Figure 1. Workflow for Generating Blood Vessel Organoids Using the STEMdiff™ Blood Vessel Organoid Kit
Human pluripotent stem cells (hPSCs) maintained in mTeSR™1 or mTeSR™ Plus are seeded as single cells in 6-well ultra-low attachment plates using STEMdiff™ Blood Vessel Organoid Aggregation Medium. mTeSR™1-maintained hPSCs are seeded at 0.2 - 0.4 x 10⁶ cells/well and mTeSR™ Plus-maintained hPSCs at 0.1 - 0.2 x 10⁶ cells/well. After 1 - 2 days, differentiation is initiated by switching to STEMdiff™ Blood Vessel Organoid Mesoderm Medium for 3 days. On day 4 - 5, vascular induction is performed by replacing the medium with STEMdiff™ Blood Vessel Organoid Vascular Induction Medium and culturing for 2 days. Resulting aggregates are embedded in a collagen-Matrigel® sandwich, where they sprout and mature into vascular networks in STEMdiff™ Blood Vessel Organoid Maturation Medium over 5 days. Free-floating organoids can be collected at day 15 for downstream assays.
Figure 2. Vascular Aggregates Sprout into Vascular Networks and Form Mature Blood Vessel Organoids in STEMdiff™ Blood Vessel Growth Medium
(A) Vascular aggregates generated using the STEMdiff™ Blood Vessel Organoid Kit sprout into interconnected vascular networks after 5 days in STEMdiff™ Blood Vessel Maturation Medium within a collagen-Matrigel® sandwich. Sprouts mature into blood vessel organoids with continued culture in STEMdiff™ Blood Vessel Maturation Medium. Magnification: 10X; Inset: 25X; anti-human CD31 (red). (B) 3D reconstruction of z-stack planes shows complex vasculature is formed after 22 days in STEMdiff™ Blood Vessel Organoid Maturation Medium and free-floating conditions. Magnification: 10X; anti-human CD31 (green). (C) hPSC-derived blood vessel organoids are composed of hCD31+ cells (green) and hPDGFRβ+ cells (magenta). The inset highlights tight endothelial-pericyte interactions. (D) hPSC-derived organoids contain hCD31⁺ endothelial cells (red) surrounded by collagen IV (green). 3D reconstruction of optical z-stacks reveals a defined vessel lumen as shown in the inset. (E) hPSC-derived organoids also include hCD31⁺ endothelial cells (blue) and α-smooth muscle actin⁺ cells (magenta).
Figure 3. Blood Vessel Organoids Contain Both Endothelial Cells and Pericytes
(A) Generated blood vessel organoids contain endothelial cells (42.92 ± 3.97% CD31+, n = 3 - 4) and pericytes (43.10 ± 3.31% CD140b+, n = 3 - 4) in mTeSR™ 1-maintained cell lines WLS-1C (hiPSC), STiPS-M001 (hiPSC), H9 (hESC), H7 (hESC) and H1 (hESC). (B) hiPSC- and hESC-derived blood vessel organoids contain endothelial cells (54.29 ± 3.72% CD31+, n = 3) and pericytes in mTeSR™ Plus-maintained cell lines WLS-1C (iPSC), STiPS-M001 (hiPSC) and H7 (hESC).
Figure 4. Blood Vessel Organoids Generated with the STEMdiff™ Blood Vessel Organoid Kit Exhibit Extracellular Matrix Remodeling Under Diabetic Conditions
(A) Blood vessel organoids generated using the STEMdiff™ Blood Vessel Organoid Kit show increased collagen IV deposition under diabetic conditions. Confocal imaging reveals CD31⁺ endothelial tubes (red) surrounded by expanded collagen IV basement membrane (green). Among the small molecules tested, only DAPT and forskolin significantly reduced collagen IV expansion. (B) Collagen IV thickness is increased in diabetic versus non-diabetic organoids (n = 100 vessels per condition). Data represent one independent experiment across two iPSC and two ESC lines (mean ± SD; p < 0.0001). (C) Collagen IV thickness was measured across non-diabetic organoids (n = 50), diabetic vehicle controls (n = 73), and diabetic organoids treated with SB431542, Y-27632, CHIR99021, DAPT, or forskolin (n = 100). Only forskolin and DAPT reduced collagen IV expansion (mean ± SD; p < 0.0001).
Modeling Aberrant Angiogenesis in Arteriovenous Malformations Using Endothelial Cells and Organoids for Pharmacological Treatment
E. J. Oh et al.
Cells 2025 Jul
Abstract
Arteriovenous malformations (AVMs) are congenital vascular anomalies defined by abnormal direct connections between arteries and veins due to their complex structure or endovascular approaches. Pharmacological strategies targeting the underlying molecular mechanisms are thus gaining increasing attention in an effort to determine the mechanism involved in AVM regulation. In this study, we examined 30 human tissue samples, comprising 10 vascular samples, 10 human fibroblasts derived from AVM tissue, and 10 vascular samples derived from healthy individuals. The pharmacological agents thalidomide, U0126, and rapamycin were applied to the isolated endothelial cells (ECs). The pharmacological treatments reduced the proliferation of AVM ECs and downregulated miR-135b-5p, a biomarker associated with AVMs. The expression levels of angiogenesis-related genes, including VEGF , ANG2 , FSTL1 , and MARCKS , decreased; in comparison, CSPG4 , a gene related to capillary networks, was upregulated. Following analysis of these findings, skin samples from 10 AVM patients were reprogrammed into induced pluripotent stem cells (iPSCs) to generate AVM blood vessel organoids. Treatment of these AVM blood vessel organoids with thalidomide, U0126, and rapamycin resulted in a reduction in the expression of the EC markers CD31 and α-SMA. The establishment of AVM blood vessel organoids offers a physiologically relevant in vitro model for disease characterization and drug screening. The authors of future studies should aim to refine this model using advanced techniques, such as microfluidic systems, to more efficiently replicate AVMs’ pathology and support the development of personalized therapies.
Reassessment of marker genes in human induced pluripotent stem cells for enhanced quality control
Nature Communications 2024 Oct
Abstract
Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, “hiPSCore”, trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications. Quality control, including pluripotency testing of human iPSCs lacks standardization. Here, authors identify and validate gene markers to develop the machine learning-based hiPSCore to streamline pluripotency testing and elevate iPSC quality.
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