Selective in vitro expansion and efficient retroviral transduction of human CD34⁺ CD38^– haematopoietic stem cells

Abstract

Summary. Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38– cells from purified CD34+ cells upon stimulation with Flt3‐ligand, stem cell factor and thrombopoietin. Over a 100‐fold (range 80 to 128‐fold) expansion of CD34+CD38– cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38– cells remained negative for lineage‐specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B‐lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38– cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38– cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine‐stimulated CD34+ cells were increased by 20‐fold. Expanded CD34+CD38– cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long‐lasting expression of retroviral‐encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3‐ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38– cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.

Radiation Sensitivity of Human CD34+Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects

“…Those results are in line with an NDI of 1.33 6 0.08 for isolated lymphocytes previously reported by Baeyens et al (49) and in accordance with Fenech, who proposed an anticipated NDI range of 1.3-2.2 for normal peripheral blood lymphocytes cultured under optimal conditions (18). The strict culture conditions of CD34 þ cells required different growth media with early acting cytokine (FL/SCF/TPO) stimulation and different well plates than the T lymphocytes (51)(52)(53). Under these cell culture conditions, an NDI of 1.58 6 0.13 was obtained, which was not significantly different from the NDI of newborn and adult T-lymphocyte cultures.…”

CD34 + Cells Home, Proliferate, and Participate in Capillary Formation, and in Combination With CD34 − Cells Enhance Tube Formation in a 3-Dimensional Matrix

“…23,35,36 Our observation that human CD34 To study the proliferation of LNGFR + -cells in situ, consecutive sections were stained with anti-LNGFR antibody or an antibody against Ki67 nuclear-antigen, which is expressed on all proliferating cells during late G1, S, G2, and M-phases of the cell-cycle 3 .…”

“…Genetic marking was performed as described previously 23 using a retrovirus encoding the truncated low-affinity nerve growth factor receptor (LNGFR) as a marker gene. After transduction, CD34…”

Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells

“…The extended transduction protocol was evaluated, as improved cycling and retroviral transduction of NOD/SCID repopulating cells has been described using longer periods of in vitro manipulation (Bjorgvinsdottir et al 2002;Dorrell et al 2000;Ng et al 2002), and similar data have been reported in a primate transplantation and gene therapy model (Sellers et al 2004;Takatoku et al 2001), or with lentiviral vectors (Piacibello et al 2002. In addition, other groups addressing the relationship between cell cycling and engraftment eYciency, have postulated an engraftment defect for cells in active cycle (Ahmed et al 2004;Gothot et al 2003), and along this line Takatoku et al (2001) recently demonstrated, a beneWcial eVect of extended posttransduction culture on engraftment.…”