Different Sets Of Transcription Factors Research Articles

Dysregulation of early gene response to a mixed meal in skeletal muscle in obesity and type 2 diabetes.

Obesity- and type 2 diabetes mellitus-induced changes in the expression of protein-coding genes in human skeletal muscle were extensively examined at baseline (after an overnight fast). We aimed to compare the early transcriptomic response to a typical single meal in skeletal muscle of metabolically healthy subjects and obese individuals without and with type 2 diabetes. Transcriptomic response (RNA-seq) to a mixed meal (nutritional drink, ∼25 kJ/kg of body mass) was examined in the vastus lateralis muscle (1 h after a meal) in 7 healthy subjects and 14 obese individuals without or with type 2 diabetes. In all obese individuals, the transcriptome response to a meal was dysregulated (suppressed and altered) and associated with different biological processes compared with healthy control. To search for potential transcription factors regulating transcriptomic response to a meal, the enrichment of transcription factor-binding sites in individual promoters of the human skeletal muscle was examined. In obese individuals, the transcriptomic response is associated with a different set of transcription factors than that in healthy subjects. In conclusion, metabolic disorders are associated with a defect in the regulation of mixed meal/insulin-mediated gene expression-insulin resistance in terms of gene expression. Importantly, this dysregulation occurs in obese individuals without type 2 diabetes, i.e., at the first stage of the development of metabolic disorders.NEW & NOTEWORTHY In skeletal muscle of metabolically healthy subjects, a typical single meal normalized to body mass induces activation of various transcription factors, expression of numerous receptor tyrosine kinases associated with the insulin signaling cascade, and transcription regulators. In skeletal muscle of obese individuals without and with type 2 diabetes, this signaling network is poorly regulated at the transcriptional level, indicating dysregulation of the early gene response to a mixed meal.

Abstract 1470: ASCL1 dimerizes with TCF12 protein and activates BMP-SMAD1 pathway during subtype switching from mesenchymal towards adrenergic neuroblastoma

Abstract Recently, several studies have demonstrated two semi-stable and interchangeable subtypes of neuroblastoma, namely mesenchymal (MES) and adrenergic (ADRN) subtypes, which are associated with the early neural crest and committed adrenergic neural progenitor stages, respectively. Each of these subtypes is controlled by a different set of transcription factors forming the core regulatory circuit (CRC) and exhibits different enhancer and transcription profiles. However, transcription factors that drives transition of the subtype switching and the corresponding regulated signaling pathways remain to be investigated. In this study, we demonstrate that ASCL1, a critical member of the ADRN CRC, can induce a subtype switching from MES to ADRN neuroblastoma. Our study shows that a forced expression of ASCL1 in MES neuroblastoma cells results in cell cycle arrest and cell differentiation towards the ADRN lineage with induction of ADRN gene expression, such as DLL3 and DBH. Furthermore, we have identified TCF12 as an important interaction partner of ASCL1 via SILAC analysis. ASCL1 forms a complex with TCF12 and regulates the expression of downstream target genes. On the other hand, overexpression of ASCL1 inhibits existing MES specific TGFβ-SMAD2/3 signaling pathway, while it activates BMP-SMAD1 signaling pathway. Interestingly, activation of BMP-SMAD1 signaling pathway results in the upregulation of the ID proteins, which can form dimer with TCF12 protein and sequester it away from ASCL1 protein. Thus, the BMP SMAD1-ID protein signaling cascade in turn serves as a negative feedback loop to prevent the cells from terminal differentiation via equilibration of the amount of ASCL1-TCF12 dimer. In summary, ASCL1 can induce the subtype switching and partially define the ADRN transcription program. ASCL1 serves as a lineage master initiator that characterizes ADRN neuroblastoma. Citation Format: Lu Wang, Tze King Tan, Hyoju Kim, Dennis Kappei, Shi Hao Tan, A. Thomas Look, Takaomi Sanda. ASCL1 dimerizes with TCF12 protein and activates BMP-SMAD1 pathway during subtype switching from mesenchymal towards adrenergic neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1470.

Comprehensive characterization of three classes of Arabidopsis SWI/SNF chromatin remodelling complexes.

Although SWI/SNF chromatin remodelling complexes are known to regulate diverse biological functions in plants, the classification, compositions and functional mechanisms of the complexes remain to be determined. Here we comprehensively characterized SWI/SNF complexes by affinity purification and mass spectrometry in Arabidopsis thaliana, and found three classes of SWI/SNF complexes, which we termed BAS, SAS and MAS (BRM-, SYD- and MINU1/2-associated SWI/SNF complexes). By investigating multiple developmental phenotypes of SWI/SNF mutants, we found that three classes of SWI/SNF complexes have both overlapping and specific functions in regulating development. To investigate how the three classes of SWI/SNF complexes differentially regulate development, we mapped different SWI/SNF components on chromatin at the whole-genome level and determined their effects on chromatin accessibility. While all three classes of SWI/SNF complexes regulate chromatin accessibility at proximal promoter regions, SAS is a major SWI/SNF complex that is responsible for mediating chromatin accessibility at distal promoter regions and intergenic regions. Histone modifications are related to both the association of SWI/SNF complexes with chromatin and the SWI/SNF-dependent chromatin accessibility. Three classes of SWI/SNF-dependent accessibility may enable different sets of transcription factors to access chromatin. These findings lay a foundation for further investigation of the function of three classes of SWI/SNF complexes in plants.

The Iron Deficiency-Regulated Small Protein Effector FEP3/IRON MAN1 Modulates Interaction of BRUTUS-LIKE1 With bHLH Subgroup IVc and POPEYE Transcription Factors.

In light of climate change and human population growth one of the most challenging tasks is to generate plants that are Fe-efficient, resilient to low Fe supply and Fe-biofortified. For such endeavors, it is crucial to understand the regulation of Fe acquisition and allocation in plants. One open question is how identified Fe-regulatory proteins comprising positive and negative regulators act together to steer Fe homeostasis. bHLH transcription factors (TFs) belonging to the subgroups IVb and IVc can initiate a bHLH cascade controlling the –Fe response in roots. In Arabidopsis thaliana, the –Fe-induced genes are sub-divided into several gene co-expression clusters controlled by different sets of TFs. Some of the co-expressed genes encode regulatory E3 ligase proteins BRUTUS (BTS)/BTS-LIKE (BTSL) and small proteins belonging to the group of FE UPTAKE-INDUCING PEPTIDE/IRON MAN (FEP/IMA). Recently, it was described that FEP1/IMA3 and FEP3/IMA1 proteins inhibit the repression of bHLH factors by BTS. We had postulated that –Fe-regulated co-expression clusters provide new information about regulatory protein interaction complexes. Here, we report a targeted yeast two-hybrid screen among 23 proteins of the –Fe response. This identified a novel protein interactome involving another E3 ligase, namely BTSL1, basic helix-loop-helix (bHLH) protein POPEYE (PYE) and transcription factors of the subgroup IVc as well as FEP3/IMA1. Because of the difficulty in stable BTSL1 protein expression in plant cells, we used a yeast two hybrid-based deletion mapping, homology modeling and molecular docking, to pinpoint interaction sites in BTSL1 and FEP3/IMA1. bHLH IVc TFs have similar residues at their C-terminus as FEP3/IMA1 interacting sites. FEP3/IMA1 attenuated interaction of BTSL1 and bHLH proteins in a yeast three-hybrid assay, in line with physiological data pointing to enhanced Fe acquisition and allocation in FEP3/IMA1 overexpression and btsl1 btsl2 mutant plants. Hence, exploiting –Fe-induced gene co-expression networks identified FEP3/IMA1 as a small effector protein that binds and inhibits the BTSL1 complex with PYE and bHLH subgroup IVc proteins. Structural analysis resolved interaction sites. This information helps improving models of Fe regulation and identifying novel targets for breeding of Fe-efficient crops.

Gentian FLOWERING LOCUS T orthologs regulate phase transitions: floral induction and endodormancy release.

Perennial plants undergo a dormant period in addition to the growth and flowering phases that are commonly observed in annuals and perennials. Consequently, the regulation of these phase transitions in perennials is believed to be complicated. Previous studies have proposed that orthologs of FLOWERING LOCUS T (FT) regulate not only floral initiation but also dormancy. We, therefore, investigated the involvement of FT orthologs (GtFT1 and GtFT2) during the phase transitions of the herbaceous perennial gentian (Gentiana triflora). Analysis of seasonal fluctuations in the expression of these genes revealed that GtFT1 expression increased prior to budbreak and flowering, whereas GtFT2 expression was induced by chilling temperatures with the highest expression occurring when endodormancy was released. The expression of FT-related transcription factors, reportedly involved in flowering, also fluctuated during each phase transition. These results suggested the involvement of GtFT1 in budbreak and floral induction and GtFT2 in dormancy regulation, implying that the two gentian FT orthologs activated a different set of transcription factors. Gentian ft2 mutants generated by CRISPR/Cas9-mediated genome editing had a lower frequency of budbreak and budbreak delay in overwintering buds caused by an incomplete endodormancy release. Our results highlighted that the gentian orthologs of FRUITFULL (GtFUL) and SHORT VEGETATIVE PHASE-like 1 (GtSVP-L1) act downstream of GtFT2, probably to prevent untimely budbreak during ecodormancy. These results suggest that each gentian FT ortholog regulates a different phase transition by having variable responses to endogenous or environmental cues, leading to their ability to induce the expression of distinct downstream genes.

Androgen and Luteinizing Hormone Stimulate the Function of Rat Immature Leydig Cells Through Different Transcription Signals.

The function of immature Leydig cells is regulated by hormones, such as androgen and luteinizing hormone (LH). However, the regulation of this process is still unclear. The objective of this study was to determine whether luteinizing hormone (LH) or androgens contribute to this process. Immature Leydig cells were purified from 35-day-old male Sprague Dawley rats and cultured with LH (1 ng/ml) or androgen (7α-methyl-19- nortestosterone, MENT, 100 nM) for 2 days. LH or MENT treatment significantly increased the androgens produced by immature Leydig cells in rats. Microarray and qPCR and enzymatic tests showed that LH up-regulated the expression of Scarb1, Cyp11a1, Cyp17a1, and Srd5a1 while down-regulated the expression of Sult2a1 and Akr1c14. On the contrary, the expression of Cyp17a1 was up-regulated by MENT. LH and MENT regulate Leydig cell function through different sets of transcription factors. We conclude that LH and androgens participate in the regulation of rat immature Leydig cell function through different transcriptional pathways.

Perception and Signaling of Ultraviolet-B Radiation in Plants.

Ultraviolet-B (UV-B) radiation is an intrinsic fraction of sunlight that plants perceive through the UVR8 photoreceptor. UVR8 is a homodimer in its ground state that monomerizes upon UV-B photon absorption via distinct tryptophan residues. Monomeric UVR8 competitively binds to the substrate binding site of COP1, thus inhibiting its E3 ubiquitin ligase activity against target proteins, which include transcriptional regulators such as HY5. The UVR8-COP1 interaction also leads to the destabilization of PIF bHLH factor family members. Additionally, UVR8 directly interacts with and inhibits the DNA binding of a different set of transcription factors. Each of these UVR8 signaling mechanisms initiates nuclear gene expression changes leading to UV-B-induced photomorphogenesis and acclimation. The two WD40-repeat proteins RUP1 and RUP2 provide negative feedback regulation and inactivate UVR8 by facilitating redimerization. Here, we review the molecular mechanisms of the UVR8 pathway from UV-B perception and signal transduction to gene expression changes and physiological UV-B responses.

Transcriptomic Signatures and Upstream Regulation in Human Skeletal Muscle Adapted to Disuse and Aerobic Exercise.

Inactivity is associated with the development of numerous disorders. Regular aerobic exercise is broadly used as a key intervention to prevent and treat these pathological conditions. In our meta-analysis we aimed to identify and compare (i) the transcriptomic signatures related to disuse, regular and acute aerobic exercise in human skeletal muscle and (ii) the biological effects and transcription factors associated with these transcriptomic changes. A standardized workflow with robust cut-off criteria was used to analyze 27 transcriptomic datasets for the vastus lateralis muscle of healthy humans subjected to disuse, regular and acute aerobic exercise. We evaluated the role of transcriptional regulation in the phenotypic changes described in the literature. The responses to chronic interventions (disuse and regular training) partially correspond to the phenotypic effects. Acute exercise induces changes that are mainly related to the regulation of gene expression, including a strong enrichment of several transcription factors (most of which are related to the ATF/CREB/AP-1 superfamily) and a massive increase in the expression levels of genes encoding transcription factors and co-activators. Overall, the adaptation strategies of skeletal muscle to decreased and increased levels of physical activity differ in direction and demonstrate qualitative differences that are closely associated with the activation of different sets of transcription factors.

Sex Differences in Gene Expression and Regulatory Networks across 29 Human Tissues

Sex differences manifest in many diseases and may drive sex-specific therapeutic responses. To understand the molecular basis of sex differences, we evaluated sex-biased gene regulation by constructing sample-specific gene regulatory networks in 29 human healthy tissues using 8,279 whole-genome expression profiles from the Genotype-Tissue Expression (GTEx) project. We find sex-biased regulatory network structures in each tissue. Even though most transcription factors (TFs) are not differentially expressed between males and females, many have sex-biased regulatory targeting patterns. In each tissue, genes that are differentially targeted by TFs between the sexes are enriched for tissue-related functions and diseases. In brain tissue, for example, genes associated with Parkinson's disease and Alzheimer's disease are targeted by different sets of TFs in each sex. Our systems-based analysis identifies a repertoire of TFs that play important roles in sex-specific architecture of gene regulatory networks, and it underlines sex-specific regulatory processes in both health and disease.

Prokaryotic sigma factors and their transcriptional counterparts in Archaea and Eukarya.

RNA polymerases (RNAPs) carry out transcription in the three domains of life, Bacteria, Archaea, and Eukarya. Transcription initiation is highly regulated by a variety of transcription factors, whose number and subunit complexity increase during evolution. This process is regulated in Bacteria by the σ factor, while the three eukaryotic RNAPs require a complex set of transcription factors (TFs) and a TATA-binding protein (TBP). The archaeal transcription system appears to be an ancestral version of the eukaryotic RNAPII, requiring transcription factor B (TFB), TBP, and transcription factor E (TFE). The function of the bacterial sigma (σ) factor has been correlated to the roles played by the eukaryotic RNAP II and the archaeal RNAP. In addition, σ factors, TFB, and TFIIB all contain multiple DNA binding helix-turn-helix (HTH) structural motifs; although TFIIB and TFB display two HTH domains, while the bacterial σ factor spans 4 HTH motifs. The sequence similarities and structure alignments of the bacterial σ factor, eukaryotic TFIIB, and archaeal TFB evidence that these three proteins are homologs.Key Points• Transcription initiation is highly regulated by TFs.• Transcription is finely regulated in all domains of life by different sets of TFs.• Specific TFs in Bacteria, Eukarya and Archaea are homologs.

Spatial Genome Re-organization between Fetal and Adult Hematopoietic Stem Cells

SUMMARYFetal hematopoietic stem cells (HSCs) undergo a developmental switch to become adult HSCs with distinct functional properties. To better understand the molecular mechanisms underlying the developmental switch, we have conducted deep sequencing of the 3D genome, epigenome, and transcriptome of fetal and adult HSCs in mouse. We find that chromosomal compartments and topologically associating domains (TADs) are largely conserved between fetal and adult HSCs. However, there is a global trend of increased compartmentalization and TAD boundary strength in adult HSCs. In contrast, intra-TAD chromatin interactions are much more dynamic and wide-spread, involving over a thousand gene promoters and distal enhancers. These developmental-stage-specific enhancer-promoter interactions are mediated by different sets of transcription factors, such as TCF3 and MAFB in fetal HSCs, versus NR4A1 and GATA3 in adult HSCs. Loss-of-function studies of TCF3 confirm the role of TCF3 in mediating condition-specific enhancer-promoter interactions and gene regulation in fetal HSCs.

Gene expression signatures for HOXA4, HOXA9, and HOXD10 reveal alterations in transcriptional regulatory networks in colon cancer.

We previously reported that HOXA4, HOXA9, and HOXD10 are selectively expressed in colonic stem cells (SCs) and their overexpression contributes to colorectal cancer (CRC). Our goals here were to determine how these HOX genes are transcriptionally regulated and whether transcriptional dysregulation of HOX genes occurs in CRC. Accordingly, we used correlation analysis to identify genes that are expression-correlated or anticorrelated with HOXA4, HOXA9, and HOXD10. We then used Gene Ontology (GO) analysis to functionally classify these genes. The GO results for both HOXA4 and HOXD10 correlated gene sets for normal colon and CRC show functions mostly classified as developmental, transcriptional regulation, and DNA binding. This raised the question: Are these gene sets regulated by the same transcription factors (TFs)? Consequently, we used promoter analysis and interaction network toolset (PAINT) to identify commonly shared transcription response elements. The results indicated that completely different sets of TFs coregulate HOXA4 and HOXD10 (but not HOXA9) and their expression-correlated genes. And predicted TFs are altered in CRC compared with normal colon. Taken together, analysis of gene signatures correlated with expression of HOXA4 and HOXD10 indicates how these HOX genes are: (a) transcriptionally regulated in the normal colon; (b) dysregulated in CRC. This discovery provides a mechanism for targeting CRC SCs.

Sex-related gonadal gene expression differences in the Russian sturgeon (Acipenser gueldenstaedtii) grown in stable aquaculture conditions

The Russian sturgeon (Acipenser gueldenstaedtii) is a primitive freshwater fish and a source of caviar. In the present study, the gonadal transcriptomes of male and female Russian sturgeons grown in stable aquaculture conditions were analyzed. RNA sequencing of whole-gonad transcriptomes from pools of 4-year old fish (five females, four males), resulted in the identification of 28,170 unique transcripts. Of these, 16,191 could be annotated by similarity to gene sequences from other species. There were 392 transcripts that showed differential abundance by a factor of 20-fold or more between the sexes; 272 of these were annotated; of these, 175 and 97 were in greater abundance in ovaries and testes, respectively. Functional annotation and clustering of the genes with differential abundances of mRNA allowed for identification of several clusters. Thus, a group of transcriptional regulators and factors involved in cell division, especially septins, were in greater abundance in the ovaries; while a different set of transcription factors (including sox6 and sox30) and a group of protein kinases were in greater abundance in the testes. The transcript abundances of nine highly abundant candidate transcripts, as well as of two additional genes previously known to be involved in reproduction, cyp19 (p450 aromatase) and foxl2, were assessed in the individual samples by qRT-PCR. Of these, five (including cyp19 and foxl2) were in greater abundance in ovaries, while the abundance of ighm1 mRNA was greater in testes. Phylogenetic analysis based on the k1c18 keratin gene placed the sturgeon sequence nearest those of other primitive fish species, supporting the ancient origin of the sturgeon. In conclusion, this study details transcriptome differences between male and female sturgeon and identifies key genes that may contribute to sexual determination and differentiation.

Differential transcriptome profiling of chilling stress response between shoots and rhizomes of Oryza longistaminata using RNA sequencing.

Rice (Oryza sativa) is very sensitive to chilling stress at seedling and reproductive stages, whereas wild rice, O. longistaminata, tolerates non-freezing cold temperatures and has overwintering ability. Elucidating the molecular mechanisms of chilling tolerance (CT) in O. longistaminata should thus provide a basis for rice CT improvement through molecular breeding. In this study, high-throughput RNA sequencing was performed to profile global transcriptome alterations and crucial genes involved in response to long-term low temperature in O. longistaminata shoots and rhizomes subjected to 7 days of chilling stress. A total of 605 and 403 genes were respectively identified as up- and down-regulated in O. longistaminata under 7 days of chilling stress, with 354 and 371 differentially expressed genes (DEGs) found exclusively in shoots and rhizomes, respectively. GO enrichment and KEGG pathway analyses revealed that multiple transcriptional regulatory pathways were enriched in commonly induced genes in both tissues; in contrast, only the photosynthesis pathway was prevalent in genes uniquely induced in shoots, whereas several key metabolic pathways and the programmed cell death process were enriched in genes induced only in rhizomes. Further analysis of these tissue-specific DEGs showed that the CBF/DREB1 regulon and other transcription factors (TFs), including AP2/EREBPs, MYBs, and WRKYs, were synergistically involved in transcriptional regulation of chilling stress response in shoots. Different sets of TFs, such as OsERF922, OsNAC9, OsWRKY25, and WRKY74, and eight genes encoding antioxidant enzymes were exclusively activated in rhizomes under long-term low-temperature treatment. Furthermore, several cis-regulatory elements, including the ICE1-binding site, the GATA element for phytochrome regulation, and the W-box for WRKY binding, were highly abundant in both tissues, confirming the involvement of multiple regulatory genes and complex networks in the transcriptional regulation of CT in O. longistaminata. Finally, most chilling-induced genes with alternative splicing exclusive to shoots were associated with photosynthesis and regulation of gene expression, while those enriched in rhizomes were primarily related to stress signal transduction; this indicates that tissue-specific transcriptional and post-transcriptional regulation mechanisms synergistically contribute to O. longistaminata long-term CT. Our findings provide an overview of the complex regulatory networks of CT in O. longistaminata.

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes.

Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs. As a proof of principle, we showed that the PB-CRISPRa system could accelerate the differentiation of human induced pluripotent stem cells into neurons and astrocytes by triggering endogenous expression of different sets of transcription factors. The PB-CRISPRa system has the potential to become a convenient and robust tool in neuroscience, which can meet the needs of a variety of in vitro and in vivo gain-of-function applications.

Contractile Differences in Left and Right Ventricles of Healthy Human Hearts

Left ventricle (LV) and right ventricle (RV) differ in embryology, structure and function. Left ventricle originates from the primary heart field while right ventricle originates from the secondary heart field. The two heart fields express different sets of transcription factors and signaling molecules. At the molecular level, the gene expression in response to the pressure loading and failure is different in both ventricles. They also exhibit differences in response to adrenergic stimulation. Adrenergic agonist increase LV contractility but RV contractility may be reduced. Performing different functions, these ventricles have differences in the resistance offered by the systems they pump blood to. The force and tension developed in each ventricle also varies. Although numerous studies have been performed at macroscopic (whole organ) level previously, none (to my knowledge) address the differences at a mesoscopic level, where only a few molecules are investigated. This becomes important, as in macroscopic studies there may be differences due to basic fiber structures, differences in orientation of fibers as well as molecular crowding. These differences were investigated at the level of few molecules by sparsely labeling the myosin lever arm with SeTau 647 Maleimide. The kinetics and distribution of cross-bridges were examined in healthy ex-vivo human ventricles and differences were compared in LV and RV. The results suggest that there is a significance difference in the cross bridge order of left and right ventricles. In particular, the cross bridges of left ventricles were better ordered when compared to those of right ventricles.

Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

Direct Reprogramming-The Future of Cardiac Regeneration?

Today, the only available curative therapy for end stage congestive heart failure (CHF) is heart transplantation. This therapeutic option is strongly limited by declining numbers of available donor hearts and by restricted long-term performance of the transplanted graft. The disastrous prognosis for CHF with its restricted therapeutic options has led scientists to develop different concepts of alternative regenerative treatment strategies including stem cell transplantation or stimulating cell proliferation of different cardiac cell types in situ. However, first clinical trials with overall inconsistent results were not encouraging, particularly in terms of functional outcome. Among other approaches, very promising ongoing pre-clinical research focuses on direct lineage conversion of scar fibroblasts into functional myocardium, termed “direct reprogramming” or “transdifferentiation.” This review seeks to summarize strategies for direct cardiac reprogramming including the application of different sets of transcription factors, microRNAs, and small molecules for an efficient generation of cardiomyogenic cells for regenerative purposes.

Induction of pluripotency in human umbilical cord mesenchymal stem cells in feeder layer-free condition.

Induced Pluripotent Stem Cells (iPSCs) has been produced by the reprogramming of several types of somatic cells through the expression of different sets of transcription factors. This study consists of a technique to obtain iPSCs from human umbilical cord mesenchymal stem cells (UC-MSCs) in a feeder layer-free process using a mini-circle vector containing defined reprogramming genes, Lin28, Nanog, Oct4 and Sox2. The human MSCs transfected with the minicircle vector were cultured in iPSCs medium. Human embryonic stem cell (ESC)-like colonies with tightly packed domelike structures appeared 7-10 days after the second transfection. In the earliest stages, the colonies were green fluorescence protein (GFP)-positive, while upon continuous culture and passage, genuine hiPSC clones expressing GFP were observed. The induced cells, based on the ectopic expression of the pluripotent markers, exhibited characteristics similar to the embryonic stem cells. These iPSCs demonstrated in vitro capabilities for differentiation into the three main embryonic germ layers by embryoid bodies formation. There was no evidence of transgenes integration into the genome of the iPSCs in this study. In conclusion, this method offers a means of producing iPSCs without viral delivery that could possibly overcome ethical concerns and immune rejection in the use of stem cells in medical applications.