In utero transplantation (IUT) has the potential to cure or ameliorate many disorders before birth.
Animal models for studying IUT have fundamental differences from human models in regard to immunologic ontogeny and placentation.
Naturally occurring events during pregnancy result in chimerism in large animals and in humans and support the concept of IUT.
Using mesenchymal stem cells for IUT may be possible in disorders with a functioning immune system.
Development of advanced medicinal therapy products for use in IUT is complicated and requires large resources and knowledge but holds great promise for the future.
The first successful in utero transplantation (IUT) was reported by Touraine et al . in 1989. The report was followed by a limited number of IUTs in human fetuses with severe combined immune deficiency (SCID). Most of these reports claim partial engraftment of the transplants. Several researchers tried a similar approach with haematopoietic stem cell (HSC) transplantation in utero for nonimmunologic disorders; unequivocally, these cases failed. HSC is the stem cell that give rise to all blood cells, both of the myeloid and lymphoid lineages. The arguments for IUT, as summarised in Table 45.1 , are well known and have repeatedly been broadcasted in different reviews on this topic for almost 3 decades. However, we are still lacking knowledge on why IUT with HSC in fetuses with normal immunologic function do not engraft, and the evidence supporting the arguments are, to put it cautiously, not very strong.
|Arguments for IUT with stem cells||Evidence HSC||Evidence MSC|
|Right-to-left heart shunting that enhances systemic distribution of the cells|
|Small size of the fetus allows big dose|
|Treat before severe consequences of the disease|
|In fetal life, large-scale migration of stem cells takes place|
|Allows transplantation across HLA barriers|
|Donor-specific tolerance induction|
|Niches for exogenous stem cells|
|Less expensive than postnatal transplantation|
A number of reviews on this topic have been published, with extensive examination of past literature on animal work and previous human experience. Recently two excellent reviews on IUT with HSC were published, and we will therefore in the present review focus mainly on IUT with mesenchymal stem cells (MSCs). MSCs are the stem cells that give rise to cell types of the mesodermal lineage, such as osteoblasts, chondrocytes, myocytes and adipocytes. Before embarking on such undertaking, we will briefly comment on the most current experience of IUT in animal models and whether we can learn more from nature regarding engraftment of foreign stem cells in human fetuses.
What Can We Learn From Animal Models?
General problems with animal models for IUT are the fundamental differences among different species models and differences between animals and humans with regard to immunologic ontogeny and placentation. These are crucial questions that need to be kept in mind in the interpretation of the current literature.
Achieving engraftment in the mouse model is complicated without a great advantage for donor cells. In 1953, Billingham and colleagues published a pioneer report on donor specific tolerance to skin grafts after IUT in mice. Three decades later, Fleischman and colleagues reported on successful haematopoietic chimerism after IUT that could ameliorate a genetic disorder in a mouse model. These studies were directed to study stem cell biology rather than exploring the therapeutic possibility of IUT with stem cells. Several researchers embarked on similar studies, but when using immunocompetent recipients, the results were poor with regard to donor chimerism and not until immunodeficient mice were studied was significant chimerism obtained. Recently, Flake and associates have used the mouse model to extensively explore the fetal mouse immune system. Their studies indicate clonal deletion, anergy (absence of a normal immune response) and immune tolerance after IUT. It seems that a low level of chimerism is associated with postnatal tolerance across full major histocompatibility complex (MHC) barriers. Furthermore, recent studies on IUT in mice have revealed evidence of maternal alloimmunisation and that maternal–fetal T-cell trafficking (bidirectional transfer of cells between the mother and the fetus) could be an explanation for loss of chimerism after birth. Arguments against this hypothesis have been raised by Alhajjat and colleagues, who have pointed out that in the context of the human experience of IUT, there has been no evidence of immunisation. Although this might be true, we are not aware of any studies on maternal immunology in conjunction with these IUTs. Shaaban’s research group has focused on the significance of the natural killer (NK) cells of the innate immune system and has identified a subset of cells within the fetal liver that may pose a barrier for early engraftment. Interestingly these naïve NK cells were recently also found in early human tissues and in amniotic fluid. Kim and colleagues have recently explored other means to enhance engraftment by mobilising fetal HSCs by inhibition of the integrin α4β1/7.
The most commonly used model for studies of IUT is the fetal lamb model. After allogenic IUT with fetal liver–derived HSCs, significant levels of chimerism have been obtained. Even transplantations of xenogenic cells from humans have been successful. A concern with these studies, that to a large extent has formed the base for the arguments for IUT, is that several research groups have tried to repeat these studies but have unequivocally failed. Thus caution is important when evaluating these data. Other animal models used for studies of IUT are pigs, goats, canines and nonhuman primates. Studies on nonhuman primates are, of course, of special value, but it has been notoriously difficult to attain engraftment.
Therefore, although different animal models provide support for IUT with HSCs, there are limitations in most models with regard to species specific differences, and results are often difficult to comprehensively evaluate. The main question of how we can extrapolate from these experiments into the human situation remains.
Can We Learn From Nature?
Dizygotic twin cows, goats and marmosets all provide evidence that naturally occurring chimerism of haematopoietic cells early during pregnancy occurs through placental blood vessel anastomosis between the two fetal circulations. It is important to note that transmission through placental anastomosing vessels is very different from IUT. With placental anastomosis, the mixing of fetal blood most probably occurs very early in pregnancy, and the mixing of fetal blood is likely to be continuous. Dizygotic twin cattle demonstrate inter twin tolerance when measured using mixed lymphocyte cultures (MLC), accept renal grafts from their co-twin and have delayed rejection to skin grafts transplants. Similar evidence of intertwin tolerance has been demonstrated in dizygotic twin marmosets using cytotoxic T-lymphocyte (CTL) assays. Marmosets typically show high degrees of haematopoietic chimerism (28%–82% by peripheral blood karyotype).
Although the natural chimeras noted in dizygotic large animals are highly suggestive that IUT should be successful, the most encouraging data comes from dizygotic human twins. Although the frequency of chimerism in dizygotic twins in these animals at birth is relatively high, the level of chimerism in humans is quite low. These data are consistent with placental architecture studies showing that intertwin placental anastomosis does not occur between most normal dizygotic human twins. However, more than 30 cases of opposite-sex dizygotic twins have been reported and show much higher levels of haematopoietic chimerism. In these cases, early placental fusion and anastomosis presumably occurred. The level of chimerism in these cases is generally high, and in one well-documented case, chimerism has persisted for more than 25 years. Immune tolerance (as assessed by MLC and skin grafting) has also been demonstrated. These naturally occurring ‘experiments’ in large animals, including primates as well as humans, support the concept of IUT.
Another naturally occurring model is the passage of maternal cells across the placenta to the fetal recipient. These cells can engraft, and we and others have shown that maternal cells of lymphoid and myeloid lineages as well as haematopoietic progenitors are widely distributed in human second trimester fetuses. Furthermore, after birth, some cells remain and are found in the child’s lymphoid tissue. Later in life, maternal chimerism is very rare, so it might be that there is some low-grade immunologic mechanism that is responsible for the loss of chimerism.
To summarise, induction of tolerance and engraftment of HSCs seems to be possible but is complicated to achieve in immunocompetent animal models, and it is still an open question how this will be accomplished in human fetal recipients. Several hurdles seem to be in play, such as the fetal immune system, the maternal immune system and host cell competition.
In Utero Transplantation
Some disorders result in irreversible damage or even perinatal lethality, and treatment should or must be initiated before birth. Therefore it is desirable to introduce treatment as early as possible before additional pathology occurs and at a time of rapid development. Additional reasons in favour for IUT include:
The right-to-left heart shunting that enhances systemic distribution of the cells instead of the cells being trapped in the lungs as in childhood and adult life. Two clinical studies show that after postnatal intravenous infusion of MSCs, the cells accumulated in the lungs within 20 minutes, and after 48 hours, the cells were distributed to other organs such as the liver and spleen. In several animal studies, MSCs were found in the lungs within seconds after intravenous infusion with redistribution to the liver, spleen, kidney, heart and bone marrow after 24 to 48 hours. In contrast, after intraperitoneal IUT in mouse models of osteogenesis imperfecta (OI) and muscular dystrophy, the transplanted human fetal MSCs were identified at all time points in all tissues examined, except in the lungs at birth.
The rapid growth of the fetus providing an opportunity for engraftment and expansion and subsequent migration and distribution of the donor cells to different anatomical compartments. During fetal life, naturally occurring stem cells expand and migrate to seed and populate anatomical compartments. One example is the parallel migration of HSCs and MSCs from the aorta-gonad-mesonephros and the yolk sac to the liver and last to the bone marrow. These compartments provide a potent and specialised supportive environment for proliferation and differentiation of fetal cells, especially when a fetal-to-fetal transplantation approach is applied.
The relatively naïve fetal immune system that may permit the development of immune tolerance towards donor cells. The immunologic naivety in the early gestational fetus has given rise to the concept of fetal tolerance (i.e., the inability to raise an immunologic response against foreign antigens). During fetal life, the developing immune system is educated to distinguish between autologous and foreign antigens, and if introduced early enough, foreign antigens can be recognised as ‘self’ and not be rejected.
The far better psychosocial situation for the mother and father resulting from the birth of a child who has already been treated. No comparative studies are available, but from our extensive experience in regard to fetal therapy, we are convinced that the parents prefer an active approach including a therapeutic strategy rather than being subjected to an observational wait-and-see attitude. These parents have often refrained from termination and are ready to do as much as possible for their unborn child. Thus they look at their fetus as a potential unborn patient who should receive optimal care and management.
The possible advantages of IUT are summarised in Table 45.1 .
Pre- and Postnatal Transplantation With Fetal Mesenchymal Stem Cells for Treatment of Osteogenesis Imperfecta
Our research group has during the past decade been involved in projects aiming to treat OI before and after birth. OI, often called brittle bone disease, is a heterogeneous genetically inherited disease with a prevalence at birth estimated to 1 in 10,000 to 20, 000. OI is caused by more than 1400 different dominant and more than 150 recessive mutations. The most common cause (>90%) is a dominant mutation in one of the two genes encoding collagen type I ( COL1A1 and COL1A2 ), which shows an autosomal dominant pattern of inheritance. Four types of OI, types I to IV, were originally described in 1979 by Sillence and colleagues based on clinical, radiologic and hereditary findings. OI is heterogeneous and range from the mild type I that may only become evident in adulthood to the perinatally lethal type II A/C. Type III OI is the most severe form that is compatible with survival into adulthood. Recently, the classification was expanded with OI types V to VIII because of distinct clinical features or different causative gene mutations, but these types are commonly not used in the clinical diagnosis. The pathology of OI develops in fetal life, and sonographic signs of severe forms of OI can be detected in the first trimester, for example, with increased nuchal translucency and bony malformations. Later during gestation, fetuses affected by OI type III often exhibit a typical phenotype with sonographic signs of multiple fractures at various stages of healing; very short, deformed long bones with bowing and angulation; and sometimes reduced echogenicity. Conclusive prenatal diagnosis of OI usually requires an invasive test to obtain fetal material for molecular analysis. The major clinical manifestations of OI are atypical skeletal development, osteopenia, multiple painful fractures and short stature, but individuals with OI also have brittle teeth, hearing loss and hypermobile joints and have a higher risk for heart valve insufficiency, aneurysms and bleeding as a result of coagulation deficiencies throughout their lifetimes. Life expectancy is not affected in milder OI types but may be shortened for those with more severe types. Individuals affected by OI type III may experience hundreds of fractures in a lifetime,
Finding suitable cell sources is one of the main challenges in regenerative medicine. In addition to improving the dysfunctional tissue requiring reconstruction, low immunogenicity is beneficial. MSCs are multipotent stromal cells, originally identified in adult bone marrow, displaying colony-forming capacity and are nonhaematopoietic and nonendothelial. MSCs can be easily isolated and expanded and differentiate along the osteogenic, chondrogenic, myogenic and adipogenic lineages. MSCs do not express HLA class II antigens, are generally considered to be nonimmunogenic, do not elicit an immune response and inhibit proliferating allogeneic lymphocytes in vitro . MSCs, when grown under normal culture conditions, are commonly considered as safe to transplant, and there are no reports of ectopic tissue formation or malignant transformation. Because of these characteristics, they have been tested in clinical trials for a diverse variety of disorders, ranging from the treatment of inflammatory, autoimmune and cardiovascular diseases and in orthopaedic applications. The first MSC transplantation was performed more than 15 years ago, and adult MSCs have been used in a diverse range of conditions in thousands of individuals without adverse reactions.
Our research group at Karolinska Institutet were one of the first in the world to isolate and characterise MSCs from human fetal tissues. These primitive MSC types are found at a higher frequency, with greater colony-forming capacity, have longer telomeres (cells are more long lived if the telomeres are not shortened at each cell division) and a superior proliferative potential compared with MSC from adult sources. Furthermore, fetal MSCs differentiate more readily into bone, muscle and oligodendrocytes compared with adult MSCs. Like their adult counterparts, fetal MSC are also nonimmunogenic.
The potential of MSC transplantation for treatment of OI was first demonstrated in mouse models of OI disease. Postnatal transplantation of allogeneic adult mouse MSCs resulted in donor cell homing to the bones, where they contributed to the osteoprogenitor population, with improvement in collagen content and bone mineralisation. In a later study, Panaroni and colleagues demonstrated that IUT of allogeneic mouse adult bone marrow transplantation in the perinatally lethal dominant BrtIIV model of OI rescued the neonatal mouse from perinatal death and improved the mechanical properties of bones. Donor cells engrafted in haematopoietic and nonhaematopoietic tissues and differentiated to bone cells. The donor cells synthesised up to 20% of the total collagen type I content of bone, despite an engraftment of only 2%. IUT of human fetal MSC in the oim mouse, a naturally occurring recessive mouse model approximating to OI type III with progressive deformities and skeletal fractures showed that intraperitoneal injection of human fetal MSC resulted in engraftment in the bones (∼5%). Furthermore, there were a 67% reduction in long bone fractures and increased bone strength, thickness and length, and normal human type I collagen could be measured.
Postnatal stem cell transplantation has been attempted in a few children with severe OI. The first clinical proof-of-principle came from Horwitz and colleagues more than a decade ago when five children with type III OI underwent transplantation with matched adult whole bone marrow. Linear growth increased and fracture frequency reduced despite low level (<2%) donor osteoblast engraftment. Subsequent infusions of adult MSCs isolated from the bone marrow of donors to the recipients showed similar results as the bone marrow transplantation with donor cell engraftment in the bones and an acceleration of the growth velocity. Data from mouse studies suggest that significant amounts of normal collagen can be deposited by a relatively small population of donor cells, explaining the marked improvements despite low-level engraftment. Engraftment is likely to be higher after IUT for the reasons discussed earlier and may therefore provide improved phenotypic disease correction.
Encouraged by the data from preclinical and clinical studies, we performed in 2002 the first IUT of human fetal liver–derived MSCs in an immunocompetent fetus with OI type III ( COL1A2 c.3008G>A; p.Gly1003Asp; Gly913Asp in the triple helical domain) using cells developed by us at Karolinska Institutet with a successful outcome. IUT of 6.5 × 10 6 /kg fetal MSCs into the umbilical vein under ultrasound guidance was performed in gestational week 31. The transplantation showed promising clinical results with long-term donor cell engraftment and site-specific differentiation of completely HLA-mismatched MSC in the bone. Until 8 years of age, the patient had only had one fracture and one compression fracture per year. Remarkably, she continued to grow and follow her own height and weight curve at −5 standard deviations (SDs), until from age 6 to 8 years when it deteriorated to −6.5 SDs. As a result and because of an increased fracture rate, the patient was retransplanted with the same-donor MSC intravenously. Over the following 2 years, she did not acquire any fractures, and her linear growth and mobility improved.
We have followed this patient for more than 15 years, and she further received yearly infusions for 3 years with same-donor cells in an attempt to improve her height (during years 2013–2015 at 11, 12 and 13 years of age, respectively). After each infusion, a clinical effect was noted (reduced frequency of fractures, increased growth and better quality of life according to the parents). A child with an identical mutation was identified in a worldwide OI registry. This child did not receive MSC transplantation before or after birth and exhibited a very severe phenotype of OI and died as a result of the disease at 5 months of age. This is the first suggestion that IUT with fetal MSCs can improve severe OI in humans, although we acknowledge that the two patients had overall different genetic backgrounds despite identical OI-causing mutation.
Through international collaborations, we have in 2009 and 2014 performed two additional IUT with fetal liver MSCs for treatment of OI. In the first case, we transported clinical grade human fetal liver MSCs to National University Hospital of Singapore, where local fetal medicine experts performed an ultrasound-guided intrahepatic umbilical vein infusion at 31 weeks’ gestation in a fetus with a confirmed OI type IV mutation ( COL1A2 c.659G>A; p.Gly220Asp; Gly130Asp in the triple helical domain). The patient has received one retransplantation with same-donor cells at 1.5 years of age with a good effect and is doing better than expected with boosted height and reduced frequency of fractures. In the most recent case, another international patient with a molecularly confirmed prenatal diagnosis of OI type III ( COL1A1 c.3469G>C p.Gly1157Arg) was transplanted at Karolinska University Hospital in gestational week 28. The baby was born in October 2014 and is doing better than expected.
The clinical effect after MSC transplantation in patients with OI is not permanent and probably regular yearly or biannual infusions are needed. This has previously been noted in the clinical trial on OI using HLA-matched MSCs postnatally. Even though a single IUT may not be clinically sufficient for permanent phenotype correction, an IUT approach is still justifiable because the immunologic naïveté of the fetus may allow for the development of immune tolerance towards the donor cells, rendering postnatal transplantations more efficient. An early treatment of this severe disorder is also beneficial, both for the child and for the parents.
Building on the work carried out at Karolinska Institutet for the past 15 years, we are now preparing an international multicentre EU Horizon 2020–funded clinical trial on pre- and postnatal transplantation of fetal MSCs for ameliorating severe types of OI (BOOSTB4.EU, grant agreement 681045). In Table 45.2 , an example of implications of a successful MSC therapy for OI patients and health care systems is illustrated.