1. Poly-L-Lysine Increases the Ex Vivo Expansion and Erythroid Differentiation of Human Hematopoietic Stem Cells, as Well as Erythroid Enucleation Efficacy

2. Ex vivo expansion and differentiation of HSCs on PLL substrates

3. Tissue culture plates were coated with various concentrations of PLL (0.001, 0.01, and 0.1 weight%), and the oxygen, nitrogen, and carbon contents on the coated surfaces were quantified by X-ray photoelectron spectroscopy amine groups

4. FIG. 1. Poly-L-lysine (PLL) substrates (0.001%, 0.01%, and 0.1%) enhance the ex vivo expansion of hematopoietic stem cells (HSCs). Ex vivo expansion was analyzed by cell counting (A), cell division assay of CD34+ cells (B), and fluorescence-activated cell sorting analysis of CD34+ Lin - cells (C) at day 1 and 3. FIG. 2. The PLL substrate (0.01%) does not alter the multipotency of HSCs. The total colony number (A) and the percentage of various types of colonies (B) were observed at 14 days in culture using the colony-forming assay. These results demonstrate that PLL, at a coating concentration of 0.01% or more, increased the total cell number of CD34+ cells through an increase in cell division, without altering the percentage of HSCs with the CD34+ Lin - surface marker expression pattern or the differentiation potential of CD34+ cells.

5. Erythroid differentiation and RBC production on PLL substrates

6. FIG. 3. PLL substrates (0.001%, 0.01%, and 0.1%) increase erythroid differentiation of CD34+ cells. The fold increase of erythroid cells (A), the percentage of CD71+ GPA+ cells (B) and enucleated cells (C) for 21 days are shown. GPA+SYTO64- cells were regarded as enucleated red blood cells. CD71 and Glycophorin A (GPA) are cell surface markers of erythroid cells. 7-130-713-1717-21 These results suggest that the 0.01% PLL substrate enhanced the expansion of differentiating HSCs at early but not late phases. These results suggests that the PLL substrates mainly induced enucleation of erythroid cells at phase III rather than at phase IV

7. FIG. 4. Effect of the PLL substrate (0.01%) on erythroid differentiation, as shown by benzidine staining (A), RT-PCR with erythropoiesis-related primers (B) and Oxygen-carry abilities (C) of hemoglobin in the erythroid cells at 17 days. Black arrowheads indicate benzidine+ /hematoxylin- cells as enucleated erythrocytes. Oxygen equilibrium curves were determined using an automated apparatus. CB and PB indicate umbilical cord blood and adult peripheral blood, respectively. More These analyses confirmed that the 0.01% PLL substrate enhanced the enucleation of erythroid cells and, in addition, elevated the expression of erythropoiesis-related genes during erythroid differentiation. PLL substrate is able to enhance oxygen- carrying ability of erythroid cells.

8. Enucleation efficacy and direction of enucleation on the 0.01% PLL substrate

10. The interaction between HSCs and the PLL substrate (0.01%) stimulates downward enucleation. phosphoinositide 3-kinase (PI3K) activity of erythroid cells was visualized by anti-PIP3 (green) and DAPI (blue) Enucleating cells were observed via confocal imaging in Z-section by using an anti-GPA antibody (green), phalloidin (red), and DAPI (blue, left panel), concluding which an enucleation mechanistic model is also shown (right panel) This result strongly supports the fact that the PLL substrate is able to increase PI3K activity and cellular polarization of erythroid cells followed by an increase in enucleation.

11. Conclusions 1. PLL substrates increased the total number of HSCs without altering their expression of surface markers related to stemness. 2. Higher coating concentrations of PLL had a greater influence on the ex vivo expansion of CD34+ cells, with no change in the percentage of CD34+ Lin - cells. 3. The number of cells that underwent erythroid differentiation followed by enucleation increased on PLL substrate versus the control. 4. These results indicate that PLL might serve as an important material to enhance the ex vivo expansion and erythroid differentiation of HSCs, and especially the enucleation of erythroid cells to form RBCs.