JOURNAL OF HEMATOTHERAPY & STEM CELL RESEARCH 11:617–631 (2002) © Mary Ann Liebert, Inc.
State-of-the-Art Review Fetal Hematopoietic Stem Cell Transplantation: A Challenge for the Twenty-First Century LAURENCE E. SHIELDS,1 BIM LINDTON,2 ROBERT G. ANDREWS,3 and MAGNUS WESTGREN2
ABSTRACT Successful in utero hematopoietic stem cell transplantation will likely represent a major step forward in the management of patients with congenital hematological, metabolic, and immunological disorders. We review the naturally occurring models of hematopoietic chimerism in animals and humans, as well as available experimental animal data and human clinical attempts of fetal transplantation. Data available from naturally occurring models and experimental models of fetal transplantation suggest that this technique should be translatable to the human fetus. However, to date, the success of human fetal hematopoietic stem cell therapy has been limited to fetuses with severe immunologic defects. Evaluation of successful attempts of human transplantation, the ontogeny of fetal immune development, and data available from animals provide insights into innovative approaches to fetal therapy that may bring the reality of successful fetal transplantation closer.
INTRODUCTION
S
UCCESSFUL
IN
UTERO
HEMATOPO IETIC
STEM
CELL
will likely represent a major step forward in the management of patients with congenital hematological, metabolic, and immunological disorder. The rationale for in utero stem cell transplantation is based on three primary assumptions. First, treatment of certain diseases is only possible in utero because the target disease is lethal for the fetus (e.g., a-thalassemia) or the disease results in early fetal and neonatal central nervous system morbidity, making postnatal therapy less efficacious (e.g., globoid leukodystrophy). Second, unlike postnatal settings, the immature fetal immune system may be relatively more permissive for the transplantation of partial or non-HLA-matched donor cells that will ulTRANSPLANTATION
timately be recognized as “self” by the fetus. Third, fetal growth should create abundant hematopoietic niches or homing sites facilitating engraftment and in vivo expansion of donor cells. In addition, in utero treatment of some diseases, where fetal morbidity does not occur, may be preferable due to improved long-term outcomes (1). Successful allogeneic in utero transplantation of hematopoietic stem cells has been reported in several animal models (reviewed below). The first attempted human fetal transplant, using fetal bone marrow for severe Rh disease, was reported in 1967 (2). Unfortunately, this fetus did not survive. Twenty-two years later, the first successful transplantation in a human fetus with Bare lymphocyte syndrome was reported (3). Since then, 39 in utero transplantations have been published or reported at national or international meetings (see Table 1). The
1 Obstetrics
and Gynecology and 3Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Washington School of Medicine, Seattle WA 98195-6460. 2 Center for Fetal Medicine, Department of Obstetrics and Gynecology, Karolinska Institute, Huddinge, Sweden. 3 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle WA 98193.
617
618
19
19 14,16
10. b-Thalassemia
11. b-Thalassemia 12. b-Thalassemia 13. b-Thalassemia
? ?
16
9. b-Thalassemia
16. Sickle cell anemia 17. Sickle cell anemia
15
8. b-Thalassemia
13
14 18
6. b-Thalassemia 7. b-Thalassemia
12,14,16
18 25 14
3. b-Thalassemia 4. b-Thalassemia 5. b-Thalassemia
14. b-Thalassemia Other red cell disorders 15. Sickle cell anemia
15,31
13 19 24
GA weeks
2. a-Thalassemia
Thalassemia 1. a-Thalassemia
Indication
FL (n 5 5; 6–10 wks) BM BM
Pat.BM
OF
Cell number
? ?
16.7 3 10*8/kg
XX XX 2.9 3 10*8/kg 2 3 10*7/kg T cells: 1.7 3 10*5/kg 3 3 10*6 CD341
5.2 3 10*3 CD341
8 3 10*8/kg
9 3 10*8/kg
XX 8.6 3 10*8/kg
No No
No
No
No ? Yes
No
?
?
? No
Yes No Yes
No
Yes
Engrafted
NA NA
Transfusion-dependent
Transfusion-dependent, immunization Transfusion-dependent Procedure-related death Female fetus at birth BM 5 4%; metaphases1 at 4 months all test negative Transfusion-dependent
Elective termination
Procedure-related death
Septic abortion Transfusion dependent
Elective termination Born with disease 0.9% HbA @ 6 months
Transfusion dependent
Microchimerism noted at birth, evidence of tolerance to donor, transfusion dependent
Comments
HUMAN IN UTERO STEM CELL TRANSPLANTATIONS
3 3 106/kg CD341 3 3 106/kg CD341 3 3 106/kg CD341 Total T cells/kg ,1.5 3 105 20.4 3 10*8/kg 1.2 3 10*8/kg XX 6 3 10*9/kg 1.5 3 10*10/kg
SUMMARY
FL (n 5 7; 5– 10 wks) Mat. BM Sib BM FL.(9.5 wks) 1 thymic cells FL FL (n 5 5; 6–10 wks) FL (n 5 5; 8–11 wks) FL (n 5 6; 9–11 wks) FB (HLA identical twin) FB FL (not stated) BM HLA match sib
Pat.BM
Tissue
TABLE 1.
Detroit (unpublished) Philadelphia (unpublished)
Westgren et al. 1996 (77)
Monni et al. 1998 (84)
Singapore (unpublished) Touraine et al. 1992 (80) Peschle et al. 1997 (83)
Orlandi et al. 1996 (82)
Palermo, Italy (unpublished) Palermo, Italy (unpublished)
Cowan et al. 1994 (81) Westgren et al. 1996 (77)
Diukman et al. 1992 (78) Slavin et al. 1992 (79) Touraine et al. 1991 (80)
Westgren et al. 1996 (77)
Hayward et al. 1998 (61)
Reference (ref. number)
SHIELDS ET AL.
619
Chediack-Higashi
31.
32. Omenn Syndrome Storage diseases 33. Globoid cell leukodystrophy 34. Globoid cell leukodystrophy 35. Globoid cell leukodystrophy
CGD
30.
Mat.BM
Pat.BM
Pat.BM
Pat.BM
14
13
13
Mat.BM
Pat.BM
BM CD341
FL (10 wk) FL (not stated)
Pat.BM
Mat. BM Pat.BM
22, 23
26
15
14 17, 21 21 13.5
27. X-SCID 28. CGD
CGD
21 22
26. X-SCID
29.
20 16, 17.5, 18.5
24. SCID 25. X-SCID
28 23, 24
22. SCID 23. SCID (Rak)
Mat. BM
12
FL (7 and 7.5 wks) and thymic cells FL (7.5 wks) Pat BM
Mat. BM
17
30
Fetal BM
24
Immunodeficiencies 21. BLS
18. Rh isoimmunization 19. Rh isoimmunization 20. Rh isoimmunization
5 3 10*8/kg
CD341 : 5 3 10*9/kg CD31 : 2.7 3 10*8/kg 5 3 10*8/kg
40 3 10*6/kg 3 2
XX
XX
2.9 3 10*8/kg
XX 1.1 3 10*8/kg 8.9 3 10*6/kg 6.2 3 10*6/kg 3.9 3 10*7/kg 1.1 3 10*7/kg T cells: 3.4 3 10*5/kg 7 3 10*7/kg XX
3.7 3 10*7/kg 1.7 3 10*7/kg 8.3 3 10*6/kg
1.2 3 10*7/kg
1.5 3 10*10/kg 7.6 3 10*8/kg 5 CD3 4
7.5 3 10*8/kg
X
No
No
Yes
Yes
No
No
No
Yes ?
Yes
No Yes
Yes Yes
Yes
No
No
No
Born with disease; no chimerism Born with disease; no chimerism
Fetal death at 20 weeks
Born with disease; no chimerism Born with disease; no chimerism Born with disease; no chimerism Born alive; split chimerism
Born alive, split chimerism Procedure-related death
Born alive, split chimerism
Alive and well At birth negative; postbirth retested and positive Elective termination Born alive Split chimerism
Alive and well
Not tolerant to maternal antigens Some tolerance to maternal antigens
Postnatal death
Leung et al. 1999 (94)
Leung et al. 1999 (94)
Bambach et al. 1997 (57)
Porta et al. 2000 (93)
Diukman et al. 1992 (78)
Flake et al. 1999 (92)
Muench et al. 2001 (91)
Westgren et al. (89) Touraine et al. 1996 (90)
Lanfranchi et al. 1998 (88)
Diukman et al. 1992 (78) Flake et al. 1996 (87)
Touraine et al. 1989 (80) Wengler et al. 1996 (86)
Touraine et al. 1989 (80)
Thilaganthan et al. 1993 (62)
Linch et al. 1986 (85)
Davies 1967 (2)
FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION
36. Hurler’s Syndrome 37. Niemann-Pick Dz 38. Metachromatic leukodystrophy 39. Metachromatic leukodystrophy
Indication
620
23
14 12,13 37
GA weeks
SUMMARY
Pat.BM
FL FL (not stated) Pat.BM
Tissue
TABLE 1. Cell number
4.3 3 10*9/kg
No
No ? No
Engrafted
Died of disease No data Born with disease; no chimerism Born with disease; no chimerism
Comments
H UMAN IN U TERO STEM CELL TRANSPLANTATIONS (CONT ’D )
? XX 1 3 10*9/kg
OF
Slavin et al. 1992 (79)
Flake et al. 1997 (51) Touraine et al. 1996 (90) Slavin et al. 1992 (79)
Reference (ref. number)
SHIELDS ET AL.
FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION transplants that have been attempted have come from a number of centers around the world, each with their own protocols. Unfortunately, the number of fetuses treated at any single center has been small and, to date, there are no universally established target diseases, protocols, or multicenter collaborative efforts. As such, the human transplantations have been performed for a variety of indications, at various gestational ages, and with different donor cells sources. All reported cases of in utero transplantation performed in fetuses with normal immunological function have clinically failed and unequivocal engraftment has been demonstrated only in fetuses suffering from severe immunodeficiency disorders. Given the failure of in utero stem cell transplantation in the immunologically intact fetus, the assumption that the fetus is an ideal candidate for stem cell therapy must be reevaluated. This review analyzes the experimental and clinical progress in this field with a focus on preclinical animal experiments.
NATURALLY OCCURRING AND EXPERIMENTAL MODELS OF IN UTERO STEM CELL TRANSPLANTATION Experiments of nature (large animal) Dizygotic twin cows (4,5), goats (6), and marmosets (7) provide evidence suggesting that in utero hematopoietic therapy should result in clinically relevant levels of chimerism, correct many inherited fetal diseases, and induce transplantation tolerance if the donor cells are given to the fetus early in gestation. It is assumed in all of these examples of naturally occurring chimerism that mixing of hematopoietic cells early in gestation occurs through placental blood vessel anastomosis between the two fetal circulations (7,8). It is important to note that transplantation through placental anastomosing vessels is different from experimental fetal transplantation. With placental anastomosis, the mixing of fetal blood likely occurs very early in pregnancy and the mixing of fetal blood is likely to be continuous. Dizygotic twin cows mismatched for the presence of a-mannosidosis (4) and Pompe’s disease (9) and goats mismatched for b-mannosidosis (6) have shown reductions in the severity of their storage disease. Additionally, dizygotic twin cattle demonstrate intertwin tolerance when measured by mixed lymphocyte cultures (MLCs), accept renal grafts from their co-twin, and have delayed rejection to skin grafts transplants (10). Similar evidence of intertwin tolerance has been demonstrated in dizygotic twin marmosets using cytotoxic T lymphocyte assays (CTL) (7). Marmosets typically show high degrees of hematopoietic chimerism (28–82% by peripheral blood karyotype).
Experiments of nature (humans) While the natural chimeras noted in dizygotic large animals are highly suggestive that in utero transplantation should be successful, the most encouraging data comes from dizygotic human twins. While the frequency of transplantation chimerism in dizygotic twins at birth is relatively high (8% for twins and 21% for triplets), the level of chimerism is extremely low (11). These data are consistent with placental architecture studies showing that intertwin placental anastomosis does not occur between most dizygotic human twins. However, more than 30 cases of opposite-sex dizygotic twins have been reported that show much higher levels of hematopoietic chimerism (12–16). Many of these cases have been previously reviewed (12). Reports where the sex of the twins was different or where HLA typing confirms nonidentical twinning are listed in Table 2. In these cases, early placental fusion and anastomosis presumably occurred. The level of chimerism in these reports is generally high and, in one well-documented case, chimerism has persisted for greater than 25 years (14). Immune tolerance (MLC and skin grafting) has also been demonstrated (15,17,18). These naturally occurring “experiments” in large animals, including primates as well as humans, provide the first evidence that in utero stem cell therapy may be successful and have led to a number of studies in various animal models.
Mouse models In utero stem cell transplantation in mice has been studied in strains with and without lymphohematopoietic defects and allogeneic models. In utero transplantation in mice with congenital defects of their lymphohematopoietic system (WW, SCID, and NOD-SCID) suggests that, when a competitive advantage exists for donor cells, high rates of engraftment and levels of chimerism will be produced (19–21). In the SCID and NOD-SCID mouse models, the level of chimerism appears to be related to the severity of the immune defect. It is possible that donor cell engraftment was improved in the more severely affected NOD-SCID model due to increased available “space” from absent cells [T cell, B cell, natural killer (NK) cells, and macrophages], a reduction in the fetus’s ability to mount an immune response secondary to the more severe immune defect, or a combination of these two factors. Allogeneic in utero transplantation in fetal mice without stem cell or immune defects results in variable degrees of engraftment, ranging from ,1 to 50%. However, in general, the level of chimerism has been disappointingly low (,2%) and would not be expected to ameliorate or cure most diseases of the lymphohe-
621
SHIELDS ET AL. TABLE 2. Sex 1 2 3 4 5 6 7 8 9 10 11 12
13
M F M F M F M F M F M F M F M F M F M F M F Mg M F M F
HUMAN DIZYGOTIC TWINS SHOWING HIGH LEVELS Lymphocyte chimerisma
RBC chimerismb
91%/9%0 98%/2%0 NA
86%/14% 99%/1%0 61%/39% 51%/49% 85%/15% 85%/15%c 99.8%/0.2% 80%/20% 73%/27% 69%/31% 90%/10% 65%/35% 98%/2%0 50%/50% 99%/,1% 99%/,1%c 89%/11% 88%/12% 90%/10% 74%/26% 80%/20%c 85%/15% 91–93%/7–9%g
30%/70% 78%/22% 97%/3%0 22%/78% 91%/9% 70%/30% 34%/66% 87%/13% 99%/1% 53%/47% 80%/20% 46%/54% 68%/32% 60%/40% 75%/25% 66%/34% 19%/81% 18%/82% 89%/11% 92%/8%0 16%/84% 90%/10% 10%/90%
NA
OF
HEMATOPOIETIC CHIMERISM HLA type
Reference
NA
(12)
NA
(12)
NA
(12)
NA
(12)
DR4, DRw6 DRw8DRw6 d,e NA
(14)
NA
(12)
NA
(12)
NA
(95)
NAe
(17)
NAf
(13)
NA
(16)
DR4, DR53, DQ8, DQB1-0301h
(15)
(12)
a NA,
Not applicable; definitive HLA serologic or DNA typing was not presented. of lymphocytes that are same as phenotypic sex/percentage nonphenotypic sex lymphocytes ascertained by karyotype. c Percentages of red blood cells (RBCs) antigen subtypes that predominate/percentage RBC types that predominate in the other twin. The RBC differences are based on expression of or absence of RBC antigens (e.g., A, B, O, Jka, MS, etc.). d RBC phenotypes were identical (or nearly) in both twins. e HLA typing showing that the twins were haploidentical. fMixed lymphocyte testing showed no response to co-twin but normal response to unrelated lymphocytes. g Triplet pregnancy with two males (monozygotic) and a female. HLA typing of lymphocytes showed the three to HLA identical. HLA typing of fibroblast was not reported. Blood groups and Rhesus factor were similar in all three neonates. h HLA subtypes listed were present in the male but not the female. Karyotype evaluation of skin fibroblast were 100% sex specific for each twin. Mixed lymphocyte cultures showed no reactivity between the twins and normal responses against controls. b Percentages
matopoietic system (22–24). Despite low levels of donor cells, the presence of microchimerism has resulted in tolerance induction (22,25). In those mice showing evidence of tolerance, enhanced engraftment with postnatal booster cell dosing has also been demonstrated (22). If induction of tolerance and the ability to enhance the level of chimerism postnatally can be demonstrated in other animal models, clinical trials of in utero stem cell therapy may be appropriate for diseases that do not affect the
individual during in utero life (b-thalassemia and sickle cell disease).
Ovine and caprine models The majority of work evaluating the feasibility of in utero hematopoietic stem cell transplantation has been carried out in the ovine model (26,27). When transplanted between 0.38 and 0.45 gestation, the proportion of the
622
FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION fetuses engrafting varied from 29% (donor cells from T cell-depleted adult bone marrow) to 84% (donor cells from fetal liver). The level of chimerism ranged between 5 and 14% (27). As in postnatal transplantation, increasing the absolute number of T cells given to the fetus increased the level of chimerism. However, the risk of fetal graft-versus-host-disease (GVHD) also increased, and, at a T cell dose of approximately 1 3 108 /kg, all of the fetuses died of GVHD (28). Induction of tolerance has also been demonstrated in neonatal sheep transplanted in utero based on reduced responses to donor cells in mixed lymphocyte culture (MLC) and enhanced engraftment after a single postnatal “booster dose” from the original donor without pretransplant ablative and immune suppressive conditioning (29). Tolerance to solid organ transplantation (renal) was not observed in chimeric sheep (30), unlike the results noted in chimeric nonhuman primates (see below). Despite the encouraging results from the immunologically normal sheep fetuses attempts at in utero transplantation in genetically defective sheep (ceroid-lipofuscinosis) (31) and goat fetuses (b-D -mannosidosis) (32) have not been as encouraging. Although 9% chimerism was achieved when hemoglobin isotypes were measured, transplanted sheep had had similar clinical disease, brain weight, and histopathology as nontransplanted lambs. The authors concluded that under the conditions of the experimental protocol, transplantation of fetal hematopoietic cells was not beneficial.
Non-human primate models Non-human primates are a relevant animal model for the study of postnatal stem cell transplantation and stem cell gene therapy. Similar ontogeny of immune and hematopoietic development suggests that fetal non-human primates should also be a highly relevant model for the study of in utero stem cell transplantation (33–35). Harrison et al. (36) reported results from 5 rhesus monkeys transplanted by intraperitoneal injection of cryopreserved allogeneic fetal liver cells. Engraftment was noted in the 4 animals that survived with the level of chimerism ranging between 2.4 and 6.2% by peripheral blood by karyotype analysis. Slightly higher levels of chimerism were noted in the neonatal bone marrow progenitor population (5.0–9.3%). These studies, along with those of Roodman et al. (37) and Brent et al. (38) (see below) provided the first data suggesting chimerism in fetal primates could be achieved experimentally. In utero transplantation with adult bone marrow cells has been tested by two investigators. Brent et al. (38) attempted in utero stem cell transplantation in 22 cynomolgus monkeys between 0.30 and 0.60 gestation using T cell-depleted adult bone marrow. Eight of the 14 fetuses (57%) transplanted at less than 0.45 gestation were chi-
meric. Unfortunately, all of the chimeric animals died in utero. The authors concluded that the fetal deaths were most likely from GVHD secondary to inadequate T cell depletion. Prior to in utero fetal death, four of the eight chimeric fetuses were hydropic. One of these hydropic animals was delivered prior to in utero death and GVHD was confirmed histologically. In utero fetal death also was a problem in the studies by Roodman et al. (37) after transplantation with unfractionated adult bone marrow. All of the chimeric animals died in utero. In both studies, chimerism was not observed when transplantation was performed beyond 0.45 gestation. Thus, in two different species of non-human primates, the “window of opportunity” for fetal transplantation appears to be at less than 0.45 gestation. Cowan et al. (39) transplanted T cell (CD2)-depleted haploidentical adult bone marrow from either the sire or dam into opposite sexed rhesus fetuses. Engraftment was noted in 8 of 11 animals. Unfortunately, as in the allogeneic fetal mouse experiments, the level of engraftment was low (,0.1%). In utero induction of tolerance was demonstrated by reduced MLC responses and solid organ transplantation in some of the engrafted animals. These results are similar to those noted in studies using allogeneic fetal mice, suggesting that tolerance can occur after in utero hematopoietic stem cell/bone marrow transplantation. Unfortunately, attempts to increase chimerism by postnatal booster cell dosing were not attempted. Further investigation of in utero tolerance induction will hopefully improve our understanding of fetal immune development and may provide insights for future efforts to enhance the effectiveness of in utero treatment.
FACTORS THAT INFLUENCE IN UTERO ENGRAFTMENT Fetal hematology Fetal hematologic stem cell development is thought to begin in the para-aortic splanchnopleur and/or the yolk sac and progress to the fetal liver and, to a lesser extent, the fetal spleen. In the late second and early third trimester, fetal bone marrow becomes the primary hematopoietic organ (40). Analysis, by flow cytometry, of early fetal hematopoietic sites (liver, bone marrow, and blood) has demonstrated at high frequency of immature hematopoietic progenitor and stem cells. When compared to similar cells obtained from postnatal sources, fetal cells also demonstrate higher in vitro proliferation (33,41,42). Fetal growth and presumably fetal hematopoietic needs expand tremendously (approximately 240-fold) from the first trimester to full-term delivery. The rapidly expanding hematopoietic requirements suggest that donor cells
623
SHIELDS ET AL. TABLE 3. FOR
limit the degree of engraftment (43). The lack of clinical results of in utero stem cell transplantation in fetuses with intact fetal immune systems suggest that the concept of readily available space for donor cells must be questioned.
POTENTIAL CANDIDATE DISEASES IN UTERO FETAL THERAPY
Hematopoietic Disorders Disorders affecting lymphocytes SCID (sex linked) SCID (adenosine deaminase deficiency) Ommen syndrome Agammaglobinemia Bare lymphocyte syndrome Disorders affecting erythrocytes Sickle cell disease a-Thalassemia b-Thalassemia Hereditary spherocytosis Fanconi anemia Disorders affecting granulocytes Chronic granulomatous disease Infantile agranulocytosis Neutrophil membrane GP-180 Lazy leukocyte syndrome Chediak-Higashi syndrome Inborn Errors of Metabolism Mucopolysaccharidoses MPS I (Hurler Disease) MPS II (Hunter Disease) MPS IIIB (Sanfilippo B) MPS IV (Morquio) MPS VI (Maroteaux-Lamy) Alpha mannosidosis Mannosidosis a-Mannosidosis b-Mannosidosis Mucolipodoses Gaucher disease Metachromatic leukocystrophy Krabbe disease Niemann-Pick disease b-Glucoronidase deficiency Fabry disease Adrenal leukodystrophy
Fetal immunology
may have readily available hematopoietic space or homing sites for engraftment. The theoretical availability of homing sites or hematopoietic space has been one of the primary arguments favoring fetal stem cell therapy and theoretically allows donor cells to engraft with preconditioning/myeloablation. However, while fetal hematopoietic environment is expanding at a very rapid rate, the fetus is producing very high levels of early hematopoietic progenitor/stem cells that are likely formidable competitors for allogeneic donor cells. In addition to cell number, allogeneic homing sites for donor cells may also
Many investigators have assumed that the early second-trimester fetus is preimmune and unable to mount a successful immunological attack against donor cells (3,27,44). In light of published reports of in utero cell transplantation in humans, this assumption must be reassessed. The only clinically successful in utero transplants have been carried out in severely immunologically compromised fetuses, suggesting that the fetal immune system likely plays a vital role in the success or failure of in utero fetal transplantation. The appearance and development of differentiated fetal immune cells (T cells, B cells, and NK cells) occurs at different stages during gestation. Fetal T cell development begins with the migration of hematopoietic fetal liver cells to the fetal thymus at approximately 8.5–9.5 weeks gestation. Shortly after this time period, 20–50% of cells in the fetal thymus express the common T cell surface phenotypes (CD7, CD2). VDJ rearrangement and T cell receptor (TCR) b-chain transcripts are detectable in first-trimester fetal liver cells and these cells are strongly alloreactive against major histocompatibility complex (MHC) Class I molecules (45). These findings concur with in vitro functional testing by MLC, suggesting that early in fetal life (9.5 weeks) primitive T cells can respond to allogeneic cells, and consistent in vitro MLC responses are present by 12 weeks gestation (45–47). Recently, we have demonstrated that fetal liver cell alloresponsiveness, when studied in MLC, is mediated through the T cell (CD31 ) and NK cell (CD561 ) cell population (unpublished data). This data would support the concept that early fetal T and NK cells have allocapacity. Relative to T cell development, the ontogeny of fetal B cell development begins slightly later in gestation. Surface expression of immunoglobulin M (IgM) can be noted as early as 9–10 weeks. Cells in the fetal circulation express common B cell antigens (CD20) at 14–16 weeks gestation and secretion of IgM has been noted as early as 15 weeks. Normal serum levels are not reached until 1 year of age. Although secretion of IgG is first noted at 20 weeks gestation, levels increase until approximately 5 years of age. Due to the relatively late appearance of humoral immunity, it is unlikely that B cell function significantly affects the success of in utero stem cell transplants. NK cells are likely to play an important role in early fetal immunological responses. These cells share a num-
624
FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION ber of antigenic and functional similarities to T cells but do not rearrange or express T cell antigen receptor genes, nor do they require the presence of MCH class I or class II antigens to effect their immune response. NK cells function through direct cytotoxicity to foreign antigens, usually virus-infected cells, tumor cell, or trophoblastic cell stimulation, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). When expressed as a percentage of the total lymphocytes, the proportion of NK cells in the fetal circulation is quite high, 29% at 13 weeks gestation (48). As they do in postnatal bone marrow transplantation (49), NK cells are likely to play an important role in graft failure after in utero transplantation due to their early presence and high frequency. Unfortunately, there is not a large literature relative to the ontogeny of the fetal immune system. However, the ontogeny of fetal immune cells, particularly in vitro MLC data demonstrating early fetal allogenic responses, do not support the concept that the fetus is “preimmune” at the time of most fetal transplants (12–14 weeks). Furthermore, these data challenges the “preimmune” status of the fetus as an argument supporting fetal transplantation. These factors must be taken into account when strategies are developed for in utero therapy and potential approaches to these problems are discussed below.
Donor cell source and stem cell identification Hematopoietic stem cells have been isolated from fetal liver, umbilical cord blood, and bone marrow and are the earliest precursors of all lymphohematopoietic cells. They are best described by their function, the ability to sustain lymphohematopoiesis and to generate mature progeny of all lymphohematopoietic lineages, i.e., red blood cells (RBCs), white blood cells (WBCs), platelets, and tissue macrophages). A number of specific cell-surface markers (particularly CD34, c-kit, Sca1, Thy1, and recently KDR) have been used to isolate cells that function as definitive stem cells (50). Different stem cell sources, fetal and adult, differ in in vitro proliferation, cell-surface phenotype expression, and telomeric lengths. In utero transplantation using different cell sources has been evaluated systemically only in the ovine xenogeneic transplantation model. Using this model, the highest rate of engraftment and chimerism was noted when fetal sources of stem cells were used. However, their use in clinical practice is likely to be limited due to availability and ethical considerations regarding the use of tissue from aborted fetuses. More importantly, if postnatal “booster” therapy is going to be used as a method to improve a low level of chimerism, then donor cells must be obtained from a renewable source (51). Umbilical cord blood has many of the same advantages (immature cells with high proliferative capacity) and limitations (non-renewable) as fetal liver cells, and, as such, it is unlikely
at the present time that these cells will be useful for clinical trials of in utero stem cell transplantation. At this time it is unclear whether the differences between fetal and adult stem cells will influence the results from allogeneic in utero transplantation in other preclinical animal models or in humans. Further investigation in preclinical animal models using hematopoietic growthstimulated adult bone marrow cells and mobilized peripheral blood cells should also be evaluated based on the enhanced proliferative capacity, which may allow them to compete more effectively in the fetal environment.
Cell dose and administration Transplantation studies in both humans and animal models (mice, sheep, and non-human primates) suggest that the number of cells transplanted influences the rate of engraftment and level of chimerism (27,52–54). Experience with postnatal bone marrow transplantation, usually after pretransplantation conditioning, indicates that 1 3 106–7 CD341 cells/kg are needed to achieve successful engraftment (55). However, fetal transplantation occurs under very different conditions than traditional postnatal bone marrow transplantation. The fetus has not been subjected to conditioning regimens, and the fetal environment may actually limit the ability of allogeneic donor cells to effectively engraft. Circulating CD341 fetal hematopoietic cells are present in very high numbers (10% of mononuclear cells) and these cells show very high in vitro proliferative potential (35,41). If in utero transplantation truly takes place under permissive conditions, then transplantation of allogeneic hematopoietic stem cells should be similar to a competitive repopulating model with donor cells and autologous fetal cells competing for engraftment into the same hematopoietic niches or homing sites. Under these conditions, it is likely that a very large number of donor cells will be needed to engraft successfully in the fetus. Data from fetal sheep studies and from non-human primates suggests that engraftment is cell dose related (27) and at least 1 3 108–9 CD341 cells/kg are needed for successful fetal engraftment (39,56). Thus, relative to the postnatal setting, the number of donor cells required for fetal transplantation is approximately 1,000-fold higher. There are two potential concerns related to the use of high cell numbers as part of in utero fetal transplants. Although CD34 selection will significantly deplete donor T cells, further T cell depletion may be required to bring the absolute number of donor T cells to a level that will prevent GVHD. The one caveat of T cell depletion is that the rate of graft failure will also increase. The optimal percentage and absolute number of donor T cells that can safely be given to the fetus in both the fetal sheep (28) and non-human primate (56) models appears to be in the range of 1 3 106–7 /kg. In both animal models, an absolute
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SHIELDS ET AL. T cell number greater than 1 3 108 /kg produced GVHD. This is in contrast to the postnatal setting, where the risk of GVHD increases tremendously as the absolute number of T cells exceeds 106/kg. The second potential concern is the report of fetal “over-engraftment” (57). It should be noted that in that case tissue sampling to rule out GVHD completely was suboptimal, due to the macerated condition of the fetus, and greater than 108 /kg CD31 T cells were given to the fetus. Administration of donor cells to the fetus can be accomplished by two routes, intravenous and intraperitoneal. Although giving donor cells through intravenous injection has been shown to increase the number of donor cells in hematopoietic organs (58) higher levels of chimerism have been noted after intraperitoneal administration in the fetal sheep model (59). The reason for this difference is not known, but the intraperitoneal cavity may serve as a reservoir for transplanted cells with gradual shedding into the fetal circulation. In addition to the route of administration, the number of donor cell infusions also appears to influence the success of in utero transplantation. In the fetal sheep model, giving the same number of donor cells through three versus a single infusion also improved the level of chimerism (27). These findings are consistent with studies in postnatal syngeneic mice where five daily injections of donor cells resulted in a four-fold increase in the level of donor cell chimerism (11–39%) relative to giving the same number of donor cells as a single injection (54).
The role of HLA on in utero engraftment In postnatal bone marrow transplantation, HLA matching improves successful engraftment and significantly reduces the risk of GVHD. The rate of both graft failure and GVHD is also directly related to the degree of HLA mismatch (60). The few experiments of nature (cows, marmosets, and humans) and the ovine model of in utero transplantation have demonstrated that successful engraftment with clinically relevant levels of chimerism can occur even when significant major histocompatibility antigen barriers appear to exist. On the basis of this data, it is presumed that successful fetal engraftment will occur with less than perfect HLA matching, presumably due to the naive fetal immune system and induction of central and peripheral tolerance. Unfortunately, the levels of chimerism noted in marmosets, cows, and sheep have not been repeated in published reports of non-human primates or in human trials of in utero stem cell transplantation in the absence of immune deficiencies. To date, the role that HLA plays in the success of in utero transplantation has not been addressed. However, based on studies of postnatal stem cell transplantation, it is reasonable to assume that even in the setting of in utero
transplantation, HLA may significantly influence engraftment and the level of chimerism. Examination of the role of HLA in fetal transplantation deserves further investigation.
In utero induction of tolerance and postnatal boosting to enhance chimerism Treatment of genetic diseases affecting the lymphohematopoietic system can be categorized into those that result in significant damage to the developing fetus prior to birth (leukodystrophies and a-thalassemia) and diseases that do not affect the fetus but result in significant morbidity postnatally (b-thalassemia, sickle cell disease, and immune deficiencies). For those diseases that affect the fetus prior to birth, clinically effective levels of chimerism must be obtained in utero. For diseases that express their deleterious effects postnatally, induction of tolerance may allow more efficacious postnatal therapy without the usual risk associated with pretransplantation conditioning (chemotherapy, immune suppression, and radiation therapy). Although data from animal models are limited, they are also encouraging. Induction of tolerance has been demonstrated in mice, sheep, and non-human primates (22,29,39), and increased hematopoietic chimerism with postnatal booster dosing has been demonstrated in mice and sheep. Two cases of human intrauterine transplantation have been reported where donor-specific tolerance was tested. In both of these cases, a reduced response to the donor was noted in MLC and in cytotoxic T lymphocyte testing (61,62). If further study of the response to postnatal boosting demonstrates that clinically relevant levels of chimerism can be achieved, then the number of candidate diseases for in utero transplantation could be greatly expanded. Furthermore, in cases with tolerance induction, the use of minimally myeloablative regimens may augment the presence of microchimerism and improve clinical efficiency.
IN UTERO TRANSPLANTATION IN HUMANS Thirty-nine cases of allogeneic in utero stem cell transplantation in humans have been either described or presented at national and international meetings (Table 1). In many of these reports, details of the individual cases are scarce. Because the timing of the intervention, the source of donor cells, and the targeted diseases have varied greatly it is difficult to draw general conclusions. Detectable engraftment has been reported in 13 of the 39 cases. In all cases, with the exception of the severe immunodeficiencies, the level of chimerism has been very low and had no impact on the course of the disease be-
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FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION ing treated. In the four well-documented cases of in utero therapy of X-SCID, some may argue that successful therapy could be achieved postnatally (63,64). However, it should be noted that these infants were born with functional T cells, reducing the risk of postnatal opportunistic infection. In one case, testing for B cell function with a unique antigen (bacteriophage f174), the infant showed near normal immunoglobulin (IgM, IgG) responses (65). Additional cases of in utero-treated X-SCID will be needed to evaluate this effect fully. The success of in utero therapy for X-SCID will also have to be evaluated against the recent French report of successful postnatal gene therapy for cases of common gamma-chain defects where the treated infants had normal T and B cell function without pretreatment conditioning (64). It is unclear if the failure of most in utero allogeneic hematopoietic stem cell transplants in humans is related to fetal immune function, HLA mismatching, the lack of potential space or niches for the donor cells, the types and numbers of stem cells transplanted, or the inability of donor cells to home to sites of engraftment. The lack of information explaining the reason for the failed cases of human in utero stem cell transplantation leaves many unresolved questions. In addition, on the basis of the dismal results reported to date, a strong argument can be made that a moratorium should be placed on in utero stem cell therapy, except in cases of severe immune defects, until new, compelling, preclinical data is reported.
FUTURE DIRECTIONS: FETAL IMMUNE MODULATION AND SELECTIVE FETAL MYELOABLATION The failure of in utero stem cell transplantation, except in the cases of severe immunodeficiencies, suggests that current techniques and future research should be directed at methods that will potentially enhance the success of fetal stem cell therapy. The rationale for in utero transplantation has been based on the premise that successful treatment will occur prior to the occurrence of in utero damage or fetal death and that treatment of the fetus prior to birth will be carried out without the need for cytoablative drugs or immune suppression. However, as noted above, human trials of in utero stem cell therapy in the absence of immune deficiencies have failed. Thus, future preclinical experiments need to evaluate the protocols of fetal immune modulation and the role of fetal hematopoietic space through nontoxic methods of fetal myeloablation.
Fetal immune suppression If the fetal immune system, albeit immature, does influence graft failure, then immune suppression of the fe-
tus should improve the rate of engraftment in immunologically normal fetuses. Fetal immune suppression should be safe during pregnancy because the fetus lives in the sterile intrauterine environment and fetuses with severe inherited immune defects are usually born without detrimental effects. Fetal immune suppression could be accomplished through the use of either corticosteroids and/or antibody-mediated immune suppression (antiCD3 monoclonal antibody (OKT-3), antithymocyte antigen (ATG), or anti-asialo-GM1 (NK cells). Corticosteroid therapy could be given either transplacentally or directly to the fetus, whereas treatment through an antibody mediate approach would require direct fetal dosing. A number of investigators have demonstrated that the use of high-dose corticosteroids results in suppression of hematopoiesis in marrow and fetal liver cells from rodents and humans (66–68). In addition, when fetal and adult hematopoietic cell sources are compared, there is a greater suppressive effect on fetal erythropoiesis (68). Antibody-mediated immune suppression would likely work through mechanisms that are similar to adult cells where antibody-coated lymphocytes are removed through classic complement-mediated cytolysis, clearance through opsonization, and phagocytosis by macrophages and apoptosis (69).
Creating hematopoietic space and enhancing donor cell engraftment Creating hematopoietic space could be mediated through conventional chemotherapeutic/myeloablative agents, the use of corticosteroids (see above), or through novel approaches such as recombinant parvovirus B19 capsid. Although the use of conventional myeloablative agents would test the hypothesis that hematopoietic space is limiting allogeneic engraftment, such agents are unlikely to be used in the clinical setting. Due to the potential limitation of conventional chemotherapy, we have explored the use of recombinant parvovirus capsid as a method of inhibiting fetal hematopoiesis. Fetal infection with human parvovirus B19 is a well-recognized cause of severe fetal anemia. We have recently demonstrated that recombinant parvovirus B19 capsid produces a 60–95% reduction of in vivo hematopoiesis. This effect has been demonstrated using cells obtained from human fetal liver, umbilical cord blood, and bone marrow as well as umbilical cord and bone marrow cells from non-human primates (70,71). More importantly, the inhibitory effect of parvovirus capsid can be blocked by incubating the hematopoietic cells with monoclonal antibodies to the P antigen, the capsid binding site, suggesting that this method of therapy may provide a short-term competitive advantage to donor cells. Other methods that may improve homing and engraftment of allogeneic cells include the use of donor-specific
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SHIELDS ET AL. (MHC-matched) stromal cell co-transplantation or cotransplantation of irradiated donor lymphocytes. In vitro culturing of mouse hematopoietic stem cells with MHCmatched and -mismatched stromal cells has been shown to enhanced hematopoiesis (72). One could argue that this strategy has already been tried in humans in cases of fetal liver transplantation where the donor cell population includes hematopoietic and stromal elements. However, the kinetics of homing and expression of adhesional molecules may be significantly different with co-transplantation of cultured stromal elements than transplantation of fetal liver cells or enriched CD341 cells that contain stromal or stromal progenitor cells. This methodology has been shown to improve engraftment and longterm donor cell expression in fetal sheep (43,73). The efficacy of this method of transplantation will hopefully be tested in other animal models. An alternative method that may potentially improve engraftment and donor-specific tolerance is infusion of irradiated donor lymphocytes (74–76). Although this methodology has not been tested in the fetus, in postnatal mice the infusion of irradiated lymphocytes enhances engraftment and immunologic tolerance, presumably through enhancement of the type-2 T cell cytokine responses (75). Using irradiated donor lymphocytes may be a method that will allow a larger number of donor T cells to be given to the fetus, thus facilitating initial engraftment, without increasing the risk of GVHD.
COMMENT If safe and efficacious in utero stem cell therapy can be achieved, the potential to treat many disorders that now result in either fetal or neonatal death or significant long-term morbidity may be cured (Table 3). At the present time, there does not appear to be sufficient preclinical data to support clinical trials for in utero stem cell therapy, except in the case of severe immunodeficiencies. Hopefully, through continued research, new innovative methods will be developed that will enhance our ability to create stable chimerism through in utero treatment or tolerance induction. Finally, the complexity of fetal stem cell transplantation calls for collaboration between many fields in medicine. A multidisciplinary approach is clearly a prerequisite if progress is going to be made in this field. It is of utmost importance that cases selected for these experimental treatments have acceptable risk/benefit ratios. If the disease to be treated results in significant neonatal morbidity or in utero death, then allowable acceptable risk associated with in utero treatment should be different from a disease that produces purely postnatal morbidity or mortality. Furthermore, because experience will be limited in all centers, it is a prerequi-
site that all cases are reported through both standard journal publications and through the development of Internet-based registries.
ACKNOWLEDGMENT This work was supported by National Institutes of Health grant HL62422-02.
REFERENCES 1. Blazar BR, PA Taylor, R McElmurry, L Tian, A Panoskaltsis-Mortari, S Lam, C Lees, T Waldschmidt, and DA Vallera. (1998). Engraftment of severe combined immune deficient mice receiving allogeneic bone marrow via In utero or postnatal transfer. Blood 92:3949–3959. 2. Davies J. (1967). A case of haemolytic disease with congenital rubella. Br Med J 2:819–822. 3. Touraine JL, C Royo, MG Roncarolo, K Murray and O de Bouteiller. (1989). Unmatched stem cell transplantation as a possible alternative to bone marrow transplantation. Transplant Proc 21:3197–3198. 4. Jolly RD, KG Thompson, CE Murphy, BW Manktelow, AN Bruere and BG Winchester. (1976). Enzyme replacement therapy-an experiment of nature in chimeric mannosidosis calf. Pediatr Res 10:219–224. 5. Owen RD. (1945). Immunogenetic consequences of vascular anastomoses between bovine twins. Science 102: 400–401. 6. Jones MZ, KT Cavanagh, R Kranich, C Traviss, Y Fujita, M Ohta and F Matsuura. (1993). Possible beta-mannosidosis chimera. Altered expression of metabolic perturbations. J Inherit Metab Dis 16:1012–1023. 7. Picus J, WR Aldrich and NL Levin. (1985). A naturally occurring bone-marrow-chimeric primate. I. Integrity of its immune system. Transplantation 39:297–303. 8. Lillie FR. (1916). The theory of the free-martin. Science 43:611–613. 9. Howell JM, PR Dorling, JN Shelton, EG Taylor, DG Palmer and PN Di Marco. (1991). Natural bone marrow transplantation in cattle with Pompe’s disease. Neuromusc Disord 1:449–454. 10. Emery D and P McCullagh. (1980). Immunological reactivity between chimeric cattle twins. I. Homograft reaction. Transplantation 29:4–9. 11. van Dijk BA, DI Boomsma and AJ de Man. (1996). Blood group chimerism in human multiple births is not rare. Am J Med Genet 61:264–268. 12. Race R and R Sanger. (1975). Blood Groups in Man, 6th ed. Blackwell Scientific Publications, Oxford, pp 511–546. 13. Bird GW, M Gibson, J Wingham, P Mackintosh, W Watkins and P Greenwell. (1980). Another example of haemopoietic chimaerism in dizygotic twins. Br J Haematol 46:439–445. 14. Hansen HE, E Niebuhr and C Lomas. (1984). Chimeric twins. T.S. and M.R. reexamined. Hum Hered 34:127–130. 15. Vietor HE, BC Hamel, SP van Bree, EM van der Meer, DF Smeets, BJ Otten, RA Holl, and FH Claas. (2000). Im-
628
FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION
16.
17. 18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
munological tolerance in an HLA non-identical chimeric twin. Hum Immunol 61:190–192. Kuhl-Burmeister R, E Simeoni, K Weber-Matthiesen, A Milde, C Herwartz, J Neppert and M Suttorp. (2000). Equal distribution of congenital blood cell chimerism in dizygotic triplets after in-vitro fertilization. Hum Reprod 15:1200– 1204. Angela E, E Robinson and D North. (1976). A case of twin chimerism. J Med Genet 13:528–530. Thomsen M, HE Hansen and E Dickmeiss. (1977). MLC and CML studies in the family of a pair of HLA haploidentical chimeric twins. Scand J Immunol 6:523–528. Fleischman RA and B Mintz. (1979). Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta. Proc Natl Acad Sci USA 76:5736–5740. Blazar BR, PA Taylor and DA Vallera. (1995). In utero transfer of adult bone marrow cells into recipients with severe combined immunodeficiency disorder yields lymphoid progeny with T- and B-cell functional capabilities. Blood 86:4353–4366. Archer DR, CW Turner, AM Yeager and WH Fleming. (1997). Sustained multilineage engraftment of allogeneic hematopoietic stem cells in NOD/SCID mice after in utero transplantation. Blood 90:3222–3229. Carrier E, TH Lee, MP Busch and MJ Cowan. (1995). Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood 86: 4681–4690. Hajdu K, S Tanigawara, LK McLean, MJ Cowan and MS Golbus. (1996). In utero allogeneic hematopoietic stem cell transplantation to induce tolerance. Fetal Diagn Ther 11:241–248. Howson JK, YH Matloub, DA Vallera and BR Blazar. (1993). In utero engraftment of fully H-2-incompatible versus congenic adult bone marrow transferred into nonanemic or anemic murine fetal recipients. Transplantation 56:709–716. Kim HB, AF Shaaban, R Milner, C Fichter and AW Flake. (1999) In utero bone marrow transplantationinduces donorspecific tolerance by a combination of clonal deletion and clonal anergy. Pediatr Surg 34:726–730. Flake A. (1986). Transplantation of fetal hematopoietic stem cells in utero: the creation of hematopoietic chimeras. Science 223:776–778. Zanjani ED, G Almeida-Porada, JL Ascensao, FR MacKintosh and AW Flake. (1997). Transplantation of hematopoietic stem cells in utero. Stem Cells 15:79–93. Crombleholme TM, MR Harrison and ED Zanjani. (1990). In utero transplantation of hematopoietic stem cells in sheep: the role of T cells in engraftment and graft-versushost disease. J Pediatr Surg 25:885–892. Zanjani ED, A Ruthven, J Ruthven, D Shaft, D Smith and AW Flake. (1994). In utero hematopoietic stem cell transplantation results in donor specific tolerance and facilitates postnatal “boosting” of donor cell levels. Blood 84:100a. Hedrick MH, HE Rice, TE MacGillivray, JF Bealer, ED Zanjani and AW Flake. (1994). Hematopoietic chimerism achieved by in utero hematopoietic stem cell injection does
31.
32.
33.
34.
35.
36. 37.
38.
39.
40. 41.
42.
43.
44.
45.
46.
629
not induce donor-specific tolerance for renal allografts in sheep. Transplantation 58:110–111. Westlake V, R Jolly, B Jones, D Mellor, R Machon, E Zanjani and W Krivit. (1995). Hematopoietic cell transplantation in fetal lambs with ceroid-lipofuscinosis. Am J Med Genet 57:365. Pearce RD, D Kiehm, DT Armstrong, PB Little, JW Callahan, LR Klunder and JT Clarke. (1989). Induction of hematopoietic chimerism in the caprine fetus by intraperitoneal injection of fetal liver cells. Experientia 45:307–308. Opie TM, LE Shields and RG Andrews. (1998). Cell surface antigen expression in early and germ gestation fetal hematopoietic progenitor cells. Stem Cells 16:343–348. Winkler A, H-P Kiem, LE Shields, and RG Andrews. (1999). Gene transfer into fetal baboon hematopoietic progenitor cells. Hum Gene Ther 10:667–677. Shields LE and RG Andrews. (1998). Gestational age changes in circulating CD341 hematopoietic stem/progenitor cells in fetal cord blood. Am J Obstet Gynecol 178:931–937. Harrison M. (1989). In-utero transplantation of fetal liver hematopoietic stem cells in monkeys. Lancet 2:1425–1427. Roodman GD, TJ Kuehl, JL Vandeberg and DY Muirhead. (1991). In utero bone marrow transplantation of fetal baboons with mismatched adult baboon marrow. Blood Cells 17:367–375. Brent L, DC Linch, CH Rodeck, K Nicolaides, L DeGiorgi, RA Sherwood, L al-Imara, J Mew and W Socha. (1989). On the feasibility of inducing tolerance in man: a study in the cynomolgus monkey. Immunol Lett 21:55–61. Cowan MJ, AF Tarantal, J Capper, M Harrison and M. Garovoy. (1996). Long-term engraftment following in utero T cell-depleted parental marrow transplantation into fetal rhesus monkeys. Bone Marrow Transplant 17:1157– 1165. Tavassoli M. (1991). Embryonic and fetal hematopoiesis: an overview. Blood Cells 17:269–281. Thilaganathan B, KH Nicolaides and G Morgan. (1994). Subpopulations of CD34-positive haematopoietic progenitors in fetal blood. Br J Haematol 87:634–636. Muench MO, J Cupp, J Polakoff and MG Roncarolo. (1994). Expression of CD33, CD38, and HLA-DR on CD341 human fetal liver progenitors with a high proliferative potential. Blood 83:3170–3181. Almeida-Porada G, CD Porada, N Tran and ED Zanjani. (2000). Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood 95:3620–3627. Crombleholme TM, JC Langer, MR Harrison and ED Zanjani. (1991). Transplantation of fetal cells. Am J Obstet Gynecol 1164:218–230. Renda MC, E Fecarotta, F Dieli, L Markling, M Westgren, G Damiani, C Jakil, F Picciotto and A Maggio. (2000). Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation. Bone Marrow Transplant 25:135–141. Toivanen P, J Uksila, A Leino, O Lassila, T Hirvonen and O Ruuskanen. (1981). Development of mitogen respond-
SHIELDS ET AL.
47.
48.
49. 50.
51.
52.
53.
54.
55. 56.
57.
58.
59.
60.
61.
62.
ing T cells and natural killer cells in the human fetus. Immunol Rev 57:89–105. Stites DP, MC Carr and HH Fudenberg. (1974). Ontogeny of cellular immunity in the human fetus: development of responses to phytohemagglutinin and to allogeneic cells. Cell Immunol 11:257–271. Thilaganathan B, A Abbas and KH Nicolaides. (1993). Fetal blood natural killer cells in human pregnancy. Fetal Diagn Ther 8:149–153. Lanier LL. (1995). The role of natural killer cells in transplantation. Curr Opin Immunol 7:626–631. Ziegler BL, M Valtieri, GA Porada, R De Maria, R Muller, B Masella, M Gabbianelli, I Casella, E Pelosi, T Bock, ED Zanjani and C Peschle. (1999). KDR receptor: a key marker defining hematopoietic stem cells. Science 285:1553–1558. Flake AW and ED Zanjani. (1997). In utero hematopoietic stem cell transplantation. A status report. J Am Med Assn 278:932–937. Blomberg M, S Rao, J Reilly, C Tiarks, S Peters, E Kittler and P. Quesenberry. (1998). Repetitive bone marrow transplantation in nonmyeloablated recipients. Exp Hematol 26:320–324. Shields L and R Andrews. (1999). In utero engraftment by allogeneic CD341 marrow cells in nonhuman primates: Relationship to the absolute number of cells transplanted. J Soc Gynecol Invest 6:186A. Rao SS, SO Peters, RB Crittenden, FM Stewart, HS Ramshaw and PJ Quesenberry. (1997). Stem cell transplantation in normal nonmyeloablated host: relationship between cell dose, schedule, and engraftment. Exp Hematol 25:114–121. Burk R. (1999). Clinical utility in maximizing CD341 cell count in stem cell grafts. Stem Cells 17:373–376. Shields LE and RG Andrews. (2001). In utero stem cell transplantation in non-human primates: the role of T-cell number. Am J Obstet Gynecol 184:S2. Bambach BJ, HW Moser, K Blakemore, VL Corson, CA Griffin, SJ Noga, EJ Perlman, R Zuckerman, DA Wenger and RJ Jones. (1997). Engraftment following in utero bone marrow transplantation for globoid cell leukodystrophy. Bone Marrow Transplant 19:399–402. Westgren M, S Ek, MY Divon, A Kjaeldnard, L Marling, A Seiger, E Sundstrom, B Sarby and O Ringden. (1995). Distribution of transplanted fetal stem cells in the human fetus. Am J Obstet Gynecol 172:398. Zanjani ED, FR Mackintosh and MR Harrison. (1991). Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells. Blood Cells 17:349–363. Jankowski RA and ST Ildstad. (1997). Chimerism and tolerance: from freemartin cattle and neonatal mice to humans. Hum Immunol 52:155–161. Hayward A, D Ambruso, F Battaglia, T Donlon, K Eddelman, R Giller, J Hobbins, YE Hsia, R Quinones, E Shpall, E Trachtenberg and P Giardina. (1998). Microchimerism and tolerance following intrauterine transplantation and transfusion for alpha-thalassemia-1. Fetal Diagn Ther 13:8–14. Thilaganthan B and K Nicoliades. (1993). Intrauterine bone-marrow transplantation at 12 gestation. Lancet 342: 243.
63. Buckley RH, SE Schiff, RI Schiff, L Markert, L.W. Williams, JL Roberts, LA Myers and FE Ward. (1999). Hematopoietic stem-cell transplantationfor the treatment of severe combined immunodeficiency. N Engl J Med 340:508–516. 64. Cavazzana-Calvo M, S Hacein-Bey, G de Saint Basile, F Gross, E Yvon, P Nusbaum, F Selz, C Hue, S Certain, JL Casanova, P Bousso, FL Deist and A Fischer. (2000). Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288:669–672. 65. Flake AW and ED Zanjani. (1999). Treatment of severe combined immunodeficiency syndrome. N Engl J Med 341: 291–292. 66. Leung P and AS Gidari. (1985). Effect of aminoglutethimide on murine fetal hepatic erythroid colony formation. Experientia 41:498–500. 67. Singer JW, AI Samuels and JW Adamson. (1976). Steroids and hematopoiesis. I. The effect of steroids on in vitro erythroid colony growth: structure/activity relationships. Cell Physiol 88:127–134. 68. Roodman GD, J Lee and AS Gidari. (1983). Effects of dexamethasone on erythroid colony and burst formation from human fetal liver and adult marrow. Br J Haematol 53: 621–628. 69. Bonnefoy-Berard N and JP Revillard. (1996). Heart Lung Transplant 15:435–442. 70. Shields LE, RG Andrews, M Westgren and K Brolinden. (2000). In vitro hematopoiesis is inhibited in humans and non-human primates by recombinant parvo virus capsid. Am J Obstet Gynecol 182:S18. 71. Lindton B, T Tolfvenstam, O Norbeck, L Markling, O Ringden, M Westgren and K Broliden. (2001). Recombinant parvovirus B19 empty capsids inhibit fetal hematopoietic colony formation in vitro. Fetal Diagn Ther 16:26–31. 72. Sugiura K, H Hisha, J Ishikawa, Y Adachi, S Taketani, S Lee, T Nagahama and S Ikehara. (2001). Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vitro. Stem Cells 19:46–58. 73. Almeida-Porada G, AW Flake, HA Glimp and ED Zanjani. (1999). Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 27:1569–1575. 74. Bittencourt MC, S Perruche, E Contassot, S Fresnay, MH Baron, R Angonin, F Aubin, P Herve, P Tiberghien and P Saas. (2001). Intravenous injection of apoptotic leukocytes enhances bone marrow engraftment across major histocompatibility barriers. Blood 98:224–230. 75. Kao KJ, ES Huang and S Donahue. (2001). Characterization of immunologic tolerance induced by transfusion of UV-B-irradiated allogeneic mononuclear leukocytes. Blood 98:1239–1245. 76. Walker EK, AM Ship, S Mittelstaedt, TW Murray, R Carter, I Kakhniashvili, S Lonial, JT Holden and MW Boyer. (1999). Irradiated donor leukocytes promote engraftment of allogeneic bone marrow in major histocompatibility complex mismatched recipients without causing graft-versus-host disease. Blood 94:3222–3233. 77. Westgren M, O Ringden, S Eik Nes, S Ek, M Anvret, AM Brubakk, TH Bui, A Giambona, T Kiserud, A Kjaeldgaard, A Maggio, L Marking, A Seiger and F Orlandi. (1996).
630
FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION
78. 79.
80.
81.
82.
83.
84. 85.
86.
87.
88.
Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies. Transplantation 61:1176–1179. Diukman R and M Golbus. (1992). In utero stem cell therapy. J Reprod Med 37:515–520. Slavin S, E Naparstek, M Ziegler and A Lewin. (1992). Clinical application of intrauterine bone marrow transplantation for treatment of genetic diseases-feasibility studies. Bone Marrow Transplant 9:189–190. Touraine JL, D Raudrant, C Royo, A Rebaud, F Barbier, MG Roncarolo, F Touraine, S Laplace, L Beguhrer, H B’etuel et al. (1991). In utero transplantation of hematopoietic stem cells in humans. Transplant Proc 23:1706–1708. Cowan MJ and M Golbus. (1994). In utero hematopoietic stem cell transplants for inherited diseases. Am J Pediatr Hematol Oncol 16:35–42. Orlandi F, A Giambona, F Messana, M Marino, I Abate, R Calzolari, F Damiani, C Jakil, M Renda, F Dieli, F Buscemi, M Westgren, O Ringden and A Maggio. (1996). Evidence of induced non-tolerance in HLA-identical twins with hemoglobinopathy after in utero fetal transplantation. Bone Marrow Transplant 18:637–639. Peschle C. (1997). In utero transplantation of purified stem cells from an HLA identical sibling into a bea-thalassemia embryo. In: Second International Meeting for In Utero Stem Cell Transplantation and Gene Therapy, Nottingham, England. Monni G, RM Ibba, MA Zoppi and M. Floris. (1998). In utero stem cell transplantation. Croat Med J 39:220–223. Linch D, C Rodeck, K Nicolaides, H Jones and L Brent. (1986). Attempted bone-marrow transplantation in a 17week fetus. Lancet 27:1453. Wengler GS, A Lanfranchi, T Frusca, R Verardi, A Neva, D Brugnoni, S Giliani, M Fiorini, P Mella, F Guandalini, E Mazzoloari, S Pecorelli, LD Notarangelo, F Porta and AG Ugazio. (1996). In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with Xlinked severe combined immunodeficiency (SCIDXI). Lancet 348:1484–1487. Flake AW, MG Roncarolo, JM Puck, G Almeida Porada, MI Evans, MP Johnson, EM Abella, DD Harrison and ED Zanjani. (1996). Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow [see comments]. N Engl J Med 335: 1806–1810. Lanfranchi A, A Neva, K Tettoni, R Veradi, E Mazzollari
89.
90.
91.
92.
93.
94.
95.
and G Wengler. (1998). In utero transplantation (IUT) of parental CD341 cells in patient affected by primary immunodeficiencies. Bone Marrow Transplant 21:S127. Westgren M, O Ringden, P Bartmann, T Bui, B Lindton, J Mattsson, M Uzunel and H Zetterquist. (2002). Prenatal T cell reconstruction after in utero transplantation with fetal liver cells in a transplantation with x-linked SCID. Am J Obstet Gynecol, in press. Touraine JL, D Raudrant and S Laplace. (1997). Transplantation of hematopoietic cells from the fetal liver to treat patients with congenital diseases postnatally or prenatally. Transplant Proc 29:712–713. Muench MO, J Rae, A Barcena, T Leemhuis, J Farrell, L Humeau, JR Maxwell-Wiggins, J Capper, GB Mychaliska, CT Albanese, T Martin, A Tsukamoto, JT Curnutte and MR Harrison. (2001). Transplantation of a fetus with paternal Thy-1(1)CD34(1)cells for chronic granulomatous disease. Bone Marrow Transplant 27:355–364. Flake AW and ED Zanjani. (1999). In utero hematopoietic stem cell transplantation: Ontogenic opportunities and biologic barriers. Blood 94:2179–2191. Porta F, E Mazzolari, S Zucca, F Schumacher, A Lanfranchi, K Tettoni and LD Notarangelo. (2000). Prenatal transplant in a fetus affected by Omenn syndrome. Bone Marrow Transplant 25(S):S43. Leung W, K Blakemore, RJ Jones, HW Moser, G Mukherjee, CA Griffin, LS Rosenblum-Vos and CI Civin. (1999). A human-murine chimera model for in utero human hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 5:1–7A. Hosoi T, S Yahara, K Kunitomo, H Saji and Y Ohtsuki. (1977). Blood chimeric twins: an example of blood cell chimerism. Vox Sang 32:339–341.
Address reprint requests to: Dr. Laurence E. Shields 1959 NE Pacific St. Box 356460 Department of Obstetrics and Gynecology University of Washington Seattle, WA 98195-6460 E-mail:
[email protected] Received January 7, 2002; accepted March 27, 2002.
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