Induced pluripotent stem cells: Mechanisms, achievements and …

World J Stem Cells. 2015 Mar 26; 7(2): 315328.

Dharmendra Kumar, Thirumala R Talluri, Taruna Anand, Wilfried A Kues, Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, 31535 Mariensee, Germany

Dharmendra Kumar, Animal Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar 125001, Haryana, India

Thirumala R Talluri, Taruna Anand, National Research Centre on Equines, Hisar 125001, Haryana, India

Correspondence to: Dr. Wilfried A Kues, PhD, Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Hltystr. 10, 31535 Mariensee, Germany. ed.dnub.ilf@seuk.deirfliw

Telephone: +49-5034-871120 Fax: +49-5034-871101

Received 2014 Jul 15; Revised 2014 Dec 3; Accepted 2014 Dec 16.

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.

Keywords: Reprogramming, Large animal models, Stemness, Chimera, Germline transmission, Induced pluripotent stem cells, Gene delivery

Core tip: The generation of an induced status of pluripotency in somatic cells by ectopic expression of core transcription factors allows to extending advanced genetic modifications and reproductive techniques to species, for which the derivation of genuine embryonic stem cells was not successful till now. The commonly employed viral gene transfer may be genotoxic and therefore non-viral methods for iPS cell derivation are intensively studied. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency.

Induced pluripotent stem (iPS) cells are defined as differentiated cells that have been experimentally reprogrammed to an embryonic stem (ES) cell-like state. The first generation of murine iPS cells was achieved[1] by retroviral transduction of four core reprogramming factors: Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPS cells were produced by viral transduction of adult fibroblasts[2,3]. Also a combination of Oct4, Sox2, Nanog and Lin28, was effective for the generation of human iPS cells[4]. An overview of reprogramming cells into iPS cells is shown in Figure .

Methodological toolbox for generating induced pluripotent stem cells. iPS: Induced pluripotent stem.

Subsequently, the core reprogramming factors have been successfully used to derive pluripotent cells in various other species, including rhesus monkey[5], rat[6], pig[7], dog[8], cattle[9], horse[10], sheep[11], goat[12] and buffalo[13]. A summary of the generation of iPS cells from different species of livestock is enumerated in Table . Importantly, iPS cells could be isolated from several species, in which the isolation of authentic ES cells was not successful despite several attempts since many years[14,15]. In particular, for economically important species, such as farm animals, the availability of authentic iPS cells would have important consequences for reproductive biology and approaches for genetic modification. For agricultural purposes, iPS cells from farm animal species can serve as a valuable genetic engineering tool to boost the generation of livestock with advantageous genes that are important for economic, reproductive and disease resistant traits, or for the study of functional genomics in mammals.

Most advanced achievements in induced pluripotent stem cells from domestic animals

So far, iPS cells have been successfully produced from fibroblasts[16], pancreas cells[17], leukocytes[18], hepatocytes[19], keratinocytes[20], neural stem cells[21], cord blood cells[22], and other cell types. Together these data suggest that most cell types can be reprogrammed to a pluripotent state, and that the unidirectional lineage commitment can be experimentally overwritten. Certain cell types, such as neuronal progenitors, which exhibit basal expression of one or more of the core reprogramming factors, seem to be ideal for reprogramming[21].

Rodent iPS cells are almost identical to their ES cell counterparts, sharing typical hallmarks of pluripotency such as colony morphology, unlimited self-renewal, in vitro and in vivo differentiation potentials, and contribution to the germline[23,24]. Most iPS lines from farm animal species have not been tested in chimera complementation assays; however some preliminary reports suggest that chimeras and germline transmission can be achieved in sheep and pig[25,26]. iPS cells derived from rodents, humans, monkeys and farm animals share the features of high telomerase activity, expression of alkaline phosphatase, and expression of stemness genes, such as OCT4, SOX2, UTF1 and REX1. The epigenetic status of murine iPS cells has been analysed by bisulfite sequencing and chromatin immuno-precipitation DNA-Sequencing (ChIP-Seq)[27]. Thus the hallmarks for iPS cell characterisation can be enumerated as (1) unlimited self-renewal; (2) in vitro differentiation capacity; (3) in vivo differentiation capacity; (4) chimera contribution; and (5) subsequently germline transmission.

Apart from scientific and ethical hindrances, religious concerns restricted the derivation of human ES cells. To circumvent these concerns, alternative approaches to generate pluripotent cells have been assessed. The alternative approaches include culture of somatic cells with cell extracts isolated from ES cells[28] or oocytes[29], and fusion of somatic cell with pluripotent cell[30]. However, extremely low efficiencies, high technical difficulties and aberrant ploidies of the resulting cells[31,32] did reduce the enthusiasm for these attempts. At the moment, the derivation of iPS cells from human tissues seems to be the most promising alternative. Prior to clinical application of iPS-derivatives, cell survival, functional integration of the cellular transplant and safety of the cell products have to be assessed in informative animal models.

The progress in iPS cell development in farm animals lags behind those in rodents, but large mammalian models may be instrumental for pre-clinical tests of novel cell therapies (Table ), enhanced pharmaceutical studies and regenerative studies, including the restoration of fertility.

Achievements with induced pluripotent stem cells from rodents, farm animals and humans

Ontogenesis of an organism and cellular differentiation were thought to be a unidirectional process, where stem and progenitor cells progressively develop to terminally differentiated cells, for example neurons, muscle, and epithelial cells. During ontogenesis the nuclear DNA of most cell types is unchanged, but different epigenetic marks, such as DNA methylation and histon modifications, are set, and lock the cellular potency and cell lineage commitment. This is depicted by the epigenetic landscape proposed by Waddington[33].

Already in 1962, Gurdon[34] questioned this view by amphibian cloning; he transplanted nuclei from intestinal cells into irradiated oocytes and obtained vital tadpoles. More than three decades later, the successful cloning of a sheep (Dolly) by SCNT of a mammary epithelial cell to an enucleated oocyte, showed that even mammalian cells can be reprogrammed[35]. This success demonstrated that differentiated cells contain the genetic information to direct ontogenesis of an entire mammalian organisms, and that enucleated oocytes contain pivotal factors for reprogramming of differentiated cell nuclei. However, the identity of the oocyte reprogramming factors remained elusive.

The discoveries that ectopic expression of Antennapedia-a transcription factor was able and sufficient to induce leg structures in Drosophila[36], and that ectopic expression of the mammalian transcription factor MyoD1 converted fibroblasts into myocytes[37] led to the concept of master genes. A master gene was defined as a key transcription factor that in a hierarchical manner regulates a cascade of critical genes, which in a concerted action induce the cell commitment.

In 2006, Takahashi et al[1] proved that not a single master factor, but a a combination of four reprogramming factors, Oct4, Sox2, Klf4 and c-Myc, was sufficient to induce the pluripotent status in somatic mammalian cells. The resulting cells were called iPS cells[1]. This discovery offers new opportunities to study developmental biology, regenerative medicine, as well as reproductive biology and biotechnology of farm animals.

IPS cells from farm animals will likely serve as a bridging link between well developed rodent iPS and poorly characterised human iPS (Table ), supporting the translation of innovative cell therapies from experimental studies to curative treatments. At the moment, human iPS cell application seems to be too risky because of basic lack of knowledge and ethical consideration which forbid certain tests such as chimera assays.

In contrast, research on iPS cells derived from farm animal species is not tainted with ethical concerns. Furthermore, the methodology for generation of iPS cells is relative simple and and is thought to be easily transferable to other mammalian species. Thus farm animal models may turn out to be ideally suited to determine required cell doses, to assess long-term performance, tumorigenicity, applications methods and fate of transplanted cells[38-41].

Recent advances in genetic engineering of farm animals allow the generation of precise genetic modifications[42-47], such as the production of immunodeficient pigs[48] which will be instrumental for further advances in preclinical testings of new cell therapies. A boost of recent publications describe iPS cells from buffalo[13], cattle[9,49-53], dog[8,54-56], goat[11,57], horse[10,58-62], pig[7,63-71], rabbit[72-74] and sheep[11,75,76]. The majority of these iPS cells from farm animals showed typical hallmarks of pluripotency, such as differentiation in vivo and teratoma formation. However, most farm animal iPS cultures were not assessed for chimera contribution so far. Preliminary results that porcine iPS cells can contribute to chimera formation in blastocyst complementation were provided recently[71]. Similarly, ovine iPS cells contributed moderately to chimeric lambs after injection into eight-cell stage embryos or blastocysts[25]. These experiments represent an important step in the understanding of mechanistic nature of pluripotency in farm animals. The iPS technology may become instrumental for advanced transgenesis in large mammals (Figure ).

Application of induced pluripotent stem cells for advanced generation of transgenic animals. iPS: Induced pluripotent stem.

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