Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro

Acta Biochim Biophys Sin (Shanghai). 2008 Aug;40(8):711-20.

Abstract

A combination of extrinsic hematopoietic growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6, can induce division of quiescent hematopoietic stem cells (HSCs), but it usually impairs HSCs' self-renewal ability. However, intrinsic negative cell cycle regulators, such as p18INK4C (p18), p27Kip1 (p27) and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the removal of some extrinsic regulators and the knockdown of intrinsic negative cell cycle regulators via RNA interference (RNAi) induce ex vivo expansion of the HSCs. To address this question, a lentiviral vector-based RNAi tool was developed to produce two copies of small RNA that target multiple genes to knockdown the intrinsic negative cell cycle regulators p18, p27 and MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and engraftment assays were used to evaluate the effects of extrinsic and intrinsic regulators. Results showed that the medium with only SCF, but without IL-3 and IL-6, could maintain the sca-1+c-kit+ bone marrow cells with high LTC-IC frequency and low cell division. However, when the sca-1+c-kit+ bone marrow cells were cultured in a medium with only SCF and simultaneously knocked down the expression of p18, p27 and MAD1 via the lentiviral vector-based RNAi, the cells exhibited both high LTC-IC frequency and high cell division, though engraftment failed. Thus, the simultaneous knockdown of p18, p27 and MAD1 with a medium of only SCF can induce LTC-IC expansion despite the loss of engraftment ability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Bone Marrow Cells / cytology*
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / metabolism*
  • Bone Marrow Transplantation
  • Cell Cycle / genetics
  • Cell Cycle Proteins / antagonists & inhibitors*
  • Cell Cycle Proteins / genetics
  • Cells, Cultured
  • Colony-Forming Units Assay
  • Cyclin-Dependent Kinase Inhibitor p18 / antagonists & inhibitors*
  • Cyclin-Dependent Kinase Inhibitor p18 / deficiency
  • Cyclin-Dependent Kinase Inhibitor p18 / genetics
  • Cyclin-Dependent Kinase Inhibitor p27 / antagonists & inhibitors*
  • Cyclin-Dependent Kinase Inhibitor p27 / deficiency
  • Cyclin-Dependent Kinase Inhibitor p27 / genetics
  • DNA, Complementary / genetics
  • Female
  • Genetic Vectors
  • Hematopoiesis / genetics
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / drug effects
  • Hematopoietic Stem Cells / metabolism
  • Lentivirus / genetics
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Nuclear Proteins / antagonists & inhibitors*
  • Nuclear Proteins / deficiency
  • Nuclear Proteins / genetics
  • RNA Interference
  • RNA, Small Interfering / genetics
  • Stem Cell Factor / pharmacology

Substances

  • Cdkn1b protein, mouse
  • Cdkn2c protein, mouse
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p18
  • DNA, Complementary
  • Mad1l1 protein, mouse
  • Nuclear Proteins
  • RNA, Small Interfering
  • Stem Cell Factor
  • Cyclin-Dependent Kinase Inhibitor p27