Generation of Equine-Induced Pluripotent Stem Cells and Analysis of Their Therapeutic Potential for Muscle Injuries

Lee, Eun-Mi; Kim, Ah-Young; Lee, Eunjoo; Park, Jin‐Kyu; Park, Se-Il; Cho, Ssang-Goo; Kim, Hong Kyun; Kim, Shin‐Yoon; Jeong, Kyu‐Shik

doi:10.3727/096368916x691691

Cited by 27 publications

(35 citation statements)

References 42 publications

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“…The derivation of equine iPSCs was first reported in 2011 (44) where a piggyback transposon system was used containing murine Oct4, Sox2, Klf4, and Myc reprogramming factors. This has then been followed by several other publications over the last 5 years , the majority of which have used viral expression vectors to mediate integration of the reprogramming gene sequences. Following reprogramming to a pluripotent state the viral transgenes should become silenced .…”

Section: Induced Pluripotent Stem Cells In Companion Animalsmentioning

confidence: 99%

“…Our group has been working to understand the factors which control tendon differentiation of equine ESCs and predifferentiating them prior to use may help to reduce the risk of teratoma formation. The derivation of horse and dog iPSCs has been conducted by more groups than ESC derivation, with successful teratoma formation being described in several publications . However, there are a few reported attempts to generate teratomas with canine iPSCs which failed .…”

Section: Characterizing Pluripotent Stem Cellsmentioning

confidence: 99%

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Characterization of companion animal pluripotent stem cells

2017

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Pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate into derivatives of the three germ layers. These properties underpin their potential to be used in regenerative medicine. Originally derived from early embryos, pluripotent stem cells can now be derived by reprogramming an adult cell back to a pluripotent state. Companion animals such as horses, dogs, and cats suffer from many injuries and diseases for which regenerative medicine may offer new treatments. As many of the injuries and diseases are similar to conditions in humans the use of companion animals for the experimental and clinical testing of stem cell and regenerative medicine products would provide relevant animal models for the translation of therapies to the human field. In order to fully utilize companion animal pluripotent stem cells robust, standardized methods of characterization must be developed to ensure that safe and effective treatments can be delivered. In this review we discuss the methods that are available for characterizing pluripotent stem cells and the techniques that have been applied in cells from companion animals. We describe characteristics which have been described consistently across reports as well as highlighting discrepant results. Significant steps have been made to define the in vitro culture requirements and drive lineage specific differentiation of pluripotent stem cells in companion animal species. However, additional basic research to compare pluripotent stem cell types and define characteristics of pluripotency in companion animal species is still required. V C 2017 International Society for Advancement of Cytometry

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Section: Induced Pluripotent Stem Cells In Companion Animalsmentioning

confidence: 99%

Section: Characterizing Pluripotent Stem Cellsmentioning

confidence: 99%

Characterization of companion animal pluripotent stem cells

2017

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“…In contrast to mesenchymal stem cells that have been used for tissue regeneration in the last decade [2][3][4], iPSCs have the advantages of indefinite proliferation and higher differentiation potential [5]. In fact, equine iPSCs myogenic differentiation capability has already been probed for skeletal muscle regeneration [6].…”

Section: Introductionmentioning

confidence: 99%

“…Several groups have reported the generation of iPSCs in the horse by reprogramming adult fibroblasts, fetal fibroblasts, keratinocytes and adipose-derived stem cells [6][7][8][9]. With these reports, it is clear that equine iPSCs can be generated from different sources, with the capacity to differentiate into all three germinal layers both in vitro and in vivo.…”

Section: Introductionmentioning

confidence: 99%

188 MicroRNA characterization in equine induced pluripotent stem cells

Moro

Amín

Furmento

et al. 2019

Reprod. Fertil. Dev.

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Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. A single microRNA can regulate the expression of hundreds of mRNA targets, a property given by a short sequence (called “seed”) in positions 2 to 8 from the 5′ end that is complementary to the 3′ untranslated region (UTR) tail of specific mRNAs. We aimed to generate horse induced pluripotent stem cells (iPSC), characterise them, and evaluate the expression of different microRNAs (miR-302a, b, c, d, miR-205, miR-145, miR-9, miR-96, miR-125b, and miR-296) in pluripotency and differentiation. Both cell states were evaluated (pluripotency and differentiation) in order to understand more deeply the complex network of transcriptional regulation in different contexts but with the same genomic background. Two equine iPSC lines (named L2 and L3) were characterised after the reprogramming of equine fibroblasts with the 4 human Yamanaka factors (OCT-4, SOX-2, c-MYC, KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG, and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2, and c-MYC. In vitro differentiation to embryo bodies (EB) showed the capacity of the iPSC to differentiate into ectodermal, endodermal, and mesodermal phenotypes. MicroRNA expression was analysed by quantitative RT-PCR and resulted in higher expression of the miR-302 family, miR-9, and miR-96 in L2 and L3v. fibroblasts (P ≤ 0.05), as previously shown in human pluripotent cells. Moreover, down-regulation of miR-145 and miR-205 was observed. After differentiation to EB, greater expression of miR-96 was observed in the EB compared with iPSC, and the expression of miR-205 was induced but only in the EB-L2. In addition, we performed in silico analysis of horse and human microRNAs. First, we compared the horse-miR-302/367 cluster with the human-miR-302/367 cluster and determined a 75% homology between them. Moreover, the seed region of the horse-miR-302 family resulted complementary to the 3′ UTR of horse cell cycle regulator genes CDK2, CYCLIN D1, and E2F1, and to the 3′ UTR of the RHOC gene, which is involved in the epithelial-mesenchymal transition. The miR-145 seed sequence was complementary to the 3′ UTR region of the OCT-4 and KLF-4 horse genes. With respect to miR-9 and miR-96, the seed sequence of these genes were complementary to the HES1 and PAX-6 genes. In all cases, the same gene targets were previously demonstrated in humans. In conclusion, we report the generation and characterization of equine iPSC and determined for the first time the expression of microRNAs in equine pluripotent cells. Moreover, several results led us to think that the horse microRNAs evaluated herein are highly conserved in sequence and function with respect to the human species. It will now be necessary to generate directed differentiations to derivatives of the 3 germ layers in order to strengthen our results. This is the first report to evaluate the expression and possible targets of microRNAs in pluripotent cells from domestic animals.

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“…Compared to humans, the availability of iPSCs (and specially ESCs) from horses is limited, as is the number of studies reporting their differentiation into functional cell types (Donadeu, 2014 ). A previous study showed the generation of myosin heavy chain (MyHC)-positive myotubes from equine skeletal muscle cell-derived iPSCs (Quattrocelli et al, 2016 ), whereas another reported formation of muscle fibers after transplantation of equine adipose stem cell-derived iPSCs into injured skeletal muscle of mice (Lee et al, 2016 ). In the present study, we took a step further by generating functional myocytes from equine iPSCs and comparing their characteristics with those of myocytes produced from adult equine skeletal muscle precursors.…”

Section: Introductionmentioning

confidence: 99%

Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells

Amilon

Cortes‐Araya

Moore

et al. 2018

Cellular Reprogramming

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Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.

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Generation of Equine-Induced Pluripotent Stem Cells and Analysis of Their Therapeutic Potential for Muscle Injuries

Cited by 27 publications

References 42 publications

Characterization of companion animal pluripotent stem cells

Characterization of companion animal pluripotent stem cells

188 MicroRNA characterization in equine induced pluripotent stem cells

Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells

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