Conversion Of Mouse Embryonic Fibroblasts Research Articles

Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules.

Reprogramming of somatic cells into desired functional cell types by small molecules has vast potential for developing cell replacement therapy. Here, we developed a stepwise strategy to generate chemically induced neural crest cells (ciNCCs) and chemically induced corneal endothelial cells (ciCECs) from mouse fibroblasts using defined small molecules. The ciNCCs exhibited typical NCC features and could differentiate into ciCECs using another chemical combination in vitro. The resulting ciCECs showed consistent gene expression profiles and self-renewal capacity to those of primary CECs. Notably, these ciCECs could be cultured for as long as 30 passages and still retain the CEC features in defined medium. Transplantation of these ciCECs into an animal model reversed corneal opacity. Our chemical approach for direct reprogramming of mouse fibroblasts into ciNCCs and ciCECs provides an alternative cell source for regeneration of corneal endothelia and other tissues derived from neural crest.

SUN-589 MED1 Is a Lipogenesis Coactivator Required for Postnatal Adipose Tissue Expansion

Mediator is a multi-subunit transcription coactivator complex that controls gene activation by connecting enhancer-binding transcription factors (TFs) with RNA polymerase II (Pol II). The Med1 subunit of Mediator is required for PPARγ-stimulated conversion of mouse embryonic fibroblasts to adipocytes in culture. However, it has remained unclear whether MED1 is required for adipose tissue development in vivo. Using Med1 conditional knockout mice, here we report that MED1 is required for postnatal adipose tissue development/expansion, a process that involves dramatic induction of lipogenic enzymes such as SCD1. Mice with Myf5-Cre-mediated MED1 knockout in progenitor cells show normal embryonic development of brown adipose tissue (BAT) but become runts with reduced BAT and white adipose tissue (WAT) after birth. Furthermore, mice with Adipoq-Cre-mediated MED1 knockout in adipocytes show reduced accumulation of lipid droplets and impaired induction of SCD1 in BAT and WAT. These mice display severe lipodystrophy, cold intolerance, and insulin resistance. In culture, Med1 is dispensable for the induction of adipogenesis markers including PPARγ and C/EBPα in the early phase of preadipocyte differentiation. However, Med1 is required for SCD1 induction, lipogenesis and lipid accumulation in the late phase of adipogenesis. Our preliminary data suggest that MED1 controls the induction of SCD1 by facilitating the recruitment of Mediator and subsequently Pol II to active enhancers bound by the lipogenic TF ChREBP. Taken together, our findings indicate that MED1 is a lipogenesis coactivator required for postnatal adipose tissue expansion.

Cancer‐associated missense mutations enhance the pluripotency reprogramming activity of OCT4 and SOX17

The functional consequences of cancer-associated missense mutations are unclear for the majority of proteins. We have previously demonstrated that the activity of SOX and Pit-Oct-Unc (POU) family factors during pluripotency reprogramming can be switched and enhanced with rationally placed point mutations. Here, we interrogated cancer mutation databases and identified recurrently mutated positions at critical structural interfaces of the DNA-binding domains of paralogous SOX and POU family transcription factors. Using the conversion of mouse embryonic fibroblasts to induced pluripotent stem cells as functional readout, we identified several gain-of-function mutations that enhance pluripotency reprogramming by SOX2 and OCT4. Wild-type SOX17 cannot support reprogramming but the recurrent missense mutation SOX17-V118M is capable of inducing pluripotency. Furthermore, SOX17-V118M promotes oncogenic transformation, enhances thermostability and elevates cellular protein levels of SOX17. We conclude that the mutational profile of SOX and POU family factors in cancer can guide the design of high-performance reprogramming factors. Furthermore, we propose cellular reprogramming as a suitable assay to study the functional impact of cancer-associated mutations.

An isoflavone compound daidzein elicits myoblast differentiation and myotube growth

Preservation of muscle stem cell function is critical for muscle homeostasis and the regenerative capacity of stem cells declines with age, leading to muscle loss. Previously, daidzein, a natural isoflavone from Leguminosae, has been shown to prevent TNF-α-induced muscle atrophy. However, the molecular mechanisms of daidzein in these processes are currently unknown. In this study, we demonstrate a positive effect of black soybean extract (BS Ext) and daidzein on myoblast differentiation and myotube growth. Daidzein prevents dexamethasone-induced myotube atrophy through Akt activation. The promyogenic effect of BS Ext and daidzein is mediated through two promyogenic kinases, Akt and p38MAPK that in turn activates a key myogenic transcription factor MyoD. Additionally, BS Ext and daidzein promote myotube growth through Akt/mTOR/S6K pathway. Finally, daidzein augments MyoD-dependent myogenic conversion of mouse embryonic fibroblasts. Taken together, our data suggest that daidzein promotes myogenic differentiation and myotube hypertrophy through p38 and Akt/mTOR/S6K pathway.

Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes.

The insufficient ability of specialized cells such as neurons, cardiac myocytes, and epidermal cells to regenerate after tissue damage poses a great challenge to treat devastating injuries and ailments. Recent studies demonstrated that a diverse array of cell types can be directly derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or somatic cells by combinations of specific factors. The use of iPSCs and direct somatic cell fate conversion, or transdifferentiation, holds great promise for regenerative medicine as these techniques may circumvent obstacles related to immunological rejection and ethical considerations. However, producing iPSC-derived keratinocytes requires a lengthy two-step process of initially generating iPSCs and subsequently differentiating into skin cells, thereby elevating the risk of cellular damage accumulation and tumor formation. In this study, we describe the reprogramming of mouse embryonic fibroblasts into functional keratinocytes via the transient expression of pluripotency factors coupled with directed differentiation. The isolation of an iPSC intermediate is dispensable when using this method. Cells derived with this approach, termed induced keratinocytes (iKCs), morphologically resemble primary keratinocytes. Furthermore they express keratinocyte-specific markers, downregulate mesenchymal markers as well as the pluripotency factors Oct4, Sox2, and Klf4, and they show important functional characteristics of primary keratinocytes. iKCs can be further differentiated by high calcium administration in vitro and are capable of regenerating a fully stratified epidermis in vivo. Efficient conversion of somatic cells into keratinocytes could have important implications for studying genetic skin diseases and designing regenerative therapies to ameliorate devastating skin conditions.

Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells.

ABSTRACTNeural crest cells (NC cells) are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs) into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+) cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

Jmjd2C increases MyoD transcriptional activity through inhibiting G9a-dependent MyoD degradation

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

The Philosopher’s Stone of Reprogramming

A hierarchical transcriptional mechanism governs direct conversion of mouse embryonic fibroblasts to neurons.

Direct Reprogramming of Fibroblasts to Myocytes via Bacterial Injection of MyoD Protein

Forced exogenous gene expression has been well characterized as an effective method for directing both cellular differentiation and dedifferentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Protein-based techniques provide a safe alternative, but current protein delivery methods are quite limited by labor-intensive purification processes, low protein yield, and inefficient intracellular targeting. Such limitations may be overcome by using a naturally occurring bacterial protein injection system, called the type III secretion system (T3SS), which injects bacterial proteins directly into the eukaryotic cell cytoplasm. Using a genetically attenuated strain of Pseudomonas aeruginosa, we have previously described the ability of this system to easily deliver a high quantity of protein to both differentiated and pluripotent cells. MyoD is a key muscle regulatory factor, the overexpression of which is able to induce transdifferentiation of numerous cell types into functional myocytes. Here we demonstrate transient injection of MyoD protein by P. aeruginosa to be sufficient to induce myogenic conversion of mouse embryonic fibroblasts. In addition to clear morphological changes, muscle-specific gene expression has been observed both at mRNA and protein levels. These studies serve as a foundation for the bacterial delivery of transcription factors to efficiently modulate concentration-dependent and temporal activation of gene expression that directs cell fate without jeopardizing genomic integrity.

Glycogen Synthase Kinase 3β negatively regulates myogenic differentiation

Glycogen Synthase Kinase 3β (GSK-3β) has been implicated in the negative regulation of both cardiac and skeletal muscle hypertrophy. Recently we showed that pharmacological inactivation of GSK-3β is sufficient to stimulate myogenic differentiation. To further investigate the role of GSK-3β in this process, GSK-3β expression was suppressed in C2C12 myoblasts by RNA interference, which resulted in increased Muscle Creatine Kinase (MCK) activity after 3 days of differentiation. In addition, myogenic conversion of Mouse Embryonic Fibroblasts (MEF) by over-expression of the muscle transcription factor MyoD resulted in enhanced transactivation of a co-transfected Troponin-I promoter-reporter construct in GSK-3β-/- compared to WT MEFs. Furthermore, transient transfection of GSK-3β-/- MEFs with a GSK-3β expression construct reduced Troponin-I promoter transactivation, whereas co-transfection of a kinase inactive variant (K85R) of GSK-3β did not affect increased myogenic conversion of GSK-3β-/- MEFs. This implies that GSK-3β kinase activity is required for its negative regulatory role in myogenic differentiation. Therefore inactivation of GSK-3β can have a potential therapeutical role in prevention or treatment of muscle wasting. This abstract was sponsored by the Netherlands Asthma Foundation (320263).

Nuclear lamin A inhibits adipocyte differentiation: implications for Dunnigan-type familial partial lipodystrophy

Mutations in the LMNA gene encoding A-type lamins cause several diseases, including Emery-Dreifuss muscular dystrophy and Dunnigan-type familial partial lipodystrophy (FPLD). We analyzed differentiation of 3T3-L1 preadipocytes to adipocytes in cells overexpressing wild-type lamin A as well as lamin A with amino acid substitutions at position 482 that cause FPLD. We also examined adipogenic conversion of mouse embryonic fibroblasts lacking A-type lamins. Overexpression of both wild-type and mutant lamin A inhibited lipid accumulation, triglyceride synthesis and expression of adipogenic markers. This was associated with inhibition of expression of peroxisome-proliferator-activated receptor gamma 2 (PPARgamma2) and Glut4. In contrast, embryonic fibroblasts lacking A-type lamins accumulated more intracellular lipid and exhibited elevated de novo triglyceride synthesis compared with wild-type fibroblasts. They also had increased basal phosphorylation of AKT1, a mediator of insulin signaling. We conclude that A-type lamins act as inhibitors of adipocyte differentiation, possibly by affecting PPARgamma2 and insulin signaling.