STEMdiff™ NK Cell Kit

For expansion and differentiation of hPSCs to NK cells
STEMdiff™ NK Cell Kit

For expansion and differentiation of hPSCs to NK cells

1 Kit
Catalog # 100-0170
1,268 USD

INDIVIDUAL COMPONENTS

STEMdiff™ Hematopoietic - EB Basal Medium

290 USD
Catalog # 100-0171
120 mL

Medium for the generation of embryoid bodies for downstream lymphoid differentiation

STEMdiff™ Hematopoietic - EB Supplement A

192 USD
Catalog # 100-0172
265 µL

Supplement for mesoderm specification with downstream lymphoid potential

STEMdiff™ Hematopoietic - EB Supplement B

336 USD
Catalog # 100-0173
7 mL

Supplement for hematopoietic differentiation optimized for lymphoid potential

Required Products
  1. Y-27632 (Dihydrochloride)
    Y-27632

    RHO/ROCK pathway inhibitor; Inhibits ROCK1 and ROCK2

  2. AggreWell™400 24-well plate|34411
    AggreWell™400

    Microwell culture plates for easy and reproducible production of embryoid bodies and spheroids

  3. EasySep™ Human CD34 Positive Selection Kit II|17856
    EasySep™ Human CD34 Positive Selection Kit II

    Immunomagnetic positive selection kit

  4. UM729
    UM729

    Pyrimido-indole derivative that enhances HSC self-renewal in vitro

Overview

STEMdiff™ NK Cell Kit includes serum-free media and supplements for the feeder-free differentiation of human pluripotent stem cells (PSCs) to natural killer (NK) cells expressing CD56.

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.
Advantages
⦁ Serum-free and feeder-free formulation
⦁ Uniform generation of embryoid bodies
⦁ Robust generation of CD56+ NK cells across multiple ES and iPS cell lines
Components
  • STEMdiff™ Hematopoietic - EB Basal Medium, 120 mL (Catalog #100-0171)
  • STEMdiff™ Hematopoietic - EB Supplement A, 265 μL (Catalog #100-0172)
  • STEMdiff™ Hematopoietic - EB Supplement B, 7 mL (Catalog #100-0173)
  • 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)
Subtype
Specialized Media
Cell Type
Hematopoietic Cells, PSC-Derived, NK Cells, Pluripotent Stem Cells
Species
Human
Application
Cell Culture, Differentiation, Expansion
Brand
STEMdiff
Area of Interest
Cancer, Disease Modeling, Immunology, Stem Cell Biology
Formulation
Serum-Free

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
STEMdiff™ Hematopoietic - EB Basal Medium
Catalog #
100-0171
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
STEMdiff™ Hematopoietic - EB Supplement A
Catalog #
100-0172
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
STEMdiff™ Hematopoietic - EB Supplement B
Catalog #
100-0173
Lot #
All
Language
English
Document Type
Manual
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 6
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet 7
Product Name
STEMdiff™ NK Cell Kit
Catalog #
100-0170
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
STEMdiff™ Hematopoietic - EB Basal Medium
Catalog #
100-0171
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
STEMdiff™ Hematopoietic - EB Supplement A
Catalog #
100-0172
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
STEMdiff™ Hematopoietic - EB Supplement B
Catalog #
100-0173
Lot #
All
Language
English

Educational Materials (7)

Brochure
Hematopoietic Stem and Progenitor Cells - Products for Your Research
Brochure
Tools For Your Immunology Research
Technical Bulletin
Generation of Natural Killer Cells and T Cells from Human Pluripotent Stem Cells Using STEMdiff™ and StemSpan™ Media and Supplements
Wallchart
Derivation and Applications of Human Pluripotent Stem Cells
Wallchart
Human Immune Cytokines
Webinar
ISSCR Innovation Showcase: Differentiating Immune Cells from Human Pluripotent Stem Cells in Feeder- and Serum-Free Cultures
29:54
ISSCR Innovation Showcase: Differentiating Immune Cells from Human Pluripotent Stem Cells in Feeder- and Serum-Free Cultures
Webinar
Online Immunology Journal Club: NK Cell Expansion or Differentiation from Progenitors for Cell Therapy
46:15
Online Immunology Journal Club: NK Cell Expansion or Differentiation from Progenitors for Cell Therapy

Product Applications

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

Data

Generate CD34+ Cells from PSCs Using the STEMdiff™ Hematopoietic-EB Progenitor Differentiation Protocol

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.

PSC-Derived CD34+ Cell Characterization, Frequency, and Yield

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).

Differentiate PSC-Derived CD34+ Cells to NK Cells

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.

CD5+CD7+ Lymphoid Progenitor Cells Characterization, Frequency, and Yield

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).

CD56+ NK Cells Characterization, Frequency, and Yield

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).

Analysis of Cell Surface Markers Expressed on PSC-Derived CD56+ NK Cells

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.

Analysis of Specific Lysis by Cultured NK 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).

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