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Generation of universal natural killer cells from a cryopreserved cord blood mononuclear cell‐derived induced pluripotent stem cell library

Natural killer (NK) cells play a key role in innate immunity and are regarded as a promising candidate for cellular immunotherapy. Natural killer cells may be generated from different sources, including induced pluripotent stem cells (iPSCs); these stem cells produce an abundant amount of NK cells to meet the needs of a wide range of clinical applications. Autologous iPSCs are expensive and labor‐intensive to prepare, while allogeneic iPSCs require human leukocyte antigen (HLA) matched cells to avoid the risk of immune rejection. In the current study, we prepared HLA‐matched iPSCs using HLA common haplotype homozygous (HLAh) donors from cryopreserved human cord blood (CB) sourced from the Tianjin Cord Blood Public Bank. This approach was designed to generate a CB‐derived iPSC library from HLAh donors and use it to produce off‐the‐shelf NK cells. Starting with readily available cryopreserved CB mononuclear cells (cryoCBMCs), we produced cryoCBMC‐derived iPSCs (cryoCB‐iPSCs). These cryoCB‐iPSCs were induced to generate embryoid bodies (EBs) using an improved 3D suspension culture method, and induced NK (iNK) cells were differentiated from EBs. iNK cells expressed specific surface markers of NK cells, exhibited cytotoxicity comparable with NK cells generated from CB (CB‐NK) and peripheral blood (PB‐NK), and expressed lower levels of KIRs and HLA‐DR compared to CB‐NK and PB‐NK. Taken together, we have shown that an iPSC library can be established from HLAh cryoCBMCs, and cryoCB‐iPSCs can be used to generate a large number of ‘universal’ NK cells for future clinical applications.

Natural killer (NK) cells play a key role in innate immunity and are regarded as a promising candidate for cellular immunotherapy. Natural killer cells may be generated from different sources, including induced pluripotent stem cells (iPSCs); these stem cells produce an abundant amount of NK cells to meet the needs of a wide range of clinical applications. Autologous iPSCs are expensive and labor-intensive to prepare, while allogeneic iPSCs require human leukocyte antigen (HLA) matched cells to avoid the risk of immune rejection. In the current study, we prepared HLA-matched iPSCs using HLA common haplotype homozygous (HLAh) donors from cryopreserved human cord blood (CB) sourced from the Tianjin Cord Blood Public Bank. This approach was designed to generate a CB-derived iPSC library from HLAh donors and use it to produce off-the-shelf NK cells. Starting with readily available cryopreserved CB mononuclear cells (cryoCBMCs), we produced cryoCBMC-derived iPSCs (cryoCB-iPSCs). These cryoCB-iPSCs were induced to generate embryoid bodies (EBs) using an improved 3D suspension culture method, and induced NK (iNK) cells were differentiated from EBs. iNK cells expressed specific surface markers of NK cells, exhibited cytotoxicity comparable with NK cells generated from CB (CB-NK) and peripheral blood (PB-NK), and expressed lower levels of KIRs and HLA-DR compared to CB-NK and PB-NK. Taken together, we have shown that an iPSC library can be established from HLAh cryoCBMCs, and cryoCB-iPSCs can be used to generate a large number of 'universal' NK cells for future clinical applications.
There have been many recent developments in immune-cellular therapy targeting cancer, notably in the field of chimeric antigen receptor T (CAR-T) cell therapy. To date, five CAR-T products have been approved by the Food and Drug Administration and two CAR-T products have also been recently launched in China [1]. Natural killer (NK) cells, a unique lymphocyte subgroup, are an important participant of innate immunity and exhibit the capacity to kill virally infected cells and tumor cells. In addition, NK cells exhibit potential value in tumor immunotherapy due to their non-major histocompatibility complex (MHC) restriction and extensive tumor recognition ability [2]. Natural killer cells can recognize and lyse mutated cells with downregulated MHC-I molecules or overexpressed activated NK cell receptors [3,4]. Thus, NK cells, not only in autologous but also allogenetic, can be used to treat cancer without causing serious adverse effects, such as graft-versus-host disease (GvHD), holding promise for significantly broadening the availability of clinical application [5].
At present, there are many clinical studies on the applications of NK cells in tumor therapy [3,[5][6][7]. The clinical application of NK cells usually requires large and repeated doses to achieve therapeutic effects [8,9]. Thus, identifying sources that can be used to massproduce NK cells to meet clinical requirements has been an active area of research. Indeed, various sources have been tested to generate NK cells, including NK cell lines, peripheral blood-derived NK cells (PB-NK), and cord blood-derived NK cells (CB-NK) [5,10]. Natural killer cell lines are unstable and require irradiation before infusion, and the irradiated cells only survive for 48 h [11]. Due to this limitation, NK cell lines cannot achieve long-term therapeutic effects [12]. PB-NK and CB-NK cells are limited for individual dosing, and generating PB-NK and CB-NK cells is time-consuming and cost-inefficient [10]. Therefore, there is an urgent need to identify an appropriate source to produce a large number of allogeneic NK cells.
Induced pluripotent stem cells (iPSCs) are often generated by the forced expression of relevant pluripotent transcription factors in somatic cells [13]. iPSCs have strong self-renewal ability and exhibit embryonic stem cell (ES)-like pluripotency to differentiate into various functional cell lineages [14]. More interestingly, iPSCs have been reported to have the potential to become standard raw materials to produce a large number of NK cells [15,16]. Further, human CB represents an attractive source of cells for reprogramming iPSCs [17] due to its wide availability, non-invasive acquisition, low risk of viral contamination and mutation, and it is typically already stored in liquid nitrogen for transplantation. Moreover, the most frequent HLA haplotype homozygous (HLAh) CB selected from public CB bank is an optimal source to produce 'universal' iPSCs for a wider range of recipients [18].
In the present study, we designed a strategy to use cryopreserved CB mononuclear cells (cryoCBMCs) to generate iPSCs (cryoCB-iPSCs). We then improved a method to mass-produce induced NK (iNK) cells derived from cryoCB-iPSCs. We characterized iNK cells by evaluating the expression of surface markers of NK cells and cytotoxicity.

CryoCB and cryoCBMC isolation
Human cryoCB was obtained from the Tianjin Cord Blood Public Bank. The donors involved in the study were informed and signed written informed consents. This study was conducted in accordance with the Declaration of Helsinki for experiments involving humans and was approved by the ethical advisory board of the Institute of Hematology and Blood Diseases Hospital (YW2018001-EC-1). The CBMC and cryoCBMCs were prepared from fresh and cryopreserved CB, respectively, by Ficoll-Hypaque (G&E Healthcare, Chicago, IL, USA; 17-1440-02) as previously described [19].

Alkaline phosphate staining and immunocytochemistry
Alkaline phosphatase (AP) staining was performed using an Alkaline Phosphatase Staining Kit II (Stemgent, Cambridge, MA, USA; 00-0055). For detection of pluripotent stem cell marker antigens, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After washing with PBS, cells were incubated in 0.25% Triton X-100 (Sigma-Aldrich; X100-1L) for 10 min at room temperature, and then blocked with 5% sheep or donkey serum for 30 min at room temperature. Cells were then incubated with the following primary antibodies SSEA-4

Differentiation capacity of cryoCB-iPSCs in vitro
STEMdiff™ Trilineage Differentiation Kit (Stemcell; 5230) was used to differentiate cryoCB-iPSCs into endoderm, mesoderm, and ectoderm. Mesoderm and endoderm lineages were formed on day 5, and ectoderm lineages were formed on day 7. Immunofluorescence staining was used to detect representative markers (AFP3 for mesoderm, 1A4 for endoderm and 10C2 for ectoderm) of cryoCB-iPSCs. The Human Pluripotent Stem Cell Trilineage Differentiation quantitative polymerase chain reaction (qPCR) Array (Stemcell; 7515) was performed to characterize hPSCs and their trilineage differentiation capacity. This qPCR array was designed to detect the gene expression profile of undifferentiated hPSCs and their trilineage derivatives following in vitro directed or spontaneous differentiation. Genes were selected based on their demonstrated differential expression in ES and iPSCs compared with hPSC-derived ectodermal, mesodermal, and endodermal lineage cells.

Cytotoxicity assay of iNK cells
CD107a expression and intracellular IFN-γ and TNF-α production determined by flow cytometry were used to evaluate the cytotoxicity of iNK cells. In brief, iNK cells (i.e., effector cells) were co-cultured with K562 cells (i.e., target cells) at a ratio of 1 : 1. iNK cells that were not cocultured with target cells were used as a negative control. iNK cells were collected after 5 h incubation and stained with CD56-FITC (catalog no. 562794), CD107a-APC (catalog no.560664), IFN-γ-PE (catalog no. 559327; BD Biosciences), and TNF-α-APC (catalog no. 502912; Biolegend, San Diego, CA, USA). The expression of CD107A, IFN-γ, and TNF-α was analyzed and compared in iNK cells between the two groups.
To detect direct cytotoxicity of NK cells against target cells, a flow cytometry-based method was used [10,12]. In brief, 0.5 × 10 5 to 5 × 10 5 NK cells were cocultured with 5 × 10 4 carboxyfluorescein diacetate succinimidyl ester (CFSE)labeled cancer cells at various effector to target (E : T) ratios for 4 h. Samples were then stained on ice with 7-AAD for 10 min. After washing, target cell death was assessed with a flow cytometer by the percentage of 7-AAD-stained cells in the CFSE-positive population. To inhibit the activity of the perforin/granzyme system in NK cells, the co-culture killing assays were performed in the presence of 5/3 mM EGTA/Mg ++ . Target cells (CFSE + ) were gated, and the percent of 7-AAD + cells was used to calculate NK cell cytotoxicity using the following equation: (Experimental-Spontaneous dead cells/(100-Spontaneous dead cells) × 100%.

Statistical analysis
All analyses were performed with SPSS 25.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean AE standard error of the mean (SEM). ANOVA was used for the comparison of variables among multiple groups. Student t-test was used for comparison of variables between two groups. A P value < 0.05 was considered statistically significant.

Safety evaluation of cryoCB-iPSCs
We selected three cryoCB-iPSCs and analyzed the changes in average copies of total plasmids. Zero copies of the plasmid were detected in cells from passage 10 of the three cryoCB-iPSCs (Fig. 4A), suggesting that plasmids were depleted from these cells. Karyotype analysis was performed to evaluate the genomic stability of cryoCB-iPSCs at passage 15, and showed a normal karyotype (Fig. 4B), suggesting that long-term cultured cryoCB-iPSCs did not exhibit detectable chromosomal abnormalities.

HLAh CB in Tianjin Cord Blood Public Bank
To demonstrate the feasibility of generating a CBderived HLAh iPSC library, we investigated HLAh CB from the Tianjin Cord Blood Public Bank. We found that there were 16 homozygous CBs, including HLA-A, -B, -C, -DR, and -DQ. The genotype frequency of these 16 homozygous CBs was 6.5% in the Chinese population, covering 11.3% of the total Chinese population (Table 1)

Characteristics of iNK Cells
We first characterized iNK cells by examining the expression of surface markers. Compared to PB-NK and CB-NK cells, iNK cells had the expression of immunoglobulin-like receptors (KIRs), CD16, NKp46,  NKp44, CD94, NKG2D, and CD117 indicating that iNK cells can be generated from cryoCB-iPSCs using our EB differentiation method. In addition, we also found that iNK cells expressed lower levels of KIRs and CD16, compared to PB-NK and CB-NK cells (Fig. 5).

Immunogenicity of cryoCB-iPSC and iNK cells
We ES-H1 had a comparable expression of HLA-I (A, B, C) and HLA-II (DR; Fig. 6A). Compared to CB-NK and PB-NK cells, iNK cells expressed comparable levels of HLA-I (A, B, C) and lower levels of HLA-II (DR) (Fig. 6B). These results demonstrate that iNK cells have low immunogenicity and may become 'universal' NK cells.

Cytotoxicity of iNK cells against cancer cells
We investigated the cytotoxicity of the generated NK cells by co-culturing three types of NK cells, iNK, PB-NK, and CB-NK cells, with K562 cells (chronic myeloid leukemia cell line) at a ratio of 1 : 1, respectively, followed by flow cytometry analysis for staining of CD56-FITC, CD107a-APC, and IFN-γ-PE. The NK cells cultured without target cells were used as a control. Compared to CB-NK and PB-NK cells, iNK cells expressed higher levels of CD107a expression and TNF-α secretion, and lower levels of IFN-γ secretion (Fig. 7A-C).
We also evaluated the direct cytotoxicity against cancer cells by assessing cytotoxicity induced by NK cells on K562, MB-MDA-231 (breast cancer cell line), and Raji (lymphoma cell line). iNK cells were able to efficiently kill K562 cells and MB-MDA-231. However, PB-NK cells induced the highest toxicity on Raji cells compared with iNK or CB-NK cells (Fig. 7D).
To determine whether NK-induced cytotoxicity depends on cytotoxic granule release, cytotoxicity was measured in the presence or absence of the Ca ++ chelator EGTA, which inhibits cytotoxic granule release. EGTA completely blocked cytotoxicity, suggesting that degranulation was required (Fig. 7E).

Discussion
Natural killer cells have marked potential value in tumor immunotherapy due to characteristics such as non-MHC restriction and extensive tumor recognition [3,23]. However, the main problem hindering the clinical application of NK cells is the production of large numbers of NK cells required for therapeutic clinical applications [24,25]. Therefore, there is an urgent need for a method that can be used to mass-produce NK cells [5,25]. To address this unmet need we designed an approach to produce unlimited NK cells from cryoCB-iPSCs. Using an improved EB formation protocol, functional mature NK cells were produced from cryoCB-iPSCs. These iNK cells expressed NK cellspecific surface markers, exhibited cytotoxicity, but had less KIRs and HLA-DR. Therefore, we believe that this approach may be used to generate a large number of 'universal' functional NK cells, enabling wider clinical application of tumor immunotherapy. Currently, therapeutic NK cells are primarily derived from NK cell lines, PB and CB. In recent years, accumulating evidence has suggested that iPSCs are a promising source of NK cells [13,26]. For example, iPSC-derived NK cells have been used in several clinical studies to treat tumors [27]. Further, a large number of preclinical and clinical studies using autologous iPSCs-derived functional cells have been used to treat various diseases in regenerative medicine [28]. Notably, autologous iPSCs have been used to produce retinal pigment epithelial cells to treat age-related macular degeneration [29]. Unfortunately generating autologous iPSCs is expensive and labor-intensive which has limited the therapeutic value of this approach.
An alternative strategy is to provide iPSC products using universal HLAh donors. For this purpose, CB represents an excellent source to produce iPSCs compared with adult cells, such as PBMCs and skin fibroblasts [17]. Previous results have shown that iPSCs derived from adult tissues have higher levels of mitochondrial DNA mutations than those derived from CBMCs [30], suggesting an advantage of using CB cells to generate iPSCs. Moreover, the most common HLA CB can be selected from a CB bank to produce iPSCs for a wider range of applications. As previously reported, high-resolution HLA CB has been used to establish an iPSCs bank, and the 10 most common homozygous iPSC lines, matching 41.07% of the Korean population, have been characterized [18]. Taylor et al. [31] showed that 14 high-frequency homozygous iPSC lines could provide HLA-matched donors for 58.11% of the UK population. We assayed HLAh CB from the Tianjin Cord Blood Public Bank and found that there were 16 homozygous CBs, including HLA-A, -B, -C, -DR, and -DQ. The genotype frequency of the 16 homozygous CB was 6.5% in the Chinese population, matching 11.3% of the Chinese population.
KIRs, also known as CD158, are a group of transmembrane glycoproteins that are universally expressed on NK cells and are key regulators of NK cell cytotoxicity [32,33]. The levels of KIR expression on NK cells mediate the cytotoxicity of NK cells, and targeting KIRs has been shown to be a therapeutic approach to improve clinical outcomes. In addition, a  KIR-HLA mismatch donor has to be selected for patients to achieve optimal outcomes [34]. Previously, iPSCs derived from PBMCs were shown to generate KIR-negative NK cells, which may contribute to their clinical efficacy [12]. Consistent with the observations above, in the present study we also found that cryoCB-iPSCs were able to produce KIRs-negative iNK cells. In addition to the expression of low levels of KIRs, these iNK cells all expressed typical NK cell surface molecules. Therefore, we hypothesize that our method of using croCB-iPSCs to generate a large number of iNK cells is a viable alternative to the traditional methods which will enable additional clinical applications.
Inducing differentiation of iPSCs toward NK cells is a key step in the application of NK cells. One method is to produce CD34 + hematopoietic progenitor cells from iPSCs, then promote CD34 + hematopoietic progenitor cells to differentiate into NK cells [16,35]. That protocol is complex and reduces the yield of NK cells because of the exclusion of many other hematopoietic progenitors [16,35]. Another method is to form EBs, collect EBs, and then differentiate EBs into NK cells. The traditional method to generate EBs is to inoculate iPSCs with 3000 cells per well into a 96-well plate and then rotate it under appropriate conditions [36]. The generated EBs are then collected and distributed every six spins in one well of a 24-well plate, and differentiated into NK cells. Technically, this method is laborintensive and difficult to mass-produce NK cells for clinical applications [36]. In the present study, we established an improved protocol for EB formation by transferring cryoCB-iPSCs at a density of 8 × 10 5 cellsÁmL −1 to non-TC treated six-well plates with mTeSR™ 3D Medium. These cells were then incubated on a shaking table with a rotation speed of 70 r.p.m. at 37°C in a 5% CO 2 incubator for 4 days. Embryoid bodies were formed with approximately spherical shapes with a diameter of 150 _ 250 nm on the fourth day. Embryoid bodies were then transferred to non-TC treated six-well plates at a density of 300 _ 400 per well for differentiation into hematopoietic cells. This improved protocol is easy for large-scale production of NK cells, which we believe will meet the needs of various clinical applications.

Conclusion
We report a technique to generate NK cells, that is, 'from cryoCB to iPSCs, then back to NK cells', and characterized the resulting NK cells. We found that these iNK cells were similar to those generated from other sources with regard to surface marker expression and functions such as cytotoxicity. Our work demonstrates that this technique is a viable approach to massproducing NK cells for various clinical applications.