Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Millington, Michelle; Arndt, Allison J; Boyd, Maureen P.; Applegate, Tanya; Shen, Sylvie

doi:10.1371/journal.pone.0006461

Cited by 46 publications

(37 citation statements)

References 62 publications

(54 reference statements)

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“…Previously, certain studies have performed cell culture without FBS or FCS for pre-clinical data or embryonic stem cells, as the serum affects the cell culture performance (15)(16)(17). STK1 and STK2 are serum-free media with unclear roles in the culture of normal and cancer cells.…”

Section: Discussionmentioning

confidence: 99%

Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2

et al. 2014

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Abstract. The majority of cells are cultured with Dulbecco's modified Eagle's medium (DMEM) or RPMI supplemented with fetal bovine serum (FBS), which contains numerous factors, including cytokines, nutrients and unknown growth factors. These factors may affect cell growth, apoptosis and differentiation. The serum-free medium, STK2, has been previously reported as suitable for the cell culture of human mesenchymal stem cells. However, how STK1 or STK2 affect the cell proliferation of normal and cancer cells remains unknown. The present study examined the growth of the human gingival fibroblast (HGF-1) cell-line and the HSC-3, CA9-22 and MSTO cancer cell-lines, cultured with STK1 and STK2. STK1 increased the cell proliferation of HGF-1 compared to DMEM by assessment with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, whereas STK1 and STK2 markedly inhibited the cell proliferation of HSC-3 and MSTO. The cell proliferation rate of CA9-22 cultured with STK1 or STK2 for 96 h was ~2-fold higher than the rate for 24 h culture. The shape of the HSC-3 cells was also found to have changed to round when cultured with STK2. These results indicate that STK1 increased the cell proliferation of HGF-1 compared to DMEM, whereas the proliferation of HSC-3 and MSTO was inhibited by STK1 and STK2. Thus, STK1 and STK2 had different affects on the cell growth of HGF-1, CA9-22, HSC-3 and MSTO.

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Section: Discussionmentioning

confidence: 99%

Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2

et al. 2014

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“…+ cells were thawed and prestimulated in X-vivo 10 base media (Lonza) supplemented with SCF (10 μg/mL), TPO (5 μg/mL), IL-3 (10 μg/mL), and FLT3-L (10 μg/mL) for 48 h (39). On the day of infection, an appropriate amount of viral soup (both control and DOCK4 shRNA) to infect 100,000 cells at a multiplicity of infection of 20 was added to 0.25 mL of complete X-vivo 10 media.…”

Section: Hsc Culture Cd34mentioning

confidence: 99%

Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes

Sundaravel

Duggan

Bhagat

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.yelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders that are characterized by cytopenias caused by ineffective hematopoiesis (1-3). Even though MDS may transform to acute leukemia in one-third of patients who have MDS, cytopenias drive morbidity for most patients (4). Most of the morbidity experienced by these patients is due to low red blood cell (RBC) counts; therefore, studies on the molecular pathogenesis of dysplastic erythropoiesis are critically needed. Cytogenetic studies have shown that stem and progenitor cells in MDS contain deletions in chromosomes 5, 7, 20, and others (5). Deletions of the chromosomal 7q region are seen in 10% of cases and are associated with significantly worse survival (6). These genomic deletions are usually large, and even though some candidate pathogenic genes have been postulated (7), it is not clear which of the deleted genes contribute to the pathogenesis of ineffective erythropoiesis and dysplasia in MDS.In a previous study (8), we had observed that numerous 7q genes, including dedicator of cytokinesis 4 (DOCK4), were deleted or epigenetically silenced in MDS, thus prompting an examination of its role in erythropoiesis in the present study. DOCK4 belongs to a large family of proteins (CED5/DOCK180/MYOBLAST CITY class) that is well conserved in mammals (9-11). They are large proteins (>200 kb) that act as signaling intermediates and provide docking sites for many other signaling molecules. One of the welldescribed functions of DOCK4 is its ability to activate GTPases, such as ras-related C3 botulinum toxin substrate 1 (RAC1) and RAP1, in man...

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“…18,19 Moreover, when combining high vector doses with strong cytokine stimulation, the risk for multicopy integration and insertional mutagenesis cannot be neglected. 3,4 In an effort to achieve high-level transduction of HSCs under mild transient cytokine stimulation to preserve stem cells fitness, [20][21][22] we engineered LVs displaying stem cell factor (SCF) at their surface, able to efficiently transduce HSCs in vivo. [22][23][24] Therefore, mild cytokine stimulation allowing efficient HSC transduction is an important objective.…”

Section: Introductionmentioning

confidence: 99%

Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs

Girard-Gagnepain

Amirache

Costa

et al. 2014

Blood

119

142

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Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Cited by 46 publications

References 62 publications

Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2

Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2

Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes

Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs

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