Regulation Of Stem Cell Differentiation Research Articles

Enhancer of polycomb coordinates multiple signaling pathways to promote both cyst and germline stem cell differentiation in the Drosophila adult testis.

Stem cells reside in a particular microenvironment known as a niche. The interaction between extrinsic cues originating from the niche and intrinsic factors in stem cells determines their identity and activity. Maintenance of stem cell identity and stem cell self-renewal are known to be controlled by chromatin factors. Herein, we use the Drosophila adult testis which has two adult stem cell lineages, the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage, to study how chromatin factors regulate stem cell differentiation. We find that the chromatin factor Enhancer of Polycomb [E(Pc)] acts in the CySC lineage to negatively control transcription of genes associated with multiple signaling pathways, including JAK-STAT and EGF, to promote cellular differentiation in the CySC lineage. E(Pc) also has a non-cell-autonomous role in regulating GSC lineage differentiation. When E(Pc) is specifically inactivated in the CySC lineage, defects occur in both germ cell differentiation and maintenance of germline identity. Furthermore, compromising Tip60 histone acetyltransferase activity in the CySC lineage recapitulates loss-of-function phenotypes of E(Pc), suggesting that Tip60 and E(Pc) act together, consistent with published biochemical data. In summary, our results demonstrate that E(Pc) plays a central role in coordinating differentiation between the two adult stem cell lineages in Drosophila testes.

Praja1 E3 ubiquitin ligase promotes skeletal myogenesis through degradation of EZH2 upon p38\u03b1 activation

Polycomb proteins are critical chromatin modifiers that regulate stem cell differentiation via transcriptional repression. In skeletal muscle progenitors Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), contributes to maintain the chromatin of muscle genes in a repressive conformation, whereas its down-regulation allows the progression through the myogenic programme. Here, we show that p38α kinase promotes EZH2 degradation in differentiating muscle cells through phosphorylation of threonine 372. Biochemical and genetic evidence demonstrates that the MYOD-induced E3 ubiquitin ligase Praja1 (PJA1) is involved in regulating EZH2 levels upon p38α activation. EZH2 premature degradation in proliferating myoblasts is prevented by low levels of PJA1, its cytoplasmic localization and the lower activity towards unphosphorylated EZH2. Our results indicate that signal-dependent degradation of EZH2 is a prerequisite for satellite cells differentiation and identify PJA1 as a new player in the epigenetic control of muscle gene expression.

Human Long Noncoding RNA Regulation of Stem Cell Potency and Differentiation.

Because of their capability of differentiation into lineage-specific cells, stem cells are an attractive therapeutic modality in regenerative medicine. To develop an effective stem cell-based therapeutic strategy with predictable results, deeper understanding of the underlying molecular mechanisms of stem cell differentiation and/or pluripotency maintenance is required. Thus, reviewing the key factors involved in the transcriptional and epigenetic regulation of stem cell differentiation and maintenance is important. Accumulating data indicate that long noncoding RNAs (lncRNAs) mediate numerous biological processes, including stem cell differentiation and maintenance. Here, we review recent findings on the human lncRNA regulation of stem cell potency and differentiation. Although the clinical implication of these lncRNAs is only beginning to be elucidated, it is anticipated that lncRNAs will become important therapeutic targets in the near future.

Preparation of Cell-Derived Decellularized Matrices Mimicking Native ECM During the Osteogenesis and Adipogenesis of Mesenchymal Stem Cells.

The extracellular matrix (ECM) is an important extracellular microenvironmental factor that regulates stem cell differentiation. The ECM is remodeled according to stem cell differentiation progression to precisely regulate the differentiation. Thus, it is expected that the matrices mimicking native ECM surrounding differentiating cells at each differentiation stage provide a favorable microenvironment to promote stem cell differentiation. However, it is difficult to prepare matrices mimicking native ECM using chemical methods because the ECM has a complicated composition. The decellularization technique is useful to prepare such matrices. In this chapter, we described the protocol to prepare matrices mimicking native ECM surrounding cells that are differentiating from mesenchymal stem cells to either osteoblasts or adipocytes via stem cell differentiation culture and a detergent- and nuclease-based decellularization technique.

Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.

Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes. Oil red O uptake into fat droplets and adiponectin production were used as biomarkers of adipogenesis. Positive peptides were formulated in either collagen I or hyaluronan (Orthovisc) gels then assessed for their adipogenic potential in vivo following injection into dorsal rat skin and mammary fat pads. Fat content was quantified and characterized using micro CT imaging, morphometry, histology, RT-PCR and ELISA analyses of adipogenic gene expression. Injection of screened peptides increased dorsal back subcutaneous fat pad area (208.3 ± 10.4 mm2versus control 84.11 ± 4.2 mm2; p < 0.05) and mammary fat pad size (45 ± 11 mg above control background, p = 0.002) in female rats. This effect lasted >5 weeks as detected by micro CT imaging and perilipin 1 mRNA expression. RHAMM expression suppresses while blocking peptides promote expression of PPARγ, C/EBP and their target genes. Blocking RHAMM function by peptide injection or topical application is a novel and minimally invasive method for potentially promoting subcutaneous adipogenesis in lipodystrophic diseases and a complementary tool to subcutaneous fat augmentation techniques.

Current perspectives in Set7 mediated stem cell differentiation.

Set7 is a key regulatory enzyme involved in the methylation of lysine residues of histone and non-histone proteins. This lysine methyltransferase is induced during stem cell differentiation and regulates lineage specific gene transcription and cell fate. In this article we discuss recent experimental evidence identifying regulatory targets under the control of Set7 as well as emerging evidence of regulation in stem cell differentiation. Furthermore, we discuss the function of non-coding RNAs regulated by Set7 implicated in cell plasticity.

Perlecan Delineates Stem Cell Niches in Human Foetal Hip, Knee and Elbow Cartilage Rudiments and Has Potential Roles in the Regulation of Stem Cell Differentiation

Perlecan Delineates Stem Cell Niches in Human Foetal Hip, Knee and Elbow Cartilage Rudiments and Has Potential Roles in the Regulation of Stem Cell Differentiation

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation.

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms.

Stem Cell-Derived Exosomes, Autophagy, Extracellular Matrix Turnover, and miRNAs in Cardiac Regeneration during Stem Cell Therapy

Stem cell therapy (SCT) raises the hope for cardiac regeneration in ischemic hearts. However, underlying molecular mechanisms for repair of dead myocardium by SCT in the ischemic heart is poorly understood. Growing evidences suggest that cardiac matrix stiffness and differential expressions of miRNAs play a crucial role in stem cell survival and differentiation. However, their roles on transplanted stem cells, for myocardial repair of the ischemic heart, remain unclear. Transplanted stem cells may act in an autocrine and/or paracrine manner to regenerate the dead myocardium. Paracrine mediators such as stem cell-derived exosomes are emerging as a novel therapeutic strategy to overcome some of the limitations of SCT. These exosomes carry microRNAs (miRNAs) that may regulate stem cell differentiation into a specific lineage. MicroRNAs may also contribute to stiffness of surrounding matrix by regulating extracellular matrix (ECM) turnover. The survival of transplanted stem cell depends on its autophagic process that maintains cellular homeostasis. Therefore, exosomes, miRNAs, extracellular matrix turnover, and autophagy may have an integral role in improving the efficacy of SCT. This review elaborates the specific roles of these regulatory components on cardiac regeneration in the ischemic heart during SCT.

DNA N6-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs.

ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine (N6-mA), a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells (MSCs) was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBH1-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.

Cellular interactions regulate stem cell differentiation in tri-culture

ABSTRACTCurrently, the mechanism governing the regeneration of the soft tissue-to-bone interface, such as the transition between the anterior cruciate ligament (ACL) and bone, is not known. Focusing on the ACL-to-bone insertion, this study tests the novel hypothesis that interactions between cells from the ligament (fibroblasts) and bone (osteoblasts) initiate interface regeneration. Specifically, these heterotypic cell interactions direct the fibrochondrogenic differentiation of interface-relevant cell populations, defined here as ligament fibroblasts and bone marrow stromal cells (BMSC). The objective of this study is to examine the effects of heterotypic cellular interactions on BMSC or fibroblast growth and biosynthesis, as well as expression of fibrocartilage-relevant markers in tri-culture. The effects of cell–cell physical contact and paracrine interactions between fibroblasts and osteoblasts were also determined. It was found that, in tri-culture with fibroblasts and osteoblasts, BMSC exhibited greater fibrochondrogenic potential than ligament fibroblasts. The growth of BMSC decreased while proteoglycan production and TGF-β3 expression increased. Moreover, tri-culture regulated BMSC response via paracrine factors, and interestingly, fibroblast–osteoblast contact further promoted proteoglycan and TGF-β1 synthesis as well as induced SOX9 expression in BMSC. Collectively, the findings of this study suggest that fibroblast–osteoblast interactions play an important role in regulating the stem cell niche for fibrocartilage regeneration, and the mechanisms of these interactions are directed by paracrine factors and augmented with direct cell–cell contact.

Subcellular cell geometry on micropillars regulates stem cell differentiation.

While various material factors have been shown to influence cell behaviors, recent studies started to pay attention to the effects of some material cues on "subcellular" geometry of cells, such as self-deformation of cell nuclei. It is particularly interesting to examine whether a self deformation happens discontinuously like a first-order transition and whether subcellular geometry influences significantly the extent of stem cell differentiation. Herein we prepared a series of micropillar arrays of poly(lactide-co-glycolide) and discovered a first-order transition of nuclear shape as a function of micropillar height under the examined section area and interspacing of the pillars. The deformed state of the nuclei of mesenchymal stem cells (MSCs) was well maintained even after osteogenic or adipogenic induction for several days. The nuclear deformation on the micropillar arrays was accompanied with smaller projected areas of cells, but led to an enhanced osteogenesis and attenuated adipogenesis of the MSCs, which is different from the previously known relationship between morphology and differentiation of stem cells on flat substrates. Hence, the present study reveals that the geometry of cell nuclei may afford a new cue to regulate the lineage commitment of stem cells on the subcellular level.

Photoactivation of Noncovalently Assembled Peptide Ligands on Carbon Nanotubes Enables the Dynamic Regulation of Stem Cell Differentiation.

Stimuli-responsive hybrid materials that combine the dynamic nature self-assembled organic nanostructures, unique photophysical properties of inorganic materials, and molecular recognition capability of biopolymers can provide sophisticated nanoarchitectures with unprecedented functions. In this report, infrared (IR)-responsive self-assembled peptide-carbon nanotube (CNT) hybrids that enable the spatiotemporal control of bioactive ligand multivalency and subsequent human neural stem cell (hNSC) differentiation are reported. The switching between the ligand presented and hidden states was controlled via IR-induced photothermal heating of CNTs, followed by the shrinkage of the thermoresponsive dendrimers that exhibited lower critical solution temperature (LCST) behavior. The control of the ligand spacing via molecular coassembly and IR-triggered ligand presentation promoted the sequential events of integrin receptor clustering and the differentiation of hNSCs into electrophysiologically functional neurons. Therefore, the combination of our nanohybrid with biomaterial scaffolds may be able to further improve effectiveness, durability, and functionality of the nanohybrid systems for spatiotemporal control of stem cell differentiation. Moreover, these responsive hybrids with remote-controllable functions can be developed as therapeutics for the treatment of neuronal disorders and as materials for the smart control of cell function.

Downregulation of Nrf2 promotes autophagy-dependent osteoblastic differentiation of adipose-derived mesenchymal stem cells

Adipose derived stem cells (ADSCs) are an important source of stem cells for tissue repair and regeneration; therefore, understanding the mechanisms that regulate stem cell differentiation into a specific lineage is critical. The NF-E2-related factor 2 (Nrf2) pathway and autophagy promote cell survival in response to oxidative stress. However, the roles of Nrf2 and autophagy in bone metabolism under oxidative stress are controversial. Here, we explored the involvement of Nrf2 signaling and autophagy on the differentiation of ADSCs under conditions of oxidative stress. Exposure of ADSCs to H2O2 promoted reactive oxygen species (ROS) accumulation concomitant with the reduction of cell viability, upregulation of Nrf2, the induction of apoptosis and autophagy, and the promotion of osteogenesis. Suppression of autophagic activity at particular stages resulted in the activation of the Nrf2 pathway, whereas osteoblastic differentiation of ADSCs was inhibited upon ROS stimulation. Silencing of Nrf2 promoted autophagy and osteoblastic differentiation upon ROS stimulation in vitro, and this effect was confirmed in vivo in a mouse model, in which bone formation was enhanced in mice receiving Nrf2-knockdown ADSCs. Taken together, these findings indicate that a negative interaction between the Nrf2 pathway and autophagy may modulate oxidative stress-induced ADSC osteogenesis, and suggest that Nrf2 is a potential target to regulate the differentiation of ADSCs into a specific lineage.

Multidimensional nanomaterials for the control of stem cell fate.

Current stem cell therapy suffers low efficiency in giving rise to differentiated cell lineages, which can replace the original damaged cells. Nanomaterials, on the other hand, provide unique physical size, surface chemistry, conductivity, and topographical microenvironment to regulate stem cell differentiation through multidimensional approaches to facilitate gene delivery, cell–cell, and cell–ECM interactions. In this review, nanomaterials are demonstrated to work both alone and synergistically to guide selective stem cell differentiation. From three different nanotechnology families, three approaches are shown: (1) soluble microenvironmental factors; (2) insoluble physical microenvironment; and (3) nano-topographical features. As regenerative medicine is heavily invested in effective stem cell therapy, this review is inspired to generate discussions in the potential clinical applications of multi-dimensional nanomaterials.

Role of let-7b/Fzd4 axis in mitochondrial biogenesis through wnt signaling: In neonatal and adult megakaryocytes

BackgroundMegakaryocytes (MKs), a rare population of bone marrow cells, are responsible for the production of platelets. Sick neonates are predisposed to developing thrombocytopenia (platelet count <150×109/L) and neonates are affected by several megakaryocyte disorders as compared to adults. HypothesisMicroRNAs (miRNAs) have been shown to crucially involve in the regulation of stem-cell differentiation in normal as well as malignant hematopoiesis, but their role in regulation of biological differences between adult and neonatal megakaryopoiesis is unknown. MethodsTo study this, we cultured human cord blood (CB) and peripheral blood (PB) derived CD34+ cells in the presence of thrombopoietin for 14days and collected cultures expressing>90% CD41+ by flow cytometry and studied 88 miRNAs involved in stem cell development and differentiation. miRNA validation studies were performed in Dami cell line. ResultsOut of 88 miRNAs involved in stem cell development, let-7b was the only miRNA down regulated (∼10-fold) in neonates compared to adult-MKs. Let-7b has not been previously described in MKs, however reduced expression of let-7b was found in several human cancers, suggesting that it functions as a tumor suppressor. Our results showed the inhibitory effect of let-7b on wnt signaling pathway by regulating Fzd4 (frizzled family receptor 4) and thereby regulating proliferation as well as differentiation. Let-7b down regulation induced mitochondrial biogenesis and its markers PGC-1α and NRF1 during megakaryocyte development. ConclusionsOur findings for the first time unveil the novel role of let-7b/Fzd4 axis through wnt signaling by regulating mitochondrial biogenesis during megakaryocyte development.

Single-cell RNA-sequencing: The future of genome biology is now

ABSTRACTGenome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis.

Concise Review: Fate Determination of Stem Cells by Deubiquitinating Enzymes

Post-translational modification by ubiquitin molecules is a key regulatory process for stem cell fate determination. Ubiquitination and deubiquitination are the major cellular processes used to balance the protein turnover of several transcription factors that regulate stem cell differentiation. Deubiquitinating enzymes (DUBs), which facilitate the processing of ubiquitin, significantly influence stem cell fate choices. Specifically, DUBs play a critical regulatory role during development by directing the production of new specialized cells. This review focuses on the regulatory role of DUBs in various cellular processes, including stem cell pluripotency and differentiation, adult stem cell signaling, cellular reprogramming, spermatogenesis, and oogenesis. Specifically, the identification of interactions of DUBs with core transcription factors has provided new insight into the role of DUBs in regulating stem cell fate determination. Thus, DUBs have emerged as key pharmacologic targets in the search to develop highly specific agents to treat various illnesses. Stem Cells 2017;35:9-16.

Loss of CSL Unlocks a Hypoxic Response and Enhanced Tumor Growth Potential in Breast Cancer Cells.

SummaryNotch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL, and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1α protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1,750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a role for CSL in tumorigenesis and regulation of the cellular hypoxic response.

Sa1677 The Polycomb Histone Lysine Methyltransferase EZH2 Controls Interstitial Cell of Cajal Populations by Regulating Stem Cell Differentiation and Self-Renewal

Sa1677 The Polycomb Histone Lysine Methyltransferase EZH2 Controls Interstitial Cell of Cajal Populations by Regulating Stem Cell Differentiation and Self-Renewal