Journal: BMC Cell Biology

Article Title: Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

doi: 10.1186/1471-2121-7-30

Figure Lengend Snippet: Purity assessment of the CD34+ cell fraction by flow cytometry . The initial purity of CD34+ cells after separation through single column was 47.5%. The CD34- fraction was 99.4% pure. CD34+ and CD34- cell populations were defined by first gating on forward and side scatter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody. Percentages indicating the purity of isolated cell fractions are shown for both plots. Abbreviations: SSC, side scatter; PE, phycoerythrin.

Article Snippet: When using the Direct CD34 Progenitor Cell Isolation Kit with single column separation and the labeling protocol recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), a purity of less than 50% was reached for the CD34+ cells (Figure ).

Techniques: Flow Cytometry, Control, Labeling, Isolation

Journal: BMC Cell Biology

Article Title: Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

doi: 10.1186/1471-2121-7-30

Figure Lengend Snippet: Purity assessment of CD34+ cell fraction after one or two column separations . A) The CD34+ cell fraction was 78% pure after the first column separation. B) A 92% pure CD34+ cell faction was obtained by an additional labeling step in connection with a second column separation. CD34+ cell populations were defined by first gating on forward and side scatter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody. Percentages indicating the purity of isolated cell fractions are shown for both plots. Abbreviations: SSC, side scatter; PE, phycoerythrin.

Article Snippet: When using the Direct CD34 Progenitor Cell Isolation Kit with single column separation and the labeling protocol recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), a purity of less than 50% was reached for the CD34+ cells (Figure ).

Techniques: Labeling, Control, Isolation

Journal: BMC Cell Biology

Article Title: Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

doi: 10.1186/1471-2121-7-30

Figure Lengend Snippet: Purity assessment of CD34+/-, CD133+/- and Lin-/+ cell fractions . A) Purities for CD34+ and CD34- cell factions were 97.1% and 99.1%, respectively. B) Purities for CD133+ and CD133- fractions were 93.6% and 99.1%, respectively. C) Purities for Lin- and Lin+ cell factions were 97.0% and 99.5%, respectively. CD34+/-, CD133+/- cell populations were defined by first gating on forward and side scatter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody. Percentages indicating the purity of isolated cell fractions are shown for both plots. Abbreviations: SSC, side scatter; IgG, immunoglobulin; PE, phycoerythrin.

Article Snippet: When using the Direct CD34 Progenitor Cell Isolation Kit with single column separation and the labeling protocol recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), a purity of less than 50% was reached for the CD34+ cells (Figure ).

Techniques: Control, Labeling, Isolation

Journal: BMC Cell Biology

Article Title: Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

doi: 10.1186/1471-2121-7-30

Figure Lengend Snippet: A chart of the optimized protocols to isolate CD34+/-, CD133+/- and Lin-/+ cells from cord blood . Isolation of CD34/- and CD133+/- cells was performed using Direct CD34 Progenitor Cell Isolation Kit (#130-046-702, Miltenyi Biotec) and CD133 Cell Isolation Kit (#130-050-801, Miltenyi Biotec), respectively. Lin-/+ cells were isolated using StemSep Human Progenitor Enrichment Kit (#14056, StemCell Technologies). For all magnetic separations, MACS columns and separators (Miltenyi Biotech) were used. Abbreviations: MNC, mononuclear cells; Buffer, PBS pH 7.2 supplemented with 0.5% bovine serum albumin and 2 mM EDTA or 0.6% ACD/A.

Article Snippet: When using the Direct CD34 Progenitor Cell Isolation Kit with single column separation and the labeling protocol recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), a purity of less than 50% was reached for the CD34+ cells (Figure ).

Techniques: Isolation, Cell Isolation

Journal: BMC Cell Biology

Article Title: Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

doi: 10.1186/1471-2121-7-30

Figure Lengend Snippet: Frequency of different types of CFU colonies within CD34+, CD133+, Lin- and MNC populations.

Article Snippet: When using the Direct CD34 Progenitor Cell Isolation Kit with single column separation and the labeling protocol recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), a purity of less than 50% was reached for the CD34+ cells (Figure ).

Techniques:

Journal: bioRxiv

Article Title: Tuning of granulopoietic signaling by de novo designed agonists

doi: 10.1101/2023.11.25.568662

Figure Lengend Snippet: Representative western blot images of intracellular levels of phospho-STAT3 (Tyr705) and STAT3 (A) and phospho-STAT5 (Tyr694) and STAT5 (B) proteins after treatment of NFS-60 cells with 1 nM of different designs (saturating conditions) for 5 min (left pane) or 30 min (right pane). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining was used as a loading control. Three biological replicates of this experiment showed the same trend . (C, D) The pSTAT3 and pSTAT5 levels were quantified after 5 min and 30 min from those replicates and are represented in (C) and (D), respectively. Shown is the fold change to the GAPDH normalized rhG-CSF signal. (E, F) To probe the effect of the agonist designs (100 ng/ml) and rhG-CSF (10 ng/ml) on healthy donoŕs CD34 + HSPCs, proliferation assays were performed without (E) or with the addition of 50 ng/mL SCF and 20 ng/mL IL-3 (F). Ori0 without SCF and IL-3 was tested at three concentrations 100 ng/mL (diamond), 10 ng/mL (triangle-right), and 1 ng/mL (triangle-left). Shown is the mean (points) and standard deviation (shades) of three parallel replicates. (G, H, I) Additionally, we performed CFU assays of healthy donoŕs CD34 + HSPCs incubated on semi-solid medium supplemented with corresponding cytokines and design agonists. Shown is the fold change to rhG-CSF of the quantified colony-forming units (CFU) of granulocytes (G-CFU) (G), granulocyte-macrophages (GM-CFU) (H), and macrophages (M-CFU) (I). The circles represent the obtained values for each condition of three independent experiments with two parallel replicates each. The indicated p-values were estimated by an ordinary one-way ANOVA followed by a Tukey HSD test.

Article Snippet: Human CD34 + cells were isolated from the bone marrow mononuclear cell fraction of two healthy donors by Ficoll density gradient centrifugation with subsequent magnetic bead separation using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech Germany; #130-046-703).

Techniques: Western Blot, Staining, Control, Standard Deviation, Incubation

Journal: bioRxiv

Article Title: Identification of genome safe harbor loci for human gene therapy based on evolutionary biology and comparative genomics

doi: 10.1101/2023.09.08.556857

Figure Lengend Snippet: a Genome targeting with eGFP expressing cassettes into GSH candidates using specific CRISPR/Cas9-ssDNA editing sets. Single-stranded DNA donor templates were flanked with 300 nt homology arms to mediate homologous recombination through single stranded template repair (SSTR) pathway. b Experiments with bone marrow CD34+ HSPCs. Pre-stimulated cells were nucleofected in presence of the CRISPR/Cas9-ssDNA editing set and cultured for two days before the sorting of the GFP+ population. The GFP+ cells were cultured for an additional three days before downstream processing. Alternatively, AAV6 vectors were used to deliver templates to target Ap.102 , Dep.33 , Dep.13 , Dep.1 and Dep.35 loci. In parallel, 1 day-nucleofected cells were cultured in erythroblast expansion conditions before maturation with human EPO. c Percentage of GFP+ cells at two days post-nucleofection. Intergenic and intronic loci are depicted as red and blue bars, respectively. Open circles represent individual human donors. AAVS1 GSH (gray) served as reference control. Mean ± s.d, ANOVA followed by Dunnett test * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. n ≥ 4 independent experiments. d Cell viability determined by flow analysis of propidium iodide stained cells. Comparison control corresponds to unedited cells (mock). Open circles represent independent human donors. Mean ± s.d, ANOVA followed by Dunnette test, n ≥ 3 independent experiments. e Proliferation rate of GFP+ HSPCs three days after sorting. ANOVA followed by Dunnett test, n ≥ 3 independent experiments. f Targeting of long term-HSCs (CD34+/CD90+, magenta) and progenitor cells (CD34+/CD38+, CD34+/CD38-) within the GFP+/CD34+ fraction. n = 2-3 independent experiments using different human donors. g Targeted Integration in GFP+ cells sorted after 14 days of liquid culture. Specific primer/probe sets (arrows and bars) were utilized. The efficiency was calculated as the FAM/HEX ratio. n = 2 independent experiments. h Persistent transgene expression after 45 days of liquid culture. i AAV6 delivering of templates for Ap.102 , Dep.33 , Dep.13 , Dep.1 or Dep.35 loci show increased levels of sorted GFP+ cells without affecting cell viability and high level of integration.

Article Snippet: Human bone marrow CD34+ stem and progenitor cells (HSPCs), obtained from healthy adult donors, were purchased from Stem Cell Technologies (SCT, Cambridge, MA 02142).

Techniques: Expressing, CRISPR, Homologous Recombination, Cell Culture, Staining, Comparison

Journal: bioRxiv

Article Title: Identification of genome safe harbor loci for human gene therapy based on evolutionary biology and comparative genomics

doi: 10.1101/2023.09.08.556857

Figure Lengend Snippet: a Colony forming units (CFUs) per targeted locus after 14 days cultivation in differentiation medium (bright field and epifluorescence). The GSH name is indicated on the top, the category on the bottom. and the CFU on the left (E, erythroid; G, granulocyte; GM, granulocyte–macrophage and M, macrophage). AAVS1 , Dep.2 , Dep.3 , Dep.55 , Proto.218 , Proto.2 and Prot.181 GSHs allowed widespread transgene expression, whereas Dep.55 , Dep.1 and Ap.102 restricted expression to CFU-G, -GM, and -M. Scale bar = 200 µm. b Total colony distributions per GSH-targeted cells. Mean ± s.d., n = 3 independent experiments. Two way ANOVA followed by Dunnett test * P < 0.05, ** P < 0.01. c Erythroid (CD235a+) and myeloid (CD69+) composition of GFP expressing cells. Representative FACS plots are shown (left). d Normalized total reads detected on the transgene in sorted myeloid and erythroid populations derived from the targeting of Dep.1 or Ap.102 GSH sites. e Stages of erythroid maturation. f Erythroid maturation from AAVS1 , Dep.2 , Dep.3 , Prot.181 , Dep.55, Prot.2 , and Prot.218 unsorted cultures. Upper , 14 days-erythroblasts retain GFP+ signal. Scale bar 100 μm. Bottom , Enucleation occurs in presence (+EPO) but not in absence of human EPO (-EPO). Nuclear counterstaining with Hoechst 33342. Representative pictures of n = 2 independent human donors.

Article Snippet: Human bone marrow CD34+ stem and progenitor cells (HSPCs), obtained from healthy adult donors, were purchased from Stem Cell Technologies (SCT, Cambridge, MA 02142).

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension

doi: 10.3389/fimmu.2022.936164

Figure Lengend Snippet: (A) Human spleen and liver organoids in the mouse kidney capsule. NSG BLT mice were engrafted with fetal liver, thymus and hematopoietic cells; human engraftments were confirmed by gross inspections of the mouse kidney capsules at euthanasia, where organoids develop in humanized mice. (B) Experimental timeline. NSG mice were engrafted with either human hematopoietic CD34+ cells (cord-blood derived) or with human liver, thymus, and hematopoietic stem cells (fetal-derived). After confirmation of human engraftments by flow cytometry, humanized mice were infected with HIV and either placed in hypoxia or injected with SU5416 to induce PAH. Animals were monitored for any signs of disease including graft-vs-host disease, labored breathing or wasting throughout the course of the study. Mice were subjected to open-chest right heart catheterizations under anesthesia; specimens were collected for further analyses.

Article Snippet: Mice humanized with CD34 + stem cells were purchased from The Jackson Laboratory and showed a 31-48% level of human CD45 + cell engraftment.

Techniques: Capsules, Derivative Assay, Flow Cytometry, Infection, Injection