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In Utero Haematopoietic Stem Cell Transplantation (IUHSCT)


Maria Concetta Renda and Aurelio Maggio

U.O.C. Ematologia II con Talassemia, Ospedali Riuniti “Villa Sofia-Cervello”, Palermo

Correspondence to: Aurelio Maggio, A.O. 'V. Cervello'. Via Trabucco 180, 90146 Palermo-Italy. Tel: +39 688 5251; Fax: +39 688 0828; E-mail aureliomaggio@virgilio.it
Published: December 29, 2009
Received: December 14, 2009
Accepted: December 28, 2009
Medit J Hemat Infect Dis 2009, 1 (1): e2009031; DOI 10.4084/MJHID.2009.031
This article is available from: http://www.mjhid.org/article/view/5231
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Abstract
In utero haematopoietic stem cell transplantation (IUHSCT) is a non-myeloablative approach for the prenatal treatment of genetic disorders. However, in target disorders, where there is not a selective advantage for donor cells, a useful donor-cell chimerism has not been achieved. There are three possible barriers to engraftment following IUHSCT: limited space in the fetus due to host-cell competition; the large number of donor cells needed, and the immunological asset of recipient.


Introduction
Animal models have shown different levels of resistance to IUHSCT engraftment.  In primate, goat, rat and mouse  the levels of engraftment that has been achieved were low and not  therapeutic. Among 46 cases of  IUHSCT reported in humans, successful engraftment  was obtained only in cases of  X-SCID. Useful levels of chimerism has not been achieved in non-immunodeficiency diseases, and  a detectable engrafment , was  reported only in one case  of  -thalassemia transplanted at 12 weeks of gestation  by fetal liver cells. In one -thalassemia case, where -globin-dependent hemoglobin production and anemia are present during fetal period, microchimerism and tolerance were suggested .
To overcome the IUHSCT engraftment barriers, it is necessary to develop strategies to improve the competitive capacity of donor cells and  to define the gestational age of the possible immunological “window of opportunity” in the human fetus.
In utero haematopoietic stem cell transplantation (IUHSCT) is a non-myeloablative promising approach for the prenatal treatment of a variety of genetic disorders and  could be an alternative  option to therapeutical abortion in some congenital diseases like haematological hereditary  syndromes.
Observations of “naturally” occurring hematopoietic chimerism[1], and of experimental chimeras produced by IUHSCT, have demonstrated that allogeneic engraftment of the early gestational fetus with donor-specific tolerance is possible[2].
 Although IUHSCT has been clinically successful in severe combined immunodeficiency disease (SCID), a therapeutic donnor-cell  chimerism (more than 1%)[3], useful for the correction of most diseases, has not been achieved  in target disorders, like  hemoglobinopathies, where there is not a selective advantage for donor cells.[4]
The reasons for this failure are  unclear and reflect a limited understanding of the engraftment barriers involved in clinical IUHSCT.
There are three  possible  barriers  to allogenic engraftment following IUHSCT:  limited space in the fetus due to host-cell competition from a robust and highly proliferative fetal hematopoietic compartment; the large number of donor cells needed  to successfully compete with host cells for these sites; and the immunological asset of recipient that determines the grade of  the possible fetal tolerance which is conditional, depending on timing and appropiate presentation of antigen[4].               
The last barrier has been supported by successful allogenic IUHSCT in sheeps and monkeys[5] occurred if donor and recipient fetuses are in a preimmune condition, by  the presence of high-level engraftment after IUHSCT in disorders  where exists a competitive or survival advantage for donor cells, and by the presence of VDJ TCR chain transcript since the 7th weeks of gestation[6].
The goal of  therapeutically relevant levels of chimerism following a single in utero transplantation has been difficult to achieve. The induction of tolerance with low levels of chimerism to facilitate postnatal cellular  transplantations using minimally ablative conditioning regimens is a more realistic goal for clinical application of IUHSCT.


Cell sources:
Hematopoietic stem cells (HSC) have been isolated from fetal liver [7], umbilical cord blood, adult bone marrow [8] and mobilized peripheral blood. HSC isolated from these sources were well described for their capacity  to generate mature cells of all lymphohematopoietic lineage and their self-renewal capacity.
All these cell sources were employed to perform IUHSCT; however, if postnatal cellular therapy is needed, the donor cells must be obtained from a renewable source as adult related bone marrow or mobilized peripheral blood [9].

Animal models:
An ‘‘experiment of nature’’ first described by Owen in 1945 is the best supporting evidence that IUHSCT might work :  dizygotic cattle twins that share cross-placental circulation were born chimeric for their sibilings blood elements, and this state of ‘‘chimerism’’ persisted for life and was associated with specific transplantation tolerance[1].
The first  animal model was the sheep. Early gestational transplantation of allogenic, fetal liver–derived HSC into normal sheep fetuses resulted in a multilineage hematopoietic chimerism [10] that persisted for many years and  ranged from  10% to 15%  donor cell expression [11].
Other normal animal models  have shown different levels of  resistance to engraftment after in utero transplantation.  In the normal primate, goat, rat and mouse [12-16] the levels of engraftment that has been achieved were low and not  therapeutic: successful allogenic IUHSCT in monkeys occurred if donor and recipient fetuses are in a preimmune condition [17].
Almeida-Porada et al. 2000,  used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation [18]. In this xenogeneic model, the authors co-transplanted HSC and bone marrow-derived human stromal cells obtaining an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and  higher levels of donor cells in circulation both during gestation and after birth.
Hayashi et al 2002, obtained the  induction of  prenatal tolerance in mice where a combined approach of  IUHSCTx, performed by intraperitonel injection,  followed by postnatal donor lymphocyte infusion has converted low-level mixed hematopoietic chimerism to complete donor chimerism [19].
A mouse model described by Perenteau et al [20] strongly support the presence of  an immune barrier to allogeneic engraftment after IUHSCT . Four-teen weeks old  fetal mice were transplanted with high doses of donor cells  of either allogeneic or congenic bone marrow, using  intravascular  injection technique. Engrafment was lost in 70% of allogeneic recipients by 1 month of age; in contrast, all congenic recipients maintain stable, long-term, multilineage chimerism, thus giving a strong evidence for an  innate immune mechanism , natural killer cell or macrophage mediated,  able to eliminate  allogeneic cells  after IUHSCT [20].
However, further studies are needed to better characterize the timing of human fetal  NK-cell maturation.
The reasons for the failure  in the correction most diseases and the absence of predicting  tolerance  in  absence of immunosuppression, both in human or animal models, of IUHSCT  are  still unclear  and reflect a limited understanding of the engraftment barriers [21].

IUHSCT in Humans: 
Since Touraine et al published the first instance of intrauterine transplantation in a human fetus affected by bare lymphocyte syndrome (BLS)  in 198922, 46 cases of  IUHSCT in human for various indications have been reported [23] with different timing of transplantation, source of donor cells and the target disease. 
However, successful well-documented engraftment after in utero therapy was obtained only in cases of  X-linked SCID by Touraine , Flake  and Wengler [24-26].
Useful levels of chimerism has not been achieved in non-immunodeficiency diseases, as metachromatic leukodystrophy [27], chronic granulo-matous disease (Flake, unpublished data, 1995) or
alfa-thalassemia major [24,27-29].
Particularly, a detectable engrafment , using IUHSCT, was reported only in one case  of thalassemia major transplanted at 12 weeks of gestation by fetal liver cells [30]. In this case transitory engrafment was documented  by  the presence of the Y chromosome of donor origin  but not erythroid microchimerism [30].
Only in one -thalassemia case, transplanted at 13 weeks of gestation with paternal CD34+ enriched cells, microchimerism and tolerance were suggested [31] . However, the prenatal hematopoietic biology of the  and -thalassemia are different. In the case of - thalassemia, -globin-dependent hemoglobin production (HbF) and anemia are present during fetal period and this could determine a selective advantage for  the donor-derived  haematopoietic stem cells . Instead,   in -thalassemia the selective advantage could be not present because of globin chain synthesis impairement and  anemia do not occur until after birth .
Our experience, in  cases treated between the 13th and the 18th weeks of gestation using cryopreserved fetal liver haematopietic stem cells and without immunodepression, was also unsuccessful [29].
Particularly, a fetus, affected by -thalassemia major  who underwent a fetus-to-fetus transplantation  between the 14th  and the 20th  week of gestation, generated an alloimmune response as demonstrated by an high cytotoxic  T cell precursor (CTLp) frequency against donor cells [32].
We set-up a clinical protocol by which two female fetuses, 16-20 weeks old, were transplanted with circulating hematopoietic paternal stem cells after one week of low dose dexamethasone treatment with the aim to overcome the engraftment barrier by inducing a transient mild fetal immuno suppression and induce a donor-specific tolerance. During these procedures no adverse events were observed  both for  the mothers and the fetuses. At birth, both fetuses showed the presence of Y chromosome of donor origin but only  one,  at two months of age, showed the presence of paternal blood group allele A cDNA in peripheral blood  and an increase of HbA concentration from 3% to 14.4%, suggesting that erithroid microchimerism has been occurred. At 5 months of age haemoglobin decreased to 6.8 g/dl and the girl started a  regular blood transfusional regimen [33]. This could be explained only by a transitory engraftment of donor cells as it was previously shown in the case described by Touraine [30] .

IUHSCT future perspectives:
To overcome the barriers  that  hinder the success of IUHSCT, it is necessary to develop strategies to improve the competitive capacity of donor cells to achieve significant engraftment, and  to define the gestational age of the possible immunological “window of opportunity” in the human fetus.
A significant barrier to allogeneic engraftment following IUHSCT is limited space in the fetus due to host-cell competition from a robust and highly proliferative fetal hematopoeitic compartment in circumstances where a competitive or survival advantage does not exist for donor cells34. Additionally, the inability of HSC to home and engraft with absolute efficiency may limit donor-cell engraftment following allogeneic IUHSCT.
Thus, strategies that selectively increase donor HSC homing and engraftment in the fetal recipient may increase both the levels and frequency of engraftment following allogeneic IUHSCT. Dipeptidyl peptidase CD26 inhibition may represent a novel approach to increasing the efficacy and success of HSC/HPC transplantation [35].
Recent in vitro studies have demonstrated that increased cleavage of stromal-derived factor 1 (SDF-1) by CD26, a chemokine involved in hematopoietic cell chemotaxis, homing, mobilization and survival, results in a loss of its chemotactic effect on primitive hematopoietic cells. Instead, its inhibition results in SDF1–induced migration [36].
In vivo, postnatal studies in a murine bone marrow transplantation (BMT) model found increased short-term bone marrow (BM) homing of enriched HSC and low-density BM donor cells after inhibition or absence of CD26 activity. Reduction of CD26 activity on low-density donor BM cells also resulted in increased long-term engraftment, competitive repopulation, secondary trans-plantation, and mouse survival following lethal irradiation. Therefore, CD26 inhibition may be an important adjunct to other strategies that are directed toward overcoming the barriers to fetal engraftment [37]. The demonstration that transient manipulation of a single chemokine interaction can result in improvements in engraftment is encouraging, as there are many steps in the homing and engraftment process that could potentially be manipulated, singly or in combination, to significantly improve engraftment [38].
Other manipulations that selectively influence expression of homing receptors and engraftment include cotransplantation with stroma. The use of stromal cotransplantation has increased short- and long-term donor cell expression in the sheep model [39,40]. Human-placenta–derived mesenchymal stem cells (MSC) show a mutilineage differentiation capacity and they have a direct immunosoppressive effect on proliferation of T lymphocytes  from peripheral blood and umbilical cord blood. Even if the way that human-placenta –derived MSC modulate the immune system is unclear,  this immunoregulatory feature implies that human-placenta–derived MSC have a potential application in allograft transplantation [41].
Therefore, the recognition of an immune barrier suggests potential immune-based strategies for application to IUHSCT that might prevent loss of engraftment.
Some studies in mice [19,42] support the existence of the phenomenon of fetal tolerance  that it may be dependent on timing and appropriate presentation of antigen. Moreover, there are  extrathymic16 mechanisms of rejection  (NK – or B-cell-mediated response) that are  poorly understood.  In the human fetus there is evidence of immunocompetence from at least the 11th week of gestation  suggested by the  presence of alloreactive T lymphocytes from the 10th week of gestation that  could explain the  failure of engraftment in fetuses older than this gestation [6,28,32]. Therefore , IUHSCT should be performed before the 10th week of gestation. However, at this so early gestation it is impossible to carry out the infusion of CD34 into the umbilical cord by intravascular route or by their infusion into intraperitoneal cavity . One possible future approach could the infusion of these cells into the coelomic cavity.
Coelocentesis at 6-10 weeks of gestation  has been successfully used to obtain fetal DNA for very early prenatal diagnosis of genetic diseases [43-45]. The celomic cavity is now believed to be an important transfer interface and a reservoir of nutrients for the embryo and it is present since the fourth week after the last menstruation. Therefore, this cavity could be a new route of access to the fetus. In our study of celomic fluid from fetuses sampled from 6.6 to 10 weeks of gestation by coelocentesis we found an immunological pattern showing a very low frequency of the T, pre-B, B lymphocytes, NK and CD34+ cells. Moreover, the analysis of rearranged VDJ TCR β chain transcripts and TCR α chain transcripts showed the presence of only PreTα expression and only 40% (7 of 17) samples showed the presence of some V-subfamilies suggesting that the coelomic cavity could be a useful route of CD34+ cells infusion for “in utero transplantation” [46].
The injection of donor haematopoietic stem cells into this cavity could potentially induce tolerance in the fetus making successive booster stem cells infusions possible. This  procedure has already been evaluated in pre-immune fetal sheep in which the injection of human stem cells through the coelomic cavity was associated with a significant level of engraftment [47].



Conclusions

The intrauterine approach was criticized by several authorities in this field who claimed that this approach did not offer any advantage over postnatal transplantation. The main arguments against intrauterine transplantation were that the fetal invasive procedure carried a certain additional risk.
Conversely, promoters of intrauterine transplantation claimed that reconstitution of the immune system before birth results in reduced susceptibility to infections in the neonatal period, and to an improved psychosocial situation  for the family. Other potential advantages for an intrauterine approach include cost savings and a reduced risk of GVHD in the fetal environment.
Thus, a consensus has been difficult to reach and fetal transplantation has not been widely adopted, despite the fact that successful cases treated in utero had outcomes comparable with the best reported with postnatal transplantation48.


Acknowledgements:
We would like to thank Fondazione Roma, Rome (Italy) and Foundation Franco and Piera Cutino, Palermo (Italy).


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