A Sendai virus-based expression system directs efficient induction of chondrocytes by transcription factor-mediated reprogramming.

Cartilage rarely heals spontaneously once damaged. Osteoarthritis (OA) is the most common degenerative joint disease among the elderly; however, effective treatment for OA is currently lacking. Autologous chondrocyte implantation (ACI), an innovative regenerative technology involving the implantation of healthy chondrocytes, may restore damaged lesions.

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors.

Background: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer.

Engineering Critical Residues of SOX9 Discovers a Variant With Potent Capacity to Induce Chondrocytes.

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement.

Locus-Specific Isolation of the Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells.

Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells.

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming.

Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined.

Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency.

Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods.

Early reactivation of clustered genes on the inactive X chromosome during somatic cell reprogramming.

Reprogramming of murine female somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by X chromosome reactivation (XCR), by which the inactive X chromosome (Xi) in female somatic cells becomes reactivated. However, how Xi initiates reactivation during reprogramming remains poorly defined. Here, we used a Sendai virus-based reprogramming system to generate partially reprogrammed iPSCs that appear to be undergoing the initial phase of XCR.

Visualization of intracellular lipid metabolism in brown adipocytes by time-lapse ultra-multiplex CARS microspectroscopy with an onstage incubator.

We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80 min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.

Utilization of a novel Sendai virus vector in ex vivo gene therapy for hemophilia A.

Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro.

Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720.

Uncoupling protein 1 (UCP1) is a mitochondrial protein that is expressed in both brown and beige adipocytes. UCP1 uncouples the mitochondrial electron transport chain from ATP synthesis to produce heat via non-shivering thermogenesis. Due to their ability to dissipate energy as heat and ameliorate metabolic disorders, UCP1-expressing adipocytes are considered as a potential target for anti-obesity treatment.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome.

Mechanisms of the Metabolic Shift during Somatic Cell Reprogramming.

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold a huge promise for regenerative medicine, drug development, and disease modeling. PSCs have unique metabolic features that are akin to those of cancer cells, in which glycolysis predominates to produce energy as well as building blocks for cellular components. Recent studies indicate that the unique metabolism in PSCs is not a mere consequence of their preference for a low oxygen environment, but is an active process for maintaining self-renewal and pluripotency, possibly in preparation for rapid response to the metabolic demands of differentiation.

Live-cell imaging of subcellular structures for quantitative evaluation of pluripotent stem cells.

Pluripotent stem cells (PSCs) have various degrees of pluripotency, which necessitates selection of PSCs with high pluripotency before their application to regenerative medicine. However, the quality control processes for PSCs are costly and time-consuming, and it is essential to develop inexpensive and less laborious selection methods for translation of PSCs into clinical applications. Here we developed an imaging system, termed Phase Distribution (PD) imaging system, which visualizes subcellular structures quantitatively in unstained and unlabeled cells.

Live cell imaging of X chromosome reactivation during somatic cell reprogramming.

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the or gene on both X chromosomes.

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302.

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase.

A Role for KLF4 in Promoting the Metabolic Shift via TCL1 during Induced Pluripotent Stem Cell Generation.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is accompanied by morphological, functional, and metabolic alterations before acquisition of full pluripotency. Although the genome-wide effects of the reprogramming factors on gene expression are well documented, precise mechanisms by which gene expression changes evoke phenotypic responses remain to be determined. We used a Sendai virus-based system that permits reprogramming to progress in a strictly KLF4-dependent manner to screen for KLF4 target genes that are critical for the progression of reprogramming.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7).

The RNA binding complexes NF45-NF90 and NF45-NF110 associate dynamically with the c-fos gene and function as transcriptional coactivators.

The c-fos gene is rapidly induced to high levels by various extracellular stimuli. We used a defined in vitro transcription system that utilizes the c-fos promoter to purify a coactivator activity in an unbiased manner. We report here that NF45-NF90 and NF45-NF110, which possess archetypical double-stranded RNA binding motifs, have a direct function as transcriptional coactivators.

Association of the winged helix motif of the TFIIEα subunit of TFIIE with either the TFIIEβ subunit or TFIIB distinguishes its functions in transcription.

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and β, and plays essential roles in transcription. Structure-function studies indicate that TFIIE has three-winged helix (WH) motifs, with one in TFIIEα and two in TFIIEβ. Recent studies suggested that, by binding to the clamp region of RNA polymerase II, TFIIEα-WH promotes the conformational change that transforms the promoter-bound inactive preinitiation complex to the active complex.

Manipulation of KLF4 expression generates iPSCs paused at successive stages of reprogramming.

The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs.

The paired-box homeodomain transcription factor Pax6 binds to the upstream region of the TRAP gene promoter and suppresses receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation.

Heterogeneous nuclear ribonucleoprotein R cooperates with mediator to facilitate transcription reinitiation on the c-Fos gene.

The c-fos gene responds to extracellular stimuli and undergoes robust but transient transcriptional activation. Here we show that heterogeneous nuclear ribonucleoprotein R (hnRNP R) facilitates transcription reinitiation of the c-fos promoter in vitro in cooperation with Mediator. Consistently, hnRNP R interacts with the Scaffold components (Mediator, TBP, and TFIIH) as well as TFIIB, which recruits RNA polymerase II (Pol II) and TFIIF to Scaffold.

cAMP-response element-binding protein (CREB) controls MSK1-mediated phosphorylation of histone H3 at the c-fos promoter in vitro.

The rapid induction of the c-fos gene correlates with phosphorylations of histone H3 and HMGN1 by mitogen- and stress-activated protein kinases. We have used a cell-free system to dissect the mechanism by which MSK1 phosphorylates histone H3 within the c-fos chromatin. Here, we show that the reconstituted c-fos chromatin presents a strong barrier to histone H3 phosphorylation by MSK1; however, the activators (serum response factor, Elk-1, cAMP-response element-binding protein (CREB), and ATF1) bound on their cognate sites recruit MSK1 to phosphorylate histone H3 at Ser-10 within the chromatin.

DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation.

Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF.

Heterogeneous nuclear ribonucleoprotein R enhances transcription from the naturally configured c-fos promoter in vitro.

Transcription of a proto-oncogene c-fos is induced rapidly to high levels by various extracellular stimuli. To explore the molecular mechanism of c-fos gene induction, we established a defined in vitro transcription system for the c-fos promoter that consists of purified activators (SRF, Elk-1, cAMP-responsive element-binding protein, and ATF1), general transcription factors, and RNA polymerase II. In this reconstituted transcription system, activation of c-fos transcription was highly dependent upon coactivators such as PC4 and Mediator, indicating a very weak activation potential of the activators in the context of an unaltered promoter structure.