centro biotecnologie avanzate

LABORATORY OF: Stem Cell Biochemistry
CONTACT PERSONS:Dr. Sonia Scarfì, Prof. Elena Zocchi (University of Genova)
Phone +39 010 5737208 E-mail: soniascarfi@unige.it
E-mail: ezocchi@unige.it

Description of Laboratory and Expertise:

The main research activities of the laboratory of “Stem Cell Biochemistry” are focused on the intercellular communication network of the bone marrow (BM) stem cell niche. The BM contains multiple stem cell types, including hemopoietic stem cells (HSC) and mesenchymal stem cells (MSC). Identification and molecular characterization of the complex paracrine crosstalks between HSC and the heterogenous BM stroma cells, especially MSC, is a fundamental pre-requisite both for elucidating the stem cells’ biology and for tackling future potential applications in regenerative medicine.

Abstract of Activities:

This laboratory investigates the biology and biochemistry of hemopoietic (HSC) and mesenchymal stem cells (MSC), mainly focusing on the autocrine and paracrine signalling pathways leading to either self-renewal or differentiation, in order to characterize the fundamental role of the niche in determining HSC and MSC fate in the bone marrow (BM). MSC can be isolated from BM, expanded in vitro and induced to differentiate toward osteoblasts, adipocytes, chondrocytes, and endothelial cells. In the BM MSC play a key role in providing HSC with soluble factors essential to their proliferation and differentiation. MSC also release soluble immunosuppressive molecules reducing the graft versus host disease. Another clinical application of properly in vitro expanded MSC could be their engraftment at the insertion site of metal prostheses, to stimulate bone regeneration. Specifically, attention is paid on the proliferative signalling pathways triggered both on MSC and HSC by cyclic ADP-ribose (cADPR), a universal calcium mobilizer, and Abscisic acid (ABA), a plant hormone whose activity is strictly dependent on cADPR. Other topics include: i) comparative properties of MSC purified from alternative sources, e.g., amniotic fluid and chorionic villi; ii) molecular systems of release of NAD and ATP from MSC (e.g., connexin 43 hemichannels) and autocrine mechanisms derived therefrom potentially affecting MSC differentiation versus proliferation.

Detailed Research Activities:

Current activities in this laboratory derive from our earlier studies that led to demonstrate the occurrence of autocrine and paracrine mechanisms underlying a previously unknown subcellular/intercellular trafficking of signal metabolites. This trafficking ultimately regulates the spatio-temporal dynamics of intracellular calcium ([Ca2+]i) and accordingly calcium-related key functions in several cell types. These include granulocytes, monocytes, astrocytes, neurons, microglial cells, vascular smooth myocytes, mesenchymal stem cells (MSC), hemopoietic precursors (HP, both partially differentiated and early immature cells identifiable with hemopoietic stem cells (HSC)), pancreatic islet cells and various cell lines.
The above mechanisms are mainly centered, i) on the interplay of NAD, cyclic ADP-ribose (cADPR), ADP-ribose (ADPR), NADP, NAADP, and of three novel calcium-activated diadenosine dinucleotides shown in this laboratory to be generated by different ADP-ribosyl cyclases acting on either NAD or cADPR; ii) on sites (both ectocellular and subcellular, i.e. vesicle-bound) of their generation and degradation; iii) on their transport across both equilibrative and concentrative transporters; iv) on selective receptors (both ectocellular and intracellular) and related signalling pathways; v) on their, still little known, effects on specific calcium channels. Basically, this research aims at identifying and characterizing some hitherto poorly defined codes of intercellular communication, with the perspective of discovering new molecular targets for the development of innovative drugs or for diagnostic purposes.
With this background, the laboratory investigates a number of biological systems and structures potentially useful to functionally characterize the hemopoietic niche at the molecular level. The capacity of HSC to undergo self-renewal (horizontal expansion) in the bone marrow (BM) is a fundamental process in the physiology of hemopoiesis, and MSC are believed to play a pivotal role in its regulation through the production of either paracrine growth factors or of cytotoxic factors. This justifies an impressively growing interest worldwide toward identifying the signal metabolites that link the BM stroma to HSC, in order to tackle important clinical problems such as engraftment of hemopoietic precursors (HP) and, on the other side, BM failure (aplasia, transplant failure).
We have recently demonstrated that cADPR, a potent and universal intracellular calcium mobilizer, is a new hemopoietic growth factor. ADP-ribosyl cyclase (ADPRC) activity, responsible for cADPR synthesis from NAD, is expressed in the BM microenvironment, especially on MSC by the GPI-linked BST-1 protein. Incubation of human HSC with cADPR prior to transplantation, as well as co-infusion of the HSC with CD38-transfected stromal cells (CD38 is another transmembrane ADPRC producing ectocellular cADPR), improve the engraftment of human HSC into irradiated NOD/SCID mice. Indeed, hormone-like, nanomolar concentrations of cADPR, as those produced by a cyclase-positive feeder, significantly increase the in vitro proliferation of human HSC. The capacity of MSC to release NAD through connexin-43 (Cx43) hemichannels enables the consequent ADPRC-catalysed ectocellular production of cADPR in the BM stem cell niche. The mechanism of action of this cyclic nucleotide depends on its calcium-mobilizing activity, that follows its internalization across a concentrative nucleoside transporter identified in our laboratory.
Another topic under active investigation in our laboratory concerns Abscisic Acid (ABA). ABA is a plant hormone involved in fundamental functions in higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, control of stomatal closure and regulation of gene transcription. Endogenous ABA synthesis has recently been demonstrated in our laboratory to occur also in lower Metazoa (Porifera and Hydrozoa), where it is stimulated by temperature stress or by light exposure, respectively. ABA activates ADPRC through a PKA-mediated phosphorylation, leading to an increase of the intracellular cADPR concentration and of the [Ca2+]i. The increase of the [Ca2+]i stimulates several physiological functions in sponges and stem cell-mediated tissue regeneration in Hydroids.
Recently, in this laboratory, production and release of ABA have been demonstrated for the first time to take place also in human cells, specifically in particle-stimulated human granulocytes, where the hormone behaves as a pro-inflammatory cytokine. The signalling pathway of ABA in human granulocytes, strikingly similar to the one described in lower Metazoa, stimulates the functional effects typical of these phagocytes (phagocytosis, ROS production, cell migration).
As an increase of the intracellular cADPR and of the [Ca2+]i stimulates proliferation of human HSC and as ADPRC activity is expressed in HSC, we investigated the effect of ABA on the in vitro proliferation of human clonogenic progenitors. ABA, indeed, proved to cause the expansion of human HSC through the same signalling pathway described in granulocytes, involving a cADPR-dependent [Ca2+]i increase. Short- and long-term transcriptional effects on genes known to affect HSC proliferation occur following ABA priming of CD34 HP cells and the related triggering of the NAD/cADPR/ [Ca2+]i regulatory cascade in these precursor cells. Recent data related to the molecular characterization of the cross-talk among different signal metabolites in the putative hemopoietic niche also suggest that ABA is generated and released by MSC, following their exposure to variously stimulated lymphocytes. Moreover, addition of extracellular ABA proved to trigger the MSC-induced in vitro expansion of human HSC in a cADPR-related way. These results seem to indicate that MSC are a major target of “de novo” synthesized ABA by means of an autocrine loop that causes enhanced proliferation and self-renewal, but apparently no differentiation, of these cells. In addition to horizontal cell division, MSC respond to ABA with activation of immunomodulatory pathways, an effect that is expected to favour expansion of neighbouring HP in the BM hemopoietic niche. Therefore, a complex network of autocrine and paracrine loops centered around ABA emerges that ultimately stimulates hemopoiesis.
At present the outlines of the research on ABA in human cells and cell microenvironments are: i) identification and molecular characterization in these, and in other ABA-responsive human cells as well, of the ABA receptor, which is pertussis toxin (PTX)-inhibitable; ii) refinement of the analysis of ABA-induced signalling pathways and networks, which seem to share a common final target on ADPRC activation and [Ca2+]i increases, from sponges to human cells; iii) detailed reconstruction, in our in vitro model of the hemopoietic niche, of the autocrine and paracrine loops related to the NAD+/cADPR/[Ca2+]i system.
Another study on biochemical features of MSC in our laboratory concerns their capacity to release ATP and NAD in the extracellular milieu across Cx43 hemichannels (HC) and the potential of these signal metabolites to affect the stemness of MSC from outside. Preliminary data indicate that Cx43 HC-mediated efflux of both ATP and NAD is enhanced by mechanical stress. Current studies are centered on: i) analysis of consequent patterns of MSC biology, notably effects on proliferation/expansion versus differentiation toward individual lineages; ii) effects of extracellular NAD and ATP on calcium-related mechanisms of MSC regulation. Specifically, much attention will be paid to investigating, on one hand the ectoenzymatic ADPRC-mediated conversion of NAD to cADPR and on the other the interaction of extracellular ATP and NAD with specific purinergic receptors (e.g., P2Y11 and P2X7) known to trigger signalling pathways and/or pore opening with calcium influx.
Finally, in collaboration with other groups at ABC and outside, we have started comparative studies on different cellular sources of human MSC such as BM, femur fragments, amniotic fluid and chorionic villi. These collaborative studies should identify MSC sub-populations and provide more advanced knowledge on their biological properties and, accordingly, on underlying mechanisms of specific MSC functions, e.g. differentiation, self-renewal, immunosuppressive capacity, etc. Using this comparative strategy, the expected elucidation of the signals and mechanisms that determine different features in MSC behaviour should allow to understand the still unclear capacity of certain stem cells, mainly of embryonic and fetal origin, to maintain high proliferation rates alongside an undifferentiated phenotype characterized by an indefinite horizontal expansion. These studies will also help determine which sources represent the best choice for possible clinical applications of MSC in regenerative medicine. To this purpose, we are also currently performing in vivo experiments on athymic rats with femur discontinuites, to evaluate the ability of human, in vitro cADPR-expanded, MSC in repairing large bone fractures, in collaboration with G. Burastero from the Orthopedic Division of S. Corona Hospital, at Pietra Ligure.

Applications and Developments:

The laboratory of Stem Cell Biochemistry is expected to issue future applications and patents in the field of regenerative medicine, contributing to the definition of successful bone marrow transplantion protocols as well as to the development of innovative devices for bone regeneration. Furthemore, the ongoing studies of Abscisic in mammals and human cells hold promise for the development of new anti-inflammatory and immunomodulatory drugs.

Managed core facilities:

1. Complete cell culture equipment.
2. High standards molecular biology equipment.
3. Instrumentation for biochemical studies (spectrophotometers, HPLC, electrophoresis and blotting equipment, gel/blots quantification and imaging).

Ongoing collaborations:

Our research activities are currently developed in collaboration with:
Giorgio Burastero, Ortopedic Division of S. Corona Hospital, Pietra Ligure, Savona, Italy. Orthopedic surgery of disabled persons and development of new devices for bone reconstruction based on mesenchymal stem cells.
Franca Dagna Bricarelli, Genetic Division of Galliera Hospital, Genova, Italy. Genetic characterization of stem cells from amniotic fluid and chorionic villi.
Francesco Frassoni, Hematologic Division of S. Martino Hospital, Genova, Italy. Development of a Cell Factory for the isolation, quality control and therapeutic use of stem cells in a hospital setting.
Armando Genazzani, Department of Pharmacology, University of Piemonte Orientale “A. Avogadro” Novara, Italy. NAADP-related mechanisms of calcium signalling.
Andreas H. Guse, Centre of Experimental Medicine, Institute of Biochemistry and Molecular Biology I, Cellular Signal Transduction, University Medical Centre Hamburg-Eppendorf, Germany. Elucidation of spatio-temporal dynamics of calcium signals triggered by second messengers and cytotoxic metabolites, identification and functional characterization of TRPM channels.
Hon Cheung Lee, Department of Physiology, University of Hong Kong, H.K. H.C. Lee is the discoverer of cADPR and NAADP and recently elucidated the catalytic mechanism of CD38, the prevalent ADPRC in mammals.
Rodolfo Quarto, Laboratory of Stem Cell Biology, ABC, Genova, Italy. Cellular and molecular biology of progenitor cells for the repair of bone and cartilage and evaluation of the influence of the microenvironment on cell differentiation.
Silvia Scaglione, Laboratory of Stem Cells Bioengineering, ABC, Genova, Italy. Generation of 3D cellular systems as grafts for replacement of damaged tissues. Development of bioreactors and biomaterials for the differentiation of adult progenitors and stem cells under different physical stimuli.
Antonio Uccelli, Laboratory of Neuroimmunobiology, ABC, Genova, Italy. Immunosuppressive properties of MSC; Cellular therapies of human multiple sclerosis, using a murine model of EAE (Experimental Autoimmune Encephalomyelitis).
Timothy F. Walseth, Department of Pharmacology, University of Minnesota, Minneapolis, MN (USA). cADPR analogs and cADPR-related mechanisms of calcium signalling.

Most recent and significant publications:

Scarfì, S., Giovine, M., Pintus, R., Millo, E., Clavarino, E., Pozzolini, M., Sturla, L., Stock, R.P., Benatti, U., Damonte, G. Selective inhibition of inducible cyclo-oxygenase-2 expression by antisense peptide nucleic acids in intact murine macrophages.
Biotechnol Appl Biochem. 2003 Aug;38(Pt 1):61-9.

Cutrona, G., Carpaneto, E.M., Ponzanelli, A., Ulivi, M., Millo, E., Scarfì, S., Roncella, S., Benatti, U., Boffa, L.C., Ferrarini, M. Inhibition of the translocated c-myc in Burkitt's lymphoma by a PNA complementary to the E mu enhancer. Cancer Res. 2003 Oct 1;63(19):6144-8.

Zocchi, E., Basile, G., Cerrano, C., Bavestrello, G., Giovine, M., Bruzzone, S., Guida, L., Carpaneto, A., Magrassi, R., Usai, C.ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges. J Cell Sci. 2003 Feb 15;116(Pt 4):629-36.

Puce, S., Basile, G., Bavestrello, G., Bruzzone, S., Cerrano, C., Giovine, M., Arillo, A., Zocchi, E. Abscisic acid signaling through cyclic ADP-ribose in hydroid regeneration.
J Biol Chem. 2004 Sep 17;279(38):39783-8

Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., Zocchi, E. ADP-ribosyl cyclases generate two unusual adenine homodinucleotides with cytotoxic activity on mammalian cells. Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14509-14.

Bacigalupo, A., Valle, M., Podestà, M., Pitto, A., Zocchi, E., De Flora, A., Pozzi, S., Luchetti, S.Frassoni, F., Van Lint, M.T., Piaggio, G.T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia. Exp Hematol. 2005 Jul;33(7):819-27.

Podestà, M., Benvenuto, F., Pitto, A., Figari, O., Bacigalupo, A., Bruzzone, S., Guida, L., Franco, L., Paleari, L., Bodrato, N., Usai, C., De Flora, A., Zocchi, E. Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors. J Biol Chem. 2005 Feb 18;280(7):5343-9

De Flora, S., Scarfì, S., Izzotti, A., D’Agostini, F., Chang, C.C., Bagnasco, M., De Flora, A., Trosko, Induction by 7,12-dimethylbenz(a)anthracene of molecular and biochemical alterations in transformed human mammary epithelial stem cells, and protection by N-acetylcysteine. Int J Oncol. 2006 Sep;29(3):521-9.

Scarfì, S., Benatti, U., Pozzolini, M., Clavarino, E., Ferraris, C., Magnone, M., Valisano, L., Giovine, M.Ascorbic acid-pretreated quartz enhances cyclo-oxygenase-2 expression in RAW 264.7 murine macrophages. FEBS J. 2007 Jan;274(1):60-73.

Bruzzone, S., Moreschi, I., Usai, C., Guida, L., Damonte, G., Salis, A., Scarfì, S., Millo, E., De Flora, A., Zocchi E. Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger. Proc Natl Acad Sci U S A. 2007 Apr 3;104(14):5759-64.

Bruzzone, S., Moreschi, I., Guida, L., Usai, C., Zocchi, E., De Flora, A. Extracellular NAD regulates intracellular calcium levels and induces activation of human granulocytes. Biochem J. 2006 Feb 1;393(Pt 3):697-704.

Moreschi, I., Bruzzone, S., Melone, L., De Flora, A., Zocchi, E. NAADP synthesis from cADPRP and nicotinic acid by ADP-ribosyl cyclases. Biochem Biophys Res Commun. 2006 Jun 30;345(2):573-80.

Moreschi, I., Bruzzone, S., Nicholas, R.A., Fruscione, F., Sturla, L., Benvenuto, F., Usai, C., Meis, S., Kassack, M.U., Zocchi, E., De Flora, A. Extracellular NAD is an agonist of the human P2Y11 purinergic receptor in human granulocytes. J Biol Chem. 2006 Oct 20;281(42):31419-29. Epub 2006 Aug 22.

Franco, L., Bodrato, N., Moreschi, I., Usai, C., Bruzzone, S., Scarf ì, S., Zocchi, E., De Flora, A. Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated activation of murine N9 microglial cell line. J Neurochem. 2006 Oct;99(1):165-76.

Bruzzone, S., Dodoni, G., Kaludercic, N., Basile, G., Millo, E., De Flora, A., Di Lisa, F., Zocchi, E. Mitochondrial dysfunction induced by a cytotoxic adenine dinucleotide produced by ADP-ribosyl cyclases from cADPR. J Biol Chem. 2007 Feb 16;282(7):5045-52.

Billington, R.A., Bruzzone, S., De Flora, A., Genazzani, A.A., Koch-Nolte, F., Ziegler, M.,
Zocchi, E. Emerging functions of extracellular pyridine nucleotides. Mol Med. 2006 Nov-Dec;12(11-12):324-7.

Moreschi, I., Bruzzone, S., Bodrato, N., Usai, C., Guida, L., Nicholas, R.A., Kassack, M.U., Zocchi, E., De Flora, A. NAADP is an agonist of the human P2Y11 purinergic receptor. Cell Calcium. 2008 Apr;43(4):344-55.

Magnone, M., Basile, G., Bruzzese, D., Guida, L., Signorello, M.G., Chothi, M.P., Bruzzone, S., Millo, E., Qi, A.D., Nicholas, R.A., Kassack, M.U., Leoncini, G., Zocchi, E. Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation. J Biol. Chem. 2008 Sep 5;283(36):24460-8.

Scarfì, S., Ferraris, C., Fruscione, F., Fresia, C., Guida, L., Bruzzone, S., Usai, C., Parodi, A., Millo, E., Salis, A., Burastero, G., De Flora, A., Zocchi, E. Cyclic ADP-ribose-mediated expansion and stimulation of human mesenchymal stem cells by the plant hormone abscisic acid. Stem Cells. 2008 Nov;26(11):2855-64.

Bruzzone, S., Bodrato, N., Usai, C., Guida, L., Moreschi, I., Nano, R., Antonioli, B., Fruscione, F., Magnone, M., Scarfì, S., De Flora, A., Zocchi, E. Abscisic acid is an endogenous stimulator of insulin release from human pancreatic islets with cyclic ADP ribose as second messenger. J Biol. Chem. 2008 Nov 21;283(47):32188-97.

Bodrato, N., Franco, L., Fresia, C., Guida, L., Usai, C., Salis, A., Moreschi, I., Ferraris,
C., Verderio, C., Basile, G., Bruzzone, S., Scarfì, S., De Flora, A., Zocchi, E. Abscisic acid activates the murine microglial cell line N9 through the second messenger cyclic ADP-ribose.J Biol. Chem. 2009 Mar 27. [Epub ahead of print]

Magnone, M., Bruzzone, S., Guida, L., Damonte, G., Millo, E., Scarfì, S., Usai, C., Sturla, L., Palombo, D., De Flora, A., Zocchi, E. Abscisic acid released by human monocytes activates monocytes and vascular smooth muscle cell responses involved in atherogenesis. J Biol. Chem. 2009 Mar 30. [Epub ahead of print]

PATENTS

Cyclic ADP-ribose analogs. Walseth, T.F., De Flora, A., Zocchi, E., Podestà, M., Wong, L., Aarhus, R., Lee, H.C..University of Minnesota. USA Patent No. 6, 593, 307 B4, issued July 15, 2003.

Uso del fito-ormone Acido abscissico per la preparazione di un medicamento ad attività pro-emopoietica. Zocchi, E., Benatti, U., Bodrato, N., De Flora, A., Scarfì, S. Italian patent T02004A000803, issued November 15, 2004.

Fluridone come agente anti-infiammatorio. Zocchi, E., Guida, L., Bruzzone, S., Scarfì, S., Magnone, M., Basile, G., Benatti, U., De Flora, A., Moreschi, I., Franco, L., Salis, A. Italian patent T02005A000708, issued October 7, 2005.

Novel adenyl dinucleotides with antitumour activity and a method for preparing thereof. Inventors: Basile, G., Benatti, U., Bruzzone, S., Damonte, G., De Flora, A., Fattorusso, E., Franco, L., Guida, L., Taglialatela-Scafati, O., Zocchi, E. PCT/IB2006/051330 (International Publication No. WO 2006/117735 A1).

Fluridone as an anti-inflammatory agent. Inventors: Zocchi, E., Guida, L., Bruzzone, S., Scarfì, S., Magnone, M., Basile, G., Benatti, U., De Flora A., Moreschi, I., Franco, L., Salis, A. PCT/IB2006/053669.