BackgroundEndothelial cells have been shown to mediate angiogenesis in ischemic injury sites and contribute to the repair of damaged tissues. However, the treatment of ischemic disease requires a significant number of endothelial cells, which are difficult to isolate from patients. Embryonic stem cells have been considered a potential source of therapeutic cells due to their unlimited self-renewal and pluripotent properties. With regard to vascular development, Notch1 has been established as a key regulator of the specification of arterial endothelial cells.MethodsUsing a doxycycline-induced expression system of the intracellular domain of Notch1, we explored the role of Notch1 in the differentiation of embryonic stem cells to arterial endothelial cells. The therapeutic effect of the arterial endothelial cells was investigated in a murine hindlimb ischemia model. The blood perfusion rate in the ischemic limb was determined by laser Doppler perfusion imaging, and vasculogenesis was quantified using immunocytochemistry.ResultsInduced expression of the intracellular domain of Notch1 increased the levels of endothelial markers, such as CD31 and VE-cadherin, in differentiated endothelial cells. Induction of intracellular domain of Notch1 stimulated expression of the arterial-type endothelial cell markers (Nrp1 and Ephrin B2), but not the venous-type endothelial cell markers (Nrp2 and Coup-TFII). In addition, overexpression of intracellular domain of Notch1 resulted in increased expression of CXCR4, a chemokine receptor involved in vascular development. Induction of intracellular domain of Notch1 increased endothelial tube formation and migration of differentiated endothelial cells. Intramuscular administration of Notch1-induced arterial endothelial cells was more effective than administration of the control endothelial cells in restoring the blood flow in an ischemic hindlimb mouse model. Transplantation of Notch1-induced arterial endothelial cells augmented the number of blood vessels and incorporation of endothelial cells into newly formed blood vessels.ConclusionsThese results suggest that Notch1 promotes endothelial maturation and arterial specification during the differentiation of embryonic stem cells to endothelial cells and increases the angiogenic potential of endothelial cells.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0945-7) contains supplementary material, which is available to authorized users.
Oct4 plays a crucial role in the regulation of self-renewal of embryonic stem cells (ESCs) and reprogramming of somatic cells to induced pluripotent stem cells. However, the molecular mechanisms underlying posttranslational regulation and protein stability of Oct4 remain unclear. Using affinity purification and mass spectrometry analysis, we identified Kap1 as an Oct4-binding protein. Silencing of Kap1 reduced the protein levels of Oct4 in ESCs, whereas the overexpression of Kap1 stimulated the levels of Oct4. In addition, Kap1 overexpression stimulated the self-renewal of ESCs and attenuated the spontaneous differentiation of ESCs in response to LIF withdrawal. Kap1 overexpression increased the stability of Oct4 by inhibiting the Itch-mediated ubiquitination of Oct4. Silencing of Kap1 augmented Itch-mediated ubiquitination and inhibited the stability of Oct4. We identified the lysine 133 (K133) residue in Oct4 as a ubiquitination site responsible for the Kap1-Itch-dependent regulation of Oct4 stability. Preventing ubiquitination at the lysine residue by mutation to arginine augmented the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. These results suggest that Kap1 plays a crucial role in the regulation of the pluripotency of ESCs and somatic cell reprogramming by preventing Itchmediated ubiquitination and the subsequent degradation of Oct4.
Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.
Background Peripheral artery disease is an ischemic vascular disease caused by the blockage of blood vessels supplying blood to the lower extremities. Mesenchymal stem cells (MSCs) and endothelial colony-forming cells (ECFCs) have been reported to alleviate peripheral artery disease by forming new blood vessels. However, the clinical application of MSCs and ECFCs has been impeded by their poor in vivo engraftment after cell transplantation. To augment in vivo engraftment of transplanted MSCs and ECFCs, we investigated the effects of hybrid cell spheroids, which mimic a tissue-like environment, on the therapeutic efficacy and survival of transplanted cells. Methods The in vivo survival and angiogenic activities of the spheroids or cell suspension composed of MSCs and ECFCs were measured in a murine hindlimb ischemia model and Matrigel plug assay. In the hindlimb ischemia model, the hybrid spheroids showed enhanced therapeutic effects compared with the control groups, such as adherent cultured cells or spheroids containing either MSCs or ECFCs. Results Spheroids from MSCs, but not from ECFCs, exhibited prolonged in vivo survival compared with adherent cultured cells, whereas hybrid spheroids composed of MSCs and ECFCs substantially increased the survival of ECFCs. Moreover, single spheroids of either MSCs or ECFCs secreted greater levels of pro-angiogenic factors than adherent cultured cells, and the hybrid spheroids of MSCs and ECFCs promoted the secretion of several pro-angiogenic factors, such as angiopoietin-2 and platelet-derived growth factor. Conclusion These results suggest that hybrid spheroids containing MSCs can serve as carriers for cell transplantation of ECFCs which have poor in vivo engraftment efficiency.
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