Abstract
Chicken pluripotent stem cells (PSCs), such as embryonic stem cells and blastoderm cells, have been used to study development and differentiation in chicken. However, chicken PSCs are not widely used because they are hard to maintain in long-term culture. Recent reports suggest that chicken somatic cells can be reprogrammed to pluripotent state by defined factors to form induced pluripotent stem cells (iPSCs). These chicken iPSCs showed pluripotent differentiation potential and could be maintained in long-term culture. However, intracytoplasmic remodeling during reprogramming of chicken cells remains largely unknown. In this study, we generated chicken iPS-like cells (ciPSLCs) from chicken embryonic fibroblasts using a retroviral expression system encoding human reprogramming factors. These ciPSLCs could be maintained for more than 10 passages and expressed the endogenous chicken pluripotency markers, cNonog and cSox2. Moreover, the ciPSLCs showed higher nucleus to cytoplasm ratio and contained globular mitochondria with immature cristae. This morphology was similar to that of mammalian PSCs, but different from that of avian somatic cells, which showed lower nucleus to cytoplasm ratio and mature mitochondria. These results suggest that intracytoplasmic organelles in differentiated somatic cells could be successfully remodeled into the pluripotent state during reprogramming in chicken.
Introduction
E
The morphology of mitochondria is correlated with the energy metabolism in mammalian pluripotent stem cells (PSCs) and differentiated cells [9]. PSCs such as ESCs and iPSCs contain round immature mitochondria and use glycolytic metabolism for generation of energy. In contrast, differentiated somatic cells have more developed and elongated mitochondria, which contain mature cristae [9 –11]. Our previous reports demonstrated that a change in the cellular state causes dynamic changes in the morphology of cytoplasmic organelles, with corresponding changes in energy metabolism [9]. However, the morphological changes in the mitochondria of avian PSCs and differentiated cells remain largely unknown. In this study, we generated chicken iPS like cells (ciPSLCs) from chicken embryonic fibroblasts (CEFs) using human reprogramming factors. These chicken iPSCs (ciPSCs) could be maintained in the defined medium on passaging (10 passages), tested positive for alkaline phosphatase (AP) and SSEA1, and expressed endogenous pluripotency markers, cNanog and cSox2. This is the first study on the changes in mitochondrial morphology during the reprogramming of avian cells.
Materials and Methods
Generation of ciPSLCs
CEFs were isolated from 11-day-old embryos (Leghorn). The embryo was washed in phosphate-buffered saline (PBS; Welgene) thrice, and the embryo was dissociated into single cells by trypsin/EDTA (Gibco). These cells were maintained in the Dulbecco's modified Eagle's medium (DMEM; Gibco) with 15% fetal bovine serum (FBS) (HyClone), 1× penicillin/streptomycin/glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), and 1 mM β-mercaptoethanol (Gibco). One day before virus infection, 1 × 105 CEFs were seeded in one well of a six-well plate. These CEFs were infected with retroviruses containing the reprogramming factors OCT4, SOX2, KLF4, c-MYC, and NANOG. Twenty-four hours after transduction, the infected CEFs were plated onto MEF feeder cells in a serum replacement (SR)-based medium [DMEM (Gibco), supplemented with 20% knockout SR (Gibco), 1× penicillin/streptomycin/glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 1 mM β-mercaptoethanol (Gibco), 10 ng/mL bFGF (Invitrogen), and 1,000 U LIF (Milipore)]. Eight days after transduction, colonies were selected and plated on new feeder cells in the SR-based medium or FBS-based medium [DMEM (Gibco), supplemented with 15% FBS (HyClone), 1× penicillin/streptomycin/glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 1 mM β-mercaptoethanol (Gibco), 10 ng/mL bFGF (Invitrogen), and 1,000 U LIF].
AP staining
For AP staining, cells were fixed with 4% paraformaldehyde for 1 min at room temperature (RT), washed with 0.05% Tween-20 (Sigma)/ PBS, and treated with AP (Sigma) solution for 15 min at RT.
Immunostaining
For immunostaining, cells were fixed with 4% paraformaldehyde for 20 min at RT. After the cells were washed with PBS, they were treated with PBS containing 10% normal goat serum and 0.03% Triton X-100 (Sigma) for 45 min at RT. The primary antibodies used were anti-SSEA1 (monoclonal, 1:200; STEMCELL). For detection of primary antibodies, fluorescently labeled (Fluorescein-conjugated goat anti-mouse antibody; STEMCELL) secondary antibodies (Thermofisher Scientific) were used according to the specifications of the manufacturer.
RNA isolation and reverse transcription polymerase chain reaction analysis
The total RNA was isolated using the RNeasy Mini Kit (Qiagen) and was treated with DNase (NEB) to remove genomic DNA contamination. One microgram of total RNA was reverse-transcribed with SuperScript III Reverse Transcriptase Kit (Invitrogen) and oligo (dT) primer (Invitrogen) according to the manufacturer's instructions.
The primers used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) were as follows: cNanog (187 bp) sense 5′-CAGCAGACCTCTCCTTGACC-3′, cNanog antisense 5′-TTCCTTGTCCCACTCTCACC-3′; cSox2 (193 bp) sense 5′-GCAGAGAAAAGGGAAAAAGGA-3′, cSox2 antisense 5′-TTTCCTAGGGAGGGGTATGAA-3′; and cACTB (282 bp) sense 5′-ACGTCGCACTGGATTTCGAG-3′, cACTB antisense 5′-TGTCAGCAATGCCAGGGTAC-3′.
Electron microscopy
For transmission electron microscope (TEM) observations, the samples were fixed in 4% paraformaldehyde (Sigma) and 2.5% glutaraldehyde (Sigma) in 0.1 M phosphate (Sigma) buffer overnight. After washing in 0.1 M phosphate buffer, the samples were postfixed for 1 h in 1% osmium tetroxide (Sigma) prepared in the same buffer. The samples were dehydrated with a series of graded ethyl alcohol. The samples were embedded in Epon 812, and polymerization was performed at 60°C for 3 days. Ultrathin sections (60–70 nm) were obtained by ultramicrotome (Leica Ultracut UCT). Ultrathin sections collected on grids (200 mesh) were examined in TEM (JEM 1010) operating at 60 kV, and images in the TEM were recorded by the charge-coupled device (CCD) camera (SC1000; Gatan).
Results
Generation and characterization of ciPSLCs
We isolated CEFs from 11-day-old Leghorn embryos, which were subjected to viral induction for iPSC generation (Fig. 1A). To determine the transduction efficiency of retrovirus, we first infected CEFs with retrovirus encoding green fluorescent protein (GFP). After 2 days, almost all CEFs expressed GFP (Fig. 1A), indicating that the retroviral system could efficiently transfer transgene into CEFs. Next, CEFs were infected with retroviruses containing the five inducing human transcription factors (OCT4, SOX2, KLF4, c-MYC, and NANOG). The CEFs transformed with the human reprogramming factors showed morphological changes and formed colonies at day 8 following viral infection (Fig. 1B). ciPSC-like colonies were selected and transferred onto new feeder-layered dishes and cultured in two different media as follows: SR-based medium and FBS-based medium supplemented with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF). ciPSLCs were efficiently maintained in the FBS-based medium; however, ciPSC-like colonies progressively died upon passaging in the SR-based medium. The established ciPSLCs were morphologically similar to chicken blastodermal cells (cBLCs) (Fig. 1D). They could be maintained and propagated for more than 10 passages (Fig. 1D). The ciPSLCs were positive for AP and SSEA1 staining (Fig. 2A, B). Moreover, these ciPSLCs expressed pluripotency-related genes such as cNanog and cSox2, similar to cBLCs (Fig. 2C and Supplementary Fig. S1; Supplementary Data are available online at
Generation of chicken iPSCs from CEFs using human reprogramming factors.
Characteristics of ciPSLCs. ciPSLCs were positive for
Mitochondrial remodeling in ciPSLCs
Next, we sought to determine the changes in intracellular organelles during reprogramming of CEFs. Intracellular organelles in ciPSLCs, cBLCs, and CEFs were examined by electron microscopy (Fig. 3A, B). The TEM images showed that the proportion of nuclei in chicken PSCs were much higher than that in the CEFs. The cytoplasm to nucleus ratios of CEFs (2.341) were higher compared with ciPSLCs (1.325) and cBLCs (1.642) (Fig. 3C). The morphology of mitochondria was more distinct in CEFs and pluripotent cells. The mitochondria of CEFs were tubule-like structures and contained matured cristae (Fig. 3B). However, the reprogrammed ciPSLCs contained globular mitochondria with immature cristae, similar to that in cBLCs. The maximum (Max) and minimum (Min) axes of mitochondria (outer membrane) in ciPSLCs, cBLCs, and CEFs were measured for precise comparison between cells (Fig. 3D). The Max/Min axis ratios of mitochondria were 8.937 ± 0.215, 2.557 ± 0.744, and 1.842 ± 0.508 in CEFs, cBLCs, and ciPSLCs, respectively (Fig. 3D), indicating that tubule like and mature mitochondria in CEFs were transformed into bilobular and immature mitochondria during pluripotent reprogramming. Taken together, these results suggest that avian PSCs contain immature mitochondria, similar to those in mammalian PSCs and mature organelles in avian somatic cells, which could be modeled into pluripotent state by reprogramming.
Electron microscopy image of ciPSLCs, cBLCs, and CEFs.
Discussion
In this study, we generated ciPSLCs from CEFs using human reprogramming factors. The ciPSLCs were positive for AP staining and expressed SSEA1. Moreover, ciPSLCs expressed endogenous chicken pluripotency markers such as cNanog and cSox2. The ciPSLCs could be efficiently maintained in the FBS-based medium, but not in the SR-based medium. Since the generation of the first avian iPSC-like cells in quail, two different groups have reported ciPSCs [8,12]. ciPSCs could not be maintained in the conventional cESC medium. Lu et al. used KO-DMEM supplemented with bFGF, FBS, and chicken serum, which supported ciPSCs up to 40 passages, but ciPSCs failed to proliferate in the SR-based medium [8]. In contrast, Dai et al. used KO-DMEM supplemented with LIF, FBS, two inhibitors (PD0325901 and CHIR99021), and A83-01, which supported iPSCs for 20 passages [12]. These reports suggest that avian iPSCs fail to proliferate in long-term culture in the SR-based medium. In the present study, we also observed that the FBS-based medium could efficiently maintain ciPSLCs.
The composition and structure of intracellular organelles are associated with the cell types and developmental stage [13,14]. The main source of energy metabolism also varies according to cell types. In particular, the morphology and function of mitochondria were closely associated with the energy metabolism of a given cell. Our previous study demonstrated that cellular organelles and metabolic systems changed during reprogramming and differentiation in mammalian cells [9]. PSCs, such as mouse ESCs and iPSCs, contain round mitochondria with immature cristae and produce energy mainly through anaerobic glycolysis. In contrast, somatic cells or differentiated cells from PSCs contain elongated mitochondria with mature cristae and largely depend on aerobic metabolism for energy production. We observed the ultrastructure of chicken pluripotent (cBLCs and ciPSLCs) and somatic cells (CEFs) by electron microscopy. Interestingly, as in mammals, chicken PSCs (cBLCs and ciPSLCs) also contained globular mitochondria with immature cristae and somatic cells (CEFs) contained elongated mitochondria. The ciPSLCs established in this study formed flat colonies like mouse epiblast stem cells (EpiSCs), a primed pluripotent cell type. Primed and naive PSCs showed a difference in mitochondrial morphology; the Max/Min ratios of mitochondria were a bit higher in EpiSCs than ESCs or iPSCs [9]. Although the ciPSLCs formed flat colonies, the Max/Min ratios of mitochondria were 5 times and 1.4 times lower than that in CEFs and cBLCs, respectively. As ciPSCs contained globular mitochondria as shown in mouse PSCs, anaerobic glycolysis should be essential for maintaining ciPSCs. In this study, we demonstrated that the morphology of mitochondria dynamically changed from elongated to round shape during reprogramming from CEFs to ciPSLCs.
Footnotes
Acknowledgments
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (grant no. 2015R1A2A2A01003604) and the Next-Generation BioGreen 21 Program (grant no. PJ01133802) funded by the Rural Development Administration, Republic of Korea.
Author Disclosure Statement
No competing financial interests exist.
References
Supplementary Material
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