Transdifferentiation of adipogenically differentiated cells into osteogenically or chondrogenically differentiated cells: Phenotype switching via dedifferentiation

Reprogramming is a new wave in cellular therapies to achieve the vital goals of regenerative medicine. Transdifferentiation, whereas the differentiated state of cells could be reprogrammed into other cell types, meaning cells are no more locked in their differentiated circle. Hence, cells of choice from abundant and easily available sources such as fibroblast and adipose tissue could be converted into cells of demand, to restore the diseased tissues. Before diverting this new approach into effective clinical use, transdifferentiation could not be simply overlooked, as it challenges the normal paradigms of biological laws, where mature cells transdifferentiate not only within same germ layers, but even across the lineage boundaries. How unipotent differentiated cells reprogram into another, and whether transdifferentiation proceeds via a direct cell-to-cell conversion or needs dedifferentiation. To address such questions, MSC were adipogenically differentiated followed by direct transdifferentiation, and subsequently examined by histology, immunohistochemistry, qPCR and single cell analysis. Direct cellular conversion of adipogenic lineage cells into osteogenic or chondrogenic resulted in mixed culture of both lineage cells (adipogenic and new acquiring osteogenic/chondrogenic phenotypes). On molecular level, such conversion was confirmed by significantly upregulated expression of PPARG, FABP4, SPP1 and RUNX2. Chondrogenic transdifferentiation was verified by significantly upregulated expression of PPARG, FABP4, SOX9 and COL2A1. Single cell analysis did not support the direct cell-to-cell conversion, rather described the involvement of dedifferentiation. Moreover, some differentiated single cells did not change their phenotype and were resistant to transdifferentiation, suggesting that differentiated cells behave differently during cellular conversion. An obvious characterization of differentiated cells could be helpful to understand the process of transdifferentiation.     

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.     

Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.Stem-cell-based therapies have gradually become a hot topic due to their high plasticity and self-renewing ability; clinical investigations with stem cell products in regenerative medicine are addressing a wide spectrum of conditions using a variety of stem cell types. These pluripotent cells including embryonic stem cells (ESCs), termed induced pluripotent stem cells (iPSCs), were first tested but inhibited in their clinical applications owing to ethical and tumorigenic problems. As a promising candidate for tissue regeneration, mesenchymal stem cells (MSCs) are fibroblast-like, with high plasticity and self-renewing ability and are able to develop into diverse cell lineages.¹ Among the MSCs from different adult tissues, placental-derived mesenchymal stem cells (PDMSCs), which reside in the fetal membranes of the term placenta, are easily accessible and less invasive. Their abundance, high proliferative potency, short population doubling time, strong immunosuppression and lack of ethical concerns make them indispensable in stem cell research and therapy.²Specific growth factors, cytokines and extracellular matrix components may have an important role in the determination of stem cell fate by switching from self-renewal to a differentiation stage. During lineage alteration to a specific tissue cell type, it was thought that MSCs progressively and developmentally became lineage restricted.³ Yet some evidences have suggested that when terminally differentiated mammalian cells are cultured under special conditions, they will revert to a more primitive phenotype.^{4, 5, 6} More recently, in the presence of human embryonic stem cell medium supplemented with valproic acid, stem cells derived from amniotic fluid could be fully reprogrammed to pluripotency without genetic manipulation.⁷ This process was defined as dedifferentiation and is considered as one of the mechanisms to reroute cell fate.⁸ Furthermore, a downregulation of lineage-specific genes and an upregulation of stem genes occurred immediately after initiation of the dedifferentiation process.⁸ This phase was characterized by repression of somatic genes via methylation, increased cell proliferation, altered morphology, signal transduction changes, reactivation of telomerase activity and the mesenchymal-to-epithelial transition (MET).^{9, 10} MET includes the loss of mesenchymal characteristics, such as motility, and the acquisition of epithelial characteristics such as cell polarity and the expression of cell adhesion molecules.¹¹In addition, bone marrow mesenchymal stem cells (BMMSCs) which were induced into osteocytes, chondrocytes and adipocytes, can dedifferentiate into a primitive population on the withdrawal of stimulating culture medium.^{12, 13, 14} This new population correlated with cell cycle arrest and associated genes, had enhanced cell survival, greater efficacy in differentiation and improved therapeutic potential in vitro and in vivo compared with uncommitted BMMSCs.^{15, 16} On the other hand, a number of studies showed enhanced mitochondrial biogenesis in various stem cell differentiation models including ESCs and iPSCs.^{17, 18} The immature mitochondrial phenotype in ESCs consists of fewer mitochondria, poorly developed cristae and a perinuclear location of mitochondria.^{19, 20} These characteristics have been regarded as potential markers of pluripotency in ESCs;²⁰ however, it has not been clearly established whether the morphology and the mitochondrial network is pluripotency dependent or stem cell specific. In addition, it has been suggested that mitochondrial dynamics and oxidative phosphorylation (OXPHOS) activity can influence each other during the biological process.²¹ Consequently, we suggest that the altered OXPHOS activity will accompany the differentiation and dedifferentiation processes.In the present study, we aimed to comprehensively analyze the transition in morphology, surface antigens, metabolism and multilineage potency during PDMSCs differentiation and dedifferentiation to clarify whether unrestricted lineage exists in differentiated PDMSCs. We showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The new population proliferated faster than unmanipulated PDMSCs, and could be differentiated into adipocytes, osteocytes and hepatocytes. Gene expression profiling showed a panel of genes with significantly up- or downregulated expression between adipogenically differentiated and dedifferentiated cells. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to regenerate, proliferate and differentiate during the differentiation, dedifferentiation and redifferentiation processes.^{22, 23} These observations indicated that the dedifferentiated PDMSCs were distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies.     

Methanolic extract of Citrullus colocynthis suppresses growth and proliferation of breast cancer cells through regulation of cell cycle

Breast cancer is a major cause of cancer related deaths in women worldwide. Available treatments pose serious limitations such as systemic toxicity, metastasis, tumor recurrence, off-target effects, and drug resistance. In recent years, phytochemicals such as secondary metabolites due to their effective anticancer potential at very low concentration have gained attention. Aim of the study was to evaluate anticancer potential of Citrullus colocynthis and its possible molecular targets on MCF-7, a human breast cancer cell line. Methanolic extract of leaves was prepared and fractionated by solvents (n-hexane, chloroform, ethyl acetate and n-butanol) with increasing polarity. Bioassays and gene expression regulation was conducted to evaluate the anticancer activity, proliferation rate and cell cycle regulation of breast cancer cells treated with extract and its fractions, separately. Results showed a significant anticancer activity of methanolic extract of C. colocynthis and two of its fractions prepared with chloroform and ethyl acetate. Bioassays depicted significant decrease in proliferation and growth potential along with cell cycle arrest of treated cells compared to control untreated cells. Expression regulation of genes further confirmed the cell cycle arrest through significant upregulation of cyclin-CDK inhibitors (p21 and p27) and cell cycle checkpoint regulators (HUS1, RAD1, ATM) followed by downregulation of downstream cell cycle progression genes (Cyclin A, Cyclin E, CDK2). It is concluded that C. colocynthis arrests cell cycle in human breast cancer cells through expression regulation of cyclin-CDK inhibitors and with further research can be proposed for therapeutic interventions.     

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells

In this study, a time-course comparison of human articular chondrocytes (HAC) and bone marrow-derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14-/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1-2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3-4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index.     

Gene expression during lens transdifferentiation from pigmented epithelial cells

Pigmented epithelial cells of chicken and human dedifferentiate in the medium containing phenylthiourea and testicular hyaluronidase, and then trans-differentiate into lens cells in vitro. To understand the molecular mechanisms of transdifferentiation, gene expression during lens transdifferentiation was analyzed. As the first step, pigment cell and lens specific genes were isolated and expression of these gene was analyzed by Northern blotting . These results clearly shown that lens transdifferentiation proceeds via neutral cell state in which both pigment and lens specific genes are repressed. Oncogene expression was also analyzed. An elevated expression of the c-myc gene was observed during dedifferentiation process. It is expected that elevated expression of c-myc gene might prevent the cells from entering the G0 phase and thus lead to dedifferentiated state.     

Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary

Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and β-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage.     

BTYNB,an inhibitor of RNA binding protein IGF2BP1 reduces proliferation and induces differentiation of leukemic cancer cells

Leukemia is a group of diseases characterized by altered growth and differentiation of lymphoid or myeloid progenitors of blood. The existence of specific clusters of cells with stemness-like characteristics like differentiation, self-renewal, detoxification, and resistance to apoptosis in Leukemia makes them difficult to treat. It was recently reported that an oncofetal RNA binding protein, insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), maintains leukemic stem cell properties. BTYNB is an inhibitor of IGF2BP1 that was shown to affect the biological functions of IGF2BP1 however, the effect of BTYNB in Leukemia is not properly established. In this study, we assessed the effect of BTYNB on leukemic cell differentiation and proliferation. We performed cell viability assay to assess the effect of BTYNB in leukemic cells. We then assessed cell morphology of the leukemic cells treated with BTYNB. Further, we conducted an apoptosis assay and cell cycle assay. We found the cell viability of leukemic cells was significantly decreased post treatment with BTYNBs. Further, a noticeable morphological change was observed in BTYNB treated leukemic cells. BTYNB treated leukemic cells showed increased cell death and cell cycle arrest at S-phase. Evidence from the upregulation of BAK and p21 further confirmed apoptosis and cycle arrest. The gene expression of differentiation genes such as CD11B, ZFPM1, and KLF5 were significantly upregulated in BTYNB treated leukemic cells, therefore, confirming cell differentiation. Collectively, our study showed inhibition of IGF2BP1 function using BTYNB promotes differentiation in leukemic cells.     

Coordinated regulation of the ribosome and proteasome by PRMT1 in the maintenance of neural stemness in cancer cells and neural stem cells

Previous studies suggested that cancer cells resemble neural stem/progenitor cells in regulatory network, tumorigenicity, and differentiation potential, and that neural stemness might represent the ground or basal state of differentiation and tumorigenicity. The neural ground state is reflected in the upregulation and enrichment of basic cell machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, and epigenetic factors, in cancers and in embryonic neural or neural stem cells. However, how these machineries are concertedly regulated is unclear. Here, we show that loss of neural stemness in cancer or neural stem cells via muscle-like differentiation or neuronal differentiation, respectively, caused downregulation of ribosome and proteasome components and major epigenetic factors, including PRMT1, EZH2, and LSD1. Furthermore, inhibition of PRMT1, an oncoprotein that is enriched in neural cells during embryogenesis, caused neuronal-like differentiation, downregulation of a similar set of proteins downregulated by differentiation, and alteration of subcellular distribution of ribosome and proteasome components. By contrast, PRMT1 overexpression led to an upregulation of these proteins. PRMT1 interacted with these components and protected them from degradation via recruitment of the deubiquitinase USP7, also known to promote cancer and enriched in embryonic neural cells, thereby maintaining a high level of epigenetic factors that maintain neural stemness, such as EZH2 and LSD1. Taken together, our data indicate that PRMT1 inhibition resulted in repression of cell tumorigenicity. We conclude that PRMT1 coordinates ribosome and proteasome activity to match the needs for high production and homeostasis of proteins that maintain stemness in cancer and neural stem cells.     

Dedifferentiation of human primary thyrocytes into multilineage progenitor cells without gene introduction

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (～0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases.     

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P < 0.01) change in expression levels were screened according to the screening standard (signal log ratio ≥1 or ≤?1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335 ± 0.0116 72 h after transfection. This value was significantly (P < 0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P < 0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.     

Differential response of epiblast stem cells to Nodal and Activin signalling: a paradigm of early endoderm development in the embryo

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.     

Downregulation of POLD4 in Calu6 cells results in G1-S blockage through suppression of the Akt-Skp2-p27 pathway

Previously, we have shown that downregulation of POLD4 in lung cancer cells delays progression through the G1-S cell cycle transition and leads to increased genomic instability. To date however, detailed molecular mechanisms have not been elucidated to explain how this occurs. In the present study, we found that reduction in POLD4 by siRNA knockdown promoted downregulation of both p-Akt Ser473 and Skp2 as well as upregulation of p27. Furthermore, these protein expression levels were rescued when siRNA-resistant POLD4 was ectopically expressed in the knockdown cells. These data suggest that the POLD4 downregulation is associated with impaired Akt-Skp2-p27 pathway in lung cancer.