Human pluripotent stem cells (hPSCs), including embryonic stem and induced pluripotent stem cells, can be induced to differentiate to hematopoietic cells, including those that constitute the immune system, such as natural killer cells (NK cells). A reproducible culture system for generation of NK cells from hPSCs is valuable in studies of disease modeling, gene editing, and cell therapy development applications. The in vitro differentiation of hPSCs to NK cells has in the past been difficult, since it is often dependent on feeder cells that rely on undefined culture medium components, which cause variability.
The protocol below outlines how to generate NK cells from hPSCs using a serum-free and feeder-free culture system. First, CD34+ hematopoietic progenitor cells are differentiated from hPSCs. Then, CD56+ NK cells are generated from hPSC-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 hPSCs
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 2.5 mL of EB Formation Medium (per one well of a 6-well plate).
Harvest hPSCs and generate a single-cell suspension using ACCUTASE™.
Dilute hPSCs 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 hPSC-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:
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