stemspan sfem-ii medium #70008  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: In vivo production of an anti-HIV antibody from primate hematopoietic cells by non-viral knock-in

doi: 10.1101/2025.05.02.651933

Figure Lengend Snippet: (A) Target genomic site within the NHP IGH locus, between the VDJ genes and the constant regions, sequence verified in two NHP species, M. mulatta and M. nemestrina . All possible Cas9 and Cas12a guides were screened within this sequence. (B) CD34 + HSPC were isolated from M. nemestrina bone marrow aspirates and electroporated with RNP. Target locus was analyzed for genetic edits, including single nucleotide polymorphisms (SNP), insertions, and deletions (indel). (C) CD34 + purity following immunomagnetic separation as measured by flow cytometry. (D, E) Indel frequency of selected Cas9 (D) and Cas12a (E) guides 3 days after electroporation of 10 CD34 + cells (n = 3 biological replicates, 3 donors). Data are presented as mean ± standard deviation (SD). Significance was calculated with an ordinary one-way ANOVA with *p<0.05, and **p<0.01.

Article Snippet: Immediately following isolation, NHP HSPC were transferred to HSPC medium (StemSpan Serum-Free Expansion Medium (SFEM) II (StemCell Technologies) with 100 ng/mL recombinant human thrombopoietin (TPO), stem cell factor (SCF), and flightless 3 (FLT-3) ligand, all from CellGenix) at 1.0×10 cells/mL in tissue culture (TC)-treated flasks, incubated at 37°C and 5% CO 2 .

Techniques: Sequencing, Isolation, Immunomagnetic Separation, Flow Cytometry, Electroporation, Standard Deviation

Journal: bioRxiv

Article Title: In vivo production of an anti-HIV antibody from primate hematopoietic cells by non-viral knock-in

doi: 10.1101/2025.05.02.651933

Figure Lengend Snippet: (A) NHP HSPC electroporated with VH4-GFP Cas12a were injected into sub-lethally irradiated, neonatal MISTRG mice. Hematopoietic lineages in the primatized mice were monitored over time and at necropsy. (B) Engraftment levels in peripheral blood over time as measured by flow cytometry on NHP-CD45 + cells from mice injected with electroporated or control HSPC. n = 8 mice, n = 2 NHP donors (electroporated group, 1.39 × 10 CD34 + cells/mouse). n = 6 mice, n = 2 NHP donors (control group, 1.71 × 10 CD34 + cells/mouse). (C, D) Multilineage engraftment in peripheral blood over time as measured by frequency of B cells (CD3 - CD20 + ), T cells (CD3 + CD20 - ), and monocytes (CD3 - CD20 - CD14 + ) in NHP-derived populations (NHP-CD45 + ). (E) Engraftment levels in the bone marrow (femur) and the liver at necropsy, performed at week 20. Error bars represent SD. (F) In vivo transgene expression as measured by frequency of GFP + cells in NHP-derived populations (NHP-CD45 + ), as compared to total B cell frequency in peripheral blood over time. (G) Editing levels at the NHP IGH locus in peripheral blood as measured by high-throughput sequencing on MiSeq platform. Large insertions represented by insertions >8 bp. Lines represent means in (B, C, D, F, and G). n = 8 mice, n = 2 NHP donors (electroporated group, week 18 omitted for low engraftment levels, C-G).

Article Snippet: Immediately following isolation, NHP HSPC were transferred to HSPC medium (StemSpan Serum-Free Expansion Medium (SFEM) II (StemCell Technologies) with 100 ng/mL recombinant human thrombopoietin (TPO), stem cell factor (SCF), and flightless 3 (FLT-3) ligand, all from CellGenix) at 1.0×10 cells/mL in tissue culture (TC)-treated flasks, incubated at 37°C and 5% CO 2 .

Techniques: Injection, Irradiation, Flow Cytometry, Control, Derivative Assay, In Vivo, Expressing, Next-Generation Sequencing

Journal: bioRxiv

Article Title: In vivo production of an anti-HIV antibody from primate hematopoietic cells by non-viral knock-in

doi: 10.1101/2025.05.02.651933

Figure Lengend Snippet: (A) bnAb template was designed for expressing the full light chain and the variable heavy chain fragment of 10-1074. Following productive knock-in at the target locus, bnAb expression replaces endogenous antibody production, bypassing VDJ recombination and forming a full-length transcript via splicing with a constant heavy chain. (B) In vitro 10-1074 production as confirmed by staining of cell surface transgene linker. Flow cytometry was performed on HEK 293E cells following transduction with an expression vector containing an IgG constant heavy chain. (C) Binding of recombinant bnAb to HIV antigen, the gp120 protein of strain CN97001, as measured by bio-layer interferometry (BLI) and quantified as the dissociation constant (K D ). n = 6 technical replicates. (D) NHP HSPC were electroporated with VH4-10-1074 Cas12a , either as dsDNA or AAV, and transplanted into MISTRG mice as before. Beginning at week 8, mice were immunized with CN97001 gp120 injections once every two weeks until necropsy. Control mice were injected with unedited HSPC. (E) Engraftment levels in peripheral blood over time as measured by flow cytometry on NHP-CD45 + cells in mice injected with edited HSPC using different templates. (F) In vivo anti-HIV antibody titers were measured by ELISA with a biotinylated gp120 on diluted plasma samples. dsDNA: n = 5 mice, n = 2 NHP donors. AAV: n = 2 mice, n = 2 NHP donors. Control: n = 1 mouse, n = 2 NHP donors. n = 2 technical replicates per sample. (G) Cell surface expression of 10-1074 in peripheral blood as measured by flow cytometry with antibody against its linker. (H) Editing levels at the NHP IGH locus in peripheral blood as measured by high-throughput sequencing (MiSeq). Large insertions represent insertions >8 bp. (I) B cell populations and fraction of B cell population expressing 10-1074 at necropsy, week 18, as measured by flow cytometry on NHP-CD19 + . n = 5 mice, n = 2 NHP donors (10-1074 dsDNA group, G-J). Error bars represent SD.

Article Snippet: Immediately following isolation, NHP HSPC were transferred to HSPC medium (StemSpan Serum-Free Expansion Medium (SFEM) II (StemCell Technologies) with 100 ng/mL recombinant human thrombopoietin (TPO), stem cell factor (SCF), and flightless 3 (FLT-3) ligand, all from CellGenix) at 1.0×10 cells/mL in tissue culture (TC)-treated flasks, incubated at 37°C and 5% CO 2 .

Techniques: Expressing, Knock-In, In Vitro, Staining, Flow Cytometry, Transduction, Plasmid Preparation, Binding Assay, Recombinant, Control, Injection, In Vivo, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Next-Generation Sequencing

Journal: bioRxiv

Article Title: In vivo production of an anti-HIV antibody from primate hematopoietic cells by non-viral knock-in

doi: 10.1101/2025.05.02.651933

Figure Lengend Snippet: (A) MISTRG6 mice were xenotransplanted using the same protocol applied in MISTRG mice, this time allowing up to 72 hours from birth for HSPC injections. Necropsy was performed at an earlier timepoint to allow the detection of 10-1074-producing cells while antibody titers were still detectable. Control mice received unedited CD34 + cells. (B) NHP engraftment levels in the peripheral blood of MISTRG6 mice over time. (C) B cells (NHP-CD4 - /CD8a - /CD20 + ) as a fraction of NHP lineage in the peripheral blood of MISTRG6 mice. (D) Anti-HIV antibody titers in the plasma of MISTRG6 mice as measured by ELISA. (E-G) 10-1074-expressing cells (Strep Tag II + ), IgG-producing cells (NHP-IgG + /CD138 + ), and plasma cells (NHP-CD20 -/low /CD31 + /CD138 + ) in different tissues of MISTRG6 mice at necropsy. (H) Fraction of total 10-1074-expressing cells found across all tissues that are either B cells, antibody-producing cells, or plasma cells. (I) Editing levels at the target locus in peripheral blood. n = 8 mice, n = 3 NHP donors. Error bars represent SD. Red lines in (B-C) represent immunizations.

Article Snippet: Immediately following isolation, NHP HSPC were transferred to HSPC medium (StemSpan Serum-Free Expansion Medium (SFEM) II (StemCell Technologies) with 100 ng/mL recombinant human thrombopoietin (TPO), stem cell factor (SCF), and flightless 3 (FLT-3) ligand, all from CellGenix) at 1.0×10 cells/mL in tissue culture (TC)-treated flasks, incubated at 37°C and 5% CO 2 .

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Strep-tag