Generation of hepatic cell from placental stem cell for congenital metabolic disorders

We took human amniotic epithelial cells (hAECs) from placentae and isolated the cells with the enzyme activities that are lacking in three inherited metabolic disorders: mucopolysaccharidosis type I (MSP I, or Hurler syndrome), maple syrup urine disease and ornithine transcarbamylase deficiency (OTC). By transplanting these enzymatically-active cells into mice, we demonstrated an effective treatment for these disorders. Our group and others have demonstrated that hAECs possess unique stem cell-like qualities, such as the ability to differentiate. More importantly, hAECs are genetically stable and therefore don’t form tumors upon transplantation in mice and humans. During the first year of the project, we identified markers on the surface of hAECs that indicate the presence of the genes that code for the desired enzymes. We successfully established colonies of mice with each of the three metabolic disorders and defined the protocols for the radiological and biochemical tests, or assays. We also performed several hAEC transplantations to mice with MSP I. The first case of hAEC transplantation demonstrated a very promising result: the pathologic protein concentration in the urine of the treated MSP I mouse was dramatically decreased. We will confirm this result by investigating it further in more mice. As proposed, we have also started building a small-scale bio-bank of hAECs from 24 placentae. These hAECs will be used to determine whether hAECs retain their therapeutic potential after cryopreservation, or freezing.

In this reporting period we have conducted multiple analyses and accomplished several tasks. First, we successfully demonstrated therapeutic efficacy of placenta-derived stem cells (PDSCs) in all three proposed congenital metabolic disease model animals. A lysosome disease model Idua deficient mouse was used to test therapeutic efficacy of the PDSC for systemic congenital metabolic diseases. We demonstrated that unfractionated PDSCs express IDUA mRNA and protein at equivalent or higher levels than human hepatocytes. PDSC transplanted mice demonstrated a 15.1% and 32.5% increase of IDUA enzyme activity in the liver and lung, respectively. Interestingly, brain IDUA activity drastically improved (96.5%). We also established quantitative and qualitative bone mass evaluation methods using micro CT. Although the therapeutic efficacy on the bone phenotype of IDUA mouse was limited, the recipient demonstrated slight improvement. This data indicated that our approach to target the largest internal organ, the liver, to treat systemic metabolic disorders was reasonable and efficient. We will further study the optimal condition of PDSC transplantation and the mechanism of cell therapy. Our single cell gene expression analysis data indicated that the PDSC contains BCKDHa expressing cells. Using an intermediate Maple Syrup Urine disease model mouse, we conducted ultrasound guided cell injections to visualize transplanted cell distribution. Previous data indicated that primary PDSCs do not express the OTC gene. We conducted unfractionated PDSC transplantation into Spf/Ash mouse, which is a disease model with OTC deficiency. The urine proteomic analysis data indicated that the PDSC transplantation clearly improved the OTC phenotype. We will further increase the number of recipient mice as well as test different dosages and frequencies of cell transplantation. In conclusion, the project has been progressing very well and has demonstrated promising data in treating congenic metabolic disorder patients with placenta derived stem cells.

In this reporting period we have conducted multiple analyses and successfully demonstrated therapeutic efficacy of placenta-derived stem cells (PDSCs) in all three proposed congenital metabolic disease model animals. A lysosome disease model Idua deficient mouse was used to test therapeutic efficacy of the PDSC for Hurler disease. We demonstrated that the primary PDSCs express IDUA mRNA without in vitro manipulations. The IDUA gene and protein expressions are equivalent or higher levels than human hepatocytes. These data indicate that the primary PDSC is a suitable alternative cell source for the cell replacement therapy, hepatocyte transplantation. PDSC transplanted disease animals demonstrated increase of IDUA enzyme activity in the liver, lung and brain. Micro CT examination on the facial bone indicated that the PDSC treatment significantly improved the disease phenotype. The efficacy of PDSC transplantation was also assessed in animal models of Maple Syrup Urine disease (MSUD) and ornithine transcarbamylase deficiency (OTC). Five of 6 PDSC treated MSUD animals survived more than 100 days while all untreated MSUD animals died before 28 days of age. PDSC treatment also improved OTC disease phenotype and rescue the animals from transiently overdosed ammonia challenge. These data indicate that our approach to target the largest internal organ, the liver, to treat both systemic and liver specific congenital metabolic disorders is reasonable and efficient. In conclusion, we successfully demonstrated that the therapeutic efficacy of human placenta-derived stem cell transplantation with all three congenital metabolic diseases murine models. Additional experiments and further detailed analyses are necessary to conduct statistic validations and to define the therapeutic mechanisms.

The overall goal of this early translational research is to demonstrate the therapeutic efficacy of amniotic epithelial cells (AECs), a type of placental stem cells. AECs possess multilineage differentiation capability, low immunogenicity, and no tumorigenicity. In this project, we focused on testing the ability of these cells to treat animal models of three congenital metabolic disorders: mucopolysaccharidosis type1 (MPS-1), maple syrup urine disease (MSUD), and ornithine transcarbamylase deficiency (OTCD). Currently, there are no definitive therapies for these disorders. First, we evaluated the enzyme profile of AECs to determine whether they are suitable for treating each congenital metabolic disorder. We found that primary AECs possess the necessary enzymes to compensate for deficiencies in MPS-1 and MSUD. While primary AECs do not have ornithine transcarbamylase (OTC) enzyme activity to compensate for the deficiency in OTCD, they can acquire OTC gene expression after induction of hepatic differentiation. We utilized disease model mice to test the in vivo therapeutic efficacy for each disease. Human AECs (hAECs) transplanted into the livers of these mice improved disease phenotypes in all three mouse models. In the mouse model of MPS-1, the cell therapy improved not only the enzyme activity in the liver but also bone morphology and neural function. hAEC-treated MSUD mice survived more than 100 days, compared to control mice that only survived up to 30 days. We also successfully demonstrated improvement of the disease phenotype in the OTCD model mice. After transplantation, hAECs differentiated and acquired OTC enzyme activity in their mitochondria. Through a unique therapeutic mechanism, hAECs transferred these functional mitochondria to diseased mouse hepatocytes via tunneling nanotubes. In conclusion, we have successfully demonstrated the therapeutic efficacy of hAEC therapy for three congenital metabolic disorders. We are eager to move forward with our research in order to bring this promising stem cell therapy to patients with unmet medical needs.