Pub Date : 2024-10-24DOI: 10.1016/j.ejcb.2024.151464
Human pluripotent stem cells (hPSCs) represent an unlimited source of β-like cells for both disease modeling and cellular therapy for diabetes. Numerous protocols have been published describing the differentiation of hPSCs into β-like cells that secret insulin in response to a glucose challenge. However, among the most widely used protocols it is not clear which yield the most functional cells with reproducible glucose-stimulated insulin-secretion (GSIS). Moreover, the technical challenges in culturing and differentiating hPSCs is a barrier for many researchers. In this study, we performed a side-by-side functional comparison based on three widely used methods to generate insulin expressing cells and identified optimal stages and conditions for cryopreserving and reconstituting stem cell (SC)-derived islets with a robust GSIS. Despite the fact that each protocol yields SC-islets consisting of insulin and glucagon-expressing cells, the SC-islets obtained from the two more recent revised protocols were more functional as measured by robust and reproducible GSIS. Moreover, we demonstrate that pancreatic progenitors and differentiated endocrine cells that have been cryopreserved for up to 10 months, can be reconstituted into glucose responsive SC-islets. These findings should enable the use of human PSC-derived β-like cells technologies even by groups with minimal PSC culture experience.
{"title":"Exploring optimal protocols for generating and preserving glucose-responsive insulin-secreting progenitor cells derived from human pluripotent stem cells","authors":"","doi":"10.1016/j.ejcb.2024.151464","DOIUrl":"10.1016/j.ejcb.2024.151464","url":null,"abstract":"<div><div>Human pluripotent stem cells (hPSCs) represent an unlimited source of β-like cells for both disease modeling and cellular therapy for diabetes. Numerous protocols have been published describing the differentiation of hPSCs into β-like cells that secret insulin in response to a glucose challenge. However, among the most widely used protocols it is not clear which yield the most functional cells with reproducible glucose-stimulated insulin-secretion (GSIS). Moreover, the technical challenges in culturing and differentiating hPSCs is a barrier for many researchers. In this study, we performed a side-by-side functional comparison based on three widely used methods to generate insulin expressing cells and identified optimal stages and conditions for cryopreserving and reconstituting stem cell (SC)-derived islets with a robust GSIS. Despite the fact that each protocol yields SC-islets consisting of insulin and glucagon-expressing cells, the SC-islets obtained from the two more recent revised protocols were more functional as measured by robust and reproducible GSIS. Moreover, we demonstrate that pancreatic progenitors and differentiated endocrine cells that have been cryopreserved for up to 10 months, can be reconstituted into glucose responsive SC-islets. These findings should enable the use of human PSC-derived β-like cells technologies even by groups with minimal PSC culture experience.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142561071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-24DOI: 10.1016/j.ejcb.2024.151465
Mesoderm induction is a crucial step for vascular cell specification, vascular development and vasculogenesis. However, the cellular and molecular mechanisms underlying mesoderm induction remain elusive. In the present study, a chemically-defined differentiation protocol was used to induce mesoderm formation and generate functional vascular cells including smooth muscle cells (SMCs) and endothelial cells (ECs) from human induced pluripotent stem cells (hiPSCs). Zebrafish larvae were used to detect an in vivo function of interleukin 10 (IL10) in mesoderm formation and vascular development. A three dimensional approach was used to create hiPSC-derived blood vessel organoid (BVO) and explore a potential impact of IL10 on BVO formation. A murine model hind limb ischemia was applied to investigate a therapeutic potential of hiPSC-derived cells treated with or without IL10 during differentiation. We found that IL10 was significantly and specifically up-regulated during mesoderm stage of vascular differentiation. IL10 addition in mesoderm induction media dramatically increased mesoderm induction and vascular cell generation from hiPSCs, whereas an opposite effect was observed with IL10 inhibition. Mechanistic studies revealed that IL10 promotes mesoderm formation and vascular cell differentiation by activating signal transducer and activator of transcription 3 signal pathway. Functional studies with an in vivo model system confirmed that knockdown of IL10 using morpholino antisense oligonucleotides in zebrafish larvae caused defective mesoderm formation, angiogenic sprouting and vascular development. Additionally, our data also show IL10 promotes blood vessel organoid development and enhances vasculogenesis and angiogenesis. Importantly, we demonstrate that IL10 treatment during mesoderm induction stage enhances blood flow perfusion recovery and increases vasculogenesis and therapeutic angiogenesis after hind limb ischemia. Our data, therefore, demonstrate a regulatory role for IL10 in mesoderm formation from hiPSCs and during zebrafish vascular development, providing novel insights into mesoderm induction and vascular cell specifications.
{"title":"An unexpected role of IL10 in mesoderm induction and differentiation from pluripotent stem cells: Implications in zebrafish angiogenic sprouting, vascular organoid development, and therapeutic angiogenesis","authors":"","doi":"10.1016/j.ejcb.2024.151465","DOIUrl":"10.1016/j.ejcb.2024.151465","url":null,"abstract":"<div><div>Mesoderm induction is a crucial step for vascular cell specification, vascular development and vasculogenesis. However, the cellular and molecular mechanisms underlying mesoderm induction remain elusive. In the present study, a chemically-defined differentiation protocol was used to induce mesoderm formation and generate functional vascular cells including smooth muscle cells (SMCs) and endothelial cells (ECs) from human induced pluripotent stem cells (hiPSCs). Zebrafish larvae were used to detect an in vivo function of interleukin 10 (IL10) in mesoderm formation and vascular development. A three dimensional approach was used to create hiPSC-derived blood vessel organoid (BVO) and explore a potential impact of IL10 on BVO formation. A murine model hind limb ischemia was applied to investigate a therapeutic potential of hiPSC-derived cells treated with or without IL10 during differentiation. We found that IL10 was significantly and specifically up-regulated during mesoderm stage of vascular differentiation. IL10 addition in mesoderm induction media dramatically increased mesoderm induction and vascular cell generation from hiPSCs, whereas an opposite effect was observed with IL10 inhibition. Mechanistic studies revealed that IL10 promotes mesoderm formation and vascular cell differentiation by activating signal transducer and activator of transcription 3 signal pathway. Functional studies with an <em>in vivo</em> model system confirmed that knockdown of IL10 using morpholino antisense oligonucleotides in zebrafish larvae caused defective mesoderm formation, angiogenic sprouting and vascular development. Additionally, our data also show IL10 promotes blood vessel organoid development and enhances vasculogenesis and angiogenesis. Importantly, we demonstrate that IL10 treatment during mesoderm induction stage enhances blood flow perfusion recovery and increases vasculogenesis and therapeutic angiogenesis after hind limb ischemia. Our data, therefore, demonstrate a regulatory role for IL10 in mesoderm formation from hiPSCs and during zebrafish vascular development, providing novel insights into mesoderm induction and vascular cell specifications.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142544577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18DOI: 10.1016/j.ejcb.2024.151462
Serge Mostowy, Theresia E B Stradal
{"title":"Editorial overview: EJCB Special Issue - Cell host-pathogen interactions.","authors":"Serge Mostowy, Theresia E B Stradal","doi":"10.1016/j.ejcb.2024.151462","DOIUrl":"https://doi.org/10.1016/j.ejcb.2024.151462","url":null,"abstract":"","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142497505","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18DOI: 10.1016/j.ejcb.2024.151463
Epigenetic editing is thriving as a robust tool for manipulating transcriptional regulation and cell fate. Despite its regulatory role in gene downregulation, epigenetic editing with histone deacetylation has been sparsely studied, especially in the context of cancer. In this current study, we have reconstructed a dCas9-HDAC8-EGFP fusion to perform histone deacetylation on the promoter of the ESR1, TERT and CDKN1C genes for the first time in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in HEK293T cells. Our results demonstrated that dCas9-HDAC8-EGFP in combination with appropriate gRNAs were able to downregulate the expression of the ESR1, TERT and CDKN1C genes transcriptionally by specifically depleting the H3K9ac level on the recruitment loci. The addition of histone deacetylase inhibitors was found to neutralize the outcomes of dCas9-HDAC8-EGFP-induced epigenetic editing. Furthermore, we observed a significant downregulation of full length ERα expression in epigenetically edited MCF-7 cells with consequential alteration in cellular response toward estradiol and tamoxifen treatment due to dCas9-HDAC8-EGFP mediated epigenetic editing of the ESR1 gene. Overall, dCas9-HDAC8-EGFP is a novel circuit that enabled downregulation of crucial genes with cellular outcome in breast cancer cells by preferentially inducing H3K9 deacetylation of specific promoter regions.
{"title":"dCas9-HDAC8-EGFP fusion enables epigenetic editing of breast cancer cells by H3K9 deacetylation","authors":"","doi":"10.1016/j.ejcb.2024.151463","DOIUrl":"10.1016/j.ejcb.2024.151463","url":null,"abstract":"<div><div>Epigenetic editing is thriving as a robust tool for manipulating transcriptional regulation and cell fate. Despite its regulatory role in gene downregulation, epigenetic editing with histone deacetylation has been sparsely studied, especially in the context of cancer. In this current study, we have reconstructed a dCas9-HDAC8-EGFP fusion to perform histone deacetylation on the promoter of the <em>ESR1</em>, <em>TERT</em> and <em>CDKN1C</em> genes for the first time in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in HEK293T cells. Our results demonstrated that dCas9-HDAC8-EGFP in combination with appropriate gRNAs were able to downregulate the expression of the <em>ESR1</em>, <em>TERT</em> and <em>CDKN1C</em> genes transcriptionally by specifically depleting the H3K9ac level on the recruitment loci. The addition of histone deacetylase inhibitors was found to neutralize the outcomes of dCas9-HDAC8-EGFP-induced epigenetic editing. Furthermore, we observed a significant downregulation of full length ERα expression in epigenetically edited MCF-7 cells with consequential alteration in cellular response toward estradiol and tamoxifen treatment due to dCas9-HDAC8-EGFP mediated epigenetic editing of the <em>ESR1</em> gene. Overall, dCas9-HDAC8-EGFP is a novel circuit that enabled downregulation of crucial genes with cellular outcome in breast cancer cells by preferentially inducing H3K9 deacetylation of specific promoter regions.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142497504","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.ejcb.2024.151461
During intracellular trafficking N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins catalyze the membrane fusion by assembling into a four-helix complex. In the mouse model, loss of the endosomal SNAREs vti1a and vti1b results in a perinatal lethal phenotype and neuronal defects including decreased neurite outgrowth in cultured primary neurons.
We used a CRISPR/Cas9 system to generate a Vti1a Vti1b double knockout (DKO) in the neuroblastoma cell line N1E-115. Three different DKO cell lines were obtained and examined at genome and protein level. The double deficiency impaired proper differentiation based on lower levels of synaptic proteins as well as reduced neurite formation and elongation compared to wild type cells in differentiation medium. Neurite elongation can be induced by a variety of extracellular signals via different signaling cascades. Treatment with the Rho kinase inhibitor Y27632, which stimulates enlargeosome exocytosis, or with neurotrophic factors (BDNF, NGF and NT3) resulted in reduced stimulation of all DKO clones and in significantly shorter neurites compared to wild type cells. The loss of vti1a and vti1b disrupted Akt signaling during enlargeosome-mediated and Erk signaling during BDNF-induced neurite outgrowth.
{"title":"The double deficiency of the SNARE proteins vti1a and vti1b affects neurite outgrowth and signaling in N1E-115 neuroblastoma cells.","authors":"","doi":"10.1016/j.ejcb.2024.151461","DOIUrl":"10.1016/j.ejcb.2024.151461","url":null,"abstract":"<div><div>During intracellular trafficking N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins catalyze the membrane fusion by assembling into a four-helix complex. In the mouse model, loss of the endosomal SNAREs vti1a and vti1b results in a perinatal lethal phenotype and neuronal defects including decreased neurite outgrowth in cultured primary neurons.</div><div>We used a CRISPR/Cas9 system to generate a <em>Vti1a Vti1b</em> double knockout (DKO) in the neuroblastoma cell line N1E-115. Three different DKO cell lines were obtained and examined at genome and protein level. The double deficiency impaired proper differentiation based on lower levels of synaptic proteins as well as reduced neurite formation and elongation compared to wild type cells in differentiation medium. Neurite elongation can be induced by a variety of extracellular signals via different signaling cascades. Treatment with the Rho kinase inhibitor Y27632, which stimulates enlargeosome exocytosis, or with neurotrophic factors (BDNF, NGF and NT3) resulted in reduced stimulation of all DKO clones and in significantly shorter neurites compared to wild type cells. The loss of vti1a and vti1b disrupted Akt signaling during enlargeosome-mediated and Erk signaling during BDNF-induced neurite outgrowth.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142425283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.ejcb.2024.151459
Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.
{"title":"The expression level of VEGFR2 regulates mechanotransduction, tumor growth and metastasis of high grade serous ovarian cancer cells","authors":"","doi":"10.1016/j.ejcb.2024.151459","DOIUrl":"10.1016/j.ejcb.2024.151459","url":null,"abstract":"<div><div>Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells <em>in vitro</em> and <em>in vivo</em>. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. <em>In vitro</em> data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142389093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.ejcb.2024.151460
Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.
{"title":"Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy","authors":"","doi":"10.1016/j.ejcb.2024.151460","DOIUrl":"10.1016/j.ejcb.2024.151460","url":null,"abstract":"<div><div>Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.ejcb.2024.151458
Mesenchymal Stem Cells (MSCs) derived from the embryonic mesoderm persist as a viable source of multipotent cells in adults and have a crucial role in tissue repair. One of the most promising aspects of MSCs is their ability to trans-differentiate into cell types outside of the mesodermal lineage, such as neurons. This characteristic positions MSCs as potential therapeutic tools for neurological disorders. However, the definition of a clear MSC signature is an ongoing topic of debate. Likewise, there is still a significant knowledge gap about functional alterations of MSCs during their transition to a neural fate. In this study, our focus is on the dynamic expression of RNA in MSCs as they undergo trans-differentiation compared to undifferentiated MSCs. To track and correlate changes in cellular signaling, we conducted high-throughput RNA expression profiling during the early time-course of human MSC neurogenic trans-differentiation. The expression of synapse maturation markers, including NLGN2 and NPTX1, increased during the first 24 h. The expression of neuron differentiation markers, such as GAP43 strongly increased during 48 h of trans-differentiation. Neural stem cell marker NES and neuron differentiation marker, including TUBB3 and ENO1, were highly expressed in mesenchymal stem cells and remained so during trans-differentiation. Pathways analyses revealed early changes in MSCs signaling that can be linked to the acquisition of neuronal features. Furthermore, we identified microRNAs (miRNAs) as potential drivers of the cellular trans-differentiation process. We also determined potential risk factors related to the neural trans-differentiation process. These factors include the persistence of stemness features and the expression of factors involved in neurofunctional abnormalities and tumorigenic processes. In conclusion, our findings contribute valuable insights into the intricate landscape of MSCs during neural trans-differentiation. These insights can pave the way for the development of safer treatments of neurological disorders.
{"title":"Paving the way to a neural fate – RNA signatures in naive and trans-differentiating mesenchymal stem cells","authors":"","doi":"10.1016/j.ejcb.2024.151458","DOIUrl":"10.1016/j.ejcb.2024.151458","url":null,"abstract":"<div><div>Mesenchymal Stem Cells (MSCs) derived from the embryonic mesoderm persist as a viable source of multipotent cells in adults and have a crucial role in tissue repair. One of the most promising aspects of MSCs is their ability to trans-differentiate into cell types outside of the mesodermal lineage, such as neurons. This characteristic positions MSCs as potential therapeutic tools for neurological disorders. However, the definition of a clear MSC signature is an ongoing topic of debate. Likewise, there is still a significant knowledge gap about functional alterations of MSCs during their transition to a neural fate. In this study, our focus is on the dynamic expression of RNA in MSCs as they undergo trans-differentiation compared to undifferentiated MSCs. To track and correlate changes in cellular signaling, we conducted high-throughput RNA expression profiling during the early time-course of human MSC neurogenic trans-differentiation. The expression of synapse maturation markers, including <em>NLGN2</em> and <em>NPTX1</em>, increased during the first 24 h. The expression of neuron differentiation markers, such as <em>GAP43</em> strongly increased during 48 h of trans-differentiation. Neural stem cell marker <em>NES</em> and neuron differentiation marker, including <em>TUBB3</em> and <em>ENO1</em>, were highly expressed in mesenchymal stem cells and remained so during trans-differentiation. Pathways analyses revealed early changes in MSCs signaling that can be linked to the acquisition of neuronal features. Furthermore, we identified microRNAs (miRNAs) as potential drivers of the cellular trans-differentiation process. We also determined potential risk factors related to the neural trans-differentiation process. These factors include the persistence of stemness features and the expression of factors involved in neurofunctional abnormalities and tumorigenic processes. In conclusion, our findings contribute valuable insights into the intricate landscape of MSCs during neural trans-differentiation. These insights can pave the way for the development of safer treatments of neurological disorders.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142327003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1016/j.ejcb.2024.151457
Pancreatic ductal adenocarcinoma is an extremely incurable cancer type characterized by cells with highly proliferative capacity and resistance against the current therapeutic options. Our study reveals that IRS1 acts as a bridging molecule between EGFR and IGFR/InsR signalization providing a potential mechanism for the interplay between signaling pathways and bypassing EGFR-targeted or IGFR/InsR-targeted therapies. The analysis of IRS1 phosphorylation status in four pancreatic cell lines identified the impact of EGFR signaling on IRS1 activation in comparison with InsR/IGFR signaling. Significantly reduced viability was observed in IRS1-silenced cells even upon EGF stimulation showing the critical role of IRS1 in the EGFR signaling network in both malignant and normal pancreatic cells. This study also demonstrated that EGFR binds directly to IRS1 and at least on two tyrosine sites, Y612 and Y896, IRS1 becomes phosphorylated in response to EGF stimulation. Mechanistically, the EGFR-mediated phosphorylation of IRS1 can further activate the MAPK signaling pathway with the recruitment of GRB2 protein. Collectively, in this study, IRS1 was identified as a crucial regulator in the EGFR signaling suggesting IRS1 as a potential target for more durable responses to targeted PDAC therapy.
{"title":"Insulin receptor substrate 1 is a novel member of EGFR signaling in pancreatic cells","authors":"","doi":"10.1016/j.ejcb.2024.151457","DOIUrl":"10.1016/j.ejcb.2024.151457","url":null,"abstract":"<div><div>Pancreatic ductal adenocarcinoma is an extremely incurable cancer type characterized by cells with highly proliferative capacity and resistance against the current therapeutic options. Our study reveals that IRS1 acts as a bridging molecule between EGFR and IGFR/InsR signalization providing a potential mechanism for the interplay between signaling pathways and bypassing EGFR-targeted or IGFR/InsR-targeted therapies. The analysis of IRS1 phosphorylation status in four pancreatic cell lines identified the impact of EGFR signaling on IRS1 activation in comparison with InsR/IGFR signaling. Significantly reduced viability was observed in IRS1-silenced cells even upon EGF stimulation showing the critical role of IRS1 in the EGFR signaling network in both malignant and normal pancreatic cells. This study also demonstrated that EGFR binds directly to IRS1 and at least on two tyrosine sites, Y612 and Y896, IRS1 becomes phosphorylated in response to EGF stimulation. Mechanistically, the EGFR-mediated phosphorylation of IRS1 can further activate the MAPK signaling pathway with the recruitment of GRB2 protein. Collectively, in this study, IRS1 was identified as a crucial regulator in the EGFR signaling suggesting IRS1 as a potential target for more durable responses to targeted PDAC therapy.</div></div>","PeriodicalId":12010,"journal":{"name":"European journal of cell biology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142319543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1016/j.ejcb.2024.151456
Our previous research revealed that androgen receptor (AR) activation reduces endothelial cell proliferation via non-genomic pathways. We hypothesized that AR activation might also affect endothelial cell migration, a critical step in angiogenesis. Our data demonstrates that treatment of human umbilical vein endothelial cells (HUVECs) with AR agonists, metribolone (R1881) or dihydrotestosterone (DHT), results in a dose-dependent reduction in migration, which can be reversed by AR antagonists or AR knockdown. Mechanistically, R1881 inhibits HUVEC migration by suppressing RhoA activity through the cSrc/FAK/paxillin pathway and promoting RhoA degradation via RhoA-p27 complex formation, ultimately resulting in RhoA ubiquitination. Transfection with constitutively active RhoA-V14 rescues the inhibitory effect of R1881 on HUVEC migration. Furthermore, R1881 elevates intracellular vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) levels but reduces VEGF secretion from HUVECs. This reduction is attributed to the formation of VEGF-CTGF complexes in the cytosol induced by R1881. Transfection with RhoA-V14 reduces CTGF levels and VEGF-CTGF complex formation, leading to enhanced VEGF secretion. Pre-treatment with WP631, a CTGF inhibitor, mitigates the R1881-induced reduction in VEGF secretion and HUVECs migration. In vivo assessments using zebrafish angiogenesis and mouse matrigel plug assays validate the anti-angiogenic effects of R1881. These findings provide insight into the molecular mechanisms through which AR activation modulates endothelial cell migration and angiogenesis.
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