Cellular reprogramming in farm animals： an overview of iPSC generation in the mammalian farm animal species

Establishment of embryonic stem cell （ESC） lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells （iPSCs） represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells, iPSCs can differentiate to all cell types of an organism＇s body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven＇t received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species （cattle, pig, horse, sheep, goat, and rabbit）, compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig （13 studies）, cattle （5） horse （5）, sheep （4）, goat （3）, and rabbit （2） that are summarized in a concise, tabular format.

Potential of transposon-mediated cellular reprogramming towards cell-based therapies

Induced pluripotent stem(iPS)cells present a seminal discovery in cell biology and promise to support innovative treatments of so far incurable diseases.To translate iPS technology into clinical trials,the safety and stability of these reprogrammed cells needs to be shown.In recent years,different non-viral transposon systems have been developed for the induction of cellular pluripotency,and for the directed differentiation into desired cell types.In this review,we summarize the current state of the art of different transposon systems in iPS-based cell therapies.

Neuroregeneration using in vivo cellular reprogramming

Cellular reprogramming is an innovative technology used to artificlally convert a mature cell type into a different cell type by molecular＇manipulation. The general concept of cellular reprogramming is to use master transcription factors to override the endogenous transcriptome profile of a given cell type with the transcriptome profile of the target cell type, thereby altering the cellular function and identity.

Cellular reprogramming and inherited peripheral neuropathies: perspectives and challenges

Inherited peripheral neuropathies （or Charcot-Marie-Tooth disease, CMT） are a phenotypically and genetically heterogeneous group of disorders, which are currently untreatable. They are the most common inherited neuromuscular disorder, affecting around 1 in every 2,500 people （over 120,000 people in the US）. Based on clinical neurophysiological and histopathological features, inherited neuropathies can be divided into two major forms： demyelinating （type 1） and axonal （type 2） CMT （Saporta, 2014）.

Direct cellular reprogramming and transdifferentiation of fibroblasts on wound healing-Fantasy or reality?

Induced pluripotent stem cell(iPSC)technology is one of the de novo approaches in regeneration medicine and has led to new research applications for wound healing in recent years.Fibroblasts have attracted wide attention as the first cell line used for differentiation into iPSCs.Researchers have found that fibroblasts can be induced into different types of cells in variable mediums or microenvironments.This indicates the potential“stem”characteristics of fibroblasts in terms of direct cellular reprogramming compared with the iPSC detour.In this review,we described the morphology and biological function of fibroblasts.The stem cell characteristics and activities of fibroblasts,including transdifferentiation into myofibroblasts,osteogenic cells,chondrogenic cells,neurons,and vascular tissue,are discussed.The biological values of fibroblasts are then briefly reviewed.Finally,we discussed the potential applications of fibroblasts in clinical practice.

Role of cellular reprogramming and epigenetic dysregulation in acquired chemoresistance in breast cancer

Acquired resistance to chemotherapy is a major limitation in clinical treatment for breast cancer.Accumulating evidence from in vitro,in vivo and clinical studies suggest that acquired chemoresistance is progressive,multifactorial and involve genetic and epigenetic aberrations.Among various mechanisms that contribute to chemoresistance,cellular reprogramming has extensively been implicated in breast cancer resistance lately.Cellular reprogramming events such as acquisition of epithelial to mesenchymal transition(EMT)and cancer stemness(CSCs)not only provide cancer cells with reversible phenotypic plasticity and survival advantage against cytotoxicity but also leads to aggressiveness,metastasis,clinical resistance,tumor recurrence and poor survival.The transient and reversible nature of cellular reprogramming processes and their controlled interaction with epigenetic regulatory complexes strongly support the involvement of dynamic epigenetic regulatory network in governing the cellular reprogramming and associated acquired chemoresistance.Further,epigenetic modulations are also gaining interest as promising interventions addressing the cancer cell reprogramming machinery to overcome acquired chemoresistance.This review discusses the previous reports and our recent findings that lead to current understanding of epigenetic dysregulation dictating the cellular reprogramming processes such as acquisition of EMT and CSCs phenotype and how they co-ordinate to establish acquired drug resistance in breast cancer.

Diabetes mellitus and cellular replacement therapy: Expected clinical potential and perspectives

Diabetes mellitus(DM) is the most prevailing disease with progressive incidence worldwide. Despite contemporary treatment type one DM and type two DM are frequently associated with long-term major microvascular and macrovascular complications. Currently restoration of failing β-cell function, regulation of metabolic processes with stem cell transplantation is discussed as complements to contemporary DM therapy regimens. The present review is considered paradigm of the regenerative care and the possibly effects of cell therapy in DM. Reprogramming stem cells, bone marrowderived mononuclear cells; lineage-specified progenitor cells are considered for regenerative strategy in DM. Finally, perspective component of stem cell replacement in DM is discussed.

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.

Reprogramming cells with synthetic proteins

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to ＂read＂ genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongeneticaUy by using drug.like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore; the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

Chemical-only reprogramming to pluripotency

Direct reprogramming technology has emerged as an outstanding technique for the generation of induced pluripotent stem cells （iPSCs） and various specialized cells directly from somatic ceils of different species. Reprogramming techniques conventionally use viral vectors encoding transcription factors to induce fate conversion. However, the introduction of transgenes limits the therapeutic applications of the reprogrammed cells. To overcome safety-related concerns, small molecules offer some advantages over the existing methods for the control of gene expression and induction of cell fate conversion. Technical advances in optimizing concentrations, durations, structures, and combinations of small molecules make chemical reprogramming a safe and feasible method. This review provides a concise overview of cutting-edge findings regarding chemical-only reprogramming as one of the integration-free approaches to iPSC generation.

Reprogramming barriers and enhancers:strategies to enhance the efficiency and kinetics of induced pluripotency

Induced pluripotent stem cells are powerful tools for disease modeling,drug screening,and cell transplantation therapies.These cells can be generated directly from somatic cells by ectopic expression of defined factors through a reprogramming process.However,pluripotent reprogramming is an inefficient process because of various defined and unidentified barriers.Recent studies dissecting the molecular mechanisms of reprogramming have methodically improved the quality,ease,and efficiency of reprogramming.Different strategies have been applied for enhancing reprogramming efficiency,including depletion/inhibition of barriers(p53,p21,p57,p16Ink4a/p19Arf,Mbd3,etc.),overexpression of enhancing genes(e.g.,FOXH1,C/EBP alpha,UTF1,and GLIS1),and administration of certain cytokines and small molecules.The current review provides an in-depth overview of the cutting-edge findings regarding distinct barriers of reprogramming to pluripotency and strategies to enhance reprogramming efficiency.By incorporating the mechanistic insights from these recent findings,a combined method of inhibition of roadblocks and application of enhancing factors may yield the most reliable and effective approach in pluripotent reprogramming.

Ultrasound-triggered three dimensional hyaluronic acid hydrogel promotes in vitro and in vivo reprogramming into induced pluripotent stem cells

Cellular reprogramming technologies have been developed with different physicochemical factors to improve the reprogramming efficiencies of induced pluripotent stem cells(iPSCs).Ultrasound is a clinically applied noncontact biophysical factor known for regulating various cellular behaviors but remains uninvestigated for cellular reprogramming.Here,we present a new reprogramming strategy using low-intensity ultrasound(LIUS)to improve cellular reprogramming of iPSCs in vitro and in vivo.Under 3D microenvironment conditions,increased LIUS stimulation shows enhanced cellular reprogramming of the iPSCs.The cellular reprogramming process facilitated by LIUS is accompanied by increased mesenchymal to epithelial transition and histone modification.LIUS stimulation transiently modulates the cytoskeletal rearrangement,along with increased membrane fluidity and mobility to increase HA/CD44 interactions.Furthermore,LIUS stimulation with HA hydrogel can be utilized in application of both human cells and in vivo environment,for enhanced reprogrammed cells into iPSCs.Thus,LIUS stimulation with a combinatorial 3D microenvironment system can improve cellular reprogramming in vitro and in vivo environments,which can be applied in various biomedical fields.

Biomedical applications of mRNA nanomedicine

As an attractive alternative to plasmid DNA, messenger RNA （mRNA） has recently emerged as a promising class of nucleic acid therapeutics for biomedical applications. Advances in addressing the inherent shortcomings of mRNA and in the development of nanoparticle-based delivery systems have prompted the development and clinical translation of mRNA-based medicines. In this review, we discuss the chemical modification strategies of mRNA to improve its stability, minimize immune responses, and enhance translational efficacy. We also highlight recent progress in nanoparticle-based mRNA delivery. Considerable attention is given to the increasingly widespread applications of mRNA nanomedicine in the biomedical fields of vaccination, protein-replacement therapy, gene editing, and cellular reprogramming and engineering.