Optimal conditions for lentiviral transduction of engrafting human CD34+ cells.

Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml⁻¹) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml⁻¹) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml⁻¹) reduced engraftment of CD34+ cells. SCF alone (100 ng ml⁻¹) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.

Vitamin D deficiency in pregnant women of ethnic minority: a potential contributor to preeclampsia.

OBJECTIVE: We investigated risk for comorbidities and preeclampsia at low vitamin D levels in ethnic minorities. STUDY DESIGN: Umbilical cord vitamin D (25(OH)D) concentration was determined in urban minorities: 80.9% African American and 17% Hispanic mothers-baby pairs. To identify the correlation between vitamin D levels and high-risk comorbidities which result in preeclampsia, multivariate logistic regression analyses were performed. RESULT: Below the Institute of Medicine threshold of 25(OH)D for pregnant women (25 ng ml⁻¹), obesity (P=0.055) and pregestational diabetes (odds ratio (OR)=2.056) were observed. The study median was 16 ng ml⁻¹ (<25th percentile), at which gestational hypertension (P=0.042), chronic hypertension (OR=4.842) and pregestational diabetes (OR=3.45) became relevant. The risk for preeclampsia increased 12-fold with gestational hypertension (P=0.003) and 14-fold with combined chronic and gestational hypertension (P=0.001). CONCLUSION: Pregnant women of ethnic minority had lower median vitamin D levels which may contribute to a potential risk for preeclampsia.