To investigate whether SB4, a bone morphogenetic protein 4 (BMP4) signaling agonist, can alleviate ionizing radiation-induced damage to mouse bone marrow hematopoietic stem/progenitor cells (HSPCs).

Mouse bone marrow lineage-negative (Lin^-) cells were isolated and cultured in vitro. The optimal concentration of SB4 was determined by detecting intracellular phosphorylated mothers against decapentaplegic homolog 1/5/9 (p-SMAD1/5/9) expressions via flow cytometry and assessing cell viability via acridine orange/propidium iodide (AO/PI) staining. An in vitro ionizing radiation (IR) injury model was established by exposing Lin^- cells to 3 Gy X-ray irradiation. Cells were divided into the irradiation alone group (IR-SB4 group) and the SB4 pretreatment followed by irradiation group (IR+SB4 group). On day 1 post-irradiation, flow cytometry was used to analyze the proportion and number of HSPCs and hematopoietic stem cells (HSCs). The percentage of γ-H2A histone family member X (γ-H2AX)-positive cells, levels of reactive oxygen species (ROS), and cysteinyl aspartate-specific protease 3 (caspase-3) expressions in HSCs were also determined. Real-time quantitative PCR (qPCR) was used to detect the mRNA expression levels of antioxidant genes and the anti-apoptotic genes in HSPCs from both groups.

The BMP4 signaling agonist SB4 effectively increased the phosphorylation of SMAD1/5/9 in mouse bone marrow hematopoietic cells, and 1 μmol/L was identified as the optimal concentration for HSPCs. Compared with the IR-SB4 group, the proportions and numbers of HSPCs and HSCs were significantly larger in the IR+SB4 group, along with a lower γ-H2AX-positive rate, reduced ROS accumulation, and downregulated caspase-3 expressions in HSCs. Meanwhile, the mRNA expression levels of key genes involved in antioxidative stress and anti-apoptotic pathways in HSPCs were upregulated.

SB4 can alleviate ionizing radiation-induced damage to mouse HSPCs. The underlying mechanism is possibly associated with the activation of SMAD1/5/9 phosphorylation and the upregulation of antioxidative stress and anti-apoptotic gene expressions in HSPCs.

To establish and characterize vascular organoids based on the capacity of definitive differentiation and multiple cell self-organization of human embryonic stem cells (hESCs).

The hESCs were subjected to a stepwise induction protocol to sequentially differentiate into mesoderm progenitors, lateral plate mesoderm progenitors, and endothelial cells over a 5-day period. The resulting endothelial cell population was harvested and embedded in Matrigel to undergo three-dimensional culture of 6-8 days, allowing for self-organization into vascular organoids with branched luminal networks. The organoids were characterized via morphological analysis, flow cytometry, immunofluorescence staining, and transmission electron microscopy.

The generated organoids formed complex networks of branched lumens, containing (41.10±4.51)% CD31⁺ endothelial cells and (6.88%±0.40)% CD140b⁺ pericytes. Among the endothelial cells, (43.00±0.21)% were CD31⁺CD184⁺ arterial endothelial cells and (3.66±0.25)% were CD31⁺CD184^-CD73⁺ venous endothelial cells. Immunofluorescence analysis found that the vascular organoids were composed of VE-cadherin⁺Sox17⁺ arterial endothelial cells, VE-cadherin⁺NR2F2⁺ venous endothelial cells, PDGFR-β⁺ pericytes, α-SMA⁺ smooth muscle cells, and collagen I. Tight junctions were observed between endothelial cells, and characteristic Weibel-Palade body ultrastructures were identified within the cytoplasm that resembled the structures of human microvascular units.

We have established a robust method for generating hESCs-based vascular organoids that mimic the architecture of functional microvasculature. This three-dimensional vascular organoid can serve as a promising platform for research on vascular development and disease modeling.

To establish a human intestinal organ-on-a-chip model using a multi-array chip array to simulate the microphysiological structure of the human intestine and to investigate the impact of ionizing radiation on radiation-induced damage to human intestinal cells.

Caco-2 and human umbilical vein endothelial cells (HUVECs) were co-cultured in an organ chip. The cells were subjected to fluid shear stress via a precision shaker. After 7 days of dynamic culture,the morphological structure of intestinal epithelial cells and venous endothelial cells within the intestinal organ chip was examined using phase contrast microscopy,immunofluorescence staining,and confocal microscopy for three-dimensional (3D) imaging. γ-H2AX and TUNEL immunofluorescence staining were employed to assess DNA damage and apoptosis in intestinal epithelial cells two days post-irradiation. Villin immunofluorescence staining was used to evaluate villus height three days post-irradiation. EdU incorporation assay and Ki67 immunofluorescence staining were conducted to observe the effects of ionizing radiation on the proliferation of intestinal epithelial cells.

After 7 days of dynamic culture,phase contrast microscopy and immunofluorescence staining combined with confocal 3D imaging revealed that the upper intestinal epithelial cells in the middle compartment of the chip formed a 3D intestinal villus structure,while the vascular endothelial cells in the lower compartment developed a vascular network structure. The chip was subsequently irradiated by 10 Gy X-ray. Immunofluorescence staining results indicated that the mean fluorescence intensity of γ-H2AX and TUNEL in the irradiated group was significantly higher than in the non-irradiated group 2 days after irradiation (P<0.01),and that the proportion of EDU⁺ and Ki67⁺ cells in the irradiated group was significantly lower than in the non-irradiated group three days after irradiation (P<0.05).

Caco-2 cells and HUVECs co-culture on an organ chip can generate the biomimetic structure of human intestinal villus. Ionizing radiation has been found to shorten intestinal villus,increase DNA damage and apoptosis in intestinal epithelial cells,and inhibit the proliferation of these cells.