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Figure 1. Gating Strategy for Immunophenotype Analysis of Cord Blood-Derived CD34+ Cells Expanded for 7 Days in StemSpan™ Media with and Without StemSpan™ HSC Plus Supplement

Purified CD34+ cells derived from cord blood (CB) were cultured for 7 days in StemSpan™ SFEM II medium supplemented with StemSpan™ CD34+ Expansion Supplement (A) alone or (B) together with StemSpan™ HSC Plus Supplement. After 7 days, the cultured cells were stained with fluorescently labeled antibodies against CD34, CD45RA, CD90, EPCR, and CD133, in addition to viability dye Zombie Yellow™, and analyzed by flow cytometry. Fluorescence minus one (FMO) controls were used to set gates for HSPCs subsets. Sequential gates (orange gates) were used to determine the percentages of viable CD34+ cells, CD34+ CD45RA- CD90+ cells, and CD34+CD45RA-CD90+CD133+EPCR+ cells.

Figure 2. StemSpan™ HSC Plus Supplement Enhances Expansion of Primitive HSPCs When Used with StemSpan™ Cytokine-Enriched Supplements

Purified CD34+ cells derived from cord blood (CB) were cultured at a concentration of 10,000 cells per mL in StemSpan™ SFEM II medium supplemented with one of the StemSpan™ cytokine-based supplements alone (CD34 Expansion Supplement, CC100, or CC110; gray bars), or together with StemSpan™ HSC Plus supplement (orange bars). After 7 days of culture, flow cytometry was used to analyze the (A) total nucleated cell (TNC) expansion, (B) expansion of CD34+ cell subsets, and (C) frequency of CD34+ HSPC subsets: CD34+, CD34+CD45RA-CD90+, and CD34+CD45RA-CD90+EPCR+CD133+. StemSpan™ HSC Plus Supplement promoted greater expansion of CD34+ CD45RA- CD90+ and CD34+CD45RA-CD90+CD133+EPCR+ HSPC subsets compared to cytokine-based supplements alone. Each subsequent cell subset was progressively more enriched for phenotypic stem/progenitor cells. Data shown are ± SEM (n = 24 for the CD34 Expansion Supplement dataset and n = 9 for both the CC100 and CC110 datasets).

Figure 3. StemSpan™ HSC Plus Supplement Supports Expansion of Primitive HSPCs Derived from Mobilized Peripheral Blood

Purified CD34+ cells derived from G-CSF mobilized peripheral blood (mPB) were cultured at a concentration of 10,000 cells per mL in StemSpan™ SFEM II medium supplemented with StemSpan™ CD34 Expansion Supplement alone (gray bars) or together with StemSpan™ HSC Plus Supplement (orange bars). After 7 days of culture, flow cytometry was used to analyze the (A) total nucleated cell (TNC) expansion, (B) expansion of CD34+ cell subsets, and (C) frequency of CD34+ HSPC subsets: CD34+, CD34+CD45RA-CD90+ and CD34+CD45RA-CD90+EPCR+CD133+. StemSpan™ HSC Plus Supplement promoted higher expansion of CD34+ CD45RA- CD90+ and CD34+CD45RA-CD90+CD133+EPCR+ cells compared to StemSpan™ CD34 Expansion Supplement alone. Each subsequent cell subset was progressively more enriched for phenotypic stem/progenitor cells. Data shown are mean ± SEM (n = 6).

Figure 4. StemSpan™ HSC Plus Supplement Supports Expansion of Primitive HSPCs Derived from Bone Marrow

Purified CD34+ cells derived from bone marrow were cultured at a concentration of 10,000 cells per mL in StemSpan™ SFEM II medium supplemented with StemSpan™ CD34 Expansion Supplement alone (gray bars) or together with StemSpan™ HSC Plus Supplement (orange bars). After 7 days of culture, flow cytometry was used to analyze the (A) total nucleated cell (TNC) expansion, (B) expansion of CD34+ cell subsets, and (C) frequency of CD34+ HSPC subsets: CD34+, CD34+CD45RA-CD90+, and CD34+CD45RA-CD90+EPCR+CD133+. StemSpan™ HSC Plus Supplement promoted higher expansion of CD34+ CD45RA- CD90+ and CD34+CD45RA-CD90+CD133+EPCR+ cells compared to StemSpan™ CD34 Expansion Supplement alone. Each subsequent cell subset was progressively more enriched for phenotypic stem/progenitor cells. Data shown are mean ± SEM (n = 8).

Figure 5. StemSpan™ HSC Plus Supplement Supports Efficient Gene Knock-Out in Mobilized Peripheral Blood-Derived CD34+ Cells

Purified CD34+ cells derived from mobilized peripheral blood (mPB) were pre-cultured for 2 days in StemSpan™ SFEM II medium supplemented with StemSpan™ CD34 Expansion Supplement alone (dark gray bars) or together with StemSpan™ HSC Plus Supplement (orange bars). After two days, ~6.0 x 10^4 cells were electroporated using the Neon® Transfection System with RNP complexes targeting the B2M gene. Cells not exposed to the electroporation (untreated; light gray bars) were cultured with StemSpan™ CD34 Expansion Supplement and StemSpan™ HSC Plus Supplement.

(A) Four days post-electroporation, all conditions maintained >80% viability but (B) exhibited reduced cell recovery in RNP treated conditions. The frequency and yield of MHC-I- cells (i.e. B2M knock-out efficiency) in CD34+ cells and primitive CD34+CD45RA-CD90+EPCR+ HSPC subset were assessed via flow cytometry.

(C) The frequency and yield of B2M knock-out in CD34+ progenitor populations were similar between cultures with (orange bar) or without (dark gray) StemSpan™ HSC Plus Supplement.

(D) The yield of primitive CD34+CD45RA-CD90+EPCR+ gene-edited B2M knock-out cells was higher in cultures containing StemSpan™ HSC Plus Supplement. Data shown are mean ± SEM (n = 3).

Figure 6. Cord Blood CD34+ Cells Expanded with StemSpan™ HSC Plus Supplement Support Long-Term In Vivo Engraftment in Immunodeficient Mice

2,500 uncultured cord blood CD34+ cells or the progeny of 2,500 cord blood CD34+ cells cultured for 7 days under various conditions were transplanted into sub-lethally irradiated immunodeficient NSG mice. The cells were cultured in StemSpan™ SFEM II medium supplemented with StemSpan™ CD34 Expansion Supplement alone (dark gray bars) or together with StemSpan™HSC Plus Supplement (orange bars). Long-term multilineage engraftment was assessed in the bone marrow of transplanted NSG mice at 20 weeks post-transplantation. Data shown are mean ± SEM (n = 8 mice). Cells expanded with StemSpan™ HSC Plus Supplement demonstrated similar or higher levels of human engraftment in the bone marrow, as measured by frequency of (A) human CD45+ cells, (B) CD45+ CD34+ progenitor cells, (C) CD45+CD33+ myeloid cells, and (D) CD45+19+ B cells, relative to the other tested conditions. All transplanted cells showed lower or undetectable T cells engraftment (data not shown).