Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ cells from three SCD patient donors were electroporated with ABE8e-NRCH mRNA or RNP using an sgRNA targeting the SCD mutant HBB codon. (a) The edited region of HBB with the target A at protospacer position 7 shown in blue along with potential bystander edits in green (silent), brown (silent), and red (non-silent). (b) Editing efficiencies by HTS at target and bystander adenines, and indels after 6 days in stem-cell culture media following electroporation. (c) Proportion of β-like globin proteins by HPLC of reticulocyte lysates after 18 days in differentiation media following electroporation. (d) Representative phase-contrast images of reticulocytes derived from unedited or edited donor HSPCs incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (e) Quantification of sickled reticulocytes from counting >300 randomly selected cells by a blinded observer from images as in (d). (f) Venn diagram showing candidate off-target sites nominated by Cas-OFFinder and CIRCLE-seq, and nominated sites for which off-target editing was observed by targeted DNA sequencing in SCD patient CD34+ cells electroporated with ABE8e-NRCH mRNA. (g) Predicted genomic features of validated off-target sites. TTS, ≤1 kb from the transcription termination site; UTR, untranslated region. (h) ABE8e-NRCH-treated HSPCs from two different SCD patient donors were sequenced at 697 potential off-target sites. The histogram shows the number of validated off-target base editing sites binned by average percentage of sequencing reads for each site with any A•T-to-G•C mutations in protospacer nucleotides 4-10. Bar values in (b), (c), and (e) and error bars reflect mean±SD of three independent biological replicates, with individual values shown as dots.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Stem Cell Culture, Electroporation, Derivative Assay, Incubation, DNA Sequencing, Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Representative, immuno-flow cytometry for erythroid maturation stage markers42,43 at culture days 7 and 14. Top: gating strategy to identify single cells expressing the erythroid marker hCD235a. Bottom: gating strategy to track the progress of erythroid maturation based on expression of CD49D and Band3 in hCD235a+ cells. SSC-A: Side scatter area. SSC-W: Side scatter width. FSC-A: Forward scatter area.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Flow Cytometry, Expressing, Marker

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Reverse-phase HPLC chromatograms of erythroid cell lysates at culture day 18, with β-like globins and their associated fractions marked near the associated peak. Data from the most efficiently edited donor is shown. Red arrows indicate the start and end of globin chain peaks.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: High Performance Liquid Chromatography, Derivative Assay, In Vitro

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CIRCLE-seq read counts obtained for each verified off-target site and the alignment of each site to the guide sequence are shown. Bar graphs show the percentage of sequencing reads containing A•T-to-G•C mutations within protospacer positions 4-10 at on-and off-target sites in genomic DNA samples from patient CD34+ HSPCs treated with ABE8e-NRCH mRNA, protein, or untreated controls (n=4). Note that the mutation frequency shown is summed across all reads with one or more A•T-to-G•C mutations in this window. Sequencing errors therefore accumulate in control samples compared to standard sequencing error frequencies for a single nucleotide. Bar values and error bars reflect mean±SD.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Sequencing, Mutagenesis, Control

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Bar graph showing the percentage of sequencing reads containing alleles harboring indels at on-and off-target sites in genomic DNA samples from patient CD34+ HSPCs treated with ABE8e-NRCH mRNA, protein, or untreated controls (n=4). Bar values and error bars reflect mean±SD.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ HSPCs from three HBBS/S SCD patient donors were electroporated with ABE8e-NRCH mRNA and sgRNA targeting the SCD mutant HBB codon. 2-5x105 treated cells were transplanted into NBSGW mice via tail-vein injection. Mice were analyzed 16 weeks after transplantation. (a) Experimental workflow. (b) Engraftment measured by percentage of human CD45+ (hCD45+) cells in recipient mouse bone marrow. (c) Human B-cells (hCD19+), myeloid cells (hCD33+), and T-cells (hCD3+) cells in recipient mouse bone marrow shown as percentages of the hCD45+ population. (d) Human erythroid precursors (hCD235a+) in recipient mouse bone marrow shown as percentage of human and mouse CD45− cells, (e) HBBS-to-HBBG editing efficiencies in human donor CD34+ cell-derived lineages from recipient bone marrow. Erythroid, myeloid, B-cell, and HSPC human lineages were collected using antibodies that recognize hCD235a, hCD33, hCD19, and hCD34, respectively, (f) Clonal editing outcomes determined by single-cell 5’ RNA-seq in CD235a+ cells from the bone marrow of two edited mice. (g) Proportions of β-like globin proteins by HPLC of human donor-derived reticulocytes isolated from recipient mouse bone marrow. (h) Representative phase-contrast images of human reticulocytes from bone marrow incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (i) Quantification of sickled cells as in Fig. 1e. n=14 mice receiving edited cells and n=13 mice receiving unedited cells in b-e, g, and i. Triangle, square, and circle symbols represent HSPCs from three different SCD donors. Plotted values and error bars reflect mean±SD. Statistical significance was assessed by one-way ANOVA in i and by two-tailed Student’s t-test elsewhere; “ns”, not significant.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Injection, Transplantation Assay, Derivative Assay, RNA Sequencing, Isolation, Incubation, Two Tailed Test

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ HSPCs from three HBBS/S SCD patient donors were electroporated with ABE8e-NRCH RNP using a single guide RNA (sgRNA) targeting the SCD mutant codon, followed by transplantation of 2-5x105 treated cells into NBSGW mice via tail-vein injection. Mice were sacrificed and analyzed 16 weeks after transplantation, (a) Experimental workflow, (b) Engraftment measured by the percentage of human donor CD45+ cells (hCD45+ cells) in recipient mouse bone marrow, (c) Human B-cells (hCD19+), myeloid cells (hCD33+), and T-cells (hCD3+) cells in recipient mouse bone marrow, shown as percentages of the total hCD45+ population. (d) Human erythroid precursors (hCD235a+) in recipient mouse bone marrow shown as percentage of total human and mouse CD45−cells. (e) On-target (A7, Fig. 1a) editing efficiencies in human donor CD34+ cell-derived lineages purified from recipient bone marrow by fluorescence-activated cell sorting. Erythroid, myeloid, B-cell, and HSPC human lineages were collected using antibodies that recognize hCD235a, hCD33, hCD19, and hCD34+, respectively. Statistical significance was assessed by one-way ANOVA to compare groups; “ns”, not significant. (f) Percentages of β-like globin proteins determined by reverse-phase HPLC analysis of human donor-derived reticulocytes isolated from recipient mouse bone marrow. (g) Representative phase contrast images of human reticulocytes purified from bone marrow and incubated for 8 hours in 2% O2. Nine images of >50 cells per image were collected per sample. Scale bar=50 μm. (h) Quantification of sickled cells calculated by counting images after incubation for 8 hours in 2% O2 such as in (g). More than 300 randomly selected cells per sample were counted by a blinded observer. n=14 total mice analyzed in panels b-f; triangle, square, and circle symbols represent samples from three different SCD CD34+ HSPC donors. Negative control data is shared with Figure 2. Bar values and error bars reflect mean±SD. Statistical significance between treated and untreated samples was assessed by a two-tailed Student’s t-test; “ns”, not significant.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Transplantation Assay, Injection, Derivative Assay, Purification, Fluorescence, FACS, Isolation, Incubation, Negative Control, Two Tailed Test

Journal: Gut Microbes

Article Title: Gut microbiota-regulated unconjugated bilirubin metabolism drives renal calcium oxalate crystal deposition

doi: 10.1080/19490976.2025.2546158

Figure Lengend Snippet: UCB promotes CaOx crystals adhesion to the renal tubular epithelial MDCK cells. (A) MDCK cells were treated with UCB in 1% FBS environment for 8 hours, followed by treatment of COM crystals. The amount of COM crystals adhered to cells was counted (bar = 20 μm; quantitative data shown are mean with SD, n = 5 of each group). (B) 2D cultured- and 3D cultured MDCK cells were treated with UCB and the morphology of cell was observed using phase contrast microscope (2D-Bar = 200 μm, 3D-Bar = 50 μm). (C) MDCK cells were treated with UCB in 1% FBS environment for 24 hours. Cell death was detected using Annexin V-FITC and PI staining and flow cytometric detection (quantitative data shown are mean with SD, n = 3 of each group). (D) MDCK cells were treated with UCB in 1% FBS environment for 24 hours. Western blotting analysis was performed to assess the expression of the indicated proteins. Pro-Cas3: pro-caspase 3; Cle-cas 3: cleaved-caspase 3; Cle-PARP-1: cleaved-PARP-1; (quantitative data shown are mean with SD, n = 3 of each group). (E) MDCK cells were treated with UCB in 1% FBS environment, combining with/without Z-VAD or fer-1 for 24 hours. The morphology of MDCK cells were observed using phase contrast microscope (bar = 50 μm). (F) MDCK cells were treated with UCB in 1% FBS environment, combining with/without Z-VAD or fer-1 for 24 hours. Western blotting analysis was performed to assess the expression of the indicated proteins. (G) MDCK cells were treated with UCB in 1% FBS environment combining with/without Z-VAD for 8 hours, followed by treatment of COM crystals. The amount of COM crystals adhered to cells was counted (bar = 50 μm; quantitative data shown are median with interquartile range, n = 5 of each group). ** p < .05, *** p < 0.01, **** p < 0.001, ** p < 0.0001.

Article Snippet: Canine kidney distal tubule epithelial MDCK cells, which were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Cell Culture, Microscopy, Staining, Western Blot, Expressing

Journal: Gut Microbes

Article Title: Gut microbiota-regulated unconjugated bilirubin metabolism drives renal calcium oxalate crystal deposition

doi: 10.1080/19490976.2025.2546158

Figure Lengend Snippet: UCB increases Slc26a6 expression of renal tubular epithelial cells and promotes transmembrane oxalate excretion. (A) The diagram illustrates an in vitro cell model that simulates renal tubular epithelium and the detection of oxalate secretion. (B) MDCK cells were treated with UCB in 1% FBS environment for 10 hours, followed by oxalate treatment in basal membrane side for 2 hours. The oxalate concentrations of upper medium and lower medium were detected using ion exchange chromatography (quantitative data shown are mean with SD, n = 3 of each group). (C) 3D-cultured cysts of MDCK cells were treated with UCB in 1% FBS environment for 6 hours. The expression of Slc26a6 was detected using if (confocal) (bar = 10 μm; quantitative data shown are median with interquartile range, n = 13 of each group). (D) MDCK cells were treated with UCB in 1% FBS environment for 6 hours, combining with/without rottlerin. The expressions of indicate proteins were detected using Western blotting analysis. (E) the renal tubular Slc26a6 expression of rat was evaluated using IHC analysis (bar = 100 μm; quantitative data shown are median with interquartile range, n = 7 of each group). * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: Canine kidney distal tubule epithelial MDCK cells, which were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing, In Vitro, Membrane, Ion Exchange Chromatography, Cell Culture, Western Blot