The protocol below outlines how to generate natural killer cells (NK cells) from pluripotent stem cells (PSCs). First, CD34+ hematopoietic progenitor cells are differentiated from PSCs. Then, CD56+ NK cells are generated from PSC-derived CD34+ cells.
This procedure has been optimized for use with multiple human embryonic stem (ES) cell and induced pluripotent stem (iPS) cell lines; refer to the Technical Manual (Document #10000007537) for complete instructions.
Part I: Differentiate CD34+ Cells from PSCs
Prepare EB Medium A (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement A). Prepare EB Formation Medium by adding Y-27632 at 10 µM to EB Medium A.
Prepare an AggreWell™400 plate by rinsing with Anti-Adherence Rinsing Solution, washing with DMEM/F-12 with 15 mM HEPES, and adding a half-volume of EB Formation Medium.
Harvest PSCs and generate a single-cell suspension using ACCUTASE™.
Dilute PSCs to 1.4 x 106 cells/mL in 2.5 mL of EB Formation Medium, then seed into the AggreWell™ plate that was prepared in step 2.
Perform a half-medium change with EB Medium A on day 2.
Prepare EB Medium B (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement B).
Perform a half-medium change with EB Medium B on day 3.
Harvest EBs on day 5, then filter and elute these with EB Medium B, using a 37 µm reversible strainer.
Transfer eluted EBs to a non-tissue culture-treated plate.
Add EB Medium B on day 7.
Perform a half-medium change with EB Medium B on day 10.
Harvest EBs and dissociate into a single-cell suspension using Collagenase Type II and TrypLE™ Express. Isolate CD34+ cells using EasySep™ Human CD34 Positive Selection Kit II.
Proceed to the protocol below for NK cell generation.
Part II: Differentiate NK Cells from PSC-Derived CD34+ Cells
Coat non-tissue culture-treated plates with StemSpan™ Lymphoid Differentiation Coating Material.
Prepare StemSpan™ Lymphoid Progenitor Expansion Medium (StemSpan™ SFEM II + StemSpan™ Lymphoid Progenitor Expansion Supplement).
Dilute CD34+ cells to 5 x 104 cells/mL in StemSpan™ Lymphoid Progenitor Expansion Medium and seed onto the coated plate.
Incubate at 37°C for 7 days, following instructions in the Technical Manual (Document #10000007537) for required half-medium changes and plate transfer on day 7. On day 14, harvest lymphoid progenitor cells (containing CD5+ CD7+ cells) for further differentiation to NK cells.
Prepare StemSpan™ NK Cell Differentiation Medium (StemSpan™ SFEM II + StemSpan™ NK Cell Differentiation Supplement + UM729).
Dilute lymphoid progenitor cells to 1 x 105 cells/mL in StemSpan™ NK Cell Differentiation Medium. Seed onto a non-coated tissue culture plate, incubate at 37°C, and follow instructions in the Technical Manual for required half-medium changes.
On day 28, harvest cells containing CD56+ NK cells (see Figure 1) for use in downstream assays.
Figure 1. 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 should only be added to the StemSpan™ NK Cell Differentiation Medium (see step 5), but not the StemSpan™ Lymphoid Progenitor Expansion Medium. NK cells are harvested after 28 days. For further details see the step-by-step protocol below
Learn more about the technologies used in this protocol: