Alicia Le Du

Andrea Sabatié-Gogova

Alfred Morgenstern

Gilles Montavon
email: montavon@subatech.in2p3.fr

IS DTPA A GOOD COMPETING CHELATING AGENT FOR Th(IV) IN HUMAN SERUM AND SUITABLE IN TARGETED ALPHA THERAPY ?

Keywords: thorium; targeted alpha therapy, speciation, transferrin, DTPA

The interaction between thorium and human serum components was studied using difference ultraviolet spectroscopy (DUS), ultrafiltration and high-pressure-anion exchange chromatography (HPAEC) with external inductively conducted plasma mass spectrometry (ICP-MS) analysis. Experimental data are compared with modelling results based on the law of mass action. Human serum transferrin (HSTf) interacts strongly with Th(IV), forming a ternary complex including two synergistic carbonate anions. This complex governs Th(IV) speciation under blood serum conditions. Considering the generally used Langmuir-type model, values of 10 33.5 and 10 32.5 were obtained for strong and weak sites, respectively. We showed that trace amounts of diethylene triamine pentaacetic acid (DTPA) cannot complex Th(IV) in the blood serum at equilibrium. Unexpectedly this effect is not related to the competition with HSTf but is due to the strong competition with major divalent metal ions for DTPA. However, Th-DTPA complex was shown to be stable for a few hours when it is formed before addition in the biological medium; this is related to the high kinetic stability of the complex. This makes DTPA a potential chelating agent for synthesis of 226 Th-labeled biomolecules for application in targeted alpha therapy.

Introduction

The quantitative description of the interactions of thorium (IV) with blood serum components is of high relevance for the rational design of molecules suitable for in vivo chelation of thorium in medical applications [1,2] or for decorporation in case of accidental ingestion [3][4][5]. In the first case, the chelating agent is used to couple the radionuclide to tumor selective carrier molecules to target and destroy cancer cells [6]. Here the Th(IV)-chelate complex is formed in vitro before application to the patient. The alpha emitters 226 Th (t 1/2 =31 min) and 227 Th (t 1/2 =18,7 days) are promising radionuclides for application in targeted alpha therapy (TAT) [1,2,7]. In the second case, the ligand should target the unwanted metal ion in vivo and form soluble complexes which can be excreted via the kidneys.

Thorium distribution in rats serum was studied by Peter and Lehmann in 1981 [8]. By the means of chromatographic studies and competition experiments with iron, the authors reported that the binding site of Th(IV) was the iron-binding site of transferrin (HSTf).

Similar behaviour was reported for different actinides (IV) including Pu(IV) from various in vivo and in vitro studies [3,[9][10][11]. The interaction between Th(IV) and HSTf was further studied by Harris et al. [12] by using difference ultraviolet spectroscopy (DUS). As in the iron-transferrin complex, two Th(IV) ions are bound to HSTf protein at the physiological pH.

Two and one tyrosine groups were proposed to coordinate to thorium in strong C-terminal and weak N-terminal sites, respectively. In conclusion, HSTf appears as the main component governing Th(IV) speciation in blood serum [13]. An estimation of the complexation constants can be obtained by extrapolation from the emprical law presented by Sun et al. [14].

However, to the best of our knowledge, they have never been experimentally measured.

Furthermore, one recent study showed no interaction between Th(IV) and HSTf [15], these results being rather conflicting with the others.

The first objective of the present paper is to complete available literature data to give a better comprehensive view of Th(IV) speciation in blood serum. The interaction of Th(IV) with HSTf has been studied by Difference ultraviolet spectroscopy (DUS) to determine complexation constants. The effect of human serum albumin (HSA) on Th(IV) speciation in the blood serum, the second important metallo-protein present in the biological medium, has been also estimated. Based on these parameters, and including literature data of thermodynamic constant of Th(IV) complexation with low molecular weigh organic and inorganic ligands, a model calculation was performed to describe Th(IV) interactions in blood serum. The reliability of the simulation is considered regarding the published in vivo experimental speciation results [8] and the experimental data from the speciation studies realized by High Pressure Anion Exchange Chromatography (HPAEC) with inductively conducted plasma mass spectrometry (ICP-MS) detection.

In addition, the developped model is used in combination with in vitro studies performed by ultrafiltration, to assess the efficiency of diethylene triamine pentaacetic acid (DTPA) to form stable complexes with Th(IV) under blood serum conditions. DTPA is the chelate recommended for the decorporation of Th(IV) [START_REF] Ammerich | Guide National "Intervention médicale en cas d'événement nucléaire ou radiologique[END_REF]. It is not toxic for humans and forms a strong complex with Th(IV) [START_REF] Anderegg | [END_REF][START_REF] Smith | Version 8.0 for windows[END_REF]. According to the "hard and soft (Lewis) acids and bases" (HSAB) therory [START_REF] Pearson | [END_REF], it presents nitrogen and oxygen atoms suitable for the complexation of the "hard" Th(IV) cation and it is well preorganised for the coordination of spherical cations. Furthermore, it can be easily attached to a biological vector; this is a pre-requisite in TAT and may be useful in chelation therapy. The obtained results are discussed to assess the potential use of DTPA as a chelating agent for TAT with 226 Th.

Materials and methods

Reagents

Human serum transferrin (HSTf, apo-transferrin human ≥ 98%, Sigma) was purified by sizeexclusion chromatography on a PD-10 column using 0.1 M NaCl / 2.10 -3 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.4 as an eluent. HSTf concentration was determined spectrophotometrically at = 280 nm using 93000 cm -1 M -1 as a molar extinction coefficient [20]. Human serum albumin (HSA, albumin from human serum ≥ 96%, Sigma) was used as received. Human serum was supplied by Lonza and was diluted in 0.1 M NaCl solution buffered at pH 7.4 equilibrated with 1 % CO 2 gas. Th(IV) stock solution was prepared by dilution of a standard solution of 232 Th ( 232 Th(NO 3 ) 4 (H 2 O) 4 ) (from Prodis) to obtain a final concentration of 2.10 -3 M. Other chemicals were analytical grade. HSTf, HSA and carbonate stock solutions were freshly prepared before use.

Samples preparation

Solutions were prepared with ultrapure water (MilliQ, 18 MΩ . cm). All experiments were performed in solution of pH 7.4  0.1 and ionic strength of 0.1 (NaCl) buffered with 2.10 -3 M (HEPES) at room temperature (20 ± 2 °C). Samples in the absence of carbonate were prepared in a glove box under 100% nitrogen atmosphere. Samples in the carbonate system were prepared in a glove box under 99/1% nitrogen/carbon dioxide atmosphere. For the complexation study realized as a function of carbonate concentration, the carbonate ion was added to the solution prepared under 100% nitrogen atmosphere. The samples were then kept closed during the experiment to prevent equilibration with atmospheric CO 2 . For the complexation study realized as a function of phosphate or nitrilotriacetic acid (NTA) concentration, the samples were prepared under normal atmosphere. Inorganic carbon concentration was determined at equilibrium by total organic carbon analysis (TOC).

Analytical tools

pH measurements were performed using a PHM220 from MeterLab. Inorganic carbon analyses were done on a TOC-V CSH apparatus supplied by Shimadzu. Thorium analyses were performed by ICP-MS using a XSeries 2 provided by Thermo Fisher (sensibility limit of 0.1 ppb). UV-spectra were recorded on a UV-visible UV-2401 PC spectrophotometer from Shimadzu. The centrifugation experiments were performed using a centrifuge RC6 from Thermo Fisher at 27000 g. The HPAEC device was a Dionex UltiMate3000 system consisting of a DGP-3600 MB pump, an AS3000 auto-sampler, a TCC-3200B column oven and a diode array DAD-3000 detector. The stationary phase was a Dionex AS20 anionic exchange column (2 mm diameter x 25 cm length), with an AG20 guard column (2 mm diameter x 5 cm length). The AS20 consists of a hydrophilic polymer grafted with quaternary alkanol ammonium ions. The total capacity represented by both columns is 79 microequivalents.

Difference ultraviolet spectroscopy (DUS)

DUS is the most widely used technique to evaluate metal binding to HSTf [21]. Metal complexation was estimated from the absorbance measurements using the difference spectra of the metal-protein complexes vs. unmetallated apo-protein. Experimental conditions are reported in Table 1. The peak area in the range 220-270 nm were monitored to describe and to quantify Th(IV)-HSTf interaction. Equilibrium is established when the absorbance does not change during an interval of 2 hours. The error associated to the absorbance was estimated as 0.03 based on repetitive experiments. The main advantage of the method is that the absorbance signal of HSTf is not influenced by the signals of other ligands which might interact with Th(IV) (e.g. carbonate). The interactions of HSA, DTPA and citrate with Th(IV) were studied using competitive method with HSTf [22].

Ultrafiltration

This technique was used for Th / HSTf (1% CO 2 ) and Th / serum (1% CO 2 ) systems to differentiate the fraction of thorium bound to low-and high-molecular weight compounds.

Experiments were performed in dialysis bags (Microcon Millipore, 3 kDa) and the separation was done by centrifugation at 27000 g. Thorium concentration in the filtrates was determined by ICP-MS analysis. A blank was systematically done in parallel to assess the amount of Th(IV) adsorbed on the membrane. It was assumed that the separation time (typically 30 minutes) was not sufficient to perturb equilibrium conditions and that the concentration of Th(IV) in the filtrate represented the concentration of Th(IV) associated to the low-molecular weight compounds (e.g. OH -, CO 3

2-

).

HPAEC analysis

The eluent at pH 7.4 was a solution of 0.1 M NaCl buffered with 2.10 -3 M phosphate buffered saline (PBS, that as 10-fold concentrate, contains 0.1 M of sodium phosphate (Na 3 PO 4 ) and 9% of NaCl) equilibrated with 1 % CO 2 gas controlled by bubbling. The choice of 1 % CO 2 will be explained latter. Samples and eluent were prepared in a glove box under 99/1% nitrogen/carbon dioxide atmosphere. The anion exchanger was pre-equilibrated with the eluent before starting the experiments. Samples were injected into the column through a 50 µL sample loop injector, with a flow rate of 350 µL.min -1 . Proteins and serum were detected spectrophotometrically at 280 nm. Th(IV) concentration was determined from collected fractions by ICP-MS analysis.

Modelling

Computer simulations were performed with the PHREEQC program [23]. Stability constants for metal ion complexes in solution are reported in Table 2 and were taken from NEA-OECD compilation [START_REF] Rand | Chemical Themodynamics of Thorium[END_REF] (complexation of Th(IV) with inorganic ligands), IUPAC [START_REF] Anderegg | [END_REF], NIST database [START_REF] Smith | Version 8.0 for windows[END_REF] (Th(IV) / DTPA) and from [START_REF] Felmy | [END_REF] for citrate and [26] for NTA. The pKa values of sulphates and phosphates ions given by PHREEQC database were taken into account. The constants given in the NIST database at an ionic strength () of 0.1 M were extrapolated at zero ionic strength using the truncated Davies equation [27].

The complexation of Th(IV) with HSTf was described with the model proposed by Montavon et al. [28]. The interaction between Th(IV) and HSTf was described by using two binding sites at the protein surface ( S HSTF 1

 and S HSTF 2  ). Any metal ion considered [21], the stability constants for interaction with the binding sites differ by approximately one unit of their log K value, the difference being probably related to outer-sphere effects [22]. This ratio of 10 for K was set as a fixed parameter in the fitting of our experimental data. Consequently, the formation of the complex has been described as:

2 2 3 2 , 1 2 3 4 2 2 , 1 ) ( ) (            n n CO STh HSTF CO n Th S HSTF (1)
Where S HSTF 1

 and S HSTF 2  represent the strong and weak binding sites, respectively, and the charge of -2 relates to the two deprotonated tyrosine groups.

Alternatively modelling was also performed using the Langmuir-type model generally used in the literature. The model takes into account the metal ion speciation and the presence of two binding sites at the protein surface:

       4 1,2 1,2 4 S HSTF - Th S HSTF Th (2)
The constants log K 1 =19.3 and log K 2 =18.2 have been extrapolated from the empirical expression given in [29] and derived from the work presented in [14]. A similar Langmuirtype model was used to described Th(IV)/HSA interaction with the consideration of one binding site.

In conclusion, for the two studied systems Th(IV)/HSTf and Th(IV)/HSA, there is only one unknown parameter (complexation constant) to be determined. The fitting of experimental data was done "by hand".

Results and discussion

Binding of Th(IV) to protein

Binding of Th(IV) to HSTf

Complexation of Th(IV) with HSTf did not occur under physiological-type conditions (pH=7.4, 0.1 M NaCl) when precaution was taken for avoiding equilibrium with atmospheric CO 2 . HSTf was not able to compete with OH -in the main Th(OH) 4 and (Th) 4 (OH) 12 +4 forms.

To complex Th(IV), a synergistic anion was therefore considered in the present work, starting with the NTA, the one used by Harris et al. and Jeanson et al in previous studies [12,15]. The second one corresponds to carbonate, CO 3 2-being the synergistic anion allowing the complexation with iron and other metal ions [21]. Finally, the third one is HPO 4

2-

, phosphate ions being present in the blood serum and being known to be a strong complexing agent for Th(IV) in environmental samples [30].

Complexation as a function of NTA concentration has been first investigated. The complexation kinetic was very slow; more than 4 days were necessary to reach equilibrium between Th(IV) and HSTf. The experimental data are presented in Figure 1. The complexation with HSTf starts above 10 -6 M to reach a maximum of complexation at about 10 -4 M, corresponding to an absorbance at 242 nm of 0.28. The start of this increase coincides with the significant formation of Th(L) 2 2-species (L=deprotonated NTA) in the absence of HSTf (figure 1A). Our data are coherent with the one reported by Harris et al. (figure 1B).

Above 10 -4 M, the decrease in absorbance indicates dissociation of the Th-HSTf complex arising from the increasing competition between free NTA and HSTf for Th(IV) as NTA concentration increases.

Complexation as a function of carbonates concentration has been investigated in a second step. Kinetic of complexation was more rapid than in the presence of NTA (30 hours vs 4 days). The experimental data are presented in Figure 2B. In the carbonate concentration range from 0 to 10 -4 M, no complexation between Th(IV) and HSTf has been observed. The complexation with HSTf starts for a carbonate concentration above 10 -4 M, until a plateau is reached corresponding to an absorbance of 0.47. It is worth saying that the complexation coincides with the significant formation of the ternary complex Th(OH) 2 (CO 3 ) 2 2-in the absence of HSTf (figure 2A).

Complexation as a function of phosphate concentration has also been studied. In the concentration range from 0 to 10 -2 M, no complexation was observed between Th(IV) and HSTf even after 2 weeks of equilibration (data not shown). Unlike NTA and carbonate, phosphate does not act as synergistic anion.

A quantitative analysis of experimental data obtained with NTA and carbonates was done as follows: the shape of the curves was reproduced by varying the values of the complexation constants between Th(IV) and HSTf and the conversion of absorbance in concentration was done considering a proportionality according to the Lambert-Beer-law (Figures 1C and2C).

In the case of NTA system, we considered the possible formation of ThL 2

2-

, the main species existing under our conditions. A curve similar to that experimentally observed in the presence of carbonate is predicted with the occurrence of a plateau for the highest NTA concentrations studied. The decrease of complexation observed above NTA concentration of 10 -4 M can be quantitatively explained considering the complexation of HSTf with one Th(IV) and one ligand L 3-. A good agreement between the experiment and the modelling was obtained with K 1 = 10 31 and K 2 = 10 30 , provided that the maximum of complexation / absorbance observed corresponds to 40 % of Th(IV) complexed to the protein (figure 1C).

For the carbonate series, we first considered the formation of a ternary complex involving one carbonate ion, as it is the case for Fe(III) or U(VI) [21,28]. The increase of complexation can be relatively well reproduced till a carbonate concentration of 10 -3 M with stability constants of log K 1 = 27 and log K 2 = 26 for strong [31] and weak sites, respectively. At higher carbonate concentrations, the fraction of HSTf bound to Th(IV) is expected to decrease, due to increasing competition of carbonate with HSTf for Th(IV) complexation leading to the complex Th(OH) 2 (CO 3 ) 2 2-(dotted line in Figure 2C). A much better agreement was obtained with two carbonate ions acting as synergistic anions: both the increase of the curve and the plateau were well described by simulation with log K 1 = 33.5 and log K 2 = 32.5. This complexation might result from the complexation of the existing species Th(OH) 2 (CO 3 ) 2 2after release of two hydroxide groups. It is important to notice, that the agreement between the experiment and the theory is good provided that we consider 24% of Th(IV) not bound to the protein on the plateau (figure 2C). This was confirmed experimentally by ultrafiltration experiments; 17 ± 2 % of Th(IV) passed through the membrane and was therefore not bound to the protein.

The analysis of the data shows that the nature of the ternary complexes and the associated stability constants rely on the quantitative description of Th(IV) interaction with lowmolecular weight compounds. We can finally calculate for the complexes 2:1 Th(IV):HSTf apparent absorptivities normalized with respect to HSTf concentration ( obs ) of = 60±7 and 65±8  10 3 M -1 cm -1 for carbonate and NTA systems, respectively. The value obtained for the carbonate system was used to simulate the titration curve of HSTf with Th (Figure 2D). The determination of similar values, within experimental errors, is in agreement with the fact that the absorption band is linked to the interaction of the metal ion to the phenolic groups of the tyrosine residues which is expected similar in the two studied systems. The difference in type and number of synergistic anions coordinated in the first coordination sphere do not contribute to the extinction coefficient.

Interaction of Th(IV) with HSTf, compared to the interaction U(VI)-HSTf or Fe(III)-HSTf, seems to be different. First, two carbonates are necessary to bind to Th(IV), whereas only one synergistic ion is necessary for most the metal ions, like Fe(III) or U(VI). Secondly, kinetics of complexation at HSTf surface are slow for Th(IV), whereas they are rapid for Fe(III) or U(VI) (only 20 min for U(VI) binding vs 30 h for Th(IV)) [28,31]. It is worth saying that the absence of complexation between Th(IV) and transferrin observed by Jeanson et al [15] is certainly due to a kinetic problem; Th and HSTf were contacted only for a few hours. Several parameters may explain these differences between Th(IV) and Fe(III) binding. The ionic radius, for example, being much higher for Th (r = 105 pm) than for Fe (r = 65 pm) [32], the penetration of Th(IV) into the lobe of the protein may be more difficult resulting into slower kinetics. Moreover, the coordination number is 8 for Th(IV), whereas it is of 6 for Fe. In both the N-and C-terminal binding sites of HSTf, the metal ion is coordinated by four groups: two tyrosines, one histidine, and one aspartic acid providing four coordination sites for the metal.

Th(IV) with a coordination number of 8, needs to complete its coordination sphere with four other coordinating groups, e.g. by two synergistic carbonates, in a bidendate mode of coordination. Fe, to complete its coordination sphere, needs only one carbonate. The occurrence of only one synergistic anion coordinated in the presence of NTA may result from steric effects, NTA being larger than carbonate.

The experiments reported in the following part have been realized at a carbonate concentration of 5.10 -3 M (1 % CO 2 ), where maximal complexation between Th(IV) and HSTf occurs.

Binding of Th(IV) to HSA

The interaction between Th(IV) and HSA has been studied using the DUS competition method in the presence of HSTf. This method is applicable for [HSA]<8.10 -5 M, corresponding to about one eigth of the HSA concentration found in human serum. Above HSA concentrations of 8.10 -5 M, the DUS competition method was no more applicable because the signal to noise ratio became too low. Experimental data are shown in Figure 3. In this range of HSA concentration, no significant complexation between Th(IV) and HSA was observed; although the absorbance of the Th(IV)-HSTf complex shows a decreasing trend with increase of [HSA], the decrease was not significant within experimental errors. The data show that interaction of Th(IV) with HSTf is stronger than with HSA: in the presence of eight times more HSA than of HSTf, Th(IV) remains bound to HSTf.

Th(IV) speciation in human serum

As mentioned in the introduction, the behaviour of Th in the presence of proteins and in serum has been studied by HPAEC with external ICP-MS analysis. Usual hyphenated techniques of speciation of trace elements in biological environment were reviewed by Lobinski et al. [33]. The data with good sensitivity were obtained, more often, by using the size-exclusion or reversed phase HPLC chromatography allowing species separation. Having also interest to identify free Th or complexed Th chemical forms, we have been working on anion-exchange chromatography with the detection done by ICP-MS general method.

On the basis of the results presented in the previous section, we expect that Th(IV) is mainly interacting with HSTf in human serum. An attempt to confirm this result is presented in this section both experimentally by HPAEC and theoretically.

HPAEC-ICP-MS results

Under non-perturbing conditions (the eluent corresponds to a physiological-type medium; pH=7.4, 0.1 M NaCl), the method allows the separation of HSA and HSTf proteins: HSTf is not retained (elution time = 2.5 min) while HSA is eluted as a broad peak at 10.5 min. The method has been tested for Th(IV) speciation in blood serum using physiological-type media.

Figure 4 shows UV-and ICP-MS-chromatograms obtained with the different samples described in Table 1. Experiments were first realised in physiological-type media where the Th(OH) 2 (CO 3 ) 2 2-complex is the major Th(IV) species in solution. Only 0.5 % of Th(IV) could be eluted and no significant peak has been attributed to Th(OH) 2 (CO 3 ) 2 2-or detected in the time scale studied (1h30). A cleaning method with acidic solution (1 M HCl) was used to eliminate not restored Th(IV) after each experiment. The origin of this strong interaction is not known, but it will modify the equilibrium conditions controlling Th(IV) speciation in the injected solutions, as shown below.

For the injection of the solution of Th(IV) pre-equilibrated with HSTf, the Th(IV) peak was detected at the position corresponding to the one of HSTf (Figure 4A,D). This corresponds to the direct signature of the complex formed and identified by DUS. However, only 6% of Th(IV) could be recovered (96% of this fraction being present with HSTf), while 80% of Th(IV) was initially bound to HSTf in the injected solution. A similar result was obtained in the presence of HSA, Th(IV) being eluted at the retention time of HSA between 10 and 15 min (Figure 4A,D). 0.5% of the total concentration was recovered; 80% of this Th(IV) was found to be bound to HSA. Both Th(IV)-HSTf and Th(IV)-HSA complexes can therefore be identified by HPAEC. The interaction strength between Th(IV) and the proteins is strong enough for avoiding a complete sorption of Th(OH) 2 (CO 3 ) 2 2-by equilibrium displacement on the resin. The percentage of eluted Th(IV) being less in the presence of HSA than in the presence of HSTf indicates that the stability constant is higher for HSTf than for HSA, in agreement with the results of the previous section.

Th(IV) was next mixed with both proteins (HSTf and HSA) (Figure 4B,E). HSTf and HSA concentrations were fixed to mimic proteins concentrations in human serum (see Table 1). As in previous experiments, only a few percent of Th(IV) were eluted (10.5 %). 85% of Th(IV) content was found to be bound to HSTf, the rest was in interaction with HSA (15%). This distribution can be quantitatively calculated using the complexation constants describing Th(IV)/HSTf interaction and considering a complexation constant of 10 17. Th(IV) speciation was next studied in human blood serum corresponding to the "real" biological system. After equilibration in the biological media for 24 hours, Th(IV) was eluted at the retention time between 3 and 5 min (9% eluted). Th(IV) appears to be well in interaction with HSTf, nevertheless no interaction with HSA was determined contrary to the observation in the synthetic system of Th(IV)/HSTf/HSA. This result is in agreement with

Peter and Lehmann's work reported that the Th was bound to HSTf in the serum of rat [8].

Modelling

Both data obtained in vivo as well as the results of our study identify HSTf as the key component governing Th(IV) speciation in blood serum. Similar to the study published on speciation of uranium in human blood serum [34], we wanted to verify these experimental results for trace concentrations of Th(IV) (10 -9 M) in a predictive way by using the law of mass action principle. Due to the complexity of the human serum, the following simplifications were considered on the basis that the interaction strength of given serum components with Th(IV) will depend on their concentrations as well as on their equilibrium complexation constants:

(i) all relevant inorganic ligands were taken into account (hydroxide, carbonate, phosphate, chloride, sulphate, nitrate) with the constants given in Table 2;

(ii) for the low-molecular-weight organic components, only the citrate ion was considered (Table 2) as it is present in human serum at a relatively high concentration (1.6

 10 -4 M) and has a relatively high stability constant for Th(IV) complexation [START_REF] Felmy | [END_REF];

(iii) both HSTf and HSA proteins were considered with the constants given in Figures 2 and3 captions;

(iv) given the high affinity of iron for HSTf, we can safely consider that trace concentrations of Th(IV) can not compete with Fe(III) for HSTf binding sites. The number of available HSTf binding sites was therefore set to 70 % of the total capacity, corresponding to the fraction of HSTf not loaded with ferric ion [35];

(v) Ca was considered as a competitive metal ion for HSA sorption sites [28].

In agreement with experiments performed with blood serum, 99.9 % of Th(IV) is predicted bound to the protein pool, 99% being attached to HSTf. A less good agreement was obtained using Th(IV)/HSTf constants extrapolated from the relation of Sun et al. This shows that the extrapolation is misleading in the present case resulting in a underestimation of Th(IV)/HSTf interaction. The values to be used and which can be recalculated from our data according to Eq. ( 2) are log K 1 =21.2 and log K 2 =20.2. All the results show that HSTf is the key component governing Th(IV) speciation in blood serum. 2 and complexation constants previously determined with HSTf), DTPA starts to complex Th(IV) at the concentration of 10 -6 M to reach conditions where 100% of Th(IV) is complexed with DTPA. An attempt of quantification was also done for the DTPA series. Since the recent NEA-OECD database does not propose any stability constant for Th/DTPA interaction, we have used two different quantitative models. The first one involves one complex ThL -with log K of 33.1 (=0) [START_REF] Anderegg | [END_REF] whereas the second one considers different species proposed in the NIST database [START_REF] Smith | Version 8.0 for windows[END_REF], i.e. ThL -, ThHL and ThOHL 2-with thermodynamic constants recalculated at =0 (Table 2). The ThL -is the major species under studied experimental conditions. A good agreement between the experiment and the calculation was obtained for logK (ThL -) > 10 29 . This is in agreement with published constants and this allows an indirect validation of the complexation constants quantifying the interaction between Th(IV) and HSTf. We will next use the data from [START_REF] Anderegg | [END_REF] for the simulations.

DTPA is a stronger chelating agent than citrate and its strong affinity for Th(IV) explains the application of this ligand as a decorporation agent [START_REF] Ammerich | Guide National "Intervention médicale en cas d'événement nucléaire ou radiologique[END_REF]. Note that hydroxypyridone ligands appear more efficient than DTPA for in vivo decorporation of Th(IV) [4,5]. In such an application, the amount of chelating agent is relatively important allowing to reach high concentrations at the injection points (about 0.01 M after injection of 200 mL of saline buffer considering a common dose of 30 µmol/kg body mass and a 70 kg person) and after dilution in the blood (~ 4.10 -4 M for 5 L of blood volume) [36]. According to our simple model developed for the serum, the efficiency of complexation is predicted in vivo in both conditions with 100 % of Th(IV) bound to DTPA considering trace concentrations of Th(IV) (10 -9 M).

Taking into account the amount of ligand injected, we have neglected competition effects with trace metal ions but we have considered a possible competition with major cations using the DTPA/M 2+ (M 2+ =Mg 2+ , Fe 2+ , Ca 2+ ) constants and "free" blood concentrations (i.e. not bound to proteins) given in [START_REF] Smith | Version 8.0 for windows[END_REF], and [29], respectively. The complexation with Th(IV) can thus occur and the complex can be eliminated naturally when used immediately after the contamination.

The quantitative analysis show that the complexation becomes inefficient (~ 1% complexed)

for a DTPA concentration of ≤10 -5 M. It is worth saying that this inefficiency is not related to the HSTf vs DTPA competition but to the increasing competition with the divalent Fe 2+ , Mg 2+ and Ca 2+ ions as the ligand concentration decreases. When the Th(IV)/M(II) competition was not considered, the complexation remained efficient for trace DTPA concentration (10 -9 M) with 71 % of Th(IV) complexed with DTPA. For TAT application, the concentration of injected chelate is usually about 10 -9 M reaching a value around 5.10 -11 M after dilution in the blood serum. Taking into account this concentration, DTPA will be inefficient to complex Th(IV) in vivo.

In vitro studies

In vitro experiments have been carried out to assess the reliability of the above predictions.

Th(IV) speciation was followed by ultrafiltration allowing the separation between lowmolecular (i.e. DTPA) and high-molecular (i.e. HSTf) weight compounds. Considering the sensitivity of the ICP-MS and the dilution required for the analysis, Th and DTPA concentrations were fixed at 5.10 -7 M and 10 -6 M, respectively. The concentrations are weak enough to expect an inefficient Th(IV) complexation (see the above section).

Experimental results are shown in Figure 6. In the first experiment, Th(IV) is pre-equilibrated with serum before addition of DTPA. In agreement with the prediction, DTPA cannot catch Th(IV) in the blood serum. In the second experiment, DTPA was pre-equilibrated with Th(IV) in 0.1 M NaCl before addition in the human serum. The Th-DTPA complex appears surprisingly stable. This is explained by the strong kinetic stability of Th-DTPA complex: the reaction time is not sufficient to reach equilibrium conditions in the time scale studied (till five hours). Five hours correspond to about 10 half-lives of 226 Th, the time for the radionuclide to "disappear". Therefore, although DTPA cannot complex Th(IV) in vivo, it may be efficient to carry the radionuclide in TAT, when the biological molecule is being labelled before injection.

Conclusion

HSTf governs Th(IV) speciation in the blood serum. This was shown both experimentally and theoretically. Using the generally used Langmuir-type model, values of 10 21.2 and 10 20.2 were obtained for strong and weak sites, respectively. Although DTPA appears to be a good decorporation agent, the thermodynamic predicts that its concentration used in TAT is not sufficient to allow an efficient complexation of Th(IV). This inefficiency is surprisingly not related to the competition with HSTf but is related to the strong competition with major divalent metal ions for DTPA. However, the high kinetic stability of Th-DTPA complex allows use of the ligand for synthesis of 226 Th labeled biomolecules, when the complex is formed before injection. Studies on the stability and cytotoxicity of 226 Th-DTPA-antibody contructs in vitro and in vivo are ongoing.

Table of Abbreviations

DTPA

Acknowledgment

We thank the financial support for Alicia Le Du provided by the region Pays de la Loire (project PRORRIM 2), and the "Ecole des Mines de Nantes". 1 and2, respectively. Th(IV)/HSTf interaction was described considering two carbonates bound with log K 1 = 33.5 and log K 2 = 32.5. Th(IV)/HSA interaction was described using a Langmuir-type model with K = 10 17.5 .

UV absorbance / u.a. 1 and2, respectively. Th(IV)/HSTf interaction was described considering two carbonates bound with log K 1 = 33.5 and log K 2 = 32.5.

  5 for describing Th(IV)/HSA interaction. This estimated value was next used for the treatment of DUS experimental data (Figure3A). As shown in Fig3A, a slight decrease of the percentage of the Th(IV)-HSTf is predicted as the HSA concentration increases, although this trend could not be experimentally proven because of too high uncertainties associated to the absorbance measurements.

[ 14 ]

 14 reviewed by Ansoborlo et al.[29] (log K 1 =19.3 and log K 2 =18.2): the model predicted 48 % of Th(IV) under the form Th(OH) 2 (CO 3 ) 2 -2 , the rest being bound to HSTf.

3. 3 .

 3 Potential of DTPA as chelating agent for Th(IV) in blood serum 3.3.1. Theoretical considerations The DTPA ability to complex Th(VI) under biological conditions was first studied by DUS experiment in the physiological-type model medium (pH=7.4, 0.1 M NaCl) in the presence of HSTf. Complexation of Th(IV) with citrate was studied for comparison. The results of the titration curves are given in Figure 5. Whereas the citrate ion cannot compete with HSTf for Th(IV) in the whole range of ligand concentration studied (10 -6 M -3.5.10 -4 M) (see Th(IV)citrate constant in Table

Figure 1 :

 1 Figure 1: Complexation of Th(IV) as a function of NTA concentration. Experimental conditions and parameters used for the modelling with low-molecular weight compounds are summarized in Tables 1 and 2, respectively. (A) Th(IV) speciation calculated in the absence of HSTf. (B) Absorbance measured as a function of NTA concentration and comparison with literature data [12]. (C) Comparison between experimental data and modelling considering one NTA (solid line; log K 1 = 31, log K 2 = 30) and two NTA (dotted line; log K 1 = 41, log K 2 = 40) acting as synergistic anions. L 3-stands for deprotonated NTA.

Figure 2 :Figure 3 :

 23 Figure 2: Complexation of Th(IV) by HSTf in the presence of carbonates. Experimental conditions and parameters used for the modelling with low-molecular-weight compounds are summarized in Tables I and II, respectively. (A) Th(IV) speciation calculated in the absence of HSTf. (B) Absorbance measured as a function of carbonate concentration. (C) Comparison between experimental data and modelling considering one carbonate (dotted line; log K 1 = 27, log K 2 = 26) and two carbonates (solid line; log K 1 = 33.5, log K 2 = 32.5) acting as synergistic anions. (D) Absorbance measured as a function of the ratio (r) between total Th and HSTf concentrations. The line represents the simulation made with log K 1 = 33.5, log K 2 = 32.5 and ( obs ) = 60  10 3 M -1 cm -1 .

FFigure 4 :Figure 5 :

 45 Figure 4: UV-(A,B,C) and ICP-MS-(D,E,F) chromatograms determined by HPAEC. SeeTable 1 for the experimental conditions. Eluent = 0.1 M NaCl, 2.10 -3 M PBS, pH 7.4, 1% CO 2 .

Table 1 :

 1 Experimental conditions

			[HSTf]
	Method [Th(IV)] (M)		carbonate	Experiment	Figure
			(M)
		2.10 -5	10 -5	atmosphere	NTA titration	1(B)
		2.10 -6	10 -6	atmosphere	phosphate titration	not shown
		2.10 -5	10 -5	0 -0,05 M	carbonate titration	2(B)
	DUS	10 -6 -3,5.10 -5	10 -5	p(CO 2 ) 1%	Th(IV) titration	2(D)
		2.10 -5	10 -5	p(CO 2 ) 1%	competition with HSA (5.10 -5 -10 -4 M)	3
		2.10 -5	10 -5	p(CO 2 ) 1%	competition with DTPA (7,5.10 -7 -10 -4 M)	5
		2.10 -5	10 -5	p(CO 2 ) 1%	competition with citrate (7,5.10 -7 -5.10 -4 M)	5
		2.10 -5	10 -5	p(CO 2 ) 1%	Th(IV)-HSTf interaction	4(A,D)
	HPEAC	2.10 -5 2.10 -5	-10 -5	p(CO 2 ) 1% p(CO 2 ) 1%	Th(IV)-HSA (6.10 -5 M) interaction competition with HSA (6.10 -5 M)	4(A,D) 4(B,E)
		2.10 -5	-	p(CO 2 ) 1%	Th(IV)-serum interaction	4(C,F)
	ultra-	2.10 -5	10 -5	p(CO 2 ) 1%	Th(IV) speciation in serum	not shown
	filtration	2.10 -7	-	p(CO 2 ) 1%	in vitro studies in human serum with DTPA	6

Table 2 :

 2 Formation of complexes between Th(IV) and low-molecular-weight ligands; equillibria relevant in the experimental conditions of this work and associated constants used for the modelling. Acid-base properties of the ligands can be found in the references. All constants are given at an ionic strength of I=0, 25°C. The values in italic are recalculated using the truncated Davies equation[26].

		species	equilibrium reaction	logK	Ref
		Th(OH) 4	Th 4+ + 4 OH -= Th(OH) 4	-17.4 [24]
		(Th) 4 (OH) 12 +4	4Th 4+ + 12H 2 O = (Th) 4 (OH) 12	+4 + 12H +	-26.6 [24]
		Th(OH) 2 (CO 3 ) 2	-2 Th 4+ + 2OH -+ 2 CO 3	2-= Th(OH) 2 (CO 3 ) 2	-2	36.8	[24]
	inorganic ligands	Th(H 2 PO 4 ) 2 +2 ThSO 4 +2	+2 + 2H + +2 Th +4 + 2H 3 PO 4 = Th(H 2 PO 4 ) 2 Th +4 + SO 4 -2 = ThSO 4	6.2 6.2	[24] [24]
		ThCl +3	ThCl +3		1.7	[24]
		Th(NO 3 ) +3	Th +4 + NO 3	-= ThNO 3	+3	1.3	[24]
	citrate ion	ThL +	Th 4+ + L -3 = ThL +	15.7	[25]
		ThL -	Th +4 + L -5 = ThL -	32.9	[18]
		ThLH	ThL -+ H + = ThLH	2.37	[18]
	DTPA	ThLOH -2	Th +4 + L -5 + H 2 O= ThLOH -2 + H +	23.6	[18]
		ThL +	Th +4 + L -5 = ThL -	33.1	[17]
		ThL +	Th +4 + L -3 = ThL +	17.1	[26]
	NTA	ThL 2	-2	ThL + + L -3 = ThL 2	-2	14.2	[26]


Bernard Pessac
email: bernard.pessac@wanadoo.fr

Vamshi K Nimmagadda

Tapas Makar

Paul S Fishman

Christopher T Bever

David Trisler

Adult hematopoietic progenitors are pluripotent in chimeric mice

. Hematopoietic progenitors express embryonic stem cell and germ layer genes. Comptes Rendus Biologies 334: 300--306). This suggested that these cells may be pluripotent. Here we have transplanted these CD34+ bone marrow stem cells from adult male C56Bl/6J ROSA mice, that carry two markers: the ß--galactosidase gene and the male Y chromosome, into blastocysts of wildtype C57Bl/6J mice. These blastocysts develop normally and give rise to healthy adult chimeric mice. Each female ROSA chimeric mouse had a distinct pattern of male organs expressing ß--galactosidase derived from each of the three embryonic germ layers: ectodermal brain, dorsal root ganglia and skin; mesodermal heart, bone and bone marrow; and endodermal pancreas, intestine, and liver. Thus, adult mammals still carry cells that appear to exhibit a developmental potential comparable to ESCs and iPSCs suggesting that CD34+ cells from adult bone marrow could be used for cell therapy.

Introduction

The search for pluripotent stem cells capable of restoring damaged functions of altered organs is the Holy Grail of tissue and organ engineering in mammals. Early investigations had shown that differentiation gene products are present in tissues other that their main site of expression. Indeed, we have reported that bone stem marrow cells, as well as differentiated lineages to which they give rise, express the myelin basic protein gene that codes for a brain structural protein (1--6). In parallel, it has been claimed that cells derived from bone marrow can give rise to various cell types including neural cells, pancreatic cells or cardiac muscle cells (7--11). Taken together, these data suggested that bone marrow cells, or a subset of them, might be multipotent. Another line of research has been to engineer pluripotent cell lines: as a first step, embryonic stem cells (ESCs) were derived from the inner cell mass of early embryo blastocysts, cells that are able to form each of the three embryonic germ layers ectoderm, mesoderm and endoderm [START_REF] Evans | Establishment in culture of pluripotent cells from mouse embryos[END_REF][START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratoma stem cells[END_REF]. But, because of practical issues ESCs are problematic for human cell replacement therapy. Then an important step was the creation of a mouse clone from olfactory neurons showing that a differentiated cell could be reprogrammed back to an embryonic stem cell--like stage [START_REF] Eggan | Mice cloned from olfactory sensory neurons[END_REF]. The more recent breakthrough has been the induction of pluripotent stem cells (iPSCs) by the genetic manipulation of normal adult somatic cells such as fibroblasts [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF][START_REF] Wernig | c-Myc is dispensable for direct reprogramming of mouse fibroblasts[END_REF]. However, induction of pluripotency may evoke genetic changes leading eventually to tumorigenicity. This lead us to investigate the existence of natural adult stem cells whose pluripotency could lead to or take part in the generation of a new organ(s). Indeed, we had previously reported that a sub--population of mouse bone marrow hematopoietic progenitors with the CD34+ antigen naturally expresses an array of ESC genes including Oct--4, Rex--1, Sox--2, Klf--4 and LIN--28, genes that have been initially used to induce iPSCs. We had also found that this same subset of adult stem cells expresses ectodermal neural lineage genes, mesodermal cardiac lineage genes as well as endodermal pancreatic and intestinal genes [START_REF] Pessac | Hematopoietic progenitors express embryonic stem cell and germ layer genes[END_REF]. Taken together, the fact that these bone marrow cells express both embryonic stem cell genes and lineage genes for each of the three embryonic germ layers suggests that they may be pluripotent. Here we illustrate that when these adult bone marrow CD34+ cells are transplanted into mouse blastocysts, they give rise to organs of adult mice such as ectodermal derived brains, mesodermal cardiac cells, bone and bone marrow cells and endodermal pancreas and intestinal cells, indicating the pluripotency of these CD34+ bone marrow cells.

Results

Generation of chimeric mice. Because a subset of adult CD34+ bone marrow stem cells expresses both embryonic stem cell genes and lineage genes for each of the three embryonic germ cell layers, pluripotency of these cells was suggested. We ascertained their pluripotency by transplanting the adult CD34+ cells into mouse blastocysts. Adult CD34+ stem cells from a male C57Bl/2J transgenic mouse that expressed ß--galactosidase, B6.129S7--Gt(ROSA)26Sor/J were cultured as previously reported [START_REF] Goolsby | Hematopoietic progenitors express neural genes[END_REF]. When these ROSA CD34+ cells were treated with X-gal they stained blue, whereas wildtype CD34+ cells do not stain with X-gal (Fig. 2). These male ROSA CD34+ cells were transplanted into wildtype C57Bl/6J blastocysts that do not express βgalactosidase. As a control, wildtype CD34+ cells that do not have the β-galactosidase transgene were transplanted into separate wildtype mouse blastocysts. Eight to fifteen adult ROSA or wildtype CD34+ cells were transplanted into each E 3.5 stage blastocyst. At E 3.5, each blastocyst is composed of 64 cells including both the trophoblasts that make up a single cell layer thick wall of the blastocyst that go on to become all of the extra-embryonic structures, e.g., placenta, umbilical cord, amnion and chorion, as well as the inner cell mass of embryonic stem cells that forms the embryo itself. After transplantation of the ROSA CD34+ cells, staining of the chimeric blastocyst with X-gal revealed the blue-stained ROSA CD34+ cells within the blastocoel of the unstained wildtype blastocysts (Fig. 2,). The chimeric blastocysts then were implanted into pseudopregnant mice. The chimeric embryos were allowed to develop normally through birth to weaning. All chimeric mice were robust and behaved normally. X-gal staining of chimeric mouse organs. To determine the potency of the adult hematopoietic progenitor cells to form multiple differentiated cell types in tissues and organs from each of the embryonic germ cell layers, fourteen tissues and organs from six ROSA CD34+ chimeric mice and from four wildtype CD34+ control chimeric mice were stained with X--gal to determine if the cells of any organs of the chimeric mice were derived from the adult ROSA CD34+ cells that were transplanted into the wildtype blastocysts. The adult ROSA CD34+ cells gave rise to tissues and organs of each of the three embryonic germ cell layers, e.g., ectodermal brain, dorsal root ganglia and skin, mesodermal femur bone and bone marrow and endodermal pancreas, liver, intestine and bladder (Fig. 3). We found organs containing X--gal (+) cells derived from CD34+ cells in both male and female chimeric mice. The pattern of tissues and organs derived from the adult ROSA CD34+ cells was different in each chimeric individual. Ectodermal neural cells were derived from adult hematopoietic progenitors. Three of six ROSA chimeric mice brains had neurons that stained positively for X--gal (Fig. 3). Some but not all neural cell types stained with X--gal. For example, hippocampal pyramidal neurons of areas CA 1--3 stained positively, as did locus ceruleus neurons and Purkinje neurons of the cerebellum, while granule neurons did not stain (Fig. 4). The cerebellar cells that stained with X--gal also stained for Purkinje calbindin but not Bergmann glial GFAP. Many but not all neural cell types stain throughout the brain (not shown). Because some neural cells in the ROSA chimeras stained with X--gal and others did not, we looked at the X--gal staining pattern in the brain of the transgenic ROSA mouse itself -the mouse from which the ROSA CD34+ cells were derived for transplantation into wildtype blastocysts. To our surprise, even though all cells of the transgenic ROSA mouse contain the βgalactosidase gene, all cells do not stain for X-gal, for example in brain astroglia, oligodendroglia and many neuronal types do not stain with X-gal. We found indistinguishable X-gal staining patterns in the ROSA chimeric mice and the ROSA transgenic mice -some cells stain but others do not. In the peripheral nervous system, which is also derived from ectoderm, the neural crest, dorsal root ganglion neurons stained with X--gal. Control chimeric dorsal root ganglia did not stain with X--gal. Neither ROSA chimeric nor control chimeric spinal cord stained with X--gal.

Mesodermal bone and bone marrow cells were derived from adult hematopoietic progenitors. A band of bone marrow cells at the interface with femur bone stained with X--gal, whereas cells in more central marrow did not stain in all six ROSA chimeric mice. Three of six ROSA chimeras elicited X--gal staining in femur bone (Fig. 3); femur cartilage cells stained with X--gal, as did cells in the trabeculae of spongiform areas of femur (Fig. 5). Some cells in ROSA chimera bone stained with X--gal and others did not. Similarly some cells of ROSA transgenic bone stained with X--gal and others did not. The femurs and bone marrows of control chimeric mice were unstained with X--gal.

Endodermal intestine, pancreas and liver cells were derived from adult hematopoietic progenitors. The intestine of all six ROSA chimeras stained with X-gal (Figs. 3,6). Four of six ROSA chimeric mouse pancreases stained with X--gal (Figs, [START_REF] Grima | Identification of new human myelin basic protein transcripts in the immune and central nervous systems[END_REF][START_REF] Goolsby | Hematopoietic progenitors express neural genes[END_REF]. And two of six ROSA chimeras showed X--gal staining of liver (Figs. 3,6). The X-gal staining pattern in ROSA transgenic mouse intestine, pancreas and liver was similar to ROSA chimeras. Control chimera intestine, pancreas and liver did not stain with X--gal. X,Y Chromosome Fluorescence In Situ Hybridization (FISH). Both chimeric mice and transgenic ROSA mice had organs with the same pattern of X--gal (+) and X--gal (--) cells. Thus, the question arose whether the X--gal (--) cells in the ROSA chimeric mice were derived from the inner cell mass of the blastocyst or whether they derive from the male ROSA CD34+ cells that give rise to cell types that are under regulatory control like those X--gal (--) cells in the ROSA transgenic mice. The Y chromosome in male CD34+ cells was used to identify progeny of those CD34+ cells in female chimeric mice. We used XY--FISH to determine whether X--gal (--) cells in chimeric mouse organs were derived from male ROSA CD34+ cells in the blastocysts. Organs from anatomically female chimeric mice were examined for X,Y and X,X chromosome ratios. Images of X,Y chromosome staining are shown in Figure 7A. X,Y chromosome counts were done on random fields of brain, heart, pancreas and liver (Fig. 7B). Chimeric mouse brain #1, which contained both X--gal (+) and X--gal (--) cells in a male mouse, had an X:Y ratio of 1:1, indicating that it is a male brain. Female mice #3 and 5 brains, that had X--gal (+ and --), also had X:Y ratios of 1:1, again meaning male brains. Female mouse brain #6, that had no X--gal (+) cells, also had no Y chromosomes detected, indicating a female brain. Futhermore, regions of mouse brain like the corpus callosum, which is devoid of neurons but contains glial cells that had no X--gal (+) cells, had an X:Y ratio of 1:1 showing that they are male. Thus, the absence of X--gal does not preclude that the X--gal (--) cells were derived from the male ROSA CD34+ cells. None of the hearts from chimeric mice had X--gal (+) cells that were detected. Similarly, transgenic ROSA heart was X--gal (--). Female chimeric mouse #3 heart had a X:Y ratio of 1:1, indicating that it is a male heart derived from the male ROSA CD34+ bone marrow stem cells. Contrastingly, female chimeric mouse #5 had no detectable Y--chromosomes in heart meaning a female heart. Pancreas from both female chimeric mice # 3 and 5, with X--gal (+ and --) cells, had a X:Y ratio of 1:1, i.e., that of male pancreas from male CD34+ cells. Chimeric mouse #3 liver, that did not exhibit X--gal (+) cells, also had no Y chromosomes. Therefore, the Y chromosome marker showed that chimeric mouse organs that had X--gal (+ and --) cells, and in the case of heart, which had no X--gal (+) cells, were wholly derived from male ROSA CD34+ cells. Another question addressed by the X,Y--FISH is whether the X--gal (+) containing organs are derived from the fusion of a male ROSA CD34+ cell with a wildtype cell of the inner cell mass in the early blastocyst. We found no XXXY cells in the chimeric organs examined and the total X:Y ratios for all organs were that of an XY male or a XX female, indicating no cell fusion.

Discussion

The quest for a versatile pluripotent stem cell capable to differentiate into various cell types and therefore eventually repair damaged tissues and organs is the subject of intense investigation, which has led to very promising results. However, a major question, which has not been conclusively answered yet, is whether such a pluripotent cell type is present in the normal adult mammal. Here we show that a bone marrow cell sub-population present in normal adult mice and which expresses both an array of ESC genes and germ layer lineage genes can give rise to many somatic tissues of an apparently normal adult mouse.

A characteristic of embryonic stem cells in culture is that they manifest unlimited selfrenewal. Although the adult bone marrow CD34+ cells investigated here express ESC genes [START_REF] Pessac | Hematopoietic progenitors express embryonic stem cell and germ layer genes[END_REF] and have the potential for pluripotency, they do not exhibit the unlimited proliferation of ESCs. The CD34+ cells have a limited lifespan since they stop dividing and plateau after 30-40 doublings like diploid somatic cells [START_REF] Goolsby | Hematopoietic progenitors express neural genes[END_REF]. These CD34+ cells do not form teratomas when injected into a host [START_REF] Goolsby | Hematopoietic progenitors express neural genes[END_REF] in sharp contrast to ESCs and iPSCs.

Mice obtained from blastocysts inoculated with CD34+ cells differ from mice generated from ESCs or iPSCs where the whole individual is derived from the inoculated stem cells [START_REF] Boland | Adult mice generated from induced pluripotent stem cells[END_REF][START_REF] Stillwell | Blastocyst injection of wildtype embryonic stem cells induces global corrections in Mdx mice[END_REF] as opposed to multiple, but not all organs derived from the CD34+ cells. One possible explanation is that ESCs and iPSCs are totipotent and these CD34+ cells may be only pluripotent.

The pattern of cells derived from the transplanted male ROSA CD34+ bone marrow stem cells was different for each chimeric mouse. This is most likely due to the mechanics of implant: the number and site of implant of the adult bone marrow stem cells. As mentioned in the Results section, 8-15 CD34+ cells were injected into each 64-cell stage blastocyst. Contingent on how many CD34+ cells implanted in the early inner cell mass, which most likely contains 30 or fewer cells, and depending on cell migration and the site of implant and the time of implant during ongoing development, the transplanted CD34+ cells differentiated into distinct cell types.

A possible role of these CD34+ cells from bone marrow or circulating blood in physiological or pathological processes should be considered. Indeed it has been reported that post-mortem brain samples from women who had received bone marrow transplants from male donors did contain male cells. The two main regions examined were cerebellum and hippocampus. The donor-derived cells were identified by the presence of a Y chromosome. The proportion of male neurons in the female recipients varied between 1:100 and 1:1000. In addition to neurons, glial and mesenchymal cells were also labeled with the Y marker, often in clusters, suggesting a clonal origin [START_REF] Mezey | Transplanted bone marrow generates new neurons in human brains[END_REF][START_REF] Weimann | Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains[END_REF][START_REF] Cogle | Bone marrow transdifferentiation in brain after transplantation: A retrospective study[END_REF]. Interestingly enough, skin biopsies of female patients following allogenic male bone marrow transplantation show the presence of donor derived keratinocytes [START_REF] Nemeth | Analyses of donor-derived keratinocytes in hairy and non-hairy skin biopsies of female patients following allogenic male bone marrow transplantation[END_REF]. In these studies the bone marrow cells that eventually gave rise to neural cells containing a Y chromosome have not been identified. Since we have shown here that a subset of bone marrow CD34+ cells can generate most if not all cells present in the brain, it is tempting to speculate that bone marrow cells that differentiated into various cell types may be the subset of pluripotent stem cells we have identified.

Lately, many investigations have been carried out on iPSCs derived from individuals bearing various genetic defects to explore their mechanisms as well as to try to correct them [START_REF] Yazawa | Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome[END_REF][START_REF] Brennand | Modeling schizophrenia using human induced pluripotent stem cells[END_REF]. It is reasonable to assume that the sub-population of CD34+ stem cells should carry the same genetic defect as the iPSCs derived from their peripheral tissues such as fibroblasts or neurons

The growth and differentiation patterns of the adult CD34+ stem cells are reminiscent of adult Urodele Amphibian newt or salamander blastema cells or mature Planarian blastema cells that replace the cells and tissues that are needed to reform the missing limb or body part [START_REF] Morgan | Regeneration[END_REF][START_REF] Weiss | Unabhängigkeit der extremitäten regeneration vom skelett (bei Triton cristatus)[END_REF](28)(29).

Taken together, these readily available adult bone marrow CD34+ stem cells, are plutipotent and constitute a potential source for cell replacement therapy in multiple tissues and organs.

Materials and Methods

Bone marrow CD34+ stem cell cultures. Non-adherent CD34+ hematopoietic stem cells were cultured from adult mouse bone marrow with medium containing mouse IL-3, IL-6, SCF and β-mercaptoethanol as reported [START_REF] Goolsby | Hematopoietic progenitors express neural genes[END_REF]. CD34+ cultures from the two strains of mice were used: the wildtype C57Bl/6J mouse and the C57Bl/6J transgenic mouse that expresses β-galactosidase, B6.129S7-Gt(ROSA)26Sor/J. The University of Maryland School of Medicine OAWA approved these animal studies.

Generation of chimeric mice. Adult CD34+ cell chimeric mice were generated by the University of Maryland School of Medicine Transgenic Animal Core Facility. Briefly, 8--galactosidase, B6.129S7-Gt(ROSA)26Sor/J, were injected into each wildtype C57Bl/6J blastocyst at E 3.5, the 64-cell stage. Six to ten CD34+ injected blastocysts were delivered to the uterus of each hormonally induced pseudo-pregnant wildtype C57Bl/6J surrogate mother mouse. Offspring were grown to weaning. X-gal staining. Chimeric animals were killed and perfused with PBS followed by 2% paraformaldehyde plus 0.2% glutaraldehyde. Perfused tissues were immersed in the same fixative at 4ºC for 72 hours. Fixed tissues and organs were exchanged with PBS then 30% sucrose, frozen and sectioned. X-gal staining was performed with an In situ βgalactosidase Staining Kit (Stratagene Cat. # 200384) by the manufacture's protocol.

Immunocytochemistry. Cells were treated by standard immunochemistry methods and observed by fluorescence microscopy [START_REF] Goolsby | Hematopoietic progenitors express neural genes[END_REF]. ) by the manufacture's protocol. X-gal staining was found in some organs and not others. In some organs some cells were stained and others were not. Different ROSA chimeras had different X-gal staining patterns. Control wildtype CD34+ chimeras showed no X-gal staining. 4. Ectodermal tissues are derived from adult ROSA CD34+ stem cells. ROSA chimeric cerebellum exhibits X-gal stained cells (blue) that originate from the adult ROSA CD34+ cells that were injected into the early wildtype blastocyst. Chimeric animals were processed as in Figure 3. A. Cells in the Purkinje cell layer of some ROSA chimera brains stained with X-gal (blue). Control wildtype CD34+ chimeras did not stain. ROSA transgenic cerebellum showed the same Xgal staining pattern as did the ROSA chimeras. Some cell types stain with X-gal whereas others, e.g. astroglia, oligodendroglia and granular neurons do not. B.

X,Y-chromosome Fluorescence In Situ Hybridization (FISH

The X-gal positive cells in cerebellum co-stain for calbindin (brown stain). Both the cell bodies and the dendritic arborizations (lower right) stained for calbindin. Astroglia and Bergmann glia stained for GFAP but the stain did not co-localize with X-gal. Thus, the X-gal stained cells in cerebellum are Purkinje cells. C. Locus ceruleus and D. hippocampal CA 1-3 neurons also stain with X-gal. E. No cells in Corpus callosum were stained with X-gal. F. Peripheral nervous system neurons of the dorsal root ganglion, derived from neural crest cells stained with X-gal. These blue cells in central and peripheral nervous system indicate that they were derived from the ROSA CD34+ cells originally transplanted into the early blastocysts. 5. Mesodermal tissues are derived from adult ROSA CD34+ stem cells. Tissues were processed as in Figure 3. ROSA chimeric mouse femur bone and bone marrow were derived from ROSA CD34+ stem cells. Bone marrow cells in a band adjacent to the femur bone itself stained with X-gal (blue). Femur cartilage and spongiform bone trabeculae stained with X-gal. Thus, mesodermal derivatives formed from adult ROSA CD34+ cells. 6. Endodermal tissues are derived from adult ROSA CD34+ stem cells. Tissues were processed as in Figure 3. ROSA chimera intestine, pancreas and liver cells were stained with X-gal (blue) indicating that these endodermal tissues were derived from the adult ROSA CD34+ cells transplanted into the wildtype blastocysts. Fig. 2 ROSA Chimera Bone Marrow ROSA Chimera Femur Cartilage ROSA Chimera Femur Spongiform bone trabeculae Fig. 5 ROSA Chimera Intestine -X--gal ROSA Chimera Liver -X-gal Fig. 6

7 .

 7 XY-chromosome Fluorescence In Situ Hybridization was performed by Empire Genomics, Albany, NY using green 5-Fluroescein dUTP mouse X-chromosome probe (Cat. # CLN-10048) and red 5-ROX dUTP mouse Y-chromosome probe (Cat. # CLN-100920) on 4µm-thick sections of chimeric mouse tissues. A. Fluorescent micrograph of mouse #3 brain cell nucleus showing one Y chromosome (upper row) and mouse #6 brain cell nucleus showing two X chromosomes (lower row). B. Organs were scored for X (green) / Y (red) chromosome ratios of DAPI stained tissue sections of the chimeric mouse organs by fluorescence microscopy. The total number of X and Y chromosomes scored for each organ are shown. A ratio of 1:1 X;Y chromosomes indicated a male organ and a X:Y ratio of 2:0 indicated a female organ.

  ). XY-FISH was performed by Empire Genomics, Albany, NY using green 5-Fluroescein dUTP mouse Xchromosome probe (Cat. # CLN-10048) and red 5-ROX dUTP mouse Y-chromosome probe (Cat. # CLN-100920). Roux Arch. 132: 57-114. 29. Kragl M, Knapp D, Nacu E Khattak S, et al. (2009) Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature 406: 60-65.

	28. Schwidefsky	G	(1934)	Entwicklung	und	determination	der
	extremitätanregeneration bei den Molchen. Figure legends
	1. Adult mouse CD34+ bone marrow stem cell gene expression. Embryonic stem
	cell gene and embryonic germ layer lineage gene expression in adult CD34+ cells
	were reported previously (17).
	2. X-gal labeling of ROSA CD34+ cells in culture (A) and after injection into
	wildtype blastocysts (B). Adult wildtype CD34+ cells do not stain with X-gal
	whereas, adult transgenic ROSA CD34+ cells, that carry the β-galactosidase gene,
	do stain with X-gal (blue). CD34+ cells were fixed with 2% paraformaldehyde
	plus 0.2% glutaraldehyde for 15 min. at 4ºC then washed with PBS. X-gal
	staining was performed with a Stratagene In situ β-galactosidase Staining Kit
	(Cat. # 200384) by the manufacture's protocol. Cells were stained for 12 hr.
	Similarly, sixty-four cell stage chimeric E 3.5 mouse wildtype blastocysts that
	contained injected adult ROSA mouse CD34+ cells were fixed and stained as
	above. The X-gal stained CD34+ cells were observed in different focal planes of
	the blastocyst.
	3. Table of organs of ROSA CD34+ chimeras and control wildtype chimeras that
	stained with X-gal. Chimeric animals were killed and perfused with PBS
	followed by 2% paraformaldehyde plus 0.2% glutaraldehyde. Perfused tissues
	were immersed in the same fixative at 4ºC for 72 hours. Fixed tissues and organs
	were exchanged with PBS then 30% sucrose, frozen and sectioned. X-gal
	staining was performed with an In situ β-galactosidase Staining Kit (Stratagene
	Cat. # 200384

Acknowledgements

The authors would like to thank David Ford and Deborah Yarnell for technical assistance in this project and Valerie Stewart of the University of Maryland School of Medicine Transgenic Core Facility. This work was supported for PSF (VA REAP), CTB (VA Merit) and DT (Abraxis Biosci. and VA REAP). (-

Fig. 3 A. DAPI Y--chromosome Merge DAPI X--chromosome Merge B.

Organ