Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies.
Liver cell types derived from induced pluripotent stem cells (iPSCs) share the potential to investigate development, toxicity, as well as genetic and infectious disease in ways currently limited by the availability of primary tissue. With the added advantage of patient specificity, which can play a role in all of these areas. Many iPSC differentiation protocols focus on 3 dimensional (3D) or organotypic differentiation, as these offer the advantage of more closely mimicking in vivo systems including; the formation of tissue like architecture and interactions/crosstalk between different cell types. Ultimately such models have the potential to be used clinically and either with or more aptly, in place of animal models. Along with the development of organotypic and micro-tissue models, there will be a need to co-develop imaging technologies to enable their visualization. A variety of liver models termed “organoids” have been reported in the literature ranging from simple spheres or cysts of a single cell type, usually hepatocytes, to those containing multiple cell types combined during the differentiation process such as hepatic stellate cells, endothelial cells, and mesenchymal cells, often leading to an improved hepatic phenotype. These allow specific functions or readouts to be examined such as drug metabolism, protein secretion or an improved phenotype, but because of their relative simplicity they lack the flexibility and general applicability of ex vivo tissue culture. In the liver field these are more often constructed rather than developed together organotypically as seen in other organoid models such as brain, kidney, lung and intestine. Having access to organotypic liver like surrogates containing multiple cell types with in vivo like interactions/architecture, would provide vastly improved models for disease, toxicity and drug development, combining disciplines such as microfluidic chip technology with organoids and ultimately paving the way to new therapies.
Induced pluripotent stem cell (iPSC) technology is an emerging technique to reprogram somatic cells into iPSCs that have revolutionary benefits in the fields of drug discovery, cellular therapy, and personalized medicine. However, these applications are just the tip of an iceberg. Recently, iPSC technology has been shown to be useful in not only conserving the endangered species, but also the revival of extinct species. With increasing consumer reliance on animal products, combined with an ever-growing population, there is a necessity to develop alternative approaches to conventional farming practices. One such approach involves the development of domestic farm animal iPSCs. This approach provides several benefits in the form of reduced animal death, pasture degradation, water consumption, and greenhouse gas emissions. Hence, it is essentially an environmentally-friendly alternative to conventional farming. Additionally, this approach ensures decreased zoonotic outbreaks and a constant food supply. Here, we discuss the iPSC technology in the form of a “Frozen Ark”, along with its potential impact on spreading awareness of factory farming, foodborne disease, and the ecological footprint of the meat industry.
The molecular and phenotypic irreversibility of mammalian cell differentiation was a fundamental principle of developmental biology at least until the 1980s, despite numerous reports dating back to the 1950s of the induction of pluripotency in amphibian cells by nuclear transfer (NT). Landmark reports in the 1980s and 1990s in sheep progressively challenged this dogmatic assumption; firstly, embryonic development of reconstructed embryos comprising whole (donor) blastomeres fused to enucleated oocytes, and famously, the cloning of Dolly from a terminally differentiated cell. Thus, the intrinsic ability of oocyte-derived factors to reverse the differentiated phenotype was confirmed. The concomitant elucidation of methods for human embryonic stem cell isolation and cultivation presented opportunities for therapeutic cell replacement strategies, particularly through NT of patient nuclei to enucleated oocytes for subsequent isolation of patient-specific (autologous), pluripotent cells from the resulting blastocysts. Associated logistical limitations of working with human oocytes, in addition to ethical and moral objections prompted exploration of alternative approaches to generate autologous stem cells for therapy, utilizing the full repertoire of factors characteristic of pluripotency, primarily through cell fusion and use of pluripotent cell extracts. Stunningly, in 2006, Japanese scientists described somatic cell reprogramming through delivery of four key factors (identified through a deductive approach from 24 candidate genes). Although less efficient than previous approaches, much of current stem cell research adopts this focused approach to cell reprogramming and (autologous) cell therapy. This chapter is a quasi-historical commentary of the various aforementioned approaches for the induction of pluripotency in lineage-committed cells, and introduces transcriptional and epigenetic changes occurring during reprogramming.
Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMX) encoding either 4 (Oct-4, Sox-2, Klf-4 and cMyc) or 5 (Oct-4, Sox-2, Klf-4, cMyc and Nanog) human transcription factors. Transduction efficiency of the retrovirus was ascertained using pMX-green fluorescent protein transgene expression and averaged 96% from 3 repeated experiments. The reprogramming efficiency of initial colony formation was 0.000308% (37/120 000 cells plated) for 4-factor induction compared with 0.000517% (62/120 000) for 5-factor induction. Transduction with 4 factors resulted in the formation of small colonies of cells, which could not be maintained for more than 4 passages (P4). However, addition of Nanog to the transfection cocktail produced stable iPS cell colonies, which formed as early as Day 3. Colonies of cells were selected at Day 5 and expanded in vitro on mouse embryonic fibroblast feeder cells. The resulting cell line was positive for alkaline phosphatase, Oct-4, Nanog and stage-specific embryonic antigen-4 at P14. Also, RT-PCR confirmed that endogenous Oct-4 and Nanog were expressed by snow leopard iPS cells from P4; although all 5 human transgenes were transcribed at P4, Oct-4, Sox-2 and Nanog transgenes were silenced as early as P14, suggesting that reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the 3 germ layers. This study describes the first derivation of iPS cells from the endangered snow leopard and is also the first report on induced pluripotency in felid species. Our results demonstrate that addition of Nanog to the reprogramming cocktail was essential for derivation of iPS lines in this felid and that iPS cells provide a unique source of pluripotent cells with utility in conservation for cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.
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