Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ 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. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.

Sarcoplasmic reticulum Ca2+ decreases with age and correlates with the decline in muscle function in Drosophila.

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SRin vivo [Ca2+]SR fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca2+]SR was <400 µM. [Ca2+]SR results from the pump-leak steady state at the SR membrane. However, changes in expression of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump and of the ryanodine receptor leak were too modest to explain the large changes seen in [Ca2+]SR Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca2+]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca2+] with age in brain neurons.This article has an associated First Person interview with the first author of the paper.

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. We find that cell doses of 10, 40 and 100 million autologous cells per knee provided quite similar healing results and that much of the effect attained 1 year after cell application remained after 2 and 4 years. These results are encouraging because they indicate that, apart from safety and simplicity: (i) the beneficial effect is both significant and sizeable, (ii) it can be achieved with a single injection of cells, and (iii) the effect is perdurable for years.Trial registration: EudraCT 2009-017405-11; NCT02123368. Registered 25 April 2014-Prospectively registered, https://clinicaltrials.gov/ct2/show/NCT02123368?term=02123368&draw=2&rank=1.

Direct monitoring of ER Ca2+ dynamics reveals that Ca2+ entry induces ER-Ca2+ release in astrocytes.

Excitability in astroglia is controlled by Ca2+ fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsP3R) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca2+-gated Ca2+ release channels is less explored in astroglia. Here we address this process by monitoring Ca2+ dynamics directly in the cytosol and the ER of astroglial cells. Cultured astrocytes exhibited spontaneous and high-K-evoked cytosolic Ca2+ transients, both of them reversibly abolished by external Ca2+ removal, addition of plasma membrane channel blockers or ER Ca2+ depletion with SERCA inhibitors. Resting astrocyte [Ca2+]ER averaged 400 µM and maximal stimulation with ATP provoked a complete and reversible ER discharge. Direct monitoring of Ca2+ in the lumen of ER showed that high-K induced a Ca2+ release from the ER, and its amplitude was proportional to the [K]. Furthermore, by combining the low affinity GAP3 indicator targeted to the ER with the high affinity cytosolic Rhod-2, we simultaneously imaged ER- and cytosolic-Ca2+ signals, in astrocytes in culture and in situ. Plasma membrane Ca2+ entry triggered a fast ER Ca2+ release coordinated with an increase in cytosolic Ca2+. Thus, we identify a Ca2+-induced Ca2+-release (CICR) mechanism that is likely to participate in spontaneous astroglial oscillations, providing a graded amplification of the cytosolic Ca2+ signal.

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. TerCel has contributed during this period to boost the translational research in cell therapy in Spain, setting up a network of Good Manufacturing Practice-certified cell manufacturing facilities- and increasing the number of translational research projects, publications, patents and clinical trials of the participating groups, especially those in collaboration. TerCel pays particular attention to the public-private collaboration, which, for instance, has led to the development of the first allogeneic cell therapy product approved by the European Medicines Agency, Darvadstrocel. The current collaborative work is focused on the development of multicenter phase 2 and 3 trials that could translate these therapies to clinical practice for the benefit of patients.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca2+ 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 Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.

Measuring Ca2+ inside intracellular organelles with luminescent and fluorescent aequorin-based sensors.

GFP-Aequorin Protein (GAP) can be used to measure [Ca2+] 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 Ca2+. 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 Ca2+ measurements ex vivo and in vivo to explore animal models of health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles.

Genetically encoded calcium indicators allow monitoring subcellular Ca(2+) signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca(2+) sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg(2+), tunable Ca(2+) affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca(2+) indicators for organelles and becomes a valuable tool for in vivo Ca(2+) imaging applications.

The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

Differential calcium handling by the cis and trans regions of the Golgi apparatus.

High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo). Ca2+ homoeostasis in these compartments and in the endoplasmic reticulum (ER) has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We found that the cGo accumulates Ca2+ to high concentrations (150-300 µM) through the sarco plasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). The tGo, in turn, is divided into two subcompartments: tGo1 and tGo2. The subcompartment tGo1 contains 20% of the aequorin and has a high internal [Ca2+]; Ca2+ is accumulated in this subcompartment via the secretory pathway Ca2+-ATPase 1 (SPCA-1) at a very high affinity (K50=30 nM). The subcompartment tGo2 contains 80% of aequorin, has a lower [Ca2+] and no SPCA-1 activity; Ca2+ uptake happens through SERCA and is slower than in tGo1. The two tGo subcompartments, tGo1 and tGo2, are diffusionally isolated. Inositol trisphosphate mobilizes Ca2+ from the cGo and tGo2, but not from tGo1, whereas caffeine releases Ca2+ from all the Golgi regions, and nicotinic acid dinucleotide phosphate and cADP ribose from none.

The coupling of plasma membrane calcium entry to calcium uptake by endoplasmic reticulum and mitochondria.

Cross-talk between organelles and plasma membrane Ca(2+) channels is essential for modulation of the cytosolic Ca(2+) ([Ca(2+)]C) signals, but such modulation may differ among cells. In chromaffin cells Ca(2+) entry through voltage-operated channels induces calcium release from the endoplasmic reticulum (ER) that amplifies the signal. [Ca(2+)]C microdomains as high as 20-50 µm are sensed by subplasmalemmal mitochondria, which accumulate large amounts of Ca(2+) through the mitochondrial Ca(2+) uniporter (MCU). Mitochondria confine the high-Ca(2+) microdomains (HCMDs) to beneath the plasma membrane, where exocytosis of secretory vesicles happens. Cell core [Ca(2+)]C is much smaller (1-2 µm). By acting as a Ca(2+) sink, mitochondria stabilise the HCMD in space and time. In non-excitable HEK293 cells, activation of store-operated Ca(2+) entry, triggered by ER Ca(2+) emptying, also generated subplasmalemmal HCMDs, but, in this case, most of the Ca(2+) was taken up by the ER rather than by mitochondria. The smaller size of the [Ca(2+)]C peak in this case (about 2 µm) may contribute to this outcome, as the sarco-endoplasmic reticulum Ca(2+) ATPase has much higher Ca(2+) affinity than MCU. It is also possible that the relative positioning of organelles, channels and effectors, as well as cytoskeleton and accessory proteins plays an important role.

In vitro and in vivo calibration of low affinity genetic Ca2+ indicators.

Calcium is a universal intracellular messenger and proper Ca2+concentrations ([Ca2+]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca2+ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca2+ levels result in malfunction and disease. Selective intraorganellar Ca2+measurements are best achieved by using targeted genetically encoded Ca2+ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca2+] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca2+]ER/SR based on imaging of fluorescence after heating the tissue at 50-52 °C, since this value coincides with that obtained in the absence of Ca2+ (Rmin). Knowledge of the dynamic range (Rmax/Rmin) and the Ca2+-affinity (KD) of the indicator permits calculation of [Ca2+] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca2+ affinity green and red GECIs.

Using Fluorescent GAP Indicators to Monitor ER Ca2.

The endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+-binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence Basic Protocol 2: Monitoring ER Ca2+ Basic Protocol 3: Monitoring ER- and cytosolic-Ca2+ Support Protocol: Generation of a stable cell line expressing erGAP3.

Mitochondria and chromaffin cell function.

Chromaffin cells are an excellent model for stimulus-secretion coupling. Ca(2+) entry through plasma membrane voltage-operated Ca(2+) channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca(2+) signal. The endoplasmic reticulum amplifies the cytosolic Ca(2+) ([Ca(2+)](C)) signal by Ca(2+)-induced Ca(2+) release (CICR) and helps generation of microdomains with high [Ca(2+)](C) (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca(2+) from HCMD and stop progression of the Ca(2+) wave towards the cell core. On the other hand, the increase of [Ca(2+)] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca(2+)](C) decreases rapidly and mitochondria release the Ca(2+) accumulated in the matrix through the Na(+)/Ca(2+) exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus-secretion coupling while avoiding propagation of the Ca(2+) signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders.

Calcium homoeostasis modulator 1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers ER stress.

CALHM1 (calcium homoeostasis modulator 1), a membrane protein with similarity to NMDA (N-methyl-D-aspartate) receptor channels that localizes in the plasma membrane and the ER (endoplasmic reticulum) of neurons, has been shown to generate a plasma-membrane Ca(2+) conductance and has been proposed to influence Alzheimer's disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca(2+) handling in HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells by using targeted aequorins for selective monitorization of Ca(2+) transport by organelles. We find that CALHM1 increases Ca(2+) leak from the ER and, more importantly, reduces ER Ca(2+) uptake by decreasing both the transport capacity and the Ca(2+) affinity of SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase). As a result, the Ca(2+) content of the ER is drastically decreased. This reduction in the Ca(2+) content of the ER triggered the UPR (unfolded protein response) with induction of several ER stress markers, such as CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], ERdj4, GRP78 (glucose-regulated protein of 78 kDa) and XBP1 (X-box-binding protein 1). Thus CALHM1 might provide a relevant link between Ca(2+) homoeostasis disruption, ER stress and cell damage in the pathogenesis of neurodegenerative diseases.

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells.

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.

Cell and tissue therapy in regenerative medicine.

Cell therapy is one of the most promising future techniques in the medical arsenal for the repair of damaged or destroyed tissue. The diseases which cell therapy can target are very varied: Hormonal dysfunction, such as diabetes and growth hormone deficiency; neurodegenerative diseases, such as Parkinson's, Alzheimer's and Huntington's; and cardiovascular lesions, such as myocardial infarction, peripheral vascular ischaemia; as well as lesions in the cornea, skeletal muscle, skin, joints and bones etc. The objective of cell therapy is to restore the lost function rather than produce a new organ, which could cause duplicity and undesirable effects. Several resources of cells can be used to restore the damaged tissue, such as resident stem cells, multipotent adult progenitor cells or embryonic stem cells. Some cell therapies have been established and approved for clinical use, such as artificial skin derived from keratinocytes, derived from chondrocyte, cells of the corneal limbus or pancreatic islet transplantation. These therapies have had good results, although the scarcity of the starting material may represent a serious limitation. Other therapies under research, using pluripotent stem cells, have been modest so it is useful to review the protocols and try to improve the outcomes. In this chapter we will review the new advances made in this way.

The endoplasmic reticulum of dorsal root ganglion neurons contains functional TRPV1 channels.

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca(2+) channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1(ER)). We have studied the properties of TRPV1(ER) in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1(ER) with capsaicin or other vanilloids produced an increase of cytosolic Ca(2+) due to Ca(2+) release from the ER. The decrease of [Ca(2+)](ER) was directly revealed by an ER-targeted aequorin Ca(2+) probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1(ER) to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1(ER) was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca(2+) because the sensitivity to capsaicin was increased by loading the cells with the Ca(2+) chelator BAPTA. Decreasing [Ca(2+)](ER) did not affect the sensitivity of TRPV1(ER) to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Delta35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1(ER) for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatidylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1(ER) to agonists may be critical for neuron health, because otherwise Ca(2+) depletion of ER could lead to ER stress, unfolding protein response, and cell death.

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 ex vivo. 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. Moreover, it is very hard to find autologous studies using adipose-derived MSCs (AD-MSC) in rodents, which is the most used preclinical animal model. In this article an experimental model for basic bone tissue engineering research is described and justified, on which adult AD-MSCs are safely isolated from the rat dorsal interscapular fat pad, allowing ex vivo expansion and autogenous orthotopic reimplantation in a bilateral mandibular bone defect made in the same animal. This reliable and reproducible model provides a simple way to perform basic experimentation studies in a small animal model using autologous MSC for bone regeneration or cell therapy techniques prior to improve the research on large animal models.•Predictable and safe harvest of adipose-derived MSC. No need of animal sacrifice.•Allows for autologous studies with the most frequently used animal model: the rat. No need of allogeneic or human MSC use and, therefore, immunological concerns are avoided.•Bilateral mandibular critical size defect to allow direct control/experimental comparison.

Imaging of Endoplasmic Reticulum Ca2+ in the Intact Pituitary Gland of Transgenic Mice Expressing a Low Affinity Ca2+ 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 Ca2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER). In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.