Expansion of functional human long-term HSCs through restraining excessive cell cycle activation

Area of Science:

• Stem Cell Biology
• Hematopoiesis
• Epigenetics

Background:

• Limited availability of human hematopoietic stem cells (HSCs) hinders clinical applications.
• Current ex vivo expansion methods often impair HSC self-renewal and cause myeloid bias.
• Molecular mechanisms underlying culture-induced HSC dysfunction are not well understood.

Purpose of the Study:

• To investigate transcriptional and epigenetic changes in HSCs during ex vivo culture.
• To identify molecular drivers of functional decline and myeloid bias in cultured HSCs.
• To develop an improved ex vivo culture system for expanding functional HSCs.

Main Methods:

• Single-cell multiome sequencing (scMultiome-seq) of human umbilical cord blood-derived CD34+ cells.
• Analysis of transcriptional and epigenetic signatures during ex vivo culture.
• Development and testing of a niche-mimetic culture system (3D-NcadP-Y and 3D-NcadP-Y-UM).

Main Results:

• Ex vivo culture reduced HSC signatures and increased myeloid-associated transcription factor activity.
• A novel niche-mimetic system (3D-NcadP-Y-UM) preserved HSC repopulating capacity and multilineage potential.
• The optimized culture maintained HSC stemness signatures, balanced transcription factor activity, and limited cell-cycle activation.

Conclusions:

• Culture-induced HSC dysfunction is linked to epigenetic and transcriptional alterations.
• A niche-mimetic strategy effectively expands functional long-term HSCs ex vivo.
• This approach preserves key stemness features and balanced differentiation potential.