Figure 5.

Effect of Orientin on human hematopoietic stem cells. (a–c) Orientin induces human CD34⁺ cells differentiation towards MEP lineage. CD34⁺ cells purified from healthy donor’s PBMCs via density gradient ficol followed by magnetic bead separation were treated with or without Orientin in a serum free expansion medium (SFEM) supplemented with 100 ng/ml cytokine cocktail (CC100) for 7 days. Cells were harvested for lineage differentiation and stained with CD34-PE-Cy7, CD38-Pacblue, CD10-FITC, CD135-PE, CD45RA-APC antibodies and subjected to FACS analysis. (d) Orientin partially restores CD34⁺ cells differentiation potential after irradiation. CD34⁺ cells purified from healthy donor’s PBMCs (1 × 10⁵/ml in triplicates) were treated with or without Orientin (5 µM) and exposed to 4 Gy (fractionated dose of 2 × 2 Gy). These cells were then co-cultured on human mesenchymal stromal cell layer in SFEM medium supplemented with 100 ng/ml cytokine cocktail for normal differentiation in 7 day time period. The MEP cells were gated in lin⁻CD34⁺CD38⁺CD10⁻CD135⁻CD45RA⁻ block in each sample. (e) Colony Assay in CD34⁺ cells (1 × 10³/ml in triplicates) post 0 or 2 Gy X-ray exposure. CFU-GM and GEMM colonies were scored on day 14 by applying standard morphologic criteria.