Promote Cell Reprogramming Research Articles

The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages.

Circadian regulation of gene expression is prevalent and plays critical roles in cell differentiation. However, its roles in the reprogramming of differentiated cells remain largely unknown. Here, we found that one of the master circadian regulators PER1 promoted virus-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to induced neurons (iNs) and induced pluripotent stem cells (iPSCs). Unexpectedly, PER1 achieved this by repressing inflammatory activation of contaminating macrophages in the MEF culture, rather than by directly modulating the reprogrammability of MEFs. More specifically, we found that transduced viruses activated inflammatory genes in macrophages, such as Tnf encoding TNFα, one of the central inflammatory regulators and an autocrine activator of macrophages. TNFα inhibited iN reprogramming, whereas a TNFα inhibitor promoted iN reprogramming, connecting the inflammatory responses to iN reprogramming. In addition, macrophages were induced to proliferate and mature by non-macrophage cells serving as feeders, which also supported up-regulation of TNFα in macrophages without virus transduction. Furthermore, the 2 inflammatory responses were repressed by the circadian regulator PER1 in macrophages, making reprogrammability dependent on time-of-day of virus transduction. Similar results were obtained with iPSC reprogramming, suggesting a wide occurrence of macrophage-mediated inhibition of cell reprogramming. This study uncovers mechanistic links between cell reprogramming, bystander inflammatory macrophages, and circadian rhythms, which are particularly relevant to in vivo reprogramming and organoid formation incorporating immune cells.

Establishment of in vitro regeneration system and molecular analysis of early development of somatic callus in Capsicum chinense and Capsicum baccatum.

Regeneration is extremely important to pepper genetic development; however, the molecular mechanisms of how the callus reactivates cell proliferation and promotes cell reprogramming remain elusive in pepper. In the present study, C. baccatum (HNUCB81 and HNUCB226) and C. chinense (HNUCC22 and HNUCC16) were analyzed to reveal callus initiation by in vitro regeneration, histology, and transcriptome. We successfully established an efficient in vitro regeneration system of two cultivars to monitor the callus induction of differential genotypes, and the regenerated plants were obtained. Compared to C. chinense, there was a higher callus induction rate in C. baccatum. The phenotype of C. baccatum changed significantly and formed vascular tissue faster than C. chinense. The KEGG enrichment analysis found that plant hormone transduction and starch and sucrose metabolism pathways were significantly enriched. In addition, we identified that the WOX7 gene was significantly up-regulated in HNUCB81 and HNUCB226 than that in HNUCC22 and HNUCC16, which may be a potential function in callus formation. These results provided a promising strategy to improve the regeneration and transformation of pepper plants.

Transient nuclear deformation primes epigenetic state and promotes cell reprogramming.

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering.

ScREMOTE: Using multimodal single cell data to predict regulatory gene relationships and to build a computational cell reprogramming model.

Cell reprogramming offers a potential treatment to many diseases, by regenerating specialized somatic cells. Despite decades of research, discovering the transcription factors that promote cell reprogramming has largely been accomplished through trial and error, a time-consuming and costly method. A computational model for cell reprogramming, however, could guide the hypothesis formulation and experimental validation, to efficiently utilize time and resources. Current methods often cannot account for the heterogeneity observed in cell reprogramming, or they only make short-term predictions, without modelling the entire reprogramming process. Here, we present scREMOTE, a novel computational model for cell reprogramming that leverages single cell multiomics data, enabling a more holistic view of the regulatory mechanisms at cellular resolution. This is achieved by first identifying the regulatory potential of each transcription factor and gene to uncover regulatory relationships, then a regression model is built to estimate the effect of transcription factor perturbations. We show that scREMOTE successfully predicts the long-term effect of overexpressing two key transcription factors in hair follicle development by capturing higher-order gene regulations. Together, this demonstrates that integrating the multimodal processes governing gene regulation creates a more accurate model for cell reprogramming with significant potential to accelerate research in regenerative medicine.

Automatic identification of small molecules that promote cell conversion and reprogramming.

SummaryControlling cell fate has great potential for regenerative medicine, drug discovery, and basic research. Although transcription factors are able to promote cell reprogramming and transdifferentiation, methods based on their upregulation often show low efficiency. Small molecules that can facilitate conversion between cell types can ameliorate this problem working through safe, rapid, and reversible mechanisms. Here, we present DECCODE, an unbiased computational method for identification of such molecules based on transcriptional data. DECCODE matches a large collection of drug-induced profiles for drug treatments against a large dataset of primary cell transcriptional profiles to identify drugs that either alone or in combination enhance cell reprogramming and cell conversion. Extensive validation in the context of human induced pluripotent stem cells shows that DECCODE is able to prioritize drugs and drug combinations enhancing cell reprogramming. We also provide predictions for cell conversion with single drugs and drug combinations for 145 different cell types.

WIND1 induces dynamic metabolomic reprogramming during regeneration in Brassica napus

Plants often display a high competence for regeneration under stress conditions. Signals produced in response to various types of stress serve as critical triggers for de novo organogenesis, but the identity of these signaling molecules underlying cellular reprogramming are largely unknown. We previously identified an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION1 (WIND1), as a key regulator involved in wound-induced cellular reprogramming in Arabidopsis. In this study, we found that activation of Arabidopsis WIND1 (AtWIND1) in hypocotyl explants of Brassica napus (B. napus) enhances callus formation and subsequent organ regeneration. Gene expression analyses revealed that AtWIND1 enhances expression of B. napus homologs of ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN (ESR1/DRN), which is a direct target of WIND1 in Arabidopsis. Further, time-course hormonal analyses showed that an altered balance of endogenous auxin/cytokinin exists in AtWIND1-activated B. napus explants. Our mass spectrometry analyses, in addition, uncovered dynamic metabolomic reprogramming in AtWIND1-activated explants, including accumulation of several compounds, e.g. proline, gamma aminobutyric acid (GABA), and putrescine, that have historically been utilized as additives to enhance plant cell reprogramming in tissue culture. Our findings thus provide new insights into how WIND1 functions to promote cell reprogramming.

MeCP2 deficiency promotes cell reprogramming by stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation.

The generation of induced pluripotent stem cells (iPSCs) offers a great opportunity in research and regenerative medicine. The current poor efficiency and incomplete mechanistic understanding of the reprogramming process hamper the clinical application of iPSCs. MeCP2 connects histone modification and DNA methylation, which are key changes of somatic cell reprogramming. However, the role of MeCP2 in cell reprogramming has not been examined. In this study, we found that MeCP2 deficiency enhanced reprogramming efficiency and stimulated cell proliferation through regulating cell cycle protein expression in the early stage of reprogramming. MeCP2 deficiency enhanced the expression of ribosomal protein genes, thereby enhancing reprogramming efficiency through promoting the translation of cell cycle genes. In the end, MeCP2 deficiency stimulated IGF1/AKT/mTOR signaling and activated ribosomal protein gene expression. Taken together, our data indicate that MeCP2 deficiency promoted cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming.

Spontaneously hyperactive MEK-Erk pathway mediates paradoxical facilitation of cell proliferation in mild hypoxia

BackgroundOxygen is important for common eukaryotic cells to generate ATP. Pathophysiological conditions such as ischemic diseases cause tissue hypoxia. In addition, oxygen availability in deep tissues is supposed to be far lower than surrounding atmosphere even in healthy animals, and the oxygen partial pressures in most normal tissues are estimated to be around 40–50mmHg, so-called mild hypoxia. Recent studies have demonstrated that mild hypoxia has distinct effects on living cells from severe hypoxia. For instance, mild hypoxia was reported to promote cell reprogramming. Although severe hypoxia is known to inhibit cell proliferation, mild hypoxia has been paradoxically demonstrated to increase cell proliferation. However, it has not been clarified by which molecular mechanisms mild hypoxia evokes the discontinuous increment of cell proliferation. MethodsWe established experimental conditions showing the opposite influences of mild and severe hypoxia on cell proliferation using undifferentiated Caco2 human colon carcinoma cells in order to clarify the underlying molecular mechanism. ResultsThe basal activity of Erk, which is a typical mediator of mitogenic signals, is spontaneously increased specifically in cells exposed to mild hypoxia, and inhibition of MEK, an upstream kinase of the Erk, completely inhibited the mild hypoxia-induced enhancement of cell proliferation. ConclusionsSpontaneous hyperactivation of the MEK-Erk pathway by mild hypoxia should be the plausible molecular mechanism of the paradoxical promotion of cell proliferation. General SignificanceOur findings will provide clues to the molecular basis of mild hypoxia-evoked phenomena such as cell reprogramming.

Actin stress in cell reprogramming.

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin-cpstFRET-actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals.

Culturing of mouse and human cells on soft substrates promote the expression of stem cell markers

Substrate elasticity is a potent regulator of the cell state. Soft substrates have been shown to promote the homogeneous self-renewal of mouse embryonic stem cells through the down-regulation of cell-matrix tractions. We therefore investigated whether soft substrates promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. After retroviral infection with five factors, Oct3/4, Klf4, Sox2, Lin28 and Nanog, mouse embryonic fibroblasts (MEFs) were cultured on several artificial substrates of varying elasticity and examined for the expression of pluripotency genes. When MEFs were cultured on soft (<0.1kPa) polyacrylamide gels coated with gelatin, the expressions of Nanog and Oct3/4 genes were higher than in cells cultured on rigid plastic dishes (∼10(6)kPa). The same result was obtained at higher elasticity (0.5kPa) for adult human dermal fibroblasts (HDFa). We also examined whether reprogramming could be enhanced on soft substrates without exogenous gene introduction, finding that cells cultured on soft substrates in the presence of chemicals known to promote cell reprogramming exhibited up-regulated stem cell markers. These results suggest that controlling the substrate stiffness can enhance the initiation of cell reprogramming, which may lead to effective and reproducible iPS cell production.

Reversine promotes porcine muscle derived stem cells (PMDSCs) differentiation into female germ‐like cells

A small molecular chemical-Reversine has been shown to promote cell reprogramming and induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, and then differentiate into other cell types within mesodermal lineages as well as neuroectodermal. However, the possibilities of these cells to give rise to germ cell lineages have not been examined. The objective of the current study was to detect the effect of Reversine on PMDSCs differentiation into germ cells. PMDSCs from fetal porcine skeletal muscle and their potential of differentiation into germ cells in vitro were investigated. The phenotype, proliferation potential, characteristic markers of the first adhesion cells (pp1), and the purified 2 times cells (pp3) were analyzed by growth curve, FACS, and RT-PCR, respectively. Then, the purified cells were induced with 10% or 20% bovine follicular fluid (FF), the results showed that some of the induced pp3 cells were similar as porcine oocyte, and expressed germ cell and oocyte markers analyzed by semi-quantitative RT-PCR and immunofluorescent staining. Reversine clearly increased the potentiality of PMDSCs differentiation into large round germ-like cells in FF induction medium analyzed by morpholgogy, QRT-PCR and immunofluoresce. The BrdU labeled PMDSCs might differentiate into female germ-like cells in recipient's kidney capsule, which were positive for germ cell and meiotic markers (Dazl, Vasa, Figla, Stra8, Scp3) and oocyte markers (Zp2, Zp3). These findings provided an efficient model to study the mechanism of cell proliferation and germ cell differentiation in livestock promoted by Reversine.