Extensive progress has been made in the field of fetal interventions for congenital disorders. Since its first description in 1982 (Harrison et al., 1982), fetal surgeries have successfully treated various anatomical anomalies; however, they are limited to the correction of structural anomalies. For some congenital hematopoietic disorders, postnatal hematopoietic stem cell transplantation (HSCT) remains the only therapy; however, it is often restricted by limited histocompatible donors and severe treatment-associated morbidity and mortality. In recent years, remarkable advances in prenatal screening and molecular diagnostics have improved the possibility of diagnosing congenital disorders early during gestation. (Flake and Zanjani, 1999a). In utero hematopoietic cell transplantation is a non-myeloablative approach that can avoid the concerns of postnatal treatment and potentially cure various congenital disorders (Surbek et al., 2001). Compared to postnatal therapy, IUHCT allowing physicians to intervene in the treatment of diseases before birth. Furthermore, certain biological advantages unique to the fetal environment provide compelling reasons for favoring prenatal therapy over postnatal therapy (Vrecenak and Flake, 2013). However, the clinical success of IUHCT has been limited to severe combined immunodeficiency (SCID) over the last few decades (Vrecenak and Flake, 2013). Herein, we review the rationale, current progress, and potential clinical applications of IUHCT. We also discuss the barriers to engraftment and potential strategies to overcome them.

The IUHCT notion is supported by the first report of a natural experiment studied by Owen in 1945, when he found that dizygotic cattle twins with shared placental circulation were chimeric postnatally (Owen, 1945). In 1953, Billingham showed that introduction of foreign antigen into early gestation fetuses resulted in the development of immunologic tolerance towards the foreign antigen (Billingham et al., 1953). The most compelling rationale for IUHCT is the induction of fetal specific immunologic tolerance to donor cells due to the naive immune system of the fetus. During the early gestation period, the fetal immune system undergoes a self-education process which occurs primarily in the fetal thymus. The outer region of thymus (cortex) supports the positive selection of T lineage progenitors whose TCRs react to self-major histocompatibility complex (MHC) molecules in a proper strength. The survival cells then travel to the inner thymus (medulla), where negative selection induces apoptosis of the progenitors that possess potential to response with self-antigens, including self-MHC, presented on thymic antigen presenting cells (APCs). The process results in deletion of self-reactive T cells and in a state of self-tolerance (central tolerance) (Palmer, 2003; Takahama, 2006). However, the thymic deletion is incomplete, self-reactive T cells that escape the deletion are suppressed by regulatory T cells (Tregs) mediated peripheral tolerance (Vrecenak and Flake, 2013). Central and peripheral tolerance leaves the fetus to recognize self-antigens and eliminate foreign antigens (Tai-MacArthur et al., 2021). Theoretically, introduction of allogenic cells by IUHCT ahead of the formation of functional fetal adaptive immune system could result in deletion of alloreactive T cells and induction of Treg cells, resulting in complete donor-specific immune tolerance (Vrecenak and Flake, 2013).

Another rationale for IUHCT is the potential hematopoietic niches available for donor cell homing and engraftment during the large-scale migration of hematopoietic stem cells (HSCs) during fetal development (Vrecenak and Flake, 2013). Hematopoiesis emerges from the yolk sac and aorto-gonadal-mesonephros regions. HSCs then circulate to the fetal liver, where HSCs undergo a dramatic expansion. Finally, HSCs colonize the bone marrow (BM), where hematopoiesis takes place throughout the adult life (Dzierzak and Speck, 2008; Bertrand et al., 2010; Gao et al., 2018). Moreover, the fetal environment also supports the expansion and differentiation of donor stem cells (Sagar et al., 2019). Manipulation of regular migration to selectively favor donor HSCs may help overcome competition from the host hematopoietic compartment and improve donor engraftment after IUHCT (Almeida-Porada et al., 2016). In addition, before and during the second trimester of gestation, the fetal BM is relatively empty, allowing donor cell engraftment without requiring myelosuppression, compared to postnatal HSCT (Surbek et al., 2001).

The third advantage of IUHCT is the small fetus size. At 12–13 weeks of gestation, when IUHCT is ideally performed, the human fetus weighs less than 50 g. Therefore, it is possible to deliver a much larger donor cell dosage on a fetal weight basis than could be provided postnatally.

IUHCT has been performed in many different animal models, of which murine models are most extensively used (Table 1). In the late 1970s, Fleischman and Mintz reported the first study of IUHCT (Fleischman and Mintz, 1979), in which an intraplacental injection of donor BM cells was administered at gestational day 11 (E11) into fetal mice with genetic anemia based on c-kit deficiency. The results showed complete substitution with donor erythroid cells in homozygous anemic mice. Later, studies conducted by Mintz and Blazar reported that even a single normal donor HSC was sufficient to engraft and reconstruct normal hematopoiesis in a c-kit-deficient mouse model (Mintz et al., 1984; Blazar et al., 1995a). Blazer also demonstrated only lymphoid reconstitution (split chimerism) in a severe combined immunodeficiency (SCID) mouse model in which donor lymphoid cells showed proliferative and survival advantages (Blazar et al., 1995b). In normal mice without any immune or stem cell deficiency, competitive pressure from the host prevented donor cell engraftment and the level of chimerism remained very low (Carrier et al., 1995; Kim et al., 1998; Donahue et al., 2001). These studies highlight the importance of host cell competition and the engraftment advantage achieved by immune deficiency. The low level of chimerism in normal mice resulted in donor-specific immune tolerance, which might form the basis of postnatal cell transplantation (Carrier et al., 1995; Kim et al., 1999). Further studies have shown that intravenous injection allows for the delivery of much higher cell numbers, resulting in increased chimerism (Peranteau et al., 2007; Boelig et al., 2016).

Murine models are also used to study the mechanisms of donor-specific tolerance induction by IUHCT and various strategies to postnatally improve donor cell engraftment. Previous studies have shown that clonal deletion, anergy, and induction of donor-specific Tregs are essential for the induction of immune tolerance (Kim et al., 1999; Hayashi et al., 2004; Merianos et al., 2009; Nijagal et al., 2011a; Nijagal et al., 2011b). Further studies that administered low-dose total body irradiation (TBI) or busulfan to chimeric recipients followed by T-cell-depleted bone marrow transplantation (BMT) resulted in complete donor cell chimerism without the graft-versus-host disease (GVHD) (Peranteau et al., 2002; Ashizuka et al., 2006). Moreover, another strategy of using postnatal donor-specific lymphocyte infusion combined with IUHCT without BMT also resulted in complete donor cell chimerism without GVHD (Hayashi et al., 2002). These results highlight the crucial roles of host immune barrier and resident hematopoietic stem cell competition in compromising IUHCT efficacy, and implicate potential strategies to promote postnatal chimerism after successful induction of donor specific tolerance by IUHCT in clinical applications.

In addition to murine models, large animal models are also valuable and necessary preclinical tools for IUHCT study. The sheep model was the first large animal model to demonstrate sustained allogeneic engraftment after in utero transplantation of fetal stem cells (Flake et al., 1986) (Table 2). Moreover, ex vivo incubation of donor cells with growth factors enhanced engraftment of allogeneic stem cells (Zanjani et al., 1992a). Increasing the proportion of donor T cells also resulted in increased level of allogeneic chimerism, and approximately 1% donor T cells allowed significant engraftment without GVHD (Crombleholme et al., 1990). In sheep model, donor specific immune tolerance could also be induced by IUHCT, and postnatal injection of cells from the same donor enhanced the engraftment of donor cells (Zanjani, 1994). However, though 3%–5% stable chimerism was achieved, tolerance to renal transplantation was not observed in chimeric sheep (Hedrick et al., 1994). The sheep model also demonstrated persistent xenogeneic engraftment after transplantation of human HSCs (Zanjani et al., 1992b; Srour et al., 1992; Srour et al., 1993; Zanjani et al., 1994; Liechty et al., 2000; Noia et al., 2003; Noia et al., 2008; Tanaka et al., 2010; Goodrich et al., 2014; Jeanblanc et al., 2014). In addition, intracelomic transplantation of human CD34⁺ cells into fetal sheep resulted in more significant engraftment than that described in peritoneal transplantation (Noia et al., 2003). However, unlike the murine model results, the efficacies of intravenous and intraperitoneal transplantation were not significantly different in the sheep model (Tanaka et al., 2010). Moreover, cotransplation of stromal cell with HSC resulted in an increased level of chimerism in both xenogeneic and allogeneic sheep models (Almeida-Porada et al., 1999; Almeida-Porada et al., 2000).

The canine model is an attractive large-animal model. The dog model demonstrated microchimerism after in utero transplantation of paternal dog BM or human HSCs (Omori et al., 1999; Blakemore et al., 2004) (Table 3). Moreover, IUHCT combined with postnatal same-donor boosting strategy using a low-dose busulfan conditioning regimen increased the level of chimerism from 1% to 35–45%, and cured leukocyte adhesion deficiency in a canine model (Peranteau et al., 2009). Vrecenak et al. (2014) demonstrated that intracardiac injection resulted in much higher levels of chimerism than intraperitoneal injection in normal canine models, without any conditioning or evidence of GVHD. Studies also showed that high doses of donor T cells with CD34⁺ resulted in microchimerism without GVHD (Petersen et al., 2007). More recently, Vrecenak et al. (2018) reported that a clear threshold of 1%–3% donor T cells allowed excellent engraftment without GVHD.

Stable allogeneic and xenogeneic multilineage engraftment was also achieved in swine models (Lee et al., 2005b; Abellaneda et al., 2012; Fisher et al., 2013) (Table 4). IUHCT-induced chimerism in fetal swine resulted in donor-specific tolerance to renal and vascularized composite allograft transplantation without conditioning (Lee et al., 2005a; Mathes et al., 2005; Mathes et al., 2014). These studies support the possibility of using this strategy to cure fetuses that require postnatal organ transplantation.

Finally, low levels of allogeneic chimerism were also achieved in non-human primates (Michejda et al., 1992; Asano et al., 2003; Shields et al., 2003; Mahieu-Caputo et al., 2004; Shields et al., 2004; Shields et al., 2005) (Table 5). Fetal immune suppression resulted in an increased level of chimerism, but the level remained low (Shields et al., 2004). Moreover, the level of long-term chimerism was not improved with postnatal donor cell infusion (Shields et al., 2004). The non-human primate model also allowed multilineage engraftment of human HSCs (Tarantal et al., 2000). In addition to these large animal models, chimerism was also observed in rats (Chen et al., 2009a; Li et al., 2012; Lazow et al., 2021), rabbits (Wengler et al., 2005; Martínez-González et al., 2012; Moreno et al., 2012), and cats (Abkowitz et al., 2009). These studies in mice and large-animal models established the foundation for clinical application of IUHCT.

Despite the compelling rationale of IUHCT, poor engraftment in most animal models suggests the existence of significant barriers to successful engraftment after IUHCT (Figure 1).

During the last few decades, efforts have been made to overcome the barriers to successful IUHCT in normal recipients and improve the engraftment of donor cells without GVHD. These strategies fall into three categories: 1) providing competitive advantage for donor cells, 2) increasing receptive niches for donor cells, 3) overcoming immune barriers (Figure 2).

Since the first clinical application of IUHCT in a bare lymphocyte syndrome fetus (Touraine et al., 1989), there have been approximately 50 reported cases of IUHCT during the past 30 years, targeting various diseases with different donor cell sources and transplantation protocols. Unfortunately, the success of this process has been limited mainly to fetuses with SCID (Flake et al., 1996; Wengler et al., 1996; Westgren et al., 2002). However, in successful IUHCT cases, the patients manifested engraftment only in the T-cell lineage (split chimerism), similar to postnatal results in HSCT treatment (Buckley et al., 1999). Thus far, there is little evidence demonstrating the advantages of prenatal treatment over postnatal treatment in patients with X-linked SCID (Flake and Zanjani, 1999b). As for other immunodeficiency disorders, such as chronic granulomatous disease and Chediak-Higashi syndrome, no detectable engraftment was achieved in the treated children (Cowan and Golbus, 1994; Flake and Zanjani, 1997; Muench et al., 2001).

Similarly, the use of IUHCT to treat hemoglobinopathies has mostly been unsuccessful. There have been 12 attempts to treat β-thalassemia with IUHCT, and only two have showed detectable postnatal engraftment (Slavin et al., 1992; Orlandi et al., 1996; Westgren et al., 1996; Sanna et al., 1999; Vrecenak and Flake, 2013). In α-thalassemia, three attempts at IUHCT were made, where only one demonstrated microchimerism and donor-specific immune tolerance (Cowan and Golbus, 1994; Westgren et al., 1996; Hayward et al., 1998). Currently, Horvei et al. (2021) are performing a phase I clinical trial (NCT02986698) of IUHCT using maternal cells as donors for treatment of α-thalassemia. As discussed previously, the presence of maternal cells in the fetus result in fetal tolerance, transplantation of maternal cells provides the highest likelihood of success. If the one-step protocol results in low levels of chimerism, a combined strategy would be performed with postnatal maternal cells transplantation.

In sickle cell anemia, fetal liver cells were injected to a female fetus at 13 weeks of gestation, however, engraftment was not detected at 3 months after her birth (Westgren et al., 1996). In another two unpublished clinic cases that applied IUHCT for sickle cell anemia therapy also failed in donor cell engraftment (Westgren, 2006). Metabolic storage diseases, another type of inherited disorder, may also benefit from IUHCT. Seven attempts were made and two cases showed engraftment (Bambach et al., 1997; Flake and Zanjani, 1997; Touraine et al., 1997; Touraine et al., 2004); however, one patient showed no improvement, while the other died prenatally, probably due to GVHD. For other genetic disorders, such as osteogenesis imperfecta, two attempts resulted in microchimerism and transient clinical effects (Le Blanc et al., 2005; Götherström et al., 2014). To date, the results of clinical cases demonstrated that clinical IUHCT, along with current methods, is not able to establish therapeutic levels of engraftment in recipients without significant immunodeficiency. Because of this, only a few recent clinical attempts at IUHCT have been reported. These clinical cases used various donor cell sources and IUHCT was performed at different times during gestation. These inconsistencies have made it impossible to determine the specific factors responsible for the low donor engraftment or failure in clinical practice. Therefore, it is vital to perform more controlled IUHCT in animal models to optimize the protocols before clinical application. In 2015, MacKenzie et al. (2015) produced an international consensus statement describing guidelines for IUSCT clinical trials.

Currently, two strategies can be used in clinical applications. The first is to perform IUHCT alone to achieve therapeutic engraftment levels; however, such a strategy might only be possible for diseases that require very low levels of engraftment for therapeutic success. The most favorable target disease for this approach is X-linked SCID, and other diseases, such as chronic granulomatous disease, hyper-IgM syndrome, and leukocyte adhesion deficiency, which are characterized by an SCID phenotype, may benefit from such a strategy. However, further studies on such diseases are required to optimize the current strategy. For diseases requiring high levels of engraftment for therapeutic success, such as hemoglobinopathies, the most compelling strategy is performing IUHCT to induce donor-specific tolerance, followed by postnatal minimal or non-toxic conditioning of HSCTs from the same donor to increase donor engraftment. As mentioned before, this strategy significantly reduces the initial chimerism required for clinical success to 1%–2%. From the studies of murine, sheep, and canine models discussed earlier (Zanjani, 1994; Hayashi et al., 2002; Peranteau et al., 2002; Ashizuka et al., 2006; Peranteau et al., 2009), on the basis of the initial chimerism after IUHCT, donor engraftment can be enhanced dramatically to complete or near-complete levels. More importantly, this strategy lowers the chimerism threshold, which may be required for clinical success.

Recently, Peranteau et al. (2015) showed that IUHCT combined with the same donor non-myeloablative allogeneic BM transplants corrected the disease phenotype in mice with β-thalassemia and sickle cell disease. Moreover, IUHCT-induced donor-specific tolerance may allow for postnatal organ transplantation without immunosuppression. Chen et al. (2010) demonstrated that the success of postnatal donor skin transplantation was dependent on the level of donor cell chimerism after IUHCT in murine models. Studies in swine and canine models support the combined strategy for successful postnatal renal transplants from the same donor without immunosuppression (Lee et al., 2005a; Mathes et al., 2005; Vrecenak et al., 2014). These results highlight the therapeutic potential of IUHCT combined with postnatal same-donor “boosting” transplantation in the clinical setting.

IUHCT has great potential for the treatment of numerous congenital hematological, genetic, and immunological disorders. However, this therapy has so far only been successfully achieved in fetuses with SCID. There are many hurdles remaining for IUHCT to overcome before it can be used as a therapeutic alternative for specific diseases. Challenges for IUHCT are mainly related to overcoming the competitive barriers to engraftment in the fetus and to better understand the maternal and fetal immune barriers to engraftment in large animal models and humans. Studies in murine and large animal models suggest that, under limited circumstances, a single IUHCT may result in high levels of donor cell engraftment to ameliorate the target disease. However, it seems unlikely to reach a therapeutic level of engraftment without the development of safe myeloablative drugs or options in the human fetus. Hopefully, through a greater understanding of induction and maintenance of immune tolerance and stem cell biology, new innovative methods and diverse source of donor cells can be applied to achieve high donor chimerism. Although limited clinical studies at the current time, the strategy of prenatal tolerance induction followed by postnatal HSC transplantation from the same donor to enhance engraftment is promising in the future. Further insights into the fetal hematopoietic ontogeny will further facilitate the improvement of therapeutic strategies based on IUHCT in the future.