Recent progress in hair follicle stem cell markers and their regulatory roles

Hair follicle stem cells(HFSCs)in the bulge are a multipotent adult stem cell population.They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing.An increasing number of biomarkers have been used to isolate,label,and trace HFSCs in recent years.Considering more detailed data from single-cell transcriptomics technology,we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

The culture and establishment of embryonic germ(EG)cell lines from Chinese mini swine

As a part of a basic research project on Xeno-transplantion, we have been engaged in the derivation ofembryonic stem cell lines from Chinese mini swine. Here, we reported for the first time the establishmentof two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 anda 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, markercharacterization as well as by in vitro and in vivo differentiation. These porcine EG cells are potentiallyuseful for further basic studies.

Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

Background:NANOG is a core transcription factor(TF)in embryonic stem cells(ESCs)and primordial germ cells(PGCs).Regulation of the NANOG gene by TFs,epigenetic factors,and autoregulatory factors is well characterized in ESCs,and transcriptional regulation of NANOG is well established in these cells.Although NANOG plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied.Therefore,we investigated the mechanism that regulates transcription of the chicken NANOG(cNANOG)gene in PGCs and ESCs.Results:We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis.Then,we measured the promoter activity of various 5′flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay.cNANOG expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in PGCs.The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ESCs.Furthermore,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of cNANOG.Conclusions:We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner.This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

Autophagy regulation combined with stem cell therapy for treatment of spinal cord injury

Stem cells are a group of cells with unique self-renewal and differentiation abilities that have great prospects in the repair of spinal cord injury. However, stem cell renewal and differentiation require strict control of protein turnover in the stem cells to achieve cell remodeling. As a highly conserved “gatekeeper” of cell homeostasis, autophagy can regulate cell remodeling by precisely controlling protein turnover in cells. Recently, it has been found that the expression of autophagy markers changes in animal models of spinal cord injury. Therefore, understanding whether autophagy can affect the fate of stem cells and promote the repair of spinal cord injury is of considerable clinical value. This review expounds the importance of autophagy homeostasis control for the repair of spinal cord injury from three aspects—pathophysiology of spinal cord injury, autophagy and stem cell function, and autophagy and stem cell function in spinal cord injury—and proposes the synergistic therapeutic effect of autophagy and stem cells in spinal cord injury.

Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

AIM To investigate the role of embryonic liver fordin(ELF) in liver fibrosis by regulating hepatic stellate cells(HSCs) glucose glycolysis.METHODS The expression of ELF and the glucose glycolysisrelated proteins were evaluated in activated HSCs. si RNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed.RESULTS The expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-si RNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix(ECM) components. Moreover, p Akt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysisrelated proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs. CONCLUSION ELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling.

Common stemness regulators of embryonic and cancer stem cells

Pluripotency of embryonic stem cells(ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal trans-ducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors(cancer stem cells), provides a common conceptual and research frame-work for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Wheat Germ Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells in nude mice was established to evaluate anticarcinogenic activities, and the apoptosis-regulated mechanism effect of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1 （LFWGE）. Methods The HT-29 cells were transplanted via subcutaneous injection of 1×10^7cells into the right flank of each nude mouse. Then, nude mice were treated for 30 d with LFWGE （high-dose 2 g/kg/d; low-dose 1 g/kg/d） and for 7 d with 5-fluorouracil （5-FU, 25 mg/kg/d） by gavage and intraperitoneal injection, respectively. An inhibition of tumor growth was observed. Results Tumor volume and weights decreased significantly in both groups of nude mice treated with LFWGE. In addition, the cell apoptosis rate of the LFWGE group （2 g/kg/d, 60.2%＋4.4%; 1 g/kg/d, 58.6%＋6.9%） was significantly higher than that of the control group （11.5%＋1.6%） and 5-FU group （32.1%＋3.5%） as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot method further confirmed these enhancing apoptosis and growth inhibition effects. The involvement of LFWGE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, Caspase-3, and CyclinD1. Conclusion The results showed that LFWGE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be as a natural nutrient supplements or chemopreventive agent in the treatment of human colon cancer.

Targeting lentiviral vectors to primordial germ cells(PGCs):An efficient strategy for generating transgenic chickens

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

Effect of VEGF on Neural Differentiation of Human Embryonic Stem Cells in vitro

The effects of vascular endothelial growth factor （VEGF） on neural differentiation of human embryonic stem cells （hESCs） in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups： group A, routine induction; group B, routine induction＋10 ng/mL VEGF; group C, routine in- duction＋10 ng/mL VEGF＋10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de- tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C （P〈0.01）. There was no significant difference between groups A and C （P〉0.05）. It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid(LPA)is a small molecule glycerophospholipid,which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.In this study,sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.At first,the maturation medium containing estrus female sheep serum and synthetic oviduct fluid(SOF)were optimized for sheep IVF,and then the effects of LPA were investigated.From 0.1 to 10μmol L^(–1),LPA had no significant effect on the cleavage rate(P>0.05),but the maturation rate and blastocyst rate increased dependently with LPA concentration(P<0.05),and the blastocyst morphology was normal.When the LPA concentration was 15μmol L^(–1),the maturation rate,cleavage rate and blastocyst rate decreased significantly(P<0.05),and the blastocyst exhibited abnormal morphology and could not develop into highquality blastocyst.Besides,the exogenous LPA increases the expression of LPAR2,LPAR4,TE-related gene CDX-2and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10μmol L^(–1).The expression of LPAR2,LPAR4,CDX-2 and OCT-4 from the LPA-0.1μmol L^(–1)to LPA-10μmol L^(–1)groups in early embryos were extremely significant(P<0.05),while the expression of these genes significantly decreased in 15μmol L^(–1)LPA-treated embryos compared with LPA-10μmol L^(–1)group(P<0.05).The inner cell mass in 15μmol L^(–1)LPA-treated embryos was also disturbed,and the blastocysts formation was abnormal.Secondly,the sheep IVF blastocysts were applied to establish embryonic stem cells.The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10μmol L^(–1),while 15μmol L^(–1)LPA decreased OCT-4 and CDX-2 expression in the derived cells.The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1μmol L^(–1)group and LPA-10μmol L^(–1)group extremely significantly increased(P<0.05),but there was significant decrease in LPA-15μmol L^(–1)group compared with LPA-10μmol L^(–1)group(P<0.05).Meanwhile,the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10μmol L^(–1)concentration.This study references the IVF embryo production and embryonic stem cell research of domestic animals.

Tissue factor expression and methylation regulation in differentiation of embryonic stem cells into trophoblast

Objective:To explore tissue factor(TF)expression and methylation regulation in differentiation of human embryonic stem cells(hESCs)into trophoblast.Methods:Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein 4(BMP4).Expression of gene,protein of TF and DNA methylation at different time points during induction process was detected by RTPCT,Western blot,flow cytometry and MSP-PCR method.Results:The expression of mRNA,protein level of TF could be detected during directional differentiation of hESCs to trophoblast cells,semi methylation-semi non methylation expression appeared at TF DNA promoter region,and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression of TF.Conclusions:It shows that during differentiation of hESCs into trophoblast,the differential expression of TF is related with DNA methylation level,and it is changed with the methylation or non methylated degree.It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.

The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

Primordial germ cells (PGCs), as precursors of mam-malian germ lineage, have been gaining more attention as anew resource of pluripotent stem cells, which bring a greatpossibility to study developmental events of germ cell invitro and at animal level. EG4 cells derived from 10.5 dayspost coitum (dpc) PGCs of l29/svJ strain mouse wereestablished and maintained in an undifferentiated state.With an attempt to study the differentiation capability ofEG4 cells with a reporter protein: green fluorescence pro-tein, and the possible application of EG4 cells in the re-search of germ cell development, we have generated severalEG4-GFP cell lines expressing enhanced green fluorescenceprotein (EGFP) and still maintaining typicaI characteris-tics of pluripotent stem cells. Then, the differentiation ofEG4-GFP cells in vitro as well as their developmental fatein chimeric embryos which were produced by aggregatingEG4-GFP cells to 8-cell stage embryos were studied. Theresults showed that EG4 cells carrying green fluorescencehave a potential use in the research of germ cell develop-ment and other related studies.

Unilateral post-chemotherapy robot-assisted retroperitoneal lymph node dissection in Stage II non-seminomatous germ cell tumor:A tertiary care experience

Objective Post-chemotherapy retroperitoneal lymph node dissection(PC-RPLND)represents an integral component of the management of patients with non-seminomatous germ cell tumor(NSGCT).Modified templates have been proposed to minimize the surgical morbidity of the procedure.Moreover,the implementation of robotic surgery in this setting has been explored.We report our experience with unilateral post-chemotherapy robot-assisted retroperitoneal lymph node dissection(PC-rRPLND)for clinical Stages IIA and IIB NSGCTs.Methods A retrospective single institution review was performed including 33 patients undergoing PC-rRPLND for Stages IIA and IIB NSGCTs between January 2015 and February 2019.Following orchiectomy,patients were scheduled for chemotherapy with three cycles of bleomycin-etoposide-cisplatin.Patients with a residual tumor of<5 cm and an ipsilateral metastatic disease on pre-and post-chemotherapy CT scans were eligible for a unilateral template in absence of rising tumor markers.Descriptive statistics were provided for demographics,clinical characteristics,intraoperative and postoperative parameters.Perioperative,oncological,and functional outcomes were recorded.Results Overall,7(21.2%)patients exhibited necrosis or fibrosis;14(42.4%)had mature teratoma;and 12(36.4%)had viable tumor at final histology.The median lymph node size at surgery was 25(interquartile range[IQR]21-36)mm.Median operative time was 180(IQR 165-215)min and no major postoperative complications were observed.Anterograde ejaculation was preserved in 75.8%of patients.Median follow-up was 26(IQR 19-30)months and a total of three recurrences were recorded.Conclusion PC-rRPLND is a reliable and technically reproducible procedure with safe oncological outcomes and acceptable postoperative ejaculatory function in well selected patients with NSGCTs.

Testicular mixed germ cell tumor:A case report

BACKGROUND Testicular mixed germ cell tumors(TMGCTs)are rare malignant tumors that are more common in men aged 20–40 years.TMGCTs comprise two or more types of germ cell tumors that primarily affect the testis.Their onset is undetectable;thus,early diagnosis is challenging.However,early recognition and diagnosis substantially improve patient prognosis.CASE SUMMARY We evaluated a rare case of TMGCT in a male patient presenting with recurrent fever and left supraclavicular lymphadenectasis instead of testicular enlargement and pain,which may easily lead to misdiagnosis.We report the clinical signs and symptoms,histopathological characteristics,and immunohistochemical results of this case of malignant TMGCT.CONCLUSION Our case,which was typical with multiple components,along with a literature review,may serve as a basis for early diagnosis.

Regenerative potential of mouse embryonic stem cell-derived PDGFRα+ cardiac lineage committed cells in infarcted myocardium

BACKGROUND Pluripotent stem cell-derived cardiomyocytes(CMs) have become one of the most attractive cellular resources for cell-based therapy to rescue damaged cardiac tissue.AIM We investigated the regenerative potential of mouse embryonic stem cell(ESC)-derived platelet-derived growth factor receptor-α(PDGFRα)+ cardiac lineagecommitted cells(CLCs), which have a proliferative capacity but are in a morphologically and functionally immature state compared with differentiated CMs.METHODSWe induced mouse ESCs into PDGFRα+ CLCs and αMHC+ CMs using a combination of the small molecule cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197.We implanted PDGFRα+ CLCs and differentiated αMHC+ CMs into a myocardial infarction(MI) murine model and performed functional analysis using transthoracic echocardiography(TTE) and histologic analysis.RESULTS Compared with the untreated MI hearts, the anterior and septal regional wall motion and systolic functional parameters were notably and similarly improved in the MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs based on TTE.In histologic analysis, the untreated MI hearts contained a thinner ventricular wall than did the controls, while the ventricular walls of MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs were similarly thicker compared with that of the untreated MI hearts. Furthermore, implanted PDGFRα+ CLCs aligned and integrated with host CMs and were mostly differentiated into α-actinin+ CMs,and they did not convert into CD31+ endothelial cells or αSMA+ mural cells.CONCLUSION PDGFRα+ CLCs from mouse ESCs exhibiting proliferative capacity showed a regenerative effect in infarcted myocardium. Therefore, mouse ESC-derived PDGFRα+ CLCs may represent a potential cellular resource for cardiac regeneration.

Histological Observation of Germ Cell Development and Discovery of Spermatophores in Ovoviviparous Black Rockfish(Sebastes schlegeli Hilgendorf) in Reproductive Season

Black rockfish(Sebastes schlegeli) is an important species for culture; however, its reproductive characteristics have not been fully documented. In this study, we investigated the morphology and developmental process of germ cells in this ovoviviparous rockfish in reproductive season(October 2011–November 2012) with histological methods. We found that the gonad of mature fish showed notable seasonal changes in developmental characteristics and morphological structure. The sperm cells matured during a period lasting from October to December, significantly earlier than the oocytes did. A large number of spermatozoa and other cells occurred in testis at different developmental stages. Vitellogenesis in oocytes began in October, and gestation appeared in April next year. Spermatophores were discovered for the first time in Sebastes, which assembled in testis, main sperm duct, oviduct and genital tract, as well as ovarian cavity in October and April. These organs may serve either as production or hiding places for spermatophores and spermatozoa which were stored and transported in form of spermatophores. Testicular degeneration started from the distal part of testis in April, with spermatophores assembled in degenerating testis and waiting for transportation. The copulation probably lasted for a long period, during which the spermatozoa were discharged in batches as spermatophores. These spermatophores were coated with sticky materials secreted from the interstitial areas of testis and the main sperm duct, then transported into ovary.

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

单性生殖的胚胎的茎(足) 细胞为学习位于印的 genomic 下面的分子的机制提供一个珍贵试管内模型系统。然而，足房间的 thepluripotency 和父亲一般地表示的印的基因的表示侧面一直不是充分探索了。在这研究，三根老鼠足房间线被建立，在扩大文化的这些房间的区别潜力被评估。无差别的房间有正常核型和同型结合的染色体，并且表示了 ES-cell-specific 分子的标记。房间在超过 50 个段落以后仍然保持无差别并且展出了 pluripotentdifferentiation 能力。确定的 ES 房间的所有三根线生产了 teratomas；ES 房间的二根线生产了怪物和细菌线传播。而且，在这些房间的父亲一般地表示的印的基因 Snrpn， U2af1-rs1， Peg3，影响， Zfp127， Dlk1 和 Mest 的激活被检测。父亲一般地表示的印的基因被发现在单性生殖的联盟者的胚囊阶段被表示的一些激活胚胎试管内，他们的表示水平与扩大的足细胞培养增加了。而且，我们在足房间的这些父亲一般地表示的印的基因的激活在 ther 的甲基化与一个变化被联系的数据表演鼓舞了差别 methylated 区域。这些调查结果为足房间的 thepluripotency 提供直接证据并且在足房间表明在脱氧核糖核酸甲基化模式和父亲一般地表示的印的基因的激活之间的协会。因此，确定的 ES 房间线为在哺乳动物的开发学习 epigenetic 规定提供一个珍贵模型。

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

POU 抄写因素 OCT4 不仅在维持 pluripotent 和房间而且幕作为通过基因剂量的一个房间命运决定因素完成的胚胎的茎(ES ) 的自我更新的状态起一个必要作用。然而，控制细胞内部的 OCT4 蛋白质水平的分子的机制留下逃犯。这里，我们报导那人的 WWP2， E3 ubiquitin (Ub ) 蛋白质 ligase，通过它的 WW 领域明确地与 OCT4 交往并且在 vitro 并且在 vivo 提高 OCT4 的 Ub 修正。我们首先证明在人的 ES 房间的内长的 OCT4 能被 Ub post-translationally 修改。而且，我们发现 WWP2 以一种剂量依赖者方式，和 WWP2 的活跃地点半胱氨酸残余通过 26S proteasome 支持了 OCT4 的降级在 OCT4 上为它的酶的活动和解朊的效果被要求。显著地，我们当 WWP2 表示是由特定的 RNA 干扰(RNAi ) 的 downregulated 时，内长的 OCT4 蛋白质水平显著地被提高的数据表演，建议那 WWP2 是为在人的 ES 房间维持合适的 OCT4 蛋白质水平的一个重要管理者。而且，北污点分析证明 WWP2 抄本在多样的人的织物 / 器官是广泛地在场的并且高度在无差别的人的 ES 房间表示了。然而，它的表示水平快速在区分的人的 ES 房间以后被减少，显示 WWP2 表示力量发展地被调整。我们的调查结果证明 WWP2 是在人的 ES 房间的 OCT4 蛋白质水平的一个重要管理者。