Transient characteristics of universal cells on human‐induced pluripotent stem cells and their differentiated cells derived from foetal stem cells with mixed donor sources

Abstract Introduction It is important to prepare ‘hypoimmunogenic’ or ‘universal’ human pluripotent stem cells (hPSCs) with gene‐editing technology by knocking out or in immune‐related genes, because only a few hypoimmunogenic or universal hPSC lines would be sufficient to store for their off‐the‐shelf use. However, these hypoimmunogenic or universal hPSCs prepared previously were all genetically edited, which makes laborious processes to check and evaluate no abnormal gene editing of hPSCs. Methods Universal human‐induced pluripotent stem cells (hiPSCs) were generated without gene editing, which were reprogrammed from foetal stem cells (human amniotic fluid stem cells) with mixing 2‐5 allogenic donors but not with single donor. We evaluated human leucocyte antigen (HLA)‐expressing class Ia and class II of our hiPSCs and their differentiated cells into embryoid bodies, cardiomyocytes and mesenchymal stem cells. We further evaluated immunogenic response of transient universal hiPSCs with allogenic mononuclear cells from survival rate and cytokine production, which were generated by the cells due to immunogenic reactions. Results Our universal hiPSCs during passages 10‐25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21‐22 survived and continued beating even after treatment with allogenic mononuclear cells.


| INTRODUC TI ON
Millions of people suffer from loss and injury of tissue and organs each year because of accidents, diseases and birth defects. Stem cells are an attractive source of cells for cell therapy. [1][2][3][4] Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), have the potential to differentiate into any of the cell types derived from the 3 germ layers. [5][6][7] However, the disadvantage of human ESC (hESCs) therapy is that large numbers of different types of hESCs with specific human leucocyte antigen (HLA) class I and class II types should be prepared and banked for therapy. This is because the hESCs used for transplantation should match the HLA type of the recipient, which generates an extremely high cost for hESC therapy; this will lead to a high cost in the National Insurance Budget once hESC therapy is covered by National Insurance in each country.
However, it takes time to generate mature hiPSCs, and hiPSCs require testing to verify that there is no gene abnormality and no contamination with viruses or other pathogens, which leads to a high cost of hiPSC therapy, with laborious and time-consuming processes to generate patient-specific hiPSCs from somatic cells from each patient. However, the hiPSCs derived from each patient do not generate immunogenicity-related problems in general after the transplantation of differentiated cells from these hiPSCs.
To reduce the high cost of preparation for hESCs and hiPSCs that match the HLA types of specific patients, the banking of hPSC lines, which are clinically approved, is proposed to provide a source of hPSCs. Although it has been reported that hESCs show relatively low expression levels of major histocompatibility complex (MHC, HLA class I and class II) proteins compared to those of normal somatic cells, [12][13][14] Taylor and colleagues suggested that the development of approximately 150 homozygous hESC lines would be necessary to provide sufficient HLA types to match hESC derivatives for most patients in the UK. 15, 16 Nakajima and his colleagues also suggested that 170 homozygous hESC lines would support HLA matching for 80% of patients in Japan (but not 100% of patients). 17 It is necessary to develop hPSCs that do not or less express HLA class Ia (HLA-A, -B and -C) and class II (universal or hypoimmunogenic hPSCs) even after differentiation into specific lineages of cells, as a single or few hPSC cell lines could theoretically be used to treat every patient using stem cell therapy. In this case, universal or hypoimmunogenic hPSCs should not or less express HLA class Ia and class II even after they are differentiated into several specific lineages, which are used for stem cell therapy.
However, it should be noted that hPSCs expressing no HLA class I such as the cells knocked out β2-microglobulin (an important component of HLA class I) leads to the missing-self response, which activate the lysing of the cells by natural killer (NK) cells. 15,[18][19][20][21][22][23] Therefore, several researchers developed (a) hPSCs where HLA-A, -B and/or -C expression were edited to be homozygous or knocked out, 18,20,[23][24][25] (b) hPSCs where HLA class Ia (HLA-A, HLA-B and HLA-C) was knocked out and HLA-E, HLA-G, PD-L1 and/or CD47 genes were knocked in 19,21 or (c) hPSCs, which express HLA-G, PD-L1, CTLA4-Ig and/or CD47 genes extensively 26,27 for universal or hypoimmunogenic hPSCs to be covered for any patient treatment with few cell lines of hPSCs. These universal or hypoimmunogenic hPSCs developed previously were all genetically modified, which makes laborious processes to evaluate and check no abnormal gene editing of hPSCs.
Here, we report generation method of universal hiPSCs from multiple sources of human amniotic fluids without gene editing, which have immune privilege from allogenic mononuclear cells containing CD8 + killer T cells, NK cells, CD4 + helper T cells and macrophages during passage 10-25 even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Our universal hiPSCs during passage 10-25 and their differentiated cells do not express HLA class Ia and class II and may theoretically be used to treat any patient using only 1 cell line in clinical application in future.

| Ethical statement
All experiments in this study were approved by the ethics committees of Cathay General Hospital (CGH-P108082), Taiwan Landseed Hospital (LSIHIRB 18-009-A2) and National Central University.
All animal procedures were performed in strict accordance to the Results: Our universal hiPSCs during passages 10-25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21-22 survived and continued beating even after treatment with allogenic mononuclear cells.
Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (National Central University, Jhongli, Taiwan). All experiments were performed in accordance with all relevant and applicable governmental and institutional guidelines and regulations.

| Materials
Human ESCs (H9, WiCell Research Institute, Inc) and hiPSCs (HPS0077, Riken BioResource Center) were used as control cell sources in this research. The materials used in this project are shown in Table S1. The other chemicals that were used in this research were obtained from Sigma-Aldrich.

| Isolation and culture of human amniotic fluid stem cells from amniotic fluid
Second-trimester amniotic fluid (AF) derived from a single donor (a) or second-trimester AF derived from multiple donors (2 or 5 pregnant women), which were mixed and stored for more than 2 days in 25°C (b), were used. The AF was centrifuged at 255 × g for 300 seconds, and the supernatant was discarded (Figure 1).
The AF cells (cell pellets) were suspended in medium composed of MCDB 201/DMEM (60%/40%) supplemented with 10 ng/mL FGF-2 and 20% foetal bovine serum (FBS) and cultivated on tissue culture polystyrene (TCPS) plates in a CO 2 incubator at 37°C to obtain human amniotic fluid stem cells (hAFSCs). 28 After reaching approximately 78%-82% confluence, the cells (hAFSCs) were F I G U R E 1 Outline of the process of reprogramming hAFSCs into transient universal (hypoimmunogenic) hiPSCs prepared from multiple AF donors (A) and conventional hiPSCs prepared from a single AF donor (B). The immunotolerance of transient universal (hypoimmunogenic) hiPSCs and their differentiated cardiomyocytes or embryoid bodies (EBs) is evaluated after treatment with mononuclear cells donated by different AF donors detached with a 0.25% trypsin-EDTA solution, centrifuged and inoculated into TCPS dishes according to a conventional passage procedure ( Figure 1). 28 hAFSCs at passage 3-4 were used for the following reprogramming processes.

| Isolation and culture of human adiposederived stem cells from human fat tissue
Human adipose-derived stem cells (hADSCs) were isolated from the fat pads from the omentum of 4 human patients (46-67 years old) after each patient provided his or her informed consent in writing. The adipose tissue cell solution (stromal vascular fraction; SVF) was obtained following a conventional method. 29,30 The cells in the adipose tissue cell solution were seeded in medium composed of DMEM supplemented with 10% FBS to obtain hADSCs and cultured for 4 passages. 29,30 The medium was changed every other day.

| Reprogramming of hAFSCs into hiPSCs
The CytoTune R -iPS 2.0 Sendai Reprogramming Kit that included 3 SeV-based reprogramming vectors was utilized for safe reprogramming from hAFSCs, which were obtained by mixing AF from 2-5 donors or a single donor, into hiPSCs in this study. The protocol for hiPSC generation by reprogramming of hAFSCs using Sendai virus vector followed the manufacturer's protocol. 31

| Generation of transient universal hESCs
Timeline for the generation method of transient universal hESCs is shown in Figure S7A. After obtaining informed consent, mononuclear cells were isolated from blood from volunteers using Ficoll-Paque solution and a conventional method. 32

| hPSC differentiation into cardiomyocytes
The hPSCs were differentiated into cardiomyocytes using a method developed by Sharma and colleagues 37 with some modifications. On day −4, hPSCs were inoculated into Matrigel-coated plates and cultured in Essential 8 medium until day 0 by exchanging the Essential 8 medium every day. On day 0, the hPSCs were approximately 80%-85% confluent. The Essential 8 medium was changed to Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 2 wt% B27 minus insulin and 6 μM CHIR99021 (a GSK-3β inhibitor). The hPSCs were incubated for 2 days. On day 2, the medium was changed to RPMI-1640 medium supplemented with 2 wt% B27 minus insulin, and the cells were incubated for 2 days. On day 4, the medium was changed to RPMI-1640 medium supplemented with 5 μM IWR-1 (a Wnt inhibitor) and 2 wt% B27 minus insulin, and the hPSCs were incubated for 2 days. On day 6, the medium was changed to RPMI-1640 medium supplemented with 2 wt% B27 minus insulin, and the hPSCs were cultured for 1 day. On day 7, the medium was changed to RPMI-1640 medium supplemented with 2 wt% B27. The hPSCs were cultured until day 18, with the medium replaced every other day.

| hPSC differentiation into mesenchymal stem cells
The hPSCs were differentiated into mesenchymal stem cells (MSCs) using a method developed by Li and colleagues 38 with some modifications. On day −4, hPSCs were inoculated into Matrigel-coated plates and cultured in Essential 8 medium until day 1 by exchanging the Essential 8 medium every day. On day 1, the hPSCs were approximately 80%-90% confluent. The Essential 8 medium was exchanged to Essential 6 medium supplemented with 10 ng/mL BMP4 and 1 μM/L A83-01. At day 3, the cells were washed with phosphate-buffered saline (PBS) and the medium was exchanged with MSC medium (αMEM containing 20% FBS). The cells were cultured in MSC medium for 10 passages. The medium was exchanged every other day.

| Survival and cytokine production of hPSCs and hPSC-derived cardiomyocytes after treatment with mononuclear cells
After obtaining informed consent, mononuclear cells were isolated from blood from male volunteers using Ficoll-Paque solution and a conventional method. 32,33 Mononuclear cells were inserted into each cell culture plate of hAFSCs, hESCs (H9), HPS0077 (hiPSCs) and universal hiPSCs (mix) (10 4 mononuclear cells/cm 2 on TCPS dishes with a 3.5 cm diameter), and the cells were cultivated for 2 days.
Cell survival was analysed with a live/dead staining kit following the manufacturer's recommended protocol. Live (green colour) and dead (red colour) cells were evaluated by fluorescence microscopy (Eclipse Ti-U inverted fluorescence microscope; Nikon Instruments, Inc). The cell survival rate of the cells was also analysed by flow cytometry after the cells were stained with 7-AAD. 32,33

| Statistical analysis
All of the quantitative results were obtained from 4 samples. The data were presented as the means ± standard deviations (SDs).
Statistical analysis was performed utilizing unpaired Student's t tests in Excel (Microsoft Corporation). Probability values (P) less than .05 were regarded as statistically significant.

| HLA class Ia and class II expression of several human stem cells and cancer cells
To prepare universal or hypoimmunogenic hiPSCs, we hypothesized that hiPSCs derived from some cell lines, such as foetal cells, for example, hAFSCs, might show some characteristics of universal hiP-SCs. Therefore, the HLA class Ia and class II expression of several cell lines that we have stocked in our laboratory, such as hADSCs, LoVo cells, HT29 cells, SW480 cells, CoLo205, hESCs (H9), hiPSCs derived from fibroblasts (HPS0077) and hAFSCs, was investigated using flow cytometry to evaluate the potential use of these cells as mother cells to generate hiPSCs ( Figure 2). hESCs and hiPSCs showed no HLA class II expression but expressed HLA class Ia, as reported by several other researchers. [12][13][14] Furthermore, the differ-

| Generation of transient universal hiPSCs
We generated transient universal hiPSCs from hAFSCs derived from multiple sources of AF, with AF from several pregnant women (2)(3)(4)(5) women) mixed to generate hAFSCs [hAFSCs (mix)] ( Figure 1). We hypothesized that not all of the cells among the foetal stem cells express HLA class Ia and class II after reprogramming into hiPSCs.
Furthermore, we expected that hAFSCs, which do not express HLA class Ia and class II after reprogramming, can be selected by treatment with AF derived from other donors and/or mononuclear cells that are contained in the AF derived from other donors.
We succeeded in generating hiPSCs using (a) hAFSCs derived from mixed AF [hiPSCs (mix)] and (b) hAFSCs from AF from a single donor [hiPSCs (single)] (Figures 3 and S1-S4). We generated 2 types of transient universal hiPSCs, which were prepared using 2 different AF donors (hiPSCs (mix-2)) and 5 different AF donors (hiP-SCs (mix-5)); these hiPSCs were generated from the transfection of pluripotency-related genes (Yamanaka factors) using the Sendai virus vector. The hiPSC colonies were typically observed 7-10 days after transfection of pluripotency-related genes (Figures 3(B), S1B and S2B). The hiPSCs generated from hAFSCs derived from mixed AF and from single AF expressed pluripotency-related proteins such as Oct3/4, Sox2, Nanog and SSEA-4 (Figures 3(C), S1C, S2C and S3), as indicated by immunostaining evaluation of pluripotency-related proteins. The hiPSCs had the ability to differentiate into cells derived from 3 germ layers in vitro (Figures 3(D), S1D, S2D and S4) and in vivo (Figures 3(E), S1E, and S2E), which are the main characteristics of hPSCs. We also evaluated karyotyping of the transient universal hiPSCs (mix-5), and the results are shown in Figure 3(F).
The karyotypes of the cells were found to be normal, suggesting that there were no genetic abnormalities on the cells during their

| Differentiation of mixed AF-derived hiPSCs into cardiomyocytes and their HLA class I and class II expression
The hiPSCs (mix) at passage 21-22, which were derived from mixed AF from different women, and hiPSCs (single) at passage 20 from a single AF donor were induced to differentiate into cardiomyocytes (Figures 4 and S5). We used this approach because we wanted to evaluate the expression of HLA class Ia and class II after differentiation of our transient universal hiPSCs into a specific lineage of cells. , an ectodermal protein (iii, GFAP, red) and an endodermal protein (iv, AFP, green) in the cells shown by immunostaining, with nuclear staining with Hoechst 33342 (ii, blue), after culturing for 21 passages. The scale bar indicates 500 μm (i) and 100 μm (ii-iv). E, A teratoma was formed by injecting transient universal hiPSCs (mix-5) cultured on recombinant vitronectin-coated dishes after 22 passages (i). Tissues including the gland duct consisting of the columnar epithelium (ii, endoderm), cartilage (iii, mesoderm) and immature neuroepithelium (iv, ectoderm) can be observed. The scale bar indicates 100 μm (ii) and 200 μm (iii, iv). F, Karyotyping of universal hiPSC (mix-5) after culturing for 21 passages. G, Dependence of HLA class Ia expression of hiPSCs (single) (closed circle), hiPSCs (mix-2) (closed square) and hiPSCs (mix-5) (closed triangle) after reprogramming from hAFSCs on passage of the cells Cardiomyocytes derived from hiPSCs (mix) and hiPSCs (single) expressed the cardiac-specific proteins MLC2a, cTnT and α-actinin ( Figures 4C,D and S5C). Beating cells were also obtained (Video S1 (cardiomyocytes derived from hiPSCs (mix-2)) and Video S2 (cardiomyocytes derived from hiPSCs (mix-5)). We evaluated the HLA class Ia and class II expression of hAFSCs (mix), hAFSCs (single), hiP-SCs (mix), hiPSCs (single) and cardiomyocytes derived from hiPSCs (mix) and hiPSCs (single) as well as that of hESC (H9)-derived cardiomyocytes and hiPSC (HPS0077)-derived cardiomyocytes at day 20 of the differentiation and the results are shown in Figure 5. As we expected, cardiomyocyte-derived hiPSCs (mix) at day 20 of the differentiation as well as undifferentiated hiPSCs (mix) at passage 20 did not show HLA class Ia and Class II expression ( Figure 5C,D).
On the other hand, hiPSCs (single) expressed distinct HLA class Ia, and cardiomyocytes derived from hiPSCs (single) also showed HLA class Ia expression ( Figure 5B). We found that the mixing of AF from different donors to generate hAFSCs is a key technique to generate transient universal hiPSCs. In particular, we succeeded in generating transient universal hiPSCs by mixing AF from a minimum of 2 different pregnant women, as hiPSCs (mix-2) and cardiomyocytes differentiated from hiPSCs (mix-2) did not express HLA class Ia and class II (Figure 5C,D).

| Differentiation of transient universal hiPSCs into MSCs and their HLA class Ia and class II expression
We further differentiated hiPSCs (mix-5) at passage 25 into MSCs, which expressed MSC markers of CD44, CD73, CD90 and CD105 after differentiation into MSCs at passage 5 ( Figure S6). MSCs derived from hiPSCs (mix-5) at passage 25 also did not show HLA class Ia and class II. for EBs derived from hESCs (H9) and hiPSCs (HPS0077) ( Figure 6F). Therefore, the cells derived from 3 germ layers (endoderm, mesoderm and ectoderm) in EBs and cardiomyocytes, which were differentiated from hiPSCs (mix) as well as undifferentiated hiPSCs (mix), were found to have universal characteristics from these results during specific passages (10-25 passages).

| No immunogenic response of transient universal hiPSCs with allogenic mononuclear cells
The survival rate under each condition was evaluated using flow cytometry after staining the cells with 7-AAD, and the results are shown in Figure 7A. The survival rate was found to be almost Surprisingly, the cardiomyocytes differentiated from hiPSCs (mix) continued beating even after treatment with the mononuclear cells for more than 5 days (Videos S3 and S4 for cardiomyocytes derived from hiPSCs (mix-2) and hiPSCs (mix-5), respectively, after treatment with mononuclear cells), whereas the cardiomyocytes differentiated from conventional hESCs (H9), hiPSCs (HPS0077) and hiPSCs (single) stopped beating after treatment with the mononuclear cells.
We also evaluated cytokines generated by the cells due to immunogenic reactions between hESCs or hiPSCs and mononuclear cells derived from individuals different than those from whom the hESCs and hiPSCs originated. Figure 7B,C show interferonγ and interleukin 6 production, respectively, in the cell culture medium before and after the addition of mononuclear cells.

| D ISCUSS I ON
We hypothesize that any AF contains few 'hypoimmunogenic or universal hAFSCs' and many typical cells that are not hypoimmunogenic or universal hAFSCs and that hypoimmunogenic or universal hAF-SCs do not express HLA class Ia and class II transiently after reprogramming of hypoimmunogenic or universal hAFSCs into hiPSCs as well as after the differentiation of transient universal hiPSCs. If this hypothesis is correct, the key point is how to select these 'hypoim- To verify our idea, conventional hESCs (H9, 10 5 cells) were treated with allogenic mononuclear cells (10 5 cells) for several times (1-3 times) ( Figure S7). HLA class Ia expression was gradually decreased with increase of mononuclear cell treatment where hESCs and mononuclear cells were incubated for 2 days in each mononuclear cell treatment ( Figure S7B). hESCs after treated with mononuclear cells more than twice did not show HLA class Ia and class II (hypoimmunogenic or universal hESCs). Therefore, we verified that conventional hESCs contain hypoimmunogenic or universal hESCs, which do not show HLA class Ia and class II and we can isolate hypoimmunogenic or universal hESCs by allogenic mononuclear cell treatment in this study. However, it should be noted that hESCs treated with mononuclear cells start to express HLA class Ia after the passage without treatment of mononuclear cells. Therefore, this phenomenon is also found to be a transient issue.
Several researchers have considered developing universal or hypoimmunogenic hPSCs that do not express HLA Class Ia and Class II. [18][19][20][21][23][24][25][26][27][40][41][42][43][44] However, all of these methods except the method presented in our present study require knocking out, knocking in or editing specific genes, which indicate the need for genetic mod- It is known that hPSCs expressing no HLA class I such as hPSCs knocked out β2-microglobulin lead to the missing-self response, which activate the lysing of the cells by NK cells. [18][19][20][21]23 Several researchers knocked in HLA-E, HLA-G, PD-L1 and/or CD47 genes to escape NK cell responses. 19,21 Han et al knocked out β-2 microglobulin to prepare hESCs expressing no HLA type and knocked in PD-L1 and HLA-G on the AAVS1 safe harbour locus, which are immunomodulatory factors to escape NK cell response. 19 They further knocked in CD47 genes, which is 'donot-eat me' signal for macrophage on the AAVS1 safe harbour locus.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.