Roberta Squecco

Isolation and Characterization of Multipotent Progenitor Cells from the Bowman’s Capsule of Adult Human Kidneys

Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowmans capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell-specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24+CD133+ PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24+CD133+ PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24+CD133+ PEC but not of CD24-CD133- renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24+CD133+ PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases.

Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue

Adipose tissue is a dynamic endocrine organ with a central role in metabolism regulation. Functional differences in adipose tissue seem associated with the regional distribution of fat depots, in particular in subcutaneous and visceral omental pads. Here, we report for the first time the isolation of human adipose‐derived adult stem cells from visceral omental and subcutaneous fat (V‐ASCs and S‐ASCs, respectively) from the same subject. Immunophenotyping shows that plastic culturing selects homogeneous cell populations of V‐ASCs and S‐ASCs from the corresponding stromal vascular fractions (SVFs), sharing typical markers of mesenchymal stem cells. Electron microscopy and electrophysiological and real‐time RT‐PCR analyses confirm the mesenchymal stem nature of both V‐ASCs and S‐ASCs, while no significant differences in a limited pattern of cytokine/chemokine expression can be detected. Similar to S‐ASCs, V‐ASCs can differentiate in vitro toward adipogenic, osteogenic, chondrogenic, muscular, and neuronal lineages, as demonstrated by histochemical, immunofluorescence, real‐time RT‐PCR, and electrophysiological analyses, suggesting the multipotency of such adult stem cells. Our data demonstrate that both visceral and subcutaneous adipose tissues are a source of pluripotent stem cells with multigermline potential. However, the visceral rather than the subcutaneous ASC could represent a more appropriate in vitro cell model for investigating the molecular mechanisms implicated in the pathophysiology of metabolic disorders such as obesity.—Baglioni, S., Francalanci, M., Squecco, R., Lombardi, A., Cantini, G., Angeli, R., Gelmini, S., Guasti, D., Benvenuti, S., Annunziato, F., Bani, D., Liotta, F., Francini, F., Perigli, G., Serio, M., Luconi, M. Characterization of human adult stem‐cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J. 23, 3494–3505 (2009). www.fasebj.org

Functional Differences in Visceral and Subcutaneous Fat Pads Originate from Differences in the Adipose Stem Cell

Metabolic pathologies mainly originate from adipose tissue (AT) dysfunctions. AT differences are associated with fat-depot anatomic distribution in subcutaneous (SAT) and visceral omental (VAT) pads. We address the question whether the functional differences between the two compartments may be present early in the adipose stem cell (ASC) instead of being restricted to the mature adipocytes. Using a specific human ASC model, we evaluated proliferation/differentiation of ASC from abdominal SAT-(S-ASC) and VAT-(V-ASC) paired biopsies in parallel as well as the electrophysiological properties and functional activity of ASC and their in vitro-derived adipocytes. A dramatic difference in proliferation and adipogenic potential was observed between the two ASC populations, S-ASC having a growth rate and adipogenic potential significantly higher than V-ASC and giving rise to more functional and better organized adipocytes. To our knowledge, this is the first comprehensive electrophysiological analysis of ASC and derived-adipocytes, showing electrophysiological properties, such as membrane potential, capacitance and K+-current parameters which confirm the better functionality of S-ASC and their derived adipocytes. We document the greater ability of S-ASC-derived adipocytes to secrete adiponectin and their reduced susceptibility to lipolysis. These features may account for the metabolic differences observed between the SAT and VAT. Our findings suggest that VAT and SAT functional differences originate at the level of the adult ASC which maintains a memory of its fat pad of origin. Such stem cell differences may account for differential adipose depot susceptibility to the development of metabolic dysfunction and may represent a suitable target for specific therapeutic approaches.

Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation.

Transient receptor potential canonical (TRPC) channels provide cation and Ca2+ entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channels activity and stretch-induced Ca2+ influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

Cytoskeleton/stretch-activated ion channel interaction regulates myogenic differentiation of skeletal myoblasts.

In the present study, we investigated the functional interaction between stress fibers (SFs) and stretch‐activated channels (SACs) and its possible role in the regulation of myoblast differentiation induced by switch to differentiation culture in the presence or absence of sphingosine 1‐phosphate. It was found that there was a clear temporal correlation between SF formation and SAC activation in differentiating C2C12 myoblasts. Inhibition of actin polymerization with the specific Rho kinase inhibitor Y‐27632, significantly decreased SAC sensitivity in these cells, suggesting a role for Rho‐dependent actin remodeling in the regulation of the channel opening. The alteration of cytoskeletal/SAC functional correlation had also deleterious effects on myogenic differentiation of C2C12 cells as judged by combined confocal immunofluorescence, biochemical and electrophysiological analyses. Indeed, the treatment with Y‐27632 or with DHCB, an actin disrupting agent, inhibited the expression of the myogenic markers (myogenin and sarcomeric proteins) and myoblast‐myotube transition. The treatment with the channel blocker, GdCl3, also affected myogenesis in these cells. It impaired, in fact, myoblast phenotypic maturation (i.e., reduced the expression of α‐sarcomeric actin and skeletal myosin and the activity of creatine kinase) but did not modify promoter activity and protein expression levels of myogenin. The results of this study, together with being in agreement with the general idea that cytoskeletal remodeling is essential for muscle differentiation, describe a novel pathway whereby the formation of SFs and their contraction, generate a mechanical tension to the plasma membrane, activate SACs and trigger Ca2+‐dependent signals, thus influencing the phenotypic maturation of myoblasts. J. Cell. Physiol. 211: 296–306, 2007.

Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is abundantly present in the serum and mediates multiple biological responses. With the aim of extending our knowledge on the role played by S1P in the regulation of cytoskeletal reorganization, native as well as C2C12 myoblasts stably transfected with green fluorescent protein (GFP)-tagged α- and β-actin constructs were stimulated with S1P (1 μM) and observed under confocal and multiphoton microscopes. The addition of S1P induced the appearance of actin stress fibres and focal adhesions through Rho- and phospholipase D (PLD)-mediated pathways. The cytoskeletal response was dependent on the extracellular action of S1P through its specific surface receptors, since the intracellular delivery of the sphingolipid by microinjection was unable to modify the actin cytoskeletal assembly. Interestingly, it was revealed by whole-cell patch-clamp that S1P-induced stress fibre formation was associated with increased ion currents and conductance through stretch-activated channels (SACs), thereby suggesting a possible regulatory role for organized actin in channel sensitivity. Experiments aimed at stretching the plasma membrane of C2C12 cells, using the cantilever of an atomic force microscope, indicated that there was a Ca2+ influx through putative SACs. In conclusion, the present data suggest novel mechanisms of S1P signalling involving actin cytoskeletal reorganization and Ca2+ elevation through SACs that might influence myoblastic functions.

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells

Abstract.Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as α-smooth muscle actin (αSMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated αSMA. More importantly, S1P challenge was responsible for the functional appearance of Ca2+ currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P2 turned out to be critical for the pro-differentiating effect of S1P, while S1P3 appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Youngs modulus and hysteresis) and electrical passive (membrane capacitance, C(m)) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, C(m), used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce C(m) and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.

Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors.

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C(2)C(12) myoblasts SPP elicited significant Ca(2+) mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca(2+) response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca(2+) transients performed with a spectrophotofluorimeter showed that the rise in Ca(2+) was strictly dependent on availability of extracellular Ca(2+). Cell treatment with pertussis toxin partially prevented the Ca(2+) response induced by SPP, indicating that G(i)-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca(2+) mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca(2+), although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca(2+) increase, supporting the notion that Ca(2+) mobilization in C(2)C(12) cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Photoactivation of bone marrow mesenchymal stromal cells with diode laser: Effects and mechanisms of action†

Mesenchymal stromal cells (MSCs) are a promising cell candidate in tissue engineering and regenerative medicine. Their proliferative potential can be increased by low‐level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. With the aim of expanding the therapeutic application of LLLI to MSC therapy, in the present study we investigated the effects of 635 nm diode laser on mouse MSC proliferation and investigated the underlying cellular and molecular mechanisms, focusing the attention on the effects of laser irradiation on Notch‐1 signal activation and membrane ion channel modulation. It was found that MSC proliferation was significantly enhanced after laser irradiation, as judged by time lapse videomicroscopy and EdU incorporation. This phenomenon was associated with the up‐regulation and activation of Notch‐1 pathway, and with increased membrane conductance through voltage‐gated K+, BK and Kir, channels and T‐ and L‐type Ca2+ channels. We also showed that MSC proliferation was mainly dependent on Kir channel activity, on the basis that the cell growth and Notch‐1 up‐regulation were severely decreased by the pre‐treatment with the channel inhibitor Ba2+ (0.5 mM). Interestingly, the channel inhibition was also able to attenuate the stimulatory effects of diode laser on MSCs, thus providing novel evidence to expand our knowledge on the mechanisms of biostimulation after LLLI. In conclusions, our findings suggest that diode laser may be a valid approach for the preconditioning of MSCs in vitro prior cell transplantation. J. Cell. Physiol. 228: 172–181, 2013.