Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Summary

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Abstract

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Introduction

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are undifferentiated, pluripotent cells capable of unlimited self-renewal and multi-lineage differentiation. hESCs have been successfully differentiated into mature and functional subsets of each germ layer, including cells of the hematopoietic system^1-3. Natural killer (NK) cells are lymphocytes of the innate immune system that can be derived from hESCs by formation of embryoid bodies (EBs)^4,5 or co-culture with stromal cell lines^1,2,6-8. NK cells possess anti-viral and anti-tumor capabilities and have the potential to be effective against a broad range of m....

Protocol

1. Adapting hESCs or iPSCs in TrypLE for Spin EB Cultures

1. The initial dissociation with TrypLE works best if the ES/iPS colonies from the collagenase-passaged cultures are relatively small. The starting ES/iPS populations should be cells passed no longer than 4-5 days previous. 4-5 days before initiation of TrypLE passage, pass ES cells at a density that will allow the cells to be ~70% confluent in 4 days time. Use regular ES media for culture of TrypLE-passaged ES cells. Here, we are using hESCs stably modified with a luciferase reporter construct (Materials Table). The protocol also works with iPSCs, using unmodified iPSCs for comparison....

Results

The generation of hematopoietic progenitor cells using the spin EB approach allows for optimal NK cell development from hESCs and iPSCs. As demonstrated in Figure 2, day 11 spin EBs contain high percentages of progenitor cells expressing CD34, CD45, CD43, and CD31. High levels of CD34 and CD45 allows direct transfer to NK conditions without need for sorting or supporting stromal cells. If there is suboptimal spin EB differentiation, it is recommended that stromal cells such as EL08-1D2 are used in the se.......

Discussion

hESCs are an ideal platform to study diverse cell types and hold remarkable potential for clinical translation. We use a defined, spin EB approach to differentiate hESC/iPSCs to hematopoietic progenitor cells. The spin EB approach has yielded consistent derivation of hematopoietic progenitor cells and differentiation to NK cells; yet, variation still exists in differentiation efficiency across cell lines and may need to be modified for generation of other hematopoietic cell lineages. While comparable results can be obtai.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Media
BPEL media for Spin EB generation
Iscove's Modified Dulbecco's Medium (IMDM)	Fisher Scientific	SH3022801
F-12 Nutrient Mixture w/ Glutamax I	Invitrogen	31765035
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A3311	Commercially available BSA can be cytotoxic to ES cells. Deionizing the solution can reduce the potential for cytotoxicity.
Poly(vinyl alcohol)	Sigma-Aldrich	P8136
Linoleic Acid	Sigma-Aldrich	L1012
Linolenic Acid	Sigma-Aldrich	L2376
Synthechol 500X solution	Sigma-Aldrich	S5442
a-monothioglycerol (a-MTG)	Sigma-Aldrich	S5442
Protein-free hybridoma mix II	Invitrogen	12040077
Ascorbic Acid 2-phosphate	Sigma-Aldrich	A8960
Glutamax I	Invitrogen	35050061
Insulin, Transferrin, Selenium 100X solution (ITS)	Invitrogen	41400-045
Pen-Strep	Invitrogen	15140122
NK Differentiation Media
Dubecco's Modified Eagle Medium (DMEM)	Invitrogen	11965-118
F-12 Media	Invitrogen	CX30315
15% Human AB serum	Valley Biomed	HP1022HI
5 ng/ml Sodium Selenite	Sigma-Aldrich	S5261
50 uM ethanolamine	Sigma-Aldrich	E9508
20 ng/ml ascorbic acid	Sigma-Aldrich	A8960
25 uM 2-mercapt–thanol (BME)	Gibco	21985
2 mM L-glutamine	Gibco	CX30310
1% Pen-Strep	Invitrogen	15140122
Cytokines
(all cytokines used fresh from frozen aliquots)
SCF	PeproTech, Inc.	300-07	40 ng/ml in BPEL media; 20 ng/ml in NK medium). As noted by the Elefanty protocol, and as we discovered with a bad lot of BMP4, there can be lot differences with cytokines in regards to hematopoietic differentiation.Especially if buying cytokines in bulk, obtain a sample of the lot to test prior to purchase. Compare head-to-head with old lot.
rhBMP-4	R & D Systems	314-BP	20 ng/ml in BPEL media
rhVEGF	R&D Systems	293-VE	20 ng/ml in BPEL media
IL-2	PeproTech, Inc.	200-02	1 x 105 U/ml given to mice after NK cell injection; 50 U/ml used in NK cell expansion protocol
IL-15	PeproTech, Inc.	200-15	10 ng/ml given to mice after NK cell injection (first 7 days only)
IL-3	PeproTech, Inc.	200-03	5 ng/ml in NK media
IL-7	PeproTech, Inc.	200-07	20 ng/ml in NK media
Flt-3-Ligand	PeproTech, Inc.	300-19	10 ng/ml in NK media
In vivo Imaging
D-Luciferin Sodium Salt	Gold BioTechnology	Lucna-500
TurboFP650 plasmid	Evrogen, Moscow, Russia	FP731	Subcloned into a Sleeping Beauty transposon based plasmid driven by the mCAGs promoter. Cells were then sorted on their expression of turboFP650 by FACS. Cells and plasmids can be obtained from our lab.
Equipment
IVIS Spectrum Imaging System	Caliper Life Sciences
Cells
Membrane bound IL-21 expressing artificial antigen presenting cells	MD Anderson, Houston, TX		contact: Dean A. Lee. http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood
Firefly luciferase expressing hESCs	University of Minnesota, Minneapolis, MN		contact: Dan S. Kaufman . H9 cells modified with a Sleeping Beauty transposon based method (references 13 and 14). Expression of firefly luciferase is driven by the mCAGGS promoter. Following the firefly luciferase gene is an IRES element at the 5' end of a GFP:zeocin fusion construct.
TurboFP650 expressing K562 cells	University of Minnesota, Minneapolis, MN		contact: Dan S. Kaufman. Description under plasmid comments section
Materials Table.