We would like to thank Dr. Handi Cao, Sanxing GAO, David Xiang, Yiming Chao, Ritika Jogani, Owen Chan, Stephanie Cheung, and Celine Chan for their technical assistance and useful discussions. This work was supported by the Platform Technology Fund. Figure 1 has been created with BioRender.com.

The present study involved in vitro experiments; therefore, ethics approval is not applicable. The established cell lines used in this study were obtained from commercial sources (see the Table of Materials).
1. Generation of 3D organoid structures from hEPSCs
<ol>
	<li>Maintain hEPSCs by culturing them with 1 mL of EPSC medium (12-well plate) on SNL feeder cells1. 
	NOTE: Dome-shaped colonies are observed (Figure 2A) if their expanded potency remains.</li>
	<li>Perform pre-differentiation of the hEPSCs.
	<ol>
		<li>Remove the hEPSC medium, and add 1 mL of DFK medium (DMEM/F12 + 10% serum replacement medium, see the Table of Materials). Incubate for 2-3 days at 37 &#176;C and 5% CO2. Perform a medium change on the second day if continuing the incubation for 3 days. 
		NOTE: Under the microscope, the dome-shaped colonies are flattened (Figure 2B).</li>
	</ol>
	</li>
	<li>Check the formation of embryoid bodies by the hanging drop technique7,8.
	<ol>
		<li>Remove the DFK medium, and wash with 1 mL of PBS once. Add 500 &#181;L of 0.05% trypsin, and incubate the plate at 37 &#176;C and 5% CO2 for 3-7 min based on the morphology. 
		NOTE: The hEPSCs must be able to be detached from the feeder cells when the plate is slightly disturbed.</li>
		<li>Remove the trypsin, and add 2 mL of DFK medium to harvest the cells. Spin down the cells at 300 x g for 3 min at room temperature.</li>
		<li>Remove the supernatant with a pipette. Add 1 mL of DFK medium and 1 &#181;L of Y27632 (see the Table of Materials) to resuspend the cells by flicking.</li>
		<li>Count the number of cells within the cell suspension with a hemocytometer9. Dilute the cell suspension with DFK medium and Y27632 (1,000x) so that there are 4,000 cells/25 &#181;L of DFK medium.</li>
		<li>Fill the cap of a 10 cm Petri dish with around 30-40 drops of 25 &#181;L of cell suspension per cap. Pour some PBS into the lower dish to prevent the evaporation of the droplets. Gently invert the cap, and cover the dish. Incubate the dish at 37 &#176;C and 5% CO2 for 3 days.</li>
	</ol>
	</li>
</ol>
2. Mesoderm and hemato-endothelial patterning of the embryoid body (EB)
<ol>
	<li>Collect EBs within the droplet.
	<ol>
		<li>Collect all the EBs from the droplet into a 15 mL tube using a 1 mL pipette and some PBS. Pump a little PBS into the droplet, and then aspirate out the medium, including the EBs, with the pipette. 
		NOTE: Droplets that are more yellow indicate more successful formation and growth than pink droplets.</li>
		<li>Spin down the collected EBs at 100 x g for 1 min at room temperature, and use a pipette to remove the supernatant. Add 1 mL of medium A (from a commercially available cell differentiation kit, see the Table of Materials) to transfer the collected EBs to a non-adherent 24-well plate. Label this day as Day 0 (Figure 1). 
		NOTE: Usually, all the EBs collected from one 10 cm dish are grouped and transferred to one well of a 24-well plate.</li>
	</ol>
	</li>
	<li>Perform mesoderm patterning of the EBs.
	<ol>
		<li>Incubate the plate for 3 days at 37 &#176;C with 5% CO2. On Day 2, remove 500 &#181;L of medium A (from the kit, step 2.1.2) while avoiding pipetting the EBs. Then, add 500 &#181;L of fresh medium A.</li>
	</ol>
	</li>
	<li>Perform haemato-endothelial specification of the embryoid body.
	<ol>
		<li>Remove 700 &#181;L of medium A from the well, and add 700 &#181;L of medium B (from the kit, step 2.1.2). Culture for 7 days. Perform a half-change of medium B on Day 5, Day 7, and Day 9.</li>
	</ol>
	</li>
</ol>
3. NK cell differentiation
<ol>
	<li>Transfer the patterned EBs onto an air-liquid interface.
	<ol>
		<li>Slightly tilt the plate so the EBs aggregate on the bottom. Use a 1 mL pipette to remove as much of medium B as possible while avoiding the EBs.</li>
		<li>Pick up the EBs with a pipette by aspirating the remaining medium. Transfer them to a transwell in a 24-well plate (see the Table of Materials). 
		NOTE: Restrict the numbers to two to three EBs per transwell for optimal density.</li>
	</ol>
	</li>
	<li>Perform lymphoid progenitor expansion.
	<ol>
		<li>Add 500 &#181;L of NK-1 medium (commercially available lymphoid expansion medium + 14 IU/mL IL-3, 4,500 IU/mL IL-15, 8,800 IU/mL IL-7, 26 IU/mL SCF, and 12 IU/mL ftl3L, see the Table of Materials) to the lower compartment of the transwell. Culture for 14 days, and perform a medium change every other day.</li>
	</ol>
	</li>
	<li>Harvest and transfer the cells on Days 7-10 to a coated plate.
	<ol>
		<li>To coat the plate, dilute the coating material (commercially available lymphoid differentiation coating material, 100x, see the Table of Materials) in PBS. Add 1 mL of diluted coating solution to each well of a 12-well plate, and incubate the plate at 4 &#176;C overnight. Cover the opening of the plate with wrapping film.</li>
		<li>Add 200 &#181;L of PBS into the transwell, and slowly pipette up and down around five to six times to harvest the cells released from the organoids. Avoid pipetting the organoids. 
		NOTE: Round cells are released from the organoids continuously (Figure 2D).</li>
		<li>Transfer the harvested cell suspension to a 2 mL tube, and spin down the cells at 500 x g for 5 min at room temperature. Remove the supernatant with a pipette, and add 1 mL of NK-1 medium (step 3.2.1) to resuspend the cells by pipetting.</li>
		<li>Remove all the coating solution from the well, and wash each well of a 12-well plate with 1 mL of PBS twice.</li>
		<li>Split the harvested cells at a 1:2 ratio. Transfer the cell suspension onto a coated 12-well plate (step 3.3.1). Add 1 mL of NK-1 medium so that each well has 1.5 mL of medium.</li>
		<li>To perform a medium change, let the cells sit for 1-2 min to allow the cells to sink to the bottom. Carefully aspirate out 750 &#181;L of medium. Spin down the collected supernatant at 500 x g for 5 min at room temperature.</li>
		<li>Discard most of the supernatant, and resuspend in 750 &#181;L of NK-1 medium with pipetting five to six times. Add the cells resuspended in the fresh medium into the original well. 
		NOTE: This parallel system is referred to as the 2D system in Figure 1. Follow the medium change schedule of the well of the organoids the cells originated from.</li>
	</ol>
	</li>
	<li>Perform NK cell maturation following the steps below.
	<ol>
		<li>Remove the old medium of the 3D system, and add 500 &#181;L of NK-2 (commercially available NK cell differentiation medium + 4,500 IU/mL IL-15, 8,800 IU/mL IL-7, 26 IU/mL SCF, and 12 IU/mL ftl3L, see the Table of Materials) into the lower compartment of the transwell. Incubate at 37 &#176;C and 5% CO2 for 14 days. 
		NOTE: Ensure that the medium is touching the bottom of the transwell.</li>
		<li>Perform a full medium change on the cells in the 2D system. Collect all 1.5 mL of cells from one well, and centrifuge it at 500 x g for 5 min at room temperature. Remove most of the supernatant with a pipette, and resuspend the pellet with 1.5 mL of NK-2 medium (step 3.4.1).</li>
		<li>Perform a medium change every alternate day. Slightly tilt the plate of the 3D system, and remove all the old medium outside the transwell. Add 500 &#181;L of NK-2 medium outside of the transwell.</li>
		<li>To perform a half medium change on one well of a 12-well plate for the 2D system, let the cells sit for 1-2 min to allow the cells to sink to the bottom. Carefully aspirate out 750 &#181;L of the medium.</li>
		<li>Spin down the collected supernatant at 500 x g for 5 min at room temperature to prevent cell loss. Discard most of the supernatant, and resuspend in 750 &#181;L of NK-2 medium by pipetting five to six times. Add the cells resuspended in fresh medium into the original well.</li>
	</ol>
	</li>
</ol>
4. Harvesting mature cells
<ol>
	<li>Harvest the cells cultured in the 2D system.
	<ol>
		<li>Harvest the cells by pipetting up and down during Days 38-45. Wash with 1 mL of PBS twice. Centrifuge the cell suspension at 500 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant with a pipette, and resuspend it in the buffer of choice (i.e., PBS + 2% FBS for flow cytometry, see the Table of Materials).</li>
	</ol>
	</li>
	<li>Harvest the cells embedded in the organoid.
	<ol>
		<li>Collect the medium from outside of the transwell into a 15 mL tube during Days 38-45. Wash the organoid twice by adding 200 &#181;L of PBS inside the transwell and pipetting up and down five to six times. Transfer the suspension to the 15 mL tube.</li>
		<li>Add 500 &#181;L of PBS into a well of a 12-well plate. Transfer the organoid from the transwell to that well of the 12-well plate with forceps.</li>
		<li>Cut the organoid 20 times using a pair of scissors and forceps. Flush the remaining cells on the forceps and scissors into the well with PBS. Collect as much PBS as possible, and transfer it to the 15 mL tube used in step 4.2.1 without aspirating the organoid structure.</li>
		<li>Add 500 &#181;L of the dissociation reagent (see the Table of Materials) into the transwell to digest the organoid enzymatically. Incubate with a commercial cell detachment solution (see the Table of Materials) for 7-10 min at 37 &#176;C and 5% CO2. Quench the reaction by adding 1 mL of PBS with 2% FBS, and collect the solution in another 15 mL tube. Wash the well with 1 mL of PBS with 2% FBS twice. 
		NOTE: Determine the length of the incubation based on the morphology. Try to achieve the point at which single cells start to be released from the membrane of the 3D structure.</li>
		<li>Add 1 mL of PBS with 2% FBS, and pipette up and down 8-10 times. Collect all the solutions, including the organoid structures. Transfer them to the 15 mL tube used in step 4.2.3 with a cell strainer.</li>
		<li>Centrifuge both of the tubes at 500 x g for 5 min at room temperature. Discard the supernatant with a pipette. Resuspend the cell pellet with the buffer of choice (step 4.1.2).</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+produce+clinical-scale+natural+killer+cells+from+human+pluripotent+stem+cells.">An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells.</a> Methods in Molecular Biology. 2048, 107-119 (2019).</li><li>Li, Y., Hermanson, D. L., Moriarity, B. S., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+natural+killer+cells+engineered+with+chimeric+antigen+receptors+enhance+anti-tumor+activity.">Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity.</a> Cell Stem Cell. 23 (2), 181-192 (2018).</li><li>Vivier, E., Tomasello, E., Baratin, M., Walzer, T., Ugolini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+natural+killer+cells.">Functions of natural killer cells.</a> Nature Immunology. 9 (5), 503-510 (2008).</li><li>Dhar, P., Wu, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+and+its+ligands+in+cancer.">NKG2D and its ligands in cancer.</a> Current Opinion in Immunology. 51, 55-61 (2018).</li><li>Dang, S. M., Kyba, M., Perlingeiro, R., Daley, G. Q., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+embryoid+body+formation+and+hematopoietic+development+from+embryonic+stem+cells+in+different+culture+systems.">Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems.</a> Biotechnology and Bioengineering. 78 (4), 442-453 (2002).</li><li>Rungarunlert, S., Techakumphu, M., Pirity, M. K., Dinnyes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoid+body+formation+from+embryonic+and+induced+pluripotent+stem+cells:+Benefits+of+bioreactors.">Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors.</a> World Journal of Stem Cells. 1, 11-21 (2009).</li><li>Counting cells using a hemocytometer. abcam Available from: <a target="_blank" href="https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer">https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer</a> (2022)</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> Journal of Immunological Methods. 294, 15-22 (2004).</li><li>Tremblay-McLean, A., Coenraads, S., Kiani, Z., Dupuy, F. P., Bernard, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+ligands+for+activating+natural+killer+cell+receptors+on+cell+lines+commonly+used+to+assess+natural+killer+cell+function.">Expression of ligands for activating natural killer cell receptors on cell lines commonly used to assess natural killer cell function.</a> BMC Immunology. 20, 8 (2019).</li><li>Inoue, T., Swain, A., Nakanishi, Y., Sugiyama, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+analysis+of+cell+surface+marker+of+human+leukemia+cell+lines+using+flow+cytometry.">Multicolor analysis of cell surface marker of human leukemia cell lines using flow cytometry.</a> Anticancer Research. 34 (8), 4539 (2014).</li><li>Mhatre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+flow+cytometry+based+cytotoxicity+assay+for+evaluation+of+NK+cell+function.">Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function.</a> Indian Journal of Experimental Biology. 52 (10), 983-988 (2014).</li><li>Medvinsky, A., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+hematopoiesis+is+autonomously+initiated+by+the+AGM+region.">Definitive hematopoiesis is autonomously initiated by the AGM region.</a> Cell. 86 (6), 897-906 (1996).</li><li>Motazedian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+RAG1++progenitors+emerge+directly+from+haemogenic+endothelium+in+human+pluripotent+stem+cell-derived+haematopoietic+organoids.">Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids.</a> Nature Cell Biology. 22 (1), 60-73 (2020).</li><li>Gong, J. 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The authors declare no conflicts of interest.

There are a few pivotal steps to ensure the successful differentiation of CD45+CD56+ cells from hEPSCs. The pre-differentiation (step 1.2) is crucial since the EPSC medium contains inhibitors of lineage commitment1. After pre-differentiation, the dome-shaped colonies of hEPSCs are flattened (Figure 2B). The addition of Y27632, a Rho-kinase inhibitor (ROCKi), during the formation of EBs from hEPSCs (step 1.3) is indispensable for the survival of the hEPSCs after dissociation. Another important consideration is the number of EBs planted on each transwell. Since it is a small-scale system, two to three EBs per transwell is the optimal density to prevent overconsumption of the media. The air-liquid interface is maintained during the whole course of NK cell differentiation for the 3D system, which is believed to maintain the polarity of the stromal cells and support the expansion of the AGM-derived hematopoietic progenitors14,15.
As mentioned before, the density of EBs on the transwell is a determining factor for successful differentiation. The key sign for this is the color of the medium 1 day after the implantation of EBs on the transwell. If the color turns yellow quickly, this indicates either contamination or overcrowding. To solve the issue, a 1 mL pipette should be used to manually pick up extra EBs and transfer them into another transwell.
A major limitation of this protocol is the purity of CD3&#8722;/CD45+ CD56+ cells in the IVD products, which is believed to be partially responsible for the inferior cytotoxicity compared to pure NK92mi cells. A 3D culture system was employed to induce hematopoietic progenitors via the generation of an EB that resembles the three embryonic germ layers. This strategy mimics the development of blood cells in the bone marrow microenvironment, thus removing the need for stromal cells to support the differentiation3. Without a sorting step to purify the hematopoietic progenitors, the purity of the IVD products is anticipated to be reduced. A strategy to retain the complex differentiation niche while increasing the purity of the endpoint product is developing an expansion method for the sorted CD3&#8722;CD56+ IVD products. For example, the sorted CD3&#8722;CD56+ IVD products can be cultured on artificial antigen-presenting cells (aAPCs), such as irradicated K562 cells engineered with membrane-bound IL-21. With the supply of IL-2 in the culture, this method can achieve a 1,000-fold increase in NK cell numbers3.
Another limitation is the limited access to bona fide human NK cells as a positive reference. NK92mi is the positive reference employed in the coculture assay, but it is not accurate enough. NK92mi cells are engineered from NK92 cells, a permanent cell line that expresses activated NK cell phenotypes16, to autonomously produce IL-2 to meet the culture requirements. NK92 cells demonstrate superior killing activity against K562 cells in vitro than human primary NK cells17. A fairer comparison of the tumor-killing capacity of IVD products could be achieved by assaying the cytotoxicity of peripheral blood NK cells in parallel, but unfortunately, access to blood banks is lacking.
This two-system culture protocol aims to generate CD3&#8722;/CD45+CD56+ cells from hEPSCs. The assessment of the surface markers and cytotoxicity with FACS can be routinely performed on cells from 2D systems without disrupting the differentiation niche. Despite the differences in the percentages of CD3&#8722;CD56+ cells, the 3D and 2D systems can derive CD3&#8722;CD56+ cells with similar variations in CD56 expression (Figure 3), and the 2D system reflects the existence of lymphoid progenitors from the 3D culture condition.
The significance of utilizing the 3D culture system is that it allows the use of high-resolution profiling assays, such as spatial transcriptomics or imaging mass spectrometry, to research the spatial relationships and cell-cell interactions within the complex differentiation niche.

Compared to conventional pluripotent stem cells like human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), human expanded potential stem cells (hEPSCs) are closer to the state of totipotency, as they can differentiate into both extra-embryonic and embryonic lineages1. For example, hEPSCs can be differentiated into trophoblasts1 and yolk-sac-like cells2. To achieve the unique potency of hEPSCs, an individual blastomere is cultured in a medium containing several small molecules that inhibit lineage commitment signaling1, which is referred to as EPSC medium (EPSCM). Culturing hESCs and hiPSCs in the EPSCM expands their previously restricted potency to differentiate them into trophoblast cells1.
Pluripotent stem cells are a valuable research tool for experimenting with novel genetic modifications. Due to the self-renewal and differentiation capacities of pluripotent stem cells, one transformed clone of a pluripotent stem cell can produce differentiated cellular products that possess the same genetic modification at the same locus. Zhu and Kaufman established the standard for NK cell differentiation from conventional pluripotent stem cells (hESCs and hiPSCs)3. First, they induced hematopoietic stem cells (HSCs) from an embryoid body derived from pluripotent stem cells. The addition of stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Ftl3L), interleukin (IL)-7, IL-15, and an early supplement of IL-3 biased the HSCs to develop into natural killer (NK) cells. Subsequently, they expanded the NK cells with artificial antigen-presenting cells (aAPCs), which present membrane-bound IL-21, with the continuous supplementation of IL-2. Researchers have applied this approach to immunotherapy in order to generate iPSC-derived natural killer cells endowed with the chimeric antigen receptor (CAR-iNK)4.
Human NK cells are defined as CD3&#8722;CD56+ leukocytes in the peripheral blood. They are effectors against virus-infected cells and tumor cells5. Some inhibitory receptors on NK cells recognize molecules expressed ubiquitously in normal cells. As an example, the NKG2A/CD94 heterodimer expressed on NK cells recognizes MHC class I molecules5. Meanwhile, the activating receptors on NK cells recognize stress-induced ligands, like how NKG2D recognizes transformation-induced MHC class I polypeptide-related sequence A (MICA/MICB)6. Some transformed cells downregulate their &#34;self-ligands&#34; to escape immune surveillance and upregulate aberrant ligands, which triggers the NK cells to execute their lytic machinery. The intrinsic anti-tumor abilities of NK cells has brought attention to this immune cell type.
The ability to simultaneously give rise to both embryonic and extra-embryonic lineages may render hEPSCs a more faithful recapitulation of the embryonic development niche than conventional pluripotent stem cells. From experience, it is easier to maintain the potency of hEPSCs than hiPSCs. In the present study, a protocol was developed (Figure 1) to bias hEPSCs to develop into hematopoietic lineages and later differentiate progenitor cells into natural killer (NK) cells in vitro. In the protocol, commercial media are employed to avoid serum inconsistencies, followed by the stepwise addition of cytokines to bias differentiation into lymphoid lineages. This protocol has two systems to preserve the complex 3D microenvironment while deriving CD3&#8722;CD56+/CD45+CD56+ cells that phenotypically and functionally resemble human NK cells.
This protocol may be useful in studying the interaction of immune cells with their differentiation niches and may have the potential to purify cellular products for immunotherapeutic uses.

<table><tbody><tr><td>30 w/v% Albumin Solution, from Bovine Serum(BSA), Fatty Acid Free</td><td>Wako Chemicals</td><td>017-22231</td><td>For resuspending cytokines.&amp;nbsp;</td></tr><tr><td>ACCUMAX</td><td>STEMCELL Technologies</td><td>&amp;nbsp;07921</td><td></td></tr><tr><td>Anti-human-CD159a-PE</td><td>BD</td><td>375104</td><td>During antibody staining, 0.5 &amp;micro;L of antibodies is added into the staining buffer (PBS + 5% FBS)</td></tr><tr><td>Anti-human-CD3-APC antibodies</td><td>BD</td><td>555355</td><td>During antibody staining, 0.5 &amp;micro;L of antibodies is added into the staining buffer (PBS + 2% FBS)&amp;nbsp;</td></tr><tr><td>Anti-human-CD45-APC antibodies</td><td>BD</td><td>555485</td><td>During antibody staining, 0.5 &amp;micro;L of antibodies is added into the staining buffer (PBS + 3% FBS)</td></tr><tr><td>Anti-human-CD56-PE antibodies</td><td>BD</td><td>555516</td><td>During antibody staining, 0.5 &amp;micro;L of antibodies is added into the staining buffer (PBS + 4% FBS)</td></tr><tr><td>Anti-human-CD56-PEcy7 antibodies</td><td>BD</td><td>335826</td><td>During antibody staining, 0.5 &amp;micro;L of antibodies is added into the staining buffer (PBS + 5% FBS)</td></tr><tr><td>Anti-human-CD94-APC&amp;nbsp;</td><td>BD</td><td>559876</td><td>During antibody staining, 0.5 &amp;micro;L of antibodies is added into the staining buffer (PBS + 5% FBS)</td></tr><tr><td>Bemis Parafilm M Laboratory Wrapping Film</td><td>Thermo Scientific</td><td>11772644</td><td></td></tr><tr><td>Cd244 Monoclonal Antibody (eBioC1.7 (C1.7)), Functional Grade, eBioscience</td><td>Thermo Scientific</td><td>16-5838-85</td><td></td></tr><tr><td>Costar 6.5 mm Transwell, 0.4 &amp;micro;m Pore Polyester Membrane Inserts</td><td>STEMCELL Technologies</td><td>3470</td><td></td></tr><tr><td>Dapi Solution, 1 mg/mL</td><td>BD</td><td>564907</td><td>It is resuspended with ddH&lt;sub&gt;2&lt;/sub&gt;O so the concentration is 10 &amp;micro;M/mL. Add 1 &amp;micro;L of the aliquot in 300 &amp;micro;L of FACS buffer (PBS + 2% FBS)&amp;nbsp;</td></tr><tr><td>DMEM</td><td>CPOS-Bioreagent core</td><td>11965092</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo Scientific</td><td>11320033</td><td></td></tr><tr><td>FcX, human</td><td>Biolengend</td><td>422302</td><td></td></tr><tr><td>Fetal Bovine Serum(FBS) qualified E.U.-approved South America</td><td>CPOS-Bioreagent core</td><td>10270106</td><td></td></tr><tr><td>FlowJo v10.8.0</td><td>BD</td><td></td><td></td></tr><tr><td>Gibco&amp;nbsp; PBS (10x) pH 7.4</td><td>CPOS-Bioreagent core</td><td>70011044</td><td>10x concentrated PBS is manufactured as follows: without calcium, magnesium or phenol red</td></tr><tr><td>K562 cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CCL-243</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Thermo Scientific</td><td>10828-028</td><td></td></tr><tr><td>MyeloCult H5100 medium&amp;nbsp;</td><td>STEMCELL Technologies</td><td>5150</td><td></td></tr><tr><td>NK92mi cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CRL-2408</td><td>Lot: 70045208. Once thawed, they are cultured in MyeloCult H5100 medium, and maintained the density between 2 x 10&lt;sup&gt;5&lt;/sup&gt; to 1 x 10&lt;sup&gt;6&lt;/sup&gt; cell/mL.</td></tr><tr><td>Nunc Cell-Culture Treated Multidishes 12 well plate</td><td>Thermo Scientific</td><td>150628</td><td>flat bottom</td></tr><tr><td>Nunc Cell-Culture Treated Multidishes 24 well plate</td><td>Thermo Scientific</td><td>142475</td><td></td></tr><tr><td>Nunc EasYDish Dishes</td><td>Thermo Scientific</td><td>150460</td><td></td></tr><tr><td>pLenti-GFP Lentiviral Control Vector&amp;nbsp;</td><td>CELL BIOLABS</td><td>LTV-400</td><td>It is packaged as the manufactuer suggested (https://www.cellbiolabs.com/sites/default/files/LTV-400-gfp- lentiviral-plasmid.pdf). 40 &amp;mu;L of 50x lentivirus is added with 1 &amp;mu;L 10 mg/mL polybrene per 1 mL of cell suspension. Complete medium change is performed 24 h after the addition of lentivirus. Cells are incubated undisputedly for 3 days at 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;.</td></tr><tr><td>Polybrene&amp;nbsp;</td><td>EMD Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td>Recombinant Human Flt-3 Ligand/FLT3L Protein</td><td>R&amp;amp;D Systems</td><td>308-FK-005</td><td>It is resuspend with PBS + 0.2% BSA. Thr working concentration is 10 ng/mL or 12 IU/mL.</td></tr><tr><td>Recombinant Human IL-15 Protein</td><td>R&amp;amp;D Systems</td><td>247-ILB-005</td><td>It is resuspended with PBS + 0.2% BSA. The working concentration is 10 ng/mL or 4500 IU/mL.&amp;nbsp;</td></tr><tr><td>Recombinant Human IL-18/IL-1F4 Protein</td><td>R&amp;amp;D Systems</td><td>9124-IL-010</td><td>It is resuspended with PBS + 0.2% BSA. The working concentration is 50 ng/mL. The IU is not provided by the company.&amp;nbsp;</td></tr><tr><td>Recombinant Human IL-2 Protein</td><td>R&amp;amp;D Systems</td><td>202-IL-010</td><td>It is resuspended with PBS + 0.2% BSA. The working concentration is 50 ng/mL or 455 IU/mL.&amp;nbsp;</td></tr><tr><td>Recombinant Human IL-3 Protein, 50ug</td><td>PeproTech</td><td>200-03</td><td>It is resuspended with PBS + 0.2% BSA. The working concentration is 5 ng/mL or 14 IU/mL.&amp;nbsp;</td></tr><tr><td>Recombinant Human IL-7 Protein</td><td>R&amp;amp;D Systems</td><td>207-IL-010</td><td>It is resuspended with PBS + 0.2% BSA. The working concentration is 20 ng/mL or 8800 IU/mL.&amp;nbsp;</td></tr><tr><td>Recombinant Human SCF Protein</td><td>R&amp;amp;D Systems</td><td>255-SC-010</td><td>It is resuspended with PBS + 0.2% BSA. The working concentration is 20 ng/mL or 26 IU/mL.&amp;nbsp;</td></tr><tr><td>STEMdiff Hematopoietic Kit</td><td>STEMCELL Technologies</td><td>5310</td><td>Medium A: 45 mL STEMdiff Hematopoietic Basal Medium + STEMdiff Hematopoietic Supplement A ; Medium B: 75 STEMdiff Hematopoietic Basal Medium + STEMdiff Hematopoietic Supplement B</td></tr><tr><td>StemSpan lymphoid differentiation coating material</td><td>STEMCELL Technologies</td><td>9950</td><td>It is resuspended in PBS (100x)</td></tr><tr><td>StemSpan NK cell differentiation medium&amp;nbsp;</td><td>STEMCELL Technologies</td><td>9960</td><td>It is prepared by adding 500 &amp;micro;L StemSpan NK Cell Differentiation Supplement (100x)&amp;nbsp; into 49.5 mL of SFEM II. Both are provided in StemSpan NK Cell Generation Kit.&amp;nbsp;</td></tr><tr><td>StemSpan lymphoid expansion medium&amp;nbsp;</td><td>STEMCELL Technologies</td><td>9960</td><td>It is prepared by adding 5 mL StemSpan Lymphoid Progenitor Expansion Supplement (10x) into 45 mL of StemSpan SFEM II. Both are provided in StemSpan NK Cell Generation Kit.&amp;nbsp;</td></tr><tr><td>StemSpan NK Cell Generation Kit</td><td>STEMCELL Technologies</td><td>9960</td><td>Thaw the medium and materials in room temperature and the medium is stored in 4 &amp;deg;C once thawed.&amp;nbsp;</td></tr><tr><td>The BD LSRFortessa cell analyzer</td><td>BD</td><td></td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Scientific</td><td>25300054</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td>working concentration: 10 &amp;micro;M</td></tr></tbody></table>

generation of natural killer cells from human expanded potential stem cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

It was hypothesized that the present in vitro differentiation (IVD) products would possess similar surface markers and anti-tumor abilities to human NK cells.
The defining marker of human NK cells was accessed to test whether the differentiation products from the 3D culture system yielded cells that phenotypically resembled human NK cells. About 15% of the cells dissociated from the 3D culture system from two batches induced at different time points (n = 2) were CD3&#8722;CD56+ (Figure 3A). CD3 was replaced with CD45 (a pan-leukocyte marker) in the test panel due to the absence of CD3 and the difficulty inducing T cells in vitro. Approximately 16% of the cells dissociated from the 3D structure were CD45+CD56+ (Figure 3B, n = 1), which is in line with the percentages of CD3&#8722;CD56+ cells seen in earlier trials. The percentages of CD3&#8722;CD56+ cells in the cells harvested from the 2D culture system ranged from 30% to 6%, from more successful inductions (Figure 3C, n = 3) to less successful inductions (Figure 3D, n = 2). The functionality and phenotypes of the cells harvested from the 2D culture system were validated. During the Day 18 culture from the 2D system, about 12% of the cells expressed both ectopic CD56 and CD107a after 2 h of 50 ng/mL IL-18, 455 IU/mL IL-2, and 20 ng/mL anti-CD244 antibody stimulation (Figure 4C, n = 1). About 28% of the harvested cells were CD94+CD159a+ (Figure 4B, n = 1). The ectopic expression of CD107a, a protein lining on the membrane of granules, indicates degranulation10, while the CD94/NKG2A(CD159a) heterodimer is another defining marker of NK cells. This provides an example of the phenotypic and functionality validation of IVD products.
The functionality of the IVD products was tested by their cytotoxicity against human erythroleukemia cells-K562 cells. Comprehensive ligand profiling on K562 cells reveals their downregulated major histochemistry complex I (MHC-I) and comparatively strong NKG2D ligand expression (such as ULBP-1, ULBP-2/5/6, and ULBP-3)11; thus, K562 cells are susceptible to the lytic activity of NK cells12.&#160;Both IVD endpoint products and NK92mi cells were primed with 50 ng/mL IL-18, 455 IU/mL IL-2, and 20 ng/mL anti-CD244 antibodies overnight. The GFP+ K562 cells were cocultured with either harvested cells from IVD or NK92mi cells at different effector-to-target ratios (E:T) in the presence of 50 ng/mL (IU not provided) IL-18, 455 IU/mL IL-2, and 20 ng/mL anti-CD244 antibodies in the same volume of H5100 medium for 3 h. The readout of NK cell killing activity was the expression of the dead cell marker on GFP+ subsets. The K562 cells were transduced with a commercially available control vector (see the Table of Materials) to express GFP constitutively, and the efficiency of the transduction was about 80% (Supplementary Figure 1A). The percentages of GFP signal added were proportional to the number of GFP+ K562 cells added, suggesting specific GFP expression from the transduced K562 cells (Supplementary Figure 1B).
The percentages of dying target cells shown in Figure 5 were calculated with the following formula13:
Percentage of dying cells = (FITC + DAPI + % of coculture [for example, Figure 5A]) - (FITC + DAPI + % of transduced K562 cells)
Of all three E:T ratios assessed (0.1:1, 0.33:1, 1:1), the percentages of dying GFP+ cells were positively correlated with the E:T ratios when cocultured with IVD products (Figure 5B, hEPSC-NK) or NK92mi cells (Figure 5B, NK92mi), indicating the specific killing of tumor targets. However, the cytotoxicity of the IVD products was comparatively modest within the tested timeframe (Figure 5C).
Finally, the lymphoid progenitor cell generation was compared between the 3D and 2D cultures. It was found that the 3D culture condition generated more progenitor cells (55,000 cells) than the 2D culture condition (36,000 cells) (Supplementary Figure 2). This suggests that the context of tissue architecture and cellular components in the 3D culture supported the generation of lymphoid progenitor cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64608/64608fig01.jpg" /> 
Figure 1: Schematic diagram showing the differentiation strategy to differentiate NK cells from hEPSCs. (A) Here, the timeline of the 3D culture systems is shown, detailing the culture conditions and brief keywords from each step. The air-liquid interface of the 3D system is maintained during the NK cell induction. (B) Here, the timeline of the 2D culture systems is shown. The released cells harvested from Days 7-10 from structures in the 3D system are seeded on a coated plate without the air-liquid interface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/64608fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64608/64608fig02.jpg" /> 
Figure 2: Morphology of the cells observed at distinct stages of differentiation. (A) The morphology of hEPSCs when maintained in EPSCM. The hEPSCs form domed-shaped colonies. (B) The morphology of hEPSCs after pre-differentiation. The domed-shaped colonies are flattened. The hEPSCs are harvested and form EBs with the hanging drop technique. The EBs are collected and cultured in medium A and then medium B. (C) The morphology of an EB on Day 10. During this period, the EB starts to expand and inflate, forming membranous structures. (D) The morphology of a cell-releasing EB. After seeding on the transwell, the EB grows and constantly releases round cells to the surroundings. Some of the released cells are collected and cultured on a 12-well plate coated with lymphoid differentiation coating materials. The cell populations are morphologically heterogeneous and tend to aggregate together. (E) The morphology of cells observed at the endpoint of the IVD. Small, circular suspension cells (black arrow) are observed. Scale bars: (A,B,E) = 100 &#181;m; (C,D) = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/64608fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64608/64608fig03.jpg" /> 
Figure 3: Representative FACS plots of hEPSC-derived products at the endpoint. Cells are incubated with fluoroscope-conjugated antibodies on ice for 30 min. The samples are stained with DAPI before being loading into the FACS sample injection port. Unstained cells are used to establish the negative gating. (A) Representative FACS plot on CD3 and CD56 expression in cells dissociated from the 3D culture system from two batches (n = 2). (B) Representative FACS plot on CD45 and CD56 expression in cells dissociated from the 3D culture (n = 1). (C) Representative FACS plot on CD3 and CD56 expression in cells harvested from the coated plate from a successful induction (n = 3). (D) Representative FACS plot on CD3 and CD56 expression from the coated plate when the induction is suboptimal (n = 2). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/64608fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64608/64608fig04.jpg" /> 
Figure 4: Representative FACS plot on hEPSC-derived products during Day 18 culture (n = 1). (A) FACS plot on CD56 and CD107a expression after 2 h of stimulation with IL2, IL-18, and anti-CD244 antibodies. (B) FACS plot on CD159a and CD94 expression.&#160;The samples are stained withDAPI before being loaded into the FACS sample injection port. Unstained cells are used to establish the negative gating. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/64608fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64608/64608fig05.jpg" /> 
Figure 5: Plot of the percentages of dying cells against different E:T ratios from the coculture of GFP+ K562 cells with either the IVD endpoint product or NK92mi cells (n = 1). The expression of&#160;FITC+ DAPI+ cells was assessed with a cell analyzer, and the data analysis was performed with compatible software. (A) A representative FACS plot showing the gating of FITC + DAPI + subsets from the coculture of IVD products and K562 cells with an E:T ratio of 1. (B) Individual plots from the coculture experiment with GFP+ IVD products (left) or NK92mi cells (right) for 3 h. Both reflect a positive proportional relationship between the percentage of dying cells and the E:T ratio. (C) A plot showing the killing curves of IVD products and NK92mi cells on the same scale. The IVD products displayed mild cytotoxicity as compared to the NK92mi cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/64608fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Functional assay of NK cells from hEPSCs. (A) FACS histogram of transduced K562 cells. The FITC+ K562 cells expressed GFP. (B) FACS histograms from the coculture of GFP+ K562 cells and the endpoint IVD products at three E:T ratios (n = 1). The percentages of FITC+ cells in the coculture corresponded to the number of added transduced K562 cells. The gatings were established with untransduced K562 cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/Supplementay Figure 1_revised.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Lymphoid progenitor differentiation in 2D versus 3D culture. The present organoid-based protocol was used to induce lymphoid progenitors from human expanded potential stem cells. Two conditions were set up: (1) lymphoid progenitors were kept in culture within the organoids (3D), and (2) lymphoid progenitors were isolated and kept on the culture dish (2D). After 2 weeks of additional culture, the cell numbers were determined to compare the 2D versus 3D culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64608/Supplementary Figure 2.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Generation of Natural Killer Cells from Human Expanded Potential Stem Cells at JoVE.com

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

generation-natural-killer-cells-from-human-expanded-potential-stem

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.0K Views.  The University of Hong Kong. Over significant gesproken, het genereren van NK-cellen uit een uitbreidbare bron is van cruciaal belang voor toepassingen zoals kankerimmunotherapie. Het voordeel van deze technologie is dat uitgebreide potentiële stamcellen met een bredere differentiatiecapaciteit kunnen worden gebruikt in plaats van gewone IP-cellen. Verwijder voor predifferentiatie van hEPSC's het hEPSC-medium waarin cellen werden onderhouden en voeg één milliliter DFK-medium toe.Incubeer gedurende twee tot drie...

Video: Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Immunology and Infection

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

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

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 ...

Bioengineering

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

Cancer Research

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

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