Figure 5. Production of CD13⁺ and CD15⁺ Myeloid Cells by the Expansion and Lineage-Specific Differentiation of Human BM-Derived Cells Cultured in StemSpan™ SFEM II Containing Myeloid Expansion Supplement

Flow cytometry dot plots for 14-day cultures of human BM-derived CD34⁺ cells in StemSpan™ SFEM II containing Myeloid Expansion Supplement showing expression of the HSPC marker CD34, and pan-hematopoietic marker CD45 (A) before and (B) after 14 days of culture, and expression of myeloid markers CD13 and CD15 (C) after 14 days of culture. The frequency of CD34⁺ cells declined from 83% before culture to ~1% after 14 days. In parallel, myeloid CD13⁺CD15⁺ cells gradually accumulated from <10% on day 0 to 46% by day 14. The frequency of CD14⁺ cells after culture was low (typically <10%).

Figure 6. Production of CD14⁺ Monocytes by the Expansion and Lineage-Specific Differentiation of Human CB CD34⁺ Cells Cultured in StemSpan™ SFEM II Containing Myeloid Expansion Supplement II

Flow cytometry dot plots for 14-day cultures of human CB CD34⁺ cells in StemSpan™ SFEM II containing Myeloid Expansion Supplement II. The plots show expression of CD34 and CD45 (A) before and (B) after culture, and (C) expression of CD13 and CD14 on the expanded cells. The frequency of CD34⁺ cells declined from 87% before culture to less than 1% after 14 days. CD14⁺ monocytes increased from < 5% on day 0 to 90% by day 14.

Figure 7. Frequency and Yield of CD4 ISP and CD4⁺CD8⁺ DP Cells After 42 Days of Culture

CB-derived CD34⁺ cells (freshly isolated or frozen) were cultured with the StemSpan™ T Cell Generation Kit (Catalog #09940) for 42 days and (A) analysed by flow cytometry for the expression of CD4, CD8, CD3 and TCRαβ. The (B) frequency and (C) yield of CD4 ISP, double-positive (CD4⁺CD8⁺) and CD3⁺TCRαβ⁺-expressing double-positive cells (CD4⁺CD8⁺CD3⁺TCRαβ⁺) are shown. On average, 38% of the total viable population were DP (CD4⁺⁺), of which 35% co-expressed CD3 and TCRαβ. The yields of total DP cells and CD3⁺TCRαβ⁺ DP cells per input CD34⁺ cell were ~23,000 and ~9,000, respectively. Shown are means with 95% confidence intervals (n = 31).

Figure 8. Frequency and Yield of CD8 SP T Cells After 49 Days of Culture

DP cells were further matured into CD8 SP T cells by culturing for an additional 7 days in StemSpan™ SFEM II with T Cell Progenitor Maturation Supplement (Catalog #09930), IL-15 (Catalog #78031) and ImmunoCult™ CD3/CD28/CD2 T Cell Activator (Catalog #10970) on coated plates. On day 49, cells were (A) analyzed by flow cytometry for the expression of CD3, TCRαβ, CD4 and CD8. The (B) frequency and yield of CD3⁺TCRαβ⁺-expressing cells and their subsets are shown. On average, 54% of the CD3⁺TCRαβ⁺ cells were DP (CD4⁺CD8⁺) and 38% were CD8 SP (CD4^-CD8⁺). The average yield of CD8 SP T cells per input CD34⁺ cell was ~6,000. CD3⁺TCRαβ⁺ CD4 SP (CD4⁺CD8^-) T cells were detected at very low frequencies (data not shown). Shown are means with 95% confidence intervals (n = 12).

Figure 9. Frequency and Yield of CD56⁺ NK Cells After 28 Days of Culture

CB-derived CD34⁺ cells (freshly isolated or frozen) were cultured with the StemSpan™ T Cell Generation Kit (Catalog #09940) for 42 days and (A) analysed by flow cytometry for the expression of CD4, CD8, CD3 and TCRαβ. The (B) frequency and (C) yield of CD4 ISP, double-positive (CD4⁺CD8⁺) and CD3⁺TCRαβ⁺-expressing double-positive cells (CD4⁺CD8⁺CD3⁺TCRαβ⁺) are shown. On average, 38% of the total viable population were DP (CD4⁺⁺), of which 35% co-expressed CD3 and TCRαβ. The yields of total DP cells and CD3⁺TCRαβ⁺ DP cells per input CD34⁺ cell were ~23,000 and ~9,000, respectively. Shown are means with 95% confidence intervals (n = 31).

Figure 10. 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™ media (SFEM, SFEM II, AOF, and XF, orange bars), and in five media from other suppliers (Commercial Alternative, grey bars). All media were supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM171*. The (A) frequency and (B) cell expansion of viable CD34⁺CD90⁺CD45RA^- (solid) and CD34^brightCD90⁺CD45RA^- (dotted overlay) cells in culture were analyzed by flow cytometry as described in Figure 1. Compared to the competitor media tested, StemSpan™ media showed similar or significantly higher expansion of CD34^brightCD90⁺CD45RA^- cells (P < 0.05 when comparing StemSpan™ SFEM II and XF to five media from other suppliers, calculated using one-way ANOVA followed by Dunnett’s post hoc test). The CD34^brightCD90⁺CD45RA^- cell population is highly enriched for functional stem/progenitor cells. Data shown are mean ± SEM (n = 8).

*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.

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.

Figure 11. 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™ media (StemSpan™ SFEM II or StemSpan™-AOF, orange bars), or five 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).

*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.

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.

Figure 12. Colony-Forming Potential of CD34⁺ CML Cells is Maintained During Culture

CML cells were assayed in colony assays using MethoCult™ H4435 Enriched medium directly after CD34⁺ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 2). After 14 days of culture in StemSpan™ SFEM II with CD34⁺ expansion supplement (Exp) with or without UM171, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34⁺ cell. Numbers above each of the individual bars indicate the proportion of BCR-ABL positive colonies, measured by qRT-PCR on individual plucked colonies across 6 different samples (8-12 colonies were plucked for each sample per condition). SFEM II supplemented with CD34⁺ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. UM171 further promotes colony forming progenitor cell output (~3.5-fold expansion at day 7 and ~8-fold at day 14). Single-colony qRT-PCR analysis revealed that colonies generated from samples on day 0, and colonies generated from cells expanded for 7 and 14 days, were predominantly BCR-ABL⁺ but also that normal BCR-ABL^- progenitor cells were present at low frequencies. Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).

Figure 13. Colony-Forming Potential of CD34⁺ AML Cells is Maintained During Culture

AML cells, after CD34⁺ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 3), were plated in colony assays with MethoCult™ H4435 Enriched medium. After 14 days of incubation, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34⁺ cell. SFEM II supplemented with CD34⁺ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. Addition of UM171 further promotes colony-forming progenitor cell output (~3-fold expansion at day 7 and ~4-fold at day 14). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).