Johanna Frick

Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome

Recent studies have suggested that ex vivo expansion of autologous hematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. We investigated the potential of a combined infusion of autologous ex vivo expanded hematopoietic cells with mesenchymal (MSCs) for the treatment of multi‐organ failure syndrome following irradiation in a non‐human primate model.

Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs : A study of their quantitative distribution after irradiation damage

Mesenchymal stem cells (MSCs) have been shown to migrate to various tissues. There is little information on the fate and potential therapeutic efficacy of the reinfusion of MSCs following total body irradiation (TBI). We addressed this question using human MSC (hMSCs) infused to nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice submitted to TBI. Further, we tested the impact of additional local irradiation (ALI) superimposed to TBI, as a model of accidental irradiation. NOD/SCID mice were transplanted with hM‐SCs. Group 1 was not irradiated before receiving hMSC infusion. Group 2 received only TBI at a dose of 3.5 Gy, group 3 received local irradiation to the abdomen at a dose of 4.5 Gy in addition to TBI, and group 4 received local irradiation to the leg at 26.5 Gy in addition to TBI. Fifteen days after irradiation, quantitative and spatial distribution of the hMSCs were studied. Histological analysis of mouse tissues confirmed the presence of radio‐induced lesions in the irradiated fields. Following their infusion into nonirradiated animals, hMSCs homed at a very low level to various tissues (lung, bone marrow, and muscles) and no significant engraftment was found in other organs. TBI induced an increase of engraftment levels of hMSCs in the brain, heart, bone marrow, and muscles. Abdominal irradiation (AI) as compared with leg irradiation (LI) increased hMSC engraftment in the exposed area (the gut, liver, and spleen). Hind LI as compared with AI increased hMSC engraftment in the exposed area (skin, quadriceps, and muscles). An increase of hMSC engraftment in organs outside the fields of the ALI was also observed. Conversely, following LI, hMSC engraftment was increased in the brain as compared with AI. This study shows that engraftment of hMSCs in NOD/ SCID mice with significantly increased in response to tissue injuries following TBI with or without ALI. ALI induced an increase of the level of engraftment at sites outside the local irradiation field, thus suggesting a distant (abscopal) effect of radiation damage. This work supports the use of MSCs to repair damaged normal tissues following accidental irradiation and possibly in patients submitted to radiotherapy.

Mesenchymal Stem Cells Increase Self-Renewal of Small Intestinal Epithelium and Accelerate Structural Recovery after Radiation Injury

Patients who undergo pelvic or abdominal radiotherapy may develop side effects that can be life threatening. Tissue complications caused by radiation-induced stem cell depletion may result in structural and functional alterations of the gastrointestinal (GI) tract. Stem cell therapy using mesenchymal stem cells (MSC) is a promising approach for replenishment of the depleted stem cell compartment during radiotherapy. There is little information on the therapeutic potential of MSC in injured-GI tract following radiation exposure. In this study, we addressed the ability of MSC to support the structural regeneration of the small intestine after abdominal irradiation. We isolated MSC from human bone marrow and human mesenchymal stem cells (hMSC) were transplanted into immunotolerent NOD/SCID mice with a dose of 5.10(6) cells via the systemic route. Using a model of radiation-induced intestinal injury, we studied the link between damage, hMSC engraftment and the capacity of hMSC to sustain structural recovery. Tissue injury was assessed by histological analysis. hMSC engraftment in tissues was quantified by PCR assay. Following abdominal irradiation, the histological analysis of small intestinal structure confirms the presence of partial and transient (three days) mucosal atrophy. PCR analysis evidences a low but significant hMSC implantation in small intestine (0.17%) but also at all the sites of local irradiation (kidney, stomach and spleen). Finally, in presence of hMSC, the small intestinal structure is already recovered at three days after abdominal radiation exposure. We show a structural recovery accompanied by an increase of small intestinal villus height, three and fifteen days following abdominal radiation exposure. In this study, we show that radiation-induced small intestinal injury may play a role in the recruitment of MSC for the improvement of tissue recovery. This work supports, the use of MSC infusion to repair damaged GI tract in patients subjected to radiotherapy. MSC therapy to avoid extended intestinal crypt sterilization is a promising approach to diminish healthy tissue alterations during the course of pelvic radiotherapy.

PLASMA Flt-3 LIGAND CONCENTRATION CORRELATED WITH RADIATION-INDUCED BONE MARROW DAMAGE DURING LOCAL FRACTIONATED RADIOTHERAPY

PURPOSE To determine whether variations in the plasma Flt-3 ligand (FL) concentration after radiotherapy (RT) may serve as a biomarker for radiation-induced bone marrow damage. METHODS AND MATERIALS Twenty-seven patients were followed during RT. The irradiated bone marrow volume was determined. The blood cell counts and plasma FL concentrations were evaluated before and after RT. The expression of membrane-bound FL and mRNA expression were also defined in circulating blood cells. RESULTS We found a negative correlation between the plasma FL concentration and the number of circulating white blood cells and platelets during RT. Moreover, the overall amount of FL in the blood of patients during RT correlated directly with both the cumulated radiation dose and the proportion of irradiated bone marrow. CONCLUSIONS We demonstrated that the variations in plasma FL concentration directly reflect the radiation-induced bone marrow damage during fractionated local RT. We suggest a possible use for FL monitoring as a means to predict the occurrence of Grade 3-4 leukopenia or thrombocytopenia during the course of RT.

Radiation-Induced Increase in Plasma Flt3 Ligand Concentration in Mice: Evidence for the Implication of Several Cell Types

Abstract Prat, M., Demarquay, C., Frick, J., Thierry, D., Gorin, N. C. and Bertho, J. M. Radiation-Induced Increase in Plasma Flt3 Ligand Concentration in Mice: Evidence for the Implication of Several Cell Types. Radiat. Res. 163, 408–417 (2005). Circulating T lymphocytes were proposed as the main producer of Flt3 ligand. However, during aplasia, there is a drastic reduction in the number of T lymphocytes, while plasma Flt3 ligand concentration is increased. This contradiction prompted us to compare variations in plasma Flt3 ligand during radiation-induced aplasia in BALB/c mice and in T-lymphocyte-deficient NOD-SCID mice to delineate the role of T lymphocytes in the increase in Flt3 ligand concentration. The results showed that plasma Flt3 ligand concentration was increased similarly in the two strains of mice, and that Flt3 ligand concentration was negatively correlated to the number of residual hematopoietic progenitors. Moreover, the Flt3 ligand mRNA expression and Flt3 ligand protein concentration were similar in the two strains of mice in all organs tested, i.e. thymus, spleen, bone marrow, liver, brain and blood cells. These results confirm that Flt3 ligand concentration in the blood is a reflection of bone marrow function and that T lymphocytes are not the main regulator of Flt3 ligand variations during aplasia.

Use of Flt3 Ligand to Evaluate Residual Hematopoiesis after Heterogeneous Irradiation in Mice

Abstract Prat, M., Demarquay, C., Frick, J., Dudoignon, N., Thierry, D. and Bertho, J. M. Use of Flt3 Ligand to Evaluate Residual Hematopoiesis after Heterogeneous Irradiation in Mice. Radiat. Res. 166, 504–511 (2006). We evaluated the possibility of using plasma Flt3 ligand (FL) concentration as a biological indicator of bone marrow function after heterogeneous irradiation. Mice were irradiated with 4, 7.5 or 11 Gy with 25, 50, 75 or 100% of the bone marrow in the field of irradiation. This model of irradiation resulted in graded and controlled damage to the bone marrow. Mice exhibited a pancytopenia correlated with both the radiation dose and the percentage of bone marrow irradiated. The FL concentration in the blood increased with the severity of bone marrow aplasia. Nonlinear regression analysis showed that the FL concentration was strongly correlated with the total number of residual colony-forming cells 3 days after irradiation, allowing a precise estimate of residual hematopoiesis. Moreover, the FL concentration on day 3 postirradiation was correlated with the duration and severity of subsequent pancytopenia, suggesting that variations in FL concentrations might be used as a predictive indicator of bone marrow aplasia, especially by the use of linear regression equations describing these correlations. Our results provide a rationale for the use of FL concentration as a biological indicator of residual hematopoiesis after heterogeneous irradiation.

Application of Autologous Hematopoietic Cell Therapy to a Nonhuman Primate Model of Heterogeneous High-Dose Irradiation

Abstract Bertho, J-M., Prat, M., Frick, J., Demarquay, C., Gaugler, M-H., Dudoignon, N., Clairand, I., Chapel, A., Gorin, N-C., Thierry, D. and Gourmelon, P. Application of Autologous Hematopoietic Cell Therapy to a Nonhuman Primate Model of Heterogeneous High-Dose Irradiation. Radiat. Res. 163, 557– 570 (2005). We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.

Feasibility and limits of bone marrow mononuclear cell expansion following irradiation

Purpose: To define the ability of bone marrow mononuclear cells (BMMNC) to expand after irradiation and to determine the amount of apoptosis in irradiated expanded cells. Materials and methods: Non‐human primate BMMNC were irradiated in vitro at doses ranging from 0 to 4 Gy and were cultured during 1 week in the presence of interleukin 3, interleukin 6, stem cell factor, thrombopoietin and fms‐like tyrosine kinase‐3 ligand. The expansion yield of BMMNC, colony‐forming cells and CD34+ cells were compared with non‐irradiated control cultures. Apoptosis in expanded cells was also defined by annexin V/propidium iodine staining. Results: Irradiation of BMMNC up to 1 Gy did not modify the ability of haematopoietic cells to expand. At higher doses, expansion of haematopoietic cells is reduced as compared with non‐irradiated cultures but it remains significant. This reduction in expansion of BMMNC was related to radiation‐induced apoptosis. Conclusion: The results suggest that it is possible to expand haematopoietic cells after irradiation doses at least up to 2 Gy. This suggests a possible use of cell therapy for the treatment of radiation accident victims.

Reinjection of Ex Vivo–Expanded Primate Bone Marrow Mononuclear Cells Strongly Reduces Radiation-Induced Aplasia

To assess the therapeutic efficacy of ex vivo-expanded hematopoietic cells in the treatment of radiation-induced pancytopenia, we have set up a non-human primate model. Two ex vivo expansion protocols for bone marrow mononuclear cells (BMMNC) were studied. The first consisted of a 7-day culture in the presence of stem cell factor (SCF), Flt3-ligand, thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6, which induced preferentially the expansion of immature hematopoietic cells [3.1 +/- 1.4, 10.0 +/- 5.1, 2.2 +/- 1.9, and 1.0 +/- 0.3-fold expansion for mononuclear cells (MNC), colony-forming units-granulocyte-macrophage (CFU-GM), burst-forming units erythroid (BFU-E), and long-term culture initiating cells (LTC-IC) respectively]. The second was with the same cytokine combination supplemented with granulocyte colony-stimulating factor (G-CSF) with an increased duration of culture up to 14 days and induced mainly the production of mature hematopoietic cells (17.2 +/- 11.7-fold expansion for MNC and no detectable BFU-E and LTC-IC), although expansion of CFU-GM (13.7 +/- 18.8-fold) and CD34+ cells (5.2 +/- 1.4-fold) was also observed. Results showed the presence of mesenchymal stem cells and cells from the lymphoid and the megakaryocytic lineages in 7-day expanded BMMNC. To test the ability of ex vivo-expanded cells to sustain hematopoietic recovery after radiation-induced aplasia, non-human primates were irradiated at a supralethal dose of 8 Gy and received the product of either 7-day (24 h after irradiation) or 14-day (8 days after irradiation) expanded BMMNC. Results showed that the 7-day ex vivo-expanded BMMNC shortened the period and the severity of pancytopenia and improved hematopoietic recovery, while the 14 day ex vivo-expanded BMMNC mainly produced a transfusion-like effect during 8 days, followed by hematopoietic recovery. These results suggest that ex vivo expanded BMMNC during 7 days may be highly efficient in the treatment of radiation-induced aplasia.

Kinetics of plasma FLT3 ligand concentration in hematopoietic stem cell transplanted patients

The present study aimed to follow-up variations in plasma Flt3 ligand (FL) concentration after hematopoietic stem cell transplantation and to compare the influence of conditioning regimens on variations in FL concentration. Ten patients undergoing a conditioning regimen, including BEAM, cyclophosphamide (Cy) + total body irradiation or Cy + anti-thymocyte globulins (ATG), which was then followed by hematopoietic stem cell transplantation, were studied. Plasma FL concentrations, white blood cell (WBC) expression of both FL mRNA and the membrane-bound form of FL were carried out at different times post-treatment. The results indicated that plasma FL concentration increased rapidly after the conditioning regimen in all patients, in correlation with the decrease in number of WBCs. The area under the curve of FL according to time was directly correlated with the duration of pancytopenia, except when ATG was included in the conditioning regimen. Although the number of patients was limited in this study, the comparison of ATG-treated patients and other patients suggests that plasma FL concentration is regulated by a complex mechanism partly involving circulating blood cells.