Cell reprogramming therapy for Parkinson’s disease

Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta.Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson’s disease.The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson’s disease,which could substantially alleviate the symptoms of Parkinson’s disease in clinical practice.However,ethical issues and tumor formation were limitations of its clinical application.Induced pluripotent stem cells can be acquired without sacrificing human embryos,which eliminates the huge ethical barriers of human stem cell therapy.Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons,without the need for intermediate proliferation states,thus avoiding issues of immune rejection and tumor formation.Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson’s disease.However,there are also ethical concerns and the risk of tumor formation that need to be addressed.This review highlights the current application status of cell reprogramming in the treatment of Parkinson’s disease,focusing on the use of induced pluripotent stem cells in cell replacement therapy,including preclinical animal models and progress in clinical research.The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson’s disease,as well as the controversy surrounding in vivo reprogramming.These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson’s disease.

Dual-targeting AAV9P1-mediated neuronal reprogramming in a mouse model of traumatic brain injury

Traumatic brain injury results in neuronal loss and glial scar formation.Replenishing neurons and eliminating the consequences of glial scar formation are essential for treating traumatic brain injury.Neuronal reprogramming is a promising strategy to convert glial scars to neural tissue.However,previous studies have reported inconsistent results.In this study,an AAV9P1 vector incorporating an astrocyte-targeting P1 peptide and glial fibrillary acidic protein promoter was used to achieve dual-targeting of astrocytes and the glial scar while minimizing off-target effects.The results demonstrate that AAV9P1 provides high selectivity of astrocytes and reactive astrocytes.Moreover,neuronal reprogramming was induced by downregulating the polypyrimidine tract-binding protein 1 gene via systemic administration of AAV9P1 in a mouse model of traumatic brain injury.In summary,this approach provides an improved gene delivery vehicle to study neuronal programming and evidence of its applications for traumatic brain injury.

Post-transcriptional mechanisms controlling neurogenesis and direct neuronal reprogramming

Neurogenesis is a tightly regulated process in time and space both in the developing embryo and in adult neurogenic niches.A drastic change in the transcriptome and proteome of radial glial cells or neural stem cells towards the neuronal state is achieved due to sophisticated mechanisms of epigenetic,transcriptional,and post-transcriptional regulation.Understanding these neurogenic mechanisms is of major importance,not only for shedding light on very complex and crucial developmental processes,but also for the identification of putative reprogramming factors,that harbor hierarchically central regulatory roles in the course of neurogenesis and bare thus the capacity to drive direct reprogramming towards the neuronal fate.The major transcriptional programs that orchestrate the neurogenic process have been the focus of research for many years and key neurogenic transcription factors,as well as repressor complexes,have been identified and employed in direct reprogramming protocols to convert non-neuronal cells,into functional neurons.The post-transcriptional regulation of gene expression during nervous system development has emerged as another important and intricate regulatory layer,strongly contributing to the complexity of the mechanisms controlling neurogenesis and neuronal function.In particular,recent advances are highlighting the importance of specific RNA binding proteins that control major steps of mRNA life cycle during neurogenesis,such as alternative splicing,polyadenylation,stability,and translation.Apart from the RNA binding proteins,microRNAs,a class of small non-coding RNAs that block the translation of their target mRNAs,have also been shown to play crucial roles in all the stages of the neurogenic process,from neural stem/progenitor cell proliferation,neuronal differentiation and migration,to functional maturation.Here,we provide an overview of the most prominent post-transcriptional mechanisms mediated by RNA binding proteins and microRNAs during the neurogenic process,giving particular emphasis on the interplay of specific RNA binding proteins with neurogenic microRNAs.Taking under consideration that the molecular mechanisms of neurogenesis exert high similarity to the ones driving direct neuronal reprogramming,we also discuss the current advances in in vitro and in vivo direct neuronal reprogramming approaches that have employed microRNAs or RNA binding proteins as reprogramming factors,highlighting the so far known mechanisms of their reprogramming action.

In situ direct reprogramming of astrocytes to neurons via polypyrimidine tract-binding protein 1 knockdown in a mouse model of ischemic stroke

In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.e B-GFAP-sh PTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-sh PTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.

Glucose metabolic reprogramming-related parameters for the prediction of 28-day neurological prognosis and all-cause mortality in patients after cardiac arrest:a prospective single-center observational study

BACKGROUND:We aimed to observe the dynamic changes in glucose metabolic reprogrammingrelated parameters and their ability to predict neurological prognosis and all-cause mortality in cardiac arrest patients after the restoration of spontaneous circulation(ROSC).METHODS:Adult cardiac arrest patients after ROSC who were admitted to the emergency or cardiac intensive care unit of the First Aflliated Hospital of Dalian Medical University from August 1,2017,to May 30,2021,were enrolled.According to 28-day survival,the patients were divided into a non-survival group(n=82) and a survival group(n=38).Healthy adult volunteers(n=40) of similar ages and sexes were selected as controls.The serum levels of glucose metabolic reprogrammingrelated parameters(lactate dehydrogenase [LDH],lactate and pyruvate),neuron-specific enolase(NSE) and interleukin 6(IL-6) were measured on days 1,3,and 7 after ROSC.The Acute Physiology and Chronic Health Evaluation II(APACHE II) score and Sequential Organ Failure Assessment(SOFA) score were calculated.The Cerebral Performance Category(CPC) score was recorded on day 28 after ROSC.RESULTS:Following ROSC,the serum LDH(607.0 U/L vs.286.5 U/L),lactate(5.0 mmol/L vs.2.0 mmol/L),pyruvate(178.0 μmol/L vs.70.9 μmol/L),and lactate/pyruvate ratio(34.1 vs.22.1) significantly increased and were higher in the non-survivors than in the survivors on admission(all P<0.05).Moreover,the serum LDH,pyruvate,IL-6,APACHE II score,and SOFA score on days 1,3 and 7 after ROSC were significantly associated with 28-day poor neurological prognosis and 28-day all-cause mortality(all P<0.05).The serum LDH concentration on day 1 after ROSC had an area under the receiver operating characteristic curve(AUC) of 0.904 [95% confidence interval [95% CI]:0.851–0.957]) with 96.8% specificity for predicting 28-day neurological prognosis and an AUC of 0.950(95% CI:0.911–0.989) with 94.7% specificity for predicting 28-day all-cause mortality,which was the highest among the glucose metabolic reprogramming-related parameters tested.CONCLUSION:Serum parameters related to glucose metabolic reprogramming were significantly increased after ROSC.Increased serum LDH and pyruvate levels,and lactate/pyruvate ratio may be associated with 28-day poor neurological prognosis and all-cause mortality after ROSC,and the predictive eflcacy of LDH during the first week was superior to others.

RARRES2 regulates lipid metabolic reprogramming to mediate the development of brain metastasis in triple negative breast cancer

Background Triple negative breast cancer(TNBC),the most aggressive subtype of breast cancer,is characterized by a high incidence of brain metastasis(BrM)and a poor prognosis.As the most lethal form of breast cancer,BrM remains a major clinical challenge due to its rising incidence and lack of effective treatment strategies.Recent evidence suggested a potential role of lipid metabolic reprogramming in breast cancer brain metastasis(BCBrM),but the underlying mechanisms are far from being fully elucidated.Methods Through analysis of BCBrM transcriptome data from mice and patients,and immunohistochemical validation on patient tissues,we identified and verified the specific down-regulation of retinoic acid receptor responder 2(RARRES2),a multifunctional adipokine and chemokine,in BrM of TNBC.We investigated the effect of aberrant RARRES2 expression of BrM in both in vitro and in vivo studies.Key signaling pathway components were evaluated using multi-omics approaches.Lipidomics were performed to elucidate the regulation of lipid metabolic reprogramming of RARRES2.Results We found that downregulation of RARRES2 is specifically associated with BCBrM,and that RARRES2 deficiency promoted BCBrM through lipid metabolic reprogramming.Mechanistically,reduced expression of RARRES2 in brain metastatic potential TNBC cells resulted in increased levels of glycerophospholipid and decreased levels of triacylglycerols by regulating phosphatase and tensin homologue(PTEN)-mammalian target of rapamycin(mTOR)-sterol regulatory element-binding protein 1(SREBP1)signaling pathway to facilitate the survival of breast cancer cells in the unique brain microenvironment.Conclusions Our work uncovers an essential role of RARRES2 in linking lipid metabolic reprogramming and the development of BrM.RARRES2-dependent metabolic functions may serve as potential biomarkers or therapeutic targets for BCBrM.

In vivo astrocyte-to-neuron reprogramming for central nervous system regeneration:a narrative review

The inability of damaged neurons to regenerate within the mature central nervous system(CNS)is a significant neuroscientific challenge.Astrocytes are an essential component of the CNS and participate in many physiological processes including blood-brain barrier formation,axon growth regulation,neuronal support,and higher cognitive functions such as memory.Recent reprogramming studies have confirmed that astrocytes in the mature CNS can be transformed into functional neurons.Building on in vitro work,many studies have demonstrated that astrocytes can be transformed into neurons in different disease models to replace damaged or lost cells.However,many findings in this field are controversial,as the source of new neurons has been questioned.This review summarizes progress in reprogramming astrocytes into neurons in vivo in animal models of spinal cord injury,brain injury,Huntington’s disease,Parkinson’s disease,Alzheimer’s disease,and other neurodegenerative conditions.

Epigenetic drug library screening reveals targeting DOT1L abrogates NAD^(+)synthesis by reprogramming H3K79 methylation in uveal melanoma

Uveal melanoma(UM)is the most frequent and life-threatening ocular malignancy in adults.Aberrant histone methylation contributes to the abnormal transcriptome during oncogenesis.However,a comprehensive understanding of histone methylation patterns and their therapeutic potential in UM remains enigmatic.Herein,using a systematic epi-drug screening and a high-throughput transcriptome profiling of histone methylation modifiers,we observed that disruptor of telomeric silencing-1-like(DOT1L),a methyltransferase of histone H3 lysine 79(H3K79),was activated in UM,especially in the high-risk group.Concordantly,a systematic epi-drug library screening revealed that DOT1L inhibitors exhibited salient tumor-selective inhibitory effects on UM cells,both in vitro and in vivo.Combining Cleavage Under Targets and Tagmentation(CUT&Tag),RNA sequencing(RNA-seq),and bioinformatics analysis,we identified that DOT1L facilitated H3K79 methylation of nicotinate phosphoribosyltransferase(NAPRT)and epigenetically activated its expression.Importantly,NAPRT served as an oncogenic accelerator by enhancing nicotinamide adenine dinucleotide(NAD^(+))synthesis.Therapeutically,DOT1L inhibition epigenetically silenced NAPRT expression through the diminishment of dimethylation of H3K79(H3K79me2)in the NAPRT promoter,thereby inhibiting the malignant behaviors of UM.Conclusively,our findings delineated an integrated picture of the histone methylation landscape in UM and unveiled a novel DOT1L/NAPRT oncogenic mechanism that bridges transcriptional addiction and metabolic reprogramming.

Discussion on reprogramming of tumor energy metabolism and intervention of traditional Chinese medicine based on the theory of“collateral Q i stagnation”

The theory of stagnation of collateral Qi(Chinese medicine refers to the most fundamental and subtle substances thatconstitute the human body and maintain life activities,and also has the meaning of physiological functions)originates from the theory of collateral disease,which refers to the deficiency of Qi in the body’s collaterals,the loss of Qi and blood,and the failure of stagnation of collateral Qi,which leads to the loss of Qi,blood and body fluid,and the formation of pathological products such as deficiency,depression,phlegm,blood stasis in the local area,and ultimately damage the pathological process of collaterals.Based on the in-depth study of the pathogenesis of collateral Qi stagnation and the previous study of meridian channels,we believe that the key pathogenesis of the formation,evolution and spread of malignant tumors is“collateral Qi deficiency stagnation,collateral Qi stagnation and collateral Qi decay”.As an important energy resonance channel of the body,meridians play a key role in the process of material transformation and energy metabolism.It is believed that the small focus caused by the pathogenesis of stagnation is the cause of malignant transformation of tumor,the reprogramming of energy metabolism induced by the lesion of collateral Qi is the basis of the progress of tumor pathogenesis,and the formation of tumor microenvironment regulated by the tumor toxin vena is the root of alienation of tumor development.Guided by this theory,focusing on the correlation between collateral Qi and tumor energy metabolism,using Professor Hua Baojin's treatment method of“Regulating Qi and detoxifying”to prescribe drugs can adjust collateral Qi function,achieve the relative balance of internal environment,and then inhibit the progress of tumor.Based on the above understanding,this study tries to enlighten new diagnosis and treatment ideas under the guidance of“stagnation of collateral Qi”in traditional Chinese medicine,in order to provide some theoretical support for the intervention of traditional Chinese medicine in the process of tumor development.

Expression level of pluripotent genes in incomplete reprogramming

Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.Methods:four genes(Oct4,Sox2,Klf4,C-Myc) were introduced into human foreskin fibroblasts(HFFs) by retroviruses.The HFFs were induced to reprogramming.Different forms of colonies were picked up,analyzed,and compared with iPSCs from different aspects,including the morphology of clones,alkaline phosphatase(AP) staining,immuno-fluorescence,and Q-PCR.Results:In the reprogramming process,different colonies were emerged,some of them exhibited typical human embryonic stem cell morphology(eg.,compact colonies,high nucleus-to-cytoplasm ratios,and prominent nucleoli).However,these colonies couldn't maintain these characters after passage.There was an intermediate state,named partially reprogramming.Through analysis and identification,AP staining results were weakly positive,compared with iPSC colonies.The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4.Q-PCR indicated that the expression of exogenous transcription factors was inappropriate,either at a high level or at a low level.Most of the endogenous pluripotency genes were expressed at a low level.Conclusions:It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed,and successful reprogramming may depend on a specific stoichiometric balance of Oct4,Sox2,Klf4 and c-Myc.

Differential neuronal reprogramming induced by NeuroD1 from astrocytes in grey matter versus white matter

A new technology called in vivo glia-to-neuron conversion has emerged in recent years as a promising next generation therapy for neural regeneration and repair. This is achieved through reprogramming endogenous glial cells into neurons in the central nervous system through ectopically expressing neural transcriptional factors in glial cells. Previous studies have been focusing on glial cells in the grey matter such as the cortex and striatum, but whether glial cells in the white matter can be reprogrammed or not is unknown. To address this fundamental question, we express NeuroD1 in the astrocytes of both grey matter(cortex and striatum) and white matter(corpus callosum) to investigate the conversion efficiency, neuronal subtypes, and electrophysiological features of the converted neurons. We discover that NeuroD1 can efficiently reprogram the astrocytes in the grey matter into functional neurons, but the astrocytes in the white matter are much resistant to neuronal reprogramming. The converted neurons from cortical and striatal astrocytes are composed of both glutamatergic and GABAergic neurons, capable of firing action potentials and having spontaneous synaptic activities. In contrast, the few astrocyte-converted neurons in the white matter are rather immature with rare synaptic events. These results provide novel insights into the differential reprogramming capability between the astrocytes in the grey matter versus the white matter, and highlight the impact of regional astrocytes as well as microenvironment on the outcome of glia-toneuron conversion. Since human brain has large volume of white matter, this study will provide important guidance for future development of in vivo glia-to-neuron conversion technology into potential clinical therapies. Experimental protocols in this study were approved by the Laboratory Animal Ethics Committee of Jinan University(approval No. IACUC-20180321-03) on March 21, 2018.

Endogenous retinal neural stem cell reprogramming for neuronal regeneration

In humans, optic nerve injuries and associated neurodegenerative diseases are often followed by perma- nent vision loss. Consequently, an important challenge is to develop safe and effective methods to replace retinal neurons and thereby restore neuronal functions and vision. Identifying cellular and molecular mechanisms allowing to replace damaged neurons is a major goal for basic and translational research in regenerative medicine. Contrary to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system, including retina. This regenerative process depends on endogenous retinal neural stem cells, the Miiller glial cells. Following injury, zebrafish Miiller cells go back into cell cycle to proliferate and generate new neurons, while mammalian Mtiller cells undergo reactive gliosis. Recently, transcription factors and microRNAs have been identified to control the formation of new neurons derived from ze- brafish and mammalian Mtiller cells, indicating that cellular reprogramming can be an efficient strategy to regenerate human retinal neurons. Here we discuss recent insights into the use of endogenous neural stem cell reprogramming for neuronal regeneration, differences between zebrafish and mammalian Mtiller cells, and the need to pursue the identification and characterization of new molecular factors with an instructive and potent function in order to develop theurapeutic strategies for eye diseases.

Reprogramming of glucose metabolism in hepatocellular carcinoma: Progress and prospects

Hepatocellular carcinoma(HCC) is one of the most lethal cancers, and its rate of incidence is rising annually. Despite the progress in diagnosis and treatment, the overall prognoses of HCC patients remain dismal due to the difficulties in early diagnosis and the high level of tumor invasion, metastasis and recurrence. It is urgent to explore the underlying mechanism of HCC carcinogenesis and progression to find out the specific biomarkers for HCC early diagnosis and the promising target for HCC chemotherapy. Recently, the reprogramming of cancer metabolism has been identified as a hallmark of cancer. The shift from the oxidative phosphorylation metabolic pathway to the glycolysis pathway in HCC meets the demands of rapid cell proliferation and offers a favorable microenvironment for tumor progression. Such metabolic reprogramming could be considered as a critical link between the different HCC genotypes and phenotypes. The regulation of metabolic reprogramming in cancer is complex and may occur via genetic mutations and epigenetic modulations including oncogenes, tumor suppressor genes, signaling pathways, noncoding RNAs, and glycolytic enzymes etc. Understanding the regulatory mechanisms of glycolysis in HCC may enrich our knowledge of hepatocellular carcinogenesis and provide important foundations in the search for novel diagnostic biomarkers and promising therapeutic targets for HCC.

Neuronal reprogramming in treating spinal cord injury

Spinal cord injury represents a devastating central nervous system injury that could impair the mobility and sensory function of afflicted patients.The hallmarks of spinal cord injury include neuroinflammation,axonal degeneration,neuronal loss,and reactive gliosis.Furthermore,the formation of a glial scar at the injury site elicits an inhibitory environment for potential neuroregeneration.Besides axonal regeneration,a significant challenge in treating spinal cord injury is to replenish the neurons lost during the pathological process.However,despite decades of research efforts,current strategies including stem cell transplantation have not resulted in a successful clinical therapy.Furthermore,stem cell transplantation faces serious hurdles such as immunorejection of the transplanted cells and ethical issues.In vivo neuronal reprogramming is a recently developed technology and leading a major breakthrough in regenerative medicine.This innovative technology converts endogenous glial cells into functional neurons for injury repair in the central nervous system.The feasibility of in vivo neuronal reprogramming has been demonstrated successfully in models of different neurological disorders including spinal cord injury by numerous laboratories.Several reprogramming factors,mainly the pro-neural transcription factors,have been utilized to reprogram endogenous glial cells into functional neurons with distinct phenotypes.So far,the literature on in vivo neuronal reprogramming in the model of spinal cord injury is still small.In this review,we summarize a limited number of such reports and discuss several questions that we think are important for applying in vivo neuronal reprogramming in the research field of spinal cord injury as well as other central nervous system disorders.

Cellular reprogramming and hepatocellular carcinoma development

Hepatocellular carcinoma(HCC)is one of the most common cancers,and is also the leading cause of death worldwide.Studies have shown that cellular reprogramming contributes to chemotherapy and/or radiotherapy resistance and the recurrence of cancers.In this article,we summarize and discuss the latest findings in the area of cellular reprogramming in HCC.The aberrant expression of transcription factors OCT4,KLF4,SOX2,c-MYC,NANOG,and LIN28 have been also observed,and the expression of these transcription factors is associated with unfavorable clinical outcomes in HCC.Studies indicate that cellular reprogramming may play a critical role in the occurrence and recurrence of HCC.Recent reports have shown that DNA methylation,miRNAs,tumor microenvironment,and signaling pathways can induce the expression of stemness transcription factors,which leads to cellular reprogramming in HCC.Furthermore,studies indicate that therapies based on cellular reprogramming could revolutionize HCC treatment.Finally,a novel therapeutic concept is discussed:reprogramming control therapy.A potential reprogramming control therapy method could be developed based on the reprogramming demonstrated in HCC studies and applied at two opposing levels:differentiation and reprogramming.Our increasing understanding and control of cellular programming should facilitate the exploitation of this novel therapeutic concept and its application in clinical HCC treatment,which may represent a promising strategy in the future that is not restricted to liver cancer.

Metabolic reprogramming in triple-negative breast cancer

Since triple-negative breast cancer(TNBC)was first defined over a decade ago,increasing studies have focused on its genetic and molecular characteristics.Patients diagnosed with TNBC,compared to those diagnosed with other breast cancer subtypes,have relatively poor outcomes due to high tumor aggressiveness and lack of targeted treatment.Metabolic reprogramming,an emerging hallmark of cancer,is hijacked by TNBC to fulfill bioenergetic and biosynthetic demands;maintain the redox balance;and further promote oncogenic signaling,cell proliferation,and metastasis.Understanding the mechanisms of metabolic remodeling may guide the design of metabolic strategies for the effective intervention of TNBC.Here,we review the metabolic reprogramming of glycolysis,oxidative phosphorylation,amino acid metabolism,lipid metabolism,and other branched pathways in TNBC and explore opportunities for new biomarkers,imaging modalities,and metabolically targeted therapies.

Cell signalling pathways underlying induced pluripotent stem cell reprogramming

Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.

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.

Small molecules facilitate single factor-mediated sweat gland cell reprogramming

Background: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands(SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.Methods: The expression of the SG markers cytokeratin 5(CK5), cytokeratin 10(CK10), cytokeratin 18(CK18), carcinoembryonic antigen(CEA), aquaporin 5(AQP5) and α-smooth muscle actin(α-SMA) was assessed with quantitative PCR(qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells(iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs.BALB/c nude mice were randomly divided into normal group, SGM treatment group and iSGC transplantation group.Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.Results: Ectodermal dysplasia antigen(EDA) overexpression drove human dermal fibroblast(HDF) conversion into i SGCs in SG culture medium(SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18and CEA in iSGCs, and flow cytometry data demonstrated(4.18±0.04)% of iSGCs were CK5 positive and(4.36±0.25)%of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails,(23.05±2.49)% of iSGCs expressed CK5^(+) and(55.79±3.18)% of iSGCs expressed CK18^(+), respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79±7.71)% vs.(70.59±0.34)%, ns]. In vivo transplantation experiments showed approximately(5.2±1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.Conclusions: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.

Therapeutic potential of glial cell line-derived neurotrophic factor and cell reprogramming for hippocampal-related neurological disorders

Hippocampus serves as a pivotal role in cognitive and emotional processes,as well as in the regulation of the hypothalamus-pituitary axis.It is known to undergo mild neurodegenerative changes during normal aging and severe atrophy in Alzheimer's disease.Furthermore,dysregulation in the hippocampal function leads to epilepsy and mood disorders.In the first section,we summarized the most salient knowledge on the role of glial cell-line-derived neurotrophic factor and its receptors focused on aging,cognition and neurodegenerative and hippocampal-related neurological diseases mentioned above.In the second section,we reviewed the therapeutic approaches,particularly gene therapy,using glial cell-line-derived neurotrophic factor or its gene,as a key molecule in the development of neurological disorders.In the third section,we pointed at the potential of regenerative medicine,as an emerging and less explored strategy for the treatment of hippocampal disorders.We briefly reviewed the use of partial reprogramming to restore brain functions,non-neuronal cell reprogramming to generate neural stem cells,and neural progenitor cells as source-specific neuronal types to be implanted in animal models of specific neurodegenerative disorders.