Osteogenic Differentiation from Mouse Embryonic Stem Cells.

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. We have studied signaling pathways and their contributions to osteogenic differentiation. In addition to our previously published protocol where we recommended the addition of retinoic acid with later addition of dexamethasone to boost osteogenic lineage cells, here we describe an optimized protocol for osteogenic differentiation from R1 ESCs with suggestions for inhibition of Src activity.

c-Src kinase inhibits osteogenic differentiation via enhancing STAT1 stability.

The proto-oncogene Src is ubiquitously expressed and is involved in cellular differentiation. However, the role of Src in embryonic stem (ES) cell osteogenic differentiation is largely unknown. Using the small molecule inhibitor PP2, c-Src specific siRNAs, and tet-inducible lentiviral vectors overexpressing active c-Src, we delineated an inhibitory role of c-Src in osteogenic differentiation of mouse embryonic stem cells (mESCs) and mouse MC3T3-E1s preosteoblasts.

Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells.

Calreticulin, the major Ca buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis.

Calreticulin regulates a switch between osteoblast and chondrocyte lineages derived from murine embryonic stem cells.

Calreticulin is a highly conserved, ubiquitous Ca-buffering protein in the endoplasmic reticulum that controls transcriptional activity of various developmental programs and also of embryonic stem cell (ESC) differentiation. Calreticulin activates calcineurin, which dephosphorylates and induces the nuclear import of the osteogenic transcription regulator nuclear factor of activated T cells 1 (NFATC1). We investigated whether calreticulin controls a switch between osteogenesis and chondrogenesis in mouse ESCs through NFATC1.

Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells.

Calreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells.

Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro.

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. One such lineage is osteoblasts, which are responsible for forming bone tissue in the body. There are many osteogenic differentiation protocols in the literature utilizing different soluble factors.

Osteogenic Differentiation from Embryonic Stem Cells.

Embryonic stem (ES) cells have been widely studied due to their pluripotency and their potential of self-renewal. Murine ES cells are useful in investigating the molecular pathways underlying their differentiation to various mature cell types in the body. This chapter describes the maintenance of murine ES cells in culture and a routine ES cell osteogenic differentiation protocol utilized in our laboratory.

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1.

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression.

Both chondroinduction and proliferation account for growth of cartilage nodules in mouse limb bud cultures.

High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules.

Calreticulin induces dilated cardiomyopathy.

Background: Calreticulin, a Ca(2+)-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Expression of survivin in human oocytes and preimplantation embryos.

Objective: To determine whether [1] survivin is expressed in human oocytes and embryos; [2] embryos grown in vitro secrete survivin protein; and [3] survivin levels are correlated with embryo cleavage rates.

Design: Experimental.

Setting: University-affiliated IVF clinic.

Adaptive-weighted cubic B-spline using lookup tables for fast and efficient axial resampling of 3D confocal microscopy images.

Confocal laser scanning microscopy has become a most powerful tool to visualize and analyze the dynamic behavior of cellular molecules. Photobleaching of fluorochromes is a major problem with confocal image acquisition that will lead to intensity attenuation. Photobleaching effect can be reduced by optimizing the collection efficiency of the confocal image by fast z-scanning.

An automatic segmentation algorithm for 3D cell cluster splitting using volumetric confocal images.

With the rapid advance of three-dimensional (3D) confocal imaging technology, more and more 3D cellular images will be available. Segmentation of intact cells is a critical task in automated image analysis and quantification of cellular microscopic images. One of the major complications in the automatic segmentation of cellular images arises due to the fact that cells are often closely clustered.

Cytoskeletal disassembly and cell rounding promotes adipogenesis from ES cells.

Biomechanical signals such as cell shape and spreading play an important role in controlling stem cell commitment. Cell shape, adhesion and spreading are also affected by calreticulin, a multifunctional calcium-binding protein, which influences several cellular processes, including adipogenesis. Here we show that cytoskeletal disruption in mouse embryonic stem cells using cytochalasin D or nocodazole promotes adipogenesis.

Evidence for calreticulin attenuation of cardiac hypertrophy induced by pressure overload and soluble agonists.

While calreticulin has been shown to be critical for cardiac development, its role in cardiac pathology is unclear. Previous studies have shown the detrimental effects on the heart of sustained germline calreticulin overexpression, yet without calreticulin, the heart does not develop normally. Thus, carefully balanced calreticulin levels are required for the heart to develop and to function properly into adulthood.

Calreticulin and focal-contact-dependent adhesion.

Cell adhesion is regulated by a variety of Ca2+-regulated pathways that depend on Ca2+-binding proteins. One such protein is calreticulin, an ER-resident protein. Calreticulin signalling from within the ER can affect processes outside the ER, such as expression of several adhesion-related genes, most notably vinculin and fibronectin.

3D boundary extraction of confocal cellular images using higher order statistics.

In recent years, cell biologists have benefited greatly from using confocal microscopy to study intracellular organelles. For high-level image analysis, 3D boundary extraction of cell structure is a preliminary requisite in confocal cellular imaging. To detect the object boundaries, most investigators have used gradient/Laplacian operator as a principal tool.

Embryonic stem cell-derived cardiomyogenesis: a novel role for calreticulin as a regulator.

A role for calreticulin, an endoplasmic reticulum (ER)-resident, Ca(2+)-binding chaperone, has recently emerged in the context of cardiomyogenesis. We previously proposed calreticulin to be a novel cardiac fetal gene, because calreticulin knockout causes embryonic lethality in mice as a result of cardiac defects, it is transiently activated during heart development, and heart-targeted overexpression of constitutively active calcineurin in calreticulin-null mice rescues the lethal phenotype. Calreticulin affects Ca(2+) homeostasis and expression of adhesion-related genes.

Cell adhesion and spreading affect adipogenesis from embryonic stem cells: the role of calreticulin.

Calreticulin is an endoplasmic reticulum-resident multifunctional protein, which has been shown to influence numerous cellular processes, including cell adhesion. In this study, we characterized the adhesive properties of embryonic stem cells (ESCs) lacking calreticulin and showed that adipogenesis from ESCs is directly and reciprocally controlled by the adhesive status of a cell, which in turn is modulated by calreticulin. Calreticulin-deficient ESCs are not only highly adipogenic but also show elevated calmodulin/CaMKII signaling and poor adhesiveness compared with the wild-type ESCs.

Endoplasmic and sarcoplasmic reticulum in the heart.

The concept of the presence of sarcoplasmic reticulum (SR) membrane in the heart is widely accepted and has been considered merely to be a different name for the endoplasmic reticulum (ER) in muscle tissues. Cardiac SR membranes are specialized in the regulation of Ca(2+) transport and control of excitation-contraction coupling. By contrast, the ER is responsible for protein synthesis, modification, secretion, lipid and steroid synthesis, and modulation of Ca(2+) signaling.

Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum.

Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development.

Expression of endoplasmic reticulum chaperones in cardiac development.

To determine if cardiogenesis causes endoplasmic reticulum stress, we examined chaperone expression. Many cardiac pathologies cause activation of the fetal gene program, and we asked the reverse: could activation of the fetal gene program during development induce endoplasmic reticulum stress/chaperones? We found stress related chaperones were more abundant in embryonic compared to adult hearts, indicating endoplasmic reticulum stress during normal cardiac development. To determine the degree of stress, we investigated endoplasmic reticulum stress pathways during cardiogenesis.

Tamoxifen-inducible Cre-mediated calreticulin excision to study mouse embryonic stem cell differentiation.

Embryonic stem cells are useful to study the functional aspects of lineage commitment. In this study, we report that using the Cre/loxP system provides a useful tool for studying multifunctional proteins that are involved in stem cell differentiation, such as calreticulin. Calreticulin is a chaperone and a major calcium buffer of the endoplasmic reticulum and it functions during both adipogenesis and cardiomyogenesis.

Calreticulin inhibits commitment to adipocyte differentiation.

Calreticulin, an endoplasmic reticulum (ER) resident protein, affects many critical cellular functions, including protein folding and calcium homeostasis. Using embryonic stem cells and 3T3-L1 preadipocytes, we show that calreticulin modulates adipogenesis. We find that calreticulin-deficient cells show increased potency for adipogenesis when compared with wild-type or calreticulin-overexpressing cells.