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In Utero Cell Transplantation as a Tool for Fixing Liver-based Inherited Metabolic Disorders

Early Technical Evidence Supporting Potential Feasibility

Mas, Valeria R. PhD, FAST1,2; Maluf, Daniel G. MD1,2

doi: 10.1097/TP.0000000000002736

1 Methodist University Transplant Institute, Memphis, TN.

2 Transplant Research Institute, University of Tennessee Health Science Center, Memphis, TN.

Received 20 March 2019.

Accepted 24 March 2019.

The authors declare no conflicts of interest

V.R.M. and D.G.M. participated in reviewing the supporting data and writing of the commentary.

Correspondence: Valeria R. Mas, PhD, FAST, Endowed Professor of Transplant Research, Professor of Surgery, Professor of Genetics, Genomics, and Informatics, Director of Transplant Research Institute, Methodist University Transplant Institute, University of Tennessee Health Science Center, 910 Madison Ave, Memphis, TN 38163. (

Liver transplantation has been established as a curative therapy for acute and chronic liver failure, as well as liver-based inherited metabolic diseases. Congenital metabolic disorders result from the absence or abnormality of an enzyme or its cofactor, leading to either accumulation or deficiency of a specific metabolite. However, as consequence of the complexity associated with organ transplantation and the worldwide shortage of donor organs, hepatocyte transplantation has been developed as a bridging therapy until donor organs become available, or for amelioration of inherited liver-based diseases.1

Despite clinical trials of hepatocyte transplantation revealing the long-term safety of the procedure, only partial correction of metabolic disorders has been achieved. Although hepatocyte transplantation can be performed safely in humans, its applicability remains restricted by several concerns.2 The lack of human hepatocytes source has brought considerable restriction to the clinical application of hepatocyte transplantation. Hepatocytes are primarily acquired from livers that are not considered for transplantation. As it is well known from animal experiments, only about 30% of the transplanted cells engraft permanently. This would mean that beneficial effects have been observed at cell doses of about 1.5% of the recipient’s calculated total liver cell count.3 Monitoring of cell engraftment is difficult, as for most conditions it requires multiple liver biopsies.

One liver-based inherited metabolic diseases in which transient improvement has been demonstrated following this treatment is Crigler-Najjar syndrome (CNS).4-7 CNS is a congenital defect in bilirubin conjugation caused by complete or partial deficiency of with a lifelong risk of neurological damage and death. It is of two types, namely CN type I and CN type II. In Crigler-Najjar patients, the effect of transplanted cells can be easily monitored by reduction of plasma bilirubin. This has been shown very convincingly in the first patient (CNS 1), a 10-year-old girl, whose case is frequently regarded as the breakthrough in human liver cell transplantation and prompted the initiation of several clinical programs.4 The initial success was followed by treatment of four additional children that experienced substantial reduction of plasma bilirubin and duration of daily phototherapy.4-7 Data on the long-term clinical courses in these children are incomplete.

Advances in prenatal screening and molecular diagnosis allow the detection of many genetic diseases early in gestation. Early diagnosis and preemptive treatment of anticipated postnatal disease by in utero cell transplantation (IUCT) may have an important clinical impact and applicability for liver-based inherited metabolic diseases.

It is known that the amniotic fluid (AF) provides a source of stem cells with potential use for in utero cell gene therapy to treat congenital disease.7-10 Furthermore, human amniotic epithelial cells (hAECs) which are isolated from the amniotic membrane have stem cell-like properties and immunomodulatory effects.8 hAECs from term placenta express surface markers and gene characteristics of embryonic stem cells and can differentiate into all three germ layers, including tissues of endodermal origin (ie, liver).11 Previously, several protocols have been proposed for isolation of hAECs, with significant concerns associated with isolation quality, number of cells, and purity.12 However, hAECs are available in abundance via noninvasive resources and do not associate with ethical concerns as embryonic and fetal-derived stem cells.12 Thus, AECs represent a useful and noncontroversial source for liver-based regenerative medicine, as they can differentiate into hepatocytes upon transplantation into the liver.

Hereby, Grubbs et al13 test the use of hAEC as an alternative cell type for the treatment of hepatocyte dysfunction. The authors take advantage of the relatively easier handling and large variety of available congenital metabolic disorder model rodents for testing the therapeutic potential of IUCT using a murine model of CNS. Specifically, the authors used direct injection of these cells into the midgestation fetal Gunn rat liver via ultrasound-guided IUCT (embryonic day 16). Safety and impact of IUCT on live birth and postnatal survival was evaluated. Human cell engraftment was immunohistochemically analyzed on postnatal day 21 and detected in the liver of recipient rats.

Although the innovative aspects of the approach are exciting and promising, the current short communication results are limited by the small sample size and the inability to draw conclusions regarding the efficacy of prenatal hAEC therapy for bilirubin reduction. The main finding of the current study was the presence of engrafted hAECs on postnatal day 21 following intrauterine delivery of these cells, as evidenced by the presence of antihuman mitochondria staining. The authors describe that the pups survived to day 21 after birth (although a higher than anticipated number of pup’s death preparturition was observed). As an important note, the rat’s immune system is still in development currently, with the follicles and germinal centers still developing the spleen and some lymph nodes have yet to form. Consequently, of importance would be to analyze why there was such a high incidence of pup’s death pre-parturition. The authors mention as part of the discussion that poor stress tolerance of homozygous Gunn rats is recognized in the husbandry notes provided by the RRRC, inferring that this is the cause of the poor viability of the Gunn rat pups in this set of experiments. However, it can be wondered if this outcome is consequence of technical challenge or may be related to immune system activation. This last point deserves further exploration as the lack of alloimmune activation represents an advantage of the current presented approach compared with hepatocyte transplantation. A longer follow-up period is necessary to confirm the tolerance of these cells without immunosuppression, although the immunological advantages of IUCT have been already reported.

Clearly, utilization of a different and a more robust animal model, longer observational evaluation period, and additional assessments for evaluating recovery of function will likely allow to better test the therapeutic potential of IUCT of hAECs. However, this preliminary study represents a potential groundbreaking approach to intra uterus gene therapies to ameliorate liver-based inherited metabolic diseases. With advances being done in molecular techniques, genetic manipulation, and gene editing, further evaluation of mechanisms and approaches moving forward these types of approaches is guaranteed.

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