This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
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.
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 system1-3. 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 lines1,2,6-8. NK cells possess anti-viral and anti-tumor capabilities and have the potential to be effective against a broad range of malignancies, as they do not require prior antigen stimulation to perform their effector functions. Thus, hESC-derived NK cells are an attractive source of cells for immunotherapy. Additionally, the derivation of NK cells from hESCs provides a genetically amenable system to study normal development in vitro.
Because they provide a genetically tractable system, hESC-derived NK cells can be experimentally modified to express fluorescent and bioluminescent reporters providing an optimal model to study NK cell effector functions in vitro and in vivo. hESC-derived NK cells possess activity against a range of targets including HIV 9, leukemia (K562) and other cancer types 7,8. However, the ability to efficiently derive enough NK cells capable of treating patients remains an important barrier for clinical translation and is, to a lesser degree, a limitation for extensive pre-clinical in vivo studies of NK cell development and anti-cancer functions. Here, we use a spin EB approach to derive hematopoietic progenitors from hESCs 4,5,10. Following 11 days the spin EBs are transferred to NK cell culture with or without feeders for 28 days. After 4 weeks in NK cell culture medium, NK cells are transferred to co-culture with K562 cells modified to express membrane-bound interleukin 21 (IL-21), which serve as artificial antigen presenting cells (aAPCs). Adapting a protocol for the expansion of peripheral blood NK cells using these artificial APCs 11,12, we are able to expand NK cells 2-logs while retaining a mature phenotype and cytotoxic capabilities.
This process of development and expansion provides sufficient hESC-derived NK cells for extensive in vivo characterization. For in vivo studies, we are able to non-invasively monitor long term engraftment and kinetics of injected firefly luciferase expressing (Fluc+), hESC-derived NK cells using bioluminescence imaging. Furthermore, we are able to follow NK cell interactions with tumor cells using a dual, bioluminescent or fluorescent imaging scheme. An earlier study by our group used bioluminescence imaging in an anti-tumor model to follow tumor progression and clearance of Fluc+ K562 cells in vivo 7. Now, by engineering our hESCs to express firefly luciferase 13,14 we can follow the biodistribution and trafficking of NK cells to K562 tumor cells that express the recently characterized fluorescent protein, turboFP650 15. We have chosen this dual reporter system in order to simultaneously follow the two cell populations in vivo (Figure 1). Most dual imaging models have been dual-luciferase systems, but these systems can be technically challenging due to the delivery requirements of coelenterazine, the substrate required for expression of most Renilla and Gaussia luciferase reporters 16-18. Fluorescent reporters have allowed easy monitoring of many cell lines and constructs in vitro, but has had limited success for in vivo imaging due to the overlap between tissue and fur autofluorescence and the emission spectra of many commonly used fluorescent reporters including GFP, DsRed, and TdTomato 15,19. This concern has encouraged the development of far-red fluorescent proteins, which allow for better tissue penetrance and higher specific signal compared to background 15,19. TurboFP650, the fluorescent protein shown in this system, is far-red shifted and overcomes many of the issues involved with imaging fluorescent proteins in living animals.
This method for developing and expanding NK cells derived from hESCs has allowed us to further characterize hESC-derived NK cells in vitro and in vivo, which is necessary to better understand NK cell function and clinically important to improve current NK cell adoptive therapies. It is also amenable to the derivation and expansion of iPSC-derived NK cells. The dual fluorescent and bioluminescent imaging scheme is broadly applicable to systems other than the anti-tumor model we have shown here.
1. Adapting hESCs or iPSCs in TrypLE for Spin EB Cultures
2. Prepare Stock Solutions for Setting Up Spin EB Cultures
3. Assembly of BPEL Media (for ~200 ml Total Volume)
4. Setting up TrypLE-passaged ES Cells into Stage I Spin EBs
Use ES/iPS cells that have been adapted to TrypLE passage on lower-density MEFs(i.e. ES/iPS cells that have been passaged with TrypLE at least 5-7 times). If cells can be passed at ~1:3 and become 70-80% confluent in 3-4 days, the cells should be good to use.
5. Dissociating Stage I EBs for FACS Analysis
Collect around18-36 spin EB for FACS analysis. If analyzing cells before day 6, more wells are needed to obtain sufficient cell numbers.
6. Development, Expansion, and Characterization of NK Cells from hPSC-derived Progenitor Cells
Because the spin EBs contain a high percentage of hematopoietic progenitor cells, they can be directly transferred to NK cell culture on day 11. The spin EBs may be transferred to 24 well plates with or without feeders. We have shown no difference in phenotype or function between NK cells derived in each condition. Therefore, we generally transfer to cultures without feeders to have a completely defined system for NK cell development. If using an hESC/iPSC line with suboptimal hematopoietic differentiation the use of feeder layers is recommended 7,8.
7. In vivo Fluorescent and Bioluminescent Imaging to Monitor hESC-derived NK Cells and Tumor Cells in Immunodeficient Mice
We use nonobese diabetic/severe combined immunodeficiency with gamma-chain knockout (NOD/SCID/ γC-/-) mice that are 6 to 8 weeks old in all of our experiments. We use a dual imaging model to simultaneously track tumor progression (turboFP650 reporter) and hESC-NK cells kinetics (Fluc reporter expressed in parent ES cells) in vivo. This imaging scheme is broadly applicable to systems other than the anti-tumor model shown here. The IVIS Spectrum (Caliper Life Sciences) is optimal for simultaneous fluorescent and bioluminescent in vivo imaging.
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 secondary NK conditions. Following 4 weeks of culture the spin EBs give rise to large numbers of NK cells (approximately 1-2 x 106 cells per well of a 24-well plate). Cultures can also be phenotyped at earlier time points to see the gradual progression of NK cell development in this system 7. NK cells maintain the expression of reporter genes that were modified into the parent ES/iPS line and therefore can then be followed in vivo. Here, we have used the IVIS Spectrum to demonstrate simultaneous imaging of both NK cells and tumor cells in the same mouse (Figure 3). Using the spectral unmixing feature in Living Image software, tissue autofluorescence can be subtracted to optimize signal from fluorescently labeled cells.
Figure 1. Dual imaging scheme and developmental timeline. A) Schematic of in vivo model. Mice are injected IP with luciferin, and following proper incubation time, firefly luciferase+ cells are imaged for bioluminescent signal. Following acquisition of the bioluminescent image, fluorescent signal from TurboFP650+ cells can be imaged. B) Timeline for the development and testing of hESC-derived NK cells.
Figure 2. Phenotype of spin EBs and NK cells derived from hESCs and iPSCs. A) Following 11 days in spin EB culture, cells can be analyzed by flow cytometry. The day 11 time point gives high percentages of CD34, CD43, CD45, and CD31 expressing cells optimal for NK cell differentiation. B) NK cells can be detected using flow cytometry following 4 weeks of NK cell culture. Cells within the lymphocyte gate (FSC/SSC plot) are analyzed for the expression of CD56. CD56+ cells can be further analyzed for co-expression of markers such as CD117, CD94, NKp46, or others. Click here to view larger figure.
Figure 3. Fluorescent and bioluminescent imaging in vivo using the IVIS Spectrum. In vivo monitoring is the primary aim of this protocol. Therefore, mice are not followed out for tumor clearance. Our previous report (using 200,000 K562 cells) demonstrates that tumor clearance occurs at three weeks 7. A) Bioluminescent imaging of Fluc+hESC-derived NK cells on day 0, 7, and 14 after NK cell injection is shown in the top row. Fluorescent imaging of TurboFP650+ K562 cells on day 0, 7, and 14 after NK cell injection is shown in the bottom row. Fluorescent images were acquired immediately following bioluminescent image acquisition. B) Sequence showing tissue autofluorescence and fluorescent signal from turboFP650+ cells, generated using the spectral unmixing feature in Living Image software. The mouse on the far left in each image is a non-injection control and only shows the background signal. Click here to view larger figure.
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 obtained from derivation in other EB or stromal culture methods, this system is serum-free and represents a more defined approach for producing hematopoietic progenitors compared to other methods. We have also standardized an efficient method of deriving NK cells from these hematopoietic progenitors in a similarly feeder-free manner, which overcomes a major concern for clinical translation. It also provides a defined system to study NK cell development in vitro. Extensive pre-clinical, in vivo characterization of hematopoietic cells, such as NK cells, is also necessary to specifically define these experimentally derived cell types and their implementation to clinical trials.
Advances in imaging and microscopy techniques have enabled long-term and non-invasive in vivo monitoring of cells expressing bioluminescent and fluorescent reporters15-17,19,21. Most notable is the use of the firefly luciferase construct, which has been used to visualize diverse cell types since its initial characterization. This is an ideal reporter for in vivo imaging, but is limited to only monitoring one cell population. The biggest advantage of a dual-reporter system is the ability to follow the interactions between two distinct cell populations. However, few reporters have been developed that are both easy to image in vivo and have sufficient and quantifiable signal above background.
TurboFP650, the fluorescent protein used in our dual-imaging scheme, is far-red shifted, which gives signal distinct from background autofluorescence. However, utilization of spectral unmixing methods in Living Image software (Caliper Life Sciences) was necessary to maximize the signal to background ratio in our system. There are many features in the Living Image software able to help with the intrinsic challenges of fluorescent protein imaging. Still, signal penetrance remains a challenge. The system needs further optimization for imaging of small cell populations or imaging of dispersed cells, as would be the case if the tumor cells were injected IP. However, the consistency and ease of fluorescent imaging makes its use an ideal platform to use in a dual imaging model. The animals are under anesthesia for less time and there is no need to deliver a second substrate as with most dual-luciferase systems.
This method of derivation, expansion, and dual-imaging in vivo provides a consistent approach for producing hESC-derived or iPSC-derived NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies. The dual fluorescent and bioluminescent imaging model is broadly applicable for long-term study of two distinct cell populations other than anti-tumor model we have shown.
The authors have nothing to disclose.
The authors would like to thank Melinda Hexum for initiation of the spin EB protocol within our lab. We would like to thank other members of the lab, including Laura E. Bendzick, Michael Lepley, and Zhenya Ni for their technical assistance with this work. The authors would also like to thank Brad Taylor at Caliper Life Sciences for his expert technical advice.
Name of Reagent | 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-mercaptoethanol (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.