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Introduction: Duchenne muscular dystrophy (DMD) is a progressive disease that leads to damage of muscle and myocardium due to genetic abnormalities in the dystrophin gene. In utero cell transplantation that might facilitate allogenic transplantation is worth considering to treat this disease. Methods: We performed allogeneic in utero transplantation of GFP-positive myoblasts and adipose-derived mesenchymal stem cells into murine DMD model animals. The transplantation route in this study was fetal intraperitoneal transplantation and transplacental transplantation. Transplanted animals were examined at 4-weeks old by immunofluorescence staining and RT-qPCR. Results: No GFP-positive cells were found by immunofluorescence staining of skeletal muscle and no GFP mRNA was detected by RT-qPCR in any animal, transplantation method and cell type. Compared with previous reports, myoblast transplantation exhibited an equivalent mortality rate, but adipose-derived stem cell (ASC) transplantation produced a higher mortality rate. Conclusions: In utero transplantation of myoblasts or ASCs to murine models of DMD does not lead to engraftment and, in ASC transplantation primarily, frequently results in fetal death.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach.

In utero hematopoietic cell transplantation (IUHCT) is considered a potentially efficient therapeutic approach with relatively few side effects, compared to adult hematopoietic cell transplantation, for various hematological genetic disorders. The principle of IUHCT has been extensively studied in rodent models and in some large animals with close evolutionary similarities to human beings. However, IUHCT has only been used to rebuild human T cell immunity in certain patients with inherent immunodeficiencies. This review will first summarize the animal models utilized for IUHCT investigations and describe the associated outcomes. Recent advances and potential barriers for successful IUHCT are discussed, followed by possible strategies to overcome these barriers experimentally. Lastly, we will outline the progress made towards utilizing IUHCT to treat inherent disorders for patients, list out associated limitations and propose feasible means to promote the efficacy of IUHCT clinically. [ABSTRACT FROM AUTHOR]

We sought to determine the pathway through which syngeneic hematopoietic stem cells (HSCs) delivered into the amniotic fluid can reach the fetal circulation. Lewis rat fetuses were divided in two groups based on the content of intra-amniotic injections performed on gestational day 17 (E17; term=E21–22): either a suspension of luciferase-labeled syngeneic HSCs (n = 137), or acellular luciferase (n = 44). Samples from placenta, chorion, amnion, amniotic fluid, umbilical cord, and 8 fetal sites were procured at 5 daily time points thereafter until term for analysis. When controlled by acellular luciferase, donor HSCs were identified in the amnion, chorion, placenta, and amniotic fluid of fetuses receiving cells at all time points (p = 0.033 to <0.001), peaking first at the amnion and subsequently at the chorion and placenta. Cells could be detected in the fetal liver as early as day 1, progressively expanding to all the other fetal sites over time, in parallel to their increased presence in the chorion and placenta. The chronology of syngeneic donor hematopoietic stem cell trafficking after intra-amniotic injection is suggestive of controlled routing through the gestational membranes and placenta. Hematogenous donor cell routing is a constituent of transamniotic hematopoietic stem cell therapy, significantly expanding its potential applications. [ABSTRACT FROM AUTHOR]

We sought to determine whether intrauterine growth restriction (IUGR) could be a target for mesenchymal stem cell (MSC)-based transamniotic stem cell therapy (TRASCET). Pregnant dams subjected to hypoxia (10.5% O 2) cycles had their fetuses divided into four groups: untreated (n = 24) and three groups receiving volume-matched intra-amniotic injections of either saline (sham; n = 16), or suspensions of luciferase-labeled, syngeneic amniotic fluid-derived MSCs that were either native (TRASCET-unprimed; n = 29), or primed by exposure to IFNγ and IL-1β (TRASCET-primed; n = 31). Normal fetuses served as additional controls (n = 22). Multiple analyses were performed at term. Compared to normal, fetal weights were significantly decreased in all hypoxia groups (p = 0.002 to <0.001), except for TRASCET-primed. Placental efficiency (fetal/placental weight) was significantly decreased in all hypoxia groups (p = 0.002 to <0.001), but normalized in both TRASCET groups. A significant increase in metrial expression of IFNγ in both the untreated and sham groups (p = 0.04 to 0.02) was reversed only in the TRASCET-primed group. Luciferase DNA was present in both TRASCET groups' placentas. Transamniotic stem cell therapy with primed mesenchymal stem cells reverses some of the effects of intrauterine growth restriction in a rat model. Further study into this novel approach for the treatment of this disease is warranted. N/A (Animal and Laboratory Study) [ABSTRACT FROM AUTHOR]

In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to circumvent the challenges of postnatal hematopoietic stem cell (HSC) transplantation. The goal of IUHCTx is to introduce donor cells into a naïve host prior to immune maturation, thereby inducing donor-specific tolerance. Thus, this technique has the potential of avoiding host myeloablative conditioning with cytotoxic agents. Over the past two decades, several attempts at IUHCTx have been made to cure numerous underlying congenital anomalies with limited success. In this review, we will briefly review the history of IUHCTx and give a perspective on alpha thalassemia major, one target disease for its clinical application.

In utero administration of hematopoietic stem cells (HSCs) has a variety of actual or potential clinical applications but is hindered by invasive, morbid administration techniques. We sought to determine whether donor HSCs could reach the fetal circulation after simple intra-amniotic delivery in a syngeneic rat model of transamniotic stem cell therapy (TRASCET). Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all fetuses (n = 90) on gestational day 17 (E17; term=E21-22) of a suspension of commercially available syngeneic Lewis rat HSCs labeled with luciferase (n = 37 fetuses) or an acellular suspension of recombinant luciferase (n = 53). HSC phenotype was confirmed by flow cytometry. Fetuses were euthanized at term for screening of luciferase activity at select anatomical sites. Statistical comparisons were by Fisher's exact test. Among survivors (47/90; 52.2%), donor HSCs were identified selectively in the placenta (p = 0.003), umbilical cord (p < 0.001), bone marrow (p < 0.001), thymus (p = 0.009), bowel (p = 0.003), kidney (p = 0.022), and skin (p < 0.001) when compared with acellular luciferase controls. Donor hematopoietic stem cells undergo hematogenous routing and can reach the fetal bone marrow after simple intra-amniotic delivery in a syngeneic rat model. Transamniotic stem cell therapy may become a practicable, minimally invasive strategy for the prenatal administration of these cells. [ABSTRACT FROM AUTHOR]

In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to circumvent the challenges of postnatal hematopoietic stem cell (HSC) transplantation. The goal of IUHCTx is to introduce donor cells into a naïve host prior to immune maturation, thereby inducing donor-specific tolerance. Thus, this technique has the potential of avoiding host myeloablative conditioning with cytotoxic agents. Over the past two decades, several attempts at IUHCTx have been made to cure numerous underlying congenital anomalies with limited success. In this review, we will briefly review the history of IUHCTx and give a perspective on alpha thalassemia major, one target disease for its clinical application.

Sickle Cell Disease (SCD) is an autosomal recessive disorder resulting from a β-globin gene missense mutation and is among the most prevalent severe monogenic disorders worldwide. Haematopoietic stem cell transplantation remains the only curative option for the disease, as most management options focus solely on symptom control. Progress in prenatal diagnosis and fetal therapeutic intervention raises the possibility of in utero treatment. SCD can be diagnosed prenatally in high-risk patients using chorionic villus sampling. Among the possible prenatal treatments, in utero stem cell transplantation (IUSCT) shows the most promise. IUSCT is a non-myeloablative, non-immunosuppressive alternative conferring various unique advantages and may also offer safer postnatal management. Fetal immunologic immaturity could allow engraftment of allogeneic cells before fetal immune system maturation, donor-specific tolerance and lifelong chimerism. In this review, we will discuss SCD, screening and current treatments. We will present the therapeutic rationale for IUSCT, examine the early experimental work and initial human experience, as well as consider primary barriers of clinically implementing IUSCT and the promising approaches to address them.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative procedure that leads to donor cell chimerism and donor-specific tolerance. However, most clinical applications of IUHCT have failed because of low levels or even no engraftment of donor cells in immunologically normal fetuses. It is likely that the competition from the host hematopoietic compartment is the primary barrier to successful IUHCT, suggesting that conditioning methods that provide a competitive advantage to donor cells may lead to higher-level engraftment following IUHCT. This study aimed to research whether maternal administration of low-dose total body irradiation (TBI) or busulfan (BU) before IUHCT may result in increased donor cell chimerism in postnatal bone marrow transplantation in a congenic murine model. We first determined the birth and mortality rates after maternal administration of low-dose TBI (0, 2 or 4 Gy) or BU (5, 10, 15, or 20 mg/kg) before IUHCT in B6 mice. The mice that received 2 Gy TBI plus IUHCT showed significantly lower birth rate (23.3%) and a 100% 3-day mortality rate. The mice that received 10 mg/kg BU plus IUHCT had similar birth and 3-day mortality rates (58.6% and 0%) compared to mice that received IUHCT alone (61.1% and 4.55%). We then performed maternal administration of BU at 1 of 3 dosages (5, 10, or 15 mg/kg) at 24 hours before intrauterine transplantation of 2.5 × 10 5 B6GFP Sca-1 + bone marrow cells (BMCs) or 2.5 × 10 6 B6GFP BMCs on gestational day 14 (E14). Green fluorescent protein (GFP) chimerism in peripheral blood mononuclear cells (PBMCs), RBCs, and platelets of mice at 4 weeks of age was enhanced significantly with an increase in BU dose. Moreover, GFP chimerism of PBMCs from the B6GFP BMC group was significantly higher than that of the B6GFP Sca-1 + BMC group (22.56% versus 7.20%; P = .018). Finally, the pregnant mice were treated with 10 mg/kg of BU at E13, E14, or E15, followed by intrauterine transplantation of 2.5 × 10 6 B6GFP BMCs 24 hours later. Except for the short-term level of chimerism in PBMCs, which showed no significant difference among the 3 study groups, the results indicate that both short-term (age 4 weeks) and long-term (age 14 weeks) engraftment in PBMCs, RBCs, and platelets was higher in group E16 compared with groups E14 and E15. We also discovered that the engraftment was stable, multilineage, and increased with time. In conclusion, maternal administration of BU, but not of TBI, along with IUHCT could significantly enhance engraftment in a congenic murine model., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Hematopoiesis, a process which generates blood and immune cells, changes significantly during mammalian development. Definitive hematopoiesis is marked by the emergence of long-term hematopoietic stem cells (HSCs). Here, we will focus on the post-transcriptional differences between fetal liver (FL) and adult bone marrow (ABM) HSCs. It remains unclear how or why exactly FL HSCs transition to ABM HSCs, but we aim to leverage their differences to revive an old idea: in utero HSC transplantation. Unexpectedly, the expression of certain RNA-binding proteins (RBPs) play an important role in HSC specification, and can be employed to convert or reprogram adult HSCs back to a fetal-like state. Among other features, FL HSCs have a broad differentiation capacity that includes the ability to regenerate both conventional B and T cells, as well as innate-like or unconventional lymphocytes such as B-1a and marginal zone B (MzB) cells. This chapter will focus on RNA binding proteins, namely LIN28B and IGF2BP3, that are expressed during fetal life and how they promote B-1a cell development. Furthermore, this chapter considers a potential clinical application of synthetic co-expression of LIN28B and IGF2BP3 in HSCs., (Copyright © 2024. Published by Elsevier Inc.)

Amniotic fluid-derived progenitor cells (AFPCs) are oligopotent and shed from the fetus into the amniotic fluid. It was reported that AFPCs express stem cell-like markers and are capable of differentiating into specific cell type in in vitro experiments. However, no study has fully investigated the potentiality and destiny of these cells in in vivo experiments. Methods: Ds-red transgenic mice (on Day 13.5 of pregnancy) were transplanted in utero with enhanced green fluorescent protein-labeled mouse AFPC (EGFP-mAFPCs). After birth, baby mice were euthanized at 3-week intervals beginning 3 weeks postnatally, and the specimens were examined by polymerase chain reaction, histology, and flow cytometry. Results: Our results demonstrate the transplantability of mAFPCs into all three germ layers and the potential of mAFPCs in the study of progenitor cell homing, differentiation, and function. Engraftment of EGFP-mAFPCs was detected in the intestine, kidney, muscle, skin, bladder, heart, stomach, etc., at 3 weeks after delivery. Conclusion: This model using EGFP-mAFPCs injected in utero may provide an ideal method for determining the fate of transplanted cells in recipients and these findings may justify a clinical trial of in utero transplantation during gestation for patients who have inherited genetic disorders.

Placental stem cells are of growing interest for a variety of clinical applications due to their multipotency and ready availability from otherwise frequently discarded biomaterial. Stem cells derived from the placenta have been investigated in a number of disease processes, including wound healing, ischemic heart disease, autoimmune disorders, and chronic lung or liver injury. Fetal intervention for structural congenital defects, such as spina bifida, has rapidly progressed as a field due to advances in maternal-fetal medicine and improving surgical techniques. In utero treatment of structural, as well as non-structural, congenital disorders with cell-based therapies is of particular interest given the immunologic immaturity and immunotolerant environment of the developing fetus. A comprehensive literature review was performed to assess the potential utilization of placenta-derived stem cells for in utero treatment of congenital disorders. Most studies are still in the preclinical phase, utilizing animal models of common congenital disorders. Future research endeavors may include autologous transplantation, gene transfers, induced pluripotent stem cells, or cell-free therapies derived from the stem cell secretome. Though much work still needs to be done, placental stem cells are a promising therapeutic agent for fetal intervention for congenital disease. [ABSTRACT FROM AUTHOR]

The primary benefits of in utero hematopoietic cellular transplantation (IUHCT) arise from transplanting curative cells prior to the immunologic maturation of the fetus. However, this approach has been routinely successful only in the treatment of congenital immunodeficiency diseases that include an inherent NK cell deficiency despite the existence of normal maternal immunity in either setting. These observations raise the possibility that fetal NK cells function as an early barrier to allogeneic IUHCT. Herein, we summarize the findings of previous studies of prenatal NK cell allospecific tolerance in mice and in humans. Cumulatively, this new information reveals the complexity of the fetal immune response in the setting of rejection or tolerance and illustrates the role for fetal NK cells in the final endorsement of allospecific prenatal tolerance.

In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to circumvent the challenges of postnatal hematopoietic stem cell (HSC) transplantation. The goal of IUHCTx is to introduce donor cells into a naïve host prior to immune maturation, thereby inducing donor–specific tolerance. Thus, this technique has the potential of avoiding host myeloablative conditioning with cytotoxic agents. Over the past two decades, several attempts at IUHCTx have been made to cure numerous underlying congenital anomalies with limited success. In this review, we will briefly review the history of IUHCTx and give a perspective on alpha thalassemia major, one target disease for its clinical application.

Hemophilia A (HA) is the most frequent inheritable defect of the coagulation proteins. The current standard of care for patients with HA is prophylactic factor infusion, which is comprised of regular (2-3 times per week) intravenous infusions of recombinant or plasma-derived FVIII to maintain hemostasis. While this treatment has greatly increased the quality of life and lengthened the life expectancy for many HA patients, its high cost, the need for lifelong infusions, and the fact that it is unavailable to roughly 75% of the world’s HA patients make this type of treatment far from ideal. In addition, this lifesaving therapy suffers from a high risk of treatment failure due to immune response to the infused FVIII. There is thus a need for novel treatments, such as those using stem cells and/or gene therapy, which have the potential to mediate long-term correction or permanent cure following a single intervention. In the present review, we discuss the clinical feasibility and unique advantages that an in utero approach to treating HA could offer, placing special emphasis on a new sheep model of HA we have developed and on the use of mesenchymal stromal cells (MSC) as cellular vehicles for delivering the FVIII gene.

Regenerative medicine is a rapidly expanding and promising field for many diseases and injuries. Stem cells for regenerative therapies have originally been obtained from bone marrow, but are now readily extracted from a variety of adult tissues. Fetal tissue has recently garnered interest for its ease of differentiation into a variety of phenotypes and its relative abundance of pluripotent-linked transcription factors. However, much ethical concern surrounds the methods of obtaining fetal cells. The placenta has emerged as a potential source of fetal derived cells due to its favorable technical and ethical characteristics, as well as its promising therapeutic properties. This preview focuses on providing on overview on the derivation and characteristics of placental derived stem cells as well as delving into their various clinical applications and potential future directions. [ABSTRACT FROM AUTHOR]

Osteogenesis Imperfecta (OI) can be a severe disorder that can be diagnosed before birth. Transplantation of mesenchymal stem cells (MSC) has the potential to improve the bone structure, growth and fracture healing. In this review we give an introduction to OI and MSC, and the basis for prenatal and postnatal transplantation in OI. We also summarize the two patients with OI who has received prenatal and postnatal transplantation of MSC.The findings suggest that prenatal transplantation of allogeneic MSC in OI is safe. The cell therapy is of likely clinical benefit with improved linear growth, mobility and reduced fracture incidence. Unfortunately, the effect is transient. For this reason postnatal booster infusions using same-donor MSC have been performed with clinical benefit, and without any adverse events.So far there is limited experience in this specific field and proper studies are required to accurately conclude on clinical benefits of MSC transplantation to treat OI.

The primary benefits of in utero hematopoietic cellular transplantation (IUHCT) arise from transplanting curative cells prior to the immunologic maturation of the fetus. However, this approach has been routinely successful only in the treatment of congenital immunodeficiency diseases that include an inherent NK cell deficiency despite the existence of normal maternal immunity in either setting. These observations raise the possibility that fetal NK cells function as an early barrier to allogeneic IUHCT. Herein, we summarize the findings of previous studies of prenatal NK cell allospecific tolerance in mice and in humans. Cumulatively, this new information reveals the complexity of the fetal immune response in the setting of rejection or tolerance and illustrates the role for fetal NK cells in the final endorsement of allospecific prenatal tolerance. [ABSTRACT FROM AUTHOR]

In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to circumvent the challenges of postnatal hematopoietic stem cell (HSC) transplantation. The goal of IUHCTx is to introduce donor cells into a naïve host prior to immune maturation, thereby inducing donor-specific tolerance. Thus, this technique has the potential of avoiding host myeloablative conditioning with cytotoxic agents. Over the past two decades, several attempts at IUHCTx have been made to cure numerous underlying congenital anomalies with limited success. In this review, we will briefly review the history of IUHCTx and give a perspective on alpha thalassemia major, one target disease for its clinical application. [ABSTRACT FROM AUTHOR]

Hemophilia A (HA) is the most frequent inheritable defect of the coagulation proteins. The current standard of care for patients with HA is prophylactic factor infusion, which is comprised of regular (2-3 times per week) intravenous infusions of recombinant or plasma-derived FVIII to maintain hemostasis. While this treatment has greatly increased the quality of life and lengthened the life expectancy for many HA patients, its high cost, the need for lifelong infusions, and the fact that it is unavailable to roughly 75% of the world's HA patients make this type of treatment far from ideal. In addition, this lifesaving therapy suffers from a high risk of treatment failure due to immune response to the infused FVIII. There is thus a need for novel treatments, such as those using stem cells and/or gene therapy, which have the potential to mediate long-term correction or permanent cure following a single intervention. In the present review, we discuss the clinical feasibility and unique advantages that an in utero approach to treating HA could offer, placing special emphasis on a new sheep model of HA we have developed and on the use of mesenchymal stromal cells (MSC) as cellular vehicles for delivering the FVIII gene. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) can be a severe disorder that can be diagnosed before birth. Transplantation of mesenchymal stem cells (MSC) has the potential to improve the bone structure, growth, and fracture healing. In this review, we give an introduction to OI and MSC, and the basis for pre- and postnatal transplantation in OI. We also summarize the two patients with OI who have received pre- and postnatal transplantation of MSC. The findings suggest that prenatal transplantation of allogeneic MSC in OI is safe. The cell therapy is of likely clinical benefit with improved linear growth, mobility, and reduced fracture incidence. Unfortunately, the effect is transient. For this reason, postnatal booster infusions using same-donor MSC have been performed with clinical benefit, and without any adverse events. So far there is limited experience in this specific field and proper studies are required to accurately conclude on clinical benefits of MSC transplantation to treat OI. [ABSTRACT FROM AUTHOR]

Background aims. Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. Methods. We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4+ fraction of CD34 1 HSCs, we could improve engraftment. Human cord blood-derived CD34+ cells and human bone marrow-derived MSCs were used for these studies. Results. When MSCs were transplanted 1 week before CD34 H cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34 1 cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4+ cells in the CD34+ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. Conclusions. Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4--SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus. [ABSTRACT FROM AUTHOR]

Cells isolated from foetal membranes of human term placenta display multiple properties, including some features of stem/progenitor cells, together with immunomodulatory actions and the ability to secrete bioactive soluble factors. Whilst such properties support the potential applicability of these cells in transplantation settings aimed at regenerating/repairing tissues in adults, theoretically, using these cells in prenatal treatment strategies may also be achievable. To assess the feasibility of a foetal membrane-derived cell-based therapeutic treatment during foetal development, we firstly addressed the question of whether in utero transplantation using these cells was possible. To this end, we assessed postnatal microchimerism after transplantation of amniotic membrane-derived cells (a mixture of both mesenchymal stromal/stem cells and epithelial cells) in foetal sheep. Transplantation was performed with or without human umbilical cord blood mononuclear cells and chorionic membrane-derived mesenchymal stromal/stem cells, and was followed by a postnatal booster cell injection. Lambs were euthanized 2-4 months postnatally and their organs/tissues were analysed for microchimerism through detection of human DNA. Human DNA was found in almost all tissues of all of the lambs, with the seemingly random appearance of human cells in some of the analysed tissues suggesting long-term human microchimerism and donor cell migration after in utero/postnatal booster xenotransplation. Differences in microchimerism tissue distribution between animals transplanted with different cell types are discussed. This pilot study adds to ongoing efforts by different investigators to explore the potential of in utero cellular transplantation, and warrants further investigation of using foetal membrane-derived cells for prenatal cell therapies. [ABSTRACT FROM AUTHOR]

Introduction: Duchenne muscular dystrophy (DMD) is a progressive disease that leads to damage of muscle and myocardium due to genetic abnormalities in the dystrophin gene. In utero cell transplantation that might facilitate allogenic transplantation is worth considering to treat this disease., Methods: We performed allogeneic in utero transplantation of GFP-positive myoblasts and adipose-derived mesenchymal stem cells into murine DMD model animals. The transplantation route in this study was fetal intraperitoneal transplantation and transplacental transplantation. Transplanted animals were examined at 4-weeks old by immunofluorescence staining and RT-qPCR., Results: No GFP-positive cells were found by immunofluorescence staining of skeletal muscle and no GFP mRNA was detected by RT-qPCR in any animal, transplantation method and cell type. Compared with previous reports, myoblast transplantation exhibited an equivalent mortality rate, but adipose-derived stem cell (ASC) transplantation produced a higher mortality rate., Conclusions: In utero transplantation of myoblasts or ASCs to murine models of DMD does not lead to engraftment and, in ASC transplantation primarily, frequently results in fetal death., Competing Interests: The authors declare no conflicts of interest associated with this manuscript., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

In utero hematopoietic cell transplantation (IUHCT) is considered a potentially efficient therapeutic approach with relatively few side effects, compared to adult hematopoietic cell transplantation, for various hematological genetic disorders. The principle of IUHCT has been extensively studied in rodent models and in some large animals with close evolutionary similarities to human beings. However, IUHCT has only been used to rebuild human T cell immunity in certain patients with inherent immunodeficiencies. This review will first summarize the animal models utilized for IUHCT investigations and describe the associated outcomes. Recent advances and potential barriers for successful IUHCT are discussed, followed by possible strategies to overcome these barriers experimentally. Lastly, we will outline the progress made towards utilizing IUHCT to treat inherent disorders for patients, list out associated limitations and propose feasible means to promote the efficacy of IUHCT clinically., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi, Pan and Hu.)

In utero hematopoietic cellular transplantation (IUHCT) holds great promise for the treatment of congenital diseases of cellular dysfunction such as sickle cell disease, immunodeficiency disorders and inherited metabolic disorders. However, repeated failures in clinical cases of IUHCT that do not involve an immunodeficiency disease force a closer examination of the fetal immune system. While the mechanisms regulating T cell tolerance have been previously studied, the educational mechanisms leading to NK cell tolerance in prenatal chimeras remain unknown. As a low level of donor cells (1.8%) is required to induce and maintain this tolerance, it is likely that these mechanisms employ indirect host-donor interaction. This report examines donor-to-host MHC transfer (trogocytosis) as an intrinsic mechanism regulating the development and maintenance of NK cell tolerance in prenatal chimeras. The findings demonstrate that phenotypically tolerant host NK cells express low levels of transferred donor MHC antigens during development and later as mature cytotoxic lymphocytes. Further study is needed to understand how the cis-recognition of transferred donor MHC ligand influences the selection and maintenance of tolerant NK cells in prenatal chimeras. [ABSTRACT FROM AUTHOR]

Background: Among cell suspensions from different origins, lymphocytes were reported to have the superiority of tolerance-conferring capacity in preimmune hosts. However, this belief was derived directly from murine combinations with fewer major histocompatibility complex (MHC) barriers that are exceptional in the clinical arena. Because of the potential for prenatal tolerance induction to facilitate postnatal therapies, it is important to examine the relative merits and hazards of fully MHC-mismatched naïve lymphocytes as the prenatal tolerogenic agent in the preimmune fetus to cross MHC barriers. Materials and methods: In utero injection of C57BL/6 splenic lymphocytes was conducted in gestational day 14 FVB/N fetuses. Then, FVB/N recipients were subjected to the evaluation of hematopoietic chimerism, donor-specific tolerance, and graft-versus-host disease (GVHD). Results: With a dose of ≥5 × 105 C57BL/6 lymphocytes, the recipients born alive either died unexpectedly by maternal cannibalization or succumbed to GVHD within postnatal 1 mo. GVHD mice showed significant hematopoietic chimerism that was dominated by donor CD3 T cells. It was found that allogeneic lymphocytes could rapidly damage the fetal liver within 5 d after injection. Fetal recipients could survive a dose of ≤2 × 105 allogeneic lymphocytes beyond 1 mo of age, but at best showed microchimerism that was insufficient to confer donor-specific skin tolerance. Conclusions: Fully MHC-mismatched lymphocytes injected in utero had lethal graft-versus-host effects, which might rapidly develop within 1 wk after injection in preimmune fetuses. They were incapable of conferring significant hematopoietic chimerism and graft tolerance even at bearable doses. [ABSTRACT FROM AUTHOR]

Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B. [ABSTRACT FROM AUTHOR]

Background aims. Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties. Methods. We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γγ. Results. Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γγ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC. Conclusions. Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy. [ABSTRACT FROM AUTHOR]

Abstract: A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55±1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments. [Copyright &y& Elsevier]

The human/sheep xenograft model has been imagined and used by many researchers in order to asses the hematopoietic activity of stem cells. The aim of this paper was to show engraftment of human umbilical cord blood CD34+ stem cells in sheep bone marrow following in utero transplantation. Human umbilical cord blood was collected immediately after delivery and processed in order to isolate the CD34+ cells that were transplanted by intraperitoneal injection into 60 days old fetal sheep. The pregnancies were monitored to term and bone marrow was collected from new-born lambs by iliac crest aspiration. Cells were stained with anti-human CD 45/CD 34 antibodies and chimerism was established using a BD FACS Canto II flow cytometer that showed a 3% engraftment of human hematopoietic stem cells within the sheep bone marrow. [ABSTRACT FROM AUTHOR]

Abstract: In the murine model, in utero hematopoietic cell transplantation (IUHCT) has been shown to achieve low levels of allogeneic chimerism and associated donor-specific tolerance permitting minimal conditioning postnatal hematopoietic stem cell transplantation (HSCT). In this pilot study, we investigated IUHCT in the canine leukocyte adhesion deficiency (CLAD) model. Haploidentical IUHCT resulted in stable low-level donor cell chimerism in all dogs that could be analyzed by sensitive detection methodology (4 of 10) through 18 months of follow-up. In the 2 CLAD recipients, low-level chimerism resulted in amelioration and complete reversal of the CLAD phenotype, respectively. Six recipients of IUHCT (5 carriers and 1 CLAD) subsequently received postnatal HSCT from the same haploidentical prenatal donor after minimal conditioning with busulfan 10 mg/kg. Chimerism in 2 of 5 CLAD carriers that underwent HSCT increased from < 1% pre-HSCT to sustained levels of 35% to 45%. Control animals undergoing postnatal haploidentical HSCT without IUHCT had no detectable donor chimerism. These results demonstrate that haploidentical IUHCT in the CLAD model can result in low-level donor chimerism that can prevent the lethal phenotype in CLAD dogs, and can result in donor-specific tolerance that can facilitate postnatal minimal conditioning HSCT. [Copyright &y& Elsevier]

We have recently characterized a stem cell population isolated from the rodent amniotic membrane termed amnion-derived stem cells (ADSCs). In vitro ADSCs differentiate into cell types representing all three embryonic layers, including neural cells. In this study we evaluated the neuroectodermal potential of ADSCs in vivo after in utero transplantation into the developing rat brain. A clonal line of green fluorescent protein-expressing ADSCs were infused into the telencephalic ventricles of the developing embryonic day 15.5 rat brain. At E17.5 donor cells existed primarily as spheres in the ventricles with subsets fused to the ventricular walls, suggesting a mode of entry into the brain parenchyma. By E21.5 green fluorescent protein (GFP) ADSCs migrated to a number of brain regions. Examination at postnatal time points revealed that donor ADSCs expressed vimentin and nestin. Subsets of transplanted ADSCs attained neuronal morphologies, although there was no immunohistochemical evidence of neural or glial differentiation. Some donor cells migrated around blood vessels and differentiated into putative endothelial cells. Donor ADSCs transplanted in utero were present in recipients into adulthood with no evidence of immunological rejection or tumour formation. Long-term survival may suggest utility in the treatment of disorders where differentiation to a neural cell type is not required for clinical benefit. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: The failure of in utero transplantation in immune-competent recipients suggests the existence of a fetal immune barrier. The importance of donor major histocompatibility complex (MHC) class I expression in the induction of prenatal tolerance remains undefined. We hypothesized that donor cell MHC class I expression facilitates engraftment in prenatal allogeneic recipients rather than promoting immune rejection. Methods: B6.Ly5.2 (class I+) or B6.TAP−/− (class I−) murine fetal liver cells were transplanted into age-matched allogeneic fetal recipients. Survival to weaning and subsequent growth was assessed. Engraftment rates and peripheral blood chimerism levels were measured serially. Results: The presence or absence of class I expression did not affect survival or growth of recipients and no graft-vs-host disease developed. Allogeneic recipients of B6.Ly5.2 cells exhibited significantly higher levels of donor hematopoietic chimerism when compared to recipients of B6.TAP−/− cells (27% + 10% vs 11% + 8%; P = .004) that deteriorated further over time. Conclusions: Donor class I MHC antigen expression is essential for stable long-term engraftment and maintenance of donor-specific tolerance. Further studies are needed to better characterize the role of the fetal innate immune system in prenatal allotransplantation. [Copyright &y& Elsevier]

Abstract: Fetal stem cells can be isolated from fetal blood and bone marrow as well as other fetal tissues. Fetal blood is both a source of haemopoietic stem cells, which proliferate more rapidly than those in cord blood or adult bone marrow, and a source of non-haemopoietic mesenchymal stem cells, which support haemopoiesis and can also differentiate along multiple lineages. Although the placenta was traditionally seen as a barrier separating the genetically distinct mother and fetus, it is now recognised that fetal cells pass into the maternal circulation throughout normal pregnancy. This cell traffic may help establish maternal tolerance to the fetal allograft. The principal mechanism is fetomaternal haemorrhage, as evidenced by the variety of fetal cell types, including stem cells, identified in maternal blood. Isolation of fetal cells from the maternal circulation in pregnancy has been investigated as an alternative to existing methods of genetic prenatal diagnosis. Fetal stem cells have considerable potential for non-invasive prenatal testing, as they differ in phenotype from other cells circulating in the adult and can be amplified into large numbers for analysis after enrichment from maternal blood. Stem cells isolated from the fetus may be transplanted in utero to treat genetic disease and also show promise as targets for gene therapy, which could be applied to diseases such as osteogenesis imperfecta or muscular dystrophies. The destination of fetal stem cells after trafficking across the placenta is the subject of much debate. While these cells may persist in blood in undetectable amounts, it is more likely that stem cells engraft in maternal tissues and persist for years after pregnancy, as has been demonstrated by finding fetal mesenchymal stem cells in post-reproductive tissues. Fetal microchimerism, which refers to low levels of fetal cells harboured by the mother, has been associated with the development of autoimmune disease in the mother and with repair of damaged tissues. However, fetal stem cells in maternal tissues could also act as a long-term reservoir of stem cells and may even explain why women live longer than men and why pregnancy protects against susceptibility to some diseases. Cellular trafficking in pregnancy has far reaching biological consequences. [Copyright &y& Elsevier]

Objective: To address the role that alloreactivity may play and better define the window for histoincompatible stem cell transplantation in utero. Subjects, Material and Methods: We studied 9 fetal blood specimens obtained by cardiocentesis during elective abortions in the second trimester by multicolor flow cytometry and in vitro stimulation. Results: Lymphocytes ranged from adult levels (3/9) to >90% leukocytes. Six specimens had T cells within adult range. T cells in the other specimens were reduced, while B cells were conversely elevated. This variability did not correlate with gestational age, or leukocyte composition. Following 4 h of mitogenesis, fetal CD4+ and CD8+ T cells from 1 of 5 specimens showed a response similar to that of maternal T cells, while the other 4 specimens showed a diminished response (0.3 ± 0.2-fold). This heterogeneity did not correlate with gestational age, or lymphocyte subset distribution. Following 18 h of in vitro mitogenesis, fetal T cells from 2 specimens showed a response similar to that of maternal T cells (0.8 ± 0.2-fold). Despite that, one specimen gave a 3-fold greater response in a one-way mixed lymphocyte reaction vs. maternal cells compared to the other specimen. Conclusion: We determine that fetal immunocompetence differs greatly during the second trimester and assessment of host vs. donor reactivity prior to in utero transplantation is likely to potentiate more favorable outcomes. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]