SLC38A2 provides proline to fulfil unique synthetic demands arising during osteoblast differentiation and bone formation.

Cellular differentiation is associated with the acquisition of a unique protein signature which is essential to attain the ultimate cellular function and activity of the differentiated cell. This is predicted to result in unique biosynthetic demands that arise during differentiation. Using a bioinformatic approach, we discovered osteoblast differentiation is associated with increased demand for the amino acid proline. When compared to other differentiated cells, osteoblast-associated proteins including RUNX2, OSX, OCN and COL1A1 are significantly enriched in proline. Using a genetic and metabolomic approach, we demonstrate that the neutral amino acid transporter SLC38A2 acts cell autonomously to provide proline to facilitate the efficient synthesis of proline-rich osteoblast proteins. Genetic ablation of SLC38A2 in osteoblasts limits both osteoblast differentiation and bone formation in mice. Mechanistically, proline is primarily incorporated into nascent protein with little metabolism observed. Collectively, these data highlight a requirement for proline in fulfilling the unique biosynthetic requirements that arise during osteoblast differentiation and bone formation.

Synergistic Effects of Combined Nurr1 Overexpression and Natural Inducers on the More Efficient Production of Dopaminergic Neuron-Like Cells From Stem Cells

The development of dopaminergic (DA) neurons is a very complex process, and a combination of extrinsic and intrinsic factors involves their differentiation. Transcription factor, Nurr1 plays an essential role in the differentiation and maintenance of midbrain DA neurons. Nurr1-based therapies may restore DA function in Parkinson's disease (PD) by replacing damaged cells with differentiated cells derived from stem cells. Providing tissue-specific microenvironments such as brain extract can effectively induce dopaminergic gene expression in stem cells. The present study aimed to investigate the combined effects of Nurr1 gene overexpression and a neonatal rat brain extract (NRBE) induction on dopaminergic differentiation of P19 stem cells. In order to neural differentiation induction, stably Nurr1-transfected cells were treated with 100 μg/ml of NRBE. The differentiation potential of the cells was then evaluated during a period of 1–3 weeks via various methods. The initial evaluation of the cells by direct observation under a light microscope and cresyl violet specific staining, confirmed neuron-like morphology in the differentiated cells. In addition, different molecular and cellular techniques, including real-time PCR, immunofluorescence, and flow cytometry, demonstrated that the treated cells expressed pan-neuronal and dopaminergic markers. In all experimental groups, neuronal phenotype with dopaminergic neuron-like cells characteristics mainly appeared in the second week of the differentiation protocol. Overall, the results of the present study revealed for the first time the synergistic effects of Nurr1 gene overexpression and possible soluble factors that existed in NRBE on the differentiation of P19 stem cells into dopaminergic neuron-like cells.

Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

Formation of Benign Tumours by Stem Cell Misregulation

Our tissues usually have just the right number of cells to optimally fulfil their function. Not enough cells within a tissue can lead to dysfunction, while too many cells result in a tumour. Yet, how this homeostatic balance is maintained remains poorly defined. Most differentiated cells within tissues have a finite lifespan and need to be replaced at a corresponding pace to maintain tissue homeostasis. These new differentiated cells are generated by proliferation of the stem/progenitor cells that serve the tissue. Work in simple invertebrates clearly suggests stem cells respond to at least two types of signals: niche signaling and growth factors. Niche signals promote the undifferentiated state by preventing differentiation, and thus allow for stem cell self-renewal. Growth factor sources comprise a systemic input reflecting the animal’s nutritional status, and a localized, homeostatic feedback from the tissue that the stem cells serve. That homeostatic signal couples stem cell proliferation rates to the tissue’s need for new differentiated cells. Evidence from simple organisms suggests two types of benign tumours can arise from deregulation of either niche or homeostatic signaling. Namely, constitutive niche signaling promotes the formation of undifferentiated “stem cell” tumours, while defective homeostatic signaling leads to the formation of differentiated tumours. We propose that these principles may be conserved and underlie benign tumour formation in humans, while benign tumours can evolve into cancer.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αβT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Transdifferentiation Meets Next-generation Biotechnologies

Transdifferentiation is the process of converting terminally differentiated cells to another cell type. Being less time-consuming and free from tumorigenesis, it is a promising alternative to directed differentiation, which provides cell sources for tissue regeneration therapy and disease modeling. In the past decades, transdifferentiation was found to happen within or across the cell lineages, being induced by overexpression of key transcription factors, chemical cocktail treatments, etc. Implementing next-generation biotechnologies, such as genome editing tools and scRNA-seq, improves current protocols and has the potential to facilitate discovery in new pathways of transdifferentiation, which will accelerate its application in clinical use.

The Interaction among Microbiota, Epigenetic Regulation, and Air Pollutants in Disease Prevention

Environmental pollutants can influence microbiota variety, with important implications for the general wellbeing of organisms. In subjects at high-risk of cancer, gut, and lung microbiota are distinct from those of low-risk subjects, and disease progression is associated with microbiota alterations. As with many inflammatory diseases, it is the combination of specific host and environmental factors in certain individuals that provokes disease outcomes. The microbiota metabolites influence activity of epigenetic enzymes. The knowledge of the mechanisms of action of environmental pollution now includes not only the alteration of the gut microbiota but also the interaction between different human microbiota niches such as the lung–gut axis. The epigenetic regulations can reprogram differentiated cells in response to environmental changes. The microbiota can play a major role in the progression and suppression of several epigenetic diseases. Accordingly, the maintenance of a balanced microbiota by monitoring the environmental stimuli provides a novel preventive approach for disease prevention. Metagenomics technologies can be utilized to establish new mitigation approaches for diseases induced by polluted environments. The purpose of this review is to examine the effects of particulate matter exposure on the progression of disease outcomes as related to the alterations of gut and lung microbial communities and consequent epigenetic modifications.

Unveiling Metabolic Vulnerability and Plasticity of Human Osteosarcoma Stem and Differentiated Cells to Improve Cancer Therapy

Defining the metabolic phenotypes of cancer-initiating cells or cancer stem cells and of their differentiated counterparts might provide fundamental knowledge for improving or developing more effective therapies. In this context we extensively characterized the metabolic profiles of two osteosarcoma-derived cell lines, the 3AB-OS cancer stem cells and the parental MG-63 cells. To this aim Seahorse methodology-based metabolic flux analysis under a variety of conditions complemented with real time monitoring of cell growth by impedentiometric technique and confocal imaging were carried out. The results attained by selective substrate deprivation or metabolic pathway inhibition clearly show reliance of 3AB-OS on glycolysis and of MG-63 on glutamine oxidation. Treatment of the osteosarcoma cell lines with cisplatin resulted in additive inhibitory effects in MG-63 cells depleted of glutamine whereas it antagonized under selective withdrawal of glucose in 3AB-OS cells thereby manifesting a paradoxical pro-survival, cell-cycle arrest in S phase and antioxidant outcome. All together the results of this study highlight that the efficacy of specific metabolite starvation combined with chemotherapeutic drugs depends on the cancer compartment and suggest cautions in using it as a generalizable curative strategy.

Resetting of the 24-nt siRNA landscape in rice zygotes

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells - the egg and sperm, which in plants have radically different siRNA transcriptomes from each other, and from multicellular embryos. Due to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Since siRNAs serve as both regulators and outputs of the epigenome, we performed here the successful characterization of small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibited extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation was accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ~70% of the zygote siRNA loci did not overlap any egg cell siRNA loci. Newly-detected siRNA loci in zygote are gene proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci which are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci were associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.