Stem Cells 2003;21:638-646
www.StemCells.com
© 2003 AlphaMed Press
A Novel Route of Transplantation of Human Cord Blood Stem Cells in Preimmune Fetal Sheep: The Intracelomic Cavity
Giuseppe Noiaa,*,
Luca Pierellib,*,
Giuseppina Bonannoa,
Giovanni Monegoc,
Alessandro Perilloa,
Sergio Rutellab,
Anna Franca Cavalierea,
Marco De Santisa,
Maria Serena Ligatoa,
Giuseppe Fotunatoa,
Giovanni Scambiaa,
Marinella Terzanod,
Enrico Iannacee,
Giovanni Zelanoc,
Fabrizio Michettic,
Giuseppe Leoneb,
Salvatore Mancusoa
a Department of Obstetrics and Gynecology,
b Department of Hematology,
c Department of Anatomy, Catholic University of the Sacred Heart, Rome, Italy;
d Zootecnic Experimental Institute, Monterotondo, Italy;
e Department of Pediatric Cardiology and Cardiac Surgery, Bambino Gesù Hospital, Rome, Italy
Key Words. In utero transplantation • Intracelomic route
Giuseppe Noia, M.D., Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo Agostino Gemelli 8, 00168 Rome, Italy. Telephone: 39-06-30154979; Fax: 39-06-3051160; e-mail: pi.noia{at}tin.it
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ABSTRACT
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The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined. Twelve twin ovine fetuses (gestational age: 40–45 days) received intracelomic transplants of human CD3-depleted (50 x 106 per lamb) or CD34-selected (1–2 x 105 per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human ß2-microglobulin. Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (no. 435184) and 105 (no. 915293, no. 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45+ cells in bone marrow, respectively. In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human ß2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle. This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.
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INTRODUCTION
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Human stem cell transplantation in immunocompromised, preimmune, or adult normal animals represents a reliable in vivo model to assess stem cell activity and/or tissue replacement capacity after experimental organ injury [1–3]. In adult animals, experimentally provoked organ damage can be partially repaired by syngeneic or xenogenic stem cell transplantation [3, 4]. Preimmune sheep fetuses receiving human stem cells showed human chimerism in bone marrow at birth as well as the presence of human nonhematopoietic terminally differentiated cells in organs of endodermic origin [5]. In the particular setting of prenatal transplantation, the gestational age at transplant seems to be a critical factor to permit the establishment of immune tolerance to the graft and to take advantage of the high differentiation potential of the embryo. In reference to this, a previously reported study described intraperitoneal cell transplants in prenatal sheep via the amniotic bubble injected from 55 days of gestational age onward [6]. The importance of gestational age is related to various factors, including the progressive maturation of the fetal immune system, which becomes increasingly competent and less tolerant of foreign antigens [7], and the increase in the number of hematopoietic cells produced by the fetus itself, which occupy the natural sites of hematopoiesis and compete excessively with the transplanted donor cells [8].
In humans, the extraembryonic celom, with the extracelomic cavity, develops during the fourth week after the last menstrual period [9]. At the end of the fourth week of gestation, the developing extracelomic cavity splits both the extraembryonic mesoderm, lining the trophoblast, and the splanchnic mesoderm, covering the secondary yolk sac and the embryo. There are no anatomical barriers between the mesenchyme of the placental fetal plate and the extracelomic or chorionic cavity [10]. During the following weeks, the secondary yolk sac appears as a spherical and cystic structure covered by numerous superficial small vessels emerging at the base of the vitelline duct. The extraembryonic human circulation is first established within the vitelline duct artery via the dorsal aorta. At the tenth week of gestation, the yolk sac begins to degenerate and rapidly ceases to function [10].
In the sheep model, we can distinguish the celomic cavity from the amniotic cavity by transvaginal ultrasound from 33 to 46–47 days of gestational age, and more precisely at 40 days of gestational age. Compared with human ontogenesis, this time corresponds with the end of the first trimester (12–13 weeks gestational age).
The intracelomic route of injection represents an attractive option for stem cell delivery, since, from a theoretical point of view, it allows earlier in utero stem cell injection. The present report describes the results obtained after transplantation of human cord blood CD3-depleted mononuclear cells (MNCs) or CD34+-purified stem cells in prenatal sheep via the intracelomic route.
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MATERIALS AND METHODS
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Human Cord Blood Cell Collection and Isolation
Human cord blood cells were obtained from informed mothers undergoing cesarean delivery of full-term infants in our hospital. Cord blood collection and the entire study were approved by the Hospital Investigational Review Board. Only cord blood collections with a final volume higher than 70 ml and showing a CD34+ cell frequency higher than 0.2% were processed for cell isolation. Red cells were removed from cord blood by a 90-minute sedimentation using hydroxyethyl starch at a ratio of 1:8 to cord blood. After washings, MNCs were isolated from red-cell-depleted nucleated cells by centrifugation on a Ficoll-Paque density gradient (1.077 g/ml; Pharmacia LKB; Uppsala, Sweden). Isolated MNCs were then mixed (at 108 cells/ml) with 0.5 µg/ml fluorescein isothiocyanate (FITC)-conjugated anti-human CD3 monoclonal antibody ([mAb]; Caltag Laboratories; Burlingame, CA; http://www.caltag.com) and incubated at room temperature for 30 minutes in the dark. After extensive washings, cells stained by CD3 FITC mAb were mixed with 20 µl/107 cells MultiSort anti-FITC MicroBeads (Miltenyi Biotec; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com), incubated for 15 minutes at 6–12°C and then, after washings, loaded onto a VS+ selection column in a VarioMACS magnetic instrument (Miltenyi Biotec). CD3-depleted cord blood cells were collected prior to elution of the CD3+ fraction, following the manufacturer’s guidelines. CD3 depletion produced, on average, a 90% (range, 70%-98%) removal of CD3 cells, and postimmunoselection contaminating CD3 cells contained in CD3-depleted grafts averaged 3.5 x 106 (range, 0.70–11 x 106). Particularly, CD3-depleted cord blood grafts contained, on average, 10% CD3+ cells, 55% monocytes, 15% CD19 cells, 5% CD3- lymphoid cells, 0.9% CD34+ cells, and 13% contaminating band cells and granulocytes. Some samples of CD3-depleted cord blood MNCs were subsequently incubated with Miltenyi MultiSort anti-CD34 MicroBeads (Miltenyi Biotec; 100 µl/108 cells) and, after extensive washings, processed by a VarioMACS magnetic device to remove non-CD34+ cells, as described by the manufacturer. CD34+ cord blood cells were eluted after removal of unwanted, unbound cells by discontinuation of the magnetic fields in the VarioMACS device. All CD34+ immunoselected samples had a CD34 cell purity higher than 95%, and CD3-contaminating cells were consistently less than 0.1%. Six sheep fetuses were transplanted with CD3-depleted cord blood grafts while six additional fetuses received CD34+-selected transplants.
Fetal Sheep Recipients
Comiso sheep (Ovis aries Comisana) were purchased and transferred to the Center for Animal Breeding and Use of Catholic University of Rome. The animals were grazed and fed with legume hay together with a vitamin/mineral supplement. Estrus was induced with polyurethane vaginal suppositories impregnated with fluorogestone acetate (40 mg), which were left in place for 14 days. Pregnancy was diagnosed 25–28 days after mating by means of a transvaginal ultrasonographic scan with a 6.5-MHz transducer. Twelve twin pregnancies were selected for study between day 40 and day 45 of gestation, which was determined by fetal growth curves previously determined in our institution (Fig. 1
). For each transplantation, the pregnant ewe was anesthetized with 5 ml of ketamine. Human cord blood cells were then transferred to one of the twin fetuses via its own intracelomic route between the 40th and 45th day of gestation; the other fetus was considered as control. The details of the 12 hematopoietic stem cell (HSC) transfers are shown in Table 1. The single celomic cavity of each twin fetus was visualized by ultrasound (Fig. 2), and the HSCs were injected under aseptic conditions via the transabdominal route. A 20-gauge needle was used with the free-hand double-operator technique for injection of cord blood transplants in a fixed volume of 1.5 ml saline solution. In some instances 24- or 25-gauge needles were used, but in those cases, we experienced several difficulties with our injection technique due to the flexing of the needle. In all cases the fetal heartbeat was checked immediately after transplant and at weekly intervals thereafter until birth or intrauterine death occurred.
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Table 1. Cord blood stem cell transplantation via the intracelomic route and hematopoietic engraftment in 12 sheep fetuses
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Figure 2. A) Ultrasound picture of a 37-day-old age ovine embryo with evidence of celomic cavity (arrow). B) Celomic (*) and amniotic (°) cavities in an ovine embryo under 50 days of gestational age.
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Analysis of Human Cell Engraftment
Seven sets of fetuses were sacrificed after cesarean sections were performed at various times after cord blood cell transplant (Table 1
). In four cases, abortion occurred 2 days after transplantation, and fetal material for evaluation of engraftment could not be collected. One sheep fetus was obtained at term by spontaneous delivery, analyzed after birth for human engraftment, and maintained alive for further follow-up. Livers, spleens, bone marrow, and thymuses of fetuses obtained after cesarean or standard delivery were collected, and suspensions from these organs were subjected to flow cytometry and polymerase chain reaction (PCR) analysis to evaluate engraftment of transplanted HSCs.
Flow Cytometric Analysis
Tissue samples were stored in saline immediately after collection and were processed according to standard protocols. Single-cell suspensions were prepared by mincing the sample with scissors and a scalpel in a Petri dish. Cells were centrifuged at 200 g for 10 minutes and resuspended in an isotonic buffer supplemented with 5% fetal calf serum. The entire procedure was carried out at 4°C. The cell suspension was stained with saturating amounts of FITC-anti-human CD45, phycoerythrin (PE)-conjugated-anti-human CD19, FITC-anti-human CD3 (all from Caltag Laboratories), peridinin-chlorophyll protein (PerCP)-conjugated anti-human CD34, and PerCP-anti-human CD45 (both from Becton Dickinson; San Jose, CA; http://www.bd.com) to evaluate human chimerism. After a 30-minute incubation on ice, the cell suspensions were thoroughly washed in cold Hank’s balanced salt solution, resuspended in the same medium, and subjected to flow cytometry analysis. Background fluorescence was established using isotype-matched PE-, PerCP-, or FITC-irrelevant mAbs. Controls included tissue samples from nontransplanted twin fetuses. All cytofluorimetric analyses were carried out with a FACScan (Becton Dickinson) flow cytometer. Dead cells were excluded based on light scatter characteristics; in samples not containing PerCP-conjugated mAb, 7-aminoactinomycin D (7-AAD; Molecular Probes; Eugene, OR; http://www.probes.com) was used in order to discriminate between viable cells (7-AAD negative) and dead cells (7-AAD positive).
Cloning Assay
In no. 75145, 435184, 915293, and 037568 sheep fetuses, 19-day cloning assays were performed from 105 bone marrow cells in the presence of human recombinant erythropoietin (2 IU/ml), interleukin-3 (IL-3, 5 ng/ml), and GM-CSF (5 ng/ml; all from R&D Systems; Oxon, UK; http://www.rndsystems.com) to functionally identify human progenitors. The presence of human IL-3 and GM-CSF, rather than sheep phytohemagglutinin-stimulated leukocyte-conditioned medium, maximizes the growth of human colonies at the expense of sheep colonies that have maximal growth on day 9 but are no longer present after 15 days of culture, when karyotypic analysis (carried out in the original study of Zanjani et al. [2]), consistently reveals human colonies only.
PCR Analysis
Samples for PCR were also harvested from sheep peripheral blood, heart, skeletal muscle, lung, brain, and spinal cord. High-molecular-weight genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen; West Sussex, UK; http://www.qiagen.com) according to the manufacturer’s protocol. The primers used were specific for human ß2-microglobulin (ß2M) as reported by Liechty et al. [11]. Amplification conditions were 95°C for 9 minutes followed by 43 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and a final extension at 60°C for 5 minutes. The amplification products were examined by electrophoresis on 1.8% agarose gel stained with ethidium bromide. All samples were analyzed in triplicate, and each assay included a negative control (DNA replaced with H2O), a positive control (human DNA), and samples from the nontransplanted ovine. To confirm the specific amplification of human ß2M, DNA sequencing was performed on one PCR product from a transplanted fetal liver. In order to confirm the specificity of this PCR strategy, we also tested DNA samples from transplanted fetal liver, brain, spinal cord, and skeletal muscle using a PCR for human KCNQ3 gene. The amplicons, which were digested with HaeIII enzyme, have a unique restriction site within the KCNQ3 DNA fragment (data not shown).
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RESULTS
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Transplantation Procedures via the Intracelomic Route
Figure 2 shows an ultrasound image of an ovine embryo, demonstrating its anatomical proximity to its own celomic cavity. Transplantation was performed under ultrasonic guidance, reaching the single celomic cavity in a few seconds on the first attempt, with visualization of the end of the injection needle and the bubble resulting after HSC injection. HSCs were injected via the intracelomic route in each animal after resuspension in a volume of 1.5 ml saline. In a previous set of experiments, the use of higher volumes of injectate was associated with a higher abortion rate, probably due to increased pressure generated by the injection inside the celomic cavity (data not shown). There were no significant alterations in fetal heart rate or maternal complications immediately after cord blood cell transplantation via the intracelomic route. However, 4 of 12 transplanted fetuses were aborted a few days after the procedure. Table 1 shows details of the cord blood cell transplant procedures.
Human Engraftment by Flow Cytometry
Eight of 12 animals that were transplanted via the intracelomic route were evaluable for human chimerism, and cell suspensions obtained from bone marrow, liver, spleen, and thymus were evaluated by flow cytometry for detection of cells expressing human hematopoietic surface antigens. Animals 443910, 751457, 631, and 569 were obtained by cesarean section on day 70 after transplant. These animals were transplanted with a dose of 50 x 106 T-cell-depleted cord blood MNCs (containing a dose of CD34+ cells ranging from 3–5 x 105), and their organs were evaluated by single-color human CD45 analysis. Animals 443910 and 751457 had CD45+ bone marrow in 16% and 20% of cells, while spleen, liver, and thymus were CD45+ in 6% and 7%, 14% and 13%, and 10% and 12%, respectively (Table 1 and Fig. 3). Similarly, in animals 631 and 569, human CD45+ cells were 22% and 18% in the bone marrow, 16% and 10% in the spleen, and 8.5% and 10% in the liver; thymus cell suspension was not analyzed in these two animals.
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Figure 3. A representative flow cytometric detection of human CD45 cells in the spleen, liver, thymus, and bone marrow of the 443910 sheep fetus transplanted with 50 x 106 CD3-depleted cord blood cells. Forward scatter (FSC) and side scatter (SSC) characteristics of analyzed cells are also shown. The bone marrow of sheep fetuses that were not injected with human cells was consistently negative for human CD45 cell analysis (data not shown). Background fluorescence was evaluated by isotype-matched fluorochrome-conjugated irrelevant mouse immunoglobulins. The numbers in each box indicate the percentage of positive cells.
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Animal 435184 was obtained by cesarean section on day 102 after transplantation of 1 x 105 purified cord blood CD34+ cells and was analyzed by flow cytometry and dual-color analysis for detection of a distinct subset of human HSCs, which documented human chimerism in all evaluated tissues (Table 1). An additional sheep (435240) transplanted with 1 x 105 purified cord blood CD34+ cells was born at term after spontaneous delivery and is still alive for further posttransplant follow-up. At birth and at 60 days after birth, that sheep had a detectable percentage of human CD45+ cells (10%) in bone marrow cells obtained from aspirates (Table 1). Two additional sheep fetuses (915293 and 037568) were obtained after cesarean section at 105 days gestation, and bone marrow analysis by extensive flow cytometric analysis was done (Table 1 and Fig. 4). Those animals were transplanted with higher doses of 2 x 105 CD34+ cord blood-purified cells and showed human chimerism, as revealed by the presence of 32% and 20% human CD45 cells, respectively. A subset analysis of bone marrow cells (Fig. 4) revealed the presence of relevant amounts of all human hematopoietic progeny as well as discrete subsets of human CD34+/CD45+ progenitors.
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Figure 4. Representative HSC subset analysis of bone marrow from no. 915293 and 037568 sheep fetuses transplanted with 2 x 105 purified CD34+ cord blood cells (CD34+/CD45+, progenitors; CD3+/CD45+ T lymphocytes; CD19+/CD45+, B lymphocytes). The percentages of CD3+/CD45- as well as CD19+/CD45- events could correspond to nonspecifically stained cells. The bone marrow of sheep fetuses that was not injected with human cells was consistently negative for detection of human hematopoietic markers (data not shown). In each sample, background fluorescence was evaluated by isotype-matched fluorochrome-conjugated irrelevant mouse immunoglobulins. The numbers in each box indicate the percentage of double-positive cells.
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Human Engraftment by Cloning Assay
As shown in Table 1, animal 751457 had a total of 44 day-19 hemoglobinized and nonhemoglobinized colonies per 105 seeded bone marrow cells. Sheep 435184 had 29 day-19 colonies from 105 human cytokine-exposed bone marrow cells. Cloning assay for day-19 human colonies showed the presence of 94 and 70 total colonies per 1 x 105 seeded marrow cells in sheep 915293 and 037568, respectively. From these data (sheep 915293 and sheep 037568 had 3% and 2% human CD34+/CD45+ progenitors in bone marrow, respectively), we can estimate cloning efficiencies of human progenitors of 3.1% and 3.5%, respectively, which are not far from results usually obtained from unfractionated human marrow cells. Nontreated control animals were consistently negative for any analyzed human markers (data not shown).
Human Engraftment by Molecular Analysis
Animals 443910, 751457, 631, and 569 showed positive results in DNA samples from bone marrow, liver, spleen, thymus, lung, skeletal muscle, heart, spinal cord, and brain (Fig. 5 and Fig. 6). Peripheral blood was PCR positive in 50% of cases. Human ß2M sequences were detectable in bone marrow, liver, spleen, thymus, skeletal muscle, heart, and brain but not in the peripheral blood of the ovine fetus transplanted with purified CD34+ cells (435184). ß2M PCR results were confirmed by random automated DNA sequencing, PCR, and restriction analysis for KCNQ3 gene. Samples analyzed from the lamb born at term (435240) were the following: bone marrow aspirate, liver biopsy, muscle biopsy, and peripheral blood. We identified human ß2M amplicons in DNA extracted from all these tissues except peripheral blood.
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Figure 5. A representative PCR analysis of human ß2M on DNA obtained from peripheral blood and hematopoietic organs of the 751457 sheep fetus transplanted with 50 x 106 CD3-depleted cord blood cells. A control liver of a sheep fetus that was not injected with human cells was analyzed with the same PCR conditions to exclude false-positive results. As a positive control, human DNA was also analyzed. The expected band of human ß2M was 160 bp long.
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Figure 6. A representative PCR analysis of human ß2M on DNA obtained from nonhematopoietic organs of the 751457 sheep fetus transplanted with 50 x 106 CD3-depleted cord blood cells. Negative control was a no sample control. As a positive control, human DNA was also analyzed. The expected band of human ß2M was 160 bp long.
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DISCUSSION
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In utero stem cell transplantation in preimmune sheep or stem cell injection in preirradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice are, at present, the available models for in vivo stem cell assay. In adult NOD/SCID mice or in preimmune sheep, human HSC transplantation produces hematopoietic chimeras with a degree of human cell engraftment ranging from 0.1%-35%, depending on the subset, number, and source of the stem cell injected [1, 2, 12]. From a theoretical point of view, the activities of embryogenetic factors supporting stem cell totipotency could be greater at a very early gestational age, when immune tolerance to allografts is similarly greater. The previously described intraperitoneal approach for in utero transplantation in the sheep model allows stem cell injection only beyond 55 days of gestational age [13–18], while the intracelomic route can be used from 40 days onward. On the other hand, at the present time, no data are available on the celomic route for in utero stem cell transplant. In the present study, human cord blood cell transplantation performed within 45 days of gestation via the intracelomic route produced engraftment in all evaluable fetuses with a very high degree of human chimerism in hematopoietic tissues (6%-32%), which is no lower than that described for the intraperitoneal route, which produces 0.1%-10% human chimerism [19]. Interestingly, purification of cord blood cells by selection on the basis of CD34 expression revealed that injection of CD34+-purified human stem cells at a low dose (105 per lamb) persistently produced a high degree of human chimerism beyond 100 days of gestational age and at birth. Molecular studies by PCR for detection of ß2M confirmed the presence of human cells in hematopoietic tissues as well as in lung, skeletal muscle, heart, spinal cord, and brain. The reason for absence of human cells in the peripheral blood of 50% of the fetuses transplanted with CD3-depleted cord blood cells and in two fetuses that received lower doses of 1 x 105 CD34+-purified cord blood cells awaits further clarification. The absence of cells of human origin in the peripheral blood of the two sheep fetuses (435184 and 435240) transplanted with 1 x 105 purified CD34+ cells could reflect either our inability to detect peripheral chimerism in such a small sample or a lower degree of human engraftment in these CD34+-transplanted sheep. The CD34+-transplanted fetuses also received fewer CD34+ cells than sheep injected with CD3-depleted grafts (containing about 3–5 x 105 CD34+ cells) and probably had a lower degree of human engraftment. This was revealed by a percentage of chimerism that did not exceed 10% in either animal and by a lower number of day-19 colonies responsive to human cytokines (29 colonies per 1 x 105 seeded bone marrow cells in sheep 435184). In line with this hypothesis, the two sheep fetuses that received higher doses of purified CD34+ cord blood cells (2 x 105 CD34+ cells in sheep 915293 and 037568) showed greater levels of human chimerism in bone marrow (32% and 20% CD45 human cell frequency, respectively) and a greater number of day-19 human clonogenic precursors (94 and 70 per 1 x 105 seeded bone marrow cells, respectively). It will be of interest to define, in larger studies, the optimal dose of human CD34+ cells to be transplanted at such an early gestational age through the intracelomic route. Finally, a high rate of abortion (33%) was observed in our series, while none of the delivered fetuses showed developmental abnormalities. Larger studies and a longer follow-up are required to define birth rate and to exclude the occurrence of animal defects and/or late loss of human chimerism after birth in our animal transplant model. Particularly, secondary transplants could establish the persistence of human-derived repopulating stem cells in our transplanted animals, providing the definitive validation of intracelomic stem cell transplants. In reference to the observed miscarriages, two alternative or concomitant explanations could be proposed: A) the naturally high rate of abortion in the Comiso sheep in which twinnings are prevalent, in contrast to the values of 10%, 15%, and even 20% that are recorded in the literature [20, 21]; B) the invasiveness of the intracelomic injection procedure, which could lead to the contamination of the amniotic fluid from the celomic cavity, and consequently, to the direct exposure of the fetus to higher than usual metal concentration [22].
In conclusion, we have demonstrated for the first time that intracelomic injection of human stem cells into sheep fetuses at an early gestational age is feasible and associated with a high degree of engraftment in hematopoietic tissues. The relevance of the intracelomic approach, as an in vivo assay, to evaluate human stem cell activity must be carefully addressed in further studies with longer animal follow-up and secondary stem cell transplants.
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ACKNOWLEDGMENT
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This study was supported in part by the Cord Blood Stem Cell Project "Fondazione Cassa di Risparmio," Rome, Italy.
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FOOTNOTES
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* These authors equally contributed to this work. 
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REFERENCES
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- Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci USA 1997;94:5320–5325.[Abstract/Free Full Text]
- Zanjani ED, Pallavicini MG, Ascensao JL et al. Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest 1992;89:1178–1188.
- Chen J, Sanberg PR, Li Y et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 2001;32:2682–2688.[Abstract/Free Full Text]
- Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.[CrossRef][Medline]
- Almeida-Porada G, Porada C, Zanjani ED. Adult stem cell plasticity and methods of detection. Rev Clin Exp Hematol 2001;5:26–41.[CrossRef][Medline]
- Zanjani ED, Almeida-Porada G, Flake AW. The human/sheep xenograft model: a large animal model of human hematopoiesis. Int J Hematol 1996;63:179–192.[CrossRef][Medline]
- Cowan MJ, Chou SH, Tarantal AF. Tolerance induction post in utero stem cell transplantation. Ernst Schering Res Found Workshop 2001;33:145–171.
- Shaaban AF, Flake A. Fetal hematopoietic stem cell transplantation. Semin Perinatol 1999;23:515–523.[CrossRef][Medline]
- Boyd JD, Hamilton WJ. The implantation of the blastocyst and the development of the trophoblast up to the somite stage. In: Boyd JD, Hamilton WJ, eds. The Human Placenta. Cambridge: Heffer W & Sons Ltd, 1970:36–60.
- Jones CPJ, Jauniaux E. Ultrastructure of the materno-embryonic interface in the first trimester of pregnancy. Micron 1995;26:145–173.
- Liechty KW, MacKenzie TC, Shaaban AF et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282–1286.[CrossRef][Medline]
- Wang JCY, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997;89:3919–3924.[Abstract/Free Full Text]
- McNiece IK, Almeida-Porada G, Shpall EJ et al. Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential. Exp Hematol 2002;30:612–616.[CrossRef][Medline]
- Porada CD, Tran ND, Almeida-Porada G et al. Transduction of long-term-engrafting human hematopoietic stem cells by retroviral vectors. Hum Gene Ther 2002;13:867–879.[CrossRef][Medline]
- Tran ND, Porada CD, Almeida-Porada G et al. Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses. Blood 2001;97:3417–3423.[Abstract/Free Full Text]
- Zanjani ED. The human sheep xenograft model for the study of the in vivo potential of human HSC and in utero gene transfer. STEM CELLS 2000;18:151.[Free Full Text]
- Tran ND, Porada CD, Zhao Y et al. In utero transfer and expression of exogenus genes in sheep. Exp Hematol 2000;28:17–30.[CrossRef][Medline]
- Yang EY, Cass DL, Sylvester KG et al. Fetal gene therapy: efficacy, toxicity, and immunologic effect of early gestation recombinant adenovirus. J Pediatr Surg 1999;34:235–241.[CrossRef][Medline]
- Shimizu Y, Ogawa M, Kobayashi M et al. Engraftment of cultured human hematopoietic cells in sheep. Blood 1998;91:3688–3692.[Abstract/Free Full Text]
- Gordon I. Reproduction in Sheep and Goats. Cambridge: CAB International, 1997:228.
- Egan AR. Nutrition for reproduction. In: Lindsay DR, Pearce DT, eds. Reproduction in Sheep. Cambridge: Cambridge Univerity Press, 1984:262–268.
- Wathen NC, Delves HT, Campbell DJ. The coelomic cavity—a reservoir for metals. Am J Obstet Gynecol 1995;173:1884–1888.[CrossRef][Medline]
Received January 2, 2003;
accepted for publication June 30, 2003.
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Abstract
Introduction
