Monitoring ER Ca by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca.

Direct measurements of luminal Ca with endo-lysosomal GFP-aequorin reveal functional IP receptors.

Endo-lysosomes are considered acidic Ca stores but direct measurements of luminal Ca within them are limited. Here we report that the Ca -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca dynamics in this compartment.

Using Fluorescent GAP Indicators to Monitor ER Ca.

The endoplasmic reticulum (ER) is the main reservoir of Ca of the cell. Accurate and quantitative measuring of Ca dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca indicators have been developed, including a family of fluorescent Ca indicators, dubbed GFP-Aequorin Proteins (GAPs).

In vitro and in vivo calibration of low affinity genetic Ca indicators.

Calcium is a universal intracellular messenger and proper Caconcentrations ([Ca]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca levels result in malfunction and disease. Selective intraorganellar Cameasurements are best achieved by using targeted genetically encoded Ca indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions.

Use of aequorin-based indicators for monitoring Ca in acidic organelles.

Over the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca upon cell activation. Hence, reliable recording of Ca dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca indicators (GECIs) are valuable tools to monitor Ca in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs.

Long-term efficacy of autologous bone marrow mesenchymal stromal cells for treatment of knee osteoarthritis.

Knee osteoarthritis is the most prevalent joint disease and a frequent cause of pain, functional loss and disability. Conventional treatments have demonstrated only modest clinical benefits whereas cell-based therapies have shown encouraging results, but important details, such as dose needed, long-term evolution or number of applications required are scarcely known. Here we have reanalyzed results from two recent pilot trials with autologous bone marrow-derived mesenchymal stromal cells using the Huskisson plot to enhance quantification of efficacy and comparability.

Mandibular bone regeneration with autologous adipose-derived mesenchymal stem cells and coralline hydroxyapatite: experimental study in rats.

The purpose of this paper was to investigate the bone regeneration effect of autologous adipose tissue mesenchymal stem cells (ATMSC) in a small animal model. Twelve Wistar rats were given bilateral critical-size defects in the mandible. The defects were filled with coralline hydroxyapatite alone or combined with autologous undifferentiated ATMSC obtained from the dorsal fat pad.

Imaging of Endoplasmic Reticulum Ca in the Intact Pituitary Gland of Transgenic Mice Expressing a Low Affinity Ca Indicator.

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca signals ([Ca]), which can be generated either by Ca entry through the plasma membrane and/or by Ca release from the endoplasmic reticulum (ER). In addition, Ca entry signals can eventually be amplified by ER release calcium-induced calcium release (CICR).

Bone regeneration with autologous adipose-derived mesenchymal stem cells: A reliable experimental model in rats.

The adult mesenchymal stem cell (MSC) has been proposed to be the definitive tool in regenerative medicine due to its multi-differentiation potential and expansion capacity . The use of MSCs on bone regeneration has been assessed in several studies, obtaining promising results. However, the endless combinations that can be tested and the heterogeneity in the experimental conditions become a drawback when comparing results between authors.

Direct monitoring of ER Ca dynamics reveals that Ca entry induces ER-Ca release in astrocytes.

Excitability in astroglia is controlled by Ca fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsPR) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca-gated Ca release channels is less explored in astroglia.

Sarcoplasmic reticulum Ca decreases with age and correlates with the decline in muscle function in .

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca concentration ([Ca]) is at the origin of this loss of Ca homeostasis. We engineered to express the Ca indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca] [Ca] fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca] was <400 µM.

Spanish Cell Therapy Network (TerCel): 15 years of successful collaborative translational research.

In the current article we summarize the 15-year experience of the Spanish Cell Therapy Network (TerCel), a successful collaborative public initiative funded by the Spanish government for the support of nationwide translational research in this important area. Thirty-two research groups organized in three programs devoted to cardiovascular, neurodegenerative and immune-inflammatory diseases, respectively, currently form the network. Each program has three working packages focused on basic science, pre-clinical studies and clinical application.

CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons.

Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus.

A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency.

Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca levels. Caffeine sensitizes RyR to Ca and promotes ER Ca release at basal cytosolic Ca levels.

Therapeutic potential of allogeneic mesenchymal stromal cells transplantation for lupus nephritis.

Animal and human studies have suggested the potential of mesenchymal stromal cells (MSCs) to treat systemic lupus erythematosus (SLE). Here, we present the results of compassionate MSC treatments for three SLE patients to provide the proof of concept for a randomized and controlled clinical trial. Three patients of different ethnicities who suffer from chronic SLE, and who presented with class IV active proliferative nephritis confirmed by biopsy, were treated with allogeneic MSCs from healthy donors.

Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain.

Repair of maxillary cystic bone defects with mesenchymal stem cells seeded on a cross-linked serum scaffold.

Unlabelled: Tissue engineering combining cross-linked serum scaffolds with bone-derived mesenchymal stem cells has displayed excellent results for repair of maxillofacial bone defects in animal models, but it had not been tested in humans yet. We present here a pilot clinical trial using autologous bone-derived mesenchymal stem cells (H-MSV) grown in a serum cross-linked scaffold (BioMax) for treatment of maxillary cysts in 9 patients. Cells obtained from alveolar bone were seeded in the BioMax scaffold prepared from autologous serum, expanded under GMP conditions, and subjected to osteogenic differentiation for 3-4 weeks before application.

Influence of HLA Matching on the Efficacy of Allogeneic Mesenchymal Stromal Cell Therapies for Osteoarthritis and Degenerative Disc Disease.

Background: The necessity for more effective therapies for chronic osteoarticular diseases has led to the development of treatments based on mesenchymal stem cells (MSCs), the natural precursors of musculoskeletal tissue. Treatments with autologous MSCs yielded excellent results, with nearly 70% improvement of pain and disability in osteoarthritis and degenerative disc disease. Using allogeneic MSCs is logistically more convenient and would widen the pool of eligible patients, but potential immune rejection should be considered.

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

The mitochondrial calcium uniporter complex is essential for calcium (Ca) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens.

A Microplate-Based Bioluminescence Assay of Mitochondrial Calcium Uptake.

Mitochondrial Ca homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca. Ca concentration in mitochondria ([Ca]) is low at rest (about 10 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation.

Using aequorin probes to measure Ca in intracellular organelles.

Aequorins are excellent tools for measuring intra-organellar Ca and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal.

Measuring Ca inside intracellular organelles with luminescent and fluorescent aequorin-based sensors.

GFP-Aequorin Protein (GAP) can be used to measure [Ca] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca measurements ex vivo and in vivo to explore animal models of health and disease.