechanisms for Pol Ⅱ transcription in early development. TBP-related factor 3 （TRF3） has been proposed to substitute for TBP in TBP^-/- mouse embryos. We examined the expression of TRF3 in maturing oocytes and early embryos and found that TRF3 was co-expressed with TBP in the meiotic oocytes and early embryos from the late one-cell stage onward. The amounts of TBP and TRF3 changed dynamically and correlated well with transcriptional activity. Chromatin immunoprecipitation （CHIP） assay revealed that different gene promoters in mouse embryonic stem （ES） cells recruited TRF3 and TBP selectively. Comparative analyses of TRF3 and TBP during cell cycle showed that both factors proceeded through cell cycle in a similar pace, except that TRF3 was slightly delayed than TBP in entering the nucleus when cells were exiting the M-phase. Data from expression and biochemical analyses therefore support the hypothesis that TRF3 plays a role in early mouse development. In addition, results from co-localization study suggest that TRF3 may be also involved in Pol Ⅰ transcription.

Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts

A single mammalian transcript normally encodes one protein, but the transcript of GNAS （G-protein u-subunit） contains two reading frames and produces two structurally unrelated proteins, XLas and ALEX. No other confirmed GNAS-Iike dual-coding transcripts have been reported to date, even though many such candidate genes have been predicted by bioinformatics analysis. In this study, we constructed a series of vectors to test how two protein products were translated from a single transcript in vitro. The length of the ORF （open reading frame）, position of the first AUG and the Kozak motif were found to be important factors. These factors, as well as 55-bp NMD （nonsense-mediated mRNA decay） rule, were used in a bioinformatics search for candidate dual-coding transcripts. A total of 1307, 750 and 474 two-ORF-containing transcripts were found in human, mouse and rat, respectively, of which 170, 89 and 70, respectively, were found to be potential dual-coding transcripts. Most transcripts showed low conservation among species. Interestingly, dual-coding transcripts were significantly enriched for transcripts from the zinc-finger protein family, which are usually DNA-binding proteins involved in regulation of the transcription process.

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

Sac domain-containing proteins belong to a newly identified family of phosphoinositide phosphatases （the PIPPase family）. Despite well-characterized enzymatic activity, the biological functions of this mammalian Sac domain PIPPase family remain largely unknown. We identified a novel Sac domain-containing protein, rat Sac3 （rSac3）, which is widely expressed in various tissues and localized to the endoplasmic reticulum, Golgi complex and recycling endosomes, rSac3 displays PIPPase activity with PI（3）P, PI（4）P and PI（3,5）P2 as substrates in vitro, and a mutation in the catalytic core of the Sac domain abolishes its enzymatic activity. The expression of rSac3 is upregulated during nerve growth factor （NGF）-stimulated PC 12 cell neuronal differentiation, and overexpression of this protein promotes neurite outgrowth in PC 12 cells. Conversely, inhibition ofrSac3 expression by antisense oligonucleotides reduces neurite outgrowth of NGF- stimulated PC 12 cells, and the active site mutation of rSac3 eliminates its neurite-outgrowth-promoting activity. These results indicate that rSac3 promotes neurite outgrowth in differentiating neurons through its PIPPase activity, suggesting that Sac domain PIPPase proteins may participate in forward membrane trafficking from the endoplasmic reticulum and Golgi complex to the plasma membrane, and may function as regulators of this crucial process of neuronal cell growth and differentiation.

Natural killer cells go inside： Entosis versus cannibalism

The cell-in-cell phenomenon is frequently observed in rumor samples, especially in those highly malignant or metastatic tumors. It is believed that through this mechanism, cancer cells could ingest immune cells such as neutrophils and lymphocytes, which otherwise kill tumor cells. Tumor cells could also phagocytose tumor cells--a phenomenon resembles autophagic di- gestion of cellular organelles under starvation. This cell-eat-cell phenomenon is called cannibalism [1-2].

Identification of karyopherin-alpha 2 as an Oct4 associated protein

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem （ES） cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain （containing POU domain and homeodomain） of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopberin-alpha 2 （KPNA-2）. KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal （NLS） of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

Cell–cell contact with proinflammatory macrophages enhances the immunotherapeutic effect of mesenchymal stem cells in two abortion models

Mesenchymal stem cells(MSCs),which are pluripotent cells with immunomodulatory properties,have been considered good candidates for the therapy of several immune disorders,such as inflammatory bowel diseases,concanavalin A-induced liver injury,and graft-versus-host disease.The embryo is a natural allograft to the maternal immune system.A successful pregnancy depends on the timely extinction of the inflammatory response induced by embryo implantation,followed by the switch to a tolerant immune microenvironment in both the uterus and the system.Excessive infiltration of immune cells and serious inflammatory responses are triggers for embryo rejection,which results in miscarriage.Here,we demonstrated that adoptive transfer of MSCs could prevent fetal loss in a lipopolysaccharide(LPS)-induced abortion model and immune response-mediated spontaneous abortion model.The immunosuppressive MSCs alleviated excessive inflammation by inhibiting CD4+T cell proliferation and promoting the decidual macrophage switch to M2 in a tumor necrosis factor-stimulated gene-6(TSG-6)-dependent manner.Cell-tocell contact with proinflammatory macrophages increased the TSG-6 production by the MSCs,thereby enhancing the suppressive regulation of T cells and macrophages.Moreover,proinflammatory macrophages in contact with the MSCs upregulated the expression of CD200 on the stem cells and facilitated the reprogramming of macrophages towards an anti-inflammatory skew through the interaction of CD200 with CD200R on proinflammatory macrophages.Therefore,the results demonstrate that a TSG-6-mediated paracrine effect,reinforced by cell-to-cell contact between MSCs and proinflammatory macrophages,is involved in the mechanism of MSC-mediated abortion relief through the induction of immune tolerance.Our study also indicates the potential application of MSCs in clinical recurrent miscarriages.

A tumor cell membrane-coated self-amplified nanosystem as a nanovaccine to boost the therapeutic effect of anti-PD-L1 antibody

To improve the response rate of immune checkpoint inhibitors such as anti-PD-L1 antibody in immunosup-pressive cancers like triple-negative breast cancer(TNBC),induction of immunogenic cell death(ICD)at tumor sites can increase the antigenicity and adjuvanticity to activate the immune microenvironment so that tumors become sensitive to the intervention of immune checkpoint inhibitors.Herein,a self-amplified biomimetic nanosystem,mEHGZ,was constructed by encapsulation of epirubicin(EPI),glucose oxidase(Gox)and hemin in ZIF-8 nanoparticles and coating of the nanoparticles with calreticulin(CRT)over-expressed tumor cell mem-brane.EPI acts as an ICD inducer,Gox and hemin medicate the cascade generation of reactive oxygen species(ROS)to strengthen the ICD effect,and CRT-rich membrane as“eat me”signal promote presentation of the released antigens by dendritic cells(DCs)to invoke the tumor-immunity cycle.The biomimetic delivery system displays an amplified ICD effect via Gox oxidation,hydroxyl radical generation and glutathione(GSH)depletion.The induced potent ICD effect promotes DCs maturation and cytotoxic T lymphocytes(CTLs)infiltration,reversing an immunosuppressive tumor microenvironment to an immunoresponsive one.Treatment with the nanosystem in combination with anti-PD-L1 antibody results in distinctive inhibition of tumor growth and lung metastasis,supporting that a potent ICD effect can significantly boost the therapeutic efficacy of the anti-PD-L1 antibody.This self-amplified biomimetic nanoplatform offers a promising means of raising the response rate of immune checkpoint inhibitors.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells

Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.