REGULAR ARTICLE

Echinoderm regeneration: an in vitro approach using the crinoidAntedon mediterranea

Cristiano Di Benedetto & Lorenzo Parma &

Alice Barbaglio & Michela Sugni & Francesco Bonasoro &

Maria Daniela Candia Carnevali

Received: 8 January 2014 /Accepted: 5 May 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Among echinoderms, crinoids are well known fortheir remarkable regenerative potential. Regeneration dependsmainly on progenitor cells (undifferentiated or differentiated),which migrate and proliferate in the lesion site. The crucialrole of the “progenitor” elements involved in the regenerativeprocesses, in terms of cell recruitment, sources, and fate, is acentral problem in view of its topical interest and biologicalimplications. The spectacular regenerative potential of cri-noids is used to replace lost internal and external organs. Inparticular, the process of arm regeneration in the feather starAntedon mediterranea is the regeneration model most exten-sively explored to date. We have addressed the morphologicaland functional characterization of the cell phenotypes respon-sible for the arm regenerative processes by using an in vitroapproach. This represents the first successful attempt to cul-ture cells involved in crinoid regeneration. A comparison ofthese results with others from previous in vivo investigationsconfirms the diverse cell types contributing to regenerationand underscores their involvement in migration, proliferation,and dedifferentiation processes.

Keywords Regeneration . Phenotype . Cell culture .

Echinoderms . Crinoid . Feather star (Antedon mediterranea)

Introduction

Regenerative potential is expressed to its maximum extent inechinoderms (Candia Carnevali and Bonasoro 2001a). Incrinoids (sea lilies), regenerative processes are common and

are involved in the replacement of lost internal and externalorgans. Feather stars are stalkless crinoids that extensivelyemploy regeneration to reconstruct both external appendages(arms, pinnules, and cirri) and internal organs (digestive ap-paratus, gonads, or the entire visceral mass), which are fre-quently lost following traumatic injury, predation, or autoto-my. Arm regeneration in Antedon mediterranea (Fig. 1a) isthe best established experimental model and has been suc-cessfully employed, in both classic studies (Minckert 1905;Perrier 1873; Reichensperger 1912) and recent investigations,for exploring regeneration from the macroscopic to the mo-lecular levels (Candia Carnevali and Bonasoro 2001b). Thisprocess involves typical blastemal regeneration, which is rap-id and effective, and during which new structures developfrom pluripotent proliferating cells that undergo active migra-tion through tissues (amoebocytes) or through coelomic fluids(coelomocytes).

The arm of A. mediterranea consists of a complex muscu-loskeletal system formed by a series of brachial ossiclesconnected at movable joints by segmental muscles and liga-ments (Fig. 1b). Immobile joints known as syzygies are reg-ularly distributed along the arm; these joints are specializedautotomy sites at which self-induced detachment occurs inresponse to various types of perturbations. At the level ofthese joints, the two complementary ossicles are closely ap-posed, the only interposed soft tissue being short bundles ofligaments consisting of mutable collagenous tissue (MCT)specialized for autotomy (Wilkie 1996, 2001). Other anatom-ical structures relevant to the processes of regeneration arethe central brachial nerve cord with its lateral branches,the multiple system of coelomic canals (somatocoelic andhydrocoelic), and the ambulacral groove, i.e., the mainfeeding system of crinoids, which has a specialized epi-thelium (Fig. 1b).

The process of arm regeneration in A. mediterranea occursover a period of about 4 weeks and consists of three main

C. Di Benedetto (*) : L. Parma :A. Barbaglio :M. Sugni :F. Bonasoro :M. D. Candia CarnevaliDepartment of Biosciences, University of Milan, Via Celoria 26,20133 Milano, Italye-mail: cristiano.dibenedetto@unimi.it

Cell Tissue ResDOI 10.1007/s00441-014-1915-8

phases: a repair phase, an early regenerative phase, and anadvanced regenerative phase (for further details of the regen-eration phases in A. mediterranea, see Candia Carnevali et al.1993; Candia Carnevali and Bonasoro 2001b; Candia Carnevaliand Burighel 2010; Candia Carnevali 2006; Patruno et al. 2002;2003; Thorndyke and Candia Carnevali 2001).

In terms of regeneration-competent cells, at least five dif-ferent cell types are involved in arm or visceral regeneration incrinoids: possibly, most of these progenitor cells are undiffer-entiated (amoebocytes, coelomocytes) and pluripotent ele-ments (phagocytes, granulocytes, and dedifferentiated cells;Candia Carnevali and Bonasoro 2001b; Candia Carnevali2006; Candia Carnevali and Burighel 2010; Dolmatov et al.2003; Mozzi et al. 2004, 2006; Parma et al. 2006). In thefollowing, these cells will be named according to the tradi-tional terminology proposed by Reichensperger (1912).

Amoebocytes are resident presumptive stem cells that arestored in the arm around the brachial nerve in the form ofaggregates of satellite cells. They can be activated or recruitedin response to injury signals and can rapidly migrate throughthe tissues (Candia Carnevali 2006; Candia Carnevali andBonasoro 2001b). Their contribution appears to be essentialmainly during the repair and early regenerative phases.Coelomocytes represent circulating presumptive stem cellsthat are present mainly in the coelomic fluids, but are alsoscattered in the tissues and produced by the specific prolifer-ation of the coelomic epithelium (coelothelium). They arecontinuously produced by the physiological turnover of thecoelothelium (Candia Carnevali and Bonasoro 2001b).

Phagocytes are typical phagocytic cells, involved in de-fense mechanisms and are recognizable from their largephagosomes of variable size and content. As expected, phago-cytes are present during the initial regeneration events and areespecially abundant at the level of the amputation regionduring the repair phase (Candia Carnevali et al. 1993).

Granulocytes are considered to be exclusive to crinoids(wanderzellen according to Reichensperger 1912; Smith

1981) and are recognizable from their abundant cytoplasmicgranules. During the repair phase, they migrate along thenerve or inside the coelomic canals towards the amputationregion where their chromatophilic granules are discharged byexocytosis (Candia Carnevali et al. 1993).

Dedifferentiated cells also usually contribute to regeneration.Dedifferentiation processes are particularly evident in myocytesand, to a minor extent, in other mesodermal cell types(sclerocytes, fibrocytes; Candia Carnevali et al. 1993; CandiaCarnevali and Bonasoro 2001b; Candia Carnevali 2006).

As is well known, in vitro cultures of animal cells (fromboth vertebrates and invertebrates, especially arthropods) andplant cells have been indispensable for the understanding oftissue composition and cell behavior under defined condi-tions. Although marine invertebrates (more than 30 differentphyla) represent an extremely promising and still unexploredpotential source of tissues and cell types, the low mitoticactivity of several tissues and the slow adaptation of their cellsto culture conditions have been serious obstacles to obtainingcontinuous cell lines (Odintsova et al. 2005). The majority ofattempts to develop stable cell cultures from marine inverte-brates have been ineffective to date (Rinkevich et al. 1994;Bayne 1998; Rinkevich 1999, 2011). However, Shashikumarand Desai (2011) successfully established a finite proliferativecell line from testicular tissues of the crab Scylla serrata;subsequent sub-cultures were produced intervals of 2 weeks,and the cells appeared to be healthy and proliferated for morethan 5 months.

In echinoderms, primary cell cultures have been obtainedfrom all five classes by various researchers who haveemployed specific models under limited experimental condi-tions. In echinoids, cell cultures have been produced fromcoelomic fluid (Bertheussen and Seljelid 1978; Matrangaet al. 2005; Di Benedetto et al. 2007), from embryonic cells(Odintsova et al. 1994, 1999), and from gonads (Mercurioet al. 2014). In holothurians, primary cultures obtained fromregenerating intestinal tissues (Odintsova et al. 2005). In

Fig. 1 a Specimen of Antedon mediterranea. b Arm anatomy. Repre-sentation of the basic arm anatomy of the comatulid Antedonmediterranea (lateral pinnules omitted) just after amputation (pa ampu-tation plane). Brachial ossicles (os) are connected by muscles (m) and

ligaments (l); intercalated syzygies (sz) are present along the arm. Themain continuous structures along the arm are the brachial nerve (n), thesystem of multiple coelomic canals (cc), and the ambulacral epithelium(ae). Modified from Candia Carnevali and Bonasoro (2001b)

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ophiuroids and asteroids, Moss et al. (1998) have developedcell cultures from the radial nerve cord of both the starfish andthe brittle star. Sharlaimova and Petukhova (2012) have keptcoelomic epithelium cells from starfish viable in vitro for2 months.

In crinoids, the only attempts of in vitro studies have beenperformed by our research group. Preliminary successful re-sults on primary cell cultures have been obtained from armexplants and arm regenerating tissues of A. mediterranea(Parma et al. 2005, 2006; Di Benedetto et al. 2007; CandiaCarnevali et al. 2009).

The present work is therefore intended to provide a definedbackground for the in vitro approach to crinoids, in terms ofprotocols and methods, and to complete a comprehensiveview of crinoid arm regeneration in terms of the morpholog-ical and functional characterization of the cell phenotypes

involved. A comparison of these new results with others fromprevious in vivo investigations has allowed us (1) to specifyoptimized conditions for in vitro cultures; (2) to confirm theidentification of the various progenitor cell types; and (3) tounderscore the involvement of these cell types in migration,proliferation, and dedifferentiation processes.

Some of the new results have been included in the PhDthesis of the first author (Di Benedetto 2011).

Materials and methods

Experimental animals

Specimens of A. mediterrranea (Fig. 1a) were collected in theLigurian sea, Capo Noli, at a depth of 18-30 m (depending on

Fig. 2 Representation of the various in vitro tissue treatments. a Protocol(a). Regenerating tissues. Tissues were removed from the whole armwithforceps and placed directly in a dish containing modified Leibovitz L-15medium. After a few days, undissociated regenerating tissues were re-moved from the dish; cells flowed out from the tissues to provide aprimary cell culture. b Protocol (b). As in protocol (a), except thatregenerating tissues were treated with Ca2+Mg2+-free artificial sea water(CMF-ASW). c Protocol (c). Arm explants. Explant fragments, obtainedby dividing an isolated arm into several segments (3-4 mm), were placedinto a Petri dish containing modified Leibovitz L-15 medium. Tissue

dissociation was facilitated by continuously stirring the Petri dishes. Aftera few days, cell debris and arm fragments were manually removed. dProtocol (d). As in protocol (c), except that arm explants were treated withASW. e Protocol (e). Arm explants were treated with CMF-ASW +glucose (1 mg/ml). f Protocol (f). Arm explants were dissociated inCMF-ASW + glucose (1 mg/ml). After a few days, debris was removedby sedimentation (d1), the cells were centrifuged (d2), resuspended, andreplated (d3) either in CMF-ASW + glucose (1 mg/ml) or in L-15. Theoriginal arm fragments could be re-employed several times (up to 5-6cycles). All the cell cultures were stored at 15 °C

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the season) a few days before starting the experiments. Oncetransported to the Department of Biosciences, Milan, animalswere left to acclimatize prior to the experiments and weremaintained at 16 °C in aerated 50-l aquaria filled with artificialsea water (ASW; Instant Ocean).

Autotomy induction To ensure physiological regeneration, theexperiments on arm amputation were always carried out byreproducing as closely as possible the conditions associatedwith natural autotomy, i.e., the arms were "amputated" in thedistal-intermediate region at the level of the syzygial articula-tions. Direct cutting of the tissue was avoided, and mechanicalautotomy was induced by simple blade pressure. Each animalwas subjected to 3-4 contemporary amputations and thenallowed to regenerate for a predetermined period (∼1 week).These experiments were performed under a stereomicroscope(Leica MZ75, equipped with Leica CLS 150XE illuminationand a Leica Digilux 18.102 camera).

Cell cultures

Cell collection/preparation Before collecting tissue samples,the whole animal was immersed for 2 h in filtered sea water towhich gentamicin (40 μg/ml) and penicillin (100 μg/ml) wereadded. All media and reagents employed were previouslysterilized. Various protocols (Fig. 2a–f) were tested in orderto determine which provided the best dissociated cell yield.

In (a), arm regenerating tissues (1 week post amputation) weregently and carefully removed (volume: 1-2 mm3) with forcepsand placed directly into a Petri dish containing a modifiedLeibovitz L-15 medium (see below). In order to obtain a suffi-cient number of cells in vitro, approximately 10 regenerates perPetri dish (35 mm diameter), and 4-5 regenerates per 24-multiwell (15 mm diameter) were used. Regenerates were leftin the culture dish until complete disaggregation had occurred.Procedure (b) was as in (a), except that regenerating tissues(1 week post amputation) were cultured in Ca2+Mg2+-free ASW(CMF-ASW) + glucose (1 mg/ml), instead of Leibovitz L-15.

In (c), 20–40 arm fragments (3-5 mm) obtained by dividinga non-regenerating arm into several segments were placed ineach Petri dish or approximately 10 fragments in 24-multiwells containing modified Leibovitz L-15 medium. Inorder to facilitate tissue dissociation and the exit of cells fromthe arm fragments, Petri dishes were continuously stirred for afew days after which the arm fragments were manually re-moved. The Petri dishes contained essentially the cells thathad migrated out from the arm fragments, although sometissue debris remained.

In (d), arm fragments were cultured in ASW + glucose(1 mg/ml). In (e), arm fragments were cultured in CMF-ASW+ glucose (1 mg/ml). Procedure (f) was as in (e), except that,after a few days in CMF-ASW + glucose (1 mg/ml), the cell

suspension (only a small number of cells adhered to thesubstrate) was removed, transferred to a Falcon tube, andallowed to settle in order to separate cells from gross tissuedebris and skeletal pieces. The supernatant containing the cellswas collected and centrifuged, and the pellet was resuspendedin fresh culture medium (CMF-ASW + glucose or L-15) andtransferred into a Petri dish for further culturing. The originalarm fragments could be re-employed several times (up to 5-6cycles) in order to exploit the tissues to their full potential.

All cell cultures were maintained at 15 °C and the mediumwas changed every 2–3 days.

The average cell number (per ml) in cell cultures wasestimated by direct cell counting by using a “Burker chamber”device. Cell adhesion phenomena were observed in vitro onlywith the use of phase contrast microscopy.

Culture media The following media were used: (1) LeibovitzL-15, commercially designed for mammals, was modified(1100 mOsm) for marine invertebrate cell cultures (NaCl20.2 g/l, KCl 0.54 g/l, CaCl2 0.6 g/l, NaSO4 1 g/l, MgCl23.9 g/l; additional components were added: insulin + transfer-rin 50 μg/ml, glutamine 100 μg/ml, glucose 1 mg/ml) andfiltered; (2) filtered ASW + glucose (1 mg/ml); (3) filteredCMF-ASW + glucose (1 mg/ml). In all media, a mix ofantibiotics and/or antimycotics was added (Pen-Strep100 μg/ml + Gentamicin 40 μg/ml).

Culture subtrates Various commercial substrates were tested.Poly-L-ornithine (1 % solution in distilled water) was left for2 h at room temperature (RT) on plastic dishes, followed bytwo washes with distilled water. Poly-L-lysine (0.1 mg/ml)was applied to glass slides for 2 h at RT, followed by twowashes with distilled water. Collagen type I from rat tail(1 mg/ml in acetic acid) was incubated on plastic dishes for15 min at 37 °C, followed by one wash with selected medium.Protamine (1 mg/ml in phosphate-buffered saline [PBS]) wasapplied to plastic dishes for 3 h at RT, followed by two washeswith ASW. Concanavalin (ConA; 10mg/ml in distilled water)+ bovine serum albumin (BSA) was left on plastic dishes for3 h at 37 °C and, subsequently, Con A (5 μg/ml) in PBS for3 h at 37 °C, followed by one wash with selected medium.Glass slides coated with a layer of TiO2 (titanium dioxide)were also used.

Microscopic methods

Stereomicroscopy A stereomicroscope (Leica MZ75equipped with Leica CLS 150XE lights and a Leica Digilux18.102 camera) was used for manipulation and operation ofanimals and for monitoring regeneration processes.

Light microscopy Conventional histology protocols for resinembedding and sectioning of in vitro cells were employed.

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Semithin (0.96 μm) sections (Reichert Ultracut E with glassknives) of cell pellets were stained with crystal violet-basicfuchsin and observed under a JENAVAL light microscopeprovided with a Canon S40 color camera and Canon imagingsoftware.

In order to observe primary cell cultures during the overallperiod (1-8 weeks) of in vitro observations, we used aninverted Axiovert 200 M microscope (Zeiss) provided with aAxioCam HRm HAL 100.

Transmission electron microscopy Pellets of cells were fixedwith 2 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2)for 1 h or with SPAFG fixative (for details, see Ermak andEakin 1976). Samples were washed overnight in the samebuffer and postfixed with 1 % osmic acid in 0.1 M cacodylatebuffer (pH 7.2) for 2 h. After standard dehydration in anethanol series, samples were embedded in Epon 812-Araldite. Ultrathin (0.07 μm) sections (Reichert Ultracut Ewith diamond knives) were mounted on copper grids andstained with uranyl acetate and lead citrate for electron mi-croscopy and then observed and photographed in a Jeol100SX electron microscope.

Scanning electron microscopy Cells adhering to poly-L-lysine-coated coverslips were fixed with a solution(1100 mOsm) of 2 % glutaraldehyde and artificial ASW for2 h at 4 °C. Samples were washed overnight at 4 °C withfiltered ASW (36 %) and then post-fixed with a 1 % solutionof osmic acid in ASW and glucose for 2 h. After severalwashes in distilled water, samples were dehydrated in anethanol series. Absolute ethanol was gradually substitutedwith hexamethyldisilazane. Samples were then left to dry onfilter paper, mounted on stubs, covered with a thin layer ofpure gold (Sputter Coater Nanotech) and observed with aLEO-1430 scanning electron microscope.

BrdU incorporation and detection Amodified BrdU protocolwas used to detect actively proliferating cells in vitro. Cells(1 week in culture) were exposed to culture media (modifiedL-15) in which 30 μM BrdU (bromodeoxyuridine) and FdU

(fluorodeoxyuridine; 10:1) were dissolved for 2 h. Cells werecentrifuged (800g, 1 min) and fixed in 4 % paraformaldehyde/0.5 % glutaraldehyde in 0.1 M PBS (pH 7.6) for 2 h.Following an overnight wash in the same buffer, samples weredehydrated in a graded ethanol series and embedded in Epon-Araldite 812. The fixation and embedding protocol main-tained good cell integrity and good preservation of antigenic-ity. Semithin sections were cut and processed for immunocy-tochemistry. For use with semithin Epon-Araldite sections, thestandard BrdU immunocytochemistry protocol for paraffinsection was modified according to Candia Carnevali et al.(1997). Sections were observed under a JENAVAL light mi-croscope provided with a Canon S40 color camera and Canonimaging software.

Results

Cell culture optimization

Primary cell cultures from A. mediterranea arm tissue wereobtained from two different sources: (1) arm regeneratingtissues, (2) arm explants. We present first the main resultsconcerning the suitability of the various protocols, which aresummarized in Table 1. Detailed microscopy and immunocy-tochemical results are provided in subsequent sections.

Regenerating tissues were found to be less suitable thanexplants as a source of cells (Fig. 2 protocols a, b) because oftechnical difficulties in obtaining an adequate amount of tis-sues from the former and the low cell density obtained in theculture from regenerating tissues when both L-15 and CMF-ASW were used as culture media. Although recognizablephenotypes, mainly amoebocytes and coelomocytes, wereproduced (Fig. 3a), the low cell yield led us to focus only onarm explants as a source of cells for primary cell cultures(Fig. 2 protocols c–f).

In protocol (c), explant fragments were immersed inLeibovitz L-15 (Fig. 3b). After just 2 days, phase contrastobservations revealed only a few scattered groups of cells

Table 1 Summary of the results of the various in vitro protocols. Severalparameters were considered: contamination by mold and bacteria, cellyield (cells/ml) from the various protocols, presence of proliferation

activity, cell adhesion to substrate, and the phenotypes observed in cellcultures (+ present, +/- low level, − absent, am amoebocytes, cmcoelomocytes, pg phagocytes, my myocytes, gr granulocytes)

Protocol Contamination Cell yield (cell/ml) Proliferation Cell adhesion In vitro phenotypes

a + - - +/- am, cm

b - - - - am, cm

c + 103 _ 104 - +/- am, cm, pg, my

d + - - - -

e +/- ∼106 - +/- am, cm, pg, my, gr

f - >106 - +/- am, cm, pg, my, gr

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(mainly amoebocytes and coelomocytes) adhering to the sub-strate (Fig. 3b). Phagocytes and myocytes tended to appear 3or 4 days after the cultures were set up. Myocytes were stillable to contract in vitro, even after 1 week in culture (Fig. 3c).The use of Leibovitz L-15 presented two main problems: (1)only partial tissue dissociation was achieved (Fig. 3d), andexplants tended to deteriorate before dissociation was com-plete, resulting in a relatively low cell yield, which wasevaluated to be between 103 and 104 cells/ml (Table 1); (2)the prolonged culture time period (over 2 weeks) led tounavoidable contamination in the form of mold on the ex-plants and/or bacteria on the substrate (Fig. 3e). Protocol (d),in which ASWwas used as a culture medium, did not show anappreciable improvement, even when enriched with nutritiveelements. Dissociation and migration from the stump werevery limited.

In protocol (e), fragments were placed in CFM-ASW. Asexpected, the absence of divalent cations greatly facilitatedtissue dissociation, and a large number of cells moved out ofthe tissues after 2 days. After 1 week, they were distributed

homogeneously on the substrate, and “cellular density” washigher (Fig. 3f) than that seen in the previous protocols (cellpopulation: ∼106 cells/ml, see Table 1). Therefore CMF-ASWemerged as the most suitable culture medium.

In protocols c-e, manual removal of undissociated explants(skeletal pieces and small tissue clots) was always incomplete,and the remaining debris in the culture dishes was a constantsource of contamination. In order to overcome this problem,we separated cells from the debris and replated them in freshmedium (Fig. 2, protocol f). Under these conditions, viablecell cultures could be kept for 7-8 weeks. This method pre-sented several advantages: (1) a drastic decrease in culturecontamination; (2) an increased initial cell number (more than106 cells/ml, Table 1) in vitro (Fig. 4a, b); (3) the original armfragments could be re-employed several times. We testedvarious substrates. Poly-L-ornithine was the most used sub-strate in our experiments. Unfortunately, we did not find anyimprovement in terms of cell adhesion with respect to theuncoated control plastic surface. Only a few scattered groupsof cells (mainly amoebocytes and coelomocytes) seemed to

Fig. 3 Primary cell cultures from 1-week regenerating tissues (a) andfrom arm explants (b-f, from 48 h to 10 days). Phase contrast microscopy.a Regenerating tissues (1 week post amputation), 48 h culture, protocol(a). Amoebocytes (am) and coelomocytes (cm) move out of the tissue (udundissociated tissue) and seem to adhere to the substrate. The number ofcells on the substrate is not sufficient to perform any other type ofanalysis. Bar25 μm. b After 48 h in culture, protocol (c). Coelomocytes(cm) and amoebocytes (am) migrate from undissociated tissues (ud),begin to disperse, and form a cellular network on the substrate. Bar25 μm. c After 1 week in culture, protocol (c). Myocytes (my), recogniz-able from their elongated shape and large dimensions (30-150 μm), are

still able to contract. Large roundish cells can be identified as phagocytes(pg); small coelomocytes (cm) are also present.Bar50μm.dAfter 1weekin culture, protocol (c). Undissociated tissues (ud) and skeletal fragments(sk) can be easily recognized. Bar50 μm. e After 10 days in culture,protocol (c). Evident traces of bacteria (bt) and mold (md) contaminationare present on the surface of the Petri dish. Bar50 μm. f After 1 week inculture, protocol (e). Four phenotypes are distinguishable: myocytes (my),phagocytes (pg), coelomocytes (cm,) and presumptive granulocytes (gr).Bar20 μm. Modified from Di Benedetto (2011)

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adhere on the substrate (Fig. 3b, c). Poly-L-lysine was used onglass slides for analysis by scanning electron microscopy (seebelow). However, no particular improvement in cell adhesionwas observed in comparison with the Poly-L-ornithinesubstrate.

Collagen type I from rat tail, protamine and Concanavalin+ BSA were also used on plastic surfaces but resulted in noappreciable cell adhesion (data not shown).

Glass slides coated with a layer of TiO2 were also tested forthis purpose. In mammals, a wide range of TiO2 substratesseem to possess biocompatibility and bioactivity properties.However, we encountered some difficulties in crinoid cellcultures because of the dissociation of TiO2 particles fromthe slide promoted by the high medium salinity (data notshown). TiO2 nano-sized particles are known to be toxic tovarious types of cells (Shi et al. 2013).

We therefore decided to maintain Poly-L-ornithine as afixed parameter and to vary other parameters (culture mediaand in vitro tissue treatments).

Microscopical analysis

Light microscopy In the protocols (c), (e) and (f) after 48 hrsin cell culture amoebocytes moved out of the tissues forming asort of cellular network on the substrate (Fig. 3b). Although itwas quite difficult to identify all the phenotypes present inculture by light microscopy alone, some cells could be recog-nized: myocytes were easily distinguishable because of theirelongated shape, large size (30-150μm) and ability to contract

in vitro that was still evident even after one week in culture(Fig. 3c); larger round cells were identifiable as phagocytes(Fig. 3c). Undissociated tissues and skeletal parts were stillvisible (Fig. 3d). In the cell cultures obtained with protocol (e)another phenotype was also recognizable: presumptivegranulocytes, with a morula-like shape (Fig. 3f).

Avoiding the presence of undissociated tissues and skeletalparts was an effective strategy for obtaining more sterileconditions (Fig. 4a, b).

After two weeks of culture (in CMF-ASW, which wasthe best medium) cells appeared to be healthy, the platesurface being full of identifiable cellular elements, such asmyocytes and phagocytes (Fig. 4a, b, c). After five weeksmyocytes and phagocytes were still detectable and viable(Fig. 4c). After seven/eight weeks the number of cellsin vitro tended to decrease (Fig. 4d): apparent apoptoticprocesses (see below) and clotting phenomena were ob-served (Fig. 4e) and only presumptive undifferentiatedphenotypes were present (coelomocytes and amoebo-cytes). At this point the cell culture could not be consid-ered useful any longer.

Electron microscopy All the various phenotypes culturedin vitro could be morphologically characterized in detail byusing transmission and scanning electron microscopy (TEMand SEM, respectively), and on the basis of a careful compar-ison with descriptions of the same model in vivo (CandiaCarnevali and Bonasoro 2001b; Candia Carnevali 2006;Candia Carnevali and Burighel 2010).

Fig. 4 Primary cell cultures (2–8 weeks) from arm explants. Protocol (f).Phase contrast microscopy. a After 2 weeks in culture. With CMF-ASW,cells appear to be in a healthy condition, the plate surface being coveredby identifiable cellular elements, such as myocytes (my), phagocytes (pg),and presumptive granulocytes (gr). Bar50 μm. b, c After 5 weeks inculture. Myocytes (my), phagocytes (pg), and coelomocytes (cm) are still

detectable and viable. Bars20 μm (b),50 μm (c). d After 7-8 weeks inculture. The number of cells in vitro tends to decrease substantially,undifferentiated phenotypes being the only elements still present(arrows). Bar10 μm.e After 7-8 weeks in culture. Evident apoptoticand clotting phenomena occur (arrows). Bar20 μm. Modified from DiBenedetto (2011)

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Coelomocytes and amoebocytes (Fig. 5a–d), which areconsidered to be presumptive stem cells (Candia Carnevaliand Bonasoro 2001b; Candia Carnevali 2006; CandiaCarnevali and Burighel 2010), were the most numerouscytotypes: by TEM, they were both small (∼5 μm), with alarge nucleolate and euchromatic nucleus surrounded byscarce granular cytoplasm containing a few vesicles.Amoebocytes were usually elongated in shape (Fig. 5d),whereas coelomocytes were roundish cells (Fig. 5b). Thismorphological difference was much less evident in vitro thanin vivo (Fig. 5a, c). Phagocytes were also numerous: theycould attain larger sizes (15 - 20 μm) and were distinguishedby their numerous large phagosome inclusions of variable sizeand content (Fig. 5e, f). Granulocytes were less frequent butalways present. They displayed variable shapes and sizes, buttheir phenotype was always well characterized: their distinc-tive feature was the presence of electron-dense granules,which were particularly evident by TEM (Fig. 5g, h).Several myocytes were also scattered in the cultures: theywere large and elongated, with a single and eccentric nucleus,their peculiar feature being their typical contractile apparatusfilling the whole cell (Fig. 5i, l). The myocytes showed re-markable dedifferentiation processes by TEM. Myocytes inprogressive stages of dedifferentiation were characterizedin vitro by obvious signs of disorganization in their contractileapparatus (Fig. 6a–c), which has also been described in holo-thurians (the so-called spindle-shaped structures: Dolmatovet al. 2003). The degenerative nature of at least some of theseprocesses was confirmed by TEM by the peculiar morphologyof the nuclei in some myocytes, which, in most cases, showedtypical apoptotic profiles, with central or eccentric masses ofdensely packed chromatin (Fig. 6d).

By SEM, further comparisons of in vitro and in vivo pheno-types were possible. Amoebocytes, coelomocytes (Fig. 7a, b),myocytes, and phagocytes (Fig. 7c, d) were easily recognizableon the poly-L-lysine-coated in vitro glass slides, and the variousprofiles resembled the phenotypes previously observed on thecicatricial layer (Fig. 7e, f) that appears quickly on the amputa-tion surface during the repair phase of arm regeneration (CandiaCarnevali et al. 1993; Candia Carnevali and Bonasoro 2001b;Candia Carnevali and Burighel 2010; Candia Carnevali 2006).

Immunocytochemical detection of cell proliferation

In order to monitor the presence of proliferation processes incell culture (Fig. 8a), we exposed 1-week primary cell culturesof arm explants to a BrdU solution. Nuclear BrdU incorpora-tion in actively proliferating cells was revealed in sections ofresin-embedded samples of cell cultures. However, this pre-liminary approach provided no clear evidence of proliferation(Fig. 8b, c). Only a few scattered presumptive proliferatingcells could be detected (Fig. 8b).

Discussion

The aim of this investigation was to culture and study thecellular phenotypes involved in regeneration processes of thecrinoid A. mediterranea, processes that have already beencharacterized in vivo. In crinoids, this represents the firstsignificant attempt to carry out an in vitro approach. Animportant outcome is the optimization of primary culturingconditions for crinoids cells.

Although various authors have established methodologiesfor maintaining short-term viable primary cell cultures in avariety of marine organisms, protocols for the production ofsecondary cell cultures and cell immortalization (Rinkevich2011) are still needed for echinoderm models.

In our experiments on A. mediterranea, we have been ableto maintain viable primary cell cultures for 7 weeks at least.During this period, we have provided evidence of various cellactivities and processes such as migration, adhesion, dediffer-entiation, and apoptosis.

Primary cell cultures

A first series of conclusions can be inferred from our results oncrinoid cultures. An important point is that, in principle,primary cell cultures can be produced from both regeneratingtissues (although in very small quantities) and arm explants.

As far as the former are concerned, we obtained cells thatrepresented undifferentiated phenotypes, such as amoebocytesand coelomocytes, that were comparable with cell lines.However, the number of cells obtained from regeneratingtissues was always small, and the cells were consequentlyunmanageable (Table 1). In order to develop and maintain cellcultures suitable for subsequent analysis, a large amount ofregenerating tissue would be needed. A considerable numberof animals would be required, and several arms would have tobe amputated from each animal; moreover a longer processwould be required, since a suitable regeneration period(1 week) would be necessary to obtain an adequate amountof regenerating tissues that could be easily dissected andisolated. Hence, the arm explants were a much more conve-nient source of cells. Indeed, the cell numbers derived fromexplants were satisfactory and reached quantities similar tothose obtainable in mammals (protocol [f], Table 1). Cellsspreading out from the explants can be considered as theprimary elements recruited during regeneration processes.The similarities between these cells and the phenotypes in-volved in tissue regeneration in vivo were demonstrated byTEM.

As shown by the present results, a few technical aspectsdeserve specific comments: (1) the problem related to thepresence of undissociated tissues/skeletal parts, previouslyencountered in our preliminary experiments with explant cell

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cultures (Parma et al. 2005, 2006; Di Benedetto et al. 2007;Candia Carnevali et al. 2009; Di Benedetto 2011), have beensolved by resorting to a more accurate and reliable procedure,as described in Materials and Methods; (2) in primary cellcultures from explants, at least five phenotypes are present,and therefore, in order to produce specific cell lines for thefuture progress of research, an appropriate method (such ascentrifuge gradient) to select and subdivide the various phe-notypes needs to be developed; (3) as far as the problem of themedium is concerned, in spite of reports in the literature aboutcell culture media for marine invertebrates (Moss et al. 1998;Fraser and Hall 1999), we can tentatively conclude that, forA. mediterranea cell cultures, modified Leibovitz L-15 doesnot appear to be appropriate.

CMF-ASW can be employed first as the “dissociation-solution” and then replaced by a complete culture medium.CMF-ASW can also be considered to be a culture mediumitself, thereby satisfying at least two different prerequisites byacting as a suitable dissociation solution and as a culturemedium (to which nutritive elements can be added).Furthermore, it is useful because it allowed us to effectivelycontrol bacterial contamination (protocols [e], [f], Table 1).On the other hand, the total absence or at least the low level ofCa2+ and Mg2+ in the culture medium might adversely affectcell behavior in vitro. However, we do not know the extent towhich cell viability is affected by divalent ion depletion. Afurther technical aspect is that, as stated above (see Results),none of the substrates is ideal: all the substrates employed aredeficient in that the cell populations are never firmly adherentto the substrate (Table 1) and essentially form cell“suspensions”.

�Fig. 5 Comparison of in vitro (left) and in vivo (right) isolated cellphenotypes involved in arm regeneration; the in vitro cells are from a 1-week culture from explants. Thin sections viewed by transmissionelectron microscopy (TEM). a, b Coelomocytes. These are present inthe coelomic fluid and resemble morphologically undifferentiated cells,with their roundish shape, and are interpretable as presumptive stem cells.The nucleus (n) is large, and the cytoplasm is reduced to only a thin layer(rer rough endoplasmic reticulum). Specific organelles are notdiscernible. Bars2 μm (a),1 μm (b). c, d Amoebocytes. These areresident in the tissues, are elongated, and resemble morphologicallyundifferentiated cells. Their features are similar to those ofcoelomocytes and can also be interpreted as presumptive stemelements. Bars2 μm. e, f Phagocytes (ps phagosome). Thesephenotypes are well characterized both in vivo and in vitro by thepresence of their large phagosomes. Bars3 μm (e), 2 μm (f). g, hGranulocytes. These phenotypes can be easily identified on the basis oftheir large size and the presence of many electron-dense cytoplasmicgranules (gr). Bars 3 μm (g), 2 μm (h). i, l Myocytes. Contractile cellsat various stages of dedifferentiation can be frequently observed in vitroand in vivo. Their characteristic feature is the presence of a contractileapparatus (ca) reduced to spindle-like bundles of filaments.Bars3μm (i),2 μm (l). Modified from Candia Carnevali and Bonasoro (2001b) and DiBenedetto (2011)

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Cell phenotypes

A second series of conclusions is related to the regeneration-competent cytotypes. By employing a range of microscopictechniques and protocols, a number of processes and activitiesperformed by cells in vitro could be checked and monitoredregularly in A. mediterranea. In particular, we could observecell migration phenomena involving many cell types after afew days in culture. Although light microscopy did not allow

us to readily discriminate the phenotypes to which theseactivities can be attributed, the main small elements (reason-ably identifiable as coelomocytes and amoebocytes) could beeasily distinguished from other cytotypes such as myocytes(identifiable by their elongated shapes), phagocytes (identifi-able by their large roundish shapes), and granulocytes (iden-tifiable by their granular inclusions).

In terms of convincing phenotypic identification, our ultra-structural results have allowed us to confirm that, inA. mediterranea, the same progenitor cells previously charac-terized in vivo are present in vitro. In particular, the compar-ison by TEM and SEMof the different phenotypes in vivo andin vitro has provided excellent evidence of the close corre-spondence regarding both their general structures and distinc-tive elements (see, for instance, the phagosomes of phago-cytes, electron-dense granules of granulocytes, and contractileapparatus of myocytes) and has shown that in vitro pheno-types do not undergo any major modification in comparisonwith in vivo phenotypes.

With regard to the different significance and intrinsicstemness potential of the progenitor cells during regeneration,the present data cannot give any definitive answer, but theyprovide new information and some insight into the nature,commitment, and involvement of the various elements in-volved in reconstruction and development. As far as presump-tive stem elements are concerned, we have first of all toconsider amoebocytes and coelomocytes. The in vitro resultshave confirmed their undifferentiated morphology and theiramoeboid activity, which, during arm regeneration in vivo,enables them to migrate in large numbers along the brachialnerve towards the amputation site and to undergo an extensivelocal proliferation. Indeed, the amoebocytes appear to be theprogenitor cells of the blastemal cells and consequently of allthe blastema-derived differentiated cells (Candia Carnevaliet al. 1995, 1997, 1998): in other words, they might be crinoidpluripotent stem cells.

Coelomocytes are not reserve cells present in the tissues butare new elements produced by the continuous turnover of thecoelothelium (Candia Carnevali et al. 1995, 1997, 1998).Coelomocytes can therefore be considered to be the progenitorcells of all the coelom-derived differentiated cells, includingperitoneocytes, myocytes, neurons, and free coelomocytes,although a possible extra-coelomic contribution to tissue re-growth cannot be excluded. In other words, coelomocytes canalso be regarded as pluripotent stem cells. Any differencesbetween coelomocytes and amoebocytes with regard tostemness, differentiation potential, and role have still to bedetermined. Their differentiating potential will be detailed infuture experiments. The ability to culture both phenotypesin vitro makes it easier to investigate the expression of typicalstemness markers and differentiation markers.

In contrast, phagocytes and granulocytes are well differen-tiated elements. They are randomly scattered in all tissues,

Fig. 6 Primary cell cultures (1 week) from arm explants. TEM. Variousaspects of cultured myocytes. Thin sections by TEM showing presump-tive dedifferentiation phenomena at various stages. aMyocyte in obliquesection. The dedifferentiation process is indicated by the initial disorga-nization of the contractile apparatus (ca) and the presence of scatteredmyelin figures (mf). Bar2 μm. b Myocytes in cross section showingprogressive stages of dedifferentiation. The disassembly of the contractileapparatus (ca) is emphasized by the presence of large fused bundles ofmyofilaments (dark spots). Bar1 μm. cMyocytes in longitudinal sectionshowing further differentiation stages. The contractile apparatus (ca) isalmost completely disassembled. Many myelin figures (mf) are present.Intact mitochondria (mt) are still scattered in the cytoplasm. Bar2 μm. dDetails of a myocyte. The nucleus (n) shows a typical sign of theapoptotic process (chromatin condensation). The mitochondrial structure(mt) is perfectly preserved. Bar 500 nm. Modified from Di Benedetto(2011)

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with granulocytes being preferentially distributed around thebrachial nerve. Both are involved in defense mechanisms andare clearly activated by traumatic events. They appear torepresent a source of putative growth factors (as yet, incom-pletely identified: Thorndyke and Candia Carnevali 2001),which are presumably responsible for the activation of othermigratory cells (amoebocytes, coelomocytes, or others). Thein vitro results confirm the presence of these two phenotypes,

which are easily recognizable by their large roundish shape(phagocytes) and their typical morula-l ike form(granulocytes).

As mentioned above, dedifferentiated cells also usuallycontribute to regeneration. Dedifferentiation processes areparticularly evident, in vivo, in myocytes but are also seen inother mesodermal-derived cells (sclerocytes, fibrocytes;Candia Carnevali et al. 1993; Candia Carnevali and

Fig. 7 Comparison of in vitroand in vivo cell phenotypesinvolved in arm regeneration. Thein vitro cells are from a 1-weekprimary cell culture fromexplants. SEM. a–d In vitrosystem. a Amoebocytes (am) areelongated and produce a typicalnetwork of lamellipodia (lp). Bar10 μm. b Coelomocytes (cm)with their typical roundish shapebeingmaintained, even after somedays of culture. Bar 2 μm. In vivosystem. c Myocyte (my) with itsfusiform shape. Bar10 μm. dPhagocyte (pg). This phenotypecan produce short filopodia (fp).Bar 5 μm. e, f In vivo system at24 h post-amputation. e Top viewof the regenerating arm stumpshowing the amputation surface(pa) during the repair phase. Thecicatrization processes are inprogress, and the re-epithelialization of the wound isalmost completed. Bar100 μm. fEnlargement of e showing a detailof the cells involved in the repairprocesses, namelyundifferentiated cell phenotypes(cm coelomocytes, amamoebocytes). Bar10 μm.Modified from Di Benedetto(2011)

Fig. 8 A 1-week primary cell culture from arm explants. a Light micros-copy. Semithin section (crystal violet basic-fuchsin double staining) ofin vitro untreated samples. Myocytes (my) and granulocytes (gr) can beeasily recognized. b, c BrdU immunocytochemistry. Semithin sections of

in vitro samples. Only a weak presumptive signal is detectable (n labelednuclei) with respect to the control sample (c) in which the primaryantibody (anti-BrdU) had been omitted. Bars 10 μm. Modified from DiBenedetto (2011)

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Bonasoro 2001b; Candia Carnevali 2006). Dedifferentiatingmyocytes in progressive stages of dedifferentiation are char-acterized by evident signs of disorganization in their contrac-tile apparatus; in vivo, they can be frequently found either inthe arm muscle bundles close to the amputation area or in themuscular component of the coelomic myoepithelium. In vitrodedifferentiation processes comparable with those observedin vivo are commonly found; the presence in the cultures ofmany dedifferentiating myocytes provides strong confirma-tion of the active and physiological role of muscle rearrange-ment for tissue regrowth and the intrinsic potential ofmyocytes to undergo reprogramming and supply cells andother materials required for regenerative processes.

The BrdU method for monitoring cell proliferation inregenerating arm tissue is an established protocol originallydeveloped by our research group for semithin sections(Candia Carnevali et al. 1995). In this study, we applied thesame protocol in vitro. The signal indicating the presence ofproliferating nuclei was very low compared with that obtainedfrom the labeling of regenerating tissue in vivo. This weaklabelingmight reflect the need tomodify the protocol or to testother proliferation markers. Another reason could be related tothe actual absence of strong proliferation processes in ourin vitro system (Table 1). Proliferation phenomena in culturesof marine invertebrates have been reported rarely (Fraser andHall 1999; Odintsova et al. 2005; Rinkevich 1999, 2011;Shashikumar and Desai 2011). Moreover, our preliminarydata seem to confirm the lack of proliferation activities inA. mediterranea cell cultures.

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