Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture

Area of Science:

• Epigenetics
• Stem Cell Biology
• Hematopoiesis

Background:

• Hematopoietic stem and progenitor cells (HPCs) are crucial for blood formation but lose their stemness during in vitro culture.
• Maintaining HPC stemness in vitro is essential for research and therapeutic applications.

Purpose of the Study:

• To investigate the epigenetic changes, specifically DNA methylation (DNAm) profiles, in HPCs during in vitro culture.
• To understand the molecular mechanisms underlying the loss of stemness in cultured HPCs.

Main Methods:

• Comparison of DNAm profiles between freshly isolated and cultured HPCs (CD34(+) cells).
• Assessment of DNAm changes with and without mesenchymal stromal cells (MSCs).
• Analysis of gene expression and variant transcripts related to DNA methylation.

Main Results:

• All cultured HPCs, regardless of culture conditions or CD34(+) marker retention, acquired significant DNA hypermethylation.
• DNA hypermethylation was prominent in upstream promoter regions, CpG island shores, and binding sites for transcription factors like PU.1, HOXA5, and RUNX1.
• Mesenchymal stromal cells enhanced HPC proliferation but had minimal impact on DNAm profiles.
• Differential gene expression and variant transcripts of DNMT3A were observed, correlating with DNA hypermethylation.
• Low concentrations of DNA methylation inhibitors showed a slight increase in colony-forming unit initiating cells.

Conclusions:

• HPCs acquire specific DNA hypermethylation during in vitro culture, contributing to stemness loss.
• Epigenetic modifications, particularly DNA hypermethylation, are key drivers of stemness attrition in cultured HPCs.
• Targeting DNA methylation pathways may offer strategies to preserve HPC stemness in vitro.