Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation

Abstract

A major limitation to clinical stem cellmediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34 ؉ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34 ؉ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34 ؉ /CD38 ؊/low cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34 ؉ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors. ( IntroductionGene transfer into human hematopoietic stem cells (HSCs) may be a promising tool in the correction of a wide variety of hematopoietic and genetic disorders. HSC transplantation can be used to durably deliver these genetically modified cells to the bone marrow (BM), which in turn will release mature cells with the corrected gene into the circulation throughout life. Clinical and experimental HSC transplantation procedures mimic the physiologic process of HSC migration from the circulation into the BM occurring during late embryonic development and steady-state hematopoiesis in adults throughout life. [1][2][3] One of the disadvantages of BM transplantation is the long-lasting reduced levels of immature progenitors, including long-term culture-initiating cells (LTCICs; 1 log reduction), in the BM of patients who have received transplants compared with healthy individuals. [4][5][6] Furthermore, emerging evidence exists for impaired homing 7 and low engraftment 8 of retrovirally transduced human CD34 ϩ cells. Enhanced efficacy of HSC engraftment could improve the outcome of clinical transplantations as well as gene therapy protocols and might be achieved by modulating the ability of stem cells to home to and repopulate the recipient BM.Interactions between the chemokine stromal-derived factor 1 (SDF-1), also referred to as CXCL12, and its receptor CXCR4 play an essential role in stem cell seeding of the BM during murine embryonic development. 9,10 Moreover, we have previously demonstrated in a functional preclinical model, using nonobese diabetic/ severe ...

Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium

“…SDF-1α and its unique receptor, CXCR4 play an important role in stem cell homing, chemotaxis, expression of adhesion molecules, engraftment [3], proliferation, and survival [4]. CXCR4 expression is endogenously regulated by tissue environmental factors such as cytokines, chemokines, stromal cells, adhesion molecules, myocardial ischemia and proteolytic enzymes [5]. SDF-1α, secreted by cells within ischemic myocardium, is a potent chemo-attractant for cells expressing CXCR4.…”

Functional CXCR4-Expressing Microparticles and SDF-1 Correlate with Circulating Acute Myelogenous Leukemia Cells

“…CD34 + enriched progenitors were lentivirally transfected with pHR ¶-EF1a-GFP-SIN (control vector) or pHR ¶-EF1a-CXCR4-IRES-GFP-SIN (CXCR4 vector) as described (5). Twenty-four hours after transduction of target CD34 + cells, conditioned media were discarded and changed for prefiltered (0.2 Am) fresh serum-free RPMI, and cells were incubated for additional 24 hours.…”

“…into mice, which were sacrificed 16 hours later. Samples of bone marrow cells flushed from both femur and tibia bones and spleen cell suspension were prepared, and the percentage of human cells was determined by staining with anti-human CD45-FITC mAb as described (5,11).…”

“…It is functionally expressed on a multitude of tissues and cell types, including the majority of hematopoietic cells (1). Overexpression of CXCR4 on human CD34 + progenitors increases their proliferation, migration, and repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (5). Disruption of SDF-1/CXCR4-mediated cell anchorage is followed by release of hematopoietic progenitor and mature cells from the bone marrow into the circulation, regulated by various proteolytic enzymes (3).…”

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

“…For gene transfer into HSCs, moloney-derived retrovirus vectors and lentivirus vectors are often used, although the transduction efficiencies of the retrovirus vectors are disappointingly low in immature HSC/progenitors, probably due to the quiescent state of HSCs and the lack of suitable receptors for vector binding. 1,2 Lentivirus vectors have recently shown promise, 3,4 but their safety remains to be established.…”