Induction of integration-free human-induced pluripotent stem cells under serum- and feeder-free conditions
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Human-induced pluripotent stem cells (hiPSCs) have shown great potential toward practical and scientific applications. We previously reported the generation of human dental pulp stem cells using non-integrating replication-defective Sendai virus (SeVdp) vector in feeder-free culture with serum-free medium hESF9. This study describes the generation of hiPSCs from peripheral blood mononuclear cells to increase the donor population, while reducing biopsy invasiveness. From 6-d-old primary culture of peripheral blood mononuclear cells (PBMCs) with IL-2, hiPSCs were established using SeVdp(KOSM)302L with recombinant Laminin-511 E8 fragments under serum-free condition. The established PBMC-derived hiPSCs showed pluripotency and differentiation ability both in vivo and in vitro. In addition, we evaluated microarray data from PBMC- and dental pulp–derived hiPSCs. These hiPSCs will be beneficial for characterizing the molecular mechanisms of cellular differentiation and may provide useful substrates for developing cellular therapeutics.
KeywordsHuman-induced pluripotent stem cells Peripheral blood mononuclear cells Reprogramming efficiency hESF9 serum-free defined media
Human-induced pluripotent stem cells (hiPSCs) (Takahashi and Yamanaka 2006) research holds tremendous potential for regenerative medicine, drug discovery, and disease modeling with the added advantage of circumventing the ethical concerns regarding the use of pluripotent cells derived from embryos. Ever since Yamanaka and colleagues established a cell bank of donor-derived hiPSCs, rather than making them for each patient (Andrews et al.2014), the demand for high-quality hiPSCs has risen significantly.
hiPSCs are typically induced and cultured from feeder cells in serum-containing medium in conventional culture systems. Murine-derived feeder cells are widely used to maintain the pluripotency of hiPSCs, and human-derived feeder cells are also used. However, these cells are unsuitable for stem cell maintenance (Price 2017) as the feeder cells are cultured in medium supplemented with fetal bovine serum or proprietary serum replacements. The use of culture medium containing undefined or unknown components has limited the understanding of development and cellular differentiation (Nims and Harbell 2017).
DNA-integrative retroviral and lentiviral vectors, first described by Yamanaka (Takahashi and Yamanaka 2006), have been used widely in cell reprogramming because they stably express transgenes, resulting from the chromosomal insertion of the vector (Takahashi et al.2007) (Yu et al.2007) (Lowry et al.2008). However, the therapeutic potential of hiPSCs is complicated by the potential risks posed by continuous expression of transgenes and by random genome integration of viral vectors. Recently, a number of procedures have been introduced to generate genetically non-integrative or unmodified hiPSCs. These approaches involve chemicals or plasmid, episomal, or viral vectors (Kaji et al.2009) (Woltjen et al.2009) (Zhou et al.2009) (Jia et al.2010) (Warren et al.2010) (Yu et al.2011); however, they show extremely low efficiency in generating iPSCs.
To prevent the risk of contaminating hiPSCs with unknown viruses, unknown substances, and unpredictable genome insertions, as well as to standardize a culture method under defined conditions, we previously developed and reported culture systems capable of maintaining both the undifferentiated status and pluripotency of embryonic stem cells (ESCs) and iPSCs without using serum or feeder cells or retroviruses from dental pulp cells (DPCs) (Yamasaki et al.2016), eliminating the risk of activating nearby oncogenes by gene insertions. To further simplify the generation of hiPSCs and to increase the donor population, we report here the generation of hiPSCs using peripheral blood mononuclear cells (PBMCs) from seven healthy adult donors of various ages as sources of hiPSCs. This procedure was carried out without invasive and painful biopsies.
Based on our preliminary experimental results, we performed hiPSC generation from PBMCs using Laminin-E8 in completely serum-free culture conditions after 6 d of primary culture. The system can maintain the pluripotency of the cells and retain their potential to differentiate into all three embryonic germ layers, as well as DPC-derived hiPSCs (Yamasaki et al.2016). We also compared microarray data from PBMC- and DPC-derived hiPSCs. This culture system will be beneficial for evaluating the molecular mechanisms under defined conditions. Furthermore, the combination of this system and the generation of patient-derived iPSCs will contribute to attaining new knowledge to overcome various disorders.
Materials and Methods
The list of hiPSC cell lines. Hamada et al.
Name of cell line
Isolation and culture of PBMCs in serum-free medium
We obtained human blood samples from healthy volunteers at Hiroshima University Hospital for the use of the blood to generate hiPSCs in accordance with approved guidelines. PBMCs were prepared by density gradient centrifugation in a Histopaque 1077 (Sigma-Aldrich) and cultured in RD6F serum-free medium supplemented with IL-2 (CELEUK, Takeda Pharmaceutical Co., Osaka, Japan) (Sato et al.1987; Okamoto et al.1996) for 0–6 d at 37°C in a humidified atmosphere of 95% air/5% CO2.
Induction of PBMC-hiPSCs with SeVdp(KOSM)302L
ALP staining was performed using a Fast Red substrate kit (Nichirei Biosciences, Inc., Tokyo, Japan) according to the manufacturer’s protocol. Images of the dish were acquired using LUMIX (Panasonic, Osaka, Japan) and positive areas were detected using ImageJ software (National Institutes of Health, Bethesda, MD).
In vitro and in vivo differentiation of PBMC-hiPSCs
The in vitro and in vivo differentiation of PBMC-hiPSCs was performed as described previously (Yamasaki et al.2014). Briefly, to confirm the in vitro differentiation capacity of PBMC-hiPSCs, an embryoid body assay was performed. Undifferentiated PBMC-hiPSCs were cultured in hESF6 without FGF2, heparin, and TGF-b1 or activin A in low-attachment 96-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) for 4–5 d. Then, 3–5 embryoid bodies (EBs) were transferred to gelatin-coated 35-mm dishes and further cultured for another 21 d in hESF6. The medium was changed every 3–5 d. The cells were fixed and stained with the antibodies shown in Table 1. For in vivo studies, PBMC-hiPSCs were injected into the dorsal flank of SCID (CB17/Icr-Prkdcscid/CrlCrlj) mice (1 × 106 cells/100 μL of the cell suspension). Approximately 10 wk after injection, the teratoma were surgically dissected, fixed using phosphate-buffered saline solution containing 4% formaldehyde, and embedded in paraffin. Then each section was stained with hematoxylin/eosin and Alcian blue/PAS. The histological findings were evaluated using a Nikon ECLIPSE E800 microscope (Tokyo, Japan) and photographed using a Leica DC500 camera (Leica Microsystems AG, Wetzlar, Germany).
RNA isolation and reverse transcription gene expression
The List of primer sequences for RT-PCR
Product size (bp)
Yamanaka S. et al.
Yamanaka S. et al.
Yamanaka S. et al.
Yamanaka S. et al.
Nakanishi K. et al.
Furue MK. et al.
To examine the pluripotency of the PBMC-hiPSCs, immunocytochemistry was performed as described previously (Yamasaki et al.2014). Briefly, the cells fixed with 4% paraformaldehyde were stained with antibodies against Oct4 (MAB4401, mouse monoclonal, 1/200, Millipore), Tra-1-60 (09-0010, mouse monoclonal, 1/200, Stemgent®, Cambridge, MA), and SSEA-4 (MC 813-70, mouse monoclonal, 1/100, R&D Systems Minneapolis, MN), and differentiated cells were stained with antibodies against βIII-tubulin (MAB3408/1637, mouse monoclonal, 1/300, Chemicon, Burlington, MA), α-smooth muscle actin (N1584, mouse monoclonal, pre-diluted, DAKO Cytomation, Glostrup, Denmark), and α-fetoprotein (MAB1368, mouse monoclonal, 1/100, R&D Systems) (Table 1). These primary antibodies were visualized with Alexa Fluor® 488-conjugated goat anti-mouse IgG (A11001, 1/300, Invitrogen, Carlsbad, CA). The cell nuclei and double-stranded DNA were stained with 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescence images were acquired using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Oberkochen, Germany).
Gene Expression Microarray (Agilent Technologies) was performed at Hokkaido System Science Co. Briefly, after confirming the quality of the RNA samples with the Agilent 2100 Bioanalyzer (G2940CA), microarray analysis 100 ng of total RNA was amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit, One-Color (5190-2305), and labeled RNA was hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (G4851A). Agilent Feature Extraction Image Analysis Software (Version 10.7.3) was used to extract data from raw microarray image files. Data visualization and analysis were performed using GeneSpring GX (Version 11.0) software.
DNA isolation and Short Tandem Repeat analysis
The patient’s genomic DNA was isolated from PBMCs and PBMC-hiPSCs using a QIAamp® DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Genomic DNA was used for PCR with Powerplex 16 system (Promega Corporation, Madison, WI) and analyzed by ABI PRISM 3100 Genetic analyzer and Gene Mapper v3.5 (Applied Biosystems)
Generation of integration-free PBMC-hiPSCs
Characterization of hiPSCs
Next, the gene expressions of PBMC-hiPSC isolated under different culture conditions were analyzed. WT4-PBMC iPSC induced in hESF9 displayed expression pattern more similar to that of WT3-PBMC hiPSC induced in hESF9 than that of WT4-PBMC hiPSC induced in serum-supplemented condition (Fig. 9). In other words, the gene expression pattern of PBMC-hiPSCs established under serum-free conditions is more similar to that of PBMC-hiPSCs established under serum-free conditions from another individual than that of those established under serum-supplemented conditions from the same individual. In summary, differences in culture conditions are concluded to have a greater effect on gene expression differences than differences in individuals from whom the cells were sourced.
We previously reported induction of hiPSCs from fibroblasts derived from gingiva or DPCs derived from extracted teeth (Yamasaki et al.2016; Yamasaki et al.2014). However, the extraction of skin, mucous, or dental pulp involves surgical procedure, and, in particular, target patients may not have any teeth for dental pulp extraction. Since our eventual goal is to establish a general method to induce hiPSCs from genetic disorder patients, the donor population must be bigger. Hence, it is important that we improve hiPSC induction procedure by reducing biopsy invasiveness. However, the induction efficiency of hiPSCs from PBMCs is extremely low compared to that of skin fibroblasts using conventional induction methods (Staerk et al.2010). We induced PBMC-hiPSCs multiple times using conventional methods but failed to obtain positive results. The SeVdp(KOSM)302L non-integrating viral vector used in this report encodes all four reprograming factors (Klf4, Oct3/4, Sox2, c-Myc) to achieve efficient factor expression. By incorporating this expression vector into a reprogramming protocol that included serum-free and feeder-free culture conditions, we induced and successfully recovered hiPSCs from PBMCs. The efficiency of hiPSC induction from PBMCs was approximately one half to one third of that from DPCs, but was sufficiently high for routine use. Induction of PBMC-hiPSCs has been reported using other viral expression vectors in combination with feeder cells in serum-supplemented medium. Brown et al. (2010) reported an induction efficiency of hiPSCs using retrovirus of 0.01%, and Loh et al. (2009) reported an induction efficiency of 0.0008–0.001% using lentiviruses. Nakagawa et al. (2014) used an episomal vector under serum-free culture conditions and reported an induction efficiency of 0.001–0.011%. Kishino et al. (2014) reported PBMC-hiPSC induction under serum-free conditions using Sendai virus vectors carrying one gene per vector, with an induction efficiency of 0.005%. Trokovic et al. (2014), using serum-free conditions, also reported an induction efficiency of 0.005%. In each of these cases, the efficiency of inducing PBMC-hiPSCs was extremely low as compared to the results of the present study in which induction efficiency was 0.008–0.1% using SeVdp(KOSM)302L carrying four genes in one vector.
Previously, we examined a variety of extracellular matrix molecules (ECMs) including fibronectin, type I collagen, and gelatin for their suitability for DPC-hiPSC induction and confirmed that fibronectin was the most suitable (Yamasaki et al.2014). Prior to this study, we re-examined PBMC-hiPSC induction by adding Laminin-E8 to previously characterized ECMs under serum-free condition. We successfully generated hiPSCs on all ECMs; in particular, colonies appeared more rapidly and the induction efficiency was higher in the presence of Laminin-E8 (supplement data). While Matrigel is also widely used in hiPSC induction protocols as ECM under serum-free medium, its use is potentially problematic because Matrigel contains many undefined factors that may affect cell growth and differentiation. Laminin is the main component of Matrigel and Laminin-E8 is the minimum fragment conferring integrin-binding activity. Miyazaki have reported that Laminin-E8 has the potential to promote the survival of hiPSCs by robust adhesion via integrins and phosphorylation of AKT, ERK1/2, and FAK, which are highly phosphorylated in human pluripotent stem cells (Miyazaki et al.2012). Thus, Laminin-E8 was adopted as ECM for our system.
Prior to inducing hiPSCs from blood cells, granulocyte macrophage colony-stimulating factor (GCSF) was added to stimulate the proliferation of specific T cells, B cells, and granulocytes using cytokines, such as stem cell factor and IL-3 (Loh, et al., 2009; Seki, et al., 2010). Based on a culture method similar to that used to activate lymphocytes for cell therapy performed on patients with oral cancer in our department, natural killer (NK) cells and cytotoxic T cells with strong cytotoxic activity were stimulated to proliferate with IL-2 in this study. By inducing hiPSCs from these cells, we also aimed to differentiate hiPSCs into cells with high cytotoxic activity for cell therapy. When inducing hiPSCs from PBMCs in RD6F medium containing IL-2, considerable individual differences were observed in induction efficiencies. This tendency was marked in reprogrammed cells cultured on MEF feeder cells. This suggests that lymphocytes with high cytotoxic activity attacked non-autologous mouse feeder cells or self-lymphocytes transfected with Sendai virus, resulting in reduced induction efficiency. Supporting this hypothesis, differences in marker gene expression determined by DNA microarray between individuals with the highest and lowest induction efficiencies revealed that individuals with lower induction showed higher expression of cytotoxic activity-related genes. Although hiPSC induction was carried out using activated lymphocytes with high cytotoxic activity with the goal of future cell therapy, when aiming to induce hiPSCs from PBMCs with high efficiency, induction should be performed immediately after isolation without IL-2 stimulation. In addition, the potential problems associated with isolating hiPSC from PBMC on mouse feeder cells can be avoided by using our feeder-free and serum-free culture method. Finally, our reprogramming protocol that does not use an integrating viral vector is preferable for generating hiPSCs for clinical applications.
Our defined culture system is advantageous for studying the regulation of cell differentiation and for generating and maintaining pluripotent cells for clinical applications. The combination of this system and the generation of patient-derived iPSCs will contribute to providing us with new knowledge to overcome various disorders. Furthermore, disease-relevant cells differentiated from patient-derived iPSCs will become a powerful tool for investigating pathogenesis in vitro and accelerate the development of effective therapies.
Here, we described the successful generation of PBMC-hiPSCs under serum- and feeder-free conditions. These PBMC-hiPSCs will be helpful for clinical applications and drug discovery. The ability to establish hiPSCs efficiently from PBMCs with SeVdp(KOSM)302L will enable the generation of normal and disease-specific hiPSCs from healthy people and patients with rare genetic disorders, thus facilitating studies of the mechanisms of action disease onset.
We gratefully acknowledge the work of past and present members of the Department of Molecular Oral Medicine and Maxillofacial Surgery at Hiroshima University. The authors sincerely thank Dr. J. Denry Sato, Professor Akira Shimamoto, and Professor DDS Chisa Shukunami for the helpful discussions. This research was supported in part by Grants-in-Aid for Scientific Research (B) to T. O. (grant numbers: 18H03000), Grant-in-Aid for Young Scientists (B) to S. Y. (16K20580), and Grant-in-Aid for Research Activity Start-up to A. H. (15H06435) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. H. N. was supported in part by the Hiroshima University Phoenix Leader Education Program (Hiroshima Initiative) for the “Renaissance from Radiation Disaster”, funded by the Ministry of Education, Culture, Sports, Science and Technology.
Conceived and designed the experiments: AH, EA, SY, and TO. Performed the experiments: AH, EA, HN, and FO. Analyzed the data: AH, EA, HN, ST, and TO. Contributed reagents/materials/analysis tools: AH, EA, SY, MO, KN, MN, and TO. Wrote the paper: AH and TO.
Compliance with ethical standards
This study was approved by the Ethics Committee of Human Genome/Gene Analysis Research at Hiroshima University (approval number: hi-58, hi-72). All animal experiments in this study strictly followed a protocol approved by the Institutional Animal Care and Use Committee of Hiroshima University (approval number: A-11-140).
- Hayashi Y, Chan T, Warashina M, Fukuda M, Ariizumi T, Okabayashi K, Takayama N, Otsu M, Eto K, Furue MK et al (2010) Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions. PLoS One 5:e14099CrossRefGoogle Scholar
- Nishimura K, Ohtaka M, Takada H, Kurisaki A, Tran NVK, Tran YTH, Hisatake K, Sano M, Nakanishi M (2017) Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302. Stem Cell Res 23:13–19CrossRefGoogle Scholar
- Nishimura K, Sano M, Ohtaka M, Furuta B, Umemura Y, Nakajima Y, Ikehara Y, Kobayashi T, Segawa H, Takayasu S, Sato H, Motomura K, Uchida E, Kanayasu-Toyoda T, Asashima M, Nakauchi H, Yamaguchi T, Nakanishi M (2011) Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming. J Biol Chem 286:4760–4771CrossRefGoogle Scholar
- Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7:618–630CrossRefGoogle Scholar
- Yamasaki S, Hamada A, Akagi E, Nakatao H, Ohtaka M, Nishimura K, Nakanishi M, Toratani S, Okamoto T (2016) Generation of cleidocranial dysplasia-specific human induced pluripotent stem cells in completely serum-, feeder-, and integration-free culture. In Vitro Cell Dev Biol Anim 52:252–264CrossRefGoogle Scholar
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