IL-6 promotes pluripotency of mouse embryonic stem cells and regulates cardiac differentiation in a development-dependent manner / 生理学报

Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.

Generation of developmentally competent oocytes and fertile mice from parthenogenetic embryonic stem cells

Parthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.

Cardiac fibroblast paracrine factors modulate mouse embryonic stem cells / 生理学报

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.

Splitomicin, a SIRT1 Inhibitor, Enhances Hematopoietic Differentiation of Mouse Embryonic Stem Cells

BACKGROUND AND OBJECTIVES: Embryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells. METHODS AND RESULTS: Treatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells. CONCLUSIONS: Application of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.

Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes / 浙江大学学报·医学版

OBJECTIVE@#To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs).@*METHODS@#Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot.@*RESULTS@#Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (<0.01).@*CONCLUSIONS@#Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.

Effect of Notch Signaling Pathway on Differentiation of Mouse Embryonic Stem Cells into Hematopoietic Stem Cells or Hematopoietic Progenitor Cells by VEGF / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Notch signaling pathways on differentiation of mouse embryonic stem cells(ESC) into haematopoietic stem cells or haematopoietic progenitors cells(HSC/HPC).@*METHODS@#Mouse embryonic stem cells were proliferated in vitro to form embryoid bodies; the differentiation of embryoid bodies should be induced in vitro, the experiments were divided into BE, control, VEGF, DAPT and VEGF-DAPT groups; HSC/HPC ohenotype: CD117D34Sca1 was detected by flow cytometry; the related gene expression was detected by RT-PCR.@*RESULTS@#The number of VEGF-induced HSC/HPC in VEGF group was significantey higher than that in the control and EB group (P<0.05), suggesting that VEGF promotes ESC differentiation to HSC/HPC; the number of DAPT-induced HSC/HPC in DAPT group was significanty higher than that in the Control and EB groups(P<0.05), suggesting that DAPT promotes ESC differentiation to HSC/HPC; the number of VEGF+DAPT-induced HSC/HPC in VEGF-DAPT group was significantly higher than that in VEGF and DAPT groups(P<0.05), suggesting that DAPT and VEGF play a synergistic role to promote differentiation of ESC into HSC/HPC.@*CONCLUSION@#Notch signal pathway inhibits differentiation of ESC into HSC / HPC by VEGF.

Determining Osteogenic Differentiation Efficacy of Pluripotent Stem Cells by Telomerase Activity

BACKGROUND: Bone tissue engineering based on pluripotent stem cells (PSCs) is a new approach to deal with bone defects. Protocols have been developed to generate osteoblasts from PSCs. However, the low efficiency of this process is still an important issue that needs to be resolved. Many studies have aimed to improve efficiency, but developing accurate methods to determine efficacy is also critical. Studies using pluripotency to estimate efficacy are rare. Telomerase is highly associated with pluripotency. METHODS: We have described a quantitative method to measure telomerase activity, telomeric repeat elongation assay based on quartz crystal microbalance (QCM). To investigate whether this method could be used to determine the efficiency of in vitro osteogenic differentiation based on pluripotency, we measured the pluripotency pattern of cultures through stemness gene expression, proliferation ability and telomerase activity, measured by QCM. RESULTS: We showed that the pluripotency pattern determined by QCM was similar to the patterns of proliferation ability and gene expression, which showed a slight upregulation at the late stages, within the context of the general downregulation tendency during differentiation. Additionally, a comprehensive gene expression pattern covering nearly every stage of differentiation was identified. CONCLUSION: Therefore, this assay may be powerful tools for determining the efficiency of differentiation systems based on pluripotency. In this study, we not only introduce a new method for determining efficiency based on pluripotency, but also provide more information about the characteristics of osteogenic differentiation which help facilitate future development of more efficient protocols.

Terapias alternativas para o diabetes mellitus tipo 1: caracterização funcional do gene Txnip na diferenciação ß-pancreática e desenvolvimento de biomaterial inovador para microencapsulamento celular / Alternative therapies for type 1 diabetes mellitus: functional characterization of Txnip gene during -pancreatic differentiation and generation of an innovative biomaterial for cell microencapsulation

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-ß produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação ß-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação ß-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-ß através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1

Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic ß-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the ß-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of ß-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of ß-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients

Effects of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system-Based Deletion of miR-451 in Mouse Embryonic Stem Cells on Their Self-Renewal and Hematopoietic Differentiation

Pluripotent stem cells (PSCs) are a useful source of cells for exploring the role of genes related with early developmental processes and specific diseases due to their ability to differentiate into all somatic cell types. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system has proven to be a robust tool for targeted genetic modification. Here, we generated miR-451-deficient PSCs using the CRISPR/Cas9 system with PCR-based homologous recombination donor and investigated the impact of its deletion on self-renewal and hematopoietic development. CRISPR/Cas9-mediated miR-451 knockout did not alter the gene expressions of pluripotency, cellular morphology, and cell cycle, but led to impaired erythrocyte development. These findings propose that a combination of PSCs and CRISPR/Cas9 system could be useful to promote biomedical applications of PSCs by elucidating the function and manipulating of specific miRNAs during lineage specification and commitment.

Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

Thymosin β4 impeded murine stem cell proliferation with an intact cardiovascular differentiation / 华中科技大学学报（医学）（英德文版）

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.

Mitochondrial pyruvate dehydrogenase phosphatase 1 regulates the early differentiation of cardiomyocytes from mouse embryonic stem cells

Mitochondria are crucial for maintaining the properties of embryonic stem cells (ESCs) and for regulating their subsequent differentiation into diverse cell lineages, including cardiomyocytes. However, mitochondrial regulators that manage the rate of differentiation or cell fate have been rarely identified. This study aimed to determine the potential mitochondrial factor that controls the differentiation of ESCs into cardiac myocytes. We induced cardiomyocyte differentiation from mouse ESCs (mESCs) and performed microarray assays to assess messenger RNA (mRNA) expression changes at differentiation day 8 (D8) compared with undifferentiated mESCs (D0). Among the differentially expressed genes, Pdp1 expression was significantly decreased (27-fold) on D8 compared to D0, which was accompanied by suppressed mitochondrial indices, including ATP levels, membrane potential, ROS and mitochondrial Ca²⁺. Notably, Pdp1 overexpression significantly enhanced the mitochondrial indices and pyruvate dehydrogenase activity and reduced the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate compared to a mock control. In confirmation of this, a knockdown of the Pdp1 gene promoted the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate. In conclusion, our results suggest that mitochondrial PDP1 is a potential regulator that controls cardiac differentiation at an early differentiation stage in ESCs.

Bases moleculares da depleção de glutationa sobre a pontencialidade, diferenciação e envelhecimento de células-tronco de pele / Molecular basis of glutathione depletion upon the potenciality, differentiation potential and aging of skin stem cells

A pele está em contínua auto-renovação graças a vários nichos de células-tronco presentes neste tecido. Células progenitoras epidérmicas surgem durante o desenvolvimento embrionário e contribuem para a reposição celular da epiderme durante todo o período de vida dos mamíferos. Neste trabalho, buscou-se analisar o papel da depleção de glutationa durante a estratificação da epiderme embrionária e na manutenção da homeostase no tecido adulto. Encontramos evidências de que este tiol tem um importante papel durante a proliferação da epiderme e formação do folículo capilar. As alterações observadas na ausência de GSH foram relacionados com um padrão diferencial de fosforilação dos fatores de transcrição forkhead-homeobox- tipo-O (FOXO). Em resumo, foi estabelecida uma correlação entre o estado de GSH, a fosforilação de FOXO e o desenvolvimento da epiderme. Para melhor estudar a importância do balanço de GSH, na pele do adulto, e seu papel na manutenção deste tecido, camundongos foram tratados com um inibidor da síntese de GSH e, com N-aceti-lcisteína. Foi observado um aumento da fosforilação de Akt, padrões alterados de fosforilação FOXO e aumento da expressão de genes de genes relacionados à diferenciação. Estes resultados mostram que a via Akt/FOXO desempenha um papel importante na manutenção e diferenciação de células-tronco epidermais. O envelhecimento cronológico leva a alterações morfológicas/funcionais que conduzem à diminuição da auto-renovação, o que ocorre concomitantemente com uma diminuição dos níveis de GSH na pele. Utilizamos, também, animais idosos e avaliamos quais mecanismos eram compartilhados pelo envelhecimento e a depleção deste tiol. Observou-se que uma resposta hiperproliferativa ligada à exaustão de células-tronco pode ser o elo entre a depleção de GSH e o envelhecimento. A influência desse processo também foi investigada no compartimento dérmico, através da análise do impacto da depleção de glutationa sobre a osteogênese de células-tronco mesenquimais murinas. Quando induzidas a se diferenciarem em osso (Alizarin-Red+/Von-Kossa-stain +, aumento dos níveis de mRNA para fosfatase alcalina/osteopontina/osterix), o balanço GSH/GSSG e seu sistema antioxidante correlato é diferencialmente regulado em células-tronco mesenquimais derivadas da derme. Sendo regulado de uma forma redox-dependente através da via de MAPKs. A depleção de GSH leva à diminuição nos níveis de osteogênese em favor da adipogênese, levando ao processo comumente associado ao envelhecimento denominado "adipogenic switc". Em conclusão, os dados obtidos permitem propor um papel central para a glutationa na manutenção/comprometimento de células-tronco na pele

The skin is continuously self-renewing thanks to several stem cell niches. Epidermal progenitor cells arise during embryonic development and contribute to the replenishment of the epidermis during the lifetime of mammals. We set out to analyze the glutathione (GSH) antioxidant system during embryonic epidermis stratification and follicle development and the effect of glutathione withdrawal in this process. We found that glutathione plays an important role during epidermis proliferation and hairshaft formation. The changes observed in the absence of GSH were related to a differential phosphorylation pattern of the forkhead-homeobox-type-O (FOXO) transcription factors. In brief, a correlation between GSH status, FOXO phosphorylation and skin development was established. To further study the importance of GSH in adult skin maintenance and understand the effects of ROS in the Akt/FOXO pathway, we treated cells and mice with an inhibitor of GSH synthesis, and with N-acetyl-cysteine. Increased Akt phosphorylation, altered FOXO phosphorylation patterns and increased gene expression of differentiation-related genes were observed. Our results show that the Akt/FOXO pathway plays an important role in maintenance/differentiation of epidermal stem cells. Chronological ageing leads to morphological/functional changes causing a decline in self-renewal, as well as decreased levels of GSH. We also observed that a cell cycle hyperproliferative response was the link between stem cell exaustion in GSH-depletion and ageing. Dermal mesenchymal stem cells (MSCs), are capable of adipo-chondro- and osteogenesis. Little is known about the impact of ROS in MSC differentiation. We induced murine skin MSCs to differentiate into bone (Alizarin-Red/Von-Kossastain+, increased levels of mRNA for alkalinephosphatase/ osteopontin/osterix). In brief, the balance of GSH/GSSG and related antioxidant system is differentially regulated during this process, found to be regulated in a redox-dependent fashion through the MAPK pathway. When depleted, GSH leads to an adipogenic switch in MSC differentiation. In conclusion, our data leads us to propose a central role for glutathione in the maintenance/commitment of stem cells in skin

Diferenciação de células-tronco embrionárias murinas (mESCs) em células produtoras de insulina (IPCs) e caracterização funcional do gene Purkinje cell protein 4 (Pcp4) neste processo / Differentiation of murine embryonic stem cells (mESCs) into insulin-producing cells (IPCs) and functional characterization of the Purkinje Cell Protein 4 (Pcp4) gene in this process

Fontes alternativas de células ß têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células ß-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células ß. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células ß e no processo de diferenciação ß-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoß-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células ß-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células

New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic ß-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to ß-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in ß-cells and in ß-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to ß-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic ß-cells and possibly being related to maintenance of cell viability and ß-cell maturation

Expression of imprinted genes during the course of differentiation from mouse embryonic stem cells to islet-like cells in vitro / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of in vitro inducement on the expression of SF1-G imprinted genes, Kcnq1 and Cdkn1c during the course of differentiation from mouse embryonic stem (ES) cells to islet-like cells.</p><p><b>METHODS</b>Mouse ES cells were induced to differentiate into islet-like cells in vitro. The expression of islet specific markers was tested by RT-PCR or immunofluorescence. RT-PCR/RFLP was used to test the imprinted genes parental expression in cells at different stages.</p><p><b>RESULTS</b>Islet specific genes, such as Insulin, Glucagon, Somatostatin, IAPP and Glut2, were expressed in differentiated cells. The proteins of insulin, C-peptide and Somastatin were expressed in the final stage cells. Imprinted gene Kcnq1 and Cdkn1c were biallelicly expressed in islet-like cells.</p><p><b>CONCLUSIONS</b>Mouse ES cells can be successfully induced into islet-like cells in vitro. Gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells (causing loss of imprinting) during the in vitro inducement.</p>