Romanian Society of Biological Sciences ORIGINAL PAPER

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1 Romanian Biotechnological Letters Vol. 14, No. 6, 2009, pp Copyright 2009 Bucharest University Printed in Romania. All rights reserved Romanian Society of Biological Sciences ORIGINAL PAPER Flow Cytometrical Identification and Characterization of Human Cord Blood Derived Hematopoietic Stem Cells Engrafted in Sheep Bone Marrow Following In Utero Transplantation Received for publication, April 15, 2009 Accepted, November 10, 2009 Abstract IOAN GROZA*, MIHAI CENARIU*, DARIA GROZA**, EMOKE PALL*, COSMIN PEŞTEAN* *University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, 3-5 Calea Mănăştur, Cluj-Napoca, Romania, fax: , **University of Medicine and Pharmacy Cluj-Napoca, Clinic of Obstetrics and Gynecology II Dominic Stanca The human/sheep xenograft model has been imagined and used by many researchers in order to asses the hematopoietic activity of stem cells. The aim of this paper was to prove engraftment of human umbilical cord blood derived hematopoietic stem cells in sheep bone marrow following in utero transplantation. Human umbilical cord blood was collected immediately after delivery and processed in order to isolate the CD34+ cells that were transplanted by intraperitoneal injection into 60 days old fetal sheep. The pregnancies were monitored to term and bone marrow was collected from new-born lambs by iliac crest aspiration. Cells were stained with anti-human CD 45/CD 34 antibodies and chimerism was established using a BD FACS Canto II flow cytometer that showed a 3% engraftment of human hematopoietic stem cells within the sheep bone marrow. Keywords: umbilical cord blood, stem cells, in utero transplantation, fetal sheep, engraftment, flow cytometry Introduction Allogeneic and heterogeneic in utero stem cell transplantation has been studied in human fetuses as well as in various animal models and proved to lead to variable rates of engraftment in different organs [1-4]. The technique could become an important therapeutic approach in fetuses with congenital hematologic, metabolic and immunologic disorders, as it provides many advantages, as follows: stem cells may be delivered before irreversible organ damage occurs [5]; the fetal environment is highly conductive to expansion of stem cell compartments [6,7] and, most important, the naivety of early gestation fetus induces tolerance to foreign antigens during a certain window of opportunity [8,9]. Flow cytometry represents a powerful tool that allows immunophenotyping of heterogenic cell populations, according to surface markers, following staining with fluorescent monoclonal antibodies [10]. The purpose of this paper was to elaborate an accurate technique of identification and characterization of human umbilical cord blood derived hematopoietic stem cells, engrafted in sheep bone marrow following in utero transplantation and thus to prove the chimerism resulted within the hematopoietic compartment of sheep. 4798

2 Flow Cytometrical Identification and Characterization of Human Cord Blood Derived Hematopoietic Stem Cells Engrafted in Sheep Bone Marrow Following In Utero Transplantation Materials and method I. Preparation of cells for transplantation. Human umbilical cord blood was collected from women who delivered at the Dominic Stanca maternity in Cluj-Napoca, after their written consent, using the MacoPharma CellFlex MSC 1201 DU collection kit (Slovakia). Mononuclear cells were isolated with Histopaque 1077 (Sigma), resuspended in 7 ml PBS (Sigma) and counted using a haemocytometer. The cell viability was assessed with Tripan Blue (Sigma) and CD34+ cells were separated using MACS anti-human CD34 micromagnetic beads (Miltenyi Biotec). Further, the cells were prepared for transplantation by suspending 1x10 6 CD34+ cells into 1 ml IMDM medium (Sigma) without FCS, at 37 C. II. Transplantation of cells. Transylvanian Merinos type M ewes, 2 to 5 years of age, were synchronized, artificially inseminated and diagnosed pregnant after 35 days using an ultrasound scanner. Only ewes carrying a single fetus were selected and divided into three batches, as follows: Batch 1, consisting of 3 ewes in which the in utero transplantation was performed by ultrasound-guided percutaneous transabdominal puncture; Batch 2, consisting of 3 ewes in which the in utero transplantation was performed by ultrasound-guided puncture of the uterus, exposed after median laparatomy; Batch 3, consisting of 3 control ewes that were not transplanted. Both transplantation techniques were performed on day 60, within the engraftment window (between days of gestation), established previously [8]. a. The ultrasound-guided percutaneous transabdominal puncture was performed after neuroleptanalgesia and dorsal recumbency restraint of the ewes. The position of the fetus was assessed using the ultrasound scanner and the percutaneous puncture was made using a 20 G sterile needle that was guided towards the fetal peritoneal cavity where the suspension of human umbilical cord blood CD34+ cells was injected (figure 1). Figure 1. The ultrasound-guided percutaneous transabdominal puncture Rom. Biotechnol. Lett., Vol. 14, No. 6, (2009) 4799

3 IOAN GROZA, MIHAI CENARIU, DARIA GROZA, EMOKE PALL, COSMIN PEŞTEAN b. The ultrasound-guided puncture of the exposed uterus (figure 2) was also performed after neuroleptanalgesia and dorsal recumbency restraint of the ewes. The median laparatomy was classically performed and the pregnant uterus was exposed. The fetal position was assessed using the ultrasound scanner that was placed directly on the uterine horn. Puncture of the uterine wall was made using a 20 G sterile needle that was guided towards the fetal peritoneal cavity where the suspension of human umbilical cord blood CD34+ cells was injected. Suture of the abdominal wall was finally performed by layer to layer apposition. Figure 2. The ultrasound-guided puncture of the exposed uterus In both cases, the vital signs of fetuses (heartbeat) were carefully monitored by ultrasound examination after completion of the intervention and then regularly until the end of pregnancy. III. Flow cytometrical analysis of engraftment. Five days after birth, bone marrow aspirates (from the iliac crest) were collected from lambs belonging to both experimental and control batches. Red blood cells were lysed using BD FACS Lysing Solution (BD Biosciences, Belgium) and mononuclear cells were resuspended in CellWASH (BD Biosciences, Belgium). The cells were then stained with Mouse Anti-Human CD45 FITC/CD34 PE (BD Biosciences, Belgium) and analysed using a BD FACSCanto II flow cytometer. Results and discussions The in utero transplantation succeeded in all ewes, both through ultrasound-guided percutaneous transabdominal puncture and through ultrasound-guided puncture of the exposed uterus. The vital signs were normal one hour after the procedure, in both ewes and fetuses (which had a normal cardiac rhythm with no significant modifications that would signify fetal sufferance). Forty-eight hours after the intervention, one of the ewes belonging to batch II presented the clinical signs of an acute septicemia, with hyperthermia (42 C), high heart and respiratory rate as well as elevated WBC count. Death occurred within 16 hours while necropsy revealed peritonitis, necrotic metritis and placentitis, probably following contamination during puncture of the exposed uterus. The other six transplanted ewes were clinically healthy, with a positive evolution of pregnancy, comparable to the control batch. Abortion was detected seven days after transplantation in 1 ewe belonging to batch I and 1 ewe belonging to batch 2. Necropsy of the aborted fetuses did not reveal any particular lesion that could be considered the cause of the abortion Rom. Biotechnol. Lett., Vol. 14, No. 6, (2009)

4 Flow Cytometrical Identification and Characterization of Human Cord Blood Derived Hematopoietic Stem Cells Engrafted in Sheep Bone Marrow Following In Utero Transplantation The remaining pregnancies were monitored clinically and by ultrasound examination every 10 days until full-term. The post-transplantation evolution of pregnancies can be summarized as follows (table 1): Batch no. Table 1. Post-transplantation evolution of pregnancies No. of ewes that died No. of transplanted ewes No. of ewes that aborted No. of ewes that reached fullterm pregnancy Batch I Batch II Batch III (control) Flow cytometrical analysis of the bone marrow aspirates obtained from the three lambs belonging to experimental batch I and II proved engraftment of human CD34+ cells within their hematopoietic compartment, with a percentage of chimerism that varied between 2.9% and 3.2%. The dot plots obtained for one of the lambs are presented in figure 3. The same analysis performed for the control batch showed no human CD34+ cell (figure 4), which proves the accuracy of the method. Figure 3. Dot plot showing a 3% engraftment of human CD34+ cells (Q4-3) within the bone marrow of lambs from experimental batch I Figure 4. Dot plot showing no human CD34+ cells (Q4-3) within the bone marrow of control lambs Rom. Biotechnol. Lett., Vol. 14, No. 6, (2009) 4801

5 IOAN GROZA, MIHAI CENARIU, DARIA GROZA, EMOKE PALL, COSMIN PEŞTEAN These results indicate that the preimmune sheep fetus readily allows the engraftment of human hematopoietic stem cells. The long term persistence and multilineage human cell activity in these animals has been proven by secondary transplant studies, in which human CD45+ cells isolated from bone marrow of chimeric sheep were transplanted into secondary pre-immune fetal sheep [11,12]. The demonstration of human cell activity in the secondary sheep suggests that the original graft contained human hematopoietic stem cells with long term repopulating ability. Thus, the human-sheep xenograft model provides a biologically relevant assay to determine the in vivo potential of stem cells and may also serve as a pre-clinical model for in utero gene therapy, as indicated by various studies [13, 14]. The flow cytometrical method used for identification and characterization of engrafted human hematopoietic stem cells proved to be very accurate and easy to perform, although not very cheap. Taking into consideration the benefits of studying the in utero stem cell transplantation on animal models we believe that flow cytometry is the most powerful and adequate tool that allows the identification and characterization of heterogenic cell populations within a certain environment. Conclusions The current study demonstrated that human umbilical cord blood derived hematopoietic stem cells engraft within the bone marrow compartment of fetal sheep, following in utero transplantation (chimerism of 2.9% - 3.2%). The ultrasound-guided percutaneous transabdominal puncture proved to be a better transplantation method than the ultrasound-guided puncture of the exposed uterus, providing a higher rate of survival (66.6% compared to 33.3%). The flow cytometrical method of identification and characterization of engrafted CD34+ cells proved to be very accurate and facile, which makes it recommendable to be used for the assessment of chimerism following in utero transplantation of stem cells. References 1. GRACA ALMEIDA-PORADA, E. D. ZANJANI, A large animal noninjury model for study of human stem cell plasticity, Blood Cells, Molecules, and Diseases 32, (2004). 2. E. D. ZANJANI, GRACA ALMEIDA-PORADA, A. W. FLAKE, The human/sheep xenograft model: a large animal model of human hematopoiesis, International Journal of Hematology 63, (1996). 3. M. WESTGREN, In utero stem cell transplantation. Semin.Reprod. Med 24, (2006). 4. E. D. ZANJANI, GRACA ALMEIDA-PORADA, ANNE G. LIVINGSTON, H.Q. ZENG, M. OGAWA, Reversible expression of CD34 by adult human bone marrow long-term engrafting hematopoietic stem cells, Experimental Hematology 31, (2003). 5. A. J. YOUNG, W. HOLZGREVE, LISBETH DUDLER, ANDREINA SCHOEBERLEIN, D. V. SURBEK, Engraftment of human cord blood-derived stem cells in preimmune ovine fetuses after ultrasound-guided in utero transplantation, Am J Obstet Gynecol 189, (2003). 6. CAROLYN TROEGER, D. SURBEK, ANDREINA SCHÖBERLEIN, S. SCHATT, LISBETH DUDLER, SINUHE HAHN, W. HOLZGREVE, In utero haematopoietic stem cell Transplantation, Swiss Med Wkly 136, (2006). 7. LU-LU LU, YONG-JUN LIU, SHAO-GUANG YANG, QIN-JUN ZHAO, XIN WANG, WEI GONG, ZHI- BO HAN, ZHEN-SHU XU, YONG-XIN LU, DELONG LIU, ZHI-ZHE CHEN, ZHONG-CHAO HAN, Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesissupportive function and other potentials, Haematologica 91, (2006). 8. JESSICA L. SKOPAL-CHASE, PIXLEY J.S., TORABI A., CENARIU M.C., BHAT A., THAIN D.S., FREDERICK N.M., DARIA M. GROZA, ZANJANI E.D., Immune ontogeny and engraftment receptivity in the sheep fetus, Fetal Diagnosis and Therapy 25, (2009) Rom. Biotechnol. Lett., Vol. 14, No. 6, (2009)

6 Flow Cytometrical Identification and Characterization of Human Cord Blood Derived Hematopoietic Stem Cells Engrafted in Sheep Bone Marrow Following In Utero Transplantation 9. M. O. MUENCH, ALICIA BÁRCENA, Stem Cell Transplantation in the Fetus, Cancer Control 11, (2), (2004). 10. M.O. MUENCH, In utero transplantation: baby steps towards an effective therapy, Bone Marrow Transplantation 35, (2005). 11. J.L.TOURAINE, In utero transplantation of fetal liver stem cells into human fetuses, J Hematother 5, (1996). 12. SCHOEBERLEIN ANDREINA, HOLZGREVE W, DUDLER LISBETH, HAHN S., SURBECK D. V., In utero transplantation of autologous and allogenic fetal liver stem cells in ovine fetuses. American Journal of Obstetrics and Gynecology 191, (2004). 13. E. D. ZANJANI, The Human Sheep Xenograft Model for the Study of the In Vivo Potential of Human HSC and In Utero Gene Transfer, Stem Cells 18, 151 DOI: /stemcells (2000). 14. M. VANDERSON ROCHA, ELIANE GLUCKMAN, EUROCORD AND EUROPEAN BLOOD AND MARROW TRANSPLANT GROUP, Clinical Use of Umbilical Cord Blood Hematopoietic Stem Cells. Biology of Blood and Marrow Transplantation 12, (2006). Rom. Biotechnol. Lett., Vol. 14, No. 6, (2009) 4803