Anti-CXCR4 Monoclonal antibody, clone 23H6 [PE/Cy7®]

Human pluripotent stem cells (hPSCs) are a promising source of autologous endothelial progenitor cells (EPCs) that can be used for the treatment of vascular diseases. However, this kind of treatment requires a large amount of EPCs. Therefore, a highly efficient, robust, and easily reproducible differentiation protocol is necessary. We present a novel serum-free differentiation protocol that exploits the synergy of multiple powerful differentiation effectors. Our protocol follows the proper physiological pathway by differentiating EPCs from hPSCs in three phases that mimic in vivo embryonic vascular development. Specifically, hPSCs are differentiated into (i) primitive streak, which is subsequently turned into (ii) mesoderm, which finally differentiates into (iii) EPCs. This differentiation process yields up to 15 differentiated cells per seeded hPSC in 5 days. Endothelial progenitor cells constitute up to 97% of these derived cells. The experiments were performed on the human embryonic stem cell line H9 and six human induced pluripotent stem cell lines generated in our laboratory. Therefore, robustness was verified using many hPSC lines. Two previously established protocols were also adapted and compared to our synergistic three-phase protocol. Increased efficiency and decreased variability were observed for our differentiation protocol in comparison to the other tested protocols. Furthermore, EPCs derived from hPSCs by our protocol expressed the high-proliferative-potential EPC marker CD157 on their surface in addition to the standard EPC surface markers CD31, CD144, CD34, KDR, and CXCR4. Our protocol enables efficient fully defined production of autologous endothelial progenitors for research and clinical applications.

Acute lung injury (ALI) is a highly lethal pulmonary disease that causes edema, hypoxemia and respiratory failure. Recent evidence indicates that nuclear factor-kappa B (NF-kappa B) plays a crucial role in ALI development. However, the regulatory mechanism of NF-kappa B on ALI remains enigmatic. In this study, we investigated potential molecular mechanism of NF-kappa B on ALI induced by lipopolysaccharide (LPS). BALB/c mice were subjected to intratracheal spraying of LPS to generate an ALI mode, with the activity of NF-kappa B in mice tissues being detected by enzyme linked immunosorbent assay (ELISA), and the number of inflammatory cells in bronchoalveolar lavage fluid being counted. Then, the macrophage cell line RAW264.7 exposed to LPS were treated with ammonium pyrrolidinedithiocarbamate (PDTC) (inhibitor of NF-kappa B), miR-194 mimic, or oe-chemokine receptor type 4 (CXCR4) separately or in combination. After that, ELISA and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression level of IL-1 beta, IL-6, TNF-alpha, miR-194 and CXCR4, respectively. In addition, the targeting relationship between miR-194 and CXCR4 was verified by dual-luciferase reporter gene assay. The dry/wet ratio of lung and the MPO activity were also measured to assess the inflammatory response in mice. Activation of NF-kappa B down-regulated the miR-194 expression in LPS-induced ALI. Overexpression of miR-194 alleviated LPS-induced ALI and reduced the expression of inflammatory factors IL-1 beta, IL-6 and TNF-alpha via targeting CXCR4. In LPS-induced ALI, NF-kappa B mediates the CXCR4 expression by inhibiting the expression of miR-194, thus promoting the inflammatory injury of lung.