Figure 1. Day 7 Immunophenotyping of CD34⁺ Cells Cultured in StemSpan™-AOF

CD34⁺ cells were purified from cord blood (CB) using the EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896) and cultured in StemSpan™-AOF (Catalog #100-0130) supplemented with StemSpan™ CD34⁺ Expansion Supplement (Catalog #02691) (A) without or (B) with the addition of UM729 (Catalog #72332). After 7 days, the cultured cells were stained with fluorescently labeled antibodies against CD34, CD90, and CD45RA, in addition to viability dye 7-AAD, and analyzed by flow cytometry. The horizontal dotted line in the CD34 vs FSC plots indicates the boundary between CD34^- and CD34⁺ cells as based on a fluorochrome minus one (FMO) control for CD34 expression. Orange gates on these plots indicate the population of CD34^bright cells used to generate data in Figures 2 and 3. Sequential gates were used to determine the percentages of viable CD34⁺ cells, CD34^bright cells, and CD34^brightCD90⁺CD45RA^- cells.

Figure 2. StemSpan™-AOF Supports Equivalent or Greater Expansion of Human CD34⁺ and CD34^bright Cells than Other Commercial Media

Purified cord blood (CB)-derived CD34⁺ cells were cultured for 7 days in StemSpan™-AOF (orange bar), and in four xeno-free media formulations from other suppliers (Xeno-Free Commercial Alternative, grey bars), including (in random order) SCGM (Cellgenix), X-VIVO™ 15 (Lonza), Stemline™ II (Sigma), and StemPro™-34 (Thermo). The (A) frequency and (B) cell expansion of viable CD34⁺ and CD34bright cells in culture were based on viable cell counts and flow cytometry results. StemSpan™-AOF, the only animal origin-free formulation, showed equivalent performance to all xeno-free alternative media tested. All media were supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM171*. Data shown are mean ± SEM (n = 8).

Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al., 2014.

Figure 3. StemSpan™ Media Support Equal or Greater Expansion of Primitive Human CD34^brightCD90⁺CD45RA^- Cells Than Other Commercial Media

Purified CB-derived CD34+ cells were cultured for 7 days in StemSpan™-AOF medium (orange bar), and in four xeno-free media formulations from other suppliers (Commercial Alternative, gray bars) including (in random order) SCGM (Cellgenix), X-VIVO 15 (Lonza), Stemline II (Sigma), and StemPro 34 (Thermo). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM171*. The (A) frequency and (B) cell expansion of CD34+CD90+CD45RA- (solid) and CD34brightCD90+CD45RA-(dotted overlay) cells in culture were based on viable cell counts and flow cytometry results as shown in Figure 1. StemSpan™-AOF showed similar or significantly higher expansion of CD34brightCD90+CD45RA- cells (P < 0.05 compared to four media from other suppliers, calculated using one-way ANOVA followed by Dunnett’s post hoc test) and equivalent performance to all xeno-free competitors tested. Data shown are mean ± SEM (n = 8).

Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al. 2014.

Figure 4. StemSpan™ Media Support Better CD34⁺ and Primitive CD34⁺CD90⁺CD45RA^- HSPC Expansion in a Genome Editing Application Compared with Alternative Commercial Media

Purified CB-derived CD34+ cells were cultured for 2 days in select StemSpan™-AOF, (orange bar), or four xeno-free media formulations from other suppliers (gray bars). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM171*. Cells were then electroporated using Arcitect™ CRISPR-Cas9 RNP complexes containing crRNA:tracrRNA targeting beta-2-microglobulin (B2M), and cultured for an additional 4 days in the same conditions. Knockout efficiency as measured by staining for MHC-I and analyzing by flow cytometry, was similar in all media tested, ~70-80%. (A) The percentage of CD34+ cells and (B) CD34+CD90+CD45RA- cells were quantified by flow cytometry 4 days post-electroporation. Data shown are mean + SD (n = 4 donors; **P < 0.01).

Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version of StemSpan™-AOF (Catalog #100-0130) was comparable.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1 μM. For more information including data comparing UM171 and UM729, see Fares et al., 2014.

Figure 5. StemSpan™-AOF-Expanded Cord Blood CD34⁺ Cells Engraft in NSG Mouse Recipients

Purified cord blood-derived CD34⁺ cells were cultured for 7 days in StemSpan™-AOF supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM729 (1 μM). After 7 days of expansion, progeny of 10,000 fresh or uncultured CD34⁺ cells were transplanted in sub-lethally irradiated NSG mice. (A-D) The number of human platelets and the frequency of human cells expressing the pan-leukocyte marker CD45 were measured in peripheral blood at 3 and 18 weeks post-transplantation. Data shown are mean ± SEM (n = 3 - 5 mice). (A) At 3 weeks, engraftment of human platelets was lower in recipients of cells cultured in StemSpan™-AOF than in recipients of uncultured cells. (C) At week 18, there were no significant differences in platelet engraftment between the expanded and uncultured cells. (B,D) Human CD45⁺ cell frequencies in recipients of cells expanded in StemSpan™-AOF were similar to those in recipients of uncultured cells. (E-H) At week 20, long-term multilineage engraftment was measured in bone marrow of transplanted NSG mice. Data shown are mean ± SEM (n = 3 - 4 mice). (E,F) Recipients of StemSpan™-AOF expanded cells showed similar frequencies of human CD45⁺ and CD34⁺ cells in the mouse bone marrow compared to recipients of uncultured cells. (G,H) Cells expanded with StemSpan™-AOF showed similar levels of myeloid (CD45⁺ CD33⁺ ) and lymphoid (CD45⁺ 19⁺ B cells and CD45⁺ CD3⁺ T cells) engraftment relative to uncultured cells.