Cell Renewing in Neuroblastoma: Electrophysiological and Immunocytochemical Char - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:10 |只看该作者 |倒序浏览 |打印

作者：Tiziana Biagiottia, Massimo DAmicoa, Ilaria Marzia, Paola Di Gennarob, Annarosa Arcangelia, Enzo Wankec, Massimo Olivottoa作者单位：a Departments of Experimental Pathology and Oncology andb Human Anatomy, Histology and Legal Medicine, University of Florence, Florence, Italy;c Department of Biotechnology and Biosciences, University of Milan-Bicocca, Milan, Italy
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      【摘要】' u! R) `9 i8 Z" R
   We explored the stem cell compartment of the SH-SY5Y neuroblastoma (NB) clone and its development by a novel approach, integrating clonal and immunocytochemical investigations with patch-clamp measurements of ion currents simultaneously expressed on single cells. The currents selected were the triad IHERG, IKDR, INa, normally expressed at varying mutual ratios during development of neural crest stem cells, from which NB derives upon neoplastic transformation. These ratios could be used as electrophysiological clusters of differentiation (ECDs), identifying otherwise indistinguishable stages in maturation. Subcloning procedures allowed the isolation of highly clonogenic substrate-adherent (S-type) cells that proved to be p75- and nestinpositive and were characterized by a nude electrophysiological profile (ECDS0). These cells expressed negligible levels of the triad and manifested the capacity of generating the two following lineages: first, a terminally differentiating, smooth muscular lineage, positive for calponin and smooth muscle actin, whose electrophysiological profile is characterized by a progressive diminution of IHERG, the increase of IKDR and INa, and the acquisition of IKIR (ECDS2); second, a neuronal abortive pathway (NF-68 positive), characterized by a variable expression of IHERG and IKDR and a low expression of INa (ECDNS). This population manifested a vigorous amplification, monopolizing the stem cell compartment at the expense of the smooth muscular lineage to such an extent that neuronal-like (N-type) cells must be continuously removed if the latter are to develop.
      【关键词】 Neuroblastoma stem cells Ion channels Electrophysiological cluster of differentiation
   INTRODUCTION

Recent studies have proposed a new way of thinking about cancer, considering neoplasia as an ecosystem in which polyclonality generates a cell population more or less imperfectly organized in an abortive organogenesis .
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Within this framework, the neuroblastoma (NB) represents a first-choice clinical and experimental model. It is a tumor peculiar to childhood, characterized by an unpredictable clinical course and multiform phenotypes, as well as by a propensity to differentiate in various directions, either spontaneously or as a result of different treatments .

A major cause of this plasticity is the fact that NB originates from pluripotent staminal compartments of the neural crest (NC) . This explains the striking heterogeneity displayed by neural crest cells (NCCs) when they are isolated in vitro and contributes to the phenotypic complexity of NC-derived tumors, especially of NB.

We designed an experimental protocol based on the use of the human NB adrenergic SH-SY5Y clone (hereinafter referred to as SY5Y) .
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In this paper, we report that after SY5Y subcloning, it is possible to recover¡ªand to recover constantly¡ªhighly clonogenic S-type cells that give rise both to N-committed cells and to a smooth muscular lineage committed to a terminal differentiation. The identification and development of various progenitors was made possible by a new experimental approach based on the association of classic immunocytochemical procedures with a qualitative and quantitative analysis of the currents expressed in each single cell by means of the patch-clamp technique.3 z9 m8 }: l. K- T; `' G2 h

MATERIALS AND METHODS
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Cell Culture and Cloning& |1 k# c0 ^3 N9 n8 w0 W* ]
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SH-SY5Y cells were maintained in RPMI 1640 medium (Euroclone, Milan, Italy, http://www.euroclone.net) supplemented with 10% fetal calf serum (Hyclone, Logan, UT, http://hyclone.com) in air containing 5% CO2, as previously described . To evaluate the S0 renewal capacity, a second round of cloning of S0 subclones was performed, diluting cells at clonal density and seeding them on CELLocate coverslips (Eppendorf, Hamburg, Germany, http://www.eppendorf.com), characterized by a microgrid, permitting the identification of single cells with precise coordinates.; l, R$ V9 q5 i5 u; t! ~: e

The clonal analysis was performed by successive limiting of dilutions of S colonies expanded to not more than 20 cells . At each passage, cells of the colonies were analyzed by the patch-clamp technique.
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Contrast-phase images of cell populations were acquired using a Nikon TMS microscope with x 20 or x 40 objective using a digital camera.9 D4 ?4 v% i( S0 H+ d% c6 o! ~

Clonogenic Efficiency3 d* m2 ^- x( m, n3 ^/ x
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N-type and S-type clones, obtained by the limiting dilution of SY5Y parental clone, were plated in 96-mm dishes at a dilution of 3 cells/ml and allowed to expand to form colonies of approximately 100 cells. Clones of this size were counted and the clonogenic efficiency estimated by the ratio of the number of colonies versus the number of cells seeded. When these colonies attained the number of approximately 100 cells, they were processed to start a new dilution procedure for two successive passages.8 E3 n- \5 ^9 B3 k1 X7 T
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Immunofluorescence
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Cells were seeded onto 13-mm glass coverslips and cultured as above in 24 multiwells (Corning, Cambridge, MA, http://www.corning.com). Cell cultures were fixed in 3.7% formaldehyde in PBS for 20 minutes at 4¡ãC and then incubated with Hoechst at 37¡ãC for 30 minutes to stain cell nuclei. Then cell cultures were washed with PBST (PBS 0.1% Triton X-100) and incubated with blocking buffer (3% bovine serum albumin  in PBST) for 45 minutes. This was followed by incubation with a primary antibody for 16 hours at 4¡ãC. Each cell population was tested for the following list of antibodies: mouse anti¨Csmooth muscle actin (SMA) (1:800, Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com); mouse anti¨CGlial fibrillary acidic protein (GFAP) (1:2,000; Sigma); goat anti¨Cneural cell adhesion molecule¨CL1 (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com); mouse anti-calponin (1:3,000, Sigma); mouse antinestin (1:800, BD Transduction Laboratories, Franklin Lakes, NJ, http://www.bdbiosciences.com); mouse anti-NF68 (1:1,500, Sigma); mouse anti-NF160 (1: 2,000, Sigma); mouse anti-NF200 (1:2,500, Sigma); rabbit anti-p75 (1:800, Chemicon, Temecula, CA, http://www.chemicon.com); goat anti¨CS-100 (1:1,000; Santa Cruz Bio-technology); and mouse anti-vimentin (1:3,000, Sigma). After washing with PBST, the cell cultures were incubated with anti-mouse Cy3-conjugated (Chemicon), anti-rabbit fluoresce-in-conjugated (Chemicon), or anti-goat fluorescein-conjugated (Calbiochem, San Diego, http://www.emdbiosciences.com) secondary antibody (1:800 dilution in PBST/3% BSA) for 1 hour at room temperature. Cells fixed onto coverslips were mounted on a glass slide with n-propylgallate. Preparations were observed on a Nikon TMS microscope equipped with fluorescence at x 40 objective. Cell images were acquired under appropriately filtered light using a digital camera. Except in a few cases specified in the legends, populations negative to the above antibodies are not illustrated in the figures.
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Patch-Clamp Recordings

Cells, seeded on 35-mm Petri dishes (Corning), were patched at room temperature after 2 days of culture, and traces were recorded with the patch-clamp amplifier MultiClamp 700A (Axon Instruments, Foster City, CA, http://www.moleculardevices.com) using the whole-cell configuration i, necessary for recording EAG currents, contained (in mM) K  Aspartate 130, NaCl 10, MgCl22, EGTA-KOH 10, HEPES-KOH 10, pH 7.4. For current-clamp experiments, the pipette solution contained (in mM) K  Aspartate 140, NaCl 10, MgCl22, HEPES-KOH 10, and Amphotericin B 150 µg/ml, pH 7.3.
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For a precise measurement of the current gating parameters, pipette, cell capacitance, and series resistance (up to 70%¨C80%) were carefully compensated before each voltage clamp protocol was run. The protocol used to measure the tail IHERG maximal current (IMAX) started from a holding of 0 mV and tested the current at ¨C120 mV, after preconditioning from 0 to ¨C70 mV for 15 seconds. The IKIR and IHERG currents were elicited at various Vm (from 0 to ¨C140 mV), starting with a holding potential of 0 or ¨C70 mV, according to Faravelli et al. o = 40 mM). For the activation of KDR currents, cells were preconditioned at ¨C80 mV, and test potential ranged from ¨C10 to  70 mV, with step increments of 10 mV; for the inactivation, cells were preconditioned from ¨C80 to 0 mV, with step increments of 10 mV, and test potential was at  60 mV. For activation of tetrodotoxin (TTX)-sensitive INa, cells were preconditioned at ¨C90 mV and currents were recorded from ¨C40 to 30 mV with step increments of 10 mV. This protocol also reveals IKDR, without overlapping of the two currents, which activate quite distantly on the time scale. For the activation of EAG currents, cells were preconditioned at ¨C60 and ¨C120 mV, and currents were recorded at  60 mV.4 {7 a2 z- S% _; L

RESULTS2 @; e2 y4 U: o; |3 X
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The Sequential Morphological Changes of SY5Y Subclones in Culture: h! s$ q6 x0 X$ S% Q+ Y- n
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The SY5Y bulk cells are characterized by small and dense cell bodies emitting subtle neurites (Fig. 1A). Limiting dilution experiments of this population invariably produced two types of subclones: N-type (hence referred to as NN), fast-growing and morphologically identical to the parental cells (Fig. 1B); and S-type (hence referred to as S subclones), slow-growing and composed of multiform, strongly substrate-adherent cells (Fig. 1C).. m+ l3 Y  o$ G; C  p. d

Figure 1. Phase-contrast images of SY5Y clone and subclones and clonogenic efficiency of N and S clonogenic cells. (A): SY5Y cells, displaying the typical N-type morphology. (B): N-type subclone isolated from the SY5Y parental clone (NN cells). (C): S-type subclone isolated from the SY5Y parental clone and observed within 20 days after cloning (S0 cells). (D): N-type cells generated in the above S-type subclone, starting from approximately day 60 (NS cells); white arrows indicate five S-type cells closed to the NS cell island. (E): Substrate-adherent cells appearing in culture from day 60 in the same S-type subclone (S1 cells). (F): Substrate-adherent cells generated by the same S-type subclone from approximately day 120 (S2 cells). Bars = 100 µm. (G): The clonogenic efficiency (calculated as reported in Materials and Methods) is reported as a function of the dilution passages. Closed squares represent means ¡À standard errors of mean (n = 3) of the clonogenic efficiency of N cells; open squares represent the clonogenic efficiency of S cells undergoing the fibromuscular S1-S2 differentiation in culture.& K( A5 y! {8 l9 g7 d4 Z$ o9 ?

The spontaneous development of S and NN subpopulations was followed up in long-term cultures, with no manipulation, except splitting at confluence. Whereas the NN subclones did not change substantially their morphology throughout the entire time course, the S subclones displayed a fairly constant pattern of changes that can be summarized as follows: soon after isolation, and up to day 60, cells appeared scattered and rather monomorphic, with rare small spikes protruding from the plasma membrane (Fig. 1C); because this represents the first progeny of single cells seeded, we called it S0. After day 60, cells gradually changed their shape, spreading out on the culture surface and becoming pleiomorphic (Fig. 1E); this latter aspect was assumed to identify an S1 subpopulation and remained substantially unchanged up to 120 to 140 days. Between 60 and 120 days, most S subclones produced N-type cells that gave rise to a fast-growing subpopulation (hence referred to as NS sub-clones; Fig. 1D), morphologically identical to the parental SY5Y and to NN cells. These NS cells need to be continuously removed to allow S1 cells to expand, giving rise to an S2 subpopulation (Fig. 1F), composed of larger cells. We do not have evidence to decide whether this inhibitory effect of N cell population on the S population is attributable to a direct cell-cell interaction or whether it is due to a prohibitive competition for nutrients or growing factors within the culture. We did not observe any evidence of apoptosis of S1 that appeared rarely dispersed but viable among the rapidly dividing NS cells.) A2 p4 b$ m1 X" l( i7 W2 q, j- s! s3 L# W

The above S0-S1-S2 sequence was obtained with each of 12 tested S subclones, suggesting that a self-renewing, at least bipotential, compartment persists in the SY5Y bulk.
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This issue was further explored by calculating the clonogenic efficiency of S0 and N cells (see Materials and Methods), obtaining the same value (approximately 55%) for both cell types at the first passage (Fig. 1G), which in turn enabled us to calculate the percentage of N and S0 clonogenic cells contained in the parental SY5Y clone. In this event, although the total clonogenic efficiency of this population was 30%, only 10% were S-type subclones. This leads us to estimate the percentages of clonogenic N and S0 cells in the SY5Y population to be 27% and 3%, respectively. Interestingly, the N clonogenic efficiency remained substantially constant over at least two successive passages, whereas for the S0 clonogenic cells we observed that in some cases (displaying signs of S1 differentiation), the efficiency dropped to 15% (Fig. 1G), whereas in others (with no sign of differentiation), it remained constant. Taken as a whole, these results suggest the following conclusions: the parental SY5Y population contains a large pool (approximately 27% of the entire population) of N clonogenic cells, endowed with an unlimited cell renewal; the S0 cells represent a minimal clonogenic pool (3% of the entire population), whose elements can either self-renew or undergo the S1-S2 differentiation. This finding is in keeping with the view that N cells represent the malignant abortive stage of neuronal components of the bulk tumor, whereas the S compartment is made of clonogenic cells susceptible to spontaneous terminal differentiation.% M+ T# ?$ a- O( T2 |. y
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Following Up SY5Y Subclone Development with Patch-Clamp: Use of Ion Currents as Electrophysiological Clusters of Differentiation
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Figure 2 shows the typical currents recorded in the SY5Y clone and subclones by means of the patch-clamp protocols (in Figs. 2A7¨C2C7). In SY5Y cells, IHERG (Fig. 2A1) showed the same biophysical properties previously described by Arcangeli et al. . Taken as a whole, the average SY5Y electrophysiological profile was characterized by the expression of substantial IHERG, a marked IKDR, and a small INa. At a lower scale, however, both NS and NN cells (Figs. 2A5¨C2C5 and 2A6¨C2C6, respectively) displayed a profile identical with that of SY5Y.

Figure 2. The electrophysiological clusters of differentiation (ECDs) identifying various developmental stages of SY5Y cells and subclones. The currents illustrated in (A¨CC) were measured in the same single cell by the whole cell configuration using the protocols reported in A7-C7. Currents are expressed in pA/pF (current density) and were specifically abolished by the following inhibitors, indicated in parentheses, with the respective concentrations: IHERG (2 µM WAY 123,398); IKDR (20 mM TEA); INa (2 µM tetrodotoxin).7 y2 W6 I  c3 S

Figure 3. Biophysical and pharmacological characterization of the K  currents observed in the cells belonging to the various clones and excitable properties of some S1 and S2 clone cells. (A): Clones N, NN, and NS. IKDR currents elicited by the activation protocol shown in Figure 2B7 in control (A1) and during the perfusion of solutions containing either 200 nM BDS I (A2) or 5 mM TEA (A3). (B): Clones N, NN, and NS. Same as (A) except the inactivation protocol is shown in the inset to panel (B3). (C): The activation/inactivation voltage-dependent curves obtained from recordings were similar to those shown in (A) and (B). Data derive from experiments done in the following clones: N (open squares), S1 (open circles), and S2 (open triangles). The Boltzmann curves had the following values of V1/2 and slope (mV): N (V1/2 18.02 ¡À 0.7, slope 9.76 ¡À 0.5, n = 21), S1 (V1/2 25.2 ¡À 2.4, slope 16.7 ¡À 1.1, n = 19), and S2 (V1/2 33.8 ¡À 3.0, slope 19.6 ¡À 1.5, n = 6). (D): Clone S1. Traces of IKDR currents elicited by the activation protocol shown in Figure 2B7 in control (D1) and during the perfusion of a solution containing 5 mM TEA (D2). (E): Clone S1. Traces of IKDR currents (EAG currents) elicited by the activation protocol shown in the inset. The line and symbol traces were elicited from holding potentials of ¨C60 and ¨C120 mV, respectively. (F): Clones S1 and S2. Excitable responses, recorded in current clamp, elicited with the protocol shown in the inset from a holding potential of ¨C70 mV. 0 = 40 mM.

With regard to the S subclones, the following information was obtained: S0 cells turned out to be practically devoid of currents, deserving the definition of "nude cells" (Figs. 2A2¨C2C2); as compared with SY5Y, S1 cells expressed much higher levels of IHERG and significantly higher INa but only negligible amounts of IKDR (Figs. 2A3¨C2C3; see details in Figs. 3D, 3E); S2 cells expressed low IHERG, substantial IKDR, and higher INa (Figs. 2A4¨C2C4; see details in Figs. 3F, 3G).

The simultaneous quantitative measurements of a cluster of currents in the same cell, as reported in Figure 2, emerged as a powerful tool to identify different cell types and successive stages in differentiation. We are indeed led to believe that each estimate of the cluster can be used to identify a specific cell phenotype, in the same way that clusters of differentiation (CD) antigens are widely used in cell biology and immunology . By analogy, we here propose to use the term electrophysiological cluster of differentiation# (ECD#) to describe the quantitative expression of a group of ion channels on the cell under study, where # is a signal identifying a determined stage of differentiation along a specific pathway.$ P* [# q( u  v
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Biophysical Characterization of IKDR Properties Observed in Different Clones and Properties of S1 and S2 Cells# {" x, [3 y0 P# {7 u) C) ~
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Before looking closely at the changes observed in the various clones and subclones during development time (Fig. 4), it is important to further introduce and clarify in detail some pharmacological and biophysical properties of the currents not described formerly in column B of Figure 2 under the general name IKDR.9 w1 e# V4 K: v0 c( w3 h% g% @

Figure 4. Time course of ion current expression in cell populations derived from a single S0 cell. Cells derived from a single S0 cell were expanded in culture as reported in Materials and Methods, removing NS cells generated in the interval indicated by the horizontal bar. At indicated times, cells were replated in dishes to be used for patching within 24 hours. Currents were recorded in the whole cell configuration, using the protocols reported in Figures 2A7¨C2C7, and are expressed in pA/pF. Each symbol represents the mean ¡À standard error of mean of seven cells. Closed circles = IHERG; open circles = IKDR; open squares = INa. The dashed area represents the percentage of excitable cells, responding to injected depolarizing currents as reported by D¡¯Amico et al. . (B): The normalized electrophysiological clusters of differentiation distribution in various subclones obtained from single S0 cells. Cells, taken from various S subclones at different stages of their spontaneous development, were patched in whole cell configuration. The triad of currents (IKDR, INa, IHERG) was simultaneously recorded in each cell, and IKDR and INa were expressed as a function of IHERG. The patch clamp protocols were the same as in Figures 2A7¨C2C7. Symbols represent the mean ¡À standard error of mean of current values (expressed in pA/pF) recorded in at least 15 cells taken from 10 different subclones. Closed circles = IKDR; open circles = INa.
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As shown in Figure 3C, IKDR recorded in the parental N, NS, and NN clones resulted in very similar voltage-dependent activation (open squares at right) and inactivation (open squares at left) curves . Indeed, the currents tested at  60 mV, when preconditioned by highly negative holding potentials (¨C120 mV), had a sigmoidal time course (open circles line) as opposed to the exponential time course observed when elicited from ¨C60 mV. The exemplary cell shown in Figure 3E was chosen because it was almost devoid of the TEA-sensitive current shown in Figure 3D1.

An interesting property of some of the S1 and S2 cells (see also Fig. 4) is the capability of becoming excitable. This property is shown in Figure 3F by using current-clamp recordings. This capability can be explained by the combination of a relatively high density of Na  voltage-dependent current and the low density of IKDR currents. This combination helps the regenerative development of a long AP, because IKDR currents are unable to repolarize the membrane potential and IHERG currents take over this function, as in the cardiac AP .

In the clone S2, we were able to observe the appearance of the inward rectifying K  current (IKIR) . Given that the IHERG current is also present, Figure 3G shows the IKIR biophysical and pharmacological characterization using the protocol shown in the inset. When elicited from a holding potential of 0 mV, we recorded the traces shown in panel G1; after the perfusion of 100 µM Ba2  (a specific inward rectifier blocker), the resulting current biophysically resembled a typical HERG current (G2), which disappeared completely when elicited from a holding potential of ¨C70 mV (G3). Indeed, at this potential, all HERG channels are completely closed and the test levels do not show appreciable outward currents in an inward rectifying channel. In the cell shown in Figure 3, the two currents coexisted, IKIR being much larger than IHERG and completely concealing it. The noninactivating IKDR currents (not shown) were small and characterized by a right shift of the voltage-dependent activation (Fig. 3C, open triangles; see other details in the legend).

These supplementary results, taken in their entirety, would in principle increase the variety of the ECDs identifying the NB subpopulations, but we decided, for reasons of simplicity, not to introduce novel variables in our cluster characterization.+ H' B+ S2 M( ?
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Time Course of ECDs- D/ t  i6 w8 n' P

The time course of ECDs in various populations generated in culture from one single S0 cell is shown in Figure 4A. Cells, appearing in culture within 0 to 60 days, maintained the nude ECDS0 profile; cells patched from 60 to 80 days progressively increased the expression of IHERG and INa while maintaining low IKDR; this produced the typical S1 profile (ECDS1 = high IHERG, negligible IKDR, relatively high INa), already evidenced in Figures 2A3¨C2C3. In the interval from 80 to 120 days, the IHERG and INa increase was followed by a marked decrease of both these currents, with no substantial change of IKDR. There-after, a progressive loss of IHERG occurred, with a substantial recovery of INa, a progressive increase of IKDR, and the expression of IKIR; these features characterized the S2 electrophysiological profile (ECDS2 = low IHERG, high IKDR, high INa). After 120 days, a sensible diminution of IKDR and INa took place, accompanying the exhaustion of the culture. Figure 4A also shows the time window within which S1 and S2 populations manifest their ability to elicit AP upon injection of depolarizing currents. A significant percentage (approximately 25%) of these types of cells was found only after the beginning of S1 cell generation, increasing up to 52% when the S2 cells prevailed in culture.- Q; z) g! B6 l8 j% j6 S

The generation of NS cells (see bar in Fig. 4A) started simultaneously with that of S1 cells, when the only cells present in culture were S0; when patched at days 20 to 30 after removal from the original S0 culture, NS cells displayed an electrophysiological profile (ECDNS) qualitatively identical to that of SY5Y (Figs. 2A1¨C2C1, 2A5¨C2C5). In general, although NS are morphologically indistinguishable from SY5Y cells (Figs. 1A, 1C), they appeared electrophysiologically immature compared with the bulk tumor cells.

The S0-S1-S2 developmental sequence was repeated with high consistency in various tested clones, although with shifts in the time scale from one experiment to another. We then normalized the current variations as a function of their ECDs, choosing IHERG as the independent variable. This choice was suggested by the fact that this current dominates the VREST of neuroblastoma cells throughout their fates; moreover, despite its huge variations in density at various stages of S cell development, IHERG maintained substantially unchanged in its biophysical parameters (activation and inactivation kinetics, time constants), which were similar to those previously described .

The normalized data (obtained from 10 separate experiments carried out as in Fig. 4A) are reported in Figure 4B, from which it will be seen that cells appear distributed as a function of ECDs, irrespective of their generation time. This plot allows the identification of three areas in which the following ECD kinetics are represented: the initial stages of the electrophysiological maturation of S0 cells starting from the "nude" ECDS0; the progressive achievement of ECDS1 by S1-committed cells; and the maturation of S2 cells up to their distinctive ECDS2. The limits of the areas can then be used, approximately, to assign single cells to one or the other of the S cell compartments as follows: S0 compartment = IHERG  60 pA/pF, IKDR  15 pA/pF, INa > 20 pA/pF; S2 compartment = IHERG  15 pA/pF.8 J2 L) {2 S- t% g* m4 n

To sum up, the sequential expression of ECDs allows a step-by-step study of the fluctuations of the cells through the S0-S1-S2 and neuronal differentiation pathways originating from the S0 compartment.

S0 Cell Staminal Features and NC Differentiation Markers Expressed Along with the Electrophysiological Maturation of SY5Y Cells
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The biological and oncological implications of the electrophysiological studies described above were explored by defining the correlations between tumor ECDs and immunocytochemical markers of normal NC development (Figs. 5¨C8). At first, we analyzed the immunocytochemical and self-renewal features of the S0 cells, identified above as the first cells appearing in culture and characterized by the nude profile ECDS0. These cells were 100% positive to vimentin and nestin (Figs. 5A, 5B) and 60% positive to p75 (Figs. 5C, 5D). These cells are therefore characterized by a common marker of substrate-adherent cells, vimentin , something that was never expressed elsewhere in any other cell type explored in this work.
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Figure 5. Neural crest stem cell markers and self-renewal in S0 cells. (A, B): Cells were examined at approximately 30 days after subcloning from SY5Y clone. One hundred percent of cells resulted positive to vimentin and nestin. Bars = 100 µm. (C, D): Cells were stained with anti-p75 at day 30 after cloning. The percentage of p75-positive cells in this population was 40%. Bars = 50 µm. Cell nuclei were stained with Hoechst 33258. (E¨CH) Phase-contrast images of the cell population generated by a single S0 cell. (E): The cell founder identified by the coordinates on a CELLocate coverslip. (F¨CH): The cells generated by the cell founder at days 2, 6, and 12; the asterisk indicates the original position of the cell founder at the beginning of the experiment. Bars = 100 µm.

Figure 6. The immunocytochemical profile of S1 cells. Cells were examined at approximately 90 days after subcloning from SY5Y cells. One hundred percent of cells were positive to vimentin (A), nestin (B), and smooth muscle actin (C), but only some were positive to calponin (D). Cell nuclei were stained with Hoechst 33258. Bars = 100 µm.  F! d! G7 Y( E7 P% N! u+ K: Z

Figure 7. Smooth muscle markers of S2 cells. Cells were examined at approximately 150 days after subcloning from SY5Y. S2 cells resulted in 100% positive to vimentin (A), negative to nestin (B), positive to smooth muscle actin (C), and positive to calponin (D). Cell nuclei were stained with Hoechst 33258, except for (A). Bars = 100 µm.- [! E- V7 Q' q$ T. v& e
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Figure 8. (A¨CC): The immunocytochemical profile of bulk SY5Y cells. Cells were harvested 2 days after seeding in six multiwells. Fifty percent of cell population turned out weakly positive to vimentin (A), whereas 100% of cells were strongly positive to nestin (B) and to NF-68 (C). (D¨CF): The immunocytochemical profile of NS cells. N-type cells were collected from one S0 progeny at approximately 55 days after subcloning; they resulted weakly positive to vimentin (90%) (D) and clearly positive to nestin (E) and NF-68 (F). The white arrows indicate S-type cells interspersed among the N-type. (G¨CI): The immunocytochemical profile of NN cells. SY5Y cells were subcloned and N-type single cells were isolated and followed up for 3 months. Immunocytochemistry was performed at approximately day 60 after cloning. Fifty percent of cells were weakly positive to vimentin (G), and 100% were positive to nestin (H) and NF-68 (I). Cell nuclei were stained with Hoechst 33258. Bars = 100 µm.! r. ^$ p, I9 l" T" G

The staminal features of S0 cells were further investigated by analyzing their capacity for self-renewal as follows: having obtained the first S0 population by an SY5Y-limiting dilution, we performed a second round of cloning. After 24 hours, we limited our follow up to single, isolated cells, identifying them by the coordinates on a CELLocate coverslip (see an example in Fig. 5E). The progenies of these cells were followed up for 12 days, producing in 58% of cases colonies composed of approximately 20 cells. All of these cells maintained the S0 morphology (Figs. 5F¨C5H) and the ECDS0 nude profile. Taken as a whole, these results indicate that ECDS0 identifies a self-renewal compartment (S0 cells) within the SY5Y population./ O% e7 X/ t' X7 v* Q+ \
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The staminal nature of S0 cells was further supported by their ability to generate at least two types of NC derivatives (neuronal and smooth muscular) .( ~% E/ J( ]) L+ h7 v% \& v0 x0 C) Q

A completely different immunocytochemical profile was displayed by the bulk N-type SY5Y cells (Figs. 8A¨C8C), which were generally negative to vimentin and positive to NF-68 and nestin. This profile argues for a neuronal commitment (NF-68 positivity) of cells that are scarcely able to adhere to the substrate (vimentin negative) . A similar degree of neuronal commitment was displayed in the NS and NN phenotype (Fig. 8D¨C8I), positive to NF-68 and nestin.
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Clonal Analysis of S0 and S1 Cells: b! a$ w% E! [5 h
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To assess whether the above S0-S1-S2 progression was initiated by environmental factors or whether it was a cell-autonomous process, we performed a clonal analysis of S0 and S1 cells. In these experiments, we started from an S colony morphologically displaying an S0 phenotype, isolated from the SY5Y parental clone. This colony was chosen at the stage when it consisted of not more than 20 cells, some of which were used for the electrophysiological analysis. Only after all the patched cells displayed the nude ECDS0 profile were the remaining sister cells diluted and seeded as single cells. These founder cells generated colonies of 20 to 30 cells within approximately 23 days, endowed with the enlarged body and the ECDS1 profile typical of the S1 cells. In some cases, moreover, these cells were endowed with the capability of responding with APs to injected depolarizing currents. Single cells from this S1 population were further processed with limiting dilutions to obtain single founder S1 cells. The latter were allowed to develop spontaneously for 33 days, by which time they produced 20 to 30 cell colonies and were then tested for their electrophysiological profile. All of the cells of these new colonies displayed large dimensions, the ECDS2 profile and the IKIR, typical of S2 cells. Taken as a whole, this clonal analysis confirmed the results obtained in the long-term cultures and lends support to the conclusion that the S0-S1-S2 differentiation pathway is both spontaneous and independent of cell interactions, even though it is strongly accelerated. It is interesting to record here that in these experiments we did not observe any substantial production of NS cells, a fact that suggests that the generation of the latter is initiated by progenitors only after the S0 colonies expand beyond the 20 cell number.- f" J" n. p# b. Z: L- Z. ~
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DISCUSSION
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In this work we investigated the developmental repertoire of NB cells with the principal aim of identifying the cell compartment responsible for tumor renewal. The relevance of this task for modern oncology can hardly be overstated, especially in the case of NB, which is a product of altered progenies of the staminal NC compartment .

A major technical novelty of this research is the use of patch clamp in an attempt to characterize the differentiative steps of NB elements emerging throughout the experimental time. The chosen method proved able to characterize single cells at various stages of differentiation by means of the simultaneous expression of grouped ion channels, for which we have proposed (see above) the denomination of ECD. This staging method has the following advantages: the high reliability afforded by the patch-clamp measurements; the simultaneous measurements of the currents cluster in the same cell; and the quantitative estimate of each component in the cluster, providing a new staging parameter based on the ratios among the expressed currents. This method proved to be decisive in disentangling the N and S cell maturation steps described in the present study and summarized in the model illustrated in Figure 9 (see Figure legend and text below for explanation).- _$ _; e7 R( c  ^* |/ G1 g
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Figure 9. Model for SY5Y cell renewal and commitment. Cell populations are classified according to their electrophysiological cluster of differentiation and immunocytochemical profile. The parental SY5Y clone contains a minute pool of S0 cells, endowed with self-renewal and committed to both the smooth muscle and neuronal differentiation lineage. The bulk tumor also includes neuronally committed highly clonogenic cells (NN cells). S0 cells can, in turn, generate neuronal progenitors endowed with high clonogenic potential (NS cells), whose progeny becomes identical to SY5Y bulk cells. The S0 commitment to the smooth muscle lineage is allowed to proceed only if the NS progeny is removed. This removal, while permitting the cell progressive amplification and differentiation along this smooth muscle lineage (S1-S2 cells), leads to the culture exhaustion. This implies that N cell accumulation causes the block of the S0-S1-S2 lineage and the maintenance of the S0 self-renewal. Without N cell removal, the final fate of the overall population repeats the configuration of the SY5Y parental clone, in which rare S0 cells are renewed within the overwhelming N-type bulk. A hypothetical interconversion N-S0 is represented by the dashed arrows.
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The choice of the cluster IHERG-IKDR-INa was suggested by our previous studies on NC electrophysiological maturation in vivo and in vitro, which was paced by an initial increase of IHERG, followed by a progressive loss of this current, substituted by IKDR . We here show that this cluster exhibits striking variations during the spontaneous development of NB in culture, and we present the remarkable finding that it begins with the "nude" profile displayed by the S0 cells (Fig. 2). This tiny cell population constitutes a self-renewing pool, occupying a high position in the hierarchical scale of NB staminal compartment.
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Our claim is supported by three main facts. First, S0 cells are nestin and p75 positive. Nestin is considered a marker of neural progenitors in both the central and the peripheral nervous system ; more importantly, we also showed that this negativity is shared by all the other cell populations individuated in this work, being lost before any restriction in the differentiation of NB cells occurs. Second, S0 cells maintained unvaried morphological and electrophysiological features throughout hundreds of passages in vitro of the SY5Y clone for at least four to five doublings (Fig. 5E¨C5H) and over a period of about 60 days (Fig. 4A), which implies that they are endowed with a high capacity for self-renewal. Third, S0 cells possess a high clonogenic power, generating a vast cell population that spontaneously differentiates into the two lineages so far identified (S1-S2 smooth muscle cells and NS cells). No GFAP-positive population emerged in this work, but it has to be said that we did not explore a glial-oriented pathway from S0 by adding specific growth and differentiation factors. The same consideration applies to other lineages, which still need to be explored to evaluate more exactly the differentiative potency of S0 cells. These factors were deliberately omitted in our experimental protocols because there was a danger of masking the natural fate of the tumor by forcing a commitment of the cells in predetermined directions.
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The S0-S1-S2 progression occurred with a remarkably constant sequence in each subclone examined; its smooth muscular commitment was attested by its positivity to SMA and calponin (Figs. 6, 7) as well as by its frequent (50%) excitability and IKIR expression (Figs. 3F, 3G). The clonal analysis experiments showed that this progression was spontaneous and independent of the environment. It must be observed, however, that this pathway leads to the exhaustion of the culture by a progressively restricting differentiation. This fact, together with the loss of nestin positivity, indicates that S0 cells are fully committed to a nonmalignant phenotype , but a definitive answer to this question could be given only after a study taking into account various growth and differentiation factors and lies beyond the limits of the present work.  u- g$ S& x( z- P
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The problem of the renewal of N cells within the SY5Y clone was further addressed by analyzing the electrophysiological and immunocytochemical maturation of NN and NS subclones (Figs. 2, 8). Such renewal started from NF-68¨Cpositive cells, which displayed a rather high IKDR/IHERG ratio and again produced a defective neuronally differentiated progeny with a deficient INa expression that accounts for their low excitability . This NN expansion implies the existence of N-committed, highly clonogenic cells and again raises the question of whether these cells derive from the S0 compartment.& Q# m# B/ w" Y4 f% K
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The N generation from S0 cells might well represent an example of the S-N interconversion, which has been supported by reliable experiments from Biedler¡¯s group . In summary, N cells seem necessary to maintain the conditions required for S0 renewal, but they also seem to monopolize the S0 reservoir at the expense of the S1-S2 direction.

As an alternative to the hypothesis of N-S interconversion, it might be possible to explain the necessity of N cells in maintaining the S0 compartment by drawing inferences from the fact that S0 phenotype is characterized by the expression of p75. The latter binds various neurotrophins (BDNF, NT-3/4/5, NGF) produced by neurons and NB cells . It is conceivable that in the SY5Y bulk the dominant effect of this interaction is the protection of S0 cells from mitogenic stimuli that would recruit them in the S1-S2 pathway to exhaustion. On this hypothesis, the ordinary maintenance of the tumor and its amplification would be attributable to N clonogenic progenitors produced by S0 cells at a pace compatible with their self-renewal. If these assumptions prove to be correct, they would entail important consequences for the clinical treatment of cancer in that the elimination of the N cells by available therapies, both chemotherapies and radiotherapies, would be seen to have two distinct functions: eliminating the bulk of the tumor and helping to induce S0 compartment exhaustion in the S1-S2 pathway.
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Finally, our study revealed the advantages of using the ECD description of complex cell populations based on an integrated analysis of electrophysiological and immunocytochemical studies. This procedure makes it possible to retrace the natural history of the individual cells that converge in such populations at any given time, and this provides an overall insight into the distinct stages of their maturation, which is an important parameter in developmental biology and a crucial oncological index.

ACKNOWLEDGMENTS

We thank Dr. Lukas Sommer for his scientific suggestions and discussion, Marco Cutr¨¬ for his skilled technical support, and Professor Patrick Boyde for help in the revision of the manuscript. This work was supported by grants from the Associazi-one Italiana per la Ricerca sul Cancro (AIRC), Ministero dell¡¯Istruzione, dell¡¯Universit¨¤ e della Ricerca Scientifica e Tecnologica (MIUR, COFIN 2002¨C03), Associazione Italiana contro le Leucemie (AIL) Firenze, and Ente Cassa di Risparmio di Firenze (CARIFI).1 L2 Q6 Q4 w) w0 {. p- {5 g

DISCLOSURES
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The authors indicate no potential conflicts of interest.
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