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- Alattar, AG, et al.
(author)
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Gene editing of CD34+ progenitor cells from single blood donor waste bags to create cultured early erythroid cells for study of blood group knock-outs
- 2020
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In: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 115:Suppl. s1, s. 363-363
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Conference paper (peer-reviewed)abstract
- Background: Many blood group antigens are carried by red cell surface proteins, thefunctions of which are not yet fully characterized or in some cases completelyunknown. By investigating red cells with naturally-occurring blood group variants,much has been learnt about the underlying molecules. However, interindividualvariation affecting other molecules than the one(s) of interest may confound orotherwise hamper such studies. As an alternative, a reductionistic approach whereonly a single factor differs between test and control cells significantly facilitatesinterpretation of functional studies. Using various siRNA, shRNA and various gene-editing tools blood group expression can be manipulated and useful modelsdeveloped. Applying the latter on primary hematopoietic stem and progenitor cells(HSPCs) can be challenging.Aims: We evaluated a protocol for gene editing of CD44 using a CRISPR/Cas9hybrid system on HSPCs isolated from blood donation leukocyte waste bags fromsingle donors to develop a model for study of blood group molecular function inerythropoiesis.Methods: Peripheral blood mononuclear cells (PBMCs) were from anonymizedleucocyte waste bags obtained after whole blood unit processing in the Reveosautomated blood component system. Cells were harvested following Lymphoprepgradient separation and CD34+HSPCs enriched and collected using magnetic beads.CD34+cells were cultured in 2-phase culture medium to generate erythroid cellsfrom HSPCs (Vidovic, Vox Sang 2017). For CRISPR/Cas9 gene editing, a short guideRNA (sgRNA) targeting CD44 was designed and cloned into the lentiCRISPR v2vector (Addgene plasmid #52961). A non-targeting sgRNA cloned into the vectorwas used as control. Lentivirus particles were produced in the human 293T cell lineas described previously (Galeev, Methods Mol Biol 2017). Equal number of CD34+cells were transduced 24 hours after collection using RetroNectin following theRetroNectin-Bound Virus (RBV) Infection Method according to manufacturer’sprotocol. Cells were transduced at a multiplicity of infection (MOI) of 10 with atarget transduction efficiency of 20–30%. Cells were cultured at 37°C, 5% CO2 for72 hours in phase I culture medium and then electroporated with Cas9 mRNA usingthe ECM 830 Electroporation System as described previously for cord blood-derivedCD34+cells (Backstrom, Exp Hematol 2019). GFP+CD44-edited cell frequencieswere monitored by flow cytometry at day 7 of the HSPC expansion phase and atday 14 of the erythroid expansion-differentiation phase using antibodies againsterythroid-specific cell surface markers GPA and Band3 in addition to CD49d toassess the erythroid development stage.Results: We tested the above protocol and observed that the frequencies of editedcells lacking CD44 expression within the GFP+population at day 7 of culture were10–25% while the edited frequencies were increased at day 14 of culture to 60–80%within the GFP+cells. Whilst this stage corresponds to erythroblasts, earlier or laterstages can be tested.Summary/Conclusions: The previously established hybrid system for CRISPR/Cas9gene editing in cord blood-derived CD34+HSPCs, which combines lentiviraldelivery of the sgRNA with transient delivery of Cas9 mRNA by electroporation, isalso applicable to primary human adult HSPCs from Reveos-processed single-donorwhole blood donation, resulting in a traceable high-yield gene-editing system tostudy blood group function and erythroid development
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- Dahlbom, I, et al.
(author)
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Immunoglobulin g (IgG) anti-tissue transglutaminase antibodies used as markers for IgA-deficient celiac disease patients
- 2005
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In: Clinical and Diagnostic Laboratory Immunology. - 1071-412X. ; 12:2, s. 254-258
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Journal article (peer-reviewed)abstract
- The role of immunoglobulin A (IgA) anti-tissue transglutaminase antibodies (IgA-tTG) as predictors of untreated celiac disease (CoD) is well documented, and the presence and levels of these antibodies are most accurately monitored with native or recombinant human antigens. However, IgA-deficient CoD patients are not identified by IgA serology, and conflicting results concerning the diagnostic validity of IgG antibodies against gliadin (IgG-AGA), endomysium (IgG-EmA), and tTG (IgG-tTG) have been reported. The aim of the present study was to evaluate the utility of IgG-tTG for the detection of CoD in IgA-deficient patients. Samples from 115 IgA-deficient and 200 IgA-sufficient subjects were collected and tested for the presence of IgA and IgG antibodies against tTG, EmA, and AGA. Antibodies against tTG were measured by an enzyme-linked immunosorbent assay based on recombinant human tTG, and antibodies against EmA were determined by immunofluorescence. The values for IgG-tTG showed a higher correlation (correlation coefficient [r] = 0.91) with those for IgG-EmA for the IgA-deficient subjects than for the IgA-sufficient subjects (r = 0.88). The overall concordance of the positive and negative results between IgG-tTG and IgG-EmA was 97%, and the IgG-tTG assay discriminated between IgG-EmA-positive and -negative subjects with IgA deficiency at a rate of 100%. Elevated levels of IgG-tTG and IgG-EmA were measured in 70% of the IgA-sufficient subjects. IgG-tTG detection with recombinant human tTG is a good alternative to IgG-EmA detection, and the addition of IgG-tTG assessment to present screening methods may improve the ability to identify IgA-deficient subjects with CoD.
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