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Article

Insights for Deriving Induced Pluripotent Stem Cells in Marsh Deer (Blastocerus dichotomus)

by
Luciana Diniz Rola
1,*,
Fabiana Fernandes Bressan
2,
Eluzai Dinai Pinto Sandoval
1,
Jacinthe Therrien
3,
Lawrence Charles Smith
3 and
José Maurício Barbanti Duarte
1,*
1
Deer Research and Conservation Centre (NUPECCE—Núcleo de Pesquisa e Conservação de Cervídeos), Department of Animal Science, Jaboticabal, Faculdade de Ciências Agrárias e Veterinárias (FCAV), University Estadual Paulista (UNESP), São Paulo 14884-900, SP, Brazil
2
Department of Veterinary Medicine, College of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga 13635-900, SP, Brazil
3
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 8H5, Canada
*
Authors to whom correspondence should be addressed.
J. Zool. Bot. Gard. 2025, 6(3), 43; https://doi.org/10.3390/jzbg6030043
Submission received: 13 June 2025 / Revised: 5 August 2025 / Accepted: 18 August 2025 / Published: 19 August 2025
Browse Figures
Versions Notes

Abstract

The capability to generate induced pluripotent stem cells (iPSCs) from adult somatic cells, enabling them to differentiate into any cell type, has been demonstrated in several studies. In humans and mice, iPSCs have been shown to differentiate into primordial germ cells (PGCs), spermatozoa, and oocytes. However, research on iPSCs in deer is novel. Despite the necessity for establishing germplasm banks from endangered cervid species, the collection and cryopreservation of gametes and embryos have proven complex for this group. Therefore, the focus of this study was to establish protocols for deriving stable iPSC lines from Blastocerus dichotomus (Marsh deer) using primary cells derived from antler, adipose tissue, or skin, with the ultimate goal of producing viable gametes in the future. To achieve this, two main reprogramming approaches were tested: (1) transfection using PiggyBac transposons (plasmid PB-TET-MKOS) delivered via electroporation and (2) lentiviral transduction using the STEMCCA system with either human (hOSKM) or murine (mOSKM) reprogramming factors. Both systems utilized murine embryonic fibroblasts (MEFs) as feeder cells. The PiggyBac system was further supplemented with a culture medium containing small molecules to aid reprogramming, including a GSK inhibitor, MEK inhibitor, ALK/TGF inhibitor, and thiazovivin. Initial colony formation was observed; however, these colonies failed to expand post-selection. Despite these challenges, important insights were gained that will inform and guide future studies toward the successful generation of iPSCs in deer.

Graphical Abstract

1. Introduction

Most Neotropical deer species are experiencing population declines, rendering their conservation status markedly more critical than that of other mammalian taxa globally. While approximately 25% of all mammal species are currently classified as threatened and 15% as data deficient (DD) [1], this situation is more acute for Neotropical deer: 53% are categorized under a threat level, and 17.6% are classified as DD due to insufficient comprehensive studies to ascertain their true conservation status [2]. Declining wild populations typically face reduced genetic diversity and increased rates of consanguineous mating (inbreeding), thereby elevating their extinction risk [3,4].
The Marsh deer (Blastocerus dichotomus), one of South America’s largest terrestrial mammals, has suffered a reduction of approximately 65% in its natural habitat over the past four decades [5]. Moreover, the species is potentially extinct in countries such as Uruguay and Peru [6,7,8]. Beyond habitat loss and fragmentation, other anthropogenic pressures, including close proximity to domestic livestock (which facilitates pathogen transmission) and hunting [9], have exacerbated the decline of Marsh deer populations. Consequently, B. dichotomus is classified as vulnerable (VU) on the International Union for Conservation of Nature Red List [2].
In this context, the establishment of genome resource banks (GRBs) as repositories of genetic material such as semen, oocytes, embryos, and somatic cells (e.g., fibroblasts), alongside the application of assisted reproductive technologies (ARTs), presents promising strategies for conserving endangered species [10,11]. The relative ease with which tissues and cells can be acquired and cryopreserved renders them a highly promising source of biological material. A particularly significant development in this context is the utilization of induced pluripotent stem cells (iPSCs). These cells are generated by reprogramming somatic cells to achieve a state of pluripotency analogous to that of embryonic stem cells (ESCs). Takahashi and Yamanaka [12] pioneered this methodology, successfully producing iPSCs through the induced expression of four key exogenous transcription factors: Oct4, Sox2, KLF4, and c-Myc. Advancements in stem cell technology have introduced alternative methodologies wherein iPSCs can be differentiated into functional gametes (oocytes, sperm, or spermatids) and, following fertilization, yield healthy animals [13,14].
In mammals, the efficiency of cellular reprogramming exhibits considerable variation across different cell types, being demonstrably lower in terminally differentiated cells compared to less-differentiated or undifferentiated adult progenitor cells [15,16]. Consequently, the selection of an appropriate cell source is a critical determinant in the development of iPSC lines, necessitating careful consideration of factors such as reprogramming amenability, tissue accessibility, and the facility of in vitro culture and maintenance.
In cervids, antler-derived stem cells present a unique and compelling opportunity [17]. Antlers, representing a rare instance of complete organ regeneration in mammals, serve as an invaluable model for investigating mechanisms of scar-free wound healing, resistance to tumorigenesis despite rapid cell proliferation, and potential therapeutic strategies for promoting limb regeneration [18]. These osseous structures, which undergo periodic regeneration from proliferating stem cell populations, typically originate from the frontal bone (pedicles) of adult male deer; however, in reindeer (Rangifer tarandus), antlers are present in both sexes [19]. Previous investigations in Cervus elaphus and Dama dama have demonstrated that the pedicle periosteum harbors cells expressing a panel of established stem cell markers (e.g., CD9, OCT-4, Nanog, and STRO-1) [20,21]. Nevertheless, to date, the derivation of iPSC lines from this specific tissue source has not been reported.
Adipose tissue-derived stem cells (ADSCs) represent another promising avenue, as studies have identified these cells as a highly suitable somatic cell source for the generation of iPSCs [22]. ADSCs exhibit robust proliferative capacity and have demonstrated superior efficiency in iPSC line generation when compared to other cell lineages, such as mouse embryonic fibroblasts or adult neural stem cells. Furthermore, dermal tissue (skin) constitutes a readily accessible source of biological material [23,24]. Significantly, both skin and adipose tissues can be collected from individuals irrespective of sex or developmental stage, often through minimally invasive procedures.
The successful derivation of iPSCs has been achieved in an extensive range of species. This includes laboratory animals, such as mice [12] and rats [25], and domestic species, like dogs [26], pigs [27], rabbits [28], sheep [29], horses [30], cattle [31], buffalo [32], quail [33], chickens [34], and goats [35]. Significantly, this technology has also been applied to various wild species, including rhesus monkeys (Macaca mulatta) [36], white rhinos (Ceratotherium simum) [37], mandrills (Mandrillus leucophaeus) [37], snow leopards (Panthera uncia) [38], Bengal tigers (Panthera tigris) [39], servals (Leptailurus serval) [39], jaguars (Panthera onca) [39], and bats (Myotis lucifugus) [40], among other mammals, birds, and even amphibians, reptiles, and fish [41].
Despite these widespread advancements, there are currently no published reports on iPSC derivation in any cervid species. Given that approximately one-third of global deer populations are classified as at risk of extinction [2], the development of iPSC lines with the capacity to differentiate into viable gametes could represent a pivotal conservation strategy. Therefore, the objective of the present study is to reprogram somatic cells derived from antlers, adipose, and dermal tissues of B. dichotomus in order to contribute to the conservation of this specific species and to establish a foundational model for iPSC generation applicable to other deer species.

2. Materials and Methods

This work was evaluated and approved by the Ethics and Animal Welfare Committee (CEBEA) of the Faculty of Agricultural and Veterinary Sciences (FCAV) of São Paulo State University (UNESP), Jaboticabal campus, under protocol No. 025986/13. This study complies with the ethical principles in animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA).

2.1. Animal Biopsies

Biopsies were collected from animals at the Marsh Deer Conservation Center (Centro de Conservação do Cervo-do-Pantanal—CCCP) in Promissão, SP, Brazil, and the Deer Research and Conservation Centre (Núcleo de Pesquisa e Conservação de Cervídeos-NUPECCE) at the Department of Animal Science, FCAV-UNESP, Jaboticabal, SP, Brazil. The animals were anesthetized using a combination of 7.0 mg kg−1 ketamine hydrochloride (Vetaset®-Fort Dodge, Campinas, Brazil) and 1 mg kg−1 xylazine (Rompun®-Bayer, São Paulo, Brazil) administered via intramuscular injection using anesthetic darts.
Antler biopsies were obtained from three males, with samples collected 45 to 60 days after antler growth began, approximately 0.5 cm below the tip. For skin biopsies, tissues were sampled from two females and one male; the samples were dissected from the proximal region of the pelvic limb, which had been previously shaved and disinfected with 70% alcohol, using sterile scissors and tweezers. Additionally, adipose tissue biopsies were taken from three females from the lateral area of the ear, just below the cartilage. This alternative site was selected due to the difficulty of safely obtaining fat from more commonly used regions, such as the caudal or abdominal areas, which are commonly used in other species [42,43,44].

2.2. Isolation of Somatic Cells

After collection, the tissues were stored in a cryovial containing McCoy’s solution (M4892, Sigma-Aldrich, St. Louis, MO, USA) with high concentrations of antibiotic (500 mg L−1 of gentamicin and 20 mg L−1 of amphotericin B) and kept refrigerated on ice inside a Styrofoam box. Upon arrival at the laboratory, the tissues were washed four times in PBS and mechanically separated into pieces of about 2 mm2 using a scalpel blade. After mechanical breakdown, the cells were enzymatically digested with collagenase I (200 U mL−1, Sigma) inside an incubator at 37 °C for a period of 3 h for antler and skin and 30 min for fat. After enzymatic digestion, the cells were centrifuged for 5 min at 200 G, and the pellet was suspended in 10 mL of complete culture medium composed of Dulbecco’s modified Eagle’s minimal essential medium (DMEM) with high glucose levels (4.5 g L−1), glutamine (0.6 g L−1), 1% essential amino acids, fetal bovine serum (FBS) in a proportion of 10% v/v, 50 mg L−1 of gentamicin sulfate, and 2 mg L−1 amphotericin B. The sample was centrifuged again for 5 min at 200 g, and the pellet was resuspended in 5 mL of complete culture medium containing 10% of FBS. The medium exchange was performed 48 h after the initial plating to remove non-adherent cells. For the maintenance of the culture, the medium was exchanged twice a week using complete culture medium supplemented with 10% fetal bovine serum (FBS).
The cells were cultured until they reached about 80% confluence, and, on reaching this threshold, the cells were detached using 0.25% trypsin, centrifuged, and reseeded into new flasks. The cells of the second or third passage were cryopreserved in a medium composed of 80% FBS, 10% dimethyl sulfoxide (DMSO), 10% DMEM, 100 units/mL of penicillin, and 100 ug/mL streptomycin using the slow cooling method (4 h at 4 °C, 30 min in nitrogen steam, and subsequently immersed in liquid nitrogen) and stored in cryogenic cylinders. At the time of performing the experiments, the cells were thawed in a water bath at 37 °C and cultured. The cells of the fourth and fifth passages were used in further experiments.

2.3. Pluripotency Induction

2.3.1. Electroporation Using the PiggyBac System

The electroporation protocol used for the derivation of iPSC was similar to that previously used for the derivation of equine iPSC by Nagy and collaborators [30]. Therefore, to generate iPSC in Marsh deer, we use the system based on transfections with transposon PiggyBac. The plasmids PB-TET-OSKM (an excisable polycistronic vector with murine gene sequence), PBCAG-rtTA, pCyL43 PBase, and PB-GFP were performed as previously described by Nagy and collaborators [30].
The cells were transfected using the electroporator device Neon® (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. For electroporation, we used 5 × 104 cells with a combination of DNA with equal proportions of the four plasmids (0.25 µg of each) for each well in 6-well plates. These plates were pre-gelatinized (0.1% gelatin) and plated with murine embryonic fibroblasts (MEFs) inactivated by mitomycin C for 3 h to serve as “feeders” of the transfected cells.
After transfection, the cells were placed in a medium composed of DMEM (Invitrogen, #11960-044), 2 mm glutamax (Invitrogen), 0.1 mm β-mercaptoethanol, 0.1 mm non-essential amino acids, 1 mM sodium pyruvate, 50 U/mL penicillin/streptomycin, 1000 U/mL leukemia inhibitory factor, 10 ng/mL bFGF, 5 µg/mL insulin, 1.5 µg/mL doxycycline, 3 µM GSK inhibitor (Stemgent, Beltsville, MD, USA #CHIR99021), 0.5 µM MEK inhibitor (Stemgent #PD0325901), 2.5 µM ALK/TGF inhibitor (Stemgent #A83-01), and 1 mm thiazovivin and contained either 10% FBS or 15% KnockOut Serum Replacement (KSR), depending on the tested group. We also tested two electroporation programs (see below) using Protocol 1, which consists of a pulse voltage equal to 1400, a pulse width equal to 20, and a number of pulses equal to 1, or Protocol 2, which consists of a pulse voltage equal to 1400, a pulse width equal to 30, and a number of pulses equal to 2.
The plasmid PB-GFP, which encodes the green fluorescent protein (GFP), served as a reporter gene, enabling the quantification of cells that successfully integrated the plasmid. Protocol 1, employed in this study, was previously validated for iPSC derivation in equine species [30]. In contrast, Protocol 2 was specifically optimized for the cell type under investigation in this experiment through systematic evaluation of 24 pre-existing electroporation programs on the Neon® Transfection System. These optimization tests were conducted exclusively with the PB-GFP plasmid to accurately assess the efficiency of plasmid integration into the cells.
The cells were allocated to distinct experimental groups based on the electroporation protocol and culture medium composition: (G1) Protocol 1 with a medium supplemented with fetal bovine serum (FBS); (G2) Protocol 1 with a medium supplemented with KnockOut™ Serum Replacement (KSR); (G3) Protocol 2 with a medium supplemented with FBS; and (G4) Protocol 2 with a medium supplemented with KSR. Following transfection, the cell culture plates were maintained for a minimum of 35 days to monitor for the emergence of colonies.

2.3.2. Lentiviral Transduction Using the STEMCCA System

The induction of reprogramming was performed according to the protocol previously described by Bressan and collaborators [45] for bovine cell lines. For viral production, lipofection (Lipofectamine 3000, Life Technologies, Carlsbad, CA, USA) was used in 293FT cells, which were plated in 100 mm culture plates at a concentration of 5 × 106 cells, presenting 80% confluence at the time of transfection. A total of 12 μg of the STEMCCA excisable polycistronic lentiviral vectors (murine or human factors), 2 μg of the helper vectors TAT, REV, and Hgpm2, and 2.4 μg of VSVG were used per plate.
The cells underwent lipofection for a period of 12 to 16 h (overnight), after which the transfection medium was replaced. Subsequently, at 24, 48, and 72 h post-lipofection, the conditioned medium containing lentiviral particles was collected, filtered, and utilized for transduction. For this, Marsh deer cells were cultured at a density of 2 × 105 cells per well in 6-well plates and then transduced with the STEMCCA lentiviral vector, encoding either human (hOSKM) or murine (mOSKM) reprogramming factors, in the presence of 8 µg/mL polybrene (hexadimethrine bromide, Sigma). Five to six days post-transduction, the cells were enzymatically detached using TrypLE™ (Life Technologies), and 1.25 × 104 cells were transferred onto 6-well plates containing mitotically inactivated mouse embryonic fibroblasts (MEFs). These cells were subsequently maintained in a reprogramming culture medium composed of DMEM/F12 (Invitrogen) supplemented with 20% KnockOut™ Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.055 mM β-mercaptoethanol (Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF, Sigma), and standard antibiotics. The culture medium was refreshed every two days.
The experimental design consisted of two main groups based on the origin of the reprogramming factors: one group was transduced with human STEMCCA factors (hOSKM) and another with murine STEMCCA factors (mOSKM). Following transduction, the cell culture plates were maintained for a minimum of 35 days to observe colony formation. To assess the efficacy of the lentiviral delivery system in the target cells, one of the skin cell lines was transduced solely with a GFP reporter gene. Forty-eight hours post-transduction, these GFP-transduced cells were enzymatically detached using TrypLE™ (Life Technologies) and subsequently analyzed by flow cytometry (BD FACSAria™ Cell Sorters, San Jose, CA, USA) to quantify the transduction efficiency.

3. Results

A total of twelve attempts to induce pluripotency via electroporation were conducted in six-well plates, each containing five wells with transfected cells and one negative control. These attempts were distributed as follows (Table 1): five using Protocol 1 with FBS-containing medium (three for antler-derived cells, one for adipose-derived cells, and one for skin-derived cells); three using Protocol 2 with FBS-containing medium (two for antler-derived cells and one for adipose-derived cells); one using Protocol 1 with KSR-containing medium (antler-derived cells); and three using Protocol 2 with KSR-containing medium (two for antler-derived cells and one for adipose-derived cells).
Initial attempts to induce pluripotency utilized Protocol 1, as previously described for equine cells [30], which yielded approximately 40% of GFP-positive cells. However, due to the absence of colonies that could be expanded into stable iPSC lines and a lower transfection efficiency (GFP-positive cells) compared to that observed in horses (approximately 90%), protocol optimization was undertaken. The implementation of Protocol 2 resulted in an approximate 20% increase in GFP-positive cells, reaching around 60%.
Following these attempts with Protocol 2, which still did not yield iPSC lines, FBS was replaced with the KnockOut Serum Replacement (KSR).
Despite these efforts and protocol modifications, stable iPSC lines could not be generated. Approximately 20 days post-transfection, emergent colonies were observed (Figure 1), some exhibiting irregular morphology (Figure 1A), with most being GFP-positive. These colonies were manually selected, dissected into four pieces using a scalpel blade, and transferred to new plates with feeder cells.
For attempts utilizing the STEMCCA lentiviral vector system, flow cytometry analysis of GFP reporter gene expression revealed results consistent with those previously observed using PiggyBac electroporation, with approximately 50% of cells expressing the GFP protein (Figure 2). In this lentiviral approach, each sample type (two skin, one antler, and one adipose-derived) was tested in a six-well plate format. Within each plate, three wells were dedicated to transduction with human OSKM factors, and three wells were used for murine OSKM factors (Table 1).

4. Discussion

Given the lack of colony formation during the initial iPSC line generation via the PiggyBac system, the first step was to optimize the electroporation protocol. Following these attempts with a new protocol of electroporation (Protocol 2), which still did not yield iPSC lines, FBS was replaced with the KnockOut Serum Replacement (KSR). Previous studies have demonstrated that KSR can significantly enhance the reprogramming efficiency and may even facilitate the conversion of partially reprogrammed iPSCs into a fully reprogrammed state [46,47]. Conversely, FBS used during the reprogramming phase can exert deleterious effects, such as altering the activities of H3K9 methyltransferases and demethylases, due to the presence of Bone Morphogenetic Protein (BMP) family growth factors in its composition [48].
KSR contains components such as thiamine and vitamin C, which are known to favor reprogramming. Vitamin C, in particular, has been extensively studied in the context of iPSC reprogramming. It possesses antioxidant properties [49], prevents the silencing of the Dlk1-Dio3 locus (which can be compromised by FBS, thereby impairing the generation of fully reprogrammed iPSCs from adult cells) [50], is linked to active DNA demethylation [51], and influences other epigenetic mechanisms [52]. Its application has led to improved iPSC quality in murine models, as assessed by the tetraploid complementation assay, an improvement attributed to a reduction in DNA methylation aberrations [50].
However, no subsequent cell growth was observed after selection and passage when KSR was used in Marsh deer cells. It is plausible that these cells underwent only partial reprogramming or became refractory to complete reprogramming due to various factors, including an inability to undergo mesenchymal-to-epithelial transition (MET), aberrant activation of differentiation-associated genes, or a failure to sustain pluripotency gene expression [53].
Despite applying various Marsh deer tissue sources, our attempts to generate iPSC lines using the PiggyBac protocol from Nagy et al. [30] were unsuccessful. Multiple studies confirm that the somatic cell source significantly influences the efficiency and fidelity of reprogramming into induced pluripotent stem cells (iPSCs) [54,55,56,57]. Indeed, the original protocol by Nagy et al. [30] was only effective on fetal fibroblasts, whereas it failed on adult fibroblasts (Smith, personal communication). However, obtaining fetal fibroblasts from Marsh deer presents significant logistical challenges and is considered an impractical strategy for establishing germplasm banks of this species.
For cellular reprogramming with the lentiviral system, both murine and human factors were tested. While previous studies have indicated that bovine cells reprogram more successfully with murine factors [45], equine cells show a preference for human factors [58]. Despite transduction with both human and murine OSKM vectors, no colonies were formed by Marsh deer cells.
The challenges encountered in generating deer iPSCs were expected, given the well-documented difficulties in establishing pluripotent cell lines from domestic ungulates. Success in obtaining robust embryonic stem cell (ESC) lines from these species has been limited [59,60]. Similarly, for iPSCs, numerous studies report significant obstacles [61,62,63]. These include failures in silencing exogenous transgene expression post-reprogramming, difficulties in maintaining cell cultures for extended periods, and the incomplete characterization of derived lines, often failing to meet all pluripotency criteria, particularly in vivo validation. Replicating culture conditions successfully employed for murine or human cells has not yielded comparable results in domestic ungulates, and identifying the optimal set of reprogramming factors for these species remains a major challenge [64].
Notably, iPSCs generated using the canonical Yamanaka factors (Oct4, Sox2, c-Myc, and Klf4, commonly referred to as OSKM) exhibit higher rates of aberrant DNA methylation compared to cells derived via somatic cell nuclear transfer [65]. Consequently, an efficient and reliable strategy for iPSC generation might involve the incorporation of additional or alternative expression factors. Supporting this, Wang and collaborators [66] demonstrated accelerated reprogramming of both murine and human fibroblasts by co-expressing OSKM factors with two nuclear hormone receptors, Rarc and Lrh1. This approach significantly shortened the reprogramming timeline from the typical 3–4 weeks to 4–5 days. Further evidence comes from Nie and collaborators [67], who observed no colony formation when attempting to reprogram mature murine adipocytes using only OSKM factors delivered via the PiggyBac system. However, upon employing the six-factor combination (OSKM plus Lrh1 and Rarc), as described by Wang and collaborators [66], colonies began to emerge from day 16 onward, with 86% of these successfully expanding into stable cell lines. The supplementary factors Lrh1 and Rarc are known to upregulate the expression of key pluripotency genes Oct4 and Nanog, thereby altering reprogramming kinetics and improving the efficiency of achieving a fully reprogrammed state.
In several species, OSKM alone has proven insufficient to induce pluripotency, necessitating the inclusion of additional factors. For instance, in cattle [31], wild felids such as the snow leopard [38], Bengal tiger, serval, and jaguar [39], as well as in the drill, Northern White Rhinoceros [37], and Ryukyu spiny rat [68], the addition of NANOG to the retroviral induction cocktail was required. Consistent with observations in our experiment, some of these studies reported initial colony formation using OSKM factors; however, these colonies could not be sustained through subsequent passages. In these cases, the addition of NANOG led to large, circular colonies that were expanded over multiple passages, establishing stable iPSC lines. The pluripotency of these lines was subsequently validated through in vitro and in vivo assays.
Similarly, Talluri and collaborators [69], employing the PiggyBac transposon system for bovine fetal fibroblasts, failed to generate iPSC lines using only OSKM factors, requiring the co-expression of both NANOG and LIN28 for successful reprogramming. These same factors [28] were also essential for reprogramming Northern White Rhinoceros cells via electroporation-mediated transduction [70]. West and collaborators [71] utilized this expanded cocktail to produce porcine iPSCs, and, notably, these lines demonstrated the capacity to contribute to chimeric offspring. In avian species, such as the Okinawa rail and Blakiston’s fish owl, reprogramming with OSKM factors delivered via PiggyBac transposase transduction was achieved using a modified OCT3/4 (M3O), plus the addition of NANOG, LIN28, and KLF2 [72].
However, the necessity to add or modify exogenous reprogramming factors is not attributable to a single determinant, as this requirement varies depending on the cell type, reprogramming methodology, culture medium composition, and other variables. For example, Kawaguchi and collaborators [73] successfully generated stable bovine iPSC lines from adipose-derived cells using the PiggyBac system with only OSKM factors. These lines were capable of contributing to the inner cell mass of host blastocysts and forming chimeric fetuses without the need for exogenous NANOG. However, when embryonic fibroblasts were transfected under identical conditions, the emergent colonies failed to establish stable cell lines capable of sustained propagation.
Another noteworthy observation is the apparently greater reprogramming efficiency achieved when employing two separate vectors, one encoding Oct4, Klf4, and Sox2 (OKS) and another encoding c-Myc, compared to a single polycistronic vector expressing all four factors (OSKM) [74]. In the aforementioned study on cattle by Kawaguchi and collaborators [73], it was reported that transfection with a single OSKM vector failed to yield colonies from adipose-derived cells. In contrast, co-transfection with separate OKS and c-Myc vectors resulted in the generation of iPSC lines with an efficiency of approximately 0.01%. Consequently, many specific aspects contribute to the successful generation of stable and reliable iPSC lines. This multiplicity of factors influencing cellular reprogramming, coupled with the gaps in fundamental biological knowledge regarding wild animals, highlights the considerable challenge associated with generating iPSC lines for these species. Extensive testing and research are imperative before the ultimate goal can be achieved, particularly when considering the maximization of germplasm utilization from endangered species. Future studies may deepen our understanding of the mechanisms and optimize protocols for producing stable and reliable iPSC lines in cervid species.
Furthermore, it is also of strong relevance to wildlife species to explore not only alternative combinations of transcription factors and culture medium components but also safer vector delivery methods. An aspect of particular relevance in this context is the mitigation of genomic risks associated with integrating vectors. The generation of genome-unaltered (footprint-free) cell lines is a prerequisite for conservation biology, as it ensures the genetic integrity necessary for future applications in reproductive biotechnologies, such as in vitro gametogenesis. Integrating vectors, like retroviruses and lentiviruses, carry inherent risks of insertional mutagenesis and, therefore, may not represent the primary choice [75]. The PiggyBac system, being excisable, offers an intermediate alternative; however, the possibility of imperfect excision or DNA repair errors necessitates rigorous genomic validation. In this context, non-integrating methodologies, such as the use of Sendai virus or synthetic mRNA, which induce pluripotency without modifying the genome [16,75], emerge as highly promising approaches for the future derivation of iPSC lines from wild animals.

5. Conclusions

To our knowledge, this study represents the first report in the literature on the utilization of antler-derived cells for cellular reprogramming to a pluripotent state. Although stable iPSC colonies were not obtained, our study systematically documented the initial attempts and obstacles encountered in reprogramming cells from a wild, endangered, and non-model species, such as the Marsh deer (Blastocerus dichotomus). The initial stages, including the achievement of Marsh deer cells and the exploration of diverse methodological approaches (encompassing variations in culture medium with FBS or KSR, the use of transposon-based or lentiviral vectors, and different genetic material delivery methods such as electroporation or lentiviral transduction), form an essential foundation for refining future studies and achieving more advanced outcomes. Furthermore, the low transfection efficiency observed, even with optimized parameters, suggests that effective vector delivery remains a significant challenge that needs to be overcome for successful reprogramming in this species. Consequently, future investigations can build upon the findings of this work to propose optimizations, such as the employment of different exogenous transcription factors and alternative culture medium supplementation.

Author Contributions

Conceptualization, L.D.R., L.C.S., J.M.B.D. and F.F.B.; methodology, L.D.R., L.C.S. and F.F.B.; formal analysis, L.D.R. and J.T.; investigation, L.D.R. and F.F.B.; resources, J.M.B.D., L.C.S. and F.F.B.; writing—original draft preparation L.D.R.; writing—review and editing, L.D.R., F.F.B., E.D.P.S., J.T., J.M.B.D. and L.C.S.; visualization, L.D.R., F.F.B., E.D.P.S., J.T., J.M.B.D. and L.C.S.; supervision, J.M.B.D. and L.C.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) process No. 2013/13972-0 and 2014/02439-2.

Institutional Review Board Statement

The animal study protocol was approved by the Ethics and Animal Welfare Committee (CEBEA) of the Faculty of Agricultural and Veterinary Sciences (FCAV) of São Paulo State University (UNESP), Jaboticabal campus, under protocol No. 025986/13, approved on 4 December 2013.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

We would like to thank the collaboration of the other members of the Deer Research and Conservation Center, the laboratory technician João Boer for helping with the equipment and preparing solutions, the veterinarians Eveline Zanetti dos Santos and Juliana Linardi from the Pantanal Deer Conservation Center (CCCP), and the entire team of caretakers, who kept the animals healthy and in high welfare.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Schipper, J.; Chanson, J.S.; Chiozza, F.; Cox, N.A.; Hoffmann, M.; Katariya, V.; Lamoreux, J.; Rodrigues, A.S.L.; Stuart, S.N.; Temple, H.J.; et al. The status of the world’s land and marine mammals: Diversity, threat, and knowledge. Science 2008, 322, 225–230. [Google Scholar] [CrossRef]
  2. The IUCN Red List of Threatened Species. Available online: https://www.iucnredlist.org/ (accessed on 31 March 2025).
  3. Frankham, R.; Ballou, J.D.; Briscoe, D.A. A Primer of Conservation Genetics; Cambridge University Press: Cambridge, UK, 2004; p. 220. [Google Scholar]
  4. Keller, L.F.; Biebach, I.; Ewing, S.R.; Hoeck, P.E.A. The genetics of reintroductions: Inbreeding and genetic drift. In Reintroduction Biology: Integrating Science and Management; Ewen, J.G., Armstrong, D.P., Parker, K.A., Seddon, P.J., Eds.; Wiley-Blackwell: Hoboken, NJ, USA, 2012; pp. 360–394. [Google Scholar]
  5. Weber, M.; Gonzalez, S. Latin American deer diversity and conservation: A review of status and distribution. Écoscience 2003, 10, 443–454. [Google Scholar] [CrossRef]
  6. Piovezan, U.; Tiepolo, L.M.; Tomas, W.M.; Duarte, J.B.; Varela, D.; Marinho-Filho, J.S. Marsh deer Blastocerus dichotomus (Illiger, 1815). In Neotropical Cervidology: Biology and Medicine in Latin American Deer; Duarte, J.M.B., González, S., Eds.; Funep/IUCN: Jaboticabal, Brazil, 2010; pp. 66–76. [Google Scholar]
  7. González, S.; Lessa, E.P. Historia de la mastozoología en Uruguay. In Historia de la Mastozoología en Latinoamérica, las Guayanas y el Caribe; Ortega, J., Martínez, J.L., Tirira, D.G., Eds.; Editorial Murciélago Blanco y Asociación Ecuatoriana de Mastozoología: Quito, Ecuador; México D.F., Mexico, 2014; pp. 381–404. [Google Scholar]
  8. González, S.; Aristimuño, M.P.; Moreno, F. New record in Uruguay of the marsh deer (Blastocerus dichotomus Illiger, 1815) redefines its southern geographic distribution area. Front. Ecol. Evol. 2024, 12, 1419234. [Google Scholar] [CrossRef]
  9. González, S.; Duarte, J.M.B. Speciation, evolutionary history and conservation trends of Neotropical deer. Mastozool. Neotrop. 2020, 27, 37–47. [Google Scholar] [CrossRef]
  10. Holt, W.V.; Pickard, A.R. Role of reproductive technologies and genetic resource banks in animal conservation. Rev. Reprod. 1999, 4, 143–150. [Google Scholar] [CrossRef]
  11. Wildt, D.E. Genetic resource banks for conserving wildlife species: Justification, examples and becoming organized on a global basis. Anim. Reprod. Sci. 1992, 28, 247–257. [Google Scholar] [CrossRef]
  12. Takahashi, K.; Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006, 126, 663–676. [Google Scholar] [CrossRef]
  13. Hayashi, K.; Ohta, H.; Kurimoto, K.; Aramaki, S.; Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011, 146, 519–532. [Google Scholar] [CrossRef]
  14. Hayashi, K.; Ogushi, S.; Kurimoto, K.; Shimamoto, S.; Ohta, H.; Saitou, M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 2012, 338, 971–975. [Google Scholar] [CrossRef]
  15. Smith, Z.D.; Sindhu, C.; Meissner, A. Molecular features of cellular reprogramming and development. Nat. Rev. Mol. Cell Biol. 2016, 17, 139–154. [Google Scholar] [CrossRef]
  16. González, F.; Boué, S.; Belmonte, J.C.I. Methods for making induced pluripotent stem cells: Reprogramming a la carte. Nat. Rev. Genet. 2011, 12, 231–242. [Google Scholar] [CrossRef]
  17. Wang, D.; Berg, D.; Ba, H.; Sun, H.; Wang, Z.; Li, C. Deer antler stem cells are a novel type of cells that sustain full regeneration of a mammalian organ—Deer antler. Cell Death Dis. 2019, 10, 443. [Google Scholar] [CrossRef]
  18. Colitti, M.; Allen, P.; Price, J.S. Programmed cell death in the regenerating deer antler. J. Anat. 2005, 207, 339–351. [Google Scholar] [CrossRef]
  19. Kierdorf, U.; Kierdorf, H. Deer antlers—A model of mammalian appendage regeneration: An extensive review. Gerontology 2011, 57, 53–65. [Google Scholar] [CrossRef]
  20. Li, C.; Yang, F.; Sheppard, A. Adult stem cells and mammalian epimorphic regeneration—Insights from studying annual renewal of deer antlers. Curr. Stem Cell Res. Ther. 2009, 4, 237–251. [Google Scholar] [CrossRef]
  21. Rolf, H.J.; Kierdorf, U.; Kierdorf, H.; Schulz, J.; Seymour, N.; Schliephake, H.; Napp, J.; Niebert, S.; Wolfel, H.; Wiese, K.G. Localization and characterization of STRO-1 cells in the deer pedicle and regenerating antler. PLoS ONE 2008, 3, e2064. [Google Scholar] [CrossRef]
  22. Tat, P.A.; Sumer, H.; Jones, K.L.; Upton, K.; Verma, P.J. The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Cell Transplant. 2010, 19, 525–536. [Google Scholar] [CrossRef] [PubMed]
  23. Coleman, S.R. Long-term survival of fat transplants: Controlled demonstrations. Aesthetic Plast. Surg. 1995, 19, 421–425. [Google Scholar] [CrossRef] [PubMed]
  24. Yarak, S.; Okamoto, O.K. Human adipose-derived stem cells: Current challenges and clinical perspectives. An. Bras. Dermatol. 2010, 85, 647–656. [Google Scholar] [CrossRef]
  25. Liao, J.; Cui, C.; Chen, S.; Ren, J.; Chen, J.; Gao, Y.; Li, H.; Jia, N.; Cheng, L.; Xiao, L. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 2009, 4, 11–15. [Google Scholar] [CrossRef] [PubMed]
  26. Shimada, H.; Nakada, A.; Hashimoto, Y.; Shigeno, K.; Shionoya, Y.; Nakamura, T. Generation of canine induced pluripotent stem cells by retroviral transduction and chemical inhibitors. Mol. Reprod. Dev. 2009, 77, 2. [Google Scholar] [CrossRef] [PubMed]
  27. Ezashi, T.; Telugu, B.P.; Alexenko, A.P.; Sachdev, S.; Sinha, S.; Roberts, R.M. Derivation of induced pluripotent stem cells from pig somatic cells. Proc. Natl. Acad. Sci. USA 2009, 106, 10993–10998. [Google Scholar] [CrossRef]
  28. Táncos, Z.; Nemes, C.; Varga, E.; Bock, I.; Rungarunlert, S.; Tharasanit, T.; Techakumphu, M.; Kobolák, J.; Dinnyés, A. Establishment of a rabbit induced pluripotent stem cell (RbiPSC) line using lentiviral delivery of human pluripotency factors. Stem Cell Res. 2017, 21, 16–18. [Google Scholar] [CrossRef]
  29. Bao, L.; He, L.; Chen, J.; Wu, Z.; Liao, J.; Rao, L.; Ren, J.; Li, H.; Zhu, H.; Qian, L.; et al. Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res. 2011, 21, 600–608. [Google Scholar] [CrossRef]
  30. Nagy, K.; Sung, H.K.; Zhang, P.; Laflamme, S.; Vincent, P.; Agha-Mohammadi, S.; Woltjen, K.; Monetti, C.; Michael, I.P.; Smith, L.C.; et al. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev. 2011, 7, 693–702. [Google Scholar] [CrossRef]
  31. Sumer, H.; Liu, J.; Malaver-Ortega, L.F.; Lim, M.L.; Khodadadi, K.; Verma, P.J. NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts. J Anim. Sci. 2011, 89, 2708–2716. [Google Scholar] [CrossRef] [PubMed]
  32. Deng, Y.; Liu, Q.; Luo, C.; Chen, S.; Li, X.; Wang, C.; Zhang, X.; Zhang, L.; Zhao, Y.; Shi, D. Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors. Stem Cells Dev. 2012, 21, 2485–2494. [Google Scholar] [CrossRef] [PubMed]
  33. Lu, Y.; West, F.D.; Jordan, B.J.; Mumaw, J.L.; Jordan, E.T.; Gallegos-Cardenas, A.; Willard, S.T.; Freeman, K.W.; Stice, S.L. Avian-induced pluripotent stem cells derived using human reprogramming factors. Stem Cells Dev. 2012, 21, 394–403. [Google Scholar] [CrossRef] [PubMed]
  34. Yu, P.; Lu, Y.; Jordan, B.J.; Liu, Y.; Yang, J.Y.; Hutcheson, J.M.; Nelson, T.R.; Smith, S.M.; West, F.D. Nonviral mini-circle generation of induced pluripotent stem cells compatible with production of chimeric chickens. Cell. Reprogram. 2014, 16, 366–378. [Google Scholar] [CrossRef] [PubMed]
  35. Tai, D.; Liu, P.; Gao, J.; Jin, M.; Xu, T.; Zuo, Y.; Zhang, X.; Chen, H.; Wu, J.; Liu, D. Generation of Arbas Cashmere goat induced pluripotent stem cells through fibroblast reprogramming. Cell. Reprogram. 2015, 17, 297–305. [Google Scholar] [CrossRef]
  36. Yada, R.C.; Hong, S.G.; Lin, Y.; Winkler, T.; Dunbar, C.E. Rhesus macaque iPSC generation and maintenance. Curr. Protoc. Stem Cell Biol. 2017, 41, 4A.11.1–4A.11.13. [Google Scholar] [CrossRef] [PubMed]
  37. Ben-Nun, I.F.; Montague, S.C.; Houck, M.L.; Tran, H.T.; Garitaonandia, I.; Leonardo, T.R.; Wang, Y.C.; Charter, S.J.; Laurent, L.C.; Ryder, O.A. Induced pluripotent stem cells from highly endangered species. Nat. Methods 2011, 8, 829–831. [Google Scholar] [CrossRef]
  38. Verma, R.; Holland, M.K.; Temple-Smith, P.; Verma, P.J. Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid. Theriogenology 2012, 77, 220–228. [Google Scholar] [CrossRef] [PubMed]
  39. Verma, R.; Liu, J.; Holland, M.K.; Temple-Smith, P.; Williamson, M.; Verma, P.J. Derivation of induced pluripotent stem cells from the tiger (Panthera tigris), an endangered felid. Stem Cells Dev. 2013, 22, 169–176. [Google Scholar]
  40. Mo, X.; Li, N.; Wu, S. Generation and characterization of bat-induced pluripotent stem cells. Theriogenology 2014, 82, 283–293. [Google Scholar] [CrossRef] [PubMed]
  41. Wu, Y.; Wang, C.; Fan, X.; Ma, Y.; Liu, Z.; Ye, X.; Zhang, R.; Lin, H.; Wu, C. The impact of induced pluripotent stem cells in animal conservation. Vet. Res. Commun. 2024, 48, 649–663. [Google Scholar] [CrossRef]
  42. Clerc, J.; Weller, T.; Schineller, J.B.; Szewczak, J.M. Minimally invasive collection of adipose tissue facilitates the study of eco-physiology in small-bodied mammals. Methods Ecol. Evol. 2017, 8, 109–115. [Google Scholar] [CrossRef]
  43. Huerta-Leidenz, N.O.; Cross, H.R.; Savell, J.W.; Lunt, D.K.; Baker, J.F.; Pelton, L.S.; Smith, S.B. Comparison of the fatty acid composition of subcutaneous adipose tissue from mature Brahman and Hereford cows. J. Anim. Sci. 1993, 71, 625–630. [Google Scholar] [CrossRef]
  44. Galicia, M.P.; Thiemann, G.W.; Dyck, M.G.; Ferguson, S.H. Are tissue samples obtained via remote biopsy useful for fatty acid-based diet analyses in a free-ranging carnivore? J. Mammal. 2021, 102, 1067–1078. [Google Scholar] [CrossRef]
  45. Bressan, F.F.; Bassanezze, V.; de Figueiredo Pessôa, L.V.; Sacramento, C.B.; Malta, T.M.; Kashima, S.; Meirelles, F.V. Generation of induced pluripotent stem cells from large domestic animals. Stem Cell Res. Ther. 2020, 11, 247. [Google Scholar] [CrossRef] [PubMed]
  46. Chen, Y.S.; Pelekanos, R.A.; Ellis, R.L.; Horne, R.; Wolvetang, E.J.; Fisk, N.M. Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells. Stem Cells Transl. Med. 2012, 1, 83–95. [Google Scholar] [CrossRef]
  47. Zhao, X. Establishment of highly efficient somatic cell reprogramming system to generate iPSC lines. In Studies of Pluripotency in Embryonic Stem Cells and Induced Pluripotent Stem Cells; Springer: Berlin/Heidelberg, Germany, 2014; pp. 41–52. [Google Scholar]
  48. Chen, J.; Liu, H.; Liu, J.; Qi, J.; Wei, B.; Yang, J.; Liang, H.; Chen, Y.; Chen, J.; Wu, Y.; et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat. Genet. 2013, 45, 34–42. [Google Scholar] [CrossRef] [PubMed]
  49. Esteban, M.A.; Wang, T.; Qin, B.; Yang, J.; Qin, D.; Cai, J.; Li, W.; Weng, Z.; Chen, J.; Ni, S. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 2010, 6, 71–79. [Google Scholar] [CrossRef]
  50. Stadtfeld, M.; Apostolou, E.; Ferrari, F.; Choi, J.; Walsh, R.M.; Chen, T.; Ooi, S.S.; Kim, S.Y.; Bestor, T.H.; Shioda, T.; et al. Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat. Genet. 2012, 44, 398–405. [Google Scholar] [CrossRef] [PubMed]
  51. Chung, T.L.; Brena, R.M.; Kolle, G.; Grimmond, S.M.; Berman, B.P.; Laird, P.W.; Pera, M.F.; Wolvetang, E.J. Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells. Stem Cells 2010, 28, 1848–1855. [Google Scholar] [CrossRef]
  52. Wang, T.; Chen, K.; Zeng, X.; Yang, J.; Wu, Y.; Shi, X.; Qin, B.; Zeng, L.; Esteban, M.A.; Pan, G. The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell 2011, 9, 575–587. [Google Scholar] [CrossRef] [PubMed]
  53. Polo, J.M.; Anderssen, E.; Walsh, R.M.; Schwarz, B.A.; Nefzger, C.M.; Lim, S.M.; Borkent, M.; Apostolou, E.; Alaei, S.; Cloutier, J. A molecular roadmap of cellular reprogramming into iPS cells. Cell 2012, 151, 1617–1632. [Google Scholar] [CrossRef]
  54. Aoi, T.; Yae, K.; Nakagawa, M.; Ichisaka, T.; Okita, K.; Takahashi, K.; Chiba, T.; Yamanaka, S. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 2008, 321, 699–702. [Google Scholar] [CrossRef]
  55. Maherali, N.; Ahfeldt, T.; Rigamonti, A.; Utikal, J.; Cowan, C.; Hochedlinger, K. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 2008, 3, 340–345. [Google Scholar] [CrossRef]
  56. Miura, K.; Okada, Y.; Aoi, T.; Okada, A.; Takahashi, K.; Okita, K.; Nakagawa, M.; Koyanagi, M.; Tanabe, K.; Ohnuki, M.; et al. Variation in the safety of induced pluripotent stem cell lines. Nat. Biotechnol. 2009, 27, 743–745. [Google Scholar] [CrossRef]
  57. Kim, K.; Doi, A.; Wen, B.; Ng, K.; Zhao, R.; Cahan, P.; Kim, J.; Aryee, M.J.; Ji, H.; Ehrlich, L.I.R.; et al. Epigenetic memory in induced pluripotent stem cells. Nature 2010, 467, 285–290. [Google Scholar] [CrossRef] [PubMed]
  58. Pessôa, L.V.F.; Pires, P.R.L.; Del Collado, M.; Pieri, N.C.G.; Recchia, K.; Souza, A.F.; Perecin, F.; da Silveira, J.C.; de Andrade, A.F.C.; Ambrosio, C.E.; et al. Generation and miRNA characterization of equine induced pluripotent stem cells derived from fetal and adult multipotent tissues. Stem Cells Int. 2019, 2019, 1393791. [Google Scholar] [CrossRef]
  59. Brevini, T.A.L.; Antonini, S.; Cillo, F.; Crestan, M.; Gandolfi, F. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology 2007, 68 (Suppl. S1), S206–S213. [Google Scholar] [CrossRef]
  60. Talbot, N.C.; Blomberg, L.A. The pursuit of ES cell lines of domesticated ungulates. Stem Cell Rev. 2008, 4, 235–254. [Google Scholar] [CrossRef]
  61. Tapia, N.; Schöler, H.R. Molecular obstacles to clinical translation of iPSCs. Cell Stem Cell 2016, 19, 298–309. [Google Scholar] [CrossRef]
  62. Doss, M.X.; Sachinidis, A. Current challenges of iPSC-based disease modeling and therapeutic implications. Cells 2019, 8, 403. [Google Scholar] [CrossRef] [PubMed]
  63. Madrid, M.; Sumen, C.; Aivio, S.; Saklayen, N. Autologous induced pluripotent stem cell–based cell therapies: Promise, progress, and challenges. Curr. Protoc. 2021, 1, e88. [Google Scholar] [CrossRef]
  64. Nandal, A.; Telugu, B.P.V.L. Large animal induced pluripotent stem cells as models of human diseases. In Pluripotent Stem Cells; Springer: Cham, Switzerland, 2014; pp. 215–230. [Google Scholar]
  65. Ma, H.; Morey, R.; O’Neil, R.C.; He, Y.; Daughtry, B.; Schultz, M.D.; Hariharan, M.; Nery, J.R.; Castanon, R.; Sabatini, K.; et al. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 2014, 511, 177–183. [Google Scholar] [CrossRef] [PubMed]
  66. Wang, W.; Yang, J.; Liu, H.; Lu, D.; Chen, X.; Zenonos, Z.; 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. USA 2011, 108, 18283–18288. [Google Scholar] [CrossRef] [PubMed]
  67. Nie, T.; Deng, W.; Gao, X.; Sun, W.; Hui, X.; Song, H.; Qin, D.; Xu, A.; Li, P.; Liu, P.; et al. Reprogramming mature terminally differentiated adipocytes to induced pluripotent stem cells. Sci. Bull. 2015, 60, 1752–1758. [Google Scholar] [CrossRef]
  68. Honda, A.; Choijookhuu, N.; Izu, H.; Kawano, Y.; Inokuchi, M.; Honsho, K.; Lee, A.R.; Nabekura, H.; Ohta, H.; Tsukiyama, T.; et al. Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis. Sci. Adv. 2017, 3, e1602179. [Google Scholar] [CrossRef]
  69. Talluri, T.R.; Kumar, D.; Glage, S.; Garrels, W.; Ivics, Z.; Debowski, K.; Behr, R.; Niemann, H.; Kues, W.A. Derivation and characterization of bovine induced pluripotent stem cells by transposon-mediated reprogramming. Cell. Reprogram. 2015, 17, 131–140. [Google Scholar] [CrossRef] [PubMed]
  70. Zywitza, V.; Rusha, E.; Shaposhnikov, D.; Ruiz-Orera, J.; Telugu, N.; Rishko, V.; Hayashi, M.; Michel, G.; Wittler, L.; Stejskal, J.; et al. Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction. Sci. Rep. 2022, 12, 3100. [Google Scholar] [CrossRef]
  71. West, F.D.; Terlouw, S.L.; Kwon, D.J.; Mumaw, J.L.; Dhara, S.K.; Hasneen, K.; Dobrinsky, J.R.; Stice, S.L. Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev. 2010, 19, 1211–1220. [Google Scholar] [CrossRef] [PubMed]
  72. Katayama, M.; Fukuda, T.; Kaneko, T.; Nakagawa, Y.; Tajima, A.; Naito, M.; Ohmaki, H.; Endo, D.; Asano, M.; Nagamine, T.; et al. Induced pluripotent stem cells of endangered avian species. Commun. Biol. 2022, 5, 1049. [Google Scholar] [CrossRef] [PubMed]
  73. Kawaguchi, T.; Tsukiyama, T.; Kimura, K.; Matsuyama, S.; Minami, N.; Yamada, M.; Imai, H. Generation of naïve bovine induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible transcription factors. PLoS ONE 2015, 10, e0135403. [Google Scholar] [CrossRef] [PubMed]
  74. Tsukiyama, T.; Osafune, K. A modified EpiSC culture condition containing a GSK3 inhibitor can support germline-competent pluripotency in mice. PLoS ONE 2014, 9, e95329. [Google Scholar] [CrossRef]
  75. Lou, J.; Li, W.; Chen, P.; Chen, H.; Shakoor, A.; Chen, Y.; Zhang, S. Application of induced pluripotent stem cells in the conservation of endangered animals. Stem Cell Res. Ther. 2025, 16, 261. [Google Scholar] [CrossRef]
Jzbg 06 00043 g001
Figure 1. Photomicrography illustrating the colonies obtained using the protocol with PiggyBac transposons. (A) Irregular colony obtained from antler cells (100× magnification). (B) Regular colony obtained from fat cells (40× magnification).
Figure 1. Photomicrography illustrating the colonies obtained using the protocol with PiggyBac transposons. (A) Irregular colony obtained from antler cells (100× magnification). (B) Regular colony obtained from fat cells (40× magnification).
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Figure 2. Flow cytometry analysis demonstrated that 50.3% of cells exhibited GFP reporter gene expression.
Figure 2. Flow cytometry analysis demonstrated that 50.3% of cells exhibited GFP reporter gene expression.
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Table 1. Overview of reprogramming attempts, specifying the number of trials for each technique on cells derived from antler, fat, and skin.
Table 1. Overview of reprogramming attempts, specifying the number of trials for each technique on cells derived from antler, fat, and skin.
Cell LineElectroporation Using PiggyBacLentiviral with STEMCCA Vector
FBSKSRhOSKMmOSKM
Protocol 1 *Protocol 2 **Protocol 1 *Protocol 2 **
Antler321211
Fat11 111
Skin1 22
* Protocol 1 = pulse voltage: 1400, pulse amplitude: 20, number of pulses: 1; ** Protocol 2 = pulse voltage: 1400, pulse amplitude: 30, and number of pulses: 2; hOSKM = STEMCCA vector containing human OSKM factors; mOSKM = STEMCCA vector containing murine OSKM factors.
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Rola, L.D.; Bressan, F.F.; Sandoval, E.D.P.; Therrien, J.; Smith, L.C.; Duarte, J.M.B. Insights for Deriving Induced Pluripotent Stem Cells in Marsh Deer (Blastocerus dichotomus). J. Zool. Bot. Gard. 2025, 6, 43. https://doi.org/10.3390/jzbg6030043

AMA Style

Rola LD, Bressan FF, Sandoval EDP, Therrien J, Smith LC, Duarte JMB. Insights for Deriving Induced Pluripotent Stem Cells in Marsh Deer (Blastocerus dichotomus). Journal of Zoological and Botanical Gardens. 2025; 6(3):43. https://doi.org/10.3390/jzbg6030043

Chicago/Turabian Style

Rola, Luciana Diniz, Fabiana Fernandes Bressan, Eluzai Dinai Pinto Sandoval, Jacinthe Therrien, Lawrence Charles Smith, and José Maurício Barbanti Duarte. 2025. "Insights for Deriving Induced Pluripotent Stem Cells in Marsh Deer (Blastocerus dichotomus)" Journal of Zoological and Botanical Gardens 6, no. 3: 43. https://doi.org/10.3390/jzbg6030043

APA Style

Rola, L. D., Bressan, F. F., Sandoval, E. D. P., Therrien, J., Smith, L. C., & Duarte, J. M. B. (2025). Insights for Deriving Induced Pluripotent Stem Cells in Marsh Deer (Blastocerus dichotomus). Journal of Zoological and Botanical Gardens, 6(3), 43. https://doi.org/10.3390/jzbg6030043

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