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Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells

Matthew L Hirsch1, B Matthew Fagan, Raluca Dumitru

  • 1Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States of America. mhirsch@email.unc.edu

Plos One
|November 25, 2011
PubMed

Insights

Recombinant adeno-associated virus (rAAV) transduction triggers a unique, lethal DNA damage response in human embryonic stem cells (hESCs). This p53-dependent apoptosis is caused by a telomeric sequence in the AAV origin of replication.

Area of Science:

  • Stem Cell Biology
  • Molecular Biology
  • Virology

Background:

  • Human embryonic stem cells (hESCs) are highly sensitive to genotoxic stress, leading to rapid apoptosis.
  • Recombinant adeno-associated virus (rAAV) utilizes host DNA damage responses for its lifecycle, potentially stressing pluripotent cells.

Purpose of the Study:

  • To investigate the specific DNA damage response induced by rAAV transduction in pluripotent hESCs.
  • To elucidate the molecular mechanisms underlying rAAV-induced cell death in hESCs.

Main Methods:

  • Analysis of DNA damage signaling markers (gamma-H2AX, p53 phosphorylation) post-rAAV transduction.
  • Cell cycle progression analysis using nucleotide incorporation assays.
  • Investigation of p53-dependent apoptosis pathways and toxicity of AAV components.
  • DNA microinjection experiments to identify critical AAV sequences.

Main Results:

  • rAAV transduction induced early S-phase arrest and apoptosis in hESCs, mediated by p53.
  • This lethal response was independent of canonical p53 transcriptional targets (Puma, Bax, Bcl-2).
  • Empty AAV capsids were non-toxic, but a specific telomeric repeat sequence within the AAV origin of replication was sufficient to induce hESC apoptosis.

Conclusions:

  • Pluripotent hESCs exhibit a unique, p53-dependent lethal DNA damage response to rAAV transduction.
  • A telomeric G-rich sequence within the AAV origin of replication is the primary trigger for this apoptosis.
  • Differentiated cells (neural lineage) did not show this sensitivity, highlighting pluripotency-specific responses.

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