7SK truncation at 128-179 nt suppresses embryonic stem cell proliferation / 南方医科大学学报

OBJECTIVE@#To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD).@*METHODS@#ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting.@*RESULTS@#We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6.@*CONCLUSIONS@#7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.

Research status and prospect of basic application of islet organoid / 器官移植

Organoids are tissue structures, generated from pluripotent stem cells and cultured in vitro, which form self-organize and recapitulate tissues with similar structure and function to the original organs. Organoids have similar appearance and function to the original tissues, and have been widely applied in basic research and clinical trial. At present, the organoids of liver, kidney, islet, brain, intestine and other organs have been successfully cultivated. The use of islet organoid is a hotspot in the field of organoid research. However, islet organoid is currently applied in basic research because rejection after organ transplantation and other issues remain unresolved. In this article, the origin, development and basic application of islet organoid were reviewed, aiming to provide reference for the transformation from basic research of islet organoid into clinical application as well as the treatment of diabetes mellitus.

Core pluripotency factors promote glycolysis of human embryonic stem cells by activating GLUT1 enhancer

Human embryonic stem cells (hESCs) depend on glycolysis for energy and substrates for biosynthesis. To understand the mechanisms governing the metabolism of hESCs, we investigated the transcriptional regulation of glucose transporter 1 (GLUT1, SLC2A1), a key glycolytic gene to maintain pluripotency. By combining the genome-wide data of binding sites of the core pluripotency factors (SOX2, OCT4, NANOG, denoted SON), chromosomal interaction and histone modification in hESCs, we identified a potential enhancer of the GLUT1 gene in hESCs, denoted GLUT1 enhancer (GE) element. GE interacts with the promoter of GLUT1, and the deletion of GE significantly reduces the expression of GLUT1, glucose uptake and glycolysis of hESCs, confirming that GE is an enhancer of GLUT1 in hESCs. In addition, the mutation of SON binding motifs within GE reduced the expression of GLUT1 as well as the interaction between GE and GLUT1 promoter, indicating that the binding of SON to GE is important for its activity. Therefore, SON promotes glucose uptake and glycolysis in hESCs by inducing GLUT1 expression through directly activating the enhancer of GLUT1.

Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics / 解剖学报

Objective: To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods: mESCs were cultured in vitro, and the recombinant vector pEFla-Pax6-IRES-AcGFP and the empty vector pEFlot-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1 (SSEA1) and octamer binding transcription factor 4(0CT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results: Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and 0CT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion: Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.

Isolation and identification of Oct4 RNA-binding proteins / 上海交通大学学报（医学版）

Objective: To identify the RNA binding proteins of Oct4, a key factor of embryonic stem cells. Methods: Total RNA was extracted from mouse embryonic stem cells (R1), which was reverse-transcribed into cDNA. Then PCR product of Oct4 mRNA were transcribed into Oct4 mRNA. Finally the candidate RNA-binding proteins were eluted applying the streptomycin affinity chromatography, and submitted for high-performance liquid chromatography combined with the mass spectrometry. The identified RNA binding proteins were further confirmed by RNA immunoprecipitation (RIP). Results: 121 RNA binding proteins of Oct4 3' untranslated region (UTR) and Oct4 5' UTR were identified with liquid chromatography / mass spectrometry. There were 11 proteins with the number of peptide spectrum matches more or equal to 2, and 7 of them were selected for additional confirmation using RIP method. RIP results showed that Oct4 interacted with DSP, SOD1, LMNA, NPM1, PSIP1, NCL and HDGF. Among them, HDGF had the strongest binding ability to Oct4 mRNA. Conclusion: Identification of Oct4 RNA binding proteins will provide a theoretical basis for the regulation mechanism of Oct4, and will be a basis for the further study of its function.

Pja2 Inhibits Wnt/β-catenin Signaling by Reducing the Level of TCF/LEF1

Ubiquitination of proteins plays an essential role in various cellular processes, including protein degradation, DNA repair, and cell signaling pathways. Previous studies have shown that protein ubiquitination is implicated in regulating pluripotency as well as fate determination of stem cells. To identify how protein ubiquitination affects differentiation of embryonic stem cells, we analyzed microarray data, which are available in the public domain, of E3 ligases and deubiquitinases whose levels changed during stem cell differentiation. Expression of pja2, a member of the RING-type E3 ligase family, was up-regulated during differentiation of stem cells. Wnt/β-catenin signaling is one of the most important signaling pathways for regulation of the self-renewal and differentiation of embryonic stem cells. Pja2 was shown to bind to TCF/LEF1, which are transcriptional factors for Wnt/β-catenin signaling, and regulate protein levels by ubiquitination, leading to down-regulation of Wnt signaling activity. Based on these results, we suggest that E3 ligase Pja2 regulates stem cell differentiation by controlling the level of TCF/LEF1 by ubiquitination.

Expression dynamics of lens development-associated transcription factors in human embryonic stem cell-induced lentoid body / 中华实验眼科杂志

Objective To observe the expression dynamics of lens-related transcription factors in human embryonic stem cell (hESC) differentiated into lentoid body(LB).Methods A "three-stage" protocol was used for LB directional differentiation from hESC in vitro.The hESC (D0) and three differentiation stages cells were collected to analyze the expression dynamics of lens development-associated transcription factors by high throughput RNA sequencing technology in hESC-induced LB.Western blot and cell immunofluorescence were used to observe the involved genes at protein level.Results During D0-D6,cells became more round and compact.And during D7-D18,cells morphology gradually changed to spindle.At the end of D35,three-dimensional and transparent structurelentoid body (LB) was obtained.RT-PCR results showed that the stem cell related genes reduced and the lens specific genes increased significantly,and the LB was characterized by the expression of crystallins.According to clustering analysis of high throughput sequencing,a distinct difference in transcription factors gene expression was observed between D0 and D32.Meanwhile,the difference between D6 and D18 was minimum.The expressions of preplacodal genes,including DLX3,DLX5,DLX6,HES1,HES4,OTX2 and EYA1 increased remarkably at the first induction stage and then decreased.Lens-specification gene SOX2 declined gradually and then increased.In addition,the expression of PAX6 increased during all three induction stages.Furthermore,lens-differentiation genes including MAB21L1,CMAF,PROXI and PITX3 had no significant change in the early induction stage,but increased significantly at the third induction stage.Conclusions The expression dynamics of lens development-associated transcription factors in the hESC induced LB corresponded to those in vivo,which indicate that this induction system can recapitulate early lens development well and lay the foundation of studying lens embryonic development andtranscription factor associated congenital lens diseases.

Differential MicroRNA Expression Profile of Human Embryonic Stem Cell-Derived Cardiac Lineage Cells

MicroRNAs (miRNAs) are small non-coding RNA molecules that participate in transcriptional and post-transcriptional regulation of gene expression. miRNAs have numerous roles in cellular function including embryonic development. Human embryonic stem cells (hESCs) are capable of self-renewal and can differentiate into most of cell types including cardiomyocytes (CMs). These characteristics of hESCs make them considered as an important model for studying human embryonic development and tissue specific differentiation. In this study, we tried to demonstrate the profile of miRNA expression in cardiac differentiation from hESCs. To induce differentiation, we differentiated hESCs into CMs by direct differentiation method and characterized differentiated cells. To analyze the expression of miRNAs, we distinguished (days 4, 8, 12, 16, 20, 24, 28) and isolated RNAs from each differentiation stage. miRNA specific RT-qPCR was performed and the expression profile of miR-1, -30d, -133a, -143, -145, -378a, -499a was evaluated. The expression of all miRs was up-regulated at day 8. miR-143 and -145 expression was also up-regulated at the later stage of differentiation. Only miR-378a expression returned to undifferentiated hESC levels at the other stages of differentiation. In conclusion, we elucidated the expression profile of miRNAs during differentiation into cardiomyocytes from hESCs. Our findings demonstrate the expression of miRNAs was stage-dependent during differentiation and suggest that the differentiation into CMs can be regulated by miRNAs through direct or indirect pathway.

Expression of androgen receptor and embryonic stem cell associated transcript 4 in breast cancer patients with over-expression of human epidermal growth factor receptor 2 and their clinical significances / 肿瘤研究与临床

Objective To investigate the expression and clinical significance of androgen receptor (AR) and embryonic stem cell associated transcripts 4 (NANOG) in breast cancer patients with human epidermal growth factor receptor 2 (HER-2) over-expression, and to analyze its relationship with clinicopathologic features of breast cancer. Methods 143 breast cancer patients with HER-2 over-expression were selected from the screening of 1052 cases of invasive breast cancer according to estrogen receptor (ER), progesterone receptor (PR) and HER-2 status. The protein expression of AR and NANOG was assayed by using immunohistochemistry.The relationship between AR expression and clinicopathological features was analyzed by χ2 test. The correlation between AR expression and NANOG expression was analyzed by Spearman correlation analysis. Results The positive expression of AR was 35.7 % (51/143). The AR expression was not associated with age and menstruation status (both P>0.05), and was associated with tumor size, clinical TNM staging and lymphatic metastasis (all P< 0.05). The positive rate of NANOG were 53.1 %(76/143), and NANOG proteins were negative in adjacent normal breast tissue and benign breast lesions. The positive rate of AR was 27.6%(21/76) in NANOG-positive cases, whereas the positive rate of AR was 44.8%(30/67) in NANOG-negative cases, and the difference was statistically significant (χ2=4.526, P=0.033). There was an inverse correlation between NANOG and AR expressions (r= -0.255, P= 0.002). Conclusion AR and NANOG may be new targets for endocrine therapy and molecular biological therapy.

Antitumer effect of supernatant from co-culture of human embryonic stem cells and breast cancer cells on MDA-MB-231 cells in vitro / 药学学报

This study was designed to investigate the inhibitory effect of supernatant from co-culture of human embryonic stem cells and tumor MDA-MB-231 cells on the breast cancer. The direct co-culture system of human embryonic stem cells H9 and breast cancer MDA-MB-231 cells was established, and the supernatant was tested in the inhibition of MDA-MB-231 cells. The inhibitory effects were examined in tumor cell morphology using microscope, cell proliferation with MTT assay, and cell apoptosis using the Hoechst staining and flow cytometry. Transwell assay was used to detect the migration and invasion of tumor cells. The results suggest that the supernatant significantly inhibited the proliferation, invasion and migration, and promoted cell apoptosis of MDA-MB-231 cells. However, the supernatant of H9 cells alone had little effect on MDA-MB-231 cells. Therefore, we conclude that the supernatant of co-culture cells had an inhibitory effect on tumor cells in vitro.

Directing Human Embryonic Stem Cells towards Functional Endothelial Cells Easily and without Purification

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Effects of Transfection of Mash-1 Gene on Neural Differentiation of Embryonic Stem Cell in Spinal Cord Injury Mice / 中国康复理论与实践

Objective To investigate the effects of overexpression of Mash-1 gene on functional recovery and neural differentiation of embryonic stem cells in spinal cord injury mice. Methods CE3 cell line with overexpression of Mash-1 gene was generated with murine stem cell virus. Spinal cord injury model was established with forceps compression in 4-week-old KM mice. Normal saline (model group, n=12), CE3 cells with or without overexpression Mash-1 gene (CE3-Mash-1 and CE3 groups, n=12 respectively) were transplanted into the ar-eas of injury 3 days after injury. They were assessed with the Basso Mouse Scale (BMS) 1, 7, 14, 21, and 28 days after injury. 6 mice from each group were sacrificed 14 and 28 days after injury respectively. The spinal cord area remained were observed with HE stained, and the expression of Oct3/4, nestin,β-tubulin III and glial fibrillary acidic protein (GFAP) were detected with immunofluorescence in the injured spinal cord in the CE3 and CE3-Mash-1 groups. Results The score of BMS significantly improved in CE3 and CE3-Mash-1 groups com-pared with that of the model group (F>84.471, P49.990, P0.05). Conclusion Overexpression of Mash-1 gene promotes CE3 cells to differentiate into neurons in spinal cord injury mice, and improve the motor function recovery.

Embryonic stem cell test for the study of the embryotoxicity of hydroquinone / 国际检验医学杂志

Objective To establish a model of embryonic stem cell test(EST)and utilize this model to evaluate the embryotoxici-ty of hydroquinone.Methods Mouse 3T3 fibroblasts and mouse embryonic stem(ES)cells(ES-E14TG2a)were cultured in vitro, and methyl thiazolyl tetrazolium(MTT)test was performed to detect the cytotoxicity of 3T3 cells and ES-E14TG2a cells induced by the positive control(5-fluorouracil),negative control(penicillin G)and tested compound(hydroquinone).The concentrations of the test compounds that inhibited 50% viability of ES-E14TG2a cells(IC50 ES)and 3T3 fibroblasts (IC50 3T3)were calculated.The hanging-suspension-adherent culture systems were used to induce embryonic stem cells into cardiomyocytes,and the concentrations of test compounds that caused 50% inhibition of differentiation of ES-E14TG2a cells into cardiomyocytes (ID50 ES)was calculated. The embryotoxic potential of hydroquinone was classified by prediction model of the embryonic stem cell test.Results The prolif-eration of ES-E14TG2a and 3T3 cells were inhibited by hydroquinone,of which the IC50 3T3 and IC50 ES values were (5.97±0.48) and (2.57±0.10)μg/mL respectively.The differentiation of ES-E14TG2a cells were also inhibited by hydroquinone,of which the ID50 ES was (3.77±0.31)μg/mL.Hydroquinone was evaluated as a strong embryotoxicity chemical by prediction model of EST. Conclusion Hydroquinone exhibits a strong embryotoxicity.

Sourcing human embryos for embryonic stem cell lines: Problems & perspectives.

The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ‘discarded’ or ‘spare’ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to ‘cryopreserve’ their embryos then all the embryos remaining following embryo transfer can be considered ‘spare’ or if a couple is no longer in need of the ‘cryopreserved’ embryos then these also can be considered as ‘spare’. But, the question raised by the ethicists is, “what about ‘slightly’ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ‘discarded’ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ‘discarding’ embryos. What would be the criteria for discarding embryos and the potential ‘use’ of ESC derived from the ‘abnormal appearing’ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

Enhancing effects of serum-rich and cytokine-supplemented culture conditions on developing blastocysts and deriving porcine parthenogenetic embryonic stem cells

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.

Toxic effects of methylmercury, arsanilic acid and danofloxacin on the differentiation of mouse embryonic stem cells into neural cells

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABAA-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABA(A)-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 microM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.

Recent advances in the induction of the differentiation of embryonic stem cells into corneal epithelial cells / 中华实验眼科杂志

Embryonic stem cells(ESCs)are pluripotential cells,and they can be induced to differentiate into bone cells,cartilage cells,muscle cells,blood vessels and neurogenesis cells.In addition,ESCs are able to participate in the regeneration of ocular surface.At present,many research groups,domestic and abroad,have reported that ESCs can not only differentiate into corneal epithelium-like cells,but also play an important role in the repair of corneal epithelium.However,other unsolved problems in ESCs-related corneal tissue engineering are being concerned,such as the molecular biologic mechanism underlying the directional differentiation from ESCs to corneal cells,and the potential tumorigenicity with grafting of transformed or undifferentiated ESCs.The purpose of this review was to provide a summary of research progress in the differentiation of ESCs into corneal epithelial cells.

Development of a new coating substrate for human embryonic stem cell culture / 中国实验动物学报

Objective To reduce the animal component contamination for human embryonic stem cells ( hESCs ) and to simplify hESCs culture process , we develop a new coating substrate which can support the hESCs growth without dif -ferentiation, and is easy to store and use. Methods Mouse embryonic fibroblasts(MEF)were fixed on the surface of plate by methanol.hESCs were cultured on this new substrate and were passaged every 5 to 6 days.After 10 passages, we checked the cell morphology , alkaline phosphatase expression , embryonic specific markers and the differentiation ability in vitro.Results After 10 passages , the hESCs grew well on this new substrate and maintained the typical hESCs morpholo -gy.Alkaline phosphatase staining was positive .Immunofluorescence staining showed that the expressions of Oct 4, SSEA4, Tra-1-60 were positive .The cells formed embryoid body in vitro .Conclusions This methanol-fixed MEF substrate can support the growth of undifferentiated hESCs .The coating material can be produced in large scale and stored for a long time.It provides a new and relatively easy way to amplify hESCs .

Caracterização da proteína quinase C Beta I nuclear em células tronco embrionárias / Characterization of protein kinase C beta I in embryonic stem cell nucleus

As proteína quinases C (PKC) pertencem à família das serina/treonina quinases, que vem sendo apontadas como importantes enzimas para os processos de proliferação e diferenciação das células tronco embrionárias (CTE), todavia, a função exata de cada isoforma dessa família ainda não está clara. Dados anteriores do nosso laboratório indicam que dentre as PKCs expressas em CTE, formas cataliticamente ativas da PKCßI são altamente expressas no núcleo das CTE murinas. Estas ao se diferenciarem expressam essa quinase no seu citoplasma ou deixam de expressar a mesma, e que a maioria dos alvos da PKCßI em CTE indiferenciada estão envolvidos em processos de regulação da transcrição de proteínas envolvidas em processos de proliferação/ diferenciação. Dando continuidade aos resultados anteriores do laboratório, no presente trabalho, com técnicas de proteômica e fosfoproteômica identificamos outros alvos nucleares da PKCßI em CTE indiferenciadas. Vimos que de fato inibindo-se a PKCßI diminuiu-se a fostorilação de fatores envolvidos com a indiferenciação das CTE. Dentre os alvos da PKCßI encontramos a proteína adaptadora, TIF1 que recruta proteínas remodeladoras de cromatina. Essa proteína é essencial para a manutenção do estado indiferenciado das CTE. In vitro a PKCßI foi capaz de fosforilar a TIF1ß e inibindo-se a PKCßI por RNAi vimos uma diminuição na expressão da TIF1ß e no fator de indiferenciação Nanog cuja expressão já foi demonstrada ser regulada pela TIF1ß. Além disso vimos que inibindo-se a PKCßI com o peptídeo inibidor da PKCßI aumentou a expressão de proteínas reguladas pelo c-Myc. E que o RNAi para a PKCßI aumentou a expressão de proteínas que regulam a expressão do c-Myc. Não vimos nenhum efeito na fosforilação ou expressão do c-Myc após a inibição da PKCßI o que sugere que a PKCßI ative proteínas repressoras do c-Myc. Nossos estudos sugerem que a PKCßI regula a manutenção do estado indiferenciado das CTE regulando a expressão e atividade da Tif1ß um possível alvo direto da PKCßI. Levando a modificações da cromatina e regulação da expressão de genes que mantém as CTE indiferenciadas. Outro ponto de regulação da PKCßI parece ser a nibição da atividade de c-Myc o que seria importante para a manutenção do estado indiferenciado visto que o c-Myc é um amplificador das vias de sinalização que mantém as células proliferando. Desta forma a PKCßI parece ter um papel central na regulação da expressão gênica de CTE à nível de modificações epigenéticas e a nível transcricional mantendo as CTE indiferenciadas

The Protein kinase C (PKC) family of serine/treonine kinases, are being described as important enzymes for proliferation and diferentiation of embryonic stem cells (ESC), however, the exact function of the different isoenzymes of this family still is unclear. Previous data from our laboratory indicates that amongst the PKCs expressed in ESC, catalytically active forms of PKCßI are highly expressed in nucleus of murine ESC. When these cells differentiate this kinase can be found in the cytoplasm or not expressed at all, and that the majority of PKCßI targets in undifferentiated ESC are involved in the regulation of proteins involved in transcription of proteins involved in proliferation/ diferentiation. Continuing our previous work herewith using proteomics and phosphoproteomics techniques we identified other nuclear PKCßI targets in undifferentiated ESC. We indeed saw that inhibiting PKCßI decreased the phosphorylation of factors involved with maintainance of the undifferentiated state of ESC. Amongst the targets of PKCßI we found the adaptor protein, TIF1ßI, that recruits cromatin remodeling proteins. This protein is essential for the maintenance of the undifferentiated state of ESC. In vitro PKCßI phosphorylated TIF1ß and inhibiting PKCßI with RNAi decreased the expression of TIF1ß and of the undifferentiation factor Nanog whose expression has been shown to be regulated by TIF1ß. We also saw that inhibiting PKCßI with a peptide inhibitor increased the expression of proteins regulated by c-Myc, and that RNAi for PKCßI increased the expression of proteins that regulate the expression of c-Myc. We did not see any effect on the phosphorylation or expression of c-Myc after inhibition of PKCßI suggesting that PKCßI activates c-Myc repressor proteins. Our studies sugest that PKCßI regulates the maintenance of the undiferentiated state of ESC regulating the expression and activity of Tif1ß a possibly a direct target of PKCßI, leading to chromatin modifications and regulation of genes that maintain ESC undiferentiated. Another form of regulation of PKCßI seems to be by inhibiting the activity of c-Myc which is importante to maintain ESC undifferentiated since c-Myc is na an amplifyer of signaling patheways that maintain ESC proliferating. Together PKCßI has a central role in the regulation of the gene expression of ESC at the level of epigenetic modifications and transcriptional regulation

Efficacy of Microneedling Plus Human Stem Cell Conditioned Medium for Skin Rejuvenation: A Randomized, Controlled, Blinded Split-Face Study

BACKGROUND: The use of growth factors in skin rejuvenation is emerging as a novel anti-aging treatment. While the role of growth factors in wound healing is well established, their use in skin rejuvenation has only recently been to be studied and no controlled trials have been performed. OBJECTIVE: We evaluated the anti-aging effects of secretory factors of endothelial precursor cells differentiated from human embryonic stem cells (hESC-EPC) in Asian skin. METHODS: A total of 25 women were included in this randomized, controlled split-face study. The right and left sides of each participant's face were randomly allocated to hESC-EPC conditioned medium (CM) or saline. To enhance epidermal penetration, a 0.25-mm microneedle roller was used. Five treatment sessions were repeated at 2-week intervals. RESULTS: Physician's global assessment of pigmentation and wrinkles after treatment revealed statistically significant effects of microneedling plus hESC-EPC CM compared to microneedling alone (p<0.05). Skin measurements by Mexameter and Visiometer also revealed statistically significant effects of microneedling plus hESC-EPC CM on both pigmentation and wrinkles (p<0.05). The only minimal adverse event was mild desquamation in one participant. CONCLUSION: Secretory factors of hESC-EPC improve the signs of skin aging and could be a potential option for skin rejuvenation.