Int J Stem Cells.  2020 Jul;13(2):202-211. 10.15283/ijsc20033.

Overexpression of p21 Has Inhibitory Effect on Human Hematopoiesis by Blocking Generation of CD43＋ Cells via Cell-Cycle Regulation

• Zeng J
• Zhang H
• Liu Y
• Sun W
• Yi D
• Zhu L
• Zhang Y
• Pan X
• Chen Y
• Zhou Y
• Bian G
• Lai M
• Zhou Q
• Liu J
• Chen B
• Ma F

• KMID: 2504333
• DOI: http://doi.org/10.15283/ijsc20033

Abstract

Background and Objectives
p21, an important member of the Cip/Kip family, is involved in inhibitory effects of RUNX1b overexpression during the early stage of human hematopoiesis.
Methods and Results
We established a human embryonic stem cell (hESC) line with inducible expression of p21 (p21/hESCs). Overexpression of p21 did not influence either mesoderm induction or emergence of CD34＋ cells, but it significantly decreased the production of CD43＋ cells and changed the expression profile of hematopoiesis-related factors, leading to the negative effects of p21 on hematopoiesis.
Conclusions
In RUNX1b/hESC co-cultures when RUNX1b was induced from D0, perturbation of the cell cycle caused by upregulation of p21 probably prevented the appearance of CD43＋ cells, but not CD34＋ cells. The mechanisms via which CD34＋ cells are blocked by RUNX1b overexpression remain to be elucidated.

Keyword

p21; RUNX1; CD43; Hematopoiesis; hPSC; AGM-S3; Inducible expression; PiggyBac; Cell cycle

Figure

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  Fig. 1 p21 is involved in inhibitory effects of RUNX1b on the mesoderm-hemogenesis transition. D0-induced RUNX1b/hESC co-cultures with AGM-S3 cells at D4, (A) Cell-cycle analyses revealed that the proportion of cells in S phase dropped significantly when RUNX1b was overexpressed from D0, and that these inhibitory effects could be partially rescued by RepSox. (B, C) qRT-PCR and western blotting analysis showed that p21 was upregulated when DOX was added from D0, and that this effect could be counteracted by 0.33 μM RepSox. Grayscale scanning analyses were performed using Gel-Pro Analyzer 4.

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  Fig. 2 Construction and confirmation of inducible p21/hESC lines. (A) Schematic representation of the PiggyBac constructs used to inducibly express p21. TRE, tet-on regulation element; CMV mini, cytomegalovirus minimum promoter; T2A, Thosea asigna virus 2A peptide. (B) After p21/hESCs were induced for 48 h, the cells were imaged by fluorescence microscopy to observe co-expression of GFP. (C, D) qRT-PCR and western blotting were used to confirm that inducible expression of p21 was highly stringent and efficient at the transcriptional and protein levels. (E) Pluripotency of p21/hESCs (non-induced or induced) was confirmed by western blotting for SOX2, OCT4, and NANOG.

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  Fig. 3 Overexpression of p21 blocks hematopoiesis in co-culture with AGM-S3 cells. p21/hESC co-cultures with AGM-S3 cells were treated with DOX from D0, D2, D4, D6, D8, or D10, and then subjected to FACS with antibodies against CD34/KDR and CD34/CD43 (at D6) or GPA/CD71, CD34/CD43, and CD34/CD45 (at D12) to compare non-induced co-cultures and the GFP+ fraction of co-cultures treated with DOX. (A) When p21 was overexpressed from the early stage, the abundance of D34+KDR− and CD34+CD43− cells was not influenced at D6, whereas the emergence of CD34+KDR+ and CD34+CD43+ cells was significantly blocked. (B) Most hematopoietic populations, such as CD34−CD43+, CD34+CD43+, CD34+CD45+, CD34−CD45+, and GPA+CD71+, dramatically decreased at D12, regardless of when induction of p21 started. This observation indicates that p21 has strong negative effects on hematopoietic cells (CD43+) but not their endothelial precursors (CD34+).

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  Fig. 4 Colony assays confirmed the inhibitory effects of p21 on hematopoiesis. D0-induced or non-induced co-cultures of p21/hESCs with AGM-S3 (about 3×104 cells) at D12 were subjected to colony culture assays to assess their hematopoietic potentials. (A) The number of each type of colony derived from 3×104 co-cultured cells. p<0.05 was considered significant. (B) Typical morphologies of CFU-Mix (a), CFU-E (b), BFU-E (c), and CFU-GM (d) colonies. Scale bar, 100 μm. MGG staining of cells in BFU-E colonies (e). Scale bar, 10 μm. The hematopoietic potential of p21-induced co-cultures dropped significantly.

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  Fig. 5 Induction of p21 from the early stage severely blocked the emergence of CD43+ cells. KDR+ or GFP+KDR+ cells sorted from D0-induced or non-induced p21/hESC co-cultures at D2. About 5×103 cells were re-plated on irradiated AGM-S3 in 24-well plates or further hematopoietic culture, treated with or without DOX for 8 days, and then subjected to FACS analysis using a combination of CD34/KDR or CD34/CD43 antibodies. The results indicated that induction of p21 from D0 or D2 significantly blocked the appearance of CD43+ cells, but not CD34+ cells.

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  Fig. 6 Overexpression of p21 from D0 blocked the emergence of CD43+ cells in co-cultures with AGM-S3 cells. p21/hESC co-cultures were treated with DOX from D0, and then subjected to FACS analysis using combinations of anti-CD34/CD43 antibodies to compare non-induced co-cultures and the GFP+ fraction of co-cultures at D4, D6, and D8. Production of CD34+CD43− cells was not significantly influenced, whereas production of CD34−CD43+ and CD34+CD43+ dropped significantly following induction of p21, indicating that p21 has inhibitory effects on CD43+ cells but not CD34+ cells.

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  Fig. 7 Overexpression of p21 interfered with the expression of hematopoiesis-related genes in co-cultures with AGM-S3 cells. p21/hESCs co-cultures were treated with DOX from D0. qRT-PCR analyses at D4 revealed that hematopoiesis-related genes, including GATA1, GATA2, vWF, and LOM2, were down-regulated. Expression of CD34+ was not influenced, but RUNX1b/c was up-regulated, potentially causing the inhibitory effects of p21 on hematopoiesis.

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  Fig. 8 Overexpression of p21 interferes with the cell-cycle status of KDR+ and CD34+ cells in co-culture with AGM-S3 cells. In p21/hESC co-cultures with AGM-S3 cells induced from D0, cell-cycle analysis indicated that (A) the proportion of KDR+ cells in S phase dropped significantly at D4, and this effect could be counteracted by RepSox; and that (B) the proportion of CD34+ cells in G2/M phase dropped significantly at D6, but this effect could not be counteracted by RepSox.

Reference

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