Overexpression of erythropoietin by human bone marrow endothelial cells induces long-term expansion of eryhtroid progenitors for 70 days Academic Article uri icon

Overview

MeSH Major

  • Angiogenesis Inhibitors
  • Endothelium, Vascular
  • Growth Substances
  • Neoplasms

abstract

  • We have shown, that purified human bone marrow endothclial cells (BMEC) can support hematopoiesis in vitro for more than 70 days by constitutive production of hematopoietic cytokines such as GM-CSF, G-CSF, IL-I, c-kit Ugand and others (Rafii S. et al. Blood. 10/95). However. mRNA for crythropoietin (EPO) has not been shown to be produced by BMEC. In order to evaluate whether human BMEC is capable of supporting erythropoiesis by production of EPO we infected BMEC monolayers with an adenoviral vector expressing the transgene for human EPO (AdSCMV.EPO), This approach allows a robust expression of EPO by BMEC monolayers over several months. Control experiments were conducted with human BMEC that were infected with the Null vector (Ad5CMV.Null). 4x10 CD34+ cells isolated from fetal liver tissue were incubated with one million human BMEC that had previously been infected with AdSCMV.EPO or Ad5CMV.Null respectively. CD34+ cells were cultured either in direct contact with BMEC or in parallel experiments physically separated by a 0.4 micron microporus transwell membrane. Every 10 days the non-adherant cells were demipopulated, examined for BFU-E, CFU-E and CPU-G M content using a methylcellulose assay and analysed by flow cytometry (FACS) for GlycophorinA+ and CD36+ cells. In contrast to transwell experiments, both EPO expressing and non-expressing BMEC in direct contact with the CD34+cells generated erythroid progenitors up to 70 days. The majority of erythroid progenitors were large BFU-E. By FACS, GlycophorinA+ progenitors were present for up to 40 days. Compared with the control, BMEC expressing EPO generated more phenotypically primitive erythroid progenitors (double positive for GlycophorinA and CD36) (15.1% versus 0.5% on day 40). These data suggest that direct cell/cell contact between CD34+ cells and BMEC is important for long term maintenace of clonogenic capacity of BFU-E. Furthermore, additional evidence is provided for the synergy between the cyotokines constitutively produced by human BMEC and EPO for regeneration and differentiation of erythroid progenitors.

publication date

  • December 1996

Research

keywords

  • Academic Article

Additional Document Info

start page

  • 1117

volume

  • 24

number

  • 9