Age-dependent effects of metformin on human oligodendrocyte lineage cell ensheathment capacity.

Metformin restores the myelination potential of aged rat A2B5+ oligodendrocyte progenitor cells and may enhance recovery in children with post-radiation brain injury. Human late progenitor cells (O4+A2B5+) have a superior capacity to ensheath nanofibres compared to mature oligodendrocytes, with cells from paediatric sources exceeding adults. In this study, we assessed the effects of metformin on ensheathment capacity of human adult and paediatric progenitors and mature oligodendrocytes and related differences to transcriptional changes. A2B5+ progenitors and mature cells, derived from surgical tissues by immune-magnetic separation, were assessed for ensheathment capacity in nanofibre plates over 2 weeks. Metformin (10 µM every other day) was added to selected cultures. RNA was extracted from treated and control cultures after 2 days. For all ages, ensheathment by progenitors exceeded mature oligodendrocytes. Metformin enhanced ensheathment by adult donor cells but reduced ensheathment by paediatric cells. Metformin marginally increased cell death in paediatric progenitors. Metformin-induced changes in gene expression are distinct for each cell type. Adult progenitors showed up-regulation of pathways involved in the process of outgrowth and promoting lipid biosynthesis. Paediatric progenitors showed a relatively greater proportion of down- versus up-regulated pathways, these involved cell morphology, development and synaptic transmission. Metformin-induced AMP-activated protein kinase activation in all cell types; AMP-activated protein kinase inhibitor BML-275 reduced functional metformin effects only with adult cells. Our results indicate age and differentiation stage-related differences in human oligodendroglia lineage cells in response to metformin. Clinical trials for demyelinating conditions will indicate how these differences translate in vivo.

Regulation of stress granule formation in human oligodendrocytes.

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

Cellular rejuvenation protects neurons from inflammation mediated cell death.

In multiple sclerosis (MS), the invasion of the central nervous system by peripheral immune cells is followed by the activation of resident microglia and astrocytes. This cascade of events results in demyelination, which triggers neuronal damage and death. The molecular signals in neurons responsible for this damage are not yet fully characterized. In MS, retinal ganglion cell neurons (RGCs) of the central nervous system (CNS) undergo axonal injury and cell death. This phenomenon is mirrored in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. To understand the molecular landscape, we isolated RGCs from mice subjected to the EAE protocol. RNA-sequencing and ATAC-sequencing analyses were performed. Pathway analysis of the RNA-sequencing data revealed that RGCs displayed a molecular signature, similar to aged neurons, showcasing features of senescence. Single-nucleus RNA-sequencing analysis of neurons from human MS patients revealed a comparable senescence-like phenotype., which was supported by immunostaining RGCs in EAE mice. These changes include alterations to the nuclear envelope, modifications in chromatin marks, and accumulation of DNA damage. Transduction of RGCs with an Oct4 - Sox2 - Klf4 transgene to convert neurons in the EAE model to a more youthful epigenetic and transcriptomic state enhanced the survival of RGCs. Collectively, this research uncovers a previously unidentified senescent-like phenotype in neurons under pathological inflammation and neurons from MS patients. The rejuvenation of this aged transcriptome improved visual acuity and neuronal survival in the EAE model supporting the idea that age rejuvenation therapies and senotherapeutic agents could offer a direct means of neuroprotection in autoimmune disorders.

Mechanisms of metabolic stress induced cell death of human oligodendrocytes: relevance for progressive multiple sclerosis.

Oligodendrocyte (OL) injury and loss are central features of evolving lesions in multiple sclerosis. Potential causative mechanisms of OL loss include metabolic stress within the lesion microenvironment. Here we use the injury response of primary human OLs (hOLs) to metabolic stress (reduced glucose/nutrients) in vitro to help define the basis for the in situ features of OLs in cases of MS. Under metabolic stress in vitro, we detected reduction in ATP levels per cell that precede changes in survival. Autophagy was initially activated, although ATP levels were not altered by inhibitors (chloroquine) or activators (Torin-1). Prolonged stress resulted in autophagy failure, documented by non-fusion of autophagosomes and lysosomes. Consistent with our in vitro results, we detected higher expression of LC3, a marker of autophagosomes in OLs, in MS lesions compared to controls. Both in vitro and in situ, we observe a reduction in nuclear size of remaining OLs. Prolonged stress resulted in increased ROS and cleavage of spectrin, a target of Ca2+-dependent proteases. Cell death was however not prevented by inhibitors of ferroptosis or MPT-driven necrosis, the regulated cell death (RCD) pathways most likely to be activated by metabolic stress. hOLs have decreased expression of VDAC1, VDAC2, and of genes regulating iron accumulation and cyclophilin. RNA sequencing analyses did not identify activation of these RCD pathways in vitro or in MS cases. We conclude that this distinct response of hOLs, including resistance to RCD, reflects the combined impact of autophagy failure, increased ROS, and calcium influx, resulting in metabolic collapse and degeneration of cellular structural integrity. Defining the basis of OL injury and death provides guidance for development of neuro-protective strategies.

Oncogenes and cancer associated thrombosis: what can we learn from single cell genomics about risks and mechanisms?

Single cell analysis of cancer cell transcriptome may shed a completely new light on cancer-associated thrombosis (CAT). CAT causes morbid, and sometimes lethal complications in certain human cancers known to be associated with high risk of venous thromboembolism (VTE), pulmonary embolism (PE) or arterial thromboembolism (ATE), all of which worsen patients' prognosis. How active cancers drive these processes has long evaded scrutiny. While "unspecific" microenvironmental effects and consequences of patient care (e.g., chemotherapy) have been implicated in pathogenesis of CAT, it has also been suggested that oncogenic pathways driven by either genetic (mutations), or epigenetic (methylation) events may influence the coagulant phenotype of cancer cells and stroma, and thereby modulate the VTE/PE risk. Consequently, the spectrum of driver events and their downstream effector mechanisms may, to some extent, explain the heterogeneity of CAT manifestations between cancer types, molecular subtypes, and individual cases, with thrombosis-promoting, or -protective mutations. Understanding this molecular causation is important if rationally designed countermeasures were to be deployed to mitigate the clinical impact of CAT in individual cancer patients. In this regard, multi-omic analysis of human cancers, especially at a single cell level, has brought a new meaning to concepts of cellular heterogeneity, plasticity, and multicellular complexity of the tumour microenvironment, with profound and still relatively unexplored implications for the pathogenesis of CAT. Indeed, cancers may contain molecularly distinct cellular subpopulations, or dynamic epigenetic states associated with different profiles of coagulant activity. In this article we discuss some of the relevant lessons from the single cell "omics" and how they could unlock new potential mechanisms through which cancer driving oncogenic lesions may modulate CAT, with possible consequences for patient stratification, care, and outcomes.

K27M in canonical and noncanonical H3 variants occurs in distinct oligodendroglial cell lineages in brain midline gliomas.

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations and molecular profiles. The contribution of the cell of origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte precursor cells (OPCs). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, whereas H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, whereas other epigenetic changes are mainly contingent on the cell of origin chromatin state and cycling rate.

Unraveling molecular mechanism underlying biomaterial and stem cells interaction during cell fate commitment using high throughput data analysis.

Stem cell differentiation towards various somatic cells and body organs has proven to be an effective technique in the understanding and progression of regenerative medicine. Despite the advances made, concerns regarding the low efficiency of differentiation and the remaining differences between stem cell products and their in vivo counterparts must be addressed. Biomaterials that mimic endogenous growth conditions represent one recent method used to improve the quality and efficiency of stem cell differentiation, though the mechanisms of this improvement remain to be completely understood. The effectiveness of various biomaterials can be analyzed through a multidisciplinary approach involving bioinformatics and systems biology tools. Here, we aim to use bioinformatics to accomplish two aims: 1) determine the effect of different biomaterials on stem cell growth and differentiation, and 2) understand the effect of cell of origin on the differentiation potential of multipotent stem cells. First, we demonstrate that the dimensionality (2D versus 3D) and the degradability of biomaterials affects the way that the cells are able to grow and differentiate at the transcriptional level. Additionally, according to transcriptional state of the cells, the particular cell of origin is an important factor in determining the response of stem cells to same biomaterial. Our data demonstrates the ability of bioinformatics to understand novel molecular mechanisms and context by which stem cells are most efficiently able to differentiate. These results and strategies can be used to suggest proper combinations of biomaterials and stem cells to achieve high differentiation efficiency and functionality of desired cell types.

H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.

Transcription Factors in Regulatory and Protein Subnetworks during Generation of Neural Stem Cells and Neurons from Direct Reprogramming of Non-fibroblastic Cell Sources.

Direct reprogramming of non-fibroblastic cells to the neuronal cell types including induced neurons (iNs) and induced neural stem cells (iNSCs) has provided an alternative approach for the direct reprogramming of fibroblasts to those cells. However, to increase the efficiency of the reprogramming process the underlying mechanisms should be clarified. In the current study, we analyzed the gene expression profiles of five different cellular conversions to understand the most significant molecular mechanisms and transcription factors (TFs) underlying each conversion. For each conversion, we found the list of differentially expressed genes (DEGs) and the list of differentially expressed TFs (DE-TFs) which regulate expression of DEGs. Moreover, we constructed gene regulatory networks based on the TF-binding sites' data and found the most central regulators and the most active part of the networks. Furthermore, protein complexes were identified from constructed protein-protein interaction networks for DE-TFs. Finally, we proposed a list of main regulators for each conversion; for example, in the direct conversion of epithelial-like cells (ECs) to iNSCs, combination of centrality with active modules or protein complex analyses highlighted the role of POU3F2, BACH1, AR, PBX1, SOX2 and NANOG genes in this conversion. To the best of our knowledge, this study is the first one that analyzed the direct conversion of non-fibroblastic cells toward iNs and iNSCs and we believe that the expression manipulation of identified genes may increase efficiency of these processes.

Common Gene Expression Patterns in the Generation of Induced Neurons From Fibroblasts.

In the current study, we analyzed ten gene expression data sets including RNA-sequencing and microarray experiment data during the direct reprogramming of mouse and human fibroblasts to induced neurons and found common gene expression pattern across all data sets for this conversion.

Regulating the transcriptomes that mediate the conversion of fibroblasts to various nervous system neural cell types.

Our understanding of the mechanism of cell fate transition during the direct reprogramming of fibroblasts into various central nervous system (CNS) neural cell types has been limited by the lack of a comprehensive analysis on generated cells, independently and in comparison with other CNS neural cell types. Here, we applied an integrative approach on 18 independent high throughput expression data sets to gain insight into the regulation of the transcriptome during the conversion of fibroblasts into induced neural stem cells, induced neurons (iNs), induced astrocytes, and induced oligodendrocyte progenitor cells (iOPCs). We found common down-regulated genes to be mostly related to fibroblast-specific functions, and suggest their potential as markers for screening of the silencing of the fibroblast-specific program. For example, Tagln was significantly down-regulated across all considered data sets. In addition, we identified specific profiles of up-regulated genes for each CNS neural cell types, which could be potential markers for maturation and efficiency screenings. Furthermore, we identified the main TFs involved in the regulation of the gene expression program during direct reprogramming. For example, in the generation of iNs from fibroblasts, the Rest TF was the main regulator of this reprogramming. In summary, our computational approach for meta-analyzing independent expression data sets provides significant details regarding the molecular mechanisms underlying the regulation of the gene expression program, and also suggests potentially useful candidate genes for screening down-regulation of fibroblast gene expression profile, maturation, and efficiency, as well as candidate TFs for increasing the efficiency of the reprogramming process.

Comprehensive transcriptome mining of the direct conversion of mesodermal cells.

The direct reprogramming of somatic cells is a promising approach for regenerative medicine, especially in the production of mesoderm layer-derived cells. Meta-analysis studies provide precise insight into the undergoing processes and help increase the efficiency of reprogramming. Here, using 27 high-throughput expression data sets, we analyzed the direct reprogramming of mesodermal cells in humans and mice. Fibroblast-derived cells showed a common expression pattern of up- and down-regulated genes that were mainly involved in the suppression of the fibroblast-specific gene expression program, and may be used as markers of the initiation of reprogramming. Furthermore, we found a specific gene expression profile for each fibroblast-derived cell studied, and each gene set appeared to play specific functional roles in its cell type, suggesting their use as markers for their mature state. Furthermore, using data from protein-DNA interactions, we identified the main transcription factors (TFs) involved in the conversion process and ranked them based on their importance in their gene regulatory networks. In summary, our meta-analysis approach provides new insights on the direct conversion of mesodermal somatic cells, introduces a list of genes as markers for initiation and maturation, and identifies TFs for which manipulating their expression may increase the efficiency of direct conversion.

Comprehensive Dissection of Transcriptome Data and Regulatory Factors in Pancreatic Cancer Cells.

Features of pancreatic cancers include high mortality rates caused by rapid tumor progression and a lack of effective therapy. Underpinning the molecular mechanisms involved in the alteration of the gene expression program in the pancreatic cancer remains to be understood. In the current study, we performed a comprehensive analysis using 282 pancreatic tumor and normal samples from seven independent expression data sets to provide a better view on the interactions between different transcription factors (TFs) and the most affected biological pathways in pancreatic cancer. We highlighted common differentially expressed genes (DEGs) and common affected processes within pancreatic cancer samples. We revealed 16 main DE-TFs that regulated gene expression alterations as well as the most significant processes in pancreatic cancer compared to normal cells. For example, we found the upregulated FOXM1 to be a top regulator of pancreatic cellular transformation based on results from different analyses, including from its regulation of gene regulatory networks, its presence in protein complex, its significant regulation of genes related to cancer pathways, and its regulation of most of the identified DE-TFs. Furthermore, we provided a model and assessed the role of different DE-TFs in the regulation of the most affected pancreatic- and cancer-specific processes. In conclusion, our bioinformatics meta-analysis of high throughput expression data sets, besides clarifying common affected genes and pathways, also showed the mechanisms involved in regulating these common profiles. Our results, especially for DE-TFs, could potentially be useful for screening for pancreatic cancer, and for confirming or determining novel pharmacological targets. J. Cell. Biochem. 118: 3976-3985, 2017. © 2017 Wiley Periodicals, Inc.

Meta-Analysis of Transcriptome Regulation During Induction to Cardiac Myocyte Fate From Mouse and Human Fibroblasts.

Ectopic expression of a defined set of transcription factors (TFs) can directly convert fibroblasts into a cardiac myocyte cell fate. Beside inefficiency in generating induced cardiomyocytes (iCMs), the molecular mechanisms that regulate this process remained to be well defined. The main purpose of this study was to provide better insight on the transcriptome regulation and to introduce a new strategy for candidating TFs for the transdifferentiation process. Eight mouse and three human high quality microarray data sets were analyzed to find differentially expressed genes (DEGs), which we integrated with TF-binding sites and protein-protein interactions to construct gene regulatory and protein-protein interaction networks. Topological and biological analyses of constructed gene networks revealed the main regulators and most affected biological processes. The DEGs could be categorized into two distinct groups, first, up-regulated genes that are mainly involved in cardiac-specific processes and second, down-regulated genes that are mainly involved in fibroblast-specific functions. Gata4, Mef2a, Tbx5, Tead4 TFs were identified as main regulators of cardiac-specific gene expression program; and Trp53, E2f1, Myc, Sfpi1, Lmo2, and Meis1 were identified as TFs which mainly regulate the expression of fibroblast-specific genes. Furthermore, we compared gene expression profiles and identified TFs between mouse and human to find the similarities and differences. In summary, our strategy of meta-analyzing the data of high-throughput techniques by computational approaches, besides revealing the mechanisms involved in the regulation of the gene expression program, also suggests a new approach for increasing the efficiency of the direct reprogramming of fibroblasts into iCMs. J. Cell. Physiol. 232: 2053-2062, 2017. © 2016 Wiley Periodicals, Inc.

Role of Hepatic-Specific Transcription Factors and Polycomb Repressive Complex 2 during Induction of Fibroblasts to Hepatic Fate.

Direct reprogramming using defined sets of transcription factors (TFs) is a recent strategy for generating induced hepatocytes (iHeps) from fibroblasts for use in regenerative medicine and drug development. Comprehensive studies detailing the regulatory role of TFs during this reprogramming process could help increase its efficiency. This study aimed to find the TFs with the greatest influences on the generation of iHeps from fibroblasts, and to further understand their roles in the regulation of the gene expression program. Here, we used systems biology approaches to analyze high quality expression data sets in combination with TF-binding sites data and protein-protein interactions data during the direct reprogramming of fibroblasts to iHeps. Our results revealed two main patterns for differentially expressed genes (DEGs): up-regulated genes were categorized as hepatic-specific pattern, and down-regulated genes were categorized as mesoderm- and fibroblast-specific pattern. Interestingly, hepatic-specific genes co-expressed and were regulated by hepatic-specific TFs, specifically Hnf4a and Foxa2. Conversely, the mesoderm- and fibroblast-specific pattern was mainly silenced by polycomb repressive complex 2 (PRC2) members, including Suz12, Mtf2, Ezh2, and Jarid2. Independent analysis of both the gene and core regulatory network of DE-TFs showed significant roles for Hnf4a, Foxa2, and PRC2 members in the regulation of the gene expression program and in biological processes during the direct conversion process. Altogether, using systems biology approaches, we clarified the role of Hnf4a and Foxa2 as hepatic-specific TFs, and for the first time, introduced the PRC2 complex as the main regulator that favors the direct reprogramming process in cooperation with hepatic-specific factors.

Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts.

Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs.

Signaling and Gene Regulatory Networks Governing Definitive Endoderm Derivation From Pluripotent Stem Cells.

The generation of definitive endoderm (DE) from pluripotent stem cells (PSCs) is a fundamental stage in the formation of highly organized visceral organs, such as the liver and pancreas. Currently, there is a need for a comprehensive study that illustrates the involvement of different signaling pathways and their interactions in the derivation of DE cells from PSCs. This study aimed to identify signaling pathways that have the greatest influence on DE formation using analyses of transcriptional profiles, protein-protein interactions, protein-DNA interactions, and protein localization data. Using this approach, signaling networks involved in DE formation were constructed using systems biology and data mining tools, and the validity of the predicted networks was confirmed experimentally by measuring the mRNA levels of hub genes in several PSCs-derived DE cell lines. Based on our analyses, seven signaling pathways, including the BMP, ERK1-ERK2, FGF, TGF-beta, MAPK, Wnt, and PIP signaling pathways and their interactions, were found to play a role in the derivation of DE cells from PSCs. Lastly, the core gene regulatory network governing this differentiation process was constructed. The results of this study could improve our understanding surrounding the efficient generation of DE cells for the regeneration of visceral organs. J. Cell. Physiol. 231: 1994-2006, 2016. © 2016 Wiley Periodicals, Inc.

Transcription factor regulatory network for early lung immune response to tuberculosis in mice.

Numerous transcription factors (TFs) have been suggested to have a role in Mycobacterium tuberculosis infection; however, the TFs involved in the early immune response of lung cells remains to be fully elucidated. The present study aimed to identify TFs which may have a role in the early immune response to tuberculosis and the gene regulatory networks in which they are involved. Gene expression data obtained from microarray analysis of the early lung immune response to tuberculosis (Gene Expression Omnibus; accession no. GSE23014) was integrated with data for TF binding sites and protein-protein interactions in order to construct a TF regulatory network. The role of TFs in protein complexes, active modules, topology of the network and regulation of immune processes were investigated. The results demonstrated that the constructed gene regulatory network harbored 1,270 differentially expressed (DE) genes with 4,070 regulatory and protein-protein interactions. In addition, it was revealed that 17 DE TFs were involved in the positive regulation of numerous immunological and biological processes, including T cell activation, T cell proliferation and tuberculosis-associated gene expression, in the constructed regulatory network. Signal transducer and activator of transcription 4, interferon regulatory factor 8, spleen focus-forming virus proviral integration 1, enhancer of zeste homolog 2 and kruppel-like factor 4 were predicted to be the primary TFs regulating the DE genes during early lung infection by M. tuberculosis, as determined through various analyses of the gene regulatory network. In conclusion, the present study identified novel TFs involved in the early response to M. tuberculosis infection, which may enhance current understanding of the molecular mechanism underlying tuberculosis infection and introduced potential targets for novel tuberculosis therapies.

Predicting involvement of polycomb repressive complex 2 in direct conversion of mouse fibroblasts into induced neural stem cells.

INTRODUCTION: Mouse fibroblasts could be directly converted into induced neural stem cells (iNSCs), by introducing a set of known transcription factors (TFs). This process, known as direct reprogramming, is an alternative source of NSCs production for cell therapy applications, hence, more common sources for such cells including embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs) are also in use. Despite their importance, the exact role of different TFs involved in the conversion of fibroblasts into iNSCs and the interactions between these factors has not been studied. METHODS: Here, we have used available microarray data to construct a gene regulatory network to understand the dynamic of regulatory interactions during this conversion. We have implemented other types of data such as information regarding TFs binding sites and valid protein-protein interactions to improve the network reliability. The network contained 1857 differentially expressed (DE) genes, linked by11054 interactions. The most important TFs identified based on topology analysis of the network. Furthermore, in selecting such TFs, we have also considered their role in the regulation of nervous system development. RESULTS: Based on these analyses, we found that Ezh2, Jarid2, Mtf2, Nanog, Pou5f1, Sall4, Smarca4, Sox2, Suz12, and Tcf3 are the main regulators of direct conversion of mouse fibroblasts into iNSCs. Because, members of the polycomb repressive complex 2 (PRC2) were present in the most effective TFs' list, we have concluded that this complex is one of the major factors in this conversion. Additionally, gene expression profiling of iNSCs, obtained from a different data sets, showed that Sox2 and Ezh2 are two main regulators of the direct reprogramming process. CONCLUSIONS: Our results provide an insight into molecular events that occur during direct reprogramming of fibroblasts into iNSCs. This information could be useful in simplifying the production of iNSCs, by reducing the number of required factors, for use in regenerative medicine.