Abstract
Susceptibility of human hematopoietic stem cells to infection with porcine endogenous retroviruses (PERV) is of prime importance in determining the feasibility and safety of transplanting pig organs into humans. Under the influence of lineage specific cytokines, umbilical cord blood stem cells were evaluated for their permissiveness to PERV prior to linage commitment as well as after induction into the myeloid, erythroid or megakaryocytic pathways. CD34+ cells were plated in serum-containing nutrient medium supplemented with stem cell factor (SCF) to induce proliferation - a requirement for successful retroviral infection. Various microenvironment conditions included addition of lineage specific secondary cytokines for myeloid (G-CSF), erythroid (EPO), and megakaryocytic (TPO) commitment. After seven days, one-half of each culture was exposed to retroviral pseudotypes composed of replication competent wildtype PERV-NIH virions that also carry an MLV-based retroviral vector genome encoding β-galactosidase (β-gal) (Wilson et al, J. Vir,2000); control cultures were not exposed to the virus. Analyses for PERV infection were implemented after 72 hours’ viral exposure with the following observations: (1) exposure to PERV had no detected effect on proliferation nor on terminal differentiation of all three hematopoietic lineages; (2)PERV infected (β-gal +) cells (Fig.1) were detected in all cultures exposed to PERV regardless of microenvironment variations; and (3) PERV reverse transcription (DNA) and gene expression (RNA) as analyzed by PCR were positive only in virus exposed cultures (Fig. 2). Control cultures were negative in all analyses for PERV entry and expression. Preliminary observations indicate the erythroid lineage (SCF+E) may be more efficient in supporting PERV infection than the uninduced CD34+ cells (SCF) as well as those committed to the myeloid (SCF+G) and megakaryocytic (SCF+T) lineages. These results extend the initial observation that PERV could infect established hematopoietic cells lines to now show that the in vitro tropism of PERV also includes primary hematopoietic cells representing three lineages.
Nuclear staining of PERV+ cells. Upper cell is in mitosis
qPCR Data of PERV-Exposed Cells
