Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells.

Area of Science:

• Stem cell biology
• Genetics
• Molecular biology

Background:

• Human artificial chromosomes (HAC) are valuable tools for studying chromosome structure and gene function.
• Previous attempts to create HACs were limited to tumor-derived cell lines, hindering stem cell applications.
• Efficient transfer and stable maintenance of HACs in stem cells remained a challenge.

Purpose of the Study:

• To establish a novel, non-integrating gene expression system in human embryonic stem cells (hESc) using HACs.
• To demonstrate the stable maintenance and gene expression capability of HACs in hESc.
• To overcome limitations in applying HAC technology to pluripotent stem cells for therapeutic purposes.

Main Methods:

• Modification of the herpes simplex virus type 1 amplicon system for HAC DNA transfer.
• Introduction of HAC DNA into two hESc lines.
• Assessment of gene expression (GFP), HAC stability, integration, and pluripotency markers.
• Teratoma formation assays to evaluate stem cell differentiation potential.

Main Results:

• Successfully generated gene-expressing HACs in hESc at high frequency.
• HACs were stably maintained for at least 3 months without selection.
• No integration of HAC DNA into the host genome was observed in hESc, unlike in control fibrosarcoma cells.
• hESc retained pluripotency, differentiation capacity, and teratoma formation ability.

Conclusions:

• This study reports the first successful generation of de novo gene-expressing HACs in hESc.
• The developed non-integrating system offers a safe and effective method for genetic manipulation of stem cells.
• This breakthrough advances the potential therapeutic applications of stem cells through precise genetic engineering.