Though low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method underwent senescence and lost their stem cell properties, which could be preserved by combining low-density with hypoxic culture. The mechanism was mediated through direct downregulation of E2A-p21 by HIF-1α-TWIST. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST upregulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings exhibited telomerase activity with maintained telomere length, normal karyotyping and intact genetic integrity, and do not form tumors. These results support the method of low-density hypoxic culture for efficiently expanding MSCs without losing stem cell properties and increasing tumorigenecity.

  • Submitted May 28, 2010.
  • Accepted October 3, 2010.