scholarly journals Construction and characterization of EGFP reporter plasmid harboring putative human RAX promoter for in vitro monitoring of retinal progenitor cells identity

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Atefeh Atefi ◽  
Pendar Shojaei Kojouri ◽  
Fereshteh Karamali ◽  
Shiva Irani ◽  
Mohammad Hossein Nasr-Esfahani
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Retinal Layer ◽  
Retinal Progenitor Cells ◽  
Regulatory Regions ◽  
Retina Development ◽  
Retinal Progenitor ◽  
Egfp Reporter

Abstract Background In retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene (RAX) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated. Results For this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293 T cells and then in hRPCS derived from human embryonic stem cells. The candidate region (RAX-3258 bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for in vitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells. Conclusions Furthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development.

scholarly journals Construction and Characterization of EGFP Reporter Plasmid Harboring Putative Human RAX Promoter for in vitro Monitoring of Retinal Progenitor Cells Identity

2020 ◽  
Author(s):  
Atefeh Atefi ◽  
Pendar Shojaei kojouri ◽  
Fereshteh Karamali ◽  
Shiva Irani ◽  
Mohammad Hossein Nasr Esfahani
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Retinal Layer ◽  
Retinal Progenitor Cells ◽  
Regulatory Regions ◽  
Retina Development ◽  
Retinal Progenitor ◽  
Egfp Reporter

Abstract Background : In retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene ( RAX ) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated. Results: For this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293T cells and then in hRPCS derived from human embryonic stem cells. The candidate region ( RAX -3258bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for invitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells. Conclusions: Furthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development.

scholarly journals Construction and Characterization of EGFP Reporter Plasmid Harboring Putative Human RAX Promoter for in Vitro Monitoring of Retinal Progenitor Cells Identity

2020 ◽  
Author(s):  
Atefeh Atefi ◽  
Pendar Shojaei kojouri ◽  
Fereshteh Karamali ◽  
Shiva Irani ◽  
Mohammad Hossein Nasr Esfahani
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Retinal Layer ◽  
Retinal Progenitor Cells ◽  
Regulatory Regions ◽  
Retina Development ◽  
Retinal Progenitor ◽  
Egfp Reporter

Abstract Background: In retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene ( RAX ) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated. Results: For this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293T cells and then in hRPCS derived from human embryonic stem cells. The candidate region ( RAX -3258bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for invitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells. Conclusions: Furthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development.

scholarly journals Human embryonic stem cell-derived extracellular vesicles alleviate retinal degeneration by upregulating Oct4 to promote retinal Müller cell retrodifferentiation via HSP90

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yifeng Ke ◽  
Xiaoe Fan ◽  
Rui Hao ◽  
Lijie Dong ◽  
Min Xue ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Extracellular Vesicles ◽  
Fluorescence Intensity ◽  
Embryonic Stem ◽  
Müller Cells ◽  
Muller Cells ◽  
Retinal Progenitor Cells ◽  
Retinal Progenitor ◽  
Hsp90 Expression ◽  
Rcs Rats

Abstract Objective Retinal degenerative diseases remain the dominant causes of blindness worldwide, and cell replacement is viewed as a promising therapeutic direction. However, the resources of seed cells are hard to obtain. To further explore this therapeutic approach, human embryonic stem extracellular vesicles (hESEVs) were extracted from human embryonic stem cells (hESCs) to inspect its effect and the possible mechanism on retinal Müller cells and retinal function. Methods hESEVs were extracted by multi-step differential centrifugation, whose morphologies and specific biomarkers (TSG101, CD9, CD63, and CD81) were observed and measured. After hESEVs were injected into the vitreous cavity of RCS rats, the retinal tissues and retinal functions of rats were assessed. The alteration of Müller cells and retinal progenitor cells was also recorded. Microvesicles (MVs) or exosomes (EXOs) were extracted from hESCs transfected with sh-HSP90 or pcDNA3.1-HSP9, and then incubated with Müller cells to measure the uptake of EVs, MVs, or EXOs in Müller cells by immunofluorescence. The retrodifferentiation of Müller cells was determined by measuring Vimentin and CHX10. qRT-PCR and western blot were used to detect HSP90 expression in MVs and evaluate Oct4 level in Müller cells, and Co-IP to inspect the interaction of HSP90 and Oct4. Results RCS rats at the postnatal 30 days had increased retinal progenitor cells which were dedifferentiated from Müller cells. hESEVs were successfully extracted from hESCs, evidenced by morphology observation and positive expressions of specific biomarkers (TSG101, CD9, CD63, and CD81). hESEVs promoted Müller cells dedifferentiated and retrodifferentiated into retinal progenitor cells evidenced by the existence of a large amount of CHX10-positive cells in the retinal inner layer of RCS rats in response to hESEV injection. The promotive role of hESEVs was exerted by MVs demonstrated by elevated fluorescence intensity of CHX10 and suppressed Vimentin fluorescence intensity in MVs rather than in EXOs. HSP90 in MVs inhibited the retrodifferentiation of Müller cells and suppressed the expression level of Oct4 in Müller cells. Co-IP revealed that HSP90 can target Oct4 in Müller cells. Conclusion hESEVs could promote the retrodifferentiation of Müller cells into retinal progenitor cells by regulating the expression of Oct4 in Müller cells by HSP90 mediation in MVs.

Abstract 1964: Bone Morphogenetic Protein Antagonist “Protein Related to Dan and Cerberus” Promotes Differentiation of Embryonic Stem Cells to Atrial Cardiomyocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jingbo Yan ◽  
Jianyong Hu ◽  
Iris I Mueller ◽  
William H Heaton ◽  
Wan-Der Wang ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Bmp Signaling ◽  
Cardiac Differentiation ◽  
Cardiovascular Cells

The molecular factors that regulate cardiac differentiation have been extensively studied, yet, relatively little is known about how cardiomyocytes acquire atrial versus ventricular characteristics. Embryonic stem (ES) cells, which have the potential to differentiate to a wide array of distinct cell types, including most types of cardiovascular cells, offer a pertinent in vitro model to work out the molecular mechanisms of atrial specification and differentiation. We discovered that the secreted antagonist of BMP signaling, Protein Related to Dan and Cerberus (PRDC, also called Gremlin2) leads to a surge in cardiomyocytic differentiation when applied to mouse ES-derived cardiac progenitor cells. This property is unique to PRDC among tested BMP antagonists. Lineage expansion is restricted to cardiomyocytes, with the differentiation of endodermal, blood, endothelial and neuronal cells being unaffected. Using molecular and electrophysiological analyses, we show that PRDC-induced cardiomyocytes acquire atrial characteristics. Consistent with the in vitro results, we found that injection of PRDC mRNA into the developing zebrafish embryo leads to supernumerary contracting areas. The ectopic cardiomyocytes express atrial-, but not ventricular- specific cardiac genes. We determined that PRDC treatment induces the expression of COUP-TFII, a known transcriptional regulator of atrial differentiation, but suppresses Notch signaling. Inhibition of Notch is sufficient to induce atrial-specific genes; however, blocking Notch does not expand the cardiogenic fields. Taken together, our data suggest that antagonism of BMP and Notch signaling by PRDC is a critical early step in the specification, expansion and differentiation of atrial progenitor cells. This information might be relevant for treating atrial degeneration, as well as for understanding the etiology of atrial fibrillation.

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