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 ...

Nurr1 is an orphan nuclear receptor required for the development of midbrain dopaminergic neurons. To better understand the molecular consequences of Nurr1 expression, we compared the transcriptomes of two independent control and Nurr1-expressing NSC lines using Affymetrix cDNA microarrays. These data reveal the regulation of genes involved in promoting cell survival (trophic/growth factors and stress response genes) and in preventing cell death (decreased caspase-3 and caspase-11 expression). We found that conditioned medium from Nurr1-expressing NSC lines enhanced the survival of midbrain dopaminergic neurons in primary cultures and that Nurr1-expressing NSC lines themselves were more resistant to oxidative stress. These findings are accompanied by a dynamic pattern of gene regulation that is consistent with a role for Nurr1 in promoting both the acquisition of brain-region-specific identity (Engrailed-1) and neuronal differentiation (tubulin ␤ III). Interestingly, our gene expression profiles suggested that tenascin-C was regulated by Nurr1 in developing dopaminergic neurons. This was further confirmed in vitro and in Nurr1 knockout mice where low levels of tenascin-C mRNA were observed. Analysis of tenascin-C-null mice revealed an increase in the number of Nurr1 ؉ cells that become tyrosine hydroxylase-positive (TH ؉ ) dopaminergic neurons at embryonic day 11.5, suggesting that tenascin-C normally delays the acquisition of TH by Nurr1 ؉ precursors. Thus, our results confirm the presence of both secreted and cell-intrinsic survival signals modulated by Nurr1 and suggest that Nurr1 is a key regulator of both survival and dopaminergic differentiation. STEM CELLS 2007;25:511-519