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Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

Hindawi
Stem Cells International
Authors:
Khodadad Khodadadi at Murdoch Children's Research Institute
Khodadad Khodadadi
Huseyin Sumer at Swinburne University of Technology
Huseyin Sumer
Maryam Pashaiasl at Tabriz University of Medical Sciences
Maryam Pashaiasl
Susan Lim
Susan Lim
Susan Lim
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Abstract and Figures

Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression of OCT4, SOX2 NANOG, and STAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers both in vitro by embryoid bodies (EBs) formation and in vivo by teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.
FACs analysis showing GFP fluorescence in adult equine fibroblasts following pMX-GFP viral transduction. (a) GFP fluorescence in AEFs following GP2293 mediated retroviral transduction, scale bar 200 μM. (i) Bright filed. (ii) Green filter. FACs profile of GFP fluorescence, (b) Retroviral transduction using GP2 293 packaging cell. (c) Control EAFs.
FACs analysis showing GFP fluorescence in adult equine fibroblasts following pMX-GFP viral transduction. (a) GFP fluorescence in AEFs following GP2293 mediated retroviral transduction, scale bar 200 μM. (i) Bright filed. (ii) Green filter. FACs profile of GFP fluorescence, (b) Retroviral transduction using GP2 293 packaging cell. (c) Control EAFs.
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Generation of EiPS cells. (a) Morphology of EiPS single colony growing from individual fibroblast. (b) Typical colony of EiPS cells on the MEF and colony selection. (c) Cutting individual colony for manual passaging. (d) Passaged pieces on the MEF. (e) Alkaline phosphatase activity of EiPS cells scale bar 200 μM. (f) Chromosome spread of EiPS cells.
Generation of EiPS cells. (a) Morphology of EiPS single colony growing from individual fibroblast. (b) Typical colony of EiPS cells on the MEF and colony selection. (c) Cutting individual colony for manual passaging. (d) Passaged pieces on the MEF. (e) Alkaline phosphatase activity of EiPS cells scale bar 200 μM. (f) Chromosome spread of EiPS cells.
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Gene expressions profile of EiPS cells and genomic DNA analysis. (a) Genomic PCR confirming the integration of the four transgenes. (b) Gene expression of exogenous reprogramming factors. (c) Gene expression profile of the EiPS cells compared to the parental EAFs and MEF as feeder cells.
Gene expressions profile of EiPS cells and genomic DNA analysis. (a) Genomic PCR confirming the integration of the four transgenes. (b) Gene expression of exogenous reprogramming factors. (c) Gene expression profile of the EiPS cells compared to the parental EAFs and MEF as feeder cells.
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Immunoflourescence staining of pluripotent markers in EiPS cells. Immunostaining of EiPS cells for (a) OCT4, (b) NANOG, (c) SSEA1, and (d) SSEA4, counterstained with DAPI, scale bar 200 μM.
Immunoflourescence staining of pluripotent markers in EiPS cells. Immunostaining of EiPS cells for (a) OCT4, (b) NANOG, (c) SSEA1, and (d) SSEA4, counterstained with DAPI, scale bar 200 μM.
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Differentiation potential of EiPS cells. (a) Embryoid body formation of EiPS cells grown in suspension medium in the absence of LIF. (b) Single EB, scale bar 500 μM. (c) Gene expression profile of EiPS cells following differentiation of embryoid body. (d) Histology of differentiated tissues found in the hind leg muscle of SCID mice following injection of EiPS cells included (i) endodermal differentiation, (ii) mesodermal differentiation, (iii) ectodermal (neuroblastic) differentiation, scale bar 100 μM.
Differentiation potential of EiPS cells. (a) Embryoid body formation of EiPS cells grown in suspension medium in the absence of LIF. (b) Single EB, scale bar 500 μM. (c) Gene expression profile of EiPS cells following differentiation of embryoid body. (d) Histology of differentiated tissues found in the hind leg muscle of SCID mice following injection of EiPS cells included (i) endodermal differentiation, (ii) mesodermal differentiation, (iii) ectodermal (neuroblastic) differentiation, scale bar 100 μM.
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Supplementary resource (1)

Supplementary Material
Data
March 2012
Khodadad Khodadadi · Huseyin Sumer · Maryam Pashaiasl · Susan Lim · Paul J Verma
... 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 which depend on exogenous reprogramming transcription factors have been established [20][21][22][23][24]. 6 potentially contribute to intra-or inter-species chimeras in vivo [25]. ...
... Even though many studies have generated horse iPSCs with or without exogenous genes expression on feeders [20][21][22][23][24][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]. ...
Reprogramming efficiency and pluripotency of mule iPSCs over its parents†
Article
Full-text available
  • Apr 2023
  • BIOL REPROD
The mule is the interspecific hybrid of horse and donkey and has hybrid vigour 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 (DAFs and HAFs) (each species have repeated three independent individuals) in proliferation, apoptosis and glycolysis and found significant differences. We subsequently derived mule, donkey and horse induced pluripotent stem cells (miPSCs, diPSCs and hiPSCs) from three independent individuals of each species and found the reprogramming efficiency of MAFs were significantly higher than that of cells of donkey and horse. miPSCs, diPSCs and hiPSCs all expressed 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 provide unique research material for investigation of "heterosis" and perhaps is more significant to study hybrid gamete formation.
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... PSCs exhibit both similarities and differences in their characteristics across species, highlighting the importance of understanding PSCs from various species. Derivations of PSCs have been reported in a range of species, including cows [4], pigs [5], horses [6][7][8][9][10], naked mole-rats [11], and other mammalian species [12][13][14][15][16][17][18]. In most studies, PSCs have been shown to satisfy many of the criteria for pluripotency, although some of the characteristics of the cells may differ among species [12,14]. ...
... Studies of PSCs from domestic animals, including cows [4] and pigs [5], have been reported, while research on Perissodactyla PSCs has been limited to horses [6][7][8][9][10] and the northern white rhinoceros [13,15]. Grevy's zebra (Equus grevyi) belongs to the family Equidae, a taxonomic group including horses, donkeys, and zebras. ...
Generation and Gene Expression Profiles of Grevy's Zebra Induced Pluripotent Stem Cells
Article
  • Mar 2022
Induced pluripotent stem cells (iPSCs) can serve as a biological resource for functional and conservation research for various species. This realisation has led to the generation of iPSCs from many species, including those identified as endangered. However, the understanding of species variation in mammalian iPSCs remains largely unknown. To gain insight into species variation in iPSCs, we generated iPSCs from a new species Grevy's zebra (Equus grevyi; gz-iPSCs), which has been listed as endangered in the IUCN (International Union for Conservation of Nature) Red List. We isolated primary fibroblast cells from an individual and successfully reprogrammed them into iPSCs. The generated gz-iPSCs continued to grow under primed-type culture condition and showed pluripotency and differentiation potential. To describe the molecular characteristics of gz-iPSCs, we performed RNA sequencing analysis. The gz-iPSC transcriptome showed robust expression of pluripotency associated genes reported in human and mouse, suggesting evolutionary conservation among the species. This study provides insight into the iPSCs from a rare species and helps the understanding of the gene expression basis underlying mammalian PSCs.
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... The first report of equine iPSCs was in 2011 where a piggyback transposon system was used, containing murine Oct4, Sox2, Klf4, and Myc reprogramming factors (120). Several other publications have followed this, the majority of which have used viral expression vectors to mediate genome integration of the reprogramming gene sequences (121)(122)(123)(124)(125)(126)(127). In order for an iPSC treatment to be deemed clinically safe it is important that the viral transgenes which are integrated during reprogramming are silenced (128). ...
... However, in many of the equine iPSCs generated to date, continued variable expression of the transgenes remain. In the first publication on the generation of equine iPSCs transgenes were required to be constitutively expressed to maintain pluripotency (120,(122)(123)(124)(125), with only one other study demonstrating that the transgenes were silenced (127). Although the equine iPSCs reported share many of the features of equine ESCs and iPSCs reported in other species, their differentiation potential has been shown to be limited in their ability to differentiate down the tendon lineage (121). ...
Characterising embryonic stem cell-derived tenocytes and determining the changing role of scleraxis during tendon development
Thesis
  • Jul 2021
Tendon injuries occur commonly in equine athletes. Adult tendons undergo poor natural regeneration, resulting in scar-tissue which is prone to re-injury. Fetal tendons however are capable of completely scar-less regeneration, a property which is intrinsic to the fetal cells themselves. Novel cell therapies should therefore try to recapitulate this scar-less fetal tendon regeneration. This thesis builds on previous research into the use of horse embryonic stem cells (ESCs) to aid tendon regeneration. The aim of this thesis was to determine if tendon cells derived from ESCs were more similar to fetal or adult tendon cells, as well as try to understand if scleraxis (SCX), an essential gene in tendon formation, has different roles at different stages of tendon development. Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture was compared using RNA-sequencing. No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome wide transcriptomic analysis using RNA- sequencing revealed that ESC-derived tenocytes transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this thesis adds to the growing evidence that monolayer cultured cells gene expression profiles converge, with adult and fetal tenocytes having only 10 differentially expressed (DE) genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in three- dimensional culture, large distinctions in gene expression between these two developmental stages were found, with 542 genes being DE. The effects of knocking down the expression of SCX on gene expression in adult, fetal and ESC-derived tenocytes was then determined using RNA-sequencing and qPCR. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes effected in adult tenocytes, indicating that scleraxis- dependent processes differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, novel genes that SCX differentially interacts with in adult and fetal tenocytes were identified. SCX knockdown in ESCs resulted in upregulation of cartilage markers, a result which still needs to be confirmed in further biological replicates. In summary, the data presented in this thesis provides an unprecedented insight into some of the differences between fetal regenerative and adult reparative tenocytes. It also indicated that ESC-derived tenocytes are more similar to fetal rather than adult tenocytes, highlighting their potential as a therapeutic cell source. The results presented also indicate a role for SCX in modulating ECM synthesis and breakdown and provides a useful dataset for further study into SCX gene regulation. Taken together this data is likely to be important for the future development of novel cellular or pharmacological therapeutics.
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... In addition to expressing the anticipated pluripotency markers, these cells also had the ability to form teratomas (the gold standard for pluripotency) with all three embryonic tissue layers being expressed in immunocompromised mice [44], suggesting that these cells were truly pluripotent in contrast to the earlier studies done to produce equine ES-like cells [39,40]. [45] investigated the potential to create equine iPSCs without the use of c-MYC. In their study, they induced pluripotency in equine fibroblasts through retroviral transduction with only OCT4, SOX2 and KLF4 with similar expression of pluripotency markers and ability to form teratomas as the iPSCs formed with the use of c-MYC. ...
... This gene was excluded because it has been identified as a proto-oncogene that is frequently expressed in immortalized tumor cell lines [46]. The authors hypothesized that the exclusion of this gene could lead to the formation of iPSCs that are less risky for use in autologous transplantation for equine cartilage and tendon injury repair [45]. ...
A Review of OCT4 Functions and Applications to Equine Embryos
Article
Full-text available
  • Dec 2020
  • J EQUINE VET SCI
OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine-specific extrinsic regulators of OCT4 expression that have not yet been investigated. However, an alternative hypothesis is that this restriction may not be evident in equine embryos due to our inability to culture them to the epiblast stage, preventing the observation of this restriction. In vitro studies have identified that OCT4 is expressed in the immature equine oocyte and in the early equine embryo, but OCT4 expression has not been studied after the formation of the ICM in the equine embryo. Despite the gaps in knowledge about equine-specific functions of OCT4, this factor has been used in studies assessing equine embryonic stem cells and to induce pluripotency in equine somatic cells. This review describes the role of OCT4 in the equine embryo and its applications in equine stem cell research.
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... For example, a high rate of the caregiver in the affected animals (Trela et al., 2014). This problem is mainly caused due to the low capacity of regeneration of the cartilage and tendinous tissue in horses (Khodadadi et al., 2012). In this scenario, among the promising therapeutic tools used for the treatment of the muscle-skeletal diseases, is notably the use of stem cells (SC), mainly the mesenchymal (MSCs), which consists of multipotent cells that can be obtained from several tissues of adult animals and develop an essential role in the tissue regeneration (Vidane et al., 2013), and immunomodulation. ...
Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration
Article
Full-text available
  • Jan 2023
The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.
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... Literature mining in Pubmed using the search terms: (("induced pluripotent stem cells"[MeSH Terms] OR ("induced"[All Fields] AND "pluripotent"[All Fields] AND "stem"[All Fields] AND "cells"[All Fields]) OR "induced pluripotent stem cells"[All Fields] OR "ipsc"[All Fields]) AND ("swine"[MeSH Terms] OR "swine"[All Fields] OR "swines"[All Fields])) AND ((journalarticle [Filter]) AND (english[Filter]) AND (2006:2021[pdat])), including only original articles in English language from January 2006 until October 2021, revealed over 300 studies using iPSCs and swine models, of which 103 describe the generation, maintenance, or differentiation of porcine-derived iPSCs (piPSCs), reflecting the clear clinical transfer intention [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65]. In contrast, there were only 18 original articles involving iPSCs derived from horse [60,[66][67][68][69][70][71][72][73], 15 from cattle [74][75][76][77][78][79][80], 15 from dog [81][82][83][84][85][86], and 14 from sheep/goat [87][88][89][90][91][92][93][94]. In most of these iPSC lines, pluripotency heavily depends on fibroblast growth factor and Activin/Nodal signaling (NANOG and LIN28) [80,95]. ...
iPSC Therapy for Myocardial Infarction in Large Animal Models: Land of Hope and Dreams
Article
Full-text available
  • Dec 2021
Myocardial infarction is the main driver of heart failure due to ischemia and subsequent cell death, and cell-based strategies have emerged as promising therapeutic methods to replace dead tissue in cardiovascular diseases. Research in this field has been dramatically advanced by the development of laboratory-induced pluripotent stem cells (iPSCs) that harbor the capability to become any cell type. Like other experimental strategies, stem cell therapy must meet multiple requirements before reaching the clinical trial phase, and in vivo models are indispensable for ensuring the safety of such novel therapies. Specifically, translational studies in large animal models are necessary to fully evaluate the therapeutic potential of this approach; to empirically determine the optimal combination of cell types, supplementary factors, and delivery methods to maximize efficacy; and to stringently assess safety. In the present review, we summarize the main strategies employed to generate iPSCs and differentiate them into cardiomyocytes in large animal species; the most critical differences between using small versus large animal models for cardiovascular studies; and the strategies that have been pursued regarding implanted cells’ stage of differentiation, origin, and technical application.
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... An issue seen in many domestic models is the retention of pluripotent transgene expression; a situation that allows for the maintenance of pluripotency, or in many cases a pseudo-pluripotent state, that can interfere with differentiation. Most iPSCs derived from domestic species have been generated by viral integration of human or murine reprogramming transgenes that remain expressed [82,83,85,86,91]. The continuous expression of these transgenes suggests an incomplete epigenetic remodeling with OKSM factors alone and a greater need for understanding and optimizing the pluripotency induction process in domestic species. ...
The use of induced pluripotent stem cells in domestic animals: a narrative review
Article
Full-text available
  • Dec 2020
  • BMC Vet Res
Induced pluripotent stem cells (iPSCs) are undifferentiated stem cells characterized by the ability to differentiate into any cell type in the body. iPSCs are a relatively new and rapidly developing technology in many fields of biology, including developmental anatomy and physiology, pathology, and toxicology. These cells have great potential in research as they are self-renewing and pluripotent with minimal ethical concerns. Protocols for their production have been developed for many domestic animal species, which have since been used to further our knowledge in the progression and treatment of diseases. This research is valuable both for veterinary medicine as well as for the prospect of translation to human medicine. Safety, cost, and feasibility are potential barriers for this technology that must be considered before widespread clinical adoption. This review will analyze the literature pertaining to iPSCs derived from various domestic species with a focus on iPSC production and characterization, applications for tissue and disease research, and applications for disease treatment.
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Equine induced pluripotent stem cells
Chapter
  • Jan 2021
Horses are kept throughout the world as working animals, competition athletes, or to provide pleasure and companionship to their owners. For many injuries, particularly those affecting the musculoskeletal system, horses provide a relevant model for the human conditions and could be used in preclinical trials for testing novel treatments. Horses also share hereditary conditions with humans and a one health approach to studying some of these could prove to be very valuable. Equine induced pluripotent stem cells (iPSCs) have been derived by multiple groups and differentiated into a variety of cell types that could be useful for clinical transplantation to aid tissue regeneration and/or disease modeling. In this chapter, the current status on the isolation, characterization, and differentiation of equine iPSCs is discussed along with the current challenges that must be addressed before the full potential of these cells to improve horse health and welfare can be realized.
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Bovine iPSC and applications in precise genome engineering
Chapter
  • Jan 2021
Cattle constitute one of the most commercially important livestock species. They are a significant source of nutrition as well has had great economic importance. Through thousands of years of selective breeding humans have shaped cattle into these multipurpose species that are adapted to various environments around the globe. In the past decade, the sequencing of the cattle genome has paved the way for genetic enhancement of existing breeds to increase productivity and sustainability. More recently, developments in genome editing technologies and pluripotent stem cell culture can now be combined to achieve highly commercial goals for the livestock industry. In this chapter, we discuss the basics of these cutting-edge technologies and their applications in cattle. We focus on bovine iPSCs (biPSCs) as they can be generated in theory from any individual long after their genetic value has been proven, and their phenotypic characteristics validated and regardless of their fecundity status. Furthermore, we discuss the various genome editors and how these novel tools can be used for the genetic improvement of cattle.
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Introduction to the stem cell
Chapter
  • Nov 2020
The stem cell concept is as old as the understanding of the origin of life. The concept of origin of life has developed from initial ‘spontaneous generation’ to the current concept of development through ‘zygote formation’. Understanding the concept of the development of life has paved way for the ‘stem cell’ concept. There are various types of stem cells that develop right from early phase of life to the adult life though with variable potentialities. An overview of the development of stem cell concept has been attempted in the current chapter.
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Induced Pluripotent Stem Cell Lines Derived from Equine Fibroblasts
Article
Full-text available
  • Feb 2011
  • STEM CELL REV
The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species.
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The Efficient Generation of Cell Lines from Bovine Parthenotes
Article
Full-text available
  • Oct 2010
The generation of embryonic stem cell (ESC) lines from parthenogenetically activated oocytes can provide transplantable cells, which are immunocompatible for the oocyte donors as well as an invaluable tool for genetic engineering and epigenetic studies. We report the efficient isolation of eight putative bovine parthenogenetic embryonic stem cell (bpESC) lines from 15 in vitro produced parthenotes. Five of these cell lines were maintained for more than 15 passages (>140 days) and analyzed. The cells displayed typical ESC morphology, stained positive for alkaline phosphate by histochemical staining, expressed Oct4, Nanog, and either stage-specific embryonic antigens, SSEA1, or SSEA4, detected by immunofluorescence staining. RT-PCR analysis of the cells demonstrated expression of Oct4, Rex1, SSEA1, and ALP. All the cell lines except one had a normal karyotype of 60, XX. The cells differentiated in suspension culture to form embryoid bodies (EBs) expressing markers of the three embryonic germ layers as assessed by RT-PCR. In conclusion, we report efficient derivation of putative ESCs from bovine parthenogenetic embryos. The cells express pluripotent markers, have the ability to form EBs, and differentiate into cells of the three embryonic germ layers. This is the first report of characterized putative parthenogenetic bovine ESC lines.
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Porcine Induced Pluripotent Stem Cells Produce Chimeric Offspring
Article
Full-text available
  • Apr 2010
  • STEM CELLS DEV
Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.
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Equine embryos and embryonic stem cells: Defining reliable markers of pluripotency
Article
Full-text available
  • Sep 2010
Cartilage and tendon injuries are a significant source of animal wastage and financial loss within the horse-racing industry. Moreover, both cartilage and tendon have limited intrinsic capacity for self-repair, and the functionally inferior tissue produced within a lesion may reduce performance and increase the risk of reinjury. Stem cells offer tremendous potential for accelerating and improving tissue healing, and adult mesenchymal stem cells (MSCs) are already used to treat cartilage and tendon injuries in horses. However, MSCs are scarce in the bone marrow isolates used, have limited potential for proliferation and differentiation in vitro, and do not appear to noticeably improve long-term functional repair. Embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome many of the limitations and be used to generate tissues of value for equine regenerative medicine. To date, six lines of putative ESCs have been described in the horse. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit pluripotency in vivo. Moreover, it is becoming clear that the markers used to characterize the putative ESCs were inadequate, primarily because studies in domestic species have revealed that they are not specific to ESCs or the pluripotent inner cell mass, but also because the function of most in the maintenance of pluripotency is not known. Future derivation and validation of equine embryonic or other pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing horse embryo.
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Derivation of induced pluripotent stem cells from pig somatic cells
Article
Full-text available
  • Jun 2009
  • P NATL ACAD SCI USA
For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.
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Generation of Pig Induced Pluripotent Stem Cells with a Drug-Inducible System
Article
Full-text available
  • Jul 2009
Domesticated ungulate pluripotent embryonic stem (ES) cell lines would be useful for generating precise gene-modified animals. To date, many efforts have been made to establish domesticated ungulate pluripotent ES cells from early embryos without success. Here, we report the generation of porcine-induced pluripotent stem (iPS) cells using drug-inducible expression of defined factors. We showed that porcine iPS cells expressed alkaline phosphatase, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Oct3/4, Nanog, Sox2, Rex1 and CDH1. Pig iPS cells expressed high levels of telomerase activity and showed normal karyotypes. These cells could differentiate into cell types of all three germ layers in vitro and in teratomas. Our study reveals properties of porcine pluripotent stem cells that may facilitate the eventual establishment of porcine ES cells. Moreover, the porcine iPS cells produced may be directly useful for the generation of precise gene-modified pigs.
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Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12
Article
Full-text available
  • May 2009
  • BMC CELL BIOL
Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated. In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cells express tendon-related markers including tenomodulin and decorin. Moreover we show that the same BM-MSCs can differentiate in osteocytes, as confirmed by alkaline phosphatase and von Kossa staining. As MSCs represent an attractive tool for tendon tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for therapeutic approaches.
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Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig
Article
Full-text available
  • May 2009
Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.
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Generation and characterization of reprogrammed sheep induced pluripotent stem cells
Article
  • Sep 2011
Embryonic stem cells (ESCs) from domestic species have numerous potential applications in agricultural and biomedical sciences; however, despite intensive efforts, derivation of ESCs from sheep remains elusive. The objective was to derive sheep induced pluripotent stem cells (iPSCs), as an alternative pluripotent cell type to ESCs, from sheep fibroblasts by ectopic expression of heterologous transcription factors OCT4, SOX2, KLF4, and cMYC. Sheep fibroblasts were infected with pantropic retroviruses coding the four transcription factors and reprogrammed to pluripotency at a rate of 0.002%. The sheep iPSCs (siPSCs) reactivated endogenous OCT4 and SOX2 genes assessed by qRT-PCR and immuno-cytochemistry, retained normal karyotyping, and more importantly, concurrently silenced all exogenous transgenes. The siPSCs were enzymatically dissociated to single cells, making them amenable to efficient transfection and fluorescent-activated cell sorting techniques. Further, the siPSCs differentiated in vitro to form embryoid bodies, and in vivo to form robust teratomas, containing cells representative of the three germ layers. Moreover, when injected into diploid or tetraploid sheep embryos, siPSCs contributed to the inner cell mass of resulting blastocysts, suggesting true pluripotential. These reprogrammed siPSCs may constitute a robust pluripotent alternative to elusive sheep ESCs, with great potential for use in agriculture and pharmaceutical biotechnology.
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NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts
Article
  • Apr 2011
  • J ANIM SCI
Since the first reports on isolation of pluripotent mouse embryonic stem (ES) cells 3 decades ago, there have been numerous attempts to derive ES cell lines from commercially important livestock species with limited success. The recent discovery that ectopic expression of a handful of stem cell-related genes was capable of inducing pluripotency in rodents and primates provided a novel approach to derivation of pluripotent stem cell lines. We used this approach in cattle and demonstrated that the ectopic expression of POU5F1 (also known as Oct4), SOX2, KLF4, and c-MYC alone was not sufficient for stable induction of pluripotency in bovine adult fibroblasts and that the additional expression of NANOG to the reprogramming cocktail was essential for the generation of stable bovine (b) induced pluripotent stem (iPS) cells. The resulting biPS cells were characterized by reverse-transcription PCR for a panel of ES marker genes. Immunocytochemical localization of POU5F1, SSEA-1, SSEA-4, and colorimetric alkaline phosphatase activity was measured in the iPS clones. The differentiation potential of the biPS cells was determined in vitro by expression of differentiation markers in embryoid bodies. Injection of biPS into immunocompromised mice resulted in teratomas containing cell types of the 3 germ lineages. This study reports the first generation of bovine induced pluripotent cell lines and paves the way for the use of biPS cells for biotechnological and agricultural purposes.
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