Journal of Animal Reproduction and Biotechnology 2023; 38(4): 275-290
Published online December 31, 2023
https://doi.org/10.12750/JARB.38.4.275
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Sang-Ki Baek1,#,§ , In-Won Lee1,3,#
, Yeon-Ji Lee1,3
, Bo-Gyeong Seo2,3
, Jung-Woo Choi4
, Tae-Suk Kim1
, Cheol Hwangbo3
and Joon-Hee Lee1,5,*
1Department of Animal Bioscience, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea
2Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju 52828, Korea
3Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Korea
4College of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea
5Institute of Agriculture & Life Science, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea
Correspondence to: Joon-Hee Lee
E-mail: sbxjhl@gnu.ac.kr
#These authors contributed equally to this work.
§Current affiliation: Gyeongsangnamdo Livestock Experiment Station, Sancheong 52263, Korea
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Porcine pluripotent stem cells (pPSCs) would provide enormous potential for agriculture and biomedicine. However, authentic pPSCs have not established yet because standards for pPSCs-specific markers and culture conditions are not clear. Therefore, the present study reports comparative pluripotency characteristics in porcine induced pluripotent stem cells (piPSCs) derived from different viral transduction and reprogramming factors [Lenti-iPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM)].
Methods: Porcine fibroblasts were induced into Lenti-iPSCs (OSKM) and Lenti-iPSCs (OSKMNL) by using Lentiviral vector and Sev-iPSCs (OSKM) by using Sendaiviral vector. Expressions of endogenous or exogenous pluripotency-associated genes, surface marker and in vitro differentiation in between Lenti-piPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-piPSCs (OSKM) were compared.
Results: Colonial morphology of Lenti-iPSCs (OSKMNL) closely resembles the naïve mouse embryonic stem cells colony for culture, whereas Sev-iPSCs (OSKM) colony is similar to the primed hESCs. Also, the activity of AP shows a distinct different in piPSCs (AP-positive (+) Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but AP-negative (-) Lenti-iPSCs (OSKM)). mRNAs expression of several marker genes (OCT-3/4, NANOG and SOX2) for pluripotency was increased in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but Sev-iPSCs (OSKM). Interestingly, SSEA-1 of surface markers was expressed only in Sev-iPSCs (OSKM), whereas SSEA-4, Tra-1-60 and Tra-1-81 were positively expressed in Lenti-iPSCs (OSKMNL). Exogenous reprogramming factors continuously expressed in Lenti-iPSCs (OSKMNL) for passage 20, whereas Sev-iPSCs (OSKM) did not express any exogenous transcription factors. Finally, only Lenti-iPSCs (OSKMNL) express the three germ layers and primordial germ cells markers in aggregated EBs.
Conclusions: These results indicate that the viral transduction system of reprograming factors into porcine differentiated cells display different pluripotency characteristics in piPSCs.
Keywords: induced pluripotent stem cells, Lenti-viral, pluripotency, porcine, Sendai-viral
A new type of embryonic-like stem cells had been derived from induced pluripotent stem cells (iPSCs) by reprogramming differenced somatic cells with defined factors. These cells have the self-renewal capacity that proliferates indefinitely and pluripotency that differentiates potentially into other cell types (Romito and Cobellis, 2016), and allow for patient-specific stem cells to be produced because of avoidance of rejection of their derivatives by immune system (Park et al., 2008a). Mouse iPSCs were first produced by a retrovirus vector transduction of
The reprogramming procedures developed in mouse and human have been adapted to an increasingly demand of specific specie, pig, which has an excellent resource for disease modeling, drug discovery and regenerative medicine (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). The derivation of iPSCs by using the Japanese group reprogramming procedure yields either naïve or primed stemness states, depending on the species. For instance, mouse iPSCs generally have the properties of naïve state like embryonic stem cells (ESCs), while human iPSCs so far reported have features of the primed state like epiblast stem cells (EpiSCs). Interestingly, the colonies derived from porcine iPSCs resemble human ESCs rather than mouse ESCs in morphology. Porcine iPSCs like human ESCs are totally dependent upon on basic fibroblasts growth factor (bFGF) and Activin/Nodal signaling pathway for maintaining self-renewal and pluripotency (Dahéron et al., 2004; Vallier et al., 2009; Alberio et al., 2010). On the contrary, mouse ESCs are dependent on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP-4) to maintain the undifferentiated state (Smith et al., 1988; Ying et al., 2003).
Porcine pluripotent stem cells may be especially valuable because the pig is a prime biomedical model for tissue and organ transplantation. Among domestic animals, induced pluripotent stem cells (iPSCs) have been first successfully generated from swine (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). It was clearly evidenced that the porcine iPSCs isolated have an ability to differentiate into tissue types reflective of the three germ layers (endoderm, mesoderm and ectoderm), spontaneously within either embryoid bodies or teratomas (Ezashi et al., 2012). However, it remains unclear whether or not the continued expression of reprogramming genes complicated directed differentiation along specific lineages, or if the protocols and reagents used have not been optimized for pig. Some discrepancies relating to whether or not the lines express the surface molecules stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) and the tumor rejection antigens (Tra 1-60 and Tra 1-81) have been reported. Mouse and human ESCs slightly differ in their display of cell surface antigens. For example, SSEA-1 is expressed on mESCs but it is absent on hESCs, and different surface markers (SSEA-3 and SSEA-4) are displayed on the human pluripotent stem cells (Henderson et al., 2002).
Reprogramming factors needed to make porcine iPSCs were introduced by infecting the PFFs with non-integrative methods [plasmids, Sendai virus, synthetic mRNAs and recombinant proteins] (Telugu et al., 2010). The delivery of transgenes using viral vectors, which are stably expressed, is considered the most useful tool for inducing low cytotoxicity and inserting transgenes into the host genome (Zhang and Godbey, 2006). Porcine induced pluripotent stem cells (piPSCs) generated using lentiviral vectors, often referred to as lentivirus-mediated reprogramming, involve the use of lentiviruses as a vehicle to introduce reprogramming factors into somatic cells, ultimately leading to the generation of piPSCs. This method is a variation of the commonly used induced pluripotent stem cell (iPSC) generation technique and is utilized for creating piPSCs. The Sendai virus infects the target cells and delivers the reprogramming factors into the cell’s cytoplasm. Importantly, the Sendai virus does not integrate its genetic material into the host cell’s DNA. Various lines of evidence indicate that efficient cell reprogramming requires the sustained and simultaneous expression of several exogenous factors at least 10-20 days (Jaenisch and Young, 2008). After reprogramming has been completed, these exogenous factors should be replaced promptly with their endogenous counterparts if the cells are to acquire autoregulated pluripotency (Jaenisch and Young, 2008).
Here we have transformed porcine fetal fibroblasts (PFFs) into iPSCs by delivering transcription factors with integrating (lentiviral transfection) and non-integrating (Sendai virus infection) vectors. Therefore, we have demonstrated that porcine iPSCs generated by infecting the PFFs with non-integrative method (Sendai virus infection) might carry less of an exogenous transgenes expression than porcine iPSCs generated by infecting the PFFs with integrating (lentiviral transfection) vectors.
All chemicals and reagents were purchased from SigmaAldrich Co. (St. Louis, MO, USA) unless otherwise mentioned. And all materials and methods were performed accordingly, as previously described by Lee et al. (2023).
Porcine fetal fibroblasts (PFFs) were isolated from the fetus that becomes roughly with the 30 day and cultured for 1-2 passages in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY, USA) culture medium containing 1.25% minimal essential medium (MEM) nonessential amino acids, 1.25% β-mercaptoethanol, 1% penicillin/streptomycin supplemented with 10% fetal bovine serum (FBS; GIBCO, Lot No. 2039230, Grand Island, NY, USA) at 39℃ in 5% CO2. Until the use, primary cultured cells were stored in liquid nitrogen (LN2).
Lenti-viral transduction was performed using the viPSTM Vector Kit (Thermo Fisher ScientificTM, USA) following the manufacturer’s instructions. The PFFs were thawed and cultured for 18 h at a density of 1 × 104 cells per well (4 cm2) in 12-well culture dishes (Nunc, USA). These cells were transduced with Lenti-viral vectors encoding four (
On the other hand, Sendai-viral transduction was performed using the CytoTuneTM-iPS Reprogramming Kit (Life Technologies, Frederick, MD, USA) following the manufacturer’s instructions. Approximately 1 × 105 PFFs were seeded per well in a 6-well plate (Nunc, USA) and incubated at 39℃ and 5% CO2. 2 days later, cells were transduced with the CytoTune iPS Reprogramming Kit containing Sendai-viral vectors encoding four human transcription factors (
Reprogramming efficiency of porcine induced pluripotent stem cells (piPSCs) was determined as the number of colonies formed per the number of infected cells seeded. Lenti-iPSCs and Sev-iPSCs were identified in relation to ES-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. AP staining was performed in two ways (basic AP staining and AP live stain). For the basic AP staining, the Lenti-iPSCs lines were fixed with by 4% paraformaldehyde (PFA) for 1-2 min and then washed three times with PBS. AP staining was performed using the Alkaline Phosphatase Detection Kit (Chemicon/Milipore, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, Lenti-iPSCs lines were incubated in stain solution (the ratio of Naphthol:Fast Red Violet:Water solution = 2:1:1) at room temperature until suitable staining developed. Percentage of AP positive (+) colonies was calculated as AP positive (+) Lenti-piPSCs colonies per total Lenti-piPSCs colonies. Images were observed with the LEICA microscope (TYPE 090-135 001) and captured by the Nikon’s NIS Elements microscope imaging software (version 3.0). For AP live staining, the Sev-piPSCs lines were washed three times with pre-warmed DMEM/F-12. The AP live staining was performed using the Alkaline Phosphatase Live Stain (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, Sev-piPSCs lines were incubated with the 1 × LIVE AP substrate for 20-30 min and washed twice with the basal DMEM/F-12 media to remove excess substrate. Following the final wash, images were examined using fluorescence microscope (LEICA DM 2500; Leica, Wetzlar, Germany).
For immunocytochemistry, Lenti-piPSCs and Sev-piPSCs were fixed in 4% paraformaldehyde (PFA) for 15 min and then incubated with blocking solution containing 5% bovine serum albumin (BSA) for 1 h at room temperature. These cells were incubated with primary antibodies under following conditions at 4℃ overnight: OCT-3/4 (1:100, Santa Cruz Biotechnology, catalogue number #SC-6828, Santa Cruz, CA, USA), NANOG (1:100, Abcam, catalogue number #500-p237, Cambridge, MA, USA), SOX2 (10 ng/mL, R&D System, catalogue number #MAB2018, Minneapolis, MN, USA), SSEA-1 (1:100, Santa Cruz Biotechnology, catalogue number #SC-21702), SSEA-4 (1:100, Santa Cruz Biotechnology, catalogue number #SC-59368), Tra-1-60 (1:100, Santa Cruz Biotechnology, catalogue number #SC-21705), Tra-1-81 (1:100, Santa Cruz Biotechnology, catalogue number #SC-21706) and H3K27me3 (1:200, Abcam, catalogue number #ab192985). After overnight, the stem cells were incubated with secondary antibodies under following conditions at room temperature for 1 hour: Alexa fluor® 568 Donkey Anti-Goat IgG (1:200, Invitrogen, Carlsbad, CA, USA), Alexa Fluor® 546 Goat Anti-Rabbit IgG (1:200, Invitrogen), and Alexa Fluor® 555 Donkey Anti-Mouse IgG (1:200, Invitrogen). To indicate the nuclei in cells, 5 µg/mL of Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) were treated at room temperature for 10 min. All images were examined using fluorescence microscope (LEICA DM 2500; Leica, Wetzlar, Germany).
Total RNAs of Lenti-piPSCs and Sev-piPSCs were extracted using Agilent RNA 6000 Nano Kit (Agilent Technologies, Lubbock, TX, USA) and RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions, respectively. cDNA was synthesized using an RevoscriptTM RT Premix (iNtRON Biotechnology Inc, Seongnam, Korea), and total RNA and cDNA were measured using MaestroNano® Spectrophotometer (MAESTROGEN Inc, USA). RT-PCR was performed by Maxime PCR Premix (iNtRON Biotechnology Inc, Seongnam, Korea) and conditions of RT-PCR were followed: pre-denaturation for 10 min at 95℃, denaturation for 30 sec at 94℃, annealing at a temperature specific for each primer set for 40 sec, extinction for 60 sec at 72℃ and final extension for 10 min at 72℃ for 40 cycles using Pro s6325 (Eppendorf, Germany). These PCR products were analyzed by 1.5% agarose gel in 1x TAE buffer. The primer list used for RT-PCR represents in Table 1. On the other hands, q-PCR was performed using the SYBR Green (TOYOBO LTD, Osaka, Japan) on the CFX connectTM real-time PCR detection system (BIO-RAD, USA) and conditions were followed: pre-denaturation for 30 sec at 95℃, denaturation for 5 sec at 95℃, annealing at a temperature specific for each primer set for 10 sec and extinction for 15 sec at 72℃ for 40 cycles. Data analysis was used to ΔΔCt method and gene expression was normalized relative to reference gene (
Table 1 . Reverse transcription-PCR primer lists used in this study
Gene | Sequence (5’→3’) | Target size (bp) | References | |
---|---|---|---|---|
Forward | Reverse | |||
GCAGCCTTTCTCTTGCCAAT | CCTTTGATGGCATTCCTGGG | 400 | NM_001291682.1 | |
GTCCCCATTTACGAGGGCTA | TACCAATGAAGGAGGGCTGG | 319 | NM_001170517.2 | |
ACCCTAAGTTGGAGCTGCAT | GTCCTGGTCTCTGATCTCGG | 245 | XM_005663265.2 | |
AAACCTGTGTGCAATGCTTGT | CACCTATGTACAGCCCGTCT | 330 | XM_005656114 | |
TGACCCCTTCATTGACCTCC | GGCTGACGATCTTGAGGGAGT | 343 | NM_001206359.1 |
Table 2 . Quantitative real-time PCR primer lists used in this study
Gene | Sequence (5’→3’) | Target size (bp) | References | |
---|---|---|---|---|
Forward | Reverse | |||
CATGTCCCAGCACTACCAGA | GAGAGAGGCAGTGTACCGTT | 66 | NM_001123197.1 | |
CCCGAAGCATCCATTTCCAG | GATGACATCTGCAAGGAGGC | 86 | DQ_447201.1 | |
GGATATACCCAGGCCGATGT | GTCGTTTGGCTGAACACCTT | 68 | NM_001113060.1 | |
GGTCATCATCTCTGCCCCTT | TCACGCCCATCACAAACATG | 53 | NM_001206359.1 |
For cell cycle analysis, Lenti-piPSCs and Sev-piPSCs were dissociated with 0.05% Trypsin/EDTA at 39℃ for 5 min. These cells digested to single cells were incubated with propidium iodide (PI) staining solution [50 µL/mL PI, 0.1 mg/mL RNase A, Triton X-100 in PBS] at 37℃ for 40 min. The samples were analyzed by using BD FACS Calibur flow cytometer (BD Biosciences, Becton Dickinson, NJ, USA) and cellquest software. Maximum excitation of PI bound to DNA was at 483 nm and emission was at 635 nm. The results were analyzed by using FlowJo software version 10.0.7 (TREE STAR Inc.). On the other hands, the population doubling time (PDT) of dissociated Lenti-piPSCs and Sev-piPSCs were calculated using the duration*log (2) /log (final concentration)-log (initial concentration) formula at each passage.
For karyotyping analysis, Lenti-piPSCs and Sev-piPSCs were cultured with 0.1 µL/mL of colcemide (Biological Industries Israel Beit Haemek LTD, Kibbutz Beit Haemek, Israel) in culture medium at 37℃ for 1 hour and then harvested using 0.05% Trypsin/EDTA. Harvested single cells were incubated with hypotonic solution (0.4% NaCl and 0.4% KCl in H2O) at 39℃ for 6 min and then fixed in fixative (3:1 = methanol:acetic acid). The cell pellet suspended in 1 mL of fixation solution was dropped onto cold slide and then dried. The chromosomes of metaphase stage stained with Giemsa were patterned by standard G-banding techniques.
For the production of embryoid bodies (EBs), Lenti-piPSCs and Sev-piPSCs were dissociated with 0.05% Trypsin/EDTA at 39℃ for 5 min and collected in differentiation medium [DMEM/F12 supplemented with 1% MEM nonessential amino acids, 1% penicillin/streptomycin, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol and 20% FBS]. Collected these cells were aggregated into EBs for 3 day in hanging drop at a seeding density of 1 × 103 cells/drop. EBs were transferred to low attachment dishes (Corning, USA) and kept in suspension for another 4 days. After 4 days, they were transferred to 0.1% gelatin solution (Millipore, Darmstadt, Germany) coated dishes with differentiation medium and medium were changed daily for 14 days.
At least three replicates were measured for each group. The statistical significance (
This experiment was carried out to examine the optimal induction efficiency of porcine fetal fibroblasts (PFFs) into piPSCs by using Lentiviral vectors in various culture conditions. PFFs were transduced by using Lenti-viral vectors with combinations of four (
Table 3 . Reprogramming efficiency of porcine fibroblasts by using Lentiviral vector in various culture conditions
Base medium | Supplement | Reprogramming factors delivered | Colonies obtained after TD 6 (%) | Colonies obtained after TD 16 (%) | AP-positive colonies at passage 3 (%) |
---|---|---|---|---|---|
DMEM/F12 + 20% FBS | LIF | OSKM OSKMNL | 0 0.1 | 0 1.38 | 0 68.3 |
DMEM/F12 + 20% KSR | LIF | OSKM OSKMNL | 0 0.09 | 0 1.42 | 0 68.0 |
DMEM/F12 + 10% FBS/KSR | LIF | OSKM OSKMNL | 0.08 0.12 | 1.40 1.49 | 0.8 97.0 |
Porcine fetal fibroblasts (PFFs) were reprogrammed using four (OSKM) and six (OSKMNL) human factors under multiplicity of infection (MOI) 25 condition for 24 h and then cultured on mouse embryonic feeder (MEF) cells treated with mitomycin C under three different culture conditions: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 20% fetal bovine serum (FBS) with 20 ng/mL leukemia inhibitor factor (LIF), DMEM/F12 + 20% knock-OutTM serum replacer (KSR) with 20 ng/mL LIF and DMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF. After transduction, reprogramming efficiency was determined as the percentage of colonies formed per the number of infected cells seeded (10,000 cells). Lenti-virus induced pluripotent stem cells (Lenti-iPSCs) were identified based on embryonic stem cell (ESC)-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. Reprogramming factors delivered represents OSKM or OSKMNL combination of reprogramming factors: O,
To examine the optimal induction efficiency of PFFs into piPSCs by using Sendai-viral vector in various culture conditions, PFFs were transduced by using Sendai-viral vector with combinations of four reprogramming factors (
Table 4 . Reprogramming efficiency of porcine fibroblasts by using Sendaivirus vector in various culture conditions
Base medium | Supplement | Reprogramming factors delivered | Colonies obtained after TD 6 (%) | Colonies obtained after TD 16 (%) | AP-positive colonies at passage 3 (%) |
---|---|---|---|---|---|
DMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
DMEM/F12 + 20% KSR | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
DMEM/F12 + 10% FBS/KSR | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0.02 | 0 0.02 | 0 92.5 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
Porcine fetal fibroblasts (PFFs) were reprogrammed using four human factors (OSKM) under multiplicity of infection (MOI) 3 condition for 24 h and then cultured on mouse embryonic feeder (MEF) cells treated with mitomycin C under twelve different culture conditions: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 20% fetal bovine serum (FBS) with 20 ng/mL leukemia inhibitor factor (LIF), DMEM/F12 + 20% FBS with 4 ng/mL basic fibroblast growth factor-2 (bFGF), DMEM/F12 + 20% knock-OutTM serum replacer (KSR) with 20 ng/mL LIF, DMEM/F12 + 20% KSR with 4 ng/mL bFGF, DMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF, DMEM/F12 + 10% FBS/KSR with 4 ng/mL bFGF, KnockOutDMEM/F12 + 20% FBS with 20 ng/mL LIF, KnockOutDMEM/F12 + 20% FBS with 4 ng/mL bFGF, KnockOutDMEM/F12 + 20% KSR with 20 ng/mL LIF, KnockOutDMEM/F12 + 20% KSR with 4 ng/mL bFGF, KnockOutDMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF and KnockOutDMEM/F12 + 10% FBS/KSR with 4 ng/mL bFGF. After transduction, reprogramming efficiency was determined as the percentage of colonies formed per the number of infected cells seeded (100,000 cells). Sendai-virus induced pluripotent stem cells (Sev-iPSCs) were identified based on embryonic stem cell (ESC)-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. Reprogramming factors delivered represents OSKM combination of reprogramming factors: O,
We evaluated the expression of endogenous pluripotency marker genes (
This experiment was performed in order to examine the continuous expression of exogenous reprogramming factors in piPSCs constructed by using Sendaiviral and Lentiviral vectors. We evaluated the expression of exogenous reprogramming genes (
Expression of OCT-3/4, NANOG and SOX2 proteins in the colonies of Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) was relatively higher at passage 10 and 20, demonstrating that Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) expressed the core pluripotency genes (Fig. 4A). However, OCT-3/4 and SOX2 proteins in colony of Lenti-piPSCs (OSKM) were expressed at passage 10, but not expressed at passage 20. Interestingly, there was no expression of NANOG protein in Lenti-piPSCs (OSKM) at passage 10 and 20 (Fig. 4A). On the other hand, SSEA-1 was expressed in only Sev-piPSCs (OSKM), whereas SSEA-4 was expressed in only Lenti-piPSCs (OSKMNL). However, there was no expression of TRA-1-60 and TRA-1-81 in both Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL). All of surface markers was not expressed in Lenti-piPSCs (OSKM) (Fig. 4A).
In order to examine the epigenetic mechanism of differentiation-associated gene repression at the time of conoly formation, we immunolabeled three piPSCs types (Sev-piPSCs (OSKM), Lenti-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL)) with an antibody against trimethylation of histone H3 at lysine 27 (H3K27me3) (Fig. 4B). Expression of H3K27me3 was down-regulated in the colonies of Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL), whereas was up-regulated in PFFs and Lenti-piPSCs (OSKM).
The cell cycle of Sev-piPSCs (OSKM), Lenti-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) was analyzed using FACS and FlowJo. The greater part of PFFs was remained at G0/G1 stage. Additionally Lenti-piPSCs (OSKM) were slightly higher in the G0/G1 stage than Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL). However, Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) were slightly increased in G2/M phase than Lenti-piPSCs (OSKM) (
To investigate
In the present study, we observed pluripotent characteristics of porcine induced pluripotent stem cells (piPSCs) which produced by delivering reprogramming factors with integrating Lentiviral vector and non-integrating Sendaiviral vector into the host genome. Lentivirus-mediated reprogramming is a preferentially used method for generating piPSCs because Lentiviral vector is able to integrate efficiently reprogramming factors into differentiated cell’s genome, ensuring stable and long-term expression of these factors (Ezashi et al., 2009; Kim et al., 2009). Instead, Sendaivirus-mediated reprogramming has been adapted for inducing low cytotoxicity through a type of RNA virus because the Sendaiviral vector does not integrate its reprogramming factors into the host genome unlike Lentiviruses. The ectopic reprogramming factors introduced by the Lentiviral or Sendaiviral vector induce the genetic and epigenetic changes necessary to convert the differentiated cells into pluripotent stem cells (PSCs). However, one potential problem of the methods is the risk of insertional mutagenesis, where the integration of Lentiviral DNAs into the host genome may disrupt the normal function of endogenous genes and lead to unintended genetic changes. To solve this problem, the non-integrating process may reduce the risk of genetic mutations and makes it a safer choice to produce piPSCs for research and potential therapeutic purposes in a porcine model. Therefore, the delivery of reprogramming factors using Sendai viral vectors has been considered the safest tool for inducing low cytotoxicity without inserting transgenes into the host genome.
So far, culture conditions have not been optimized to induce efficiently reprogramming and ensure the long-term stability of piPSCs depending upon the endogenous pluripotency machinery. Therefore, the effect of culture on virus-mediated reprogrammed cells is of significance for generating
They were previously reported that pluripotent characteristics of porcine embryonic stem cells (pESCs), porcine epiblast stem cells (pEpiSCs) and piPSCs are quite similar to humans, as evidenced the primed pluripotent state regarding gene expression patterns (Alberio et al., 2010; Telugu et al., 2010; Choi et al., 2013; Park et al., 2013; Baek et al., 2021). In this study, as pluripotent characteristics of piPSCs generated through different viral transduction systems, Lenti-iPSCs (OSKMNL) resembled the naïve mouse embryonic stem cells (mESCs) in colony morphology for culture, whereas Sev-iPSCs (OSKM) presented the primed human embryonic stem cells (hESCs). Although significant differences in expression of OCT-3/4 and NANOG during embryonic development were reported (Gao et al., 2010; Gao et al., 2011; Wolf et al., 2011), a pluripotent state is able to be induced in porcine cells overexpressing mouse or human reprogramming factors such as Oct-3/4 and NANOG. Endogenous
As mentioned earlier, to produce
In the PSCs, pluripotency markers including OCT-3/4, NANOG and SOX2 could be detected but the stage-specific embryonic antigens (SSEA) or Tra cell-surface markers may express specifically relying on species. In the present study, immunocytochemistry revealed that Lenti-iPSCs (OSKMNL) displayed positive expression of the pluripotent markers and also expressed positively for surface markers (SSEA-4, Tra 1-60 and Tra 1-81) but not for SSEA-1. Like Lenti-iPSCs (OSKMNL), Sev-iPSCs (OSKM) presented positive expression of the pluripotent markers but only SSEA-1 among the surface markers was expressed. As showed the naïve mESCs type in morphology, Lenti-iPSCs (OSKM) presented expression of OCT-3/4 and SOX2 proteins at initial passage, however, a strong down-regulation of pluripotent markers (OCT-3/4, NANOG and SOX2) presented at further passages. It was previously reported that expression of OCT-3/4, in contrast to that of NANOG, varies from passage to passage in porcine pluripotent stem cells (Brevini et al., 2007). On the other hand, as the characteristics of inactive X-chromosome, the activation of trimethylation of histone H3 at lysine 27 (H3K27me3) represses transcription by preventing the binding of RNA-pol II (Plath et al., 2003). In the piPSCs, H3K27me3 of Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM) was negatively expressed when compared with differentiated cells (PFFs) and Lenti-iPSCs (OSKM), thereby exhibiting their own pluripotent characteristics.
In mice, the cell-cycle of ESCs is usually characterized by a shortened G1 phase (Savatier et al., 1996; Coronado et al., 2013). Also, the cell-cycle of hESCs has shown a very short G1 phase (2-3 hours) of an abbreviated cell-cycle (16-18 hours) (Becker et al., 2006; Becker et al., 2007; Ghule et al., 2011). However, in the present study, the proportion of cell-cycle in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM) significantly decreased in G0/G1 phase but increased in G2/M phase when compared with that of the differentiated cells (PFFs). There was difference in the cell cycle of Sub G1, G0/G1, S and G2/M. These cultures showed no signs of senescence over many passages and appeared to be pluripotent as evident by their ability to form embryoid bodies (EBs). Lenti-iPSCs (OSKMNL) enhanced the reproducible ability and the efficiency of
Taken together, we have produced different porcine iPSCs lines by delivering transcription factors with integrating Lentiviral and non-integrating Sendaiviral vectors. These results suggested that the delivery system of reprogramming factors using Sendai viral vectors induces low cytotoxicity without inserting transgenes into the host genome, but Lenti-iPSCs (OSKMNL) line produced with integrating Lenti-viral vectors including six reprogramming presents the naïve mESCs type in colony morphology and pluripotent markers expression and
None.
Conceptualization, S-K.B., I-W.L. and J-H.L.; investigation, Y-J.L. and B-G.S.; methodology, T-S.K.; project administration, J-H.L.; resources, J-W.C. and J-H.L.; supervision, C.H. and J-H.L.; writing - original draft, S-K.B. and I-W.L.; writing - review & editing, C.H. and J-H.L.
This work was supported by the National Research Foundation of Korea funded by the Korean Government (2020R1l1A3072689) Republic of Korea. In-Won Lee, Yeon-Ji Lee and Bo-Gyeong Seo were supported by the scholarship from the BK21Plus Program, Ministry of Education, Republic of Korea.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2023; 38(4): 275-290
Published online December 31, 2023 https://doi.org/10.12750/JARB.38.4.275
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Sang-Ki Baek1,#,§ , In-Won Lee1,3,#
, Yeon-Ji Lee1,3
, Bo-Gyeong Seo2,3
, Jung-Woo Choi4
, Tae-Suk Kim1
, Cheol Hwangbo3
and Joon-Hee Lee1,5,*
1Department of Animal Bioscience, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea
2Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju 52828, Korea
3Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Korea
4College of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea
5Institute of Agriculture & Life Science, College of Agriculture & Life Sciences, Gyeongsang National University, Jinju 52828, Korea
Correspondence to:Joon-Hee Lee
E-mail: sbxjhl@gnu.ac.kr
#These authors contributed equally to this work.
§Current affiliation: Gyeongsangnamdo Livestock Experiment Station, Sancheong 52263, Korea
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Porcine pluripotent stem cells (pPSCs) would provide enormous potential for agriculture and biomedicine. However, authentic pPSCs have not established yet because standards for pPSCs-specific markers and culture conditions are not clear. Therefore, the present study reports comparative pluripotency characteristics in porcine induced pluripotent stem cells (piPSCs) derived from different viral transduction and reprogramming factors [Lenti-iPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM)].
Methods: Porcine fibroblasts were induced into Lenti-iPSCs (OSKM) and Lenti-iPSCs (OSKMNL) by using Lentiviral vector and Sev-iPSCs (OSKM) by using Sendaiviral vector. Expressions of endogenous or exogenous pluripotency-associated genes, surface marker and in vitro differentiation in between Lenti-piPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-piPSCs (OSKM) were compared.
Results: Colonial morphology of Lenti-iPSCs (OSKMNL) closely resembles the naïve mouse embryonic stem cells colony for culture, whereas Sev-iPSCs (OSKM) colony is similar to the primed hESCs. Also, the activity of AP shows a distinct different in piPSCs (AP-positive (+) Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but AP-negative (-) Lenti-iPSCs (OSKM)). mRNAs expression of several marker genes (OCT-3/4, NANOG and SOX2) for pluripotency was increased in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but Sev-iPSCs (OSKM). Interestingly, SSEA-1 of surface markers was expressed only in Sev-iPSCs (OSKM), whereas SSEA-4, Tra-1-60 and Tra-1-81 were positively expressed in Lenti-iPSCs (OSKMNL). Exogenous reprogramming factors continuously expressed in Lenti-iPSCs (OSKMNL) for passage 20, whereas Sev-iPSCs (OSKM) did not express any exogenous transcription factors. Finally, only Lenti-iPSCs (OSKMNL) express the three germ layers and primordial germ cells markers in aggregated EBs.
Conclusions: These results indicate that the viral transduction system of reprograming factors into porcine differentiated cells display different pluripotency characteristics in piPSCs.
Keywords: induced pluripotent stem cells, Lenti-viral, pluripotency, porcine, Sendai-viral
A new type of embryonic-like stem cells had been derived from induced pluripotent stem cells (iPSCs) by reprogramming differenced somatic cells with defined factors. These cells have the self-renewal capacity that proliferates indefinitely and pluripotency that differentiates potentially into other cell types (Romito and Cobellis, 2016), and allow for patient-specific stem cells to be produced because of avoidance of rejection of their derivatives by immune system (Park et al., 2008a). Mouse iPSCs were first produced by a retrovirus vector transduction of
The reprogramming procedures developed in mouse and human have been adapted to an increasingly demand of specific specie, pig, which has an excellent resource for disease modeling, drug discovery and regenerative medicine (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). The derivation of iPSCs by using the Japanese group reprogramming procedure yields either naïve or primed stemness states, depending on the species. For instance, mouse iPSCs generally have the properties of naïve state like embryonic stem cells (ESCs), while human iPSCs so far reported have features of the primed state like epiblast stem cells (EpiSCs). Interestingly, the colonies derived from porcine iPSCs resemble human ESCs rather than mouse ESCs in morphology. Porcine iPSCs like human ESCs are totally dependent upon on basic fibroblasts growth factor (bFGF) and Activin/Nodal signaling pathway for maintaining self-renewal and pluripotency (Dahéron et al., 2004; Vallier et al., 2009; Alberio et al., 2010). On the contrary, mouse ESCs are dependent on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP-4) to maintain the undifferentiated state (Smith et al., 1988; Ying et al., 2003).
Porcine pluripotent stem cells may be especially valuable because the pig is a prime biomedical model for tissue and organ transplantation. Among domestic animals, induced pluripotent stem cells (iPSCs) have been first successfully generated from swine (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). It was clearly evidenced that the porcine iPSCs isolated have an ability to differentiate into tissue types reflective of the three germ layers (endoderm, mesoderm and ectoderm), spontaneously within either embryoid bodies or teratomas (Ezashi et al., 2012). However, it remains unclear whether or not the continued expression of reprogramming genes complicated directed differentiation along specific lineages, or if the protocols and reagents used have not been optimized for pig. Some discrepancies relating to whether or not the lines express the surface molecules stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) and the tumor rejection antigens (Tra 1-60 and Tra 1-81) have been reported. Mouse and human ESCs slightly differ in their display of cell surface antigens. For example, SSEA-1 is expressed on mESCs but it is absent on hESCs, and different surface markers (SSEA-3 and SSEA-4) are displayed on the human pluripotent stem cells (Henderson et al., 2002).
Reprogramming factors needed to make porcine iPSCs were introduced by infecting the PFFs with non-integrative methods [plasmids, Sendai virus, synthetic mRNAs and recombinant proteins] (Telugu et al., 2010). The delivery of transgenes using viral vectors, which are stably expressed, is considered the most useful tool for inducing low cytotoxicity and inserting transgenes into the host genome (Zhang and Godbey, 2006). Porcine induced pluripotent stem cells (piPSCs) generated using lentiviral vectors, often referred to as lentivirus-mediated reprogramming, involve the use of lentiviruses as a vehicle to introduce reprogramming factors into somatic cells, ultimately leading to the generation of piPSCs. This method is a variation of the commonly used induced pluripotent stem cell (iPSC) generation technique and is utilized for creating piPSCs. The Sendai virus infects the target cells and delivers the reprogramming factors into the cell’s cytoplasm. Importantly, the Sendai virus does not integrate its genetic material into the host cell’s DNA. Various lines of evidence indicate that efficient cell reprogramming requires the sustained and simultaneous expression of several exogenous factors at least 10-20 days (Jaenisch and Young, 2008). After reprogramming has been completed, these exogenous factors should be replaced promptly with their endogenous counterparts if the cells are to acquire autoregulated pluripotency (Jaenisch and Young, 2008).
Here we have transformed porcine fetal fibroblasts (PFFs) into iPSCs by delivering transcription factors with integrating (lentiviral transfection) and non-integrating (Sendai virus infection) vectors. Therefore, we have demonstrated that porcine iPSCs generated by infecting the PFFs with non-integrative method (Sendai virus infection) might carry less of an exogenous transgenes expression than porcine iPSCs generated by infecting the PFFs with integrating (lentiviral transfection) vectors.
All chemicals and reagents were purchased from SigmaAldrich Co. (St. Louis, MO, USA) unless otherwise mentioned. And all materials and methods were performed accordingly, as previously described by Lee et al. (2023).
Porcine fetal fibroblasts (PFFs) were isolated from the fetus that becomes roughly with the 30 day and cultured for 1-2 passages in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY, USA) culture medium containing 1.25% minimal essential medium (MEM) nonessential amino acids, 1.25% β-mercaptoethanol, 1% penicillin/streptomycin supplemented with 10% fetal bovine serum (FBS; GIBCO, Lot No. 2039230, Grand Island, NY, USA) at 39℃ in 5% CO2. Until the use, primary cultured cells were stored in liquid nitrogen (LN2).
Lenti-viral transduction was performed using the viPSTM Vector Kit (Thermo Fisher ScientificTM, USA) following the manufacturer’s instructions. The PFFs were thawed and cultured for 18 h at a density of 1 × 104 cells per well (4 cm2) in 12-well culture dishes (Nunc, USA). These cells were transduced with Lenti-viral vectors encoding four (
On the other hand, Sendai-viral transduction was performed using the CytoTuneTM-iPS Reprogramming Kit (Life Technologies, Frederick, MD, USA) following the manufacturer’s instructions. Approximately 1 × 105 PFFs were seeded per well in a 6-well plate (Nunc, USA) and incubated at 39℃ and 5% CO2. 2 days later, cells were transduced with the CytoTune iPS Reprogramming Kit containing Sendai-viral vectors encoding four human transcription factors (
Reprogramming efficiency of porcine induced pluripotent stem cells (piPSCs) was determined as the number of colonies formed per the number of infected cells seeded. Lenti-iPSCs and Sev-iPSCs were identified in relation to ES-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. AP staining was performed in two ways (basic AP staining and AP live stain). For the basic AP staining, the Lenti-iPSCs lines were fixed with by 4% paraformaldehyde (PFA) for 1-2 min and then washed three times with PBS. AP staining was performed using the Alkaline Phosphatase Detection Kit (Chemicon/Milipore, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, Lenti-iPSCs lines were incubated in stain solution (the ratio of Naphthol:Fast Red Violet:Water solution = 2:1:1) at room temperature until suitable staining developed. Percentage of AP positive (+) colonies was calculated as AP positive (+) Lenti-piPSCs colonies per total Lenti-piPSCs colonies. Images were observed with the LEICA microscope (TYPE 090-135 001) and captured by the Nikon’s NIS Elements microscope imaging software (version 3.0). For AP live staining, the Sev-piPSCs lines were washed three times with pre-warmed DMEM/F-12. The AP live staining was performed using the Alkaline Phosphatase Live Stain (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, Sev-piPSCs lines were incubated with the 1 × LIVE AP substrate for 20-30 min and washed twice with the basal DMEM/F-12 media to remove excess substrate. Following the final wash, images were examined using fluorescence microscope (LEICA DM 2500; Leica, Wetzlar, Germany).
For immunocytochemistry, Lenti-piPSCs and Sev-piPSCs were fixed in 4% paraformaldehyde (PFA) for 15 min and then incubated with blocking solution containing 5% bovine serum albumin (BSA) for 1 h at room temperature. These cells were incubated with primary antibodies under following conditions at 4℃ overnight: OCT-3/4 (1:100, Santa Cruz Biotechnology, catalogue number #SC-6828, Santa Cruz, CA, USA), NANOG (1:100, Abcam, catalogue number #500-p237, Cambridge, MA, USA), SOX2 (10 ng/mL, R&D System, catalogue number #MAB2018, Minneapolis, MN, USA), SSEA-1 (1:100, Santa Cruz Biotechnology, catalogue number #SC-21702), SSEA-4 (1:100, Santa Cruz Biotechnology, catalogue number #SC-59368), Tra-1-60 (1:100, Santa Cruz Biotechnology, catalogue number #SC-21705), Tra-1-81 (1:100, Santa Cruz Biotechnology, catalogue number #SC-21706) and H3K27me3 (1:200, Abcam, catalogue number #ab192985). After overnight, the stem cells were incubated with secondary antibodies under following conditions at room temperature for 1 hour: Alexa fluor® 568 Donkey Anti-Goat IgG (1:200, Invitrogen, Carlsbad, CA, USA), Alexa Fluor® 546 Goat Anti-Rabbit IgG (1:200, Invitrogen), and Alexa Fluor® 555 Donkey Anti-Mouse IgG (1:200, Invitrogen). To indicate the nuclei in cells, 5 µg/mL of Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) were treated at room temperature for 10 min. All images were examined using fluorescence microscope (LEICA DM 2500; Leica, Wetzlar, Germany).
Total RNAs of Lenti-piPSCs and Sev-piPSCs were extracted using Agilent RNA 6000 Nano Kit (Agilent Technologies, Lubbock, TX, USA) and RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions, respectively. cDNA was synthesized using an RevoscriptTM RT Premix (iNtRON Biotechnology Inc, Seongnam, Korea), and total RNA and cDNA were measured using MaestroNano® Spectrophotometer (MAESTROGEN Inc, USA). RT-PCR was performed by Maxime PCR Premix (iNtRON Biotechnology Inc, Seongnam, Korea) and conditions of RT-PCR were followed: pre-denaturation for 10 min at 95℃, denaturation for 30 sec at 94℃, annealing at a temperature specific for each primer set for 40 sec, extinction for 60 sec at 72℃ and final extension for 10 min at 72℃ for 40 cycles using Pro s6325 (Eppendorf, Germany). These PCR products were analyzed by 1.5% agarose gel in 1x TAE buffer. The primer list used for RT-PCR represents in Table 1. On the other hands, q-PCR was performed using the SYBR Green (TOYOBO LTD, Osaka, Japan) on the CFX connectTM real-time PCR detection system (BIO-RAD, USA) and conditions were followed: pre-denaturation for 30 sec at 95℃, denaturation for 5 sec at 95℃, annealing at a temperature specific for each primer set for 10 sec and extinction for 15 sec at 72℃ for 40 cycles. Data analysis was used to ΔΔCt method and gene expression was normalized relative to reference gene (
Table 1. Reverse transcription-PCR primer lists used in this study.
Gene | Sequence (5’→3’) | Target size (bp) | References | |
---|---|---|---|---|
Forward | Reverse | |||
GCAGCCTTTCTCTTGCCAAT | CCTTTGATGGCATTCCTGGG | 400 | NM_001291682.1 | |
GTCCCCATTTACGAGGGCTA | TACCAATGAAGGAGGGCTGG | 319 | NM_001170517.2 | |
ACCCTAAGTTGGAGCTGCAT | GTCCTGGTCTCTGATCTCGG | 245 | XM_005663265.2 | |
AAACCTGTGTGCAATGCTTGT | CACCTATGTACAGCCCGTCT | 330 | XM_005656114 | |
TGACCCCTTCATTGACCTCC | GGCTGACGATCTTGAGGGAGT | 343 | NM_001206359.1 |
Table 2. Quantitative real-time PCR primer lists used in this study.
Gene | Sequence (5’→3’) | Target size (bp) | References | |
---|---|---|---|---|
Forward | Reverse | |||
CATGTCCCAGCACTACCAGA | GAGAGAGGCAGTGTACCGTT | 66 | NM_001123197.1 | |
CCCGAAGCATCCATTTCCAG | GATGACATCTGCAAGGAGGC | 86 | DQ_447201.1 | |
GGATATACCCAGGCCGATGT | GTCGTTTGGCTGAACACCTT | 68 | NM_001113060.1 | |
GGTCATCATCTCTGCCCCTT | TCACGCCCATCACAAACATG | 53 | NM_001206359.1 |
For cell cycle analysis, Lenti-piPSCs and Sev-piPSCs were dissociated with 0.05% Trypsin/EDTA at 39℃ for 5 min. These cells digested to single cells were incubated with propidium iodide (PI) staining solution [50 µL/mL PI, 0.1 mg/mL RNase A, Triton X-100 in PBS] at 37℃ for 40 min. The samples were analyzed by using BD FACS Calibur flow cytometer (BD Biosciences, Becton Dickinson, NJ, USA) and cellquest software. Maximum excitation of PI bound to DNA was at 483 nm and emission was at 635 nm. The results were analyzed by using FlowJo software version 10.0.7 (TREE STAR Inc.). On the other hands, the population doubling time (PDT) of dissociated Lenti-piPSCs and Sev-piPSCs were calculated using the duration*log (2) /log (final concentration)-log (initial concentration) formula at each passage.
For karyotyping analysis, Lenti-piPSCs and Sev-piPSCs were cultured with 0.1 µL/mL of colcemide (Biological Industries Israel Beit Haemek LTD, Kibbutz Beit Haemek, Israel) in culture medium at 37℃ for 1 hour and then harvested using 0.05% Trypsin/EDTA. Harvested single cells were incubated with hypotonic solution (0.4% NaCl and 0.4% KCl in H2O) at 39℃ for 6 min and then fixed in fixative (3:1 = methanol:acetic acid). The cell pellet suspended in 1 mL of fixation solution was dropped onto cold slide and then dried. The chromosomes of metaphase stage stained with Giemsa were patterned by standard G-banding techniques.
For the production of embryoid bodies (EBs), Lenti-piPSCs and Sev-piPSCs were dissociated with 0.05% Trypsin/EDTA at 39℃ for 5 min and collected in differentiation medium [DMEM/F12 supplemented with 1% MEM nonessential amino acids, 1% penicillin/streptomycin, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol and 20% FBS]. Collected these cells were aggregated into EBs for 3 day in hanging drop at a seeding density of 1 × 103 cells/drop. EBs were transferred to low attachment dishes (Corning, USA) and kept in suspension for another 4 days. After 4 days, they were transferred to 0.1% gelatin solution (Millipore, Darmstadt, Germany) coated dishes with differentiation medium and medium were changed daily for 14 days.
At least three replicates were measured for each group. The statistical significance (
This experiment was carried out to examine the optimal induction efficiency of porcine fetal fibroblasts (PFFs) into piPSCs by using Lentiviral vectors in various culture conditions. PFFs were transduced by using Lenti-viral vectors with combinations of four (
Table 3. Reprogramming efficiency of porcine fibroblasts by using Lentiviral vector in various culture conditions.
Base medium | Supplement | Reprogramming factors delivered | Colonies obtained after TD 6 (%) | Colonies obtained after TD 16 (%) | AP-positive colonies at passage 3 (%) |
---|---|---|---|---|---|
DMEM/F12 + 20% FBS | LIF | OSKM OSKMNL | 0 0.1 | 0 1.38 | 0 68.3 |
DMEM/F12 + 20% KSR | LIF | OSKM OSKMNL | 0 0.09 | 0 1.42 | 0 68.0 |
DMEM/F12 + 10% FBS/KSR | LIF | OSKM OSKMNL | 0.08 0.12 | 1.40 1.49 | 0.8 97.0 |
Porcine fetal fibroblasts (PFFs) were reprogrammed using four (OSKM) and six (OSKMNL) human factors under multiplicity of infection (MOI) 25 condition for 24 h and then cultured on mouse embryonic feeder (MEF) cells treated with mitomycin C under three different culture conditions: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 20% fetal bovine serum (FBS) with 20 ng/mL leukemia inhibitor factor (LIF), DMEM/F12 + 20% knock-OutTM serum replacer (KSR) with 20 ng/mL LIF and DMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF. After transduction, reprogramming efficiency was determined as the percentage of colonies formed per the number of infected cells seeded (10,000 cells). Lenti-virus induced pluripotent stem cells (Lenti-iPSCs) were identified based on embryonic stem cell (ESC)-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. Reprogramming factors delivered represents OSKM or OSKMNL combination of reprogramming factors: O,
To examine the optimal induction efficiency of PFFs into piPSCs by using Sendai-viral vector in various culture conditions, PFFs were transduced by using Sendai-viral vector with combinations of four reprogramming factors (
Table 4. Reprogramming efficiency of porcine fibroblasts by using Sendaivirus vector in various culture conditions.
Base medium | Supplement | Reprogramming factors delivered | Colonies obtained after TD 6 (%) | Colonies obtained after TD 16 (%) | AP-positive colonies at passage 3 (%) |
---|---|---|---|---|---|
DMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
DMEM/F12 + 20% KSR | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
DMEM/F12 + 10% FBS/KSR | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0.02 | 0 0.02 | 0 92.5 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
Porcine fetal fibroblasts (PFFs) were reprogrammed using four human factors (OSKM) under multiplicity of infection (MOI) 3 condition for 24 h and then cultured on mouse embryonic feeder (MEF) cells treated with mitomycin C under twelve different culture conditions: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 20% fetal bovine serum (FBS) with 20 ng/mL leukemia inhibitor factor (LIF), DMEM/F12 + 20% FBS with 4 ng/mL basic fibroblast growth factor-2 (bFGF), DMEM/F12 + 20% knock-OutTM serum replacer (KSR) with 20 ng/mL LIF, DMEM/F12 + 20% KSR with 4 ng/mL bFGF, DMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF, DMEM/F12 + 10% FBS/KSR with 4 ng/mL bFGF, KnockOutDMEM/F12 + 20% FBS with 20 ng/mL LIF, KnockOutDMEM/F12 + 20% FBS with 4 ng/mL bFGF, KnockOutDMEM/F12 + 20% KSR with 20 ng/mL LIF, KnockOutDMEM/F12 + 20% KSR with 4 ng/mL bFGF, KnockOutDMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF and KnockOutDMEM/F12 + 10% FBS/KSR with 4 ng/mL bFGF. After transduction, reprogramming efficiency was determined as the percentage of colonies formed per the number of infected cells seeded (100,000 cells). Sendai-virus induced pluripotent stem cells (Sev-iPSCs) were identified based on embryonic stem cell (ESC)-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. Reprogramming factors delivered represents OSKM combination of reprogramming factors: O,
We evaluated the expression of endogenous pluripotency marker genes (
This experiment was performed in order to examine the continuous expression of exogenous reprogramming factors in piPSCs constructed by using Sendaiviral and Lentiviral vectors. We evaluated the expression of exogenous reprogramming genes (
Expression of OCT-3/4, NANOG and SOX2 proteins in the colonies of Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) was relatively higher at passage 10 and 20, demonstrating that Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) expressed the core pluripotency genes (Fig. 4A). However, OCT-3/4 and SOX2 proteins in colony of Lenti-piPSCs (OSKM) were expressed at passage 10, but not expressed at passage 20. Interestingly, there was no expression of NANOG protein in Lenti-piPSCs (OSKM) at passage 10 and 20 (Fig. 4A). On the other hand, SSEA-1 was expressed in only Sev-piPSCs (OSKM), whereas SSEA-4 was expressed in only Lenti-piPSCs (OSKMNL). However, there was no expression of TRA-1-60 and TRA-1-81 in both Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL). All of surface markers was not expressed in Lenti-piPSCs (OSKM) (Fig. 4A).
In order to examine the epigenetic mechanism of differentiation-associated gene repression at the time of conoly formation, we immunolabeled three piPSCs types (Sev-piPSCs (OSKM), Lenti-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL)) with an antibody against trimethylation of histone H3 at lysine 27 (H3K27me3) (Fig. 4B). Expression of H3K27me3 was down-regulated in the colonies of Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL), whereas was up-regulated in PFFs and Lenti-piPSCs (OSKM).
The cell cycle of Sev-piPSCs (OSKM), Lenti-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) was analyzed using FACS and FlowJo. The greater part of PFFs was remained at G0/G1 stage. Additionally Lenti-piPSCs (OSKM) were slightly higher in the G0/G1 stage than Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL). However, Sev-piPSCs (OSKM) and Lenti-piPSCs (OSKMNL) were slightly increased in G2/M phase than Lenti-piPSCs (OSKM) (
To investigate
In the present study, we observed pluripotent characteristics of porcine induced pluripotent stem cells (piPSCs) which produced by delivering reprogramming factors with integrating Lentiviral vector and non-integrating Sendaiviral vector into the host genome. Lentivirus-mediated reprogramming is a preferentially used method for generating piPSCs because Lentiviral vector is able to integrate efficiently reprogramming factors into differentiated cell’s genome, ensuring stable and long-term expression of these factors (Ezashi et al., 2009; Kim et al., 2009). Instead, Sendaivirus-mediated reprogramming has been adapted for inducing low cytotoxicity through a type of RNA virus because the Sendaiviral vector does not integrate its reprogramming factors into the host genome unlike Lentiviruses. The ectopic reprogramming factors introduced by the Lentiviral or Sendaiviral vector induce the genetic and epigenetic changes necessary to convert the differentiated cells into pluripotent stem cells (PSCs). However, one potential problem of the methods is the risk of insertional mutagenesis, where the integration of Lentiviral DNAs into the host genome may disrupt the normal function of endogenous genes and lead to unintended genetic changes. To solve this problem, the non-integrating process may reduce the risk of genetic mutations and makes it a safer choice to produce piPSCs for research and potential therapeutic purposes in a porcine model. Therefore, the delivery of reprogramming factors using Sendai viral vectors has been considered the safest tool for inducing low cytotoxicity without inserting transgenes into the host genome.
So far, culture conditions have not been optimized to induce efficiently reprogramming and ensure the long-term stability of piPSCs depending upon the endogenous pluripotency machinery. Therefore, the effect of culture on virus-mediated reprogrammed cells is of significance for generating
They were previously reported that pluripotent characteristics of porcine embryonic stem cells (pESCs), porcine epiblast stem cells (pEpiSCs) and piPSCs are quite similar to humans, as evidenced the primed pluripotent state regarding gene expression patterns (Alberio et al., 2010; Telugu et al., 2010; Choi et al., 2013; Park et al., 2013; Baek et al., 2021). In this study, as pluripotent characteristics of piPSCs generated through different viral transduction systems, Lenti-iPSCs (OSKMNL) resembled the naïve mouse embryonic stem cells (mESCs) in colony morphology for culture, whereas Sev-iPSCs (OSKM) presented the primed human embryonic stem cells (hESCs). Although significant differences in expression of OCT-3/4 and NANOG during embryonic development were reported (Gao et al., 2010; Gao et al., 2011; Wolf et al., 2011), a pluripotent state is able to be induced in porcine cells overexpressing mouse or human reprogramming factors such as Oct-3/4 and NANOG. Endogenous
As mentioned earlier, to produce
In the PSCs, pluripotency markers including OCT-3/4, NANOG and SOX2 could be detected but the stage-specific embryonic antigens (SSEA) or Tra cell-surface markers may express specifically relying on species. In the present study, immunocytochemistry revealed that Lenti-iPSCs (OSKMNL) displayed positive expression of the pluripotent markers and also expressed positively for surface markers (SSEA-4, Tra 1-60 and Tra 1-81) but not for SSEA-1. Like Lenti-iPSCs (OSKMNL), Sev-iPSCs (OSKM) presented positive expression of the pluripotent markers but only SSEA-1 among the surface markers was expressed. As showed the naïve mESCs type in morphology, Lenti-iPSCs (OSKM) presented expression of OCT-3/4 and SOX2 proteins at initial passage, however, a strong down-regulation of pluripotent markers (OCT-3/4, NANOG and SOX2) presented at further passages. It was previously reported that expression of OCT-3/4, in contrast to that of NANOG, varies from passage to passage in porcine pluripotent stem cells (Brevini et al., 2007). On the other hand, as the characteristics of inactive X-chromosome, the activation of trimethylation of histone H3 at lysine 27 (H3K27me3) represses transcription by preventing the binding of RNA-pol II (Plath et al., 2003). In the piPSCs, H3K27me3 of Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM) was negatively expressed when compared with differentiated cells (PFFs) and Lenti-iPSCs (OSKM), thereby exhibiting their own pluripotent characteristics.
In mice, the cell-cycle of ESCs is usually characterized by a shortened G1 phase (Savatier et al., 1996; Coronado et al., 2013). Also, the cell-cycle of hESCs has shown a very short G1 phase (2-3 hours) of an abbreviated cell-cycle (16-18 hours) (Becker et al., 2006; Becker et al., 2007; Ghule et al., 2011). However, in the present study, the proportion of cell-cycle in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM) significantly decreased in G0/G1 phase but increased in G2/M phase when compared with that of the differentiated cells (PFFs). There was difference in the cell cycle of Sub G1, G0/G1, S and G2/M. These cultures showed no signs of senescence over many passages and appeared to be pluripotent as evident by their ability to form embryoid bodies (EBs). Lenti-iPSCs (OSKMNL) enhanced the reproducible ability and the efficiency of
Taken together, we have produced different porcine iPSCs lines by delivering transcription factors with integrating Lentiviral and non-integrating Sendaiviral vectors. These results suggested that the delivery system of reprogramming factors using Sendai viral vectors induces low cytotoxicity without inserting transgenes into the host genome, but Lenti-iPSCs (OSKMNL) line produced with integrating Lenti-viral vectors including six reprogramming presents the naïve mESCs type in colony morphology and pluripotent markers expression and
None.
Conceptualization, S-K.B., I-W.L. and J-H.L.; investigation, Y-J.L. and B-G.S.; methodology, T-S.K.; project administration, J-H.L.; resources, J-W.C. and J-H.L.; supervision, C.H. and J-H.L.; writing - original draft, S-K.B. and I-W.L.; writing - review & editing, C.H. and J-H.L.
This work was supported by the National Research Foundation of Korea funded by the Korean Government (2020R1l1A3072689) Republic of Korea. In-Won Lee, Yeon-Ji Lee and Bo-Gyeong Seo were supported by the scholarship from the BK21Plus Program, Ministry of Education, Republic of Korea.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Table 1 . Reverse transcription-PCR primer lists used in this study.
Gene | Sequence (5’→3’) | Target size (bp) | References | |
---|---|---|---|---|
Forward | Reverse | |||
GCAGCCTTTCTCTTGCCAAT | CCTTTGATGGCATTCCTGGG | 400 | NM_001291682.1 | |
GTCCCCATTTACGAGGGCTA | TACCAATGAAGGAGGGCTGG | 319 | NM_001170517.2 | |
ACCCTAAGTTGGAGCTGCAT | GTCCTGGTCTCTGATCTCGG | 245 | XM_005663265.2 | |
AAACCTGTGTGCAATGCTTGT | CACCTATGTACAGCCCGTCT | 330 | XM_005656114 | |
TGACCCCTTCATTGACCTCC | GGCTGACGATCTTGAGGGAGT | 343 | NM_001206359.1 |
Table 2 . Quantitative real-time PCR primer lists used in this study.
Gene | Sequence (5’→3’) | Target size (bp) | References | |
---|---|---|---|---|
Forward | Reverse | |||
CATGTCCCAGCACTACCAGA | GAGAGAGGCAGTGTACCGTT | 66 | NM_001123197.1 | |
CCCGAAGCATCCATTTCCAG | GATGACATCTGCAAGGAGGC | 86 | DQ_447201.1 | |
GGATATACCCAGGCCGATGT | GTCGTTTGGCTGAACACCTT | 68 | NM_001113060.1 | |
GGTCATCATCTCTGCCCCTT | TCACGCCCATCACAAACATG | 53 | NM_001206359.1 |
Table 3 . Reprogramming efficiency of porcine fibroblasts by using Lentiviral vector in various culture conditions.
Base medium | Supplement | Reprogramming factors delivered | Colonies obtained after TD 6 (%) | Colonies obtained after TD 16 (%) | AP-positive colonies at passage 3 (%) |
---|---|---|---|---|---|
DMEM/F12 + 20% FBS | LIF | OSKM OSKMNL | 0 0.1 | 0 1.38 | 0 68.3 |
DMEM/F12 + 20% KSR | LIF | OSKM OSKMNL | 0 0.09 | 0 1.42 | 0 68.0 |
DMEM/F12 + 10% FBS/KSR | LIF | OSKM OSKMNL | 0.08 0.12 | 1.40 1.49 | 0.8 97.0 |
Porcine fetal fibroblasts (PFFs) were reprogrammed using four (OSKM) and six (OSKMNL) human factors under multiplicity of infection (MOI) 25 condition for 24 h and then cultured on mouse embryonic feeder (MEF) cells treated with mitomycin C under three different culture conditions: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 20% fetal bovine serum (FBS) with 20 ng/mL leukemia inhibitor factor (LIF), DMEM/F12 + 20% knock-OutTM serum replacer (KSR) with 20 ng/mL LIF and DMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF. After transduction, reprogramming efficiency was determined as the percentage of colonies formed per the number of infected cells seeded (10,000 cells). Lenti-virus induced pluripotent stem cells (Lenti-iPSCs) were identified based on embryonic stem cell (ESC)-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. Reprogramming factors delivered represents OSKM or OSKMNL combination of reprogramming factors: O,
Table 4 . Reprogramming efficiency of porcine fibroblasts by using Sendaivirus vector in various culture conditions.
Base medium | Supplement | Reprogramming factors delivered | Colonies obtained after TD 6 (%) | Colonies obtained after TD 16 (%) | AP-positive colonies at passage 3 (%) |
---|---|---|---|---|---|
DMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
DMEM/F12 + 20% KSR | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
DMEM/F12 + 10% FBS/KSR | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0.02 | 0 0.02 | 0 92.5 |
KnockOutDMEM/F12 + 20% FBS | LIF bFGF | OSKM | 0 0 | 0 0 | 0 0 |
Porcine fetal fibroblasts (PFFs) were reprogrammed using four human factors (OSKM) under multiplicity of infection (MOI) 3 condition for 24 h and then cultured on mouse embryonic feeder (MEF) cells treated with mitomycin C under twelve different culture conditions: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 20% fetal bovine serum (FBS) with 20 ng/mL leukemia inhibitor factor (LIF), DMEM/F12 + 20% FBS with 4 ng/mL basic fibroblast growth factor-2 (bFGF), DMEM/F12 + 20% knock-OutTM serum replacer (KSR) with 20 ng/mL LIF, DMEM/F12 + 20% KSR with 4 ng/mL bFGF, DMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF, DMEM/F12 + 10% FBS/KSR with 4 ng/mL bFGF, KnockOutDMEM/F12 + 20% FBS with 20 ng/mL LIF, KnockOutDMEM/F12 + 20% FBS with 4 ng/mL bFGF, KnockOutDMEM/F12 + 20% KSR with 20 ng/mL LIF, KnockOutDMEM/F12 + 20% KSR with 4 ng/mL bFGF, KnockOutDMEM/F12 + 10% FBS/KSR with 20 ng/mL LIF and KnockOutDMEM/F12 + 10% FBS/KSR with 4 ng/mL bFGF. After transduction, reprogramming efficiency was determined as the percentage of colonies formed per the number of infected cells seeded (100,000 cells). Sendai-virus induced pluripotent stem cells (Sev-iPSCs) were identified based on embryonic stem cell (ESC)-like morphology, and alkaline phosphatase (AP) staining was used to facilitate the identified cation of iPSCs colonies. Reprogramming factors delivered represents OSKM combination of reprogramming factors: O,
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pISSN: 2671-4639
eISSN: 2671-4663