Transgene expression study of CXCR4 active mutants. Potential prospects in up-modulation of homing and engraftment efficiency of hematopoietic stem/progenitor cells

Abstract

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.

Keywords: Ala, Alanine; Asn, Asparagine; BM, Bone Marrow; BMEC, Bone marrow endothelial cells; BSA, Bovine Serum Albumin; CAMs, Constitutive Active Mutants; CXCR4; Conc., Concentration; ECM, Extracellular matrix; FBS, Fetal Bovine Serum; FN, Fibronectin; HSPCs; HSPCs, Hematopoietic Stem/ Progenitor Cells; HUVECs, Human Umbilical Vein Endothelial cells; IMDM, Iscove's Modified Dulbecco Media; LIF, Leukemia Inhibitory Factor; MCS, Multi Cloning Site; Ser, Serine; TM3, Transmembrane three domain; engraftment; homing.

Figure 1.

Immunofluorescence microscopy of doxycycline induced CXCR4 surface protein expression in stable transfected K-562 cells. (A) Control untransfected K-562 cells, (B) Wild type CXCR4 stable transfected K-562 cells, (C) N119ACXCR4-pTRE2hyg stable transfected K-562 cells, (D) N119SCXCR4-pTRE2hyg stable transfected K-562 cells.

Figure 2.

A bar graph depicts % cell adhesion of mutant 1 (N119ACXCR4) and mutant 2 (N119SCXCR4) expressing cells as compared with wildtype CXCR4 expressing cells on the human umbilical vein endothelial cells (HUVECs) monolayer. The results shown represent average of triplicates plus or minus SD (Error bars indicate standard deviation and * indicates P < 0.05 with respect to wild type).

Figure 3.

A bar graph showing % cell adhesion of mutant 1 (N119ACXCR4) and mutant 2 (N119SCXCR4) expressing cells as compared with wildtype CXCR4 expressing cells on fibronectin coated 24-well plate. The results shown represent average of triplicates plus or minus SD (Error bars indicate standard deviation and * indicates P < 0.05 with respect to wild type).