STEMdiff™ Hematopoietic - EB reagents are animal-component free and optimized for lymphoid differentiation potential. Using these reagents, CD34⁺ hematopoietic progenitor cells are first generated from PSCs; these can then be differentiated to NK cells using the StemSpan™ components.
⦁ Uniform generation of embryoid bodies
⦁ Robust generation of CD56+ NK cells across multiple ES and iPS cell lines
- STEMdiff™ Hematopoietic - EB Basal Medium, 120 mL
- STEMdiff™ Hematopoietic - EB Supplement A, 265 μL
- STEMdiff™ Hematopoietic - EB Supplement B, 7 mL
- StemSpan™ SFEM II, 100 mL (Catalog #09605)
- StemSpan™ Lymphoid Progenitor Expansion Supplement (10X), 5 mL (Catalog #09915)
- StemSpan™ Lymphoid Differentiation Coating Material (100X), 0.25 mL (Catalog #09925)
- StemSpan™ NK Cell Differentiation Supplement (100X), 0.5 mL (Catalog #09950)
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Data and Publications
Figure 1. STEMdiff™ Hematopoietic-EB Progenitor Differentiation Protocol
PSCs are harvested and dissociated into a single-cell suspension prior to seeding into AggreWell™ plates in EB Formation Medium (EB Medium A +10 μM Y-27632) to form 500 cell aggregates. After 3 days of mesoderm formation, the medium is changed to EB Medium B to induce hematopoietic lineage differentiation. On day 5, embryoid bodies (EBs) are transferred onto non-tissue culture-treated plates. After a total of 12 days, the EBs are harvested and dissociated, then CD34+ cells are enriched by EasySep™ positive selection.
Figure 2. PSCs Differentiate to CD34+ Hematopoietic Progenitor Cells After 12 Days of Culture
Human ES and iPS cells were induced to differentiate to CD34+ cells using a 12-day protocol shown in Figure 1. At the end of the culture period, cells were harvested, dissociated into a single-cell suspension, and analyzed by flow cytometry for CD34 and CD144 expression. Dead cells were excluded by light scatter profile and DRAQ7™ staining. (A) Representative flow cytometry plot for ES (H1)-derived cells analyzed on day 12. (B) The average frequency of viable CD34+ cells on day 12 for two ES cell lines (H1 and H9) and three iPS cell lines (WLS-1C, STiPS-M001, and STiPS-F016) ranged between 31% and 42%. The average yield of CD34+ cells produced per well of a 6-well AggreWell™400 plate ranged between 3.3 x 10^5 and 7.3 x 10^5. Data are shown as mean ± SEM (n = 7 - 22).
Figure 3. NK Cell Generation Protocol
PSC-derived CD34+ cells are seeded in StemSpan™ Lymphoid Progenitor Expansion Medium on plates coated with StemSpan™ Lymphoid Differentiation Coating Material. On day 14, cells at the lymphoid progenitor stage are harvested and reseeded in StemSpan™ NK Cell Differentiation Medium for further differentiation into NK cells. Note: UM729 (not included in the kit) should be added to the StemSpan™ NK Cell Differentiation Medium. NK cells are harvested after 28 days.
Figure 4. PSC-Derived CD34+ Cells Differentiate to CD5+CD7+ Lymphoid Progenitor Cells Over 14 Days of Culture
PSC-derived CD34+ cells were cultured for 14 days in StemSpan™ SFEM II + StemSpan™ Lymphoid Progenitor Expansion Supplement on plates coated with StemSpan™ Lymphoid Differentiation Coating Material (Figure 3). Cells were harvested and analyzed for CD7 and CD5 expression by flow cytometry. (A) Representative flow cytometry plot for ES (H1)-derived cells. (B) The average frequency of viable CD7+CD5+ lymphoid progenitor cells on day 14 ranged between 40% and 58% and the average yield of lymphoid progenitor cells produced per input PSC-derived CD34+ cell was between 11 and 22. Data are shown as mean ± SEM (n = 5 - 21).
Figure 5. PSC-Derived Lymphoid Progenitor Cells Differentiate to CD56+ NK Cells After 14 Days of Culture
PSC-derived lymphoid progenitor cells were cultured in StemSpan™ NK Cell Differentiation Medium on non-coated plates for 14 days. Cells were harvested and analyzed for expression of CD56 and CD16 by flow cytometry. Representative flow cytometry plots are shown for both (A) ES (H1)-derived and (B) iPS (WLS-1C)-derived cells. (C) After 28 days of culture, the average frequency of viable CD56+ NK cells from PSC-derived CD34+ cells ranged between 81% and 95%. The average yield of CD56+ cells produced per PSC-derived CD34+ cell was between 112 and 332. Data are shown as mean ± SEM (n = 4 - 13).
Figure 6. Cell Surface Marker Expression on PSC-Derived CD56+ NK Cells After 28 Days of Culture
PSC-derived CD34+ cells were cultured in StemSpan™ Lymphoid Progenitor Expansion Medium on plates coated with StemSpan™ Lymphoid Differentiation Coating Material for 14 days, followed by 14 days of culture in StemSpan™ NK Cell Differentiation Medium on non-coated plates to generate CD56+ NK cells. Cells were harvested and analyzed for CD56, NKp46, NKp44, NKp30, NKG2D, KIR, and CD45 expression by flow cytometry. Dead cells were excluded by light scatter profile and DRAQ7™ staining. Data shown are from a representative culture initiated with ES (H1) cells.
Figure 7. Cultured NK Cells Exhibit Cytotoxicity Toward K562 Cell Line
CD56+ NK cells were generated from PSC-derived CD34+ cells after 28 days of culture and then co-cultured with calcein AM (CAM)-labeled K562 cells at a ratio of 2.5:1 for 4 hours. Isolated peripheral blood (PB) NK cells and monocytes were also co-cultured with labeled K562 cells as positive and negative controls, respectively. Before the co-culture, frozen PB NK cells were thawed and cultured overnight with the NK Cell Differentiation Supplement and StemSpan™ SFEM II, while PB monocytes were cultured overnight in SFEM II only. To measure maximum release, the labeled K562 cells were treated with 1% Triton™ X-100. Culture supernatants were assessed for fluorescence released by dead cells after 4 hours using a SpectraMax® microplate reader (excitation 485 nm / emission 520 nm). The % specific lysis was calculated as follows: [(test release - spontaneous release) / (maximum release - spontaneous release)] X 100%. The average specific lysis by PSC-derived NK cells ranged between 51% and 75% as compared to 62% specific lysis by PB NK cells. Cultures of ES (H1 and H9) and iPS (WLS-1C, STiPS-M001, and STiPS-F016) cells are shown. Data are shown as mean ± SEM (n = 3 - 7).