Expansion and identification of primary rat aortic vascular stem cells in vitro

doi:10.12122/j.issn.1673-4254.2024.10.06

Abstract

Abstract:

Objective To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells. Methods The thoracic and abdominal aortas isolated from 2- to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes. The primary cells were further cultured to 80%-90% confluence before passaging. The morphology and growth characteristics of the cells were observed under a microscope, and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry. Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation. Results After 3 days of culture, a small number of spindle, star-shaped or polygonal cells migrated out from the peripheral of the vascular segments. At 5-6 days, island-like cell clusters occurred and the cells began to proliferate rapidly. The cell clusters expanded radially and showed signs of cell cloning. At 7-8 days, the cells fused into sheets and displayed a vortex-like distribution. The cells in the third passage presented with a uniform morphology, showing a typical fibroblast-like arrangement. Flow cytometry showed that the cells expressed predominantly CD44 (80.3%), CD73 (62.2%) and CD90 (46.8%) with low expressions of CD34 (1.1%), CD45 (0.2%) and CD11b/c (0.2%). Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro. Conclusion Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

Key words: vascular stem cells, aorta, adherent culture of the segmented outer membrane, primary culture, immunophenotype, differentiation potential, vascular remodeling

Huagen MA, Yan HUANG, Yingxin YANG, Haiqin LIU, Yuanyu TANG, Weihong CONG. Expansion and identification of primary rat aortic vascular stem cells in vitro[J]. Journal of Southern Medical University, 2024, 44(10): 1881-1886.

Figures/Tables 5

Fig.1 Morphological observation of rat aortic vascular stem cells (VSCs) in primary culture. A: Spindle-shaped, star-shaped or polygonal cells emerging from the peripheral of the vascular segments after 3 days of culture. B, C: After 5-6 days of culture, island-like cell colonies occurred and extended outward rapidly in a radial pattern. D: Cell cloning in the cell culture. E, F: After 7-8 days of culture, the cell density further increased, the cell colonies merged to exhibit a vortex-like distribution, and the cells formed a monolayer with adherent growth.

Fig.2 Morphological observation of rat aortic VSCs in the third to eighth passages. A-C: After passaging for 4-6 h, the cells became adherent and spread uniformly, exhibiting a consistent morphology. The individual cells displayed a typical fibroblast-like appearance with pseudopodia extending from both poles of the cells. D-F: After 36-48 h, the passaged cells aligned tightly along the longitudinal axis, forming a spiral pattern with a clear orientation.

Fig.3 Expression of surface marker CD molecules on cultured rat aortic VSCs. The positive rates of CD44, CD73, CD90, CD34, CD45 and CD11b/c were 80.3%, 62.2%, 46.8%, 1.1%, 0.2% and 0.2%, respectively.

Fig.4 Observation of induced adipogenesis of cultured rat aortic VSCs and oil red O staining of the cells. A, B: After 14 days of adipogenic induction, translucent lipid droplets appeared in the cytoplasm. C, D: Oil red O staining highlighting the bright red lipid droplets. E, F: Absence of translucent lipid droplets in the negative control cells before and after induction.

Fig.5 Observation of induced osteogenesis of cultured rat aortic VSCs and alizarin red staining of the cells. A, B: After 21 days of osteogenic induction, irregular dense mineralized nodules occurred at the cell aggregations. C, D: Alizarin red staining showing bright red nodules. E, F: Absence of mineralized nodules in the negative control cells before and after induction.

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