Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation

Wu, Michael H.; Smith, Stephen L.; Danet, Guénahel H.; Lin, A. T.; Williams, Stephanie; Liebowitz, David; Dolan, M. Eileen

doi:10.1038/sj.bmt.1703054

Cited by 25 publications

(19 citation statements)

References 52 publications

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“…13 Here, we hypothesize that the interaction between VCAM-1 and/or fibronectin, expressed and/or secreted by osteoblasts, and the VLA-4 integrin, expressed on HPCs, may provide a prosurvival signal for UCB-HPCs exposed to low O 2 . To test this, we plated freshly isolated UCB-CD34 ؉ progenitors onto poly-lysine (PLL)-, VCAM-1-, or fibronectin (FN)-coated plates in the presence of a cytokine cocktail (SCF ϩ FLT3L ϩ TPO), shown previously to be optimal for HPC cultures 21 ; the plates were then immediately placed in a 20% ( Figure 1A left) or 2% to 3% (Figure 1A middle) O 2 environment, and cell death was assessed after 48 hours. Compared with 20% O 2 culture, we observed a more than 2-fold increase in cell death in 2% to 3% O 2 culture.…”

Section: Resultsmentioning

confidence: 99%

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Biology of umbilical cord blood progenitors in bone marrow niches

Dao

Creer

Nolta

et al. 2007

Blood

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Within the bone marrow (BM), hematopoietic progenitor cells (HPCs) are localized in poorly oxygenated niches where they interact with the surrounding osteoblasts (OBs) through VLA4/VCAM-1 engagement, and are exposed to interleukin-6 (IL-6), stem cell factor (SCF), and chemokines such as CXCL12 (OB factors). Umbilical cord (UC) is more highly oxygenated that the BM microenvironment. When UC-HPCs are exposed to the 2% to 3% O 2 concentration found in the bone endosteum, their survival is significantly decreased. However, engagement of VLA-4 integrins on UCB-derived CD34 ؉ cells reduced cell death in 2% to 3% O 2 conditions, which was associated with an increase in phospho-Ser473 AKT and an increase in phospho-Ser9 GSK3b. Consistent with the role of GSK3b in destabilizing beta-catenin, there was more cytoplasmic beta-catenin in UC-HPCs exposed to 2% to 3% O 2 on fibronectin, compared with suspension culture. UC-HPCs cultured at 2% to 3% O 2 with OB factors showed an increase in nuclear betacatenin and persistence of a small pool of CD34 ؉ 38 ؊ HPCs. CFU assays followed by surface phenotyping of the plated colonies showed improved maintenance of mixed lineage colonies with both erythroid and megakaryocytic precursors. These studies provide a biologic perspective for how UC-derived HPCs adapt to the bone endosteum, which is low in oxygen and densely populated by osteoblasts. IntroductionThe bone marrow (BM) endosteum has been identified as the primary niche for primitive hematopoietic progenitor cells (p-HPCs), while the BM microvascular region serves as the site for committed HPCs (c-HPCs). [1][2][3][4][5] A longitudinal cross section of the bone shows that the endosteum is the farthest site from the bone vasculature. Consistent with this observation, the endosteum has the lowest oxygen pressure, compared with other regions within the bone. 6 It was therefore concluded that low oxygen plays a role in the maintenance of p-HPCs. Consistent with this notion, when p-HPCs are cultured under low oxygen conditions, they retain their full multilineage engraftment potential when transplanted into an irradiated recipient. 7 At the molecular level, the mechanisms by which low oxygen mitigates this effect are not fully understood, and to further delineate it was a major aim of the current study.Another important feature of the endosteum is its mesenchymal composition. 8 Compared with other regions of the bone, the endosteum has the highest number of osteoblasts, which play a role in the maintenance of p-HPCs. 9,10 Manipulation of osteoblast development in vivo directly affects the size of the p-HPC population. 11,12 Both adhesion molecules 13 and soluble factors 14,15 expressed by osteoblasts are important regulators of p-HPCs. These studies collectively suggest that 2% to 3% oxygen and osteoblast-derived factors play critical roles in p-HPC maintenance.Although it is not surprising that BM-derived p-HPCs are better maintained under low oxygen, which is a natural feature within the endosteum, it is somewhat surprising that sim...

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Section: Resultsmentioning

confidence: 99%

“…We did not see a further reduction of cell death on FN, compared with VCAM-1, suggesting that VLA-4 engagement alone was sufficient for the prosurvival effect under 2% to 3% O 2 . We used fibronectin (FN) 21 as the ligand throughout the remaining part of the current studies.…”

Section: Resultsmentioning

confidence: 99%

Biology of umbilical cord blood progenitors in bone marrow niches

Dao

Creer

Nolta

et al. 2007

Blood

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“…CD34 ϩ cells were plated at a density of 0.4 ϫ 10 6 cells/ml in expansion media consisting of serum-free expansion medium (SFEM; StemCell Technologies) that was supplemented with 2% penicillin/streptomycin, recombinant human thrombopoietin (50 ng/ml), SCF (25 ng/ml), and Flt-3 ligand (50 ng/ml) (27). Media were changed when cell density reached 1 ϫ 10 6 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

Functional Interactions between Erythroid Kruppel-like Factor (EKLF/KLF1) and Protein Phosphatase PPM1B/PP2Cβ

Yien

Bieker

2012

Journal of Biological Chemistry

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Background: EKLF is a transcription factor that is critical for erythroid gene expression. Results: The EKLF in vivo interaction with Ppm1b phosphatase has been identified after co-immunoprecipitation. Conclusion:The effect of the interaction is to modulate EKLF stability and activity. Significance: Our data uncover a novel interaction and reveal a multilayered and contextual difference in how Ppm1b alters EKLF regulation of its downstream targets.

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“…83,84 Regarding the requirement for active cell cycling for efficient transfection via electroporation, existing data from cell synchronization experiments are controversial. 80,[85][86][87] Prestimulation of CD34 + cells with recombinant cytokines for various time intervals prior to electroporation has been shown to enhance transfection efficiency and this effect was attributed to the increase in the subpopulation of cells in S phase. 78,83 However, others demonstrated that stimulated and unstimulated CD34 + cells are transfected with equal efficiencies.…”

Section: 79-81mentioning

confidence: 99%