Reprogramming efficiency and pluripotency of mule iPSCs over its parents

Jia Zhang; Lixia Zhao; Yuting Fu; Fangyuan Liu; Zixin Wang; Yunxia Li; Gaoping Zhao; Wei Sun; Baojiang Wu; Yongli Song; Shaohua Li; Chunxia Hao; Bilige Wuyun; Rihan Wu; Moning Liu; Guifang Cao; Buhe Nashun; M Azim Surani; Qingyuan Sun; Siqin Bao; Pentao Liu; Xihe Li

doi:10.1093/biolre/ioad041

. 2023 Apr 11;108(6):887–901. doi: 10.1093/biolre/ioad041

Reprogramming efficiency and pluripotency of mule iPSCs over its parents^{^†}

Jia Zhang ^1,^2,^#, Lixia Zhao ^3,^#, Yuting Fu ^4,^5,^#, Fangyuan Liu ^6,⁷, Zixin Wang ⁸, Yunxia Li ⁹, Gaoping Zhao ^10,¹¹, Wei Sun ^12,¹³, Baojiang Wu ^14,¹⁵, Yongli Song ^16,¹⁷, Shaohua Li ¹⁸, Chunxia Hao ^19,²⁰, Bilige Wuyun ^21,²², Rihan Wu ^23,²⁴, Moning Liu ²⁵, Guifang Cao ²⁶, Buhe Nashun ²⁷, M Azim Surani ²⁸, Qingyuan Sun ²⁹, Siqin Bao ^30,^31,^✉, Pentao Liu ^32,^33,^✉, Xihe Li ^34,^35,^36,^✉

¹ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

² Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

³ College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010110, China

⁴ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

⁵ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

⁶ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

⁷ Clinical Medical Research Center, The Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010050, China

⁸ Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China

⁹ Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China

¹⁰ Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China

¹¹ National Center of Technology Innovation for Dairy Industry, Hohhot 010020, China

¹² Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China

¹³ National Center of Technology Innovation for Dairy Industry, Hohhot 010020, China

¹⁴ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

¹⁵ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

¹⁶ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

¹⁷ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

¹⁸ College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010110, China

¹⁹ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

²⁰ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

²¹ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

²² Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

²³ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

²⁴ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

²⁵ Key Laboratory of Basic Veterinary Medicine, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

²⁶ Key Laboratory of Basic Veterinary Medicine, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

²⁷ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

²⁸ Wellcome Trust/Cancer Research UK Gurdon Institute, Henry Wellcome Building of Cancer and Developmental Biology, Cambridge CB2 1QN, UK

²⁹ Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou 510317, China

³⁰ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

³¹ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

³² School of Biomedical Science, Stem Cell and Regenerative Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong 999077, China

³³ Centre for Translational Stem Cell Biology, Building 17W, The Hong Kong Science and Technology Park, Hong Kong 999077, China

³⁴ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China

³⁵ Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China

³⁶ Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China

^✉

Correspondence: Siqin Bao, Email: baosq@imu.edu.cn; Pentao Liu, Email: pliu88@hku.hk; Xihe Li, Email: lixh@imu.edu.cn

Authors Jia Zhang, Lixia Zhao, and Yuting Fu contributed equally to this work.

PMCID: PMC10266948 PMID: 37040346

Abstract

The mule is the interspecific hybrid of horse and donkey and has hybrid vigor in muscular endurance, disease resistance, and longevity over its parents. Here, we examined adult fibroblasts of mule (MAFs) compared with the cells from their parents (donkey adult fibroblasts and horse adult fibroblasts) (each species has repeated three independent individuals) in proliferation, apoptosis, and glycolysis and found significant differences. We subsequently derived mule, donkey, and horse doxycycline (Dox)-independent induced pluripotent stem cells (miPSCs, diPSCs, and hiPSCs) from three independent individuals of each species and found that the reprogramming efficiency of MAFs was significantly higher than that of cells of donkey and horse. miPSCs, diPSCs, and hiPSCs all expressed the high levels of crucial endogenous pluripotency genes such as POU class 5 homeobox 1 (POU5F1, OCT4), SRY-box 2 (SOX2), and Nanog homeobox (NANOG) and propagated robustly in single-cell passaging. miPSCs exhibited faster proliferation and higher pluripotency and differentiation than diPSCs and hiPSCs, which were reflected in co-cultures and separate-cultures, teratoma formation, and chimera contribution. The establishment of miPSCs provides a unique research material for the investigation of “heterosis” and perhaps is more significant to study hybrid gamete formation.

Keywords: mule, donkey, horse, iPSCs, pluripotency

We found that mule adult fibroblasts have significant advantages in reprogramming efficiency compared with donkey and horse adult fibroblasts, and the establishment of mule iPSCs provides an accessible in vitro model to study hybrid gamete formation.

Graphical Abstract

Introduction

As the interspecific horse and donkey hybrid, mule has better muscular endurance, cognitive ability, body size, disease resistance, and longevity than its parents [1]. Regarding the biological phenomenon of hybrid vigor or “heterosis,” hybrid breeding has been applied to crops and livestock to improve their yield and quality [2–5]. Fibroblasts reprogramming to iPSCs has still been inefficient, which may be related to cell proliferation, metabolism, and apoptosis. Accordingly, different cell types typically exhibit different cell cycle structures and metabolism, which are critical for efficient reprogramming [6, 7]. Likewise, apoptosis occurs in the first few days after reprogramming, and this phenomenon may contribute to low efficiency of human iPSC derivation [8]. So, we want to know whether these characteristics of mule, donkey, and horse adult fibroblasts (MAFs, DAFs, and HAFs) impact their reprogramming efficiency.

The mammalian embryonic/adult fibroblasts can be converted to iPSCs by introducing defined sets of transcription factors; iPSCs exhibit similar morphology and growth properties to embryonic stem cells (ESCs) and expression of pluripotent genes as in ESCs [9–12]. The iPSCs maintain an undifferentiated state and can generate cells of all three germ layers in vivo and in vitro as ESCs [13, 14]. With self-renewal and unlimited proliferation potential, iPSCs may be easy to be genetically manipulated, for example, are used to produce transgenic, chimeric, and knockout domestic animals [15]. Fetal bovine serum, together with leukemia inhibitor factor (LIF) with the use of feeder cells, was the condition for maintaining pluripotency of first mouse iPSCs [11]. A more defined culture condition 2i/LIF is developed for the derivation and maintenance of mouse ESCs [16]. Although pig [17], bovine [18], and ovine [19] iPSCs have been established with small molecule inhibitors, it is still a challenge to establish iPSCs of other large livestock species, such as the mule. As a representative ungulate, horse iPSCs that depend on exogenous reprogramming transcription factors have been established [20–24]. Recently, Wu et al. [25] established horse ESCs and transgene-free iPSCs, named FTW-eqPSCs, which are permissive to direct induction of PGC-like cells in vitro and potentially contribute to intra- or interspecies chimeras in vivo. However, other equine iPSCs such as donkey and mule iPSCs have not been reported. Here, we determined whether there are differences in pluripotency and differentiation of iPSCs among donkey, horse, and mule.

In this study, we first compared the proliferation and the expression of apoptosis and glycolysis-related genes of MAFs, DAFs, and HAFs in all of the three independent individuals of each species. Subsequently, Dox-independent iPSCs of mule, donkey and horse (miPSCs, diPSCs, and hiPSCs) were successfully established and we characterized its pluripotency. The establishment of miPSCs provides unique research materials for the investigation of “heterosis,” and perhaps is more significant to study hybrid gamete formation.

Materials and methods

Animals

All the MAFs, DAFs, and HAFs were established from ear in our own experimental base (Inner Mongolia Saikexing Institute, Hohhot) and other cooperative farms (Table 1).

Table 1.

Information of MAFs, DAFs, and HAFs used in this study

Species	Age (years old)	Tissue origin	Cell lines	Passage for reprogramming	Establishment efficiency (%) of iPSCs
Mule #1	14	Ear	MAFs #1	5	78.3
Mule #2	19	Ear	MAFs #2	5	83.3
Mule #3	19	Ear	MAFs #3	5	79.2
Donkey #1	4	Ear	DAFs #1	5	58.2
Donkey #2	3	Ear	DAFs #2	5	62.5
Donkey #3	7	Ear	DAFs #3	5	54.2
Horse #1	4	Ear	HAFs #1	5	47.9
Horse #2	7	Ear	HAFs #2	5	50
Horse #3	10	Ear	HAFs #3	5	54.2

Open in a new tab

Ethics statement

All animal experiments were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Inner Mongolia University, China. The approval number is “NMGDX (Wu) 2022-0002.”

Reprogramming MAFs, DAFs, and HAFs to iPSCs

MAFs, DAFs, and HAFs were planted on T75 culture flask and cultured in M10 medium (Supplementary Table S1). They were dissociated with TrypLE Select (Gibco, 12,563–029) and harvested for electroporation at 70–80% confluence (~1.0 × 10⁶ cells per experiment). The transfections were performed using an Amaxa Nucleofector machine (Lonza) according to the manufacturer’s protocol (Basic Nucleofector Kit for Primary Mammalian Fibroblasts, VPI-1001, program U-023), with 6.0 μg DNA (3.0 μg PB–TRE-pOMSK (porcine OCT4, MYC, SOX2, and KLF4) [17], 1.0 μg PB–TRE–hRL (human RARG and LRH1) [26], 1.0 μg PB–EF1a–transposase, and 1.0 μg PB–EF1a–rTTA). After transfection, 0.25 million MAFs, DAFs, and HAFs were seeded on mitomycin-inactivated STO feeders in M15 medium (Supplementary Table S1) supplemented with Dox (1.0 μg ml⁻¹, Clontech, 631,311) in 10-cm dishes. The culture media was changed every other day, and the colonies were picked in M15 medium supplemented with Dox at days 10–14 and maintained in the same medium. Reprogramming efficiencies were then determined by calculating the number of colonies, respectively, in metabolite treated conditions (2-deoxy-D-glucose (2-DG; MACKLIN, D807272); D-fructose-6-phosphate (F6P; Medbio, MED21625)) relative to controls. After two passages, the colonies with endogenous core pluripotent makers OCT4, SOX2, and NANOG activated by RT-qPCR assay were selected for establishing transgene independent iPSCs lines in different culture conditions after Dox removing.

Preparing mule, horse, donkey, and STO feeders

Thaw a vial of frozen live MAFs, DAFs, and HAFs and STO SNL cells (5 million cells), respectively, to two 15-cm plates (gelatinized) with 20 ml M10 medium. After 5-day culture, the cells were passage from 2 to 32 plates (gelatinized). When the plates become confluent, add 0.37 ml Mitomycin-C (Sigma, M0503-5X2MG) solution to each plate and incubate for 3 h. The feeder cells were passaged by a brief PBS wash followed by treatment for 4 min with TrypLE Select. Then the feeder cells were dissociated and centrifuged (1300 r min⁻¹ × 3 min) in M10 medium. After removing supernatant, the collected feeder cells were counted and froze in cryopreservation medium. Before cryo-recovery, feeder plates should be prepared at least 2 days in advance at a density of 1.2 × 10⁴ cells per well (24-well plate).

Screening for the culture conditions of miPSCs, diPSCs, and hiPSCs

Dox-dependent mule, donkey, and horse iPSCs^+Dox with endogenous core pluripotent makers OCT4, SOX2, and NANOG activated were dissociated in TrypLE Select, and seeded in 24-well feeder plates at a density of 1.2 × 10⁴ cells per well. The cells were cultured in M15 medium supplemented with Dox, and then switched to candidate medium withdrawing Dox. The candidates were SERUM/LIF [27], 2i/LIF [16], t2iLIF + Gӧ [28], 4i/LIF [29], 5i/LIF/A [30], and mouse EPSC medium (mEPSCM) [31]. Small molecules and cytokines were supplemented as indicated at the following final concentrations: CHIR99021 (GSK3 inhibitor), 3 μM; PD0325901 (MEK inhibitor), 1 μM; Gӧ6983 (PKC inhibitor), 5 μM; SP600125 (JNK inhibitor), 4 μM; SB203580 (P38 inhibitor), 10 μM; Y27632 (ROCK inhibitor), 10 μM; SB590885 (BRAF inhibitor), 0.5 μM; WH-4-023 (SRC inhibitor), 0.5 μM; XAV939, 5.0 μM; vitamin C, 50 μg ml⁻¹; LIF, 1000 U ml⁻¹ and Activin A, 20.0 ng ml⁻¹. The medium was refreshed daily, and the surviving cells were passaged every 2 days. Endogenous OCT4, SOX2, and NANOG expression levels were checked after 6 days.

Culturing MAFs, DAFs, and HAFs, and miPSCs, diPSCs, and hiPSCs

MAFs, DAFs, and HAFs were cultured in M10 medium. miPSCs, diPSCs, and hiPSCs were maintained on feeder layers, and enzymatically passaged every 2–3 days by a brief PBS (Gibco, 14,190–144) wash followed by treatment for 4 min with TrypLE Select. The cells were dissociated and centrifuged (1300 r min⁻¹ × 3 min) in K10 medium (Supplementary Table S1). After removing supernatant, the miPSCs, diPSCs, and hiPSCs were resuspended and seeded in SERUM/LIF medium (Supplementary Table S1). All cell cultures in this paper were performed under conditions of 38.5°C and 5% CO₂ unless stated otherwise. MAFs, DAFs, HAFs, miPSCs, diPSCs, and hiPSCs were frozen once they are ~80% confluent using cryopreservation medium, which contains 90% FBS and 10% (vol/vol) DMSO (Sigma, D2650). Before cryorecovery, feeder plates should be prepared at least 2 days in advance at a density of 1.2 × 10⁴ cells per well (24-well plate).

AP staining

Before staining, cells were placed in 4-well plates and washed with 1 × PBS, then fixed in 4% paraformaldehyde (PFA) (Solarbio, P1110–100) at room temperature for 10 min and washed with 1 × PBS again followed by adding alkaline phosphatase (AP) staining solution. AP staining solution was prepared as following: gently mixed 50 μl sodium nitrite solution with 50 μl FRValkaline solution and placed the mixture at 37°C for 3 min, next added 2.25 ml H₂O (Sigma, W1503–500) and 50 μl naphthol-As-BI alkaline solution into the mixture, finally mixed gently and incubated staining solution with fixed cells in the dark for overnight.

Karyotype

The tested cells were incubated with 0.2 μg ml⁻¹ colchicine in culture medium for 2.5 h and dissociated by TrypLE Select, then centrifuged at 1300 r min⁻¹ for 3 min to collect the tested cells. The cells were gently resuspended in 8 ml 0.075 mol l⁻¹ KCL (Sigma) and incubated in 37°C water bath kettle for 40 min for hypotonic treatment. Stationary liquid (methanol:glacial acetic acid = 3:1) of 1 ml was subsequently added to the resuspended cells and mixed gently followed by centrifuging at 1000 r min⁻¹ for 10 min. After discarding supernatant, the cells were mixed gently in 8 ml stationary liquid and incubated in 37°C water bath kettle for 30 min for cell fixation, which repeated twice. Then resuspended the cells with 0.5 ml stationary liquid and dripped the resuspended cells on ice cold glass slides, followed by drying the glass slides for 1 h in 70°C drying oven. The glass slides were stained in Giemsa (Sigma) for 10 min and washed by distilled water, then air-dried following analyzed by LUCIA Cytogenetics.

Telomerase activity assay

The telomerase activity of cells was tested by mule, donkey, and horse (TelomeraseTE) ELISA Kit (COIBO, CB10290-ML; COIBO, CB10183-DK; and COIBO, CB10121-HS). Take the standard density as the horizontal, the OD value for the vertical, draw the standard curve on graph paper. Find out the corresponding density according to the sample OD value by the sample curve, multiplied by the dilution multiple, or calculate the straight-line regression equation of the standard curve with the standard density and the OD value. With the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.

In vitro EB formation assay of miPSCs, diPSCs, and hiPSCs

In vitro differentiation, miPSCs, diPSCs, and hiPSCs were detached from culture dishes using TrypLE Select, resuspended in M10 medium, and then seeded into ultralow cell attachment U-bottom 96-well (Corning, 7007). After 3 days in suspension culture, EBs were transferred to gelatin-coated dishes and cultured for another 3 days prior to linage genes RT-qPCR and immunostaining.

In vivo teratoma assay of miPSCs, diPSCs, and hiPSCs

miPSCs, diPSCs, and hiPSCs were resuspended in PBS supplemented with 30% Matrigel (Corning, 354,230). miPSCs, diPSCs, and hiPSCs were injected subcutaneously into 8-week-old female immunodeficiency NOD-SCID mice (Beijing Charles River) (150 μl per injection). miPSCs and diPSCs formed visible teratomas within 4 and 6 weeks. When the size of the teratomas reached ~1.2 cm³, they were collected and processed for sectioning.

Generation of chimera

In all, 10–15 tdTomato⁺ miPSCs and diPSCs were injected gently into the ICR mice (Beijing Charles River) blastocoel cavity using a piezo-assisted micromanipulator attached to an inverted microscope (Zeiss, Eppendorf) and recover the injected blastocysts in KSOM medium (Millipore), then transplant the chimeric blastocysts into the uterus of pseudopregnant ICR female mice at 2.5 days post-coitus (dpc), the protocol was performed as previously described [31]. The embryos were isolated at embryonic stage E6.5 to check chimeric contribution. And also ~20 tdTomato⁺ miPSCs and diPSCs were injected into the ICR mice gastrula-stage embryos of the epiblast. Embryos were cultured in rat serum (VivaCell, C2570) and SERUM/LIF mixture medium (1:1) as previously described [32] for 48 h in vitro at 37°C in a 5% CO₂ atmosphere.

Immunofluorescence staining

Cells for immunofluorescence assays were washed with PBS and fixed in 4% PFA for 10 min at room temperature, and following permeabilized with 0.1% Triton X-100 (Sigma) and 1% BSA (Sigma) in PBS for 30 min. Then incubated cells with primary antibodies at 4°C overnight. The cells were subsequently washed three times in 1% BSA, 0.1% Triton X-100 in PBS for 5 min per wash, and incubated with secondary antibodies for 1 h at room temperature in the dark, then washed once for 5 min in 1% BSA, 0.1% Triton X-100 in PBS, and twice for 5 min in PBS. DAPI (Bioss, C02–04002) was used to stain nuclei. The slides were imaged with a confocal microscope FLUOVIEW FV1000 (Olympus). For immunofluorescence staining of cryo-sections of mouse chimeric embryos, after 48-h culture in vitro, embryos were immersed in 4% PFA for 2 h and in 20% sucrose (Sigma) at 4°C overnight. Next, embryos were embedded in mixture of Tissue-Tek O.C.T. compound (SAKURA, 4583) and 20% sucrose for 1 h, at last for liquid nitrogen quick freezing. The frozen OCT blocks were sectioned by CRYOSTAR NX50 (Thermo) at 10 μm each section. Sections were first permeabilized with 0.1% Triton and blocked with 5% donkey serum (Solarbio, SL050) plus 1% BSA followed by incubations with primary antibodies at 4°C overnight. Fluorescence-conjugated secondary antibodies were used to incubate the slides at room temperature for 1 h. DAPI was used to stain nuclei. The antibodies are listed in Supplementary Table S2.

RT-qPCR analysis

Total RNA was isolated using a RNeasy Mini Kit (Qiagen, 74,104) for cultured cells. Complementary DNA (cDNA) was prepared using HiScript Q RT SuperMix for qPCR (Vazyme, R223–01). RT-qPCR primers are listed in Supplementary Table S3. KAPA SYBR FAST Universal qPCR Kit (Roche-KAPA, KK4601) were used for RT-qPCR assays. All RT-qPCR reactions were performed on Veriti 96 cell Thermal Cycler (Applied Biosystems, USA). Gene expression was determined relative to GAPDH using the ΔΔCt method. Data are shown as the mean and SD.

Cell cycle analysis

MAFs, DAFs, and HAFs were dissociated into single cells using TrypLE Select and collected using centrifugation. After being incubated with DAPI for 1 h at 37°C in the dark, the cell suspension was filtered using a cell strainer (FACSAria II, BD Biosciences) to remove large clumps of cells. The cells were then performed flow cytometry (FACSAria II, BD Company). Because of the different DNA content corresponding to different phases of the cell cycle, within the G1/G0 phase, normal cells usually have the DNA content of diploid cells (2 N), whereas within the G2/M phase, cells have the DNA content of tetraploid cells (4 N), and the DNA content within S phase is between diploid and tetraploid. DAPI can bind with DNA, and its fluorescence intensity directly reflects the content of DNA in cells. Therefore, when detecting the DNA content in cells by flow cytometry DAPI staining method, the cell cycle phases can be divided into G1/G0 phase, S phase, and G2/M phase. The cell percentage of each cell cycle corresponding to the obtained flow histogram can be calculated by FlowJo software.

Transcriptome analysis

Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. After quality control by the Fragment Analyzer (Advanced Analytical), 1–2 μg of RNA was used to isolate mRNA according to the NEBNext Poly (A) mRNA Magnetic Isolation Module. Then, RNA-seq libraries were constructed under the manufacturer’s instructions of the NEBNext ultra-RNA library prep kit for Illumina (NEB). The generated libraries were pooled and sequenced on Illumina NovaSeq 6000 following the vendor’s recommended protocol platforms with 150-bp paired-end mode (sequenced by Novogene). Raw data were first processed to remove reads with more than 20% low-quality bases and to remove adaptors. Then the clean data were mapped to the equus asinus and equus caballus reference genome. FeatureCounts (v1.5.0-p3) was used for reads counting, and then Fragments Per Kilobase of transcript sequence per Millions of each gene was calculated to estimate gene expression levels. Differentially expressed genes (DEGs) in different samples were determined using the DEseq2 R package (v1.16.1) with fold change ≥ 1 and adj P-value ≤ 0.05. Bar plots and bubble plots were plotted by using the ggplot2 R package (v1.0.12). Heatmaps of select genes were plotted by using the pheatmap R package (v3.3.5). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was implemented by the clusterProfiler R package (v4.1.0). Gene set enrichment analysis (GSEA) analysis was performed with the GSEA software (v4.0.3) developed by the Broad Institute.

Statistical analysis

Descriptive statistics were generated for all quantitative data, expressed as mean ± SD. The mean ± SD values were calculated from three samples per group and three technical replicas for per samples. Significance between each group was measured using unpaired two-tailed Student t-test, and a value of P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism, v.5.0 (GraphPad Software).

Results

Cell proliferation and reprogramming of mule fibroblasts compared with donkey and horse

The establishment of iPSCs from fibroblasts has been inefficient, and the reprogramming of different cell types may affect the reprogramming efficiency. Considering the “heterosis” of the mule, we wish to identify whether MAFs have advantages in reprogramming compared with the cells from their parents. It was reported that improving cell proliferation increases reprogramming efficiency, whereas cell cycle arrest inhibits successful reprogramming [33]. First, we cultured and compared different cell lines of MAFs, DAFs, and HAFs from three independent individuals of each species under the same condition, and found that MAFs proliferated faster than DAFs and HAFs (Figure 1A). Cell cycle analysis revealed that MAFs had a higher percentage of S phase (16.6%) compared with DAFs (6.99%) and HAFs (11.54%) (Figure 1B).

Furthermore, global gene expression profiles of MAFs, DAFs, and HAFs were determined using RNA-seq. To examine differences among MAFs, DAFs, and HAFs, DEGs (Supplementary Table S7) were analyzed using the Deseq2 R package (adj P < 0.05, fold change |log2Ratio| ≥ 0). As expected, KEGG and GO enrichment analysis of upregulated DEGs in MAFs revealed that biological processes and pathways such as DNA replication, cell cycle, and cell cycle phase transition were enriched, compared with DAFs and HAFs (Figure 1C and Supplementary Figure S1A). In addition, we screened out some significantly upregulated genes in MAFs compared with both DAFs and HAFs. And by KEGG online analysis, we knew that these screened representative upregulated genes may have effects on some signaling pathways associated with cell proliferation, cell cycle, and DNA repair (Figure 1D). It is well-known that HIPPO/YAP signaling is involved in regulating anti-apoptosis and pro-proliferation genes, and YAP overexpression promotes proliferation and suppresses apoptosis [34]; MAPK/ERK and PI3K-AKT signaling pathways are reported to be associated with the cell proliferation and DNA repair [35, 36] and activating WNT signaling can improve cellular survival and suppress apoptosis [37]. The differences in these signaling pathways may account for the result that MAFs proliferated significantly faster than DAFs and HAFs. Together, these results suggest that MAFs appear to have more advantages in cell viability than DAFs and HAFs.

We next examined other characteristics of fibroblasts, which may affect reprogramming efficiency. It was reported that apoptosis plays a vital role in reprogramming fibroblasts, as verified by the findings that blocking apoptosis during reprogramming enhanced the derivation of iPSCs [6, 38]. To address whether apoptosis influences the reprogramming of MAFs, DAFs, and HAFs, RT-qPCR was performed to examine some apoptosis-related genes from three independent individuals of each species. The pro-apoptotic genes BAX, FAS, DFFA, CASP3, CASP4, CASP6, and CASP8 were all highly expressed in DAFs and HAFs, compared with MAFs (Figure 1E and Supplementary Figure S1B), which may hinder the reprogramming of DAFs and HAFs.

Furthermore, Panopoulos et al. [7] identified metabolites that differ between iPSCs and ESCs, revealing novel metabolic pathways that play a critical role in regulating somatic cell reprogramming, and somatic cells with higher glycolytic metabolism displayed higher reprogramming efficiency than their highly oxidative counterparts. Here, we noticed that some glycolytic-related genes, PGM1, LDHA, PFKP, HK2, and ALDH3B2, have significantly higher expression in MAFs than in DAFs (Supplementary Figure S1C). GO analysis suggested that GO terms such as small molecule metabolic process, ATP metabolic process and glycolytic process, etc. were enriched in the DEGs between MAFs and DAFs (Figure 1F). In line with the GO analysis, GSEA revealed that glycolysis-related genes had higher expression in MAFs than DAFs (Supplementary Figure S1D). For further verification, the RT-qPCR was performed on several essential glycolysis-related genes and showed high concordance with the RNA-seq data (Supplementary Figure S1E). However, there were no significant differences in glycolysis between MAFs and HAFs. To further confirm the influences of glycolysis on proliferation, the MAFs, DAFs, and HAFs were treated with glycolysis inhibitor, 2-DG (0.1 mM), as well as glycolysis stimulator, F6P (5 μM). After F6P treatment, MAFs, DAFs, and HAFs all proliferated faster, whereas MAFs, DAFs, and HAFs all do not proliferate after 2-DG treatment. Importantly, MAFs proliferated significantly faster than DAFs and HAFs after being treated with both F6P and 2-DG (Figure 1G).

To investigate the role of cell proliferation, apoptosis, and glycolysis in the reprogramming of fibroblasts, we expressed Dox-inducible six exogenous reprogramming factors, pOMSK (porcine OCT4, MYC, SOX2, and KLF4) and hRL (human RARG and LRH1) in MAFs, DAFs, and HAFs from three independent individuals of each species, delivered via piggyBac transposition (Figure 1H). After transfection, the number of mule iPSC colonies increased gradually, reaching its maximum numbers on day 14 (~150 colonies). In comparison, the number of donkey and horse iPSC colonies increased during the first 10 days and then decreased on day 14 because of differentiation or cell death (donkey ~60 colonies and horse ~50 colonies) (Figure 1I and Supplementary Figure S1F). In addition, our data showed that mule iPSC colonies have higher establishment efficiency than donkey iPSCs colonies and horse iPSCs colonies in all of three independent individuals of each species (Table 1). Furthermore, we treated MAFs, DAFs, and HAFs with F6P and 2-DG during reprogramming. The results showed that few colonies formed after 2-DG treatment, whereas F6P-treatment led to nearly two times as many reprogrammed colonies as in the untreated groups on day 14 (Supplementary Figure S1G and Supplementary Table S4). The 2-DG-treatment decreased reprogrammed colonies in all groups, but the mule cells still produced substantially more colonies than those from DAFs and HAFs (Supplementary Table S4). These results are in line with the reported findings that stimulating glycolysis could enhance nuclear reprogramming efficiency, whereas inhibiting glycolysis reduced reprogramming efficiency [39].

These results demonstrate that the differences of MAFs, DAFs, and HAFs in proliferation, apoptosis, and glycolysis may influence their reprogramming efficiency, which account for the advantages of MAFs in reprogramming.

Identification of culture conditions for Dox-independent iPSCs of mule compared with donkey and horse

After reprogramming, the mule, donkey, and horse iPSC colonies were picked on days 10–15, and passaged in single-cell suspension in M15 medium supplemented with Dox (Figure 1H). The passaged cells of mule, donkey, and horse all expressed high levels of key endogenous pluripotency genes, such as OCT4, SOX2, and NANOG (Figure 2A), and could be maintained undifferentiated in a Dox-containing medium on STO feeders for at least 30 passages, and they were thus named as mule, donkey, and horse iPSCs^+Dox. Four iPSC^+Dox lines of each breed mule, horse, and donkey were generated and examined, respectively (Supplementary Table S5). Upon Dox removal, mule iPSCs^+Dox differentiated rapidly within 6 days, concomitant with the increased expression of both embryonic and extraembryonic cell-linage genes and with the loss of exogenous reprogramming factors and endogenous pluripotency gene expression (Supplementary Figure S2A–D). Thus, the mule iPSCs^+Dox could maintain pluripotency through Dox-induced exogenous factors expression.

We next used mule iPSCs^+Dox, which highly expressed endogenous pluripotency genes, to identify a culture condition that could maintain endogenous pluripotency gene expression independent of Dox-induced exogenous factors expression. Several culture conditions for mouse and human ESCs were tested, including SERUM/LIF [27], 2i/LIF [16], t2iLIF + Gӧ [28], 4i/LIF [29], 5i/LIF/A [30], and mEPSCM [31]. Mule iPSCs^{+Dox #}43 (passage 15) were cultured under these conditions for 6 days without Dox and was morphologically and transcriptionally examined. The mule iPSC colonies showed a differentiated morphology except for SERUM/LIF (Figure 2B). In t2iLIF + Gӧ, 4i/LIF, and mEPSCM culture conditions, mule iPSCs rapidly lost OCT4 and NANOG expression on day 6, and expressed high levels of the lineage marker genes (Figure 2C and D). Consistently, in 2i/LIF and 5i/LIF/A, significant downregulation of pluripotency gene expression and high levels of differentiated genes were observed on day 6 (Figure 2C and D). Under SERUM/LIF culture condition, the Dox-independent mule iPSCs (miPSCs) remained undifferentiated for more than 50 passages and expressed high levels of endogenous pluripotency genes comparable to the mule iPSCs^+Dox (Figure 2C and D). We also successfully derived donkey and horse Dox-independent iPSCs (diPSCs and hiPSCs) without differentiation and could be passaged stably for long-term culture in the SERUM/LIF medium.

Furthermore, undifferentiated clonal morphology of diPSCs could be maintained when LIF is removed, although lineage marker genes are expressed after five passages (Figure 2E–G). Our results showed that diPSCs were sensitive to fibroblast growth factors, as most diPSCs were eventually differentiated after five passages when bFGF was added into SERUM/LIF medium, accompanied by downregulation expression of essential pluripotency genes, upregulation expression of both embryonic and extraembryonic cell-lineage genes and differentiated clonal morphology (Figure 2E–G). In addition, we also tested different feeders and demonstrated that miPSCs, diPSCs, and hiPSCs showed higher pluripotency on their respective feeders but became differentiated on STO feeders (Supplementary Figure S2E–G). The expression of pluripotency genes was significantly decreased on STO feeders, with high levels of three germ layer genes (Supplementary Figure S2E–G). Interestingly, those iPSCs cultured on STO feeders morphologically resembled trophoblast stem cells and expressed trophoblast specific markers such as GATA3, KRT7, and CDX2 (Supplementary Figure S2H).

We therefore successfully established Dox-independent miPSCs, diPSCs, and hiPSCs in SERUM/LIF conditions on feeders.

Typical pluripotency features and cell competition of miPSCs compared with diPSCs and hiPSCs

We have established four iPSC lines for each species (Supplementary Table S5), which had high nuclear: cytoplasmic ratios and formed compact colonies with smooth colony edges in SERUM/LIF medium (Figure 3A and Supplementary Figure S3A). These miPSCs, diPSCs, and hiPSCs were AP-positive and could be recovered efficiently after multiple freeze/thaw cycles (Figure 3A and Supplementary Figure S3A). The miPSCs, diPSCs, and hiPSCs were passaged every 2–3 days at a 1:4 ratio as single cells, with no change in morphology for more than 50 passages. These iPSCs expressed high levels of key endogenous pluripotency genes (Figure 3B and Supplementary Figure S3B) and had a normal karyotype after long-term passages (Figure 3C and Supplementary Figure S3C). Notably, the leaky expression of the exogenous reprogramming factors was not readily detected in RT-PCR in our miPSCs, diPSCs, and hiPSCs (Supplementary Figure S3D). Immunofluorescence analysis showed that OCT4, SOX2, NANOG, and KLF4 were all expressed with similar patterns in miPSCs, diPSCs, and hiPSCs (Figure 3D and Supplementary Figure S3E). In addition, the telomerase activity of miPSCs, diPSCs, and hiPSCs was significantly higher than their adult fibroblasts (Figure 3E), which was consistent with the study that telomerase expression is low or absent in most human somatic tissues, with its expression principally restricted in the tissue stem cells [40]. These results indicate that miPSCs, diPSCs, and hiPSCs displayed typical pluripotency features.

Typical pluripotency features of miPSCs and cell competition among these iPSCs. (A) The morphology and AP staining of miPSCs. miPSCs: mule Dox-independent iPSCs (scale bars, 100 μm). (B) Relative expression of key pluripotency genes *OCT4*, *SOX2,* and *NANOG* in miPSCs at various passages. The relative expression is normalized to MAFs and housekeeping gene *GAPDH*. Data are represented as the mean ± SD, n = 3 independent experiments. (C) The karyotype of miPSCs at passage 32 (38 out of 50 (76%) metaphase spreads examined were normal). (D) Immunostaining of OCT4, SOX2, NANOG, and KLF4 in miPSCs at passage 32. The nuclei were stained with DAPI (scale bars, 100 μm). (E) The telomerase activation (TE) level of samples was measured using TE ELISA Kit. The concentration of TE is determined by comparing the OD of the samples to the standard curve. Data are represented as the mean ± SD, n = 3 independent experiments. P-values were calculated by two-way ANOVA, ^*^*^*P < 0.001. (F) Schematic of co-cultured of miPSCs, diPSCs, and hiPSCs. miPSCs were labeled with H2B tdTomato, diPSCs were labeled with EGFP, and hiPSCs were not labeled. hiPSCs: horse Dox-independent iPSCs. (G) Growth curves of miPSCs (red line), diPSCs (green line), and hiPSCs (blue line) in co-cultures and separate-cultures under SERUM/LIF condition. Data are represented as the mean ± SD, n = 3 independent experiments. (H) Representative fluorescence images of miPSCs (red), diPSCs (green), and hiPSCs (the rest of blue (DAPI was used to stain nuclei) colonies besides red and green) in co-cultures and separate-cultures at day 5. hiPSCs are highlighted with dashed circles. The nuclei were stained with DAPI (scale bars, 100 μm).

In order to further compare the characteristics of miPSCs, diPSCs, and hiPSCs, we co-cultured miPSCs, diPSCs, and hiPSCs in vitro to examine interspecies cell competition. We labeled miPSCs and diPSCs with H2B tdTomato and Enhanced Green Fluorescent Protein (EGFP), respectively (Figure 3F), and hiPSCs were not labeled. The cell numbers of miPSCs, diPSCs, and hiPSCs were calculated in co-cultures and separate-cultures until they grew to confluency. We found that miPSCs, diPSCs, and hiPSCs could proliferate well in co-cultures for 5 days, but miPSCs still proliferated faster than diPSCs and hiPSCs in both co-cultures and separate-cultures (Figure 3G and H and Supplementary Figure S3F). Moreover, after 5 days of co-cultures, we passed these iPSCs and found that miPSCs, diPSCs, and hiPSCs all could still proliferate robustly (Supplementary Figure S3G).

Collectively, these results demonstrate that miPSCs, diPSCs, and hiPSCs exhibit molecular and cellular pluripotent features, and miPSCs proliferated faster than diPSCs and hiPSCs in both co-cultures and separate-cultures.

Transcriptome profiling of miPSCs/MAFs compared with diPSCs/DAFs and hiPSCs/HAFs

To gain further molecular insights into miPSCs, diPSCs, hiPSCs and their respective adult fibroblasts, we determined global gene expression profiles using RNA-seq. To examine differences in three iPSCs and their adult fibroblasts, we analyzed DEGs using the DEseq2 R package (adj P < 0.05, fold change |log2Ratio| ≥ 0). Venn analysis of DEGs between miPSCs, diPSCs, hiPSCs and their respective adult fibroblasts showed that 6484 DEGs in mule, 7322 DEGs in donkey, and 5786 DEGs in horse were screened, respectively (Figure 4A). In addition, compared with their respective adult fibroblasts, the pluripotency genes in miPSCs and diPSCs, such as POU5F1 (OCT4), SOX2, and NANOG, were all significantly increased, whereas the somatic cell genes, including ZEB1 and THY1, were significantly decreased (Figure 4B and Supplementary Figure S4A). GO analysis suggested that GO terms such as translation, DNA replication, and ribosome were all enriched in the DEGs between miPSCs vs MAFs and diPSCs vs DAFs (Figure 4C and Supplementary Figure S4B). KEGG pathway enrichment analysis showed the top 10 enriched pathways (Figure 4D and Supplementary Figure S4C), including cell cycle, DNA replication, focal adhesion, etc. Similar to the bEPSCs [18], DNA methyltransferase genes, DNMT1, DNMT3A, and DNMT3B, expressed at higher levels in miPSCs, diPSCs, and hiPSCs, whereas the expression of TET methylcytosine dioxygenases (TET3) was decreased, compared with their respective adult fibroblasts (Figure 4E).

We next compared our RNA-seq data of miPSCs, diPSCs, hiPSCs, MAFs, DAFs, and HAFs with the published data of FTW-eqESCs, FTW-eqiPSCs, and equine embryonic fibroblasts (EEFs) [25]. Pearson correlation analysis and cluster dendrogram analysis revealed that miPSCs, diPSCs, and hiPSCs clustered closer to FTW-eqESCs and FTW-eqiPSCs than MAFs, DAFs, HAFs, and EEFs (Figure 4F and Supplementary Figure S4D). Moreover, miPSCs/MAFs clustered closer to diPSCs/DAFs than to hiPSCs/HAFs (Figure 4F and Supplementary Figure S4D). Similarly, principal component analysis (PCA) showed that hiPSCs clustered together with FTW-eqESCs and FTW-eqiPSCs, but were distinct from miPSCs and diPSCs (Figure 4G). In addition, the full gene expression profiles from miPSCs and diPSCs were subjected to unsupervised clustering analysis that identified discrete segregation between the miPSCs and diPSCs (Supplementary Figure S4E). GO analysis showed that GO terms such as ribosome and translation were enriched in the DEGs between miPSCs and diPSCs (Supplementary Figure S4F). In line with the GO analysis, GSEA analysis showed that miPSCs had significantly higher expression of genes functioning in ribosome and translation than diPSCs (Supplementary Figure S4G).

These results revealed the differences between miPSCs and diPSCs in transcriptomic features, and miPSCs clustered closer to diPSCs than to hiPSCs.

Early germ lineages differentiation of miPSCs compared with diPSCs and hiPSCs

To functionally evaluate the differentiation of miPSCs, diPSCs, and hiPSCs, we assessed EB formation in vitro. EB spheres were formed as early as day 3 in suspension culture. Then the EB spheres were transferred into gelatin-coated plates and continued to culture for another 3 days (Supplementary Figure S5A). RT-qPCR analysis and immunostaining showed that the miPSCs, diPSCs, and hiPSCs differentiated into cells expressing all three germ layers and trophoblast genes (Figure 5A and B and Supplementary Figure S5B and C). Furthermore, teratoma formation assays in vivo showed that miPSCs and diPSCs differentiated into tissues from all three germ lineages (Figure 5C and Supplementary Figure S5D). β-TUBLIN III-, SMA-, and SOX17-positive cells were also found in miPSCs teratoma sections by immunostaining (Figure 5D). However, hiPSCs failed to form teratoma. Our results suggested that the miPSCs have higher potential for teratoma formation than diPSCs and hiPSCs, reflected by faster formation and higher formation efficiency (Table 2). Next, we tested the differentiation of miPSCs and diPSCs in interspecies chimeras, which measures whether donor PSCs can extensively colonize all three germ layer tissues after being introduced into mouse embryos [41]. The tdTomato-labeled miPSCs and diPSCs were injected into mouse blastocysts and then transplanted into 2.5 dpc pseudopregnancy female mice. In all, 105 embryos were collected at E6.5, and no chimera contributions were observed in both miPSCs and diPSCs (data not shown). Although incapable of colonizing preimplantation ICMs, it had been reported that mouse epiblast stem cells could readily incorporate and generate chimeras when grafted into postimplantation epiblasts followed by in vitro embryo culture [42]. The tdTomato-labeled miPSCs and diPSCs were then injected into gastrula-stage mouse embryos of the epiblast. After 48-h culture in vitro, miPSCs showed a higher significant shift and proliferated more extensively than diPSCs (Figure 5E, Supplementary Figure S5E, and Supplementary Table S6). To identify the descendants of donor miPSCs and diPSCs in specific tissues, immunofluorescence analysis was performed to detect lineage marker expression in the tdTomato⁺ cells in chimeras. The results revealed that tdTomato⁺ cells of miPSCs and diPSCs expressed markers of embryonic cell lineages, such as NESTIN, β-TUBULIN III, PAX6, BRACHYURY (T), and EOMES (Figure 5F and Supplementary Figure S5F). Interestingly, diPSCs could not differentiate into endoderm, as AFP and SOX17 were not detected in chimeric embryos, whereas miPSCs could (Figure 5F and Supplementary Figure S5F). Importantly, graft-derived cells expressed PGC-specific markers, BLIMP1 and STELLA, indicating that miPSCs and diPSCs undergo PGC differentiation in vitro (Figure 5F and Supplementary Figure S5F). Using RNA-seq analysis (adj P-value < 0.05, fold change |log2Ratio| ≥ 0), some significantly upregulated genes in miPSCs compared with both diPSCs and hiPSCs were screened out (Figure 5G). We speculated that these genes could mainly regulate FOCAL ADHESION, JNK and p38 MAP Kinase, MAPK and RAS key signaling pathways by KEGG online analysis. And it is also well-known that MAPK and JNK and p38 MAP Kinase signaling is involved in proliferation and differentiation, and RAS key signaling regulates transcription and cell growth [43–45].

Early germ lineages differentiation of miPSCs in vivo and in vitro. (A) Relative expression of lineage genes in miPSCs EBs (^#43) formation at day 6. The trophoblast cell markers include *CDX2*. The relative expression levels are normalized to *GAPDH*. miPSCs: mule Dox-independent iPSCs. Data are represented as the mean ± SD, n = 3 independent experiments. P-values were calculated by two-way ANOVA, ^*^*P < 0.01, ^*^*^*P < 0.001. (B) Immunostaining of NESTIN (ectoderm), SMA (mesoderm), GATA6 (endoderm), and CDX2 (trophoblast) in cells differentiated in vitro from miPSCs (^#43) at day 6. The nuclei were stained with DAPI (scale bars, 100 μm). (C) Teratoma formation of miPSCs (^#43). H&E analysis detected the presence of keratinized epithelium derived from ectoderm (i), muscle and cartilage derived from mesoderm (ii and iii) and glandular epithelium derived from endoderm (iv). The arrows indicate representative cells (scale bars, 100 μm). (D) Immunostaining of B-TUBLIN III, SMA, and SOX17 in teratoma sections of miPSCs. The nuclei were stained with DAPI (scale bars, 100 μm). (E) Injection of tdTomato⁺ miPSCs (^#43) as the donor cells into the mouse postimplantation embryos (E6.5) and cultured in vitro for 48 h. The culture medium is the mixture of rat serum and SERUM/LIF (1:1). The arrows indicate representative cells that were donor-cell descendants (tdTomato⁺). The control embryos have no tdTomato⁺ cell injected (scale bars, 100 μm). (F) Immunostaining of three germ layers (ectoderm, mesoderm, and endoderm) and PGC markers in cryosections of chimeric embryos for miPSCs. Red indicates tdTomato⁺ donor cells. Green indicates the following lineage markers: β-TUBULIN and NESTIN for ectoderm, T and EOMES for mesoderm, AFP and SOX17 for endoderm, and BLIMP1 and STELLA for PGC. The nuclei were stained with DAPI. The boxed areas are shown below at higher magnifications. The arrows indicate representative cells that were donor-cell descendants (tdTomato⁺) (scale bars, 100 μm). (G) Heatmap showing some significantly upregulated genes in miPSCs compared with both diPSCs and hiPSCs. By KEGG online analysis, significantly enriched KEGG signaling pathways and the related biological processes in each cluster were listed on the right.

Table 2.

In vivo teratoma assay of miPSCs, diPSCs, and hiPSCs

Cell lines	No. of injected cells	No. of mouse	No. of mouse formed teratomas	No. of teratomas	Days
miPSCs	6 × 10⁶	2	1	2	34
miPSCs	5 × 10⁶	2	2	3	30
miPSCs	6 × 10⁶	3	3	4	20
diPSCs	1 × 10⁷	4	1	2	43
diPSCs	8 × 10⁶	1	1	2	36
diPSCs	5 × 10⁶	2	None	None	None
hiPSCs	6 × 10⁶	2	None	None	None
hiPSCs	8 × 10⁶	3	None	None	None
hiPSCs	8 × 10⁶	2	None	None	None

Open in a new tab

These results demonstrated the early germ lineages differentiation of miPSCs, diPSCs and hiPSCs and revealed miPSCs have significant advantages in tissue differentiation compared with diPSCs and hiPSCs.

Discussion

Cell cycle, apoptosis, and metabolism play important roles in efficient nuclear reprogramming for deriving iPSCs [6–8]. However, whether the cell cycle, apoptosis, and metabolism of hybrid fibroblast could impact the reprogramming efficiency remains unknown. Our data showed that MAFs harbor higher reprogramming efficiency than DAFs and HAFs, manifested in the following two aspects: more iPSC colonies formation on day 14 after reprogramming and higher establishment efficiency of iPSC colonies after picked and two passages. This may be related to the characteristics of MAFs as follows: first, cell cycle analysis suggested MAFs had a higher percentage in the S phase compared with DAFs and HAFs. Second, apoptosis genes were upregulated in DAFs and HAFs, compared with MAFs. Third, RNA-seq data revealed that glycolysis-related genes were significantly upregulated in MAFs compared with DAFs. Adding glycolysis inhibitor and stimulator into MAFs, DAFs, and HAFs further confirmed the previous findings that glycolysis may affect cell proliferation and reprogramming efficiency [39]. Therefore, we verified that MAFs have advantages in reprogramming efficiency by RNA-seq analysis and molecular experiments, which may confirm the “heterosis” of mule.

Even though many studies have generated horse iPSCs with or without exogenous genes expression on feeders [20–25], the establishment of other equine iPSCs is still challenging. In this study, by expressing six Dox-inducible transcription factors, which substantially improved the efficiency of reprogramming [17, 46], we established Dox (exogeneous factors)-dependent iPSCs^+Dox of mule, donkey, and horse. Most mule iPSC^+Dox lines expressed high levels of the key endogenous pluripotency genes, which supports us in testing various culture conditions. After screening, we successfully established mule, donkey, and horse Dox-independent iPSCs in a simple culture condition, SERUM/LIF medium. Compared with other mediums for mouse, human, bovine, porcine, and horse ESCs and iPSCs [16–18, 25, 28], the most significant difference was no small molecule inhibitors and cytokines except LIF were added in our culture condition to maintain miPSCs, diPSCs, and hiPSCs in an undifferentiated state.

Although mule has an odd number of chromosomes, miPSCs have a normal karyotype and could stably express high key endogenous pluripotency genes after long-term passages. These characteristics may account for the fact that miPSCs still have a stable genome after long-term passages. In addition, cell competition, first studied in Drosophila, describes the process of eliminating viable neighbor cells with lower fitness levels [47]. Zheng et al. [48] uncovered a previously unrecognized mode of cell competition between primed PSCs of different species. Here, we co-cultured miPSCs, diPSCs, and hiPSCs in vitro and showed these iPSCs all could proliferate regular in co-cultures during day 5, though miPSCs proliferated faster than diPSCs and hiPSCs, which is similar to PSC co-cultures of rat-mouse and human-rhesus [48]. The three kinds of iPSCs could proliferate regularly during co-cultures, probably because of the close genetic relationship between these species [49].

In all, 6484 DEGs between miPSCs and MAFs, 7322 DEGs between diPSCs and DAFs, and 5786 DEGs between hiPSCs and HAFs were screened out, respectively, which may reflect the complementation phenomenon between hybrid cells and its parents. Pearson correlation analysis and PCA showed that hiPSCs clustered together with FTW-eqESCs and FTW-eqiPSCs, but were distinct from miPSCs and diPSCs, which were highly associated with reprogramming and differentiation, indicating miPSCs and diPSCs represent a distinct state distinct from formative pluripotency [25].

The more stringent functional tests for pluripotency are teratoma and chimera formation [41]. Importantly, miPSCs had distinct advantages in teratoma formation, with shorter formation time and higher formation efficiency. We also found that miPSCs graft progeny differentiated into all three germ layers when grafted into E6.5 mouse epiblasts, but no chimera contribution was observed when grafted into E3.5 mouse blastocysts, whereas diPSCs could not differentiate into endoderm. The inability of miPSCs and diPSCs to form blastocyst-injection chimeras may be explained by their incompatibility with the environment of the preimplantation epiblast or species-specific differences in developmental kinetics and maternal microenvironment [42, 50]. Otherwise, some significantly upregulated genes in miPSCs compared with both diPSCs and hiPSCs were found, which are enriched in JNK and p38 MAP Kinase pathway, MAPK and RAS key signaling pathways. The differences in these signaling pathways may account for the result that miPSCs exhibited faster proliferation, higher pluripotency and differentiation than diPSCs and hiPSCs. Therefore, the advantages of miPSCs in higher pluripotency and differentiation, compared with diPSCs and hiPSCs, may further confirm the “heterosis” of mule.

In conclusion, we first examined MAFs compared with the cells from their parents in proliferation, apoptosis, and glycolysis and found MAFs have significant advantages in reprogramming efficiency (each species has repeated three independent individuals). Then, we subsequently derived miPSCs, diPSCs, and hiPSCs on feeders without exogenous genes leaky expression and characterized the molecular properties and differentiation of these iPSCs. It is worth noting that miPSCs exhibited faster proliferation and higher pluripotency and differentiation, which were reflected in proliferate in co-cultures and separate-cultures, teratoma formation and chimera contribution. The establishment of miPSCs provides unique research material and biological insights for understanding “heterosis” at the cellular and molecular levels and provides an accessible in vitro model to study hybrid gamete formation.

Supplementary Material

supplementary_file_2_supplementary_Figure_S1-S5_ioad041

Click here for additional data file.^{(40.9MB, pdf)}

supplementary_file_3_Table_S1-S6_ioad041

Click here for additional data file.^{(34.5KB, docx)}

supplementary_file_4_Table_S7_ioad041

Click here for additional data file.^{(20.3MB, xlsx)}

supplementary_file_1_supplementary_figure_legends_ioad041

Click here for additional data file.^{(22.4KB, docx)}

Acknowledgment

We would like to thank Dr Yulin Ding for assistance with teratoma treatment and H&E staining; Zhiqing Yang for the embryos collection at E6.5 and Dr Jitong Guo for the injection of miPSCs and diPSCs into mouse blastocysts. We also thank the staff members of the Research Center for Animal Genetic Resources of Mongolia Plateau of Inner Mongolia University for assistance with cell cycle analysis by flow cytometry.

Conflict of Interest: The authors have declared that no conflict of interest exists.

Footnotes

^† Grant Support: This research was funded by the Inner Mongolia Autonomous Region Science and Technology Plan of China (2020ZD0007), the National Natural Science Foundation of China (32,060,176), and the Inner Mongolia and Hohhot Science & Technology Plan (2021-National Dairy Innovation Center-3).

Contributor Information

Jia Zhang, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Lixia Zhao, College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010110, China.

Yuting Fu, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Fangyuan Liu, Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China; Clinical Medical Research Center, The Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010050, China.

Zixin Wang, Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China.

Yunxia Li, Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China.

Gaoping Zhao, Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China; National Center of Technology Innovation for Dairy Industry, Hohhot 010020, China.

Wei Sun, Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China; National Center of Technology Innovation for Dairy Industry, Hohhot 010020, China.

Baojiang Wu, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Yongli Song, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Shaohua Li, College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010110, China.

Chunxia Hao, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Bilige Wuyun, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Rihan Wu, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Moning Liu, Key Laboratory of Basic Veterinary Medicine, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.

Guifang Cao, Key Laboratory of Basic Veterinary Medicine, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.

Buhe Nashun, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China.

M Azim Surani, Wellcome Trust/Cancer Research UK Gurdon Institute, Henry Wellcome Building of Cancer and Developmental Biology, Cambridge CB2 1QN, UK.

Qingyuan Sun, Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou 510317, China.

Siqin Bao, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China.

Pentao Liu, School of Biomedical Science, Stem Cell and Regenerative Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong 999077, China; Centre for Translational Stem Cell Biology, Building 17W, The Hong Kong Science and Technology Park, Hong Kong 999077, China.

Xihe Li, The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010020, China; Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot 010020, China; Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot 011517, China.

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. RNA-seq data have been deposited to the Gene Expression Omnibus under accession number GSE201393.

Author contributions

S.B., P.L., and X.L. designed and supervised research project. J.Z. derived miPSCs, diPSCs, and hiPSCs and characterized the molecular properties of all fibroblasts and iPSCs. F.L. analyzed the RNA-seq data. J.Z., Y.L., and B.W. performed in vivo embryo experiment. J.Z., L.Z., Y.F., Z.W., G.Z., W.S., Y.S., S.L., C.H., B.W., R.W., and M.L. analyzed the molecular data. J.Z. and Y.F. wrote the manuscript with input from all authors. G.C., B.N., M.A.S., Q.S., S.B., P.L., and X.L. revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.

References

1. Proops L, Burden F, Osthaus B. Mule cognition: a case of hybrid vigour? Anim Cogn 2009; 12:75–84. [DOI] [PubMed] [Google Scholar]
2. Xiao R, Yuan Y, Zhu F, He S, Ge Q, Wang X, Taha R, Chen K. Transcriptomics and proteomics-based analysis of heterosis on main economic traits of silkworm, Bombyx mori. J Proteomics 2020; 229:103941. [DOI] [PubMed] [Google Scholar]
3. Li H, Yuan J, Wu M, Han Z, Li L, Jiang H, Jia Y, Han X, Liu M, Sun D, Chen C, Song W, et al. Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic). BMC Plant Biol 2018; 18:168. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Huang X, Yang S, Gong J, Zhao Q, Feng Q, Zhan Q, Zhao Y, Li W, Cheng B, Xia J, Chen N, Huang T, et al. Genomic architecture of heterosis for yield traits in rice. Nature 2016; 537:629–633. [DOI] [PubMed] [Google Scholar]
5. Troyer AF. Adaptedness and heterosis in corn and mule hybrids. Crop Sci 2006; 46:528–543. [Google Scholar]
6. Singh AM, Dalton S. The cell cycle and Myc intersect with mechanisms that regulate pluripotency and reprogramming. Cell Stem Cell 2009; 5:141–149. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Panopoulos AD, Yanes O, Ruiz S, Kida YS, Diep D, Tautenhahn R, Herrerias A, Batchelder EM, Plongthongkum N, Lutz M, Siuzdak G, Izpisua Belmonte JC, et al. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res 2012; 22:168–177. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Cheung HH, Liu X, Rennert OM. Apoptosis: reprogramming and the fate of mature cells. ISRN Cell Biol 2012; 2012:1–8. [Google Scholar]
9. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861–872. [DOI] [PubMed] [Google Scholar]
10. Liao J, Cui C, Chen S, Ren J, Chen J, Gao Y, Li H, Jia N, Cheng L, Xiao H, Xiao L. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 2009; 4:11–15. [DOI] [PubMed] [Google Scholar]
11. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663–676. [DOI] [PubMed] [Google Scholar]
12. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917–1920. [DOI] [PubMed] [Google Scholar]
13. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 2007; 1:55–70. [DOI] [PubMed] [Google Scholar]
14. Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, Vassena R, Bilic J, Pekarik V, Tiscornia G, Edel M, Boué S, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 2008; 26:1276–1284. [DOI] [PubMed] [Google Scholar]
15. Cao H, Yang P, Pu Y, Sun X, Yin H, Zhang Y, Zhang Y, Li Y, Liu Y, Fang F, Zhang Z, Tao Y, et al. Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins. Int J Biol Sci 2012; 8:498–511. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. The ground state of embryonic stem cell self-renewal. Nature 2008; 453:519–523. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Gao X, Nowak-Imialek M, Chen X, Chen D, Herrmann D, Ruan D, Chen ACH, Eckersley-Maslin MA, Ahmad S, Lee YL, Kobayashi T, Ryan D, et al. Establishment of porcine and human expanded potential stem cells. Nat Cell Biol 2019; 21:687–699. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Zhao L, Gao X, Zheng Y, Wang Z, Zhao G, Ren J, Zhang J, Wu J, Wu B, Chen Y, Sun W, Li Y, et al. Establishment of bovine expanded potential stem cells. Proc Natl Acad Sci U S A 2021; 118:e2018505118. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Bao L, He L, Chen J, Wu Z, Liao J, Rao L, Ren J, Li H, Zhu H, Qian L, Gu Y, Dai H, et al. Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res 2011; 21:600–608. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Nagy K, Sung HK, Zhang P, Laflamme S, Vincent P, Agha-Mohammadi S, Woltjen K, Monetti C, Michael IP, Smith LC, Nagy A. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev Rep 2011; 7:693–702. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Khodadadi K, Sumer H, Pashaiasl M, Lim S, Williamson M, Verma PJ. Induction of pluripotency in adult equine fibroblasts without c-MYC. Stem Cells Int 2012; 2012:429160. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Whitworth DJ, Ovchinnikov DA, Sun J, Fortuna PR, Wolvetang EJ. Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev 2014; 23:1515–1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Lee EM, Kim AY, Lee EJ, Park JK, Park SI, Cho SG, Kim HK, Kim SY, Jeong KS. Generation of equine-induced pluripotent stem cells and analysis of their therapeutic potential for muscle injuries. Cell Transplant 2016; 25:2003–2016. [DOI] [PubMed] [Google Scholar]
24. Pessoa LVF, Pires PRL, Del Collado M, Pieri NCG, Recchia K, Souza AF, Perecin F, da Silveira JC, de Andrade AFC, Ambrosio CE, Bressan FF, Meirelles FV. Generation and miRNA characterization of equine induced pluripotent stem cells derived from fetal and adult multipotent tissues. Stem Cells Int 2019; 2019:1393791. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Yu L, Wei Y, Sun HX, Mahdi AK, Pinzon Arteaga CA, Sakurai M, Schmitz DA, Zheng C, Ballard ED, Li J, Tanaka N, Kohara A, et al. Derivation of intermediate pluripotent stem cells amenable to primordial germ cell specification. Cell Stem Cell 2021; 28:550, e512–567. [DOI] [PubMed] [Google Scholar]
26. Wang W, Yang J, Liu H, Lu D, Chen X, Zenonos Z, Campos LS, Rad R, Guo G, Zhang S, Bradley A, Liu P. Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1. Proc Natl Acad Sci U S A 2011; 108:18283–18288. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003; 115:281–292. [DOI] [PubMed] [Google Scholar]
28. Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 2014; 158:1254–1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, Zviran A, Rais Y, Shipony Z, et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 2013; 504:282–286. [DOI] [PubMed] [Google Scholar]
30. Theunissen TW, Powell BE, Wang H, Mitalipova M, Faddah DA, Reddy J, Fan ZP, Maetzel D, Ganz K, Shi L, Lungjangwa T, Imsoonthornruksa S, et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 2014; 15:471–487. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Yang J, Ryan DJ, Wang W, Tsang JC, Lan G, Masaki H, Gao X, Antunes L, Yu Y, Zhu Z, Wang J, Kolodziejczyk AA, et al. Establishment of mouse expanded potential stem cells. Nature 2017; 550:393–397. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Glanville-Jones HC, Woo N, Arkell RM. Successful whole embryo culture with commercially available reagents. Int J Dev Biol 2013; 57:61–67. [DOI] [PubMed] [Google Scholar]
33. Ruiz S, Panopoulos AD, Herrerias A, Bissig KD, Lutz M, Berggren WT, Verma IM, Izpisua Belmonte JC. A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity. Curr Biol 2011; 21:45–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Zhao Q, Jia X, Zhang Y, Dong Y, Lei Y, Tan X, Williamson RA, Wang A, Zhang D, Ma J. Tetrandrine induces apoptosis in human neuroblastoma through regulating the Hippo/YAP signaling pathway. Biochem Biophys Res Commun 2019; 513:846–851. [DOI] [PubMed] [Google Scholar]
35. Sun Y, Liu WZ, Liu T, Feng X, Yang N, Zhou HF. Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis. J Recept Signal Transduct Res 2015; 35:600–604. [DOI] [PubMed] [Google Scholar]
36. Zhang L, Xing D, Gao X, Wu S. Low-power laser irradiation promotes cell proliferation by activating PI3K/Akt pathway. J Cell Physiol 2009; 219:553–562. [DOI] [PubMed] [Google Scholar]
37. Lin CL, Cheng H, Tung CW, Huang WJ, Chang PJ, Yang JT, Wang JY. Simvastatin reverses high glucose-induced apoptosis of mesangial cells via modulation of Wnt signaling pathway. Am J Nephrol 2008; 28:290–297. [DOI] [PubMed] [Google Scholar]
38. Kim EJY, Anko ML, Flensberg C, Majewski IJ, Geng FS, Firas J, Huang DCS, van Delft MF, Heath JK. BAK/BAX-mediated apoptosis is a Myc-induced roadblock to reprogramming. Stem Cell Reports 2018; 10:331–338. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Zhou G, Meng S, Li Y, Ghebre YT, Cooke JP. Optimal ROS signaling is critical for nuclear reprogramming. Cell Rep 2016; 15:919–925. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Sharpless NE, DePinho RA. Telomeres, stem cells, senescence, and cancer. J Clin Invest 2004; 113:160–168. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. De Los AA, Ferrari F, Xi R, Fujiwara Y, Benvenisty N, Deng H, Hochedlinger K, Jaenisch R, Lee S, Leitch HG, Lensch MW, Lujan E, et al. Hallmarks of pluripotency. Nature 2015; 525:469–478. [DOI] [PubMed] [Google Scholar]
42. Huang Y, Osorno R, Tsakiridis A, Wilson V. In vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation. Cell Rep 2012; 2:1571–1578. [DOI] [PubMed] [Google Scholar]
43. Segales J, Perdiguero E, Munoz-Canoves P. Regulation of muscle stem cell functions: a focus on the p38 MAPK signaling pathway. Front Cell Dev Biol 2016; 4:91. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Yang W, Klaman LD, Chen B, Araki T, Harada H, Thomas SM, George EL, Neel BG. An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival. Dev Cell 2006; 10:317–327. [DOI] [PubMed] [Google Scholar]
45. He H, Yin H, Yu X, Zhang Y, Ma M, Li D, Zhu Q. PDLIM5 affects chicken skeletal muscle satellite cell proliferation and differentiation via the p38-MAPK pathway. Animals 2021; 11:1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Petkov S, Glage S, Nowak-Imialek M, Niemann H. Long-term culture of porcine induced pluripotent stem-like cells under feeder-free conditions in the presence of histone deacetylase inhibitors. Stem Cells Dev 2016; 25:386–394. [DOI] [PubMed] [Google Scholar]
47. Morata G, Ripoll P. Minutes: mutants of drosophila autonomously affecting cell division rate. Dev Biol 1975; 42:211–221. [DOI] [PubMed] [Google Scholar]
48. Zheng C, Hu Y, Sakurai M, Pinzon-Arteaga CA, Li J, Wei Y, Okamura D, Ravaux B, Barlow HR, Yu L, Sun HX, Chen EH, et al. Cell competition constitutes a barrier for interspecies chimerism. Nature 2021; 592:272–276. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Jonsson H, Schubert M, Seguin-Orlando A, Ginolhac A, Petersen L, Fumagalli M, Albrechtsen A, Petersen B, Korneliussen TS, Vilstrup JT, Lear T, Myka J, et al. Speciation with gene flow in equids despite extensive chromosomal plasticity. Proc Natl Acad Sci U S A 2014; 111:18655–18660. [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Wu J, Platero-Luengo A, Sakurai M, Sugawara A, Gil MA, Yamauchi T, Suzuki K, Bogliotti YS, Cuello C, Morales Valencia M, Okumura D, Luo J, et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell 2017; 168:473, e415–486. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplementary_file_2_supplementary_Figure_S1-S5_ioad041

Click here for additional data file.^{(40.9MB, pdf)}

supplementary_file_3_Table_S1-S6_ioad041

Click here for additional data file.^{(34.5KB, docx)}

supplementary_file_4_Table_S7_ioad041

Click here for additional data file.^{(20.3MB, xlsx)}

supplementary_file_1_supplementary_figure_legends_ioad041

Click here for additional data file.^{(22.4KB, docx)}

Data Availability Statement

[ref1] 1. Proops L, Burden F, Osthaus B. Mule cognition: a case of hybrid vigour? Anim Cogn 2009; 12:75–84. [DOI] [PubMed] [Google Scholar]

[ref2] 2. Xiao R, Yuan Y, Zhu F, He S, Ge Q, Wang X, Taha R, Chen K. Transcriptomics and proteomics-based analysis of heterosis on main economic traits of silkworm, Bombyx mori. J Proteomics 2020; 229:103941. [DOI] [PubMed] [Google Scholar]

[ref3] 3. Li H, Yuan J, Wu M, Han Z, Li L, Jiang H, Jia Y, Han X, Liu M, Sun D, Chen C, Song W, et al. Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic). BMC Plant Biol 2018; 18:168. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref4] 4. Huang X, Yang S, Gong J, Zhao Q, Feng Q, Zhan Q, Zhao Y, Li W, Cheng B, Xia J, Chen N, Huang T, et al. Genomic architecture of heterosis for yield traits in rice. Nature 2016; 537:629–633. [DOI] [PubMed] [Google Scholar]

[ref5] 5. Troyer AF. Adaptedness and heterosis in corn and mule hybrids. Crop Sci 2006; 46:528–543. [Google Scholar]

[ref6] 6. Singh AM, Dalton S. The cell cycle and Myc intersect with mechanisms that regulate pluripotency and reprogramming. Cell Stem Cell 2009; 5:141–149. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref7] 7. Panopoulos AD, Yanes O, Ruiz S, Kida YS, Diep D, Tautenhahn R, Herrerias A, Batchelder EM, Plongthongkum N, Lutz M, Siuzdak G, Izpisua Belmonte JC, et al. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res 2012; 22:168–177. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref8] 8. Cheung HH, Liu X, Rennert OM. Apoptosis: reprogramming and the fate of mature cells. ISRN Cell Biol 2012; 2012:1–8. [Google Scholar]

[ref9] 9. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861–872. [DOI] [PubMed] [Google Scholar]

[ref10] 10. Liao J, Cui C, Chen S, Ren J, Chen J, Gao Y, Li H, Jia N, Cheng L, Xiao H, Xiao L. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 2009; 4:11–15. [DOI] [PubMed] [Google Scholar]

[ref11] 11. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663–676. [DOI] [PubMed] [Google Scholar]

[ref12] 12. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917–1920. [DOI] [PubMed] [Google Scholar]

[ref13] 13. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 2007; 1:55–70. [DOI] [PubMed] [Google Scholar]

[ref14] 14. Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, Vassena R, Bilic J, Pekarik V, Tiscornia G, Edel M, Boué S, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 2008; 26:1276–1284. [DOI] [PubMed] [Google Scholar]

[ref15] 15. Cao H, Yang P, Pu Y, Sun X, Yin H, Zhang Y, Zhang Y, Li Y, Liu Y, Fang F, Zhang Z, Tao Y, et al. Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins. Int J Biol Sci 2012; 8:498–511. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref16] 16. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. The ground state of embryonic stem cell self-renewal. Nature 2008; 453:519–523. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref17] 17. Gao X, Nowak-Imialek M, Chen X, Chen D, Herrmann D, Ruan D, Chen ACH, Eckersley-Maslin MA, Ahmad S, Lee YL, Kobayashi T, Ryan D, et al. Establishment of porcine and human expanded potential stem cells. Nat Cell Biol 2019; 21:687–699. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref18] 18. Zhao L, Gao X, Zheng Y, Wang Z, Zhao G, Ren J, Zhang J, Wu J, Wu B, Chen Y, Sun W, Li Y, et al. Establishment of bovine expanded potential stem cells. Proc Natl Acad Sci U S A 2021; 118:e2018505118. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref19] 19. Bao L, He L, Chen J, Wu Z, Liao J, Rao L, Ren J, Li H, Zhu H, Qian L, Gu Y, Dai H, et al. Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res 2011; 21:600–608. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref20] 20. Nagy K, Sung HK, Zhang P, Laflamme S, Vincent P, Agha-Mohammadi S, Woltjen K, Monetti C, Michael IP, Smith LC, Nagy A. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev Rep 2011; 7:693–702. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref21] 21. Khodadadi K, Sumer H, Pashaiasl M, Lim S, Williamson M, Verma PJ. Induction of pluripotency in adult equine fibroblasts without c-MYC. Stem Cells Int 2012; 2012:429160. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref22] 22. Whitworth DJ, Ovchinnikov DA, Sun J, Fortuna PR, Wolvetang EJ. Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev 2014; 23:1515–1523. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref23] 23. Lee EM, Kim AY, Lee EJ, Park JK, Park SI, Cho SG, Kim HK, Kim SY, Jeong KS. Generation of equine-induced pluripotent stem cells and analysis of their therapeutic potential for muscle injuries. Cell Transplant 2016; 25:2003–2016. [DOI] [PubMed] [Google Scholar]

[ref24] 24. Pessoa LVF, Pires PRL, Del Collado M, Pieri NCG, Recchia K, Souza AF, Perecin F, da Silveira JC, de Andrade AFC, Ambrosio CE, Bressan FF, Meirelles FV. Generation and miRNA characterization of equine induced pluripotent stem cells derived from fetal and adult multipotent tissues. Stem Cells Int 2019; 2019:1393791. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref25] 25. Yu L, Wei Y, Sun HX, Mahdi AK, Pinzon Arteaga CA, Sakurai M, Schmitz DA, Zheng C, Ballard ED, Li J, Tanaka N, Kohara A, et al. Derivation of intermediate pluripotent stem cells amenable to primordial germ cell specification. Cell Stem Cell 2021; 28:550, e512–567. [DOI] [PubMed] [Google Scholar]

[ref26] 26. Wang W, Yang J, Liu H, Lu D, Chen X, Zenonos Z, Campos LS, Rad R, Guo G, Zhang S, Bradley A, Liu P. Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1. Proc Natl Acad Sci U S A 2011; 108:18283–18288. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref27] 27. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003; 115:281–292. [DOI] [PubMed] [Google Scholar]

[ref28] 28. Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 2014; 158:1254–1269. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref29] 29. Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, Zviran A, Rais Y, Shipony Z, et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 2013; 504:282–286. [DOI] [PubMed] [Google Scholar]

[ref30] 30. Theunissen TW, Powell BE, Wang H, Mitalipova M, Faddah DA, Reddy J, Fan ZP, Maetzel D, Ganz K, Shi L, Lungjangwa T, Imsoonthornruksa S, et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 2014; 15:471–487. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref31] 31. Yang J, Ryan DJ, Wang W, Tsang JC, Lan G, Masaki H, Gao X, Antunes L, Yu Y, Zhu Z, Wang J, Kolodziejczyk AA, et al. Establishment of mouse expanded potential stem cells. Nature 2017; 550:393–397. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref32] 32. Glanville-Jones HC, Woo N, Arkell RM. Successful whole embryo culture with commercially available reagents. Int J Dev Biol 2013; 57:61–67. [DOI] [PubMed] [Google Scholar]

[ref33] 33. Ruiz S, Panopoulos AD, Herrerias A, Bissig KD, Lutz M, Berggren WT, Verma IM, Izpisua Belmonte JC. A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity. Curr Biol 2011; 21:45–52. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref34] 34. Zhao Q, Jia X, Zhang Y, Dong Y, Lei Y, Tan X, Williamson RA, Wang A, Zhang D, Ma J. Tetrandrine induces apoptosis in human neuroblastoma through regulating the Hippo/YAP signaling pathway. Biochem Biophys Res Commun 2019; 513:846–851. [DOI] [PubMed] [Google Scholar]

[ref35] 35. Sun Y, Liu WZ, Liu T, Feng X, Yang N, Zhou HF. Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis. J Recept Signal Transduct Res 2015; 35:600–604. [DOI] [PubMed] [Google Scholar]

[ref36] 36. Zhang L, Xing D, Gao X, Wu S. Low-power laser irradiation promotes cell proliferation by activating PI3K/Akt pathway. J Cell Physiol 2009; 219:553–562. [DOI] [PubMed] [Google Scholar]

[ref37] 37. Lin CL, Cheng H, Tung CW, Huang WJ, Chang PJ, Yang JT, Wang JY. Simvastatin reverses high glucose-induced apoptosis of mesangial cells via modulation of Wnt signaling pathway. Am J Nephrol 2008; 28:290–297. [DOI] [PubMed] [Google Scholar]

[ref38] 38. Kim EJY, Anko ML, Flensberg C, Majewski IJ, Geng FS, Firas J, Huang DCS, van Delft MF, Heath JK. BAK/BAX-mediated apoptosis is a Myc-induced roadblock to reprogramming. Stem Cell Reports 2018; 10:331–338. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref39] 39. Zhou G, Meng S, Li Y, Ghebre YT, Cooke JP. Optimal ROS signaling is critical for nuclear reprogramming. Cell Rep 2016; 15:919–925. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref40] 40. Sharpless NE, DePinho RA. Telomeres, stem cells, senescence, and cancer. J Clin Invest 2004; 113:160–168. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref41] 41. De Los AA, Ferrari F, Xi R, Fujiwara Y, Benvenisty N, Deng H, Hochedlinger K, Jaenisch R, Lee S, Leitch HG, Lensch MW, Lujan E, et al. Hallmarks of pluripotency. Nature 2015; 525:469–478. [DOI] [PubMed] [Google Scholar]

[ref42] 42. Huang Y, Osorno R, Tsakiridis A, Wilson V. In vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation. Cell Rep 2012; 2:1571–1578. [DOI] [PubMed] [Google Scholar]

[ref43] 43. Segales J, Perdiguero E, Munoz-Canoves P. Regulation of muscle stem cell functions: a focus on the p38 MAPK signaling pathway. Front Cell Dev Biol 2016; 4:91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref44] 44. Yang W, Klaman LD, Chen B, Araki T, Harada H, Thomas SM, George EL, Neel BG. An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival. Dev Cell 2006; 10:317–327. [DOI] [PubMed] [Google Scholar]

[ref45] 45. He H, Yin H, Yu X, Zhang Y, Ma M, Li D, Zhu Q. PDLIM5 affects chicken skeletal muscle satellite cell proliferation and differentiation via the p38-MAPK pathway. Animals 2021; 11:1016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref46] 46. Petkov S, Glage S, Nowak-Imialek M, Niemann H. Long-term culture of porcine induced pluripotent stem-like cells under feeder-free conditions in the presence of histone deacetylase inhibitors. Stem Cells Dev 2016; 25:386–394. [DOI] [PubMed] [Google Scholar]

[ref47] 47. Morata G, Ripoll P. Minutes: mutants of drosophila autonomously affecting cell division rate. Dev Biol 1975; 42:211–221. [DOI] [PubMed] [Google Scholar]

[ref48] 48. Zheng C, Hu Y, Sakurai M, Pinzon-Arteaga CA, Li J, Wei Y, Okamura D, Ravaux B, Barlow HR, Yu L, Sun HX, Chen EH, et al. Cell competition constitutes a barrier for interspecies chimerism. Nature 2021; 592:272–276. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref49] 49. Jonsson H, Schubert M, Seguin-Orlando A, Ginolhac A, Petersen L, Fumagalli M, Albrechtsen A, Petersen B, Korneliussen TS, Vilstrup JT, Lear T, Myka J, et al. Speciation with gene flow in equids despite extensive chromosomal plasticity. Proc Natl Acad Sci U S A 2014; 111:18655–18660. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref50] 50. Wu J, Platero-Luengo A, Sakurai M, Sugawara A, Gil MA, Yamauchi T, Suzuki K, Bogliotti YS, Cuello C, Morales Valencia M, Okumura D, Luo J, et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell 2017; 168:473, e415–486. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Reprogramming efficiency and pluripotency of mule iPSCs over its parents†

Jia Zhang

Lixia Zhao

Yuting Fu

Fangyuan Liu

Zixin Wang

Yunxia Li

Gaoping Zhao

Wei Sun

Baojiang Wu

Yongli Song

Shaohua Li

Chunxia Hao

Bilige Wuyun

Rihan Wu

Moning Liu

Guifang Cao

Buhe Nashun

M Azim Surani

Qingyuan Sun

Siqin Bao

Pentao Liu

Xihe Li

Abstract

Graphical Abstract

Graphical Abstract.

Introduction

Materials and methods

Animals

Table 1.

Ethics statement

Reprogramming MAFs, DAFs, and HAFs to iPSCs

Preparing mule, horse, donkey, and STO feeders

Screening for the culture conditions of miPSCs, diPSCs, and hiPSCs

Culturing MAFs, DAFs, and HAFs, and miPSCs, diPSCs, and hiPSCs

AP staining

Karyotype

Telomerase activity assay

In vitro EB formation assay of miPSCs, diPSCs, and hiPSCs

In vivo teratoma assay of miPSCs, diPSCs, and hiPSCs

Generation of chimera

Immunofluorescence staining

RT-qPCR analysis

Cell cycle analysis

Transcriptome analysis

Statistical analysis

Results

Cell proliferation and reprogramming of mule fibroblasts compared with donkey and horse

Figure 1.

Identification of culture conditions for Dox-independent iPSCs of mule compared with donkey and horse

Figure 2.

Typical pluripotency features and cell competition of miPSCs compared with diPSCs and hiPSCs

Figure 3.

Transcriptome profiling of miPSCs/MAFs compared with diPSCs/DAFs and hiPSCs/HAFs

Figure 4.

Early germ lineages differentiation of miPSCs compared with diPSCs and hiPSCs

Figure 5.

Table 2.

Discussion

Supplementary Material

Acknowledgment

Footnotes

Contributor Information

Data availability

Author contributions

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Reprogramming efficiency and pluripotency of mule iPSCs over its parents^{^†}