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Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

ABSTRACT: Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

PROVIDER: PRJNA1165675 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30805715.fastq.gz	Fastqsanger.gz
	SRR30805716.fastq.gz	Fastqsanger.gz
	SRR30805717.fastq.gz	Fastqsanger.gz
	SRR30805718.fastq.gz	Fastqsanger.gz
	SRR30805719.fastq.gz	Fastqsanger.gz

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Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

Project description:With multiple groups generating methods for differentiating induced pluripotent stem cells (hiPSCs) into endothelial cells/endothelial progenitors (ECs/EPs), there is a need to better understand the specific endothelial subtypes that different differentiation protocols produce. This is especially important for accurate tissue modeling as researchers continue to incorporate the endothelium into engineered tissues. We illustrated the heterogeneity of cells produced from different differentiation protocols by focusing on two selected protocols, one driving differentiation exclusively using small molecules (SM Protocol) and one driving differentiation primarily using growth factors (GF Protocol). We characterized the cells through a combination of vasculogenic computational analysis following encapsulation in 3D hydrogels, RNA sequencing, and measurement of soluble factor release. Vasculogenic computational analysis indicates that cells from the GF protocol formed more dense and interconnected vasculature compared to cells from the SM protocol. Likewise, RNA-seq analysis showed GF-derived cell enrichment in pathways involved in cell migration and angiogenesis. In addition, GF-derived cells favor differentiation to arterial endothelial cells as well as predisposition to undergoing a partial endothelial-to-mesenchymal transition, whereas SM-derived cells resemble immature progenitors based on higher proliferation rates as well as ECM remodeling. These trends also persisted following extended 3D culture. The results demonstrate that despite using the same starting hiPSC population and isolating EPs using the same surface marker, the two differentiation protocols yield highly distinct cell populations. This work highlights the importance of understanding the specific endothelial subtypes produced by different differentiation protocols for the creation of more accurate tissue models.

2024-09-26 | GSE278088 | GEO

Closing the mitochondrial permeability transition pore in hiPSC-derived endothelial cells induces glycocalyx formation and functional maturation.

Project description:Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSC, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable to those in primary microvascular EC. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing and signalling. We noted however, that hiPSC-EC have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species (ROS). Closure of these pores by cyclosporine-A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These indicated that mitochondrial maturation is required for proper hiPSC-EC functionality.

2019-12-14 | E-MTAB-8392 | biostudies-arrayexpress

Reliability of human retinal organoid generation from hiPSC-derived neuroepithelial cysts

Project description:The application potential of human induced pluripotent stem cells (hiPSC) derived human retinal organoids (HRO) relies on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare and optimize organoid protocols have been started to be established, but are not completed yet. To advance this, we explored the efficiency and reliability of a differentiation protocol that facilitates retina generation by formation of neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines, which reproducibly generated HROs. Histologic and ultrastructural analyses support regulated HRO differentiation and maturation. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Whereas previous reports showed that HRO in several other protocols contain a rather low numbers of cones compared to rods, HROs derived by the cyst protocol consistently are cone-richer and with an comparable ratio of cones, rods, and Müller glia. Additionally, we devised a potential strategy to systematically evaluate different protocols side-by-side through parallel differentiation from the same hiPSC batches: The cyst-protocol was compared to a conceptually different protocol based on cell aggregate formation from single hiPSCs. Comparison of four hIPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the cyst-protocol provided a higher HRO yield. Further, while cyst-derived HROs maintained stable at least up to date 250, whereas single hiPSC-derived HROs showed spontaneous pathologic changes already by day 200. Overall, our data provide insight into the efficiency, reproducibility, and stability of the cyst-protocol for HRO generation, which will be useful for further organoid system optimization, as well as basic and translational research applications.

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Project description:Five hiPSC lines from four individuals were differentiated to neural progenitor cells. The two cell types were compared in regards to marker gene expression of known cell type markers to show that the used bulk RNA-seq protocol, called prime-seq can capture known biological differences.

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Project description:Human induced pluripotent stem cells (hiPSCs) have the ability to differentiate into a variety of cells and to self-renew in vitro. Because of these two characteristics, hiPSCs have been expected to provide new applications for regenerative medicine/cell therapy. Although various in vitro differentiation protocols have been developed for the efficient derivation of specific cell types, hiPSC lines vary in their ability to differentiate into specific lineages. Therefore, surrogate markers that accurately predict the differentiation propensity of hiPSC lines could be helpful for the development and manufacture of hiPSC-derived cells for therapies and in vitro assays. Here, we tried to identify the marker genes that potentially predicts the differentiation propensity of hiPSCs into neural progenitor cells (NPCs). Using ten hiPSC lines, we searched for genes significantly correlated between expression levels in the undifferentiated state and neuronal differentiation efficiency using two differentiation induction methods, and selected genes that were commonly and predominantly correlated with neuronal differentiation. Among the genes correlated with NPC differentiation, we identified ROR2 as a novel predictive marker of NPC differentiation. ROR2 expression in hiPSCs correlates negatively with NPC differentiation capacity, and ROR2 knockdown enhances NPC differentiation. These findings suggest that ROR2 serves as a useful surrogate marker for selecting hiPSC lines appropriate for NPC differentiation.

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