Pe conjugated anti human cd34

After 7 days of the treatment with the MEISi-1 and MEISi-2, the UCB- and PB-MNCs were labeled with PE-conjugated anti-human CD34 (Biolegend, Cat.No. 343506), APC-conjugated anti-human CD133 (Miltenyibiotec, Order No. 130-090-826) antibodies as we have done previously^{14 (link),16 (link),25} . In addition, UCB mononuclear cells were treated with Aldefluor reagent according to the manufacturer’s manual (ALDEFLUOR™ Kit, Stemcell Technologies, Cat.No. 01700). The expressions of CD34, CD133 surface markers and ALDH (Aldehyde Dehydrogenase) enzyme activity in the labeled cells were analyzed by flow cytometry (BD FACSCalibur™, Cell Analyzer) as we have done previously^{14 (link),16 (link),25} .

To determine the expression of mesenchymal stem cell markers on T-MSCs, cells were collected and pelleted by centrifugation at 1300 rpm for 5 min. After washing with PBS, cells were pelleted again and stained with FITC-conjugated anti-human CD11b, PE-conjugated anti-human CD34, PerCP-conjugated anti-human CD45, APC-conjugated anti-human CD73, PE-conjugated anti-human CD90, or PE-conjugated anti-human CD105 antibody (Biolegend, San Diego, CA, USA) diluted in FACS buffer (PBS supplemented with 10% FBS, 10 mM EDTA, 20 mM HEPES, and 1 mM sodium pyruvate) for 30 min on ice. To determine the expression of megakaryocytic marker on differentiated K562 cells, Pacific Blue-conjugated anti-human CD41 antibody (Biolegend) was used. After washing cells with buffer, surface protein expression was measured using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed using NovoExpress software.