Abstract
BackgroundIschemic 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 (hADSCs), providing a potential source of cells for autologous ECs to treat ischemic vascular diseases.MethodshADSCs 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.ResultsWe found that short-term ETV2 expression combined with TGF-β inhibition is sufficient for the generation of kinase 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 promoted revascularization in a hind limb ischemic model.ConclusionsWe 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.
Highlights
Ischemic vascular diseases are the major cause of death worldwide
Cell culture Abdominal adipose tissue was obtained from five health donors and umbilical cord was obtained from three health donors in West China Hospital, Sichuan University, upon consent of its donor according to procedures approved by the Medical Ethics Committee, Sichuan University. human adipose-derived stem cells (hADSCs) and human umbilical cord mesenchymal stem cells were isolated as described [22, 23]. hADSCs and hUMSCs were cultured in mesenchymal stem cell basal medium (DAKEWE, Beijing, China) supplemented with 5% UltraGROTM (HPCFDCRL50, Helios)
We found mature endothelial-like cells (EiECs) displayed a significant induction in Endothelial cells (ECs)-related genes, including kinase insert domain receptor (KDR), CD34, and CD31 in a manner more similar to Human umbilical vein endothelial cells (hUVECs) than hADSCs (Fig. 5b)
Summary
Efficient endothelial induction from hADSCs by ETV2 transduction Untransduced hADSCs maintained spindle-shaped, fibroblast-like appearance (Additional file 2: Figure S1A). To further evaluate the functional abilities of EiECs, we collected these cells on day 30 of induction and compared them to hUVECs. Immunofluorescence staining and flow cytometry analysis revealed the EiECs were positive for endothelial-specific markers, including VE-cad, CD31, TEK, and vWF (Fig. 3d and Fig. 4a). When cultured in the EMM in the absence of DOX from day 10, hUMSC-derived KDR+ cells gradually converted into mature endothelial lineage cells with the efficiency comparable to hADSCs (Fig. 7g)
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