Levels of angiocrine factors were measured by the human VEGF ELISA kit (NeoBioscience, EHC108), the human bFGF ELISA kit (NeoBioscience, EHC130), EGF ELISA kit (NeoBioscience, EHC126), IL-8 (NeoBioscience, EHC008), and IGF ELISA kit (R&D, DG100) according to the manufacturers instructions. Abstract Background Ischemic vascular diseases are the major cause of death worldwide. In recent years, endothelial cell (EC)-based approaches to vascular regeneration are increasingly viable strategies for treating ischemic diseases, but their applications are challenged by the difficulties in their efficient generation and stable maintenance. Here, we show an alternative protocol that facilitates the generation of functional and expandable ETS variant 2 (ETV2)-induced endothelial-like cells (EiECs) from human adipose-derived stem cells Clofarabine (hADSCs), providing a potential source of cells for autologous ECs to treat ischemic vascular diseases. Methods hADSCs were obtained from fresh human adipose tissue. Passage 3 hADSCs were transduced with doxycycline (DOX)-inducible ETV2 transcription factor; purified ETV2-hADSCs were induced into endothelial-like cells using a two-stage induction culture system composed of small molecule compounds and cell factors. EiECs were evaluated for their surface markers, proliferation, gene expression, secretory capacity, and effects on vascular regeneration in vivo. Results We found that short-term ETV2 expression combined with TGF- inhibition is sufficient for the generation of kinase Clofarabine insert domain receptor (KDR)+ cells from hADSCs within 10?days. KDR+ cells showed immature endothelial characteristics, and they can gradually mature in a chemically defined induction medium at the second stage of induction. Futher studies showed that KDR+ cells deriving EC-like cells could stably self-renew and expand about 106-fold in 1?month, and they exhibited expected genome-wide molecular features of mature ECs. Functionally, these EC-like cells significantly Rabbit Polyclonal to MAGI2 promoted revascularization in a hind limb ischemic model. Conclusions We isolated highly purified hADSCs and effectively converted them into functional and expandable endothelial-like cells. Thus, the study may provide an alternative strategy to obtain functional EC-like cells with potential for biomedical and pharmaceutical applications. Electronic supplementary material The online version of this article (10.1186/s13287-018-1088-6) contains supplementary material, which is available to authorized users. test) in expression level between hADSCs and mature EiECs were selected to generate the heatmap and for GO term Clofarabine enrichment analysis. Human angiocrine factors ELISA To determine the secretion of human angiocrine factors, mature EiECs, hADSCs, or hUVECs were seeded on 6-well plates and maintained in EIM basal Clofarabine medium without angiogenic growth factors for 48?h until collection of supernatants. Levels of angiocrine factors were measured by the human VEGF ELISA kit (NeoBioscience, EHC108), the human bFGF ELISA kit (NeoBioscience, EHC130), EGF ELISA kit (NeoBioscience, EHC126), IL-8 (NeoBioscience, EHC008), and IGF ELISA kit (R&D, DG100) according to the manufacturers instructions. Serum was diluted in a range from 10- to 1000-fold to obtain values falling to the linear range of standard curve. Flow cytometry For the detection of surface markers, cells were dissociated into single-cell suspension and resuspended in PBS and then stained with fluorochrome-labeled mAbs for 30?min on ice in the dark. The flow cytometry analysis was performed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA) or a BD Bioscience Influx cell sorter; collected events were analyzed by FlowJo software (Treestar, Ashland, OR, USA). The antibodies (all from Biolegend) are listed in Additional?file?1: Table S2. Capillary-like structure formation assay To assess the formation of capillary structures, tested cells were trypsinized Clofarabine into single cells and resuspended in EGM-2 medium supplemented with 50?ng/ml VEGF. Cells were plated at a density of 5??104 cells per well in triplicate in 24-well plates coated with growth factor-reduced Matrigel (BD Biosciences), plates were incubated overnight, and tube formation was observed by phase-contrast microscope. The amount of branch points (?3 cells per branch) were counted and analyzed in five random fields per replicate. In vivo Matrigel angiogenesis assay To assess the angiogenesis potency of EiECs in vivo, about 1??106 EiECs were suspended in 100?l PBS containing 30% Matrigel and injected subcutaneously into the athymic nude mice ( em n /em ?=?5). Two weeks after implantation, the cell masses were taken out and observed. hADSCs and hUVECs were used as controls. Hind limb ischemic mouse model and angiogenesis assay All the animal care and experiments were approved by the Animal Care.