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. Analysis of the 7 days expanded Mobilized Peripheral Blood CD34+ cells by Flow Cytometry

Purified CD34+ cells derived from G-CSF mobilized peripheral blood (mPB) were cultured for 7 days in StemSpan™ AOF (Catalog #100-0130) supplemented with StemSpan™ CD34+ Expansion Supplement (Catalog #02691) and UM729 (Catalog #72332). 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 YellowTM, and analyzed by flow cytometry. The horizontal dotted line on the CD34 and FSC plot indicated the fluorescence minus one (FMO) control for CD34 marker expression. Sequential gates (orange gates ) were used to determine the percentages of viable CD34^bright cells, CD34^brightCD90+CD45RA- cells and CD34^bright CD45RA-CD90+CD133+EPCR+ cells and used to generate data in Figures 3.

Figure 3. StemSpan™-AOF Supports Equal or Greater Expansion of Mobilized Peripheral Blood HSPCs Compared to Other Commercial Media

Purified CD34+ cells derived from G-CSF mobilized peripheral blood (mPB) were cultured at a concentration of 10000 cells per mL in StemSpan™-AOF medium (orange bars) or in four xenofree media from other suppliers (Commercial Alternatives, grey bars). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM729 (1uM). After 7 days of culture, the (A) frequency and (B) cell expansion of viable CD34^bright, CD34^bright CD45RA-CD90+, and CD34^bright CD45RA-CD90+CD133+EPCR+ cells were analyzed by flow cytometry, as described in Figure 2, and fold expansion normalized to StemSpan^TM-AOF (above bar expansion value ± SD). The performance of StemSpan™-AOF, the only animal origin-free formulation, was similar to the performance of the xeno-free alternative media. Data shown are mean ± SEM (n=5).

Figure 4. 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 CD34^bright 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 5. StemSpan™ Media Support Greater Expansion of Human CD34+CD90+CD45RA- and CD34brightCD90+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 CD34^brightCD90+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 CD34^brightCD90+CD45RA- cells (P 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 6. 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 7. 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.

Figure 8. Estimation of Long-term SCID Repopulating Cell in Cord Blood CD34⁺ cells expanded in either StemSpan™-AOF or StemSpan™ SFEM II using Limiting Dilution Transplantation assay

Purified cord blood-derived CD34⁺ cells were cultured for 7 days in either StemSpan™-AOF or StemSpan™ SFEM II supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM729 (1 μM). After 7 days of expansion, progeny of 10, 100, 250 and 2500 initial CD34⁺ cells were injected intravenously into sub-lethally irradiated NSG mice. For uncultured CD34⁺ cells, 250, 500 and 2500 cells were transplanted. The frequency of human cells expressing the pan-leukocyte marker CD45 was measured in bone marrow at ~20 weeks post-transplantation. A threshold of >0.1% of CD45⁺ cells was used to consider if mice were positive or negative for engraftment. Limiting dilution analysis was performed using the ELDA software from the Walter and Eliza Hall Institute of Medical Research software. LT-HSC frequencies (red lines) and 95% confidence intervals (boxes) are presented as 1/number of original CD34⁺ cell (day 0 equivalent) for each condition; n = 2 independent experiments performed, 2-7 mice per group per experiment. Significance level * p< 0.001 (Chi-square test). StemSpan™ SFEM II and StemSpan™-AOF expanded cells results in ~36 and ~17-fold increase in LT-HSC compared to fresh CD34⁺ cells, with SRC frequencies of 1/23, 1/48, 1/835 for StemSpan™ SFEM II, StemSpan™-AOF and fresh control respectively.