Conversion Of Mouse Fibroblasts Research Articles

Abstract We033: Heart-Specific Histone Methyltransferase SMYD1 Promotes Cardiac Maturation in the Direct Conversion of Human Fibroblasts into Cardiomyocytes.

Introduction: Direct cardiac reprogramming emerges as a promising strategy for in situ conversion of fibroblasts into induced cardiomyocyte-like cells (iCMs). While the conversion of mouse fibroblasts into functional, beating iCMs has been achieved efficiently, the reprogramming of human iCMs remains challenging, particularly their maturation with existing transcription factor combinations. Given the pivotal role of epigenetic regulation in this process, our study sought to leverage heart-specific epigenetic factors alongside cardiac-enriched transcription factors to enhance cardiac maturation, thereby yielding more reliable human iCMs. Result: Through analyzing the proteomic data across various human tissues, we found that while many epigenetic factors are broadly expressed, SMYD1 is uniquely heart-specific. To determine its involvement in reprogramming, we overexpressed SMYD1 and evaluated the reprogramming efficiency and iCM quality via flow cytometry, immunofluorescence staining, and transcriptome analysis. Notably, we observed an increase in iCMs expressing sarcomere markers, such as α-myosin heavy chain and α-actinin, as well as the ventricular specific myosin-light-chain 2v in SMYD1-treated group. Gene set enrichment analysis of RNA-sequencing data further indicated that SMYD1-induced iCMs exhibit ventricular gene signatures. Additionally, we discovered moderate activation of SMYD1 in mature iCMs generated with MEF2C, GATA4, TBX5, and TBX20. TBX20 is the key factor that leads to the co-binding of MEF2C, TBX5, and TBX20 on the SMYD1 promoter region and increases active histone marks, H3K27ac and H3K4me1. Rescue assay and transcriptomic comparison confirmed SMYD1 as the primary downstream target of TBX20 in promoting iCM functional maturation. Mechanistically, SMYD1 directly interacts with MEF2C and GATA4 to modify histone marks on cardiac regulatory regions. Conclusion: Our study provides comprehensive phenotypic and molecular evidence of SMYD1’s role as a cardiac-specific regulator during direct cardiac reprogramming. By interacting with key reprogramming factors and rewriting histone marks, SMYD1 significantly advances the maturation of human iCMs towards a ventricular subtype identity.

Sox10 Functions as an Inducer of the Direct Conversion of Keratinocytes Into Neural Crest Cells.

Neural crest cells (NCCs) are highly migratory multipotent cells that play critical roles in embryogenesis. The generation of NCCs is controlled by various transcription factors (TFs) that are regulated by each other and combine to form a regulatory network. We previously reported that the conversion of mouse fibroblasts into NCCs was achieved by the overexpression of only one TF, Sox10; therefore, Sox10 may be a powerful inducer of the conversion of NCCs. We herein investigated whether Sox10 functions in the direct conversion of other somatic cells into NCCs. Sox10 directly converted bone marrow-derived mesenchymal cells, but not keratinocytes, into P75+ NCCs. However, by the co-expression of four TFs (Snail1, Snail2, Twist1, and Tcfap2a) that are involved in NCC generation, but unable convert cells into NCCs, Sox10 converted keratinocytes into P75+ NCCs. P75+ NCCs mainly differentiated into glial cells, and to a lesser extent into neuronal cells. On the other hand, when Sox10 was expressed after the four TF expression, which mimicked the expression order in in vivo NCC generation, it converted keratinocytes into multipotent NCCs. These results demonstrate that Sox10 functions as an inducer of direct conversion into NCCs in cooperation with the TFs involved in NCC generation. The sequence of expression of the inducer and cooperative factors is important for the conversion of somatic cells into bona fide target cells.

Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach.

Transplantation of oligodendrocyte progenitor cells (OPCs) is a promising way for treating demyelinating diseases. However, generation of scalable and autologous sources of OPCs has proven difficult. We previously established a chemical condition M9 that could specifically initiate neural program in mouse embryonic fibroblasts. Here we found that M9 could induce the formation of colonies that undergo mesenchymal-to-epithelial transition at the early stage of reprogramming. These colonies may represent unstable and neural lineage-restricted intermediates that have not established a neural stem cell identity. By modulating the culture signaling recapitulating the principle of OPC development, these intermediate cells could be reprogrammed towards OPC fate. The chemical-induced OPC-like cells (ciOPLCs) resemble primary neural stem cell-derived OPCs in terms of their morphology, gene expression, and the ability of self-renewal. Upon differentiation, ciOPLCs could produce functional oligodendrocytes and myelinate the neuron axons in vitro, validating their OPC identity molecularly and functionally. Therefore, our study provides a non-integrating approach to OPC reprogramming that may ultimately provide an avenue to patient-specific cell-based or in situ regenerative therapy.

Conversion of mouse fibroblasts into cardiomyocyte-like cells using small molecule treatments

The possibility of controlling cell fates by overexpressing specific transcription factors has led to numerous studies in stem cell research. Small molecules can be used, instead of transcription factors, to induce the de-differentiation of somatic cells or to induce pluripotent cells (iPSCs). Here we reported that combinations of small molecules could convert mouse fibroblasts into cardiomyocyte-like cell without requiring transcription factor expression. Treatment with specific combinations of small molecules that are enhancer for iPSC induction converted mouse fibroblasts into spontaneously contracting, cardiac troponin T-positive, cardiomyocyte-like cells. We specifically identified five small molecules that can induce mouse fibroblasts to form these cardiomyocyte-like cells. These cells are similar to primary cardiomyocytes in terms of marker gene expression, epigenetic status of cardiac-specific genes, and subcellular structure. Our findings indicate that lineage conversion can be induced not only by transcription factors, but also by small molecules.

Conversion of mouse fibroblasts to sphere cells induced by AlbuMAXI-containing medium

The reprogramming of fibroblasts to pluripotent stem cells and the direct conversion of fibroblasts to functional neurons has been successfully manipulated by ectopic expression of defined factors. We demonstrate that mouse fibroblasts can be converted into sphere cells by detaching the fibroblast cells by protease and then using the AlbuMAX I-containing culture medium without genetic alteration. AlbuMAX I is a lipid-rich albumin. Albumin-associated lipids arachidonic acid (AA) and pluronic F-68 were responsible for this effect. The converted colonies were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1) staining. Global gene expression analysis indicated that the sphere cells were in an intermediate state compared with mES cells and MEF cells. The sphere cells were able to differentiate into tissues representing all three embryonic germ layers following retinoic acid treatment, and differentiated into smooth muscle cells following treatment with vascular endothelial growth factor (VEGF). The study presented a potential novel approach to transdifferentiate mouse fibroblast cells into other cell lineages mediated by AlbuMAX I-containing culture medium.

Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy

Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.

Role of gp91phoxHomolog Nox1 in Induction of Premalignant Spindle Phenotypes of HPV 16 E6/E7—Immortalized Human Keratinocytes

The NADPH oxidase (Nox) family of superoxide- and hydrogen peroxide—producing proteins has been recognized as important for signal transduction that regulates receptor-mediated functions, including cytoskeleton remodeling, cell proliferation, migration, differentiation, and cell death. Nox1 was the first of the Nox catalytic subunits to be cloned and shown to induce tumorigenic conversion of mouse fibroblasts. While Nox1 has been shown to be expressed in human colon and prostate cancers, however, limited studies have demonstrated the involvement of Nox1 in an early step of cell transformation. The aim of this review is to provide an overview on the role of Nox1 in cancer, as well as the contribution of our studies to demonstrate the involvement of Nox1 on neoplastic progression of human keratinocytes beyond the immortalization step. The generation of spindle phenotypes concomitant with anchorage-independent growth and invasiveness will be highlighted and discussed in relation to the possible role of Nox1 in epithelial-mesenchymal transition. Understanding these mechanisms may provide insight into Nox1 and redox signaling components as potential therapeutic targets to inhibit tumor progression.

Conversion of mouse fibroblasts into adipocytes.

Conversion of mouse fibroblasts into adipocytes.