StemSpan™ NK Cell Generation Kit

For expansion and differentiation of human CD34+ hematopoietic progenitor cells to NK cells

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StemSpan™ NK Cell Generation Kit

For expansion and differentiation of human CD34+ hematopoietic progenitor cells to NK cells

1 Kit
Catalog #09960
765 USD

StemSpan™ SFEM II

Serum-free medium for culture and expansion of hematopoietic cells

100 mL
Catalog #09605
129 USD

StemSpan™ Lymphoid Progenitor Expansion Supplement (10X)

Supplement for the expansion and differentiation of lymphoid progenitor cells

5 mL
Catalog #09915
256 USD

StemSpan™ Lymphoid Differentiation Coating Material (100X)

Coating material for the differentiation of lymphoid cells

0.25 mL
Catalog #09925
199 USD

StemSpan™ NK Cell Differentiation Supplement (100X)

Supplement for the differentiation of lymphoid progenitors to NK cells

0.5 mL
Catalog #09950
200 USD

Required Products

Overview

StemSpan™ NK Cell Generation Kit has been developed to differentiate CD34+ cells isolated from cord blood (CB) and bone marrow (BM) to natural killer (NK) cells, without use of stromal cells. This kit typically promotes the expansion of thousands of CD56+ NK cells in cultures initiated with CD34+ human CB cells.
StemSpan™ Lymphoid Progenitor Expansion Supplement (10X) contains a combination of recombinant human cytokines and other additives formulated to selectively promote the expansion and differentiation of CD34+ cells isolated from human CB and BM samples to lymphoid progenitor cells when used in combination with StemSpan™ SFEM II medium, and on plates coated with StemSpan™ Lymphoid Differentiation Coating Material (100X). Subsequently, StemSpan™ NK Cell Differentiation Supplement (100X) enables differentiation of lymphoid progenitor cells to NK cells.
Components:
• StemSpan™ SFEM II, 100 mL
• StemSpan™ Lymphoid Progenitor Expansion Supplement (10X), 5 mL
• StemSpan™ Lymphoid Differentiation Coating Material (100X), 250 µL
• StemSpan™ NK Cell Differentiation Supplement (100X), 500 µL
Subtype:
Supplements; Specialized Media
Cell Type:
Hematopoietic Stem and Progenitor Cells; NK Cells
Species:
Human
Application:
Cell Culture; Differentiation; Expansion
Brand:
StemSpan
Area of Interest:
Stem Cell Biology; Immunology; Cancer Research
Formulation:
Serum-Free

Scientific Resources

Educational Materials

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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

StemSpan™ NK procedure from seeding CB-derived CD34+ cells to harvesting of differentiated NK cells

Figure 1. StemSpan™ NK Cell Generation Protocol

CB-derived CD34+ cells are seeded on day 0. Medium should be topped up after 3 - 4 days of culture followed by two half-medium changes every 3 - 4 days. On day 14, cells at the lymphoid progenitor stage are harvested and reseeded for further differentiation into NK cells. Top-up and half-medium changes should be performed every 3 - 4 days after harvest and reseed, as indicated in the figure. Note: UM729 should only be added to the NK Cell Differentiation Medium, but not the Lymphoid Progenitor Expansion Medium.

Frequency and yield of CD56- and NKp46-expressing cells produced after culture of CD34+ HSPCs with the StemSpan™ NK Cell Generation Kit

Figure 2. 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™ NK Cell Generation Kit for 28 days as described. Cells were harvested and analyzed for (A,B) CD56 and (A) NKp46 expression by flow cytometry. Dead cells were excluded by light scatter profile and viability staining. (B) The average frequency of viable CD56+ NK cells on day 28 was 77%, with ~9,000 CD56+ cells produced per input CB-derived CD34+ cell. Shown are means with 95% confidence intervals (n = 45: 23 freshly isolated and 22 frozen CD34+ cell samples). BM-derived CD34+ cells were also differentiated into NK cells using the StemSpan™ NK Cell Generation Kit. The yield of NK cells from BM HSPCs is typically lower than with CB, averaging ~75 per input CD34+ cell (n = 3, data not shown).

Flow cytometry plots showing cell surface marker expression on CD56+ NK cells after culture with the StemSpan™ NK Cell Generation Kit

Figure 3. Cell Surface Marker Expression on CD56+ NK Cells After 28 Days of Culture

CB-derived CD34+ cells were cultured with the StemSpan™ NK Cell Generation Kit for 28 days. The differentiated cells were harvested and analysed by flow cytometry for the expression of CD56, NKp44, NKp30, NKG2D, CD94, CD16, and KIR. Staining for KIR molecules was performed using a combination of two clones for the antibody, 180704 and HP-MA4, as each recognizes a distinct subset of KIR molecules.

Killing activity of NK cells toward K562 target cells compared to PB NK cells following culture with the StemSpan™ NK Cell Generation Kit

Figure 4. Cultured NK Cells Exhibit Cytotoxicity Toward K562 Cell Line

NK cells were generated from CB-derived CD34+ cells over 28 days using the protocol in Figure 1. On day 28, cells were harvested, stained for CD56, and viable CD56+ cells were counted. K562 cells were incubated with 8 μM calcein AM at 37°C for 1 hour and then washed twice. CD56+ NK cells were then combined with 10,000 of these calcein AM-labeled K562 target cells at an Effector:Target ratio of 5:1 in U-bottom 96-well plates and co-cultured at 37°C for 4 hours. Adult peripheral blood (PB) NK cells and monocytes isolated using EasySep™ were used as positive and negative controls, respectively. PB NK cells were cultured overnight with the NK Cell Differentiation Supplement and SFEM II, while PB monocytes were cultured overnight in SFEM II only. To detect spontaneous release, control wells containing only calcein AM-labeled K562 target cells were set up. The labeled K562 cells were treated with 1% Triton™ X-100 to measure maximum release. After incubation, plates were centrifuged at 500 x g for 5 minutes and 100 μL of supernatant was transferred to black plates and analyzed using a SpectraMax® microplate reader (excitation 485 nm/emission 530 nm). Results are expressed as % specific lysis: [(test release - spontaneous release) x 100] / (maximum release - spontaneous release). CB CD34+-derived NK cells show similar killing activity toward K562 target cells compared to PB NK cells. Shown are means ± SD (CB CD34+-derived NK cells: n = 18, PB NK cells and monocytes: n = 7).

STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.