Tritium contamination of hematopoietic stem cells alters long-term hematopoietic

reconstitution

Fabio Di Giacomo1, Christine Granotier2, Vilma Barroca1, David Laurent1, François D.

Boussin2, Daniel Lewandowski1, Yannick Saintigny1 and Paul-Henri Romeo1, *

1 Laboratoire de recherche sur la Réparation et la Transcription dans les cellules Souches,

2 Laboratoire de Radiopathologie,

UMR INSERM-Paris VII-Paris XI U967, Institut de Radiobiologie Cellulaire et Moléculaire,

Commissariat à l'Énergie Atomique (CEA), Direction des Sciences du Vivant (DSV), 92265

Fontenay-aux-Roses

Running Title: Effects of Tritium on hematopoietic stem cells

*Correspondence: paul-henri.romeo@cea.fr

Conflict of interest statement. None declared.

Di Giacomo, F., Granotier, C., Barroca, V., Laurent, D., Boussin, F.D., Lewandowski, D.,

Saintigny, Y. and Romeo, P.H. Tritium contamination of hematopoietic stem cells alters long-

term hematopoietic reconstitution. Radiat. Res.

Abstract

In vivo effects of manufactured tritium from nuclear weapon testing, nuclear power plant, and

industry or research are poorly documented. Here, we contaminate mouse hematopoietic stem

cells with low, medium and high concentrations of [3H] thymidine and characterize the

biological properties of these contaminated hematopoietic stem cells in vitro and in vivo.

Proliferation, viability and double strand breaks were dependent of the [3H] thymidine dose

used for contamination but the in vitro myeloid differentiation of hematopoietic stem cells

was not affected by any doses of [3H] thymidine contamination. Transplantation of

contaminated hematopoietic stem cells into lethally irradiated mice compromised their long-

term capacity of hematopoietic reconstitution even after low doses of [3H] thymidine

contamination and their differentiation potential into B-lymphoid lineage only after medium

and high doses of [3H] thymidine contamination. Finally, competition experiments showed a

diminished capacity of hematopoietic stem cells contaminated with medium or high doses of

[3H] thymidine to reconstitute hematopoiesis. These results indicate that contaminations of

hematopoietic stem cells with doses of tritium that do not result in death of these cells have

long term effects on hematopoiesis.

Introduction

Tritium is naturally produced in the atmosphere by the interaction of high-energy

cosmic radiation with oxygen and nitrogen and by ternary fission in geological formations.

Since 1954, a significant portion of tritium in the atmosphere has resulted from the release of

large amounts of manufactured tritium into the environment from nuclear weapon testing,

nuclear power plant, and industrial or research uses of tritiated compounds. Much of the

tritium that remains in the environment exists as tritiated water (HTO) but it can also be found

as organically bound tritium (OBT). Both OBT and HTO may contaminate organisms after

ingestion or inhalation and OBT can then be incorporated into numerous biochemical

compounds (1). Because of the low disintegration energy, tritium’s biological effects cannot

come from external exposure but from integration of OBT into tissue resulting in an in situ

chronic auto-irradiation of the contaminated cells. Consequently, the energy deposition is

concentrated in the sub-cellular compartment in which the OBT is incorporated.

Little information is available on long-term biological effects of contamination by OBT

of living animals but the characterization of somatic stem cells present in most adult tissues

has now opened a new field of research on the long-term effects of OBT in living animals.

The adult somatic stem cells are tissue specific, can remain quiescent, undergo apoptosis, self-

renew or differentiate into progenitors (2). In addition, the adult somatic stem cells can

reconstitute long term biological functions of an injured organ from which they are specific

(3-5). Thus, contamination of somatic stem cells by OBT can be used to study the long-term

effects of contamination of a specific tissue or organ by OBT in living animals. The best-

characterized mammalian adult somatic stem cells are the hematopoietic stem cells (HSCs).

HSCs are the only cells in the hematopoietic system that can self-renew for life and that can

produce life-long complete hematopoietic reconstitution after transplantation in a γ-lethally

irradiated recipient mouse. In vivo, the maintenance and development of HSCs in the bone

marrow are dependent on the hematopoietic stem cell niche through niche-regulating

pathways that can protect HSCs from endogenous and exogenous genotoxic stresses. Finally,

HSCs can be purified close to homogeneity making their use for experimental research easy.

Of all tissues, the hematopoietic system is the most radiosensitive and hematopoietic

stem cells have already been used to study the effects of γ-irradiation on their biological

properties both in vitro and in vivo. The bone marrow failure induced by γ-irradiation is due to

HSCs but not progenitors DNA damages (6-8) and at the molecular level, γ-irradiation

induces DNA double-strand breaks whose repair is delayed in HSCs (9) and HSCs exhibited

limiting DNA repair during ageing (7, 8). The p53 signaling is a critical pathway that

responds to ionizing radiation by regulating proliferation, DNA repair and survival (10, 11).

Puma (p53 up-regulated mediator of apoptosis) is a direct p53 target gene that is essential for

hematopoietic cell death triggered by ionizing radiation and recently deletion of Puma has

been shown to specifically protect hematopoietic stem cells against high-dose γ-irradiation

(12, 13). In contrast to γ-irradiation, no data are available on the effects of tritium

contamination on the biological properties of HSCs as studies have been limited to

contaminations of transformed hematopoietic cell lines (14). This study showed that DNA

incorporation of tritiated thymidine influenced in a dose dependent manner cell proliferation

and viability in most of the cell lines studied but did not document the effects of tritium

contamination on differentiation and/or self-renewal.

To analyze the effects of OBT on hematopoietic stem cells, we have contaminated HSCs with

different doses of [3H] thymidine and studied the effects of these contaminations both in vitro

and in vivo. Short term effects of HSCs contamination by [3H] thymidine were studied in vivo

by primary transplantations and competitive experiments and long-term effects of [3H]

thymidine contamination was studied in vivo using secondary transplantations.

Results

Efficient contamination of HSC by [3H] thymidine

A hematopoietic cell population enriched in hematopoietic stem cells (the c-kit+, lin-,

Sca+ (KLS) hematopoietic cells) was purified by cell sorting from C57Bl/6 mice bone

marrow. The KLS hematopoietic cells were grown in a culture medium containing [3H]

thymidine concentrations that ranged from 0.37 to 37.03 kBq/ml during twenty-four hours

and the amount of [3H] thymidine incorporated into DNA was determined. A dose dependent

incorporation of [3H] thymidine was found in the contaminated KLS hematopoietic cells

without any DNA saturation (Figure 1A). These [3H] thymidine incorporations in KLS cells

DNA can be due to few cells highly contaminated or to many cells contaminated with few

molecules of [3H] thymidine. Autoradiography was used to analyze [3H] thymidine

incorporation into DNA of individual KLS hematopoietic cells and showed that, at low

concentration of [3H] thymidine (0.74 kBq/ml) almost 50% of the KLS cells were positive for

radioactive DNA with an average of two (1-5) silver grains per cell (Figure 1B and Figure

1C). This percentage of positive KLS cells increased with [3H] thymidine concentration

reaching 88% of positive KLS cells with an average of up to 23 silver grains per cell when a

[3H] thymidine concentration of 7.4 kBq/ml was used (Figure 1B and Figure 1C). Finally, a

detailed analysis of the number of silver grains showed heterogeneity in the number of silver

grains present in the KLS cells even when a [3H] thymidine concentration of 7.4 kBq/ml was

used (Figure 1C) with around 10% of KLS cells without any [3H] thymidine incorporation.

These results showed that [3H] thymidine could be incorporated into the DNA of KLS cells

even at low doses of contamination and that the KLS cells is heterogeneous in term of [3H]

thymidine incorporation into DNA, heterogeneity that could not be suppress even at high

concentration of [3H] thymidine.

Proliferation and viability of KLS hematopoietic cells contaminated with low or high

doses of [3H] thymidine

Irradiation of the nucleus induced DNA damages that can arrest cell cycle and

proliferation and/or induce apoptosis. To evaluate the genotoxic effect of [3H] thymidine

contamination on KLS cells, cell proliferation and viability of contaminated KLS cells were

determined. KLS were grown in culture medium containing a concentration of 3.7, 7.4, 14.8

or 37.03 kBq/ml of [3H] thymidine and proliferation and apoptosis were determined after 24

and 48 hours of culture. At the concentration of 3.7 kBq/ml of [3H] thymidine, cell

proliferation was only lightly affected after 24 hours incubation but, after 48 hours of culture,

a two-fold difference of proliferation was detected between cells cultured in the presence of

3.7 kBq/ml of [3H] thymidine and controls (Figure 2A). Increasing [3H] thymidine

concentration resulted in a two-fold (resp. five-fold) reduced proliferation after 24 hours and a

five-fold (resp. ten-fold) reduction after 48 hours of culture in a medium containing a [3H]

thymidine concentration of 7.4 kBq/ml (resp.14.8 kBq/ml) and no detectable proliferation

when a [3H] thymidine concentration of 37.03 kBq/ml was used in the culture medium

(Figure 2A). We finally studied apoptosis of KLS cells grown in culture medium containing

3.7 or 14.8 kΒq /ml of [3H] thymidine and showed than 20% of apoptotic cells could be

detected after 24 hours of incubation with these two concentrations of [3H] thymidine whereas

45% (resp.55%) of apoptotic cells were found after 48 hours of culture of KLS cells in culture

medium containing a concentration of [3H] thymidine of 3.7 kBq/ml (resp. 14.8 kBq/ml)

(Figure 2B). Taken together, these results show that a dose dependent effect of [3H] thymidine

on proliferation of KLS hematopoietic cells, a small effect of [3H] thymidine incorporation on

apoptosis of this hematopoietic population after 24 hours of culture and a significant effect of

[3H] thymidine incorporation on apoptosis of this hematopoietic population after 48 hours of

culture.

Contamination with [3H] thymidine induces formation of γ-H2AX Foci

Since [3H] thymidine incorporation into DNA can induce double strand breaks (DSBs)

in immortalized cells (15), we analyzed the formation of γ-H2AX foci after incorporation of

[3H] thymidine into DNA of KLS cells at non-lethal doses. After generation of DSBs, H2AX

is phosphorylated (γ-H2AX) and is detectable as nuclear foci at the damaged sites (16) in a

linear relationship with the number of induced DSBs (17). The percentages of cells with γ-

H2AX foci increased significantly as a function of incorporated [3H] thymidine but required

at least a [3H] thymidine concentration of 3.7 kBq/ml in the culture medium to be significant

(Figure 3A). We then determined the mean number of γ-H2AX foci per cell after [3H]

thymidine incorporation and showed that the median number of γ-H2AX foci per KLS cell

increased as a function of [3H] thymidine concentration present in the culture medium (Figure

3B). Foci form immediately after irradiation, after which they disappear within few hours due

to DSB repair. In the present work, γ-H2AX foci were analyzed after 24 hours of culture in

the presence of [3H] thymidine and thus, the frequencies of γ-H2AX foci detected resulted

from the countervailing influences of DSB repair (focus extinction) and the generation of new

DSBs (focus formation) at the time of monitoring. Taken together, these data indicate that

[3H] thymidine incorporation into DNA of KLS cells could induce DSBs in these cells.

Myeloid potential of KLS hematopoietic cells contaminated with low or high doses of

[3H] thymidine

Colony Forming Unit Assay (CFU) was performed to study the effects of [3H]

thymidine contamination on KLS cells differentiation into the myeloid lineage. 300 purified

KLS cells were cultured on a semi-solid methylcellulose media after 24 hours incubation in

medium containing [3H] thymidine concentrations of 3.7 and 14.8 kBq/ml. After 7 days of

culture in methylcellulose, the number and quality of the myeloid colonies obtained were

analyzed. No significant difference in the total number of colonies could be detected (Figure

4A). Qualitatively, we only found a small but not significant decrease of CFU-GEMM and a

small increase of CFU-GM when KLS cells were previously grown in the presence of [3H]

thymidine concentration of 14.8 kBq/ml and no difference when KLS cells were previously

grown in the presence of [3H] thymidine concentration of 3.7 kBq/ml (Figure 4B). These

results indicated that the myeloid progeny of contaminated KLS cells is not (resp. little)

altered by low (resp.high) dose of [3H] thymidine contamination.

Hematopoietic reconstitution with KLS hematopoietic cells contaminated with low or

high doses of [3H] thymidine

Lethally γ-irradiated (11Gy) mice expressing the CD45.1 antigen (CD45.1+) were

transplanted with 20,000 KLS expressing the CD45.2 antigen (CD45.2+) and cultured during

24 hours in media containing three different concentrations of [3H] thymidine (1.48, 3.7 or

14.8 kBq/ml). Long-term reconstituted hematopoiesis was studied 4 months after

transplantation. Whatever levels of contamination, [3H] thymidine contaminated KLS cells

were able to reconstitute long-term hematopoiesis in lethally irradiated mice as shown by

more than 90% of hematopoietic cells expressing the CD45.2 antigen in the reconstituted

hematopoieisis (data not shown). This result indicated that contamination of HSCs by [3H]

thymidine didn’t alter their reconstitution abilities. Contamination with 14.8 kBq/ml was of

high interest, as less than 10% of KLS cells were not contaminated when a concentration of

7.4 kBq/ml (i.e. two times less than 14.8 kBq/ml) of [3H] thymidine was used in the KLS cells

culture. This result indicated that at most 2,000 KLS cells used for transplantation were not

contaminated at this [3H] thymidine concentration, a number of KLS cells that could not

restore hematopoiesis after lethal γ-irradiation (our personal data). Thus hematopoietic

reconstitution by KLS grown for 24 hours in medium containing a concentration of [3H]

thymidine of 14.8 kBq/ml is partly due to [3H] thymidine contaminated KLS cells. The

different mature hematopoietic cell populations derived from the [3H] thymidine

contaminated KLS cells were not affected as no difference in mature B and T lymphocytes,

monocytes/granulocytes and red blood cells could be evidenced (Figure 5A). Finally, we

could not detect any difference in the percentage of KLS cells in bone marrow of each mice

group (Figure 5B), indicating that [3H] thymidine contaminated HSC could reconstitute long-

term hematopoiesis.

To document the long-term capacity of hematopoietic reconstitution of KLS cells

contaminated with [3H] thymidine, 500,000 donor CD45.2+ bone marrow cells were isolated

from transplanted mice and injected into lethally irradiated CD45.1+ recipient mice for a

secondary transplantation. All transplanted mice survived (data not shown) indicating that

hematopoietic stem cells retained their hematopoietic reconstitution activity. Analysis of the

different mature hematopoietic populations of the secondary transplanted mice revealed a two

fold decrease of the B lymphocyte populations in mice reconstituted with bone marrow

coming from KLS cells initially contaminated with [3H] thymidine concentration of 3.7 and

14.8 kBq/ml together with a small but significant increase of red blood cells when a [3H]

thymidine concentration of 14.8 kBq/ml was initially used for KLS cells contamination and

no change in the other mature hematopoietic cells (Figure 5C). Analysis of KLS cells of these

reconstituted mice showed a 1.5 fold decrease of the KLS population whatever dose of [3H]

thymidine concentration was initially used for contamination (Figure 5D). These results

indicated that initial contamination of KLS cells with [3H] thymidine compromised their long-

term capacity of hematopoietic reconstitution and their differentiation potential in the B-

lymphoid lineage.

[3H] thymidine contaminated KLS are less efficient than non contaminated KLS cells to

reconstitute hematopoiesis

We finally determined the functional properties of [3H] thymidine contaminated KLS

hematopoietic cells in a competitive repopulation assay. 5000 KLS expressing the CD45.2

antigen were cultured for 24 hours in a medium containing [3H] thymidine (0, 1.48, 3.7 and

14.8 kBq/ml) and co-transplanted with 500,000 non-contaminated bone marrow cells

expressing the CD45.1 antigen in lethally irradiated mice. As KLS cells represent around

0.2% of total bone marrow cells, the number of CD45.2 and CD45.1 hematopoietic cells used

for this experiment resulted in a 1/5 ratio of CD45.1+ and CD45.2+ KLS cells at the time of

transplantation. Four months after transplantation, the CD45.1/CD45.2 chimerism was

determined in the reconstituted bone marrow. Mice transplanted with non-contaminated KLS

cells and mice reconstituted with KLS cells contaminated with a [3H] thymidine concentration

of 1.48 kBq/ml displayed a CD45.2/CD45.1 ratio of 6.8 and 5.1 that is the ratio of

CD45.2/CD45.1 chimerism when these cells were transplanted (Figure 6). This ratio

decreased when CD45.2 KLS cells were contaminated with [3H] thymidine concentration of

3.7, 7.4 and 14.8 kBq/ml reaching a CD45.2/CD45.1 ratio of 2.8 when concentrations of 7.4

or 14.8 kBq/ml of [3H] thymidine were used for initial KLS contamination. These results

indicated that contamination of KLS cells with [3H] thymidine diminished their hematopoietic

reconstitution capacities when compared to non-contaminated KLS cells.

Discussion

Hematopoietic cells, including both hematopoietic stem cells (HSCs) and

hematopoietic progenitors, are highly sensitive to γ-irradiation. Numerous studies have

documented the cellular and molecular effects of γ-irradiation on hematopoiesis in vitro and

in vivo (18-20) but no study has been done on the effects of tritium on primary hematopoietic

cells. In a first step for such research, we studied the biological consequences of incorporation

of [3H] thymidine into the DNA of primary hematopoietic cells. Since the average energy of

3H β particle corresponds to a range that is smaller than the diameter of mammalian cells,

[3H] thymidine incorporated into DNA will affect the nucleus more directly than γ-irradiation

and the continuous release of [3H] thymidine energy to DNA that is in close proximity might

induce DNA damages during the whole life of the contaminated cells. In this article, we

analyzed the in vitro and in vivo effects of incorporation of [3H] thymidine on a hematopoietic

cell population enriched in Hematopoietic Stem Cells (HSCs), the c-kit+/Lin-/Sca1+ (KLS)

cell population.

The [3H] thymidine incorporation into DNA of KLS cells was dose dependent and

contamination with concentrations of [3H] thymidine as low as 0.74 kBq/ml results in

contamination of 50% of the KLS cells with an average of two molecules of [3H] thymidine

incorporated into the contaminated cells whereas more than 90% of the KLS cells were

contaminated with an average of more than 25 silver grains corresponding to [3H] thymidine

incorporated into DNA when contamination was performed with a 10 times higher

concentration of [3H] thymidine. Cell proliferation and viability showed that contamination

with [3H] thymidine induced a dose dependent decrease (resp. increase) in the proliferation

(cell death) process. When a [3H] thymidine concentration of 7.4 kBq/ml was used, a dramatic

decrease of cell proliferation together with an increase of apoptotic cells was observed after

24 or 48 hours of culture. This result is in sharp contrast with the results obtained on

hematopoietic cell lines as incubation with a concentration of 7.4 kBq/ml of [3H] thymidine

scarcely affects cell proliferation and only slightly influenced cell viability even after three

days of culture (14). Assuming that the same amount of [3H] thymidine is incorporated in

primary hematopoietic cells and in hematopoietic cell lines, this result indicated that primary

hematopoietic cells might be less effective in DNA repair after the DNA damages induced by

the incorporation of [3H] thymidine into DNA and/or that hematopoietic cell lines can

proliferate even with DNA damages, a property that might be linked to a deficient p53

pathway in these transformed hematopoietic cell lines.

[3H] thymidine is a continuous source of internal radiation and may introduce mutations and

damages in the progeny of contaminated KLS. However, no significant differences were

found in the myeloid hematopoietic colonies generated by contaminated KLS even with

concentrations of [3H] thymidine that results in multiple [3H] thymidine incorporated in most

if not all the KLS cells. Indeed, little variations in the total numbers and in the composition of

the colonies were observed, contaminated KLS showing a decrease in the total number of

colonies and a slight increase in the percentage of immature CFU-GEMM colonies followed

by a decrease in CFU-GM colonies compared to control KLS cells. When KLS cells are

irradiated with high doses of γ-irradiation, a dramatic decrease of myeloid and lymphoid

potentials of these cells has been reported (21) and our data indicated that [3H] thymidine

incorporated into DNA might have a different effect on the myeloid and lymphoid progeny of

KLS cells (see below). In addition, as γ-irradiation could generate signals from cell

membranes in addition to nuclear signals whereas [3H] thymidine contamination only

generates nuclear signals, our data might indicate effects of cell membranes damages on the

differentiation potentials of KLS cells. Another explanation of the absence of effect of [3H]

thymidine incorporation into the DNA of KLS cells on the cloning capacity and myeloid

potential of KLS cells might be that the colonies obtained in semi-solid methylcellulose assay

comes from less than 40% of the KLS cells and might originated from immature progenitors

that are very efficient in DNA repair (9, 21). Thus, the apoptosis observed might be due to

death of committed hematopoietic progenitors whereas immature progenitors and

hematopoietic stem cells might be protected from the effects of [3H] thymidine into their

DNA.

Transplantation of KLS cells contaminated with [3H] thymidine into lethally irradiated mice,

did not compromise their hematopoietic recovery even when contamination was performed

with concentrations of [3H] thymidine that results in more than 80% of KLS cells that have

incorporated [3H] thymidine in their DNA indicating again a very different effect of γ-

irradiation and [3H] thymidine contamination of hematopoietic stem cells. As γ-irradiation can

induce premature aging of KLS (8), we performed secondary transplantation to monitor any

effect of [3H] thymidine contamination of KLS cells on premature senescence. After this

secondary transplantation, the KLS compartment of the transplanted mice was significantly

decreased whatever concentrations of [3H] thymidine used for contamination and significant

variations in the B and red cell population was observed but only after contamination with

high doses of [3H] thymidine. These results are in line with previous results obtained after γ-

irradiation of KLS cells (8, 22) and suggests that low [3H] thymidine contamination doses

doesn’t induce cell death and impaired hematopoietic reconstitution but could accelerate a cell

senescence process. Finally, competition experiments showed that [3H] thymidine

contamination of KLS cells decreased their capacity to restore hematopoiesis when compared

to non-contaminated KLS cells suggesting that homing and/or retention of these contaminated

KLS cells might be altered.

In conclusion, the results shown in this article indicated that [3H] thymidine contamination of

a cell population enriched in hematopoietic stem cells has significant effects on the biological

properties of the contaminated stem cells that did not died but might display (i) defect in bone

marrow homing and/or (ii) premature senescence. As much of the tritium that remains in the

environment exists as tritiated water (HTO), contamination of KLS cells with tritiated water

will definitively indicate the effects of tritium on somatic stem cells.

Materials and methods

Mice

Eight- to twelve-weeks old C57Bl/6-Ly5.2 (CD45.2) mice were obtained from Charles

River Laboratories (l’Arbresle, France) and used as donors for HSCs cells. All recipient mice

had a CD45.1 genetic background and were bred under pathogen-free conditions in the iRCM

animal facility (C.E.A. Commisariat à l’Energie Atomique, Fontenay aux Roses, France).

Recipient mice have been lethally irradiated with 11Gy using a 137Cs irradiator. Approval for

animal care was received from Services Vétérinaires de la Santé et de la Production Animale

delivered by the Ministère de l’Agriculture, France.

Flow cytometry analysis

Bone marrow (BM) cells were flushed from both tibias and femurs of donor

C57Bl/6Ly5.2 mice and treated with a 0.75% NH4Cl (Sigma-Aldrich, St Louis, MO) to

eliminate erythrocytes. BM cells were stained with antibodies conjugated to fluorescein

isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-Cyanine 7 (PE-Cy 7) and

Allophycocyanin (APC) (all from Becton Dickinson Biosciences Pharmingen [BD], San

Diego, CA): CD45.1 (A20), CD45.2 (104), B220 (RA3-6B2), CD3 (145-2C11), NK1.1

(PK136), CD11b (M1/70), Gr-1 (RB6-8C5), and TER-119 (TER-119), cKit (2B8), Sca-1

(E13-161.7). Lineage cells were labeled using a byotin-conjugated Lineage cocktail (Miltenyi

Biotech, Bergisch Gladbach, Germany). Biotinylated antibodies were revealed with

streptavidin-phycoerythrin-cyanine-7. Stained cells were analyzed using a FACSCalibur

cytometer and CellQuest software (BD).

Purification of KLS cells by Flow Cytometry

KLS cell population was labeled using Sca1, cKit and Lineage cocktail markers. The

cKit+Lin-Sca1+ (KLS) population was isolated using a MOFLO high-speed cell sorter (Dako,

Glostrup, Denmark).

[3H] thymidine incorporation into DNA

Analysis of [3H] thymidine incorporation into DNA was performed using liquid

scintillation counting. DNA of KLS cells coming from contaminated medium was

precipitated using Trichloroacetic Acid. The radioactive pellet was dissolved in 5 ml of

scintillation fluid (Ecolite, ICN Biomedicals), counted (TriCARB 1900CA, Packard

Instruments, Meridien, CT) and the incorporated radioactivity was expressed in disintegration

per minute (d.p.m.).

Autoradiography

KLS cells were cultured for 24h in a medium with different concentrations of [3H]

thymidine. Then the cells were washed and plated on polysine slides (Kindler, Freiburg,

Germany). Slides were dipped into Kodak NTB2 nuclear emulsion diluted 2:3 with distilled

water at 42°C, dried for 2 hours at room temperature, exposed at 4°C with desiccant for 7

days in a dark box and finally developed by successive baths at 13°C in Kodak developer D-

19 for 4 min 30 sec, in 2% acetic acid stop solution for 30 sec and in Kodak fixer for 10 min.

Slides were counterstained with Mayers’s hemalun (Merck) and mounted with Eukitt (Fluka).

Silver grains found on nuclei were counted under a microscope (Olympus AX70) to assess the

level of tritiated thymidine incorporation.

γ-H2AX Immunostaining

KLS cells were cultured for 24 hours in a medium with different concentration of

[3H] thymidine. KLS cells were seeded on polylysine-coated slides and incubated at 37°C for

10 minutes. Cells were immediately fixed using 1% PFA aqueous solution (E.M.S. 15714)

for 10 min and permeabilized using 0.2% Triton X-100 solution for 5 min (Sigma Aldrich

93443). Staining of γ-H2AX foci was done with antiphospho-histone H2A.X (Ser139), clone

JBW301 antibody at a 1/200 dilution (Millipore, Billerica, MA). After washing, cells were

stained with a secondary antibody at a 1/400 dilution (Alexa fluor 488 goat anti-mouse IgG

antibody, Invitrogen A11001) and 4,6-diamidino-2-phenylindole (Vector Laboratories,

Burlingame, CA). Foci were quantified by fluorescence microscopy using a Leica TCS SPE

confocal imaging microscope with an ACS APO 40X oil objective.

KLS cell culture, [3H] thymidine contamination and proliferation

Sorted KLS cells were maintained into liquid suspension culture in presence of

cytokine cocktails to stimulate proliferation. Cultures were maintained at 37ºC, 5% CO2 and

95% humidity. Cell Media contained IMDM (GIBCO), 10% Fetal Bovin Serum (FBS,

GIBCO) and 1% PSG antibiotic mix (100-U/mL penicillin, 100-g/mL streptomycin, and 2-

mmol/L L-glutamine; Invitrogen, Carlsbad, CA). The cytokine combination (STEMCELL

technologies) included 100 ng/mL murine SCF, 100 ng/mL murine flt3 ligand (FL), 10 ng/mL

murine thrombopoietin (TPO), 20 ng/mL murine IL-3, and 10 ng/mL human IL-6. [3H]

thymidine (3245-3260 GBq/mmol) was purchased from New England Nuclear (Boston, MA,

USA). KLS cells were cultured at the initial concentration of 50 000 cells/ml in a medium

containing different concentrations of [3H] thymidine. After 24 and 48 hours of culture the

total number of live cells was determined after Trypan blue staining.

Apoptosis assay

Annexin V (Becton Dickinson Biosciences Pharmingen [BD], San Diego, CA) was used in

conjunction with 7-Amino-Actinomycin (7-AAD) to identify apoptotic cells by FACS

analysis. Annexin V was used according to manufacturer protocol.

Colony Forming Unit (CFU) Assay

CFU assay (MethoCult 03434, StemCell Technologies) was performed according to

manufacturer’s instructions. Briefly 300 KLS cells were added to 2 ml of complete Methocult

and were seeded to three 35 mm culture dishes. Culture dishes were transferred to an

incubator at 37 ºC, 5% CO2 and 95% humidity and the CFU were enumerated after 7 days in

culture. Cultures were characterized for the presence of myeloid and multi-potential CFU.

Myeloid CFU include Colony-Forming Unit-Granulocyte (CFU-G), Colony-Forming Unit-

Macrophage (CFU-M) and Colony-Forming Unit-Granulocyte, Macrophages (CFU-GM).

Multi-potential CFU includes Colony-Forming Units with mixed populations of erythroid and

myeloid cells (CFU-GEMM).

Bone marrow transplantation

For non-competitive long-term reconstitution assay, recipient mice were lethally γ-

irradiated (11 Gy) 24 hours before transplantation and treated with antibiotic in drinking

water (Baytril 10%; Bayer AG, Leverkusen, Germany) for at least 4 weeks. Twenty thousands

KLS cells cultured for 24h in a medium with different concentration of [3H] thymidine were

injected via the retro-orbital vein into lethally irradiated recipients. The transplanted mice

were analyzed 4 months after transplantation. Five hundreds thousands bone marrow cells

were then isolated from transplanted mice and injected into lethally irradiated recipient mice

for a secondary transplantation. The bone marrow was analyzed 4 months later.

For competitive long-term reconstitution assay, five thousands KLS-CD45.2 cultured for 24h

in a medium with different concentration of [3H] thymidine together with five hundreds

thousands non-contaminated BM-CD45.1 cells were simultaneously injected via the retro-

orbital vein into lethally irradiated mice. The transplanted mice were analyzed 4 months later.

Data analysis and statistics

Values are presented as the mean, median or cell number ± SD. Statistical

comparisons between groups were done using the Student’s t-test, p < 0.05 and p < 0.001

were considered statistically significant (*) and highly statistically significant (**).

Acknowledgments

The authors declare that they have no conflict of interest. We acknowledge Pierre

Fouchet and Zahra Kadri for helpful discussions. We are grateful to the staff of the iRCM

animal facility for excellent support in mouse studies and to Benjelloun H., Deschamps N.

and Baijer J. of the iRCM cytometry platform for excellent support in FACS and cell sorting

experiments. Fabio di Giacomo and Vilma Barroca are supported by fellowships from Marie

Curie Research fellowship from the EU fp6 program ‘Eurythron’ MRTN-CT-2004-005499

and from Inserm. This project was supported by grants from EDF, ARC (3710), Inserm and

CEA/DSV.

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Figure Legends

Figure 1: [3H] thymidine incorporation into KLS DNA. KLS cells were purified by cell sorting

from bone marrow of healthy C57Bl/6 mice and cultured for 24 hours in a medium containing the

indicated concentrations of [3H] thymidine.

A. Dose dependent incorporation of [3H] thymidine into the DNA of KLS cells. DNA from

1000 KLS cells contaminated with the indicated concentrations of [3H] thymidine was TCA

precipitated and the amount of precipitated [3H] thymidine was counted using a liquid

scintillator counter. Data are shown as disintegration per minute (d.p.m.) (Mean ± SEM, n=5; p≤

0.05).

B. Percentage of KLS cells that have incorporated [3H] thymidine in their DNA. Silver grains

corresponding to [3H] thymidine incorporated into DNA of KLS cells were detected after

autoradiography on contaminated KLS cells. Data are expressed as percentage of KLS cells that

contained silver grains (Mean ± SEM, n=5, *p < 0.05).

C. Average number of incorporated [3H] thymidine per KLS cell. Data are expressed as average

number of silver grains per KLS cell (Mean ± SEM, n=5, **p < 0.001).

Figure 2: Proliferation and apoptosis of KLS cells grown for 24 and 48 hours in liquid medium

containing different concentrations of [3H] thymidine.

A. KLS cells were purified by cell sorting from bone marrow of healthy C57Bl/6 mice and

cultured in medium containing the indicated concentrations of [3H] thymidine. KLS cells were

cultured at an initial concentration of 50,000 cells/ml in 100μl of medium and contaminated with

different concentrations of [3H] thymidine (0; 3.7; 7.4; 14.8 and 37.03 kBq/ml). After 24 and 48

hours of culture, total cell number was measured. Results are expressed as cell number (Mean ±

SEM, n=5, *p < 0.05).

B. Percentage of apoptotic cells after 24 and 48 hours of culture of KLS cells in medium

containing various concentrations of [3H] thymidine. KLS cells were cultured in a medium

containing two different concentrations of [3H] thymidine (0; 3.7 and 14.8 kBq/ml). Apoptosis

was monitored by AnnexinV and 7-AAD staining and FACS analysis. Results are expressed as

percentage of apoptotic cells (Mean ± SEM, n=5, *p < 0.05 **p < 0.001).

Figure 3: γ-H2AX foci induced by [3H] thymidine incorporation into KLS DNA. KLS cells

were grown for 24 hours in a medium containing the indicated concentrations of [3H] thymidine,

washed, fixed and Double Strand Breaks (DSBs) were monitored by H2AX immunostaining.

A. Percentage of KLS cells containing at least one γ-H2AX focus as a function of concentrations

of [3H] thymidine in the medium where KLS cells were cultured for 24 hours. For each [3H]

thymidine concentration, a minimum of 100 nuclei was scored (Mean ± SEM, n=3, *p < 0.05).

B. Mean number of γ-H2AX foci per cell as a function of concentrations of [3H] thymidine in the

medium where KLS cells were cultured for 24 hours. For each [3H] thymidine concentration, a

minimum of 50 cells containing γ-H2AX foci were selected and the number of γ-H2AX foci in

each individual cells was scored (Mean ± SEM, n=3, *p < 0.05).

Figure 4: Colony Forming Unit (CFU) Assay on KLS cells previously cultured for 24 hours in

liquid medium containing different concentrations of [3H] thymidine. KLS were cultured for 24

hours in a medium containing the indicated concentration of [3H] thymidine (0; 3.7 and 14.8

kBq/ml). 100 KLS were then seeded on a semi-solid methylcellulose layer and hematopoietic

colonies were counted one week later.

A. Total number of myeloid and multipotential CFU colonies. Data are expressed as number of

colonies (Mean ± SEM, n=5)

B. Percentages of myeloid lineage committed colonies. Myeloid CFU include Colony Forming

Unit-Granulocyte (CFU-G), Colony Forming Unit-Macrophage (CFU-M) and Colony Forming

Unit-Granulocytes and Macrophages (CFU-GM). Multi-potential CFU includes Colony Forming

Units with mixed populations of erythroid and myeloid cells (CFU-GEMM). Data are expressed

as percentage of colonies (Mean ± SEM, n=5).

Figure 5:

A and B. Characterization of bone marrow hematopoietic cell populations of mice lethally

irradiated and transplanted with KLS cells. KLS cells were cultured for 24 hours in a medium

containing the indicated concentrations of [3H] thymidine (0; 1.48; 3.7 and 14.8 kBq/ml). 20,000

KLS were then transplanted in mice lethally irradiated at 11Gy. Mice were sacrificed 4 months

after engraftment and bone marrow cells were labeled to identify the hematopoietic

subpopulations. A. Percentage of bone marrow B-cells (B220+), Neutrophils (CD11b+ Gr1+) and

erythroid cells (Ter119+) (left panel) and T-Lymphocytes (CD3+) and Natural Killer cells

(NK1.1+) (right panel), after transplantation of contaminated KLS cells into lethally irradiated

mice (Mean ± SEM, n=5). B. Percentage of Hematopoietic Stem cells (KLS) after

transplantation of contaminated KLS cells into lethally irradiated mice (Mean ± SEM, n=5).

C and D. 500,000 bone marrow cells isolated 4 months after transplantation into lethally

irradiated mice of 20,000 KLS cells contaminated with different amounts of [3H] thymidine were

engrafted in lethally irradiated mice. Mice were sacrificed 4 months after this secondary

transplantation and bone marrow cells were characterized to identify the hematopoietic

subpopulations. C. Percentage of bone marrow B-cells (B220+), Neutrophils (CD11b+ Gr1+) and

erythroid cells (Ter119+) (left panel) and T-Lymphocytes (CD3+) and Natural Killer cells

(NK1.1+) (right panel), after secondary engraftment (Mean ± SEM, n=5, *p < 0.05). D.

Percentage of Hematopoietic Stem cells (KLS) after secondary transplantation into lethally

irradiated mice (Mean ± SEM, n=5, *p < 0.05).

Figure 6: Bone marrow CD45.2/CD45.1 chimerism four months after transplantation, in mice

lethally irradiated, of 5,000 KLS-Ly5.2 cultured for 24 hours in medium containing the indicated

concentrations of [3H] thymidine (0; 1.48; 3.7; 7.4 and 14.8 kBq/ml) and 500,000 Ly-5.1

(CD45.1+) bone marrow cells. Mice were sacrificed 4 months after transplantation and bone

marrow chimerism was determined by FACS analysis. Data are expressed as ratio

CD45.2/CD45.1 (Mean ± SEM, n=5, *p < 0.05) and 5 indicated the initial ratio of transplanted

KLS cells.

0

100

200

300

1000

2000

3000

4000

5000

0.37 0.74 1.48 7.4 14.8 37.03

kBq/ml

3.7

d.p.m./1000cells

0

20

40

60

80

100

*

0.74 1.48 3.7 7.4

kBq/ml

Silvergrain-positivecells(%)

0

10

20

30

40

**

**

**

0.74 1.48 3.7 7.4

kBq/ml

Averagenumberofsilvergrain/cell

Figure 1

A

B

C

0h 24h

48h

0

20000

40000

60000

80000

0 kBq/ml3.7 kBq/ml

7.4 kBq/ml

37.03 kBq/ml14.8 kBq/ml

5000

*

*

*

*

*

*

Cellnumber

24h

48h

0

20

40

60

80

0 kBq/ml3.7 kBq/ml14.8 kBq/ml*

**

Apoptoticcells(%)

A

B

Figure 2

0

20

40

60

80

100

0 0.74 1.48 3.7 14.8 37.03

kBq/ml

�-H2AXfoci-positivecells(%)

0

5

10

15

20

0 0.74 1.48 3.7 14.8 37.03

kBq/ml

Averagenumberof�-H2AXfoci/cell

Figure 3

A

B

*

*

0

20

40

60

0 14.83.7

kBq/ml

Numberofcolonies

CFU-G

CFU-M

CFU-GM

CFU-GEMM

0

10

20

30

40

500 kBq/ml3.7 kBq/ml14.8 kBq/ml

Colonies(%)

A

B

Figure 4

B22

0

CD11

b Gr1

Ter1

190

10

20

30

40

50

Cells

(%)

KLS

0.1

0.2

0.3

Cells

(%)

B22

0+

CD11

b+Gr1

+

Ter11

9+0

10

20

30

40

50

*

*

Cells

(%)

KLS

0.0

0.1

0.2

0.3 *

Cells

(%)

A

B

C

D

Figure 5

CD3

NK1.

10

2

4

6

Cells

(%)

CD3+

NK11

+0

1

2

3

4

Cells

(%)

0 kBq/ml

1.48 kBq/ml

3.7 kBq/ml

14.8 kBq/ml.

0

2

4

6

8

0 1.48 3.7 7.4 14.8

5

*

Figure 6

kBq/ml

RatioCD45.2/CD45.1