In Utero Transplantation of Human Cord Blood Cells into Rabbits

Abstract

We have previously shown that in utero transplantation (IUT) of haploidentical, bone marrow-derived CD34+ cells can be used as prenatal therapeutic approach for X-linked severe combined immunodeficiency (X-SCID) (1). To extend the possible applications of IUT, animal models are needed that can be used to address technical and biological questions, including optimal source of hematopoietic stem cells, timing, site and method of injection (e.g., open surgery, fetoscopy, transcutaneous ultrasound-guided injection). Different animal models were described for either surgical or transcutaneous transplantation of hematopoietic progenitor cells in utero (2). As an alternative to human bone marrow, cord blood progenitor cells were transplanted in utero in a sheep animal model applying a surgical procedure and injection under ultrasound guidance (3, 4). Ultrasound-guided IUT of human cord blood cells was also recently described in a swine model (5). We have performed IUT in four rabbit fetuses at 26 days of gestation using 80 × 106 human male cord blood mononuclear cells separated on Percoll gradient (47.5% fraction) and resuspended in PBS before transplantation. The intraperitoneal (i.p.) injection was performed under ultrasound guidance using a linear probe (10 Mhz) in a pregnant female rabbit under general anesthesia. Seven rabbit kits were born at term, two of which died shortly after birth. The surviving animals showed normal growth rate and no clinical symptoms of graft-versus-host disease (GvHD) during an observation period of three months. DNA was isolated from peripheral blood of the animals, 27 days after IUT (23 days after birth) and engraftment was determined by polymerase chain reaction amplification of the human amelogenin gene (AMGY) that is located on the Y chromosome. Human chimerism was confirmed by Southern blot analysis of the amplified sequences in four out of five available animals (Fig. 1A, lanes 4–8). Rabbit DNA quality was assessed by amplification of a rabbit specific sequence at the SAT 12 locus (Fig. 1B, lane 2–8).FIGURE 1.: (A) PCR analysis on DNA isolated from peripheral blood of rabbit kits 27 days after in utero transplantation of human male mononuclear cells isolated from cord blood. Chimerism was detected using a probe specific for the amelogenin gene (AMGY) on the Y chromosome by Southern blot analysis. The specificity of human Y chromosome signal is shown in human male DNA (lane 1) vs. male and female rabbit DNA (lane 2 and 3). Positive signals for human DNA were demonstrated in four out of five animals (lanes 4–8). H20 served as PCR negative control (lane 9). The primers used for the AMGY gene (Genbank accession number: M55419) specific amplification were: sense primer 5′-CTGATGGTTGGCCTCAAGCCTGTG -3′; antisense primer 5′- GCCCAAAGTTAGTAATTTTAC-3′. PCR amplification was performed as follows: DNA was denatured for 4 min at 94°C and amplified for 30 cycles (94°C, 20 sec; 58°C, 30 sec; 72°C, 45 sec) with a final elongation step of 72°C for 7 min, and then stored at 4°C. (B) Rabbit specific sequence amplification was performed at the Sat 12 locus (GenBank accession number X99889) to demonstrate integrity of rabbit DNA. The primers used to amplify specific rabbit sequence were: sense primer 5′-CAGACCCGGCAGTTGCAGAG-3′, antisense primer 5′-GGGAGAGAGGGATGGAGGTATG-3′. PCR conditions were as described for the AMGY gene.In summary, ultrasound-guided IUT of human hematopoietic progenitors can result in human chimerism in rabbits. In this small-size animal model, all injected animals were clearly visible on ultrasound and survived the procedure without clinical complications. Compared to the sheep (145 days of gestation), swine (114 days), and Rhesus monkey (165 days), the rabbit has the advantage of a shorter pregnancy (31 days). In addition, in contrast to mice, the size of rabbit fetuses makes transcutaneous ultrasound-guided in utero transplantation feasible. Further experiments aimed at quantifying levels of donor chimerism (i.e., percent of human CD45 cells and specific leukocyte subpopulations) and duration of engraftment will provide more detailed information on the human hematopoietic cell engraftment potential in the fetal rabbit and will determine if the rabbit IUT model can be helpful to test transplantation protocols aimed to improving engraftment, study possible applications of different sources of progenitor cells, as well as pharmacological protocols to augment donor cell engraftment (6). ACKNOWLEDGMENTS We thank Dr. Candotti for critical reviewing of the manuscript and helpful discussions. Georg S. Wengler Centro Ricerche “E. Menni” Ospedale Poliambulanza Brescia, Italy Guerino Lombardi Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna Brescia, Italy Tiziana Frusca Department of Obstetrics and Gynecology University of Brescia Brescia, Italy Daniele Alberti Department of Pediatric Surgery Ospedali Riuniti di Bergamo Bergamo, Italy Alberto Albertini Istituto di Tecnologie Biomediche CNR Milan, Italy Ornella Parolini Centro Ricerche “E. Menni” Ospedale Poliambulanza Brescia, Italy