Aging impairs the ability of vascular endothelial stem cells to generate endothelial cells in mice.

Tissue-resident vascular endothelial stem cells (VESCs), marked by expression of CD157, possess long-term repopulating potential and contribute to vascular regeneration and homeostasis in mice. Stem cell exhaustion is regarded as one of the hallmarks of aging and is being extensively studied in several types of tissue-resident stem cells; however, how aging affects VESCs has not been clarified yet. In the present study, we isolated VESCs from young and aged mice to compare their potential to differentiate into endothelial cells in vitro and in vivo. Here, we report that the number of liver endothelial cells (ECs) including VESCs was lower in aged (27-28 month-old) than young (2-3 month-old) mice. In vitro culture of primary VESCs revealed that the potential to generate ECs is impaired in aged VESCs isolated from liver and lung relative to young VESCs. Orthotopic transplantation of VESCs showed that aged VESCs and their progeny expand less efficiently than their young counterparts when transplanted into aged mice, but they are equally functional in young recipients. Gene expression analysis indicated that inflammatory signaling was more activated in aged ECs including VESCs. Using single-cell RNA sequencing data from the Tabula Muris Consortium, we show that T cells and monocyte/macrophage lineage cells including Kupffer cells are enriched in the aged liver. These immune cells produce IL-1ß and several chemokines, suggesting the possible involvement of age-associated inflammation in the functional decline of VESCs with age.

Isolation of tissue-resident vascular endothelial stem cells from mouse liver.

Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157+ vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.

Isolation of tissue-resident endothelial stem cells and their use in regenerative medicine.

BACKGROUND: During sprouting angiogenesis, stalk cells, localized behind tip cells, generate endothelial cells (ECs) for the elongation of new vessels. We hypothesized that stalk cells may have endothelial progenitor cell properties because of their highly proliferative ability. We conducted Hoechst dye DNA staining in ECs of preexisting blood vessels from hind limb muscle and found that endothelial-side population (E-SP) cells, which efflux Hoechst rapidly with abundant ABC transporters, show highly producing ability of ECs. We previously showed the existence of E-SP cells in hind limb muscle, retina, and liver, but not in other tissues such as adipose tissue, skin, and placenta. METHODS: We investigated the existence of E-SP cells and analyzed their proliferative ability among CD31+CD45- ECs from adipose tissue, skin, and placenta of adult mice. We also analyzed the neovascular formation of E-SP cells from adipose tissue in vivo. RESULTS: We detected E-SP cells in all tissues examined. However, by in vitro colony formation analysis on OP9 cells, we found that E-SP cells from adipose tissue and skin, but not from placenta, have highly proliferative ability. Moreover, E-SP cells from adipose tissue could contribute to the neovascular formation in hind limb ischemia model. CONCLUSION: The adipose tissue and skin are available sources to obtain endothelial stem cells for conducting therapeutic angiogenesis in regenerative medicine.