Inhibition of EZH2 Promotes Human Embryonic Stem Cell Differentiation into Mesoderm by Reducing H3K27me3.

Mesoderm derived from human embryonic stem cells (hESCs) is a major source of the mesenchymal stem/stromal cells (MSCs) that can differentiate into osteoblasts and chondrocytes for tissue regeneration. While significant progress has been made in understanding of molecular mechanisms of hESC differentiation into mesodermal cells, little is known about epigenetic factors controlling hESC fate toward mesoderm and MSCs. Identifying potential epigenetic factors that control hESC differentiation will undoubtedly lead to advancements in regenerative medicine. Here, we conducted an epigenome-wide analysis of hESCs and MSCs and uncovered that EZH2 was enriched in hESCs and was downregulated significantly in MSCs. The specific EZH2 inhibitor GSK126 directed hESC differentiation toward mesoderm and generated more MSCs by reducing H3K27me3. Our results provide insights into epigenetic landscapes of hESCs and MSCs and suggest that inhibiting EZH2 promotes mesodermal differentiation of hESCs

The identification of markers of macrophage differentiation in PMA-stimulated THP-1 Cells and monocyte-derived macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells

Enhanced differentiation of human embryonic stem cells towards definitive endoderm on ultrahigh aspect ratio nanopillars

Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have highlighted that the properties of the physical environment, such as substrate stiffness, affect cellular behavior. Here, mass-produced, injection molded polycarbonate nanopillars are presented, where the surface mechanical properties, i.e., stiffness, can be controlled by the geometric design of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.0003). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of definitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure population compared to planar control. By contrast, further differentiation toward the pancreatic ­endoderm is less successful on “soft” pillars when compared to “stiff” pillars or control, indicating differential cues during the different stages of differentiation. To accompany the mechanical properties of the nanopillars, the concept of surface shear modulus is introduced to describe the characteristics of engineered elastic surfaces through micro or nanopatterning. This provides a framework whereby comparisons can be drawn between such materials and bulk elastomeric materials

The Effects of Cytokinin on the Transcriptional Regulation of PIN Expression in Arabidopsis thaliana

The processes of cell division and differentiation are critical to the development of any multicellular organism. During the formation of plant roots these processes take place at a region of the root tip called the meristem. Cytokinin and auxin are two plant growth hormones that influence this process. Although these two growth hormones are both necessary they also appear in many ways to have an antagonistic relationship. As meristematic root cells undergo differentiation they cease dividing. It has been proposed that the size of the root meristem and thus the overall rate of root growth are determined by the balance between the rate of cell division, determined by auxin, and the rate of cell differentiation, determined by cytokinin. One of the ways cytokinin may have an antagonistic influence on auxin regulation is by limiting auxin transport. Directed auxin transport from one part of a plant to another is controlled by a family of auxin efflux proteins called PIN proteins. To date, studies reported in the literate are inconsistent as to whether and how cytokinin influences PIN protein production at the transcriptional and/or translational level. This study was undertaken to explore the effect of cytokinin signaling on PIN gene expression using real-time quantitative PCR

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass.

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin α4β1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength

Anticipatory adjustments to being picked up in infancy

Anticipation of the actions of others is often used as a measure of action understanding in infancy. In contrast to studies of action understanding which set infants up as observers of actions directed elsewhere, in the present study we explored anticipatory postural adjustments made by infants to one of the most common adult actions directed to them - picking them up. We observed infant behavioural changes and recorded their postural shifts on a pressure mat in three phases: (i) a prior Chat phase, (ii) from the onset of Approach of the mother's arms, and (iii) from the onset of Contact. In Study 1, eighteen 3-month-old infants showed systematic global postural changes during Approach and Contact, but not during Chat. There was an increase in specific adjustments of the arms (widening or raising) and legs (stiffening and extending or tucking up) during Approach and a decrease in thrashing/general movements during Contact. Shifts in postural stability were evident immediately after onset of Approach and more slowly after Contact, with no regular shifts during Chat. In Study 2 we followed ten infants at 2, 3 and 4 months of age. Anticipatory behavioural adjustments during Approach were present at all ages, but with greater differentiation from a prior Chat phase only at 3 and 4 months. Global postural shifts were also more phase differentiated in older infants. Moreover, there was significantly greater gaze to the mother's hands during Approach at 4 months. Early anticipatory adjustments to being picked up suggest that infants' awareness of actions directed to the self may occur earlier than of those directed elsewhere, and thus enable infants' active participation in joint actions from early in life

Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli

Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffnesschondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process

Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor y (PPARy) in the Conversion of White-to-brown Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1

A Computational Model for Understanding Stem Cell, Trophectoderm and Endoderm Lineage Determination

Background: Recent studies have associated the transcription factors, Oct4, Sox2 and Nanog as parts of a self-regulating network which is responsible for maintaining embryonic stem cell properties: self renewal and pluripotency. In addition, mutual antagonism between two of these and other master regulators have been shown to regulate lineage determination. In particular, an excess of Cdx2 over Oct4 determines the trophectoderm lineage whereas an excess of Gata-6 over Nanog determines differentiation into the endoderm lineage. Also, under/over-expression studies of the master regulator Oct4 have revealed that some self-renewal/pluripotency as well as differentiation genes are expressed in a biphasic manner with respect to the concentration of Oct4. Methodology/Principal Findings: We construct a dynamical model of a minimalistic network, extracted from ChIP-on-chip and microarray data as well as literature studies. The model is based upon differential equations and makes two plausible assumptions; activation of Gata-6 by Oct4 and repression of Nanog by an Oct4–Gata-6 heterodimer. With these assumptions, the results of simulations successfully describe the biphasic behavior as well as lineage commitment. The model also predicts that reprogramming the network from a differentiated state, in particular the endoderm state, into a stem cell state, is best achieved by over-expressing Nanog, rather than by suppression of differentiation genes such as Gata-6. Conclusions: The computational model provides a mechanistic understanding of how different lineages arise from the dynamics of the underlying regulatory network. It provides a framework to explore strategies of reprogramming a cell from a differentiated state to a stem cell state through directed perturbations. Such an approach is highly relevant to regenerative medicine since it allows for a rapid search over the host of possibilities for reprogramming to a stem cell state