Abstract
Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43− cells and derivative populations, but not that of CD34lowCD43− cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-β or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).
Highlights
Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment
Tao Cheng) were cut into small squares containing 0.5–1 × 1 03 cells each by 200 μl tips, which were inoculated into 12-well plates (25 pieces per well). hPSC maintenance medium (Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, F-12 nutrient mixture, 20% knockout serum replacement (KSR; Gibco), 1% L-glutamine, 1% non-essential amino acid solution (NEAA; Gibco), and 5 ng/ml basic FGF(b-FGF; Wako)) was used for the coculture system for the first 3 days, and replaced with hematopoiesis-inducing medium (Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum (FBS; Hyclone), 1% NEAA (Gibco), 60 ng/ml ascorbic acid (Sigma), and 20 ng/ml vascular endothelial growth factor (VEGF; Wako)); this day was defined as day 0 (D0)
HESCs (Fig. 1a) with AGM-S3 cells, overexpression of RUNX1b at the early stage can block the mesoderm– hemogenesis transition, and treatment with 0.33 μM RepSox partially alleviates this blockage1. qRT-PCR (Fig. 1b) and western blotting (Fig. 1c) revealed that when RUNX1b/hESCs were induced at day 0 (D0), expression of P18 was upregulated at D4
Summary
Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43− cells and derivative populations, but not that of CD34lowCD43− cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. Induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-β or NF-κB signaling respectively. Our findings provide the first evidence that P18 overexpression can promote hematopoiesis, providing insight into the molecular mechanisms underlying CKI activity during hematopoietic differentiation
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