Adenovirus vector-mediated efficient transduction into human embryonic and induced pluripotent stem cells

Area of Science:

• Stem Cell Biology
• Gene Therapy
• Viral Vector Technology

Background:

• Human embryonic stem (ES) and induced pluripotent stem (iPS) cells are crucial for regenerative medicine and disease modeling.
• Efficient gene delivery into these cells is essential for various research and therapeutic applications.
• Adenovirus (Ad) vectors are commonly used for gene delivery but their efficiency in stem cells requires optimization.

Purpose of the Study:

• To evaluate the transduction efficiency of adenovirus (Ad) vectors in human ES and iPS cells.
• To investigate methods for improving Ad vector-mediated gene delivery into stem cell colonies.
• To assess the impact of Ad vector transduction on the undifferentiated state of human ES and iPS cells.

Main Methods:

• RT-PCR analysis to detect the coxsackievirus and adenovirus receptor expression in ES and iPS cells.
• Transduction experiments with Ad vectors using intact and dissociated ES and iPS cell colonies.
• Passaging of dissociated cells using Rho-associated kinase inhibitor.
• Analysis of transgene expression and stem cell marker expression (Oct-3/4, Nanog, SSEA-4).

Main Results:

• Adenovirus receptor expression was confirmed in human ES and iPS cells.
• Transduction efficiency was limited to the periphery of intact cell colonies due to tight cell-cell connections.
• Dissociating cells and using Rho-associated kinase inhibitor markedly increased transduction efficiency and transgene expression throughout the colonies.
• Ad vector transduction did not affect the expression of key undifferentiated stem cell markers.

Conclusions:

• Adenovirus vectors can effectively transduce human ES and iPS cells when delivered to dissociated cells.
• Optimizing cell culture conditions, such as using Rho-associated kinase inhibitor, is critical for efficient Ad vector-mediated gene delivery.
• Ad vectors represent a viable tool for genetic manipulation of human ES and iPS cells without compromising their pluripotency.