Effect of inorganic nitrates on the morphofunctional condition of the kidney in the rat

Texte intégral

(1)W17:ZFETA692XA. SIBY. Fetal Diagn Ther 1998;13:216–222. Received: November 11, 1997 Accepted: March 26, 1998. Laboratoire d’Histologie-Embryologie II, Faculté de Médecine de Nancy, France. Effect of in utero Infusion Route on Lymphocyte Distribution in Fetal Rat Tissues. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. Key Words In utero infusion route, cells Lymphocytes Rat, infusion route. Abstract Infusion of cells into the fetus is a new form of intrauterine therapy for several genetic disorders. The effect of in utero infusion routes on labelled lymphocyte distribution in fetal rat organs was investigated. Fetuses, 14–16 days of gestation, were infused in utero with a Hoechst 33342 (bis-benzamide) labelled lymphocyte and FITC-labelled polystyrene bead mixture via four routes: intraperitoneal, intraplacental, intra-amniotic, and intravenous. The distribution within tissues was evaluated in frozen sections of placenta and fetal organs. Our results suggest that among the four routes tested, the intravenous route offered the possibility to reach easily fetal organs without any cell loss and yielded higher cell and bead concentrations in fetal organs, especially in the liver and in the kidney. In conclusion, the intravenous route seems to be appropriate for hematopoietic cell transplantation in the developing fetus.. T. Aboussaouira A. Gerard H. Gerard. OOOOOOOOOOOOOOOOOOOOOO. The infusion of normal bone marrow cells into the fetus might provide a new form of therapy for the treatment of some life-threatening disorders, such as major thalassemia [1]. Allogeneic cells injected at an early stage of development might be expected to induce immunological tolerance [2]; they could also survive in their new environment and produce normal red cells or lymphocytes that could provide protection to the affected individual. Such a procedure could ameliorate or even completely prevent the clinical manifestations of the disease without repairing the genetic defect [1]. The ability to induce hematopoietic chimerism in experimental animals without recourse to physical (i.e., irradiation) or pharmacolog-. ABC. © 1998 S. Karger AG, Basel 1015–3837/98/0134–0216$15.00/0. Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger.com. Accessible online at: http://BioMedNet.com/karger. ical manipulation of the immune system is important to our understanding of the normal prenatal development of immunocompetence [3]. Several mammalian models were proposed for injections of cells into a fetus or for inducing tolerance [3–7]. In humans, such injections are still performed for therapy [8–10]. However, a literature review shows that cell injections were carried out either by intraperitoneal intraumbilical (vein), intra-amniotic, or intravenous routes. The aim of this study was to compare four intrauterine infusion routes (intravenous, intraperitoneal, intra-amniotic, and intraplacental) of hematopoietic cells and to follow the distribution pathway in fetal organs. In addition to that of labelled of cells, the distribution of labelled beads was investigated as a control.. Dr. T. Aboussaouira Faculté de Médecine et de Pharmacie 19, rue Tarik Bnou Ziad 20000 Casablanca (Morocco) Tel. +212 27 16 30, Fax +212 26 14 53. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. Introduction.

(2) SIBY. Fetuses, 14–16 weeks of gestation, were infused with Hoechst 33342 (bis-benzamide) labelled lymphocytes and FITC (fluorescein isothiocyanate) labelled polystyrene beads via four different infusion routes: intraperitoneal, intraplacental, intra-amniotic, and intravenous. The beads and cells were counted, and their distributions were evaluated in the frozen sections of placenta and fetal organs such as the heart, the lung, the kidney, and the liver.. Fetus and Organ Sampling Six hours after the infusions, the placentas and the fetuses have been removed from pregnant rats after general anesthesia by median laparatomy and cesarean section. The placentas were fixed in 10% formol in PBS. The organs were separated from the fetuses after a large median incision and then fixed in 10% formol in PBS. Three days after fixation, the placentas and some fetal organs (heart, kidney, lung, and liver) were dropped in liquid nitrogen and stored at –30 ° C until use (1–2 weeks later).. Animals Twenty pregnant female Wistar rats used in this experiment were bred and kept in our colony under routine laboratory conditions. Five pregnant females were used for each infusion route. Fetal rats of the required ages were produced by timed matings [11]: 4–6 fetuses per pregnant female, and only half the fetuses were treated.. Histologial Technique Cryostat sections (10 Ìm) were prepared, and labelled cells and beads were counted in each section using an ultraviolet microscope (Nikon). Ten to 20 nonconsecutive sections were used for each fetus. The surface of each section was determined with an image-analyzing computer (Philips 3202) connected to a camera (Ikegami CCD).. Lymphocyte Sampling A spleen lymphocyte suspension was prepared from adult female rat. Minced spleen cells were pressed gently by Potter, and cells were suspended in phosphate-buffered saline (PBS), filtrated on cotton, and depleted of red cells by ammonium chloride (Sigma, St. Louis, Mo., USA; 0.155 M at pH 7.4).. Data Expression Labelled cell and bead densities per cubic millimeter of placental and fetal tissue were analyzed according to the equation D = N/S ! E, where N is the number of labelled cells or beads, S the total surface in square millimeters of the nonconsecutive sections of each tissue, and E the thickness in millimeters. The ‘total’ number of labelled cells or beads present in an organ is calculated according to the equation Nt = D ! V, where D is the labelled cell or bead density per cubic millimeter and V the total organ volume in cubic millimeters which was determined by measuring the displaced liquid volume by immersing the fetal organ.. Material and Methods. Labelling Lymphocytes with Hoechst 33342 (Bis-Benzamide) Lymphocytes (2 ! 107/ml) were incubated in vitro in 30 Ìg/ml Hoechst 33342 in PBS (pH 7.4) for 30 min at 37 ° C with gentle shaking. Labelled cells were washed twice through centrifugation in PBS for 10 min at 300 g, then resuspended in PBS. The labelled lymphocytes were stored at 4 ° C until infusion 1 h later. Viability Test The viability of injected cells was tested by trypan blue (4 vol trypan blue 0.2% and 1 vol of NaCl 4.25%); there were only 5% death cells after labelling. Preparation of FITC Polystyrene Beads FITC-labelled polystyrene beads (2 Ìm in diameter; Polysciences) were used. The 20-Ìl latex bead suspension was washed three times with PBS and centrifuged at 400 g for 10 min each time [12]. The last supernatant was removed, and the beads were resuspended in PBS and mixed with Hoechst 33342 labelled lymphocytes (1 vol cells/1/10 vol beads). This mixture was stored at 4 ° C until infusion 1 h later.. Statistics Using chi-square and Student t tests, we have compared the four routes of infusion, then the distribution into the organs, and finally the cell and bead distributions. The software used was Statview®. We have considered that the results were significant when p ! 0.05.. Results. Fetal Rat Cell Infusion Pregnant rats at 14–16 days of gestation were anesthetized with ether, the uterus was exteriorized after median subombilical laparatomy, and fetuses were localized by transillumination through the fetal membranes. The allogeneic cells were infused into the fetuses by the following routes: intraperitoneal, intravenous (in the fetal vessels of the ombilical cord insertion), intraplacental, or intra-amniotic. Each fetus received 2 ! 107 cells mixed with 0.5 ! 106 beads in 0.5 ml PBS, and the needle used was 30G1/2. The uterine wall and the abdomen were then closed by a series of purse-string ligatures with silk thread. Intravenous infusion has first been performed after several attempts in mice caudal veins, then into pig fetal veins at 50–60 days of gestation. Finally, we tested the intravenous infusion for the fetal rat at the latest stages of development: 19, 18, 17 and 16 days.. Six hours after infusions by intraplacental route (fig. 1), bead and cell concentrations were significantly lower (p ! 0.001) in all the organs tested, except the placenta where the number of beads was not significantly increased. Through intra-amniotic route (fig. 2) and 6 h after the infusions, the lung showed a significantly increased concentration of cells (p ! 0.001); the placenta showed only a significantly elevated concentration of beads (p ! 0.01). In the other cases the rates were too low. Through the intraperitoneal route (fig. 3) and 6 h after infusions, the liver showed a significant concentration of cells and beads (p ! 0.001), and a significant concentration of cells (p ! 0.001) was seen in the kidney. The cell and bead tissue distribution was significantly lower (p ! 0.01) in lung and heart than in liver and kidney. However, by the intraperitoneal route, the concentrations of cells and beads in the placenta were nil. The intraperitoneal. Infusion Routes Comparison. Fetal Diagn Ther 1998;13:216–222. 217. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. W17:ZFETA692XA.

(3) W17:ZFETA692XA. SIBY. Fig. 1. Colonization intensity per cubic millimeter tissue of Hoechst (bis-benzamide) labelled lymphocytes (2 ! 107) and FITC-labelled beads (0.5 ! 106) within the placenta, the liver, the lung, the heart, and the kidney in fetal rats 6 h after infusion by intraplacental route. Labelling was counted in frozen thick sections. Mean values and standard deviation are shown.. Fig. 2. Colonization intensity per cubic millimeter tissue of Hoechst (bis-benzamide) labelled lymphocytes (2 ! 107) and FITC-labelled beads (0.5 ! 106) within the placenta, the liver, the lung, the heart, and the kidney in fetal rats 6 h after infusion by intra-amniotic route. Labelling was counted in frozen thick sections. Mean values and standard deviation are shown.. 218. Fetal Diagn Ther 1998;13:216–222. intravenous infusion. The four infusion routes are compared in figure 5 for cells using the same scale and in figure 6 for beads. These two figures show a high concentration of cells (and beads in the liver) after infusion via the intravenous route. The analysis of the total cell and bead numbers in each organ for the different infusion routes (table 1) showed that the liver presented the highest numbers of beads (65,600) and cells (950,400) by intravenous route as compared with the three other routes. In the kidney, the values of beads and cells were significantly higher (p ! 0.001) by the intravenous route than by the three other routes. The numbers were smaller or nil by intra-amniotic and intraplacental routes (75.6 cells in the heart and 37.8 beads in the kidney after administration via the intra-amniotic. Aboussaouira/Gerard/Gerard. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. route showed important values as compared with intraamniotic and intraplacental routes. Through the intravenous route (fig. 4) and 6 h after infusion, the concentrations of cells and beads were significantly elevated (p ! 0.001) in all the organs studied, except the placenta where the cell number was nil. Liver and kidney presented significantly elevated values (p ! 0.001) as compared with the other organs. The cell number was higher, but not significant, than the bead number in almost all organs after intravenous infusions. The intravenous route allowed to find up to 10% of the injected cells and beads. Only 0.1–1% of cells and beads could be found in the other three routes tested. Colonization intensities of beads and cells in different organs were generally low after intraplacental infusion and high after.

(4) W17:ZFETA692XA. SIBY. Fig. 3. Colonization intensity per cubic millimeter tissue of Hoechst (bis-benzamide) labelled lymphocytes (2 ! 107) and FITC-labelled beads (0.5 ! 106) within the placenta, the liver, the lung, the heart, and the kidney in fetal rats 6 h after infusion by intraperitoneal route. Labelling was counted in frozen thick sections. Mean values and standard deviation are shown.. Fig. 4. Colonization intensity per cubic millimeter tissue of Hoechst (bis-benzamide) labelled lymphocytes (2 ! 107) and FITC-labelled beads (0.5 ! 106) within the placenta, the liver, the lung, the heart, and the kidney in fetal rats 6 h after infusion by intravenous route. Labelling was counted in frozen thick sections. Mean values and standard deviation are shown.. Fig. 5. Organ distribution of Hoechst (bis-benzamide) labelled lymphocyte (2 ! 107) in the placenta, the liver, the kidney, the lung, and the heart in fetal rats 6 h after infusion by intraplancental (IPL), intra-amniotic (IA), intraperitoneal (IP), and intravenous (IV) routes. Mean values and standard deviation are shown.. Fetal Diagn Ther 1998;13:216–222. 219. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. Infusion Routes Comparison.

(5) W17:ZFETA692XA. SIBY. Fig. 6. Organ distribution of FITC-labelled beads (0.5 ! 106) in the placenta, liver, kidney, lung, and heart in fetal rats 6 h after infusion by intraplacental (IPL), intraamniotic (IA), intraperitoneal (IP), and intravenous (IV) routes. Mean values and standard deviation are shown.. Intravenous. Intraperitoneal. Liver Cells Beads. 950,400 65,600. 18,528 5,496. Kidney Cells Beads. 69,234 26,190. 2,416.6 820.8. Lung Cells Beads. 52,848 5,536. 72 1,280. Heart Cells Beads. 26,190 76,300. 664,2 1,477.05. 75.6 103.2. 0 743.6. 5.2 1,357.72. Placenta Cells Beads. route). In the lung, the intravenous route revealed the highest numbers compared to the three other routes tested, the second highest numbers were given by intraamniotic route. In the heart the values of beads and cells were significantly higher (p ! 0.001) by intravenous route than by the other routes. In the placenta, the numbers of cells were low by all the routes tested, and the number of beads was significantly elevated only in the placenta (p ! 0.001). It appears that liver and kidney presented high colonization numbers of beads and cells, no matter which route or material was injected. Intra-amniotic and intraplacen-. 220. Fetal Diagn Ther 1998;13:216–222. 0 6,240. Intra-amniotic. 0 384. Intraplacental. 560 704. 0 37.8. 0 0. 20,952 1,780.8. 248 680 0 189.2 197.6 24,190.4. tal routes led to the detection of lower colonizations of beads and cells than intravenous and intraperitoneal routes.. Discussion Our results demonstrated that among the four routes tested, the intravenous route offered the possibility to reach easily fetal organs without any cell loss and yielded a higher concentration of cells and beads in different organs than did the other routes. Through intraplacental and. Aboussaouira/Gerard/Gerard. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. Table 1. Total number of cells and beads in the different organs after fetal rat infusion by intravenous, intraperitoneal, intra-amniotic, and intraplacental routes.

(6) SIBY. intra-amniotic routes, cell and bead rates were too low in most of the studied tissues. However, these rates increased after intraperitoneal and intravenous infusions. The intravenous injection in the early rat fetus was successful after several attempts. In the early human fetus, stem cell transplantation was successfully practiced in utero at weeks 12–13 [9, 13–15]. Immediately after intravenous infusion, the distribution of labelled beads and cells depends on the blood flow [16, 17]: tissues with a low blood flow showed low labelled cell and bead concentrations in several organs as compared with those with high flows [17]. After intra-amniotic infusion, cells were noticed especially in the lung because the fetus drinks its amniotic liquid which has an important level of labelled material. The sequestration of this material takes place first in the liver and then in the lung which is an important site of blood traffic and rapid sequestration. In the human fetus, while Westgren et al. [15] showed that the intracardiac route yields a high concentration of cells in different fetal organs and that the mode of administration of cells seems to be the determinant for successful engraftment, Carr et al. [18] used the intravascular route for intrauterine therapy of homozygous ·-thalassemia and showed that intravascular intrauterine exchange transfusions maintained appropriate fetal growth. After 6 h, one would expect phagocytosis and cell migration to occur in some organs, namely in the liver where the concentration of labelled cells is 5–9% between 1 and 90 h after infusion [16, 17]. The possible reasons for this high concentration in the liver are its high vascularization and its important purification role, especially in the fetus in whom the liver is a chief organ of sequestration and epuration. Lymphocytes migrate through most body tissues [19], generally at a high rate in lymphoid tissues and at a low rate in most nonlymphoid tissues. However, in some large nonlymphoid tissues, i.e., the liver, the alimentary wall, and the lung, the distribution represents an important proportion of the whole-body cell traffic [20]. The kidney is a highly vascularized tissue, it shows particularly a high cell entry like the liver and the lung. In the kidney, sequestration of the labelled cells is complex for other reasons [16, 21]. Initially, this tissue receives a pulse of labelled cells which is related to its high blood flow that decreases, reflecting the important role of this tissue in excretion or metabolism of dead cells. The lung is an important traffic site for blood lymphocytes [22, 23], it is also the site of rapid lymphocyte sequestration. Binns and Licence [16] noted that 3 and. 24 h after intraperitoneal infusion only 10 –5 to 10 –4 of the injected cells could be found in the lung; in our experiments this organ contained up to 10 –1 of the injected cells after intravenous infusion. The lung is an important organ of cell sequestration and purification, and the intravenous route seems to activate cell colonization into this organ because the lung could be more easily reached by the intravenous than by the intraperitoneal route. The levels of labelled cells and beads in the heart are lower, and this is in agreement with the observations of other authors [16, 17]. In the placenta, only the number of beads was high because the infusion was probably done in connective tissue where the beads were trapped; the cells have nonetheless been able to migrate. A possible reason for the different distribution pattern seemed to be that each route used reached differently the fetal compartment. These findings would help to understand the kinetics of transplanted cells in the fetal compartment and to define the fetal target organs of in utero acquired graftversus-host reaction and graft-versus-host diseases following transplacental crossing of lymphocytes [7]. Stem cells that are being injected in utero for transplantation purpose might behave differently than lymphocytes used here, and this is probably based on different surface properties or cytokine production and results in different distribution patterns [14, 24]. A fine localization of beads and cells inside each organ showed that the total number of beads was significantly higher than the number of cells in all the routes tested. This is probably due to the fact that beads are free of antigens that induce colonization of lymphocytes into the organs: lymphocyte surface molecules or ‘homing receptors’ are involved in lymphocyte recognition of high endothelial venules [20, 25], and they can discriminate between endothelial cells in different organs of the body [20, 26]. A 6-hour interval was used between injection and distribution determination in this study, because at that time cell and bead distributions became stable in preliminary assays: in fetal swine [unpubl. results], determinations after injection via intravenous and intraperitoneal routes were performed after 1, 6, and 96 h; the intraperitoneal route takes more than 6 and less than 96 h to ‘stabilize’ in fetuses. Furthermore, McCullagh [6] noticed that the success of tolerance induction in fetal rats depends on the infusion route and that the intraperitoneal route of cell administration in fetal rats was found to be less efficient than the intravenous route, even 2 weeks after injection. Immunological tolerance was induced in rat fetuses before 19 days [6, 7]. Immunological maturation in the rat. Infusion Routes Comparison. Fetal Diagn Ther 1998;13:216–222. 221. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. W17:ZFETA692XA.

(7) W17:ZFETA692XA. SIBY. begins at 14–16 weeks of gestation [27] when the fetuses are developed enough to resist in utero injections. This study demonstrated that the intravenous route leads to higher concentrations of injected cells and beads in fetal organs than the other routes tested. These findings might suggest that the intravenous route is appropriate for intrauterine cell transplantation; on the other hand, they might help knowing the kinetics of transplanted cells in. fetal compartments. The fetal target organs of in utero acquired graft-versus-host disease of transplacentally crossing lymphocytes are still unknown. Acknowledgment The authors wish to express their sincere thanks to Dr. I. Houti and Dr. M. Riyad for reading the manuscript.. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. References. 222. 11 Bertholet JY: Mating method to produce accurate timed pregnancies in rats. Lab Anim Sci 1981;31:1980–1981. 12 Lee KKH: Translocation of fibronectin-coated latex beads in avian embryonic limb buds. Anat Embryol (Berl) 1991;184:583–590. 13 Westgren M, Brubbak AM, Bui TH, et al: Fetal stem-cell transplantation in fetal ·- and ß-thalassemia (abstract). Am J Obstet Gynecol 1994; 178:398–401. 14 Westgren M, Ringden O, Eik Nes S, Ek S, Anvret M, Brubakk AM, Bui TH, Giambona A, Kiserud T, Kjældgaard A, Maggio A, Markling L, Seiger A, Arlandi F: Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies. Transplantation 1996;61:1176–1179. 15 Westgren M, Ek S, Bui TH, Jansson B, Kjældgaard A, Makling L, Nennesmo I, Seiger A, Sarby B, Thornstorm S, Ringden O: Tissue distribution of transplanted fetal liver cells in the human recipient. Am J Obstet Gynecol 1997; 176:49–53. 16 Binns RM, Licence ST: Patterns of distribution of labelled blood lymphocyte subpopulations: Evidence for two types of Peyer’s patch in the young pig; in Klaus GGB (ed): Microenvironments in the Lymphoid System. New York, Plenum Press, 1985, p 661. 17 Binns RM, Licence ST: Lymphocyte migration in the pig. II. Migration of 51Cr-labelled blood lymphocytes into lymphoid and nonlymphoid tissues. Immunology 1986;53:515–520. 18 Carr S, Rubin L, Dixon D, Star J, Dailey J: Intrauterine therapy for homozygous alpha thalassemia. Obstet Gynecol 1995;85:876– 879.. Fetal Diagn Ther 1998;13:216–222. 19 Kampmeier OF: Lymphatic system of the mammals; in Springfield T (ed): Evolution and Comparative Morphology of the Lymphatic System. New York, Raven Press, 1969, p 412. 20 Butcher EC: The regulation of lymphocyte traffic. Curr Top Microbiol Immunol 1986;128: 85–110. 21 Binns RM: The behavior of pig lymphocytes in vivo; in Tumbleson M (ed): Swine Biomedical Research. New York, Plenum Press, 1987, p 612. 22 Pabst R, Binns RM, Licence ST, Peter M: Evidence of a selective major vascular marginal pool of lymphocytes in the lung. Am Rev Respir Dis 1987;136:1213–1218. 23 Pabst R, Binns RM: Lymphocytes migrate from the branchoalveolar space to regional branchial lymph nodes. Am J Respir Crit Care Med 1995;151:495–499. 24 Jones DR, Bui TH, Anderson EM, Ek S, Liu D, Ringden O, Westgren M: In utero haematopoietic stem cell transplantation: Current perspectives and future potential. Bone Marrow Transplant 1996;18:831–837. 25 Jalkanen S, Reicher RA, Gallatin WM, Bargatze RF, Weissman IL, Butcher EC: Homing receptors and the control of lymphocyte migration. Immunol Rev 1986;91:39–60. 26 Butcher EC, Scollay RG, Weissman IL: Organ specificity of lymphocyte migration: Mediation by highly selective lymphocyte interaction with organ-specific determinants on high endothelial venules. Eur J Immunol 1980;10:556–563. 27 Aboussaouira T, Idelman S: Image analysis of cell proliferation in rat thymus throughout development. Thymus 1988;12:167–186.. Aboussaouira/Gerard/Gerard. Downloaded by: Monash University 130.194.20.173 - 10/4/2016 4:09:43 AM. 1 Brent L: An immunological approach to the treatment of inherited life-threatening bone marrow defects. Fetal Ther 1988;3:1–7. 2 Billingham RE, Brent L, Medawar PB: Quantitative studies on tissue transplantation immunity III. Actively acquired tolerance. Philos Trans R Soc Lond [B] 1956;239:357–414. 3 Pearce RD, Kiem D: Induction of hemopoietic chimerism in the carpine fetus by intraperitoneal injections of fetal liver cells. Experientia 1989;45:307–308. 4 Porter KA: Graft-versus-host reaction in the rabbit. Br J Cancer 1960;14:66–70. 5 Beer AE, Billingham RE: Maternally acquired runt disease: Immune lymphocytes from the maternal blood can traverse the placenta and cause runt disease in the progeny. Science 1973;179:240–243. 6 McCullagh P: Resistance of fetal PVG rats to induction of allograft tolerance. Eur J Immunol 1989;19:787–793. 7 Zanjani ED, Macintosh FR, Harrison MR: Hematopoietic chimerism in sheep and non-human primates by in utero transplantation of fetal hematopoietic stem-cells. Blood Cells 1991;17:349–363. 8 Touraine JL, Raudrant D, Rebaud A, Barbier F, Freycon F, Vullo C: In utero stem-cell transplantation in human fetuses. Exp Hematol 1990;18:657–662. 9 Touraine JL, Raudrant D, Rebaud A, Roncarolo MG, Laplace S: In utero transplantation of stem cells in humans: Immunological aspects and clinical follow-up of patients. Bone Marrow Transplant 1992;9(suppl 1):121–126. 10 Beavis AJ, Pennline KJ: Tracking of murine spleen cells in vivo: Detection of PKH26labelled cells in the pancreas of non-obese diabetic (NOD) mice. J Immunol Methods 1994; 170:57–65..

(8)