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.

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.

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.

Genetic and epigenetic regulators of retinal Müller glial cell reprogramming

Background:Retinal diseases characterized with irreversible loss of retinal nerve cells,such as optic atrophy and retinal degeneration,are the main causes of blindness.Current treatments for these diseases are very limited.An emerging treatment strategy is to induce the reprogramming of Müller glial cells to generate new retinal nerve cells,which could potentially restore vision.Main text:Müller glial cells are the predominant glial cells in retinae and play multiple roles to maintain retinal homeostasis.In lower vertebrates,such as in zebrafish,Müller glial cells can undergo cell reprogramming to regenerate new retinal neurons in response to various damage factors,while in mammals,this ability is limited.Interestingly,with proper treatments,Müller glial cells can display the potential for regeneration of retinal neurons in mammalian retinae.Recent studies have revealed that dozens of genetic and epigenetic regulators play a vital role in inducing the reprogramming of Müller glial cells in vivo.This review summarizes these critical regulators for Müller glial cell reprogramming and highlights their differences between zebrafish and mammals.Conclusions:A number of factors have been identified as the important regulators in Müller glial cell reprogramming.The early response of Müller glial cells upon acute retinal injury,such as the regulation in the exit from quiescent state,the initiation of reactive gliosis,and the re-entry of cell cycle of Müller glial cells,displays significant difference between mouse and zebrafish,which may be mediated by the diverse regulation of Notch and TGFβ(transforming growth factor-β)isoforms and different chromatin accessibility.

Cancer cell reprogramming:a promising therapy converting malignancy to benignity

In the past decade, remarkable progress has been made in reprogramming terminally differentiated somatic cells and cancer cells into induced pluripotent cells and cancer cells with benign phenotypes. Recent studies have explored various approaches to induce reprogramming from one cell type to another, including lineage-specific transcription factors-, combinatorial small molecules-, microRNAs- and embryonic microenvironment-derived exosome-mediated reprogramming. These reprogramming approaches have been proven to be technically feasible and versatile to enable re-activation of sequestered epigenetic regions, thus driving fate decisions of differentiated cells. One of the significant utilities of cancer cell reprogramming is the therapeutic potential of retrieving normal cell functions from various malignancies. However, there are several major obstacles to overcome in cancer cell reprogramming before clinical translation, including characterization of reprogramming mechanisms, improvement of reprogramming efficiency and safety, and development of delivery methods. Recently, several insights in reprogramming mecha-nism have been proposed, and determining progress has been achieved to promote reprogramming efficiency and feasibility, allowing it to emerge as a promising therapy against cancer in the near future. This review aims to discuss recent applications in cancer cell reprogramming, with a focus on the clinical significance and limitations of different reprogramming approaches, while summarizing vital roles played by transcription factors, small molecules, microR-NAs and exosomes during the reprogramming process.

Regulation of cell reprogramming by auxin during somatic embryogenesis

How somatic cells develop into a whole plant is a central question in plant developmental biology.This powerful ability of plant cells is recognized as their totipotency.Somatic embryogenesis is an excellent example and a good research system for studying plant cell totipotency.However,very little is known about the molecular basis of cell reprogramming from somatic cells to totipotent cells in this process.During somatic embryogenesis from immature zygotic embryos in Arabidopsis,exogenous auxin treatment is required for embryonic callus formation,but removal of exogenous auxin inducing endogenous auxin biosynthesis is essential for somatic embryo(SE)induction.Ectopic expression of specific transcription factor genes,such as "LAFL" and BABY BOOM(BBM),can induce SEs without exogenous growth regulators.Somatic embryogenesis can also be triggered by stress,as well as by disruption of chromatin remodeling,including PRC2-mediated histone methylation,histone deacetylation,and PKL-related chromatin remodeling.It is evident that embryonic identity genes are required and endogenous auxin plays a central role for cell reprogramming during the induction of SEs.Thus,we focus on reviewing the regulation of cell reprogramming for somatic embryogenesis by auxin.

Shared Gene Regulation during Human Somatic Cell Reprogramming

Human induced pluripotent stem （iPS） cells have the ability to differentiate into all somatic cells and to maintain unlimited self- renewal. Therefore, they have great potential in both basic research and clinical therapy for many diseases. To identify potentially universal mechanisms of human somatic cell reprogramming, we studied gene expression changes in three types of cells undergoing reprogramming. The set of 570 genes commonly regulated during induction of iPS cells includes known embryonic stem （ES） cell markers and pluripotency related genes. We also identified novel genes and biological categories which may be related to somatic cell reprogramming. For example, some of the down-regulated genes are predicted targets of the pluripotency microRNA cluster miR302/367, and the proteins from these putative target genes interact with the stem cell pluripotency factor POU5F1 according to our network analysis. Our results identified candidate gene sets to guide research on the mechanisms operating during somatic cell reprogramming.

Cocktail of chemical compounds robustly promoting cell reprogramming protects liver against acute injury

Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell repro- gramming favor tissue regeneration remains elusive. Here we identified combination of small chemical com- pounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical com- pounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.

Neural progenitor cells derived from fibroblasts induced by small molecule compounds under hypoxia for treatment of Parkinson’s disease in rats

Neural progenitor cells(NPCs) capable of self-renewal and differentiation into neural cell lineages offer broad prospects for cell therapy for neurodegenerative diseases. However, cell therapy based on NPC transplantation is limited by the inability to acquire sufficient quantities of NPCs. Previous studies have found that a chemical cocktail of valproic acid, CHIR99021, and Repsox(VCR) promotes mouse fibroblasts to differentiate into NPCs under hypoxic conditions. Therefore, we used VCR(0.5 mM valproic acid, 3 μM CHIR99021, and 1 μM Repsox) to induce the reprogramming of rat embryonic fibroblasts into NPCs under a hypoxic condition(5%). These NPCs exhibited typical neurosphere-like structures that can express NPC markers, such as Nestin, SRY-box transcription factor 2, and paired box 6(Pax6), and could also differentiate into multiple types of functional neurons and astrocytes in vitro. They had similar gene expression profiles to those of rat brain-derived neural stem cells. Subsequently, the chemically-induced NPCs(ciNPCs) were stereotactically transplanted into the substantia nigra of 6-hydroxydopamine-lesioned parkinsonian rats. We found that the ciNPCs exhibited long-term survival, migrated long distances, and differentiated into multiple types of functional neurons and glial cells in vivo. Moreover, the parkinsonian behavioral defects of the parkinsonian model rats grafted with ciNPCs showed remarkable functional recovery. These findings suggest that rat fibroblasts can be directly transformed into NPCs using a chemical cocktail of VCR without introducing exogenous factors, which may be an attractive donor material for transplantation therapy for Parkinson’s disease.

Transforming cancer cells for long-term living with cancer: An inspiring new approach

Cancer is the leading cause of human death and imposes a huge health burden. Currently, no matter what advanced therapeutic modalities or technologies are applied, it is still peculiarly rare for most cancers to be radically cured whereas therapy resistance and tumor recurrence are ever so common. The long-standing cytotoxic therapy is hard to achieve long-term tumor control, and produces side-effects or even promotes cancer progression. With growing understandings of tumor biology, we came to realize that it is possible to transform but not kill cancer cells to achieve long-term living with cancer, and directly altering cancer cells is a promising way. Remarkably, tissue microenvironment is involved in the fate determination of cancer cells. Of note, leveraging cell competition to combat malignant or therapy-resistant cells shows some therapeutic potentials. Furthermore, modulating tumor microenvironment to restore a normal state might help to transform cancer cells. Especially, reprogramming cancer-associated fibroblasts, and tumor-associated macrophages, or normalization of tumor vessel, tumor immune microenvironment, and tumor extracellular matrix or their combinations, et al., revealed some long-term therapeutic benefits. Despite the massive challenges ahead, it would be possible to transform cancer cells for long-term cancer control and living with cancer longevously. The related basic researches and corresponding therapeutic strategies are also ongoing.

Artemisinin, GABA signaling and cell reprogramming： when an old drug meets modern medicine

The hormone-secreting endocrine cells(α,β,δ,ε and PP cells)form pancreatic islets,which are strongly involved in regulation of metabolism.Deficiency of insulin-producingβcells is the major cause of type 1 diabetes.Recent progress in cell reprogramming demonstrates the feasibility of generating functionalβcells to treat type 1 diabetes[1].However,a non-invasive in vivo approach forβcell reprogramming is still lacking.Recently,two reports in

Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame

Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame （ORF） via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUND The development of regenerative therapy for human spinal cord injury(SCI)is dramatically restricted by two main challenges:the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing.Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge.The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIM To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells(drNPCs).METHODS Seven non-human primates with verified complete thoracic SCI were divided into two groups:drNPC group(n=4)was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury,and lesion control(n=3)was injected identically with the equivalent volume of vehicle.RESULTS Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways.Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation.Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk,migrating to areas of axon growth cones.CONCLUSION Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI,based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation.The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs.Instead,directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support,thereby further supporting the regeneration processes.

Mixed neuroendocrine and adenocarcinoma of gastrointestinal tract:A complex diagnosis and therapeutic challenge

In this editorial we comment on the manuscript describing a case of adenocarcinoma mixed with a neuroendocrine carcinoma of the gastroesophageal junction.Mixed neuroendocrine and non-neuroendocrine neoplasms of the gastrointestinal system are rare heterogeneous group of tumors characterized by a high malignant potential,rapid growth,and poor prognosis.Due to the rarity of these cancers,the standard therapy is poorly defined.The diagnosis of these tumors is based on combination of morphological features,immunohistochemical and neuroendocrine and epithelial cell markers.Both endocrine and epithelial cell components can act independently of each other and thus,careful grading of each component separately is required.These cancers are aggressive in nature and the potential of each component has paramount importance in the choice of treatment and response.Regardless of the organ of origin,these tumors portend poor prognosis with increased proportion of neuroendocrine component.Multidisciplinary services and strategies are required for the management of these mixed malignancies to provide the best oncological outcomes.The etiopathogenesis of these mixed tumors remains obscure but poses interesting question.We briefly discuss a few salient points in this editorial.

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3

Background:The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments.Clustered regularly interspaced short palindromic repeats(CRISPR)activator(CRISPRa)is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously,providing a new strategy for cell fate determination.The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.Method:The differentiation of mouse embryonic fibroblasts(MEFs)into functional induced hepatocyte-like cells(iHeps)was achieved by utilizing the CRISPRa synergistic activation mediator(SAM)system,which drove the combined expression of three endogenous transcription factors-Gata4,Foxa3,and Hnf1a-or alternatively,the expression of two transcription factors,Gata4 and Foxa3.In vivo,we injected adeno-associated virus serotype 6(AAV6)carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre^(ER);Cas9^(fl/fl)mice,effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts.The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction(RT-qPCR)and RNA-seq,and the morphology and characteristics of the induced hepatocytes were observed through microscopy.The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining,while the improvement of liver fibrosis is evaluated through Sirius red staining,alpha-smooth muscle actin(α-SMA)immunofluorescence staining,and blood alanine aminotransferase(ALT)examination.Results:Activation of only two factors,Gata4 and Foxa3,via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps.These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes,such as drug metabolism and glycogen storage.Additionally,AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis.After 8 weeks of induction,the reprogrammed hepatocytes comprised 0.87%of the total hepatocyte population in the mice,significantly reducing liver fibrosis.Conclusion:CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Plant cell totipotency: Insights into cellular reprogramming

Plant cells have a powerful capacity in their propagation to adapt to environmental change, given that a single plant cell can give rise to a whole plant via somatic embryogenesis without the need for fertilization. The reprogramming of somatic cells into totipotent cells is a critical step in somatic embryogenesis. This process can be induced by stimuli such as plant hormones, transcriptional regulators and stress. Here, we review current knowledge on how the identity of totipotent cells is determined and the stimuli required for reprogramming of somatic cells into totipotent cells. We highlight key molecular regulators and associated networks that control cell fate transition from somatic to totipotent cells. Finally,we pose several outstanding questions that should be addressed to enhance our understanding of the mechanisms underlying plant cell totipotency.

Reprogramming of ovarian granulosa cells by YAP1 leads to development of high-grade cancer with mesenchymal lineage and serous features

Understanding the cell-of-origin of ovarian high grade serous cancer(HGSC)is the prerequisite for efficient prevention and early diagnosis of this most lethal gynecological cancer.Recently,a mesenchymal type of ovarian HGSC with the poorest prognosis among ovarian cancers was identified by both TCGA and AOCS studies.The cell-of-origin of this subtype of ovarian cancer is unknown.While pursuing studies to understand the role of the Hippo pathway in ovarian granulosa cell physiology and pathology,we unexpectedly found that the Yes-associated protein 1(YAP1),the major effector of the Hippo signaling pathway,induced dedifferentiation and reprogramming of the ovarian granulosa cells,a unique type of ovarian follicular cells with mesenchymal lineage and high plasticity,leading to the development of high grade ovarian cancer with serous features.Our research results unveil a potential cell-of-origin for a subtype of HGSC with mesenchymal features.

The phenotypic convergence between microglia and peripheral macrophages during development and neuroinflammation paves the way for new therapeutic perspectives

Microglia,the tissue resident macrophages of the brain,are increasingly recognized as key players for central nervous system development and homeostasis.They are long-lived cells deriving from a transient wave of yolk-sac derived erythro-myeloid progenitors early in development.Their unique ontology has prompted the search for specific markers to be used for their selective investigation and manipulation.The first generation of genomewide expression studies has provided a bundle of transcripts(such as Olfml3,Fcrls,Tmem119,P2ry12,Gpr34,and Siglech)useful to distinguish microglia from peripheral macrophages.However,more recent reports have revealed that microglial phenotype is constantly shaped by the microenvironment in a time-,and context-dependent manner.In this article,we review data that provide additional pieces to this complex scenario and show the existence of unexpected phenotypic convergence between microglia and peripheral macrophages at certain developmental stages and under pathological conditions.These observations suggest that the two cell types act synergically boosting their mutual activities depending on the microenvironment.This novel information about the biology of microglia and peripheral macrophages sheds new light about their therapeutic potential for neuroinflammatory and neurodegenerative diseases.

Reprogramming towards endothelial cells for vascular regeneration

Endothelial damage and dysfunction are implicated in cardiovascular pathological changes and the development of vascular diseases.In view of the fact that the spontaneous endothelial cell(EC)regeneration is a slow and insufficient process,it is of great significance to explore alternative cell sources capable of generating functional ECs to repair damaged endothelium.Indeed,recent achievements of cell reprogramming to convert somatic cells to other cell types provide new powerful approaches to study endothelial regeneration.Based on progress in the research field,the present review aims to summarize the strategies and mechanisms of generating endothelial cells through reprogramming from somatic cells,and to examine what this means for the potential application of cell therapy in the clinic.

Highly efficient conversion of mouse fibroblasts into functional hepatic cells under chemical induction

Previous studies have shown that hepatocyte-like cells can be generated from fibroblasts using either lineage-specific transcription factors or chemical induction methods.However,these methods have their own deficiencies that restrict the therapeutic applications of such induced hepatocytes.In this study,we present a transgene-free,highly efficient chemical-induced direct reprogramming approach to generate hepatocyte-like cells from mouse embryonic fibroblasts(MEFs).Using a small molecule cocktail(SMC)as an inducer,MEFs can be directly reprogrammed into hepatocyte-like cells,bypassing the intermediate stages of pluripotent and immature hepatoblasts.These chemical-induced hepatocyte-like cells(ciHeps)closely resemble mature primary hepatocytes in terms of morphology,biological behavior,gene expression patterns,marker expression levels,and hepatic functions.Furthermore,transplanted ciHeps can integrate into the liver,promote liver regeneration,and improve survival rates in mice with acute liver damage.ciHeps can also ameliorate liver fibrosis caused by chronic injuries and enhance liver function.Notably,ciHeps exhibit no tumorigenic potential either in vitro or in vivo.Mechanistically,SMC-induced mesenchymal-to-epithelial transition and suppression of SNAI1 contribute to the fate conversion of fibroblasts into ciHeps.These results indicate that this transgene-free,chemical-induced direct reprogramming technique has the potential to serve as a valuable means of producing alternative hepatocytes for both research and therapeutic purposes.Additionally,this method also sheds light on the direct reprogramming of other cell types under chemical induction.