Approximately 1 in 1,500 children has a congenital metabolic disorder. These inborn errors of metabolism are caused by deficiencies of different enzymes and result in accumulation of various substances inside cells. These substances affect the function of vital organs, and in many cases are lethal. Transplantation of cells that possess the particular deficient enzyme carries the potential to cure these diseases. Currently, a shortage of human liver cells for transplantation prohibits clinical use of this therapy. The human placenta contains cells that may acquire hepatic function. Following delivery of a baby, these cells can be collected from the placenta which is in most cases is treated as medical waste and discarded. The therapeutic potential of this cell type has been shown in animal models. We propose to first develop a method to separate these cells from non liver like cells, and secondly use these cells to treat multiple mouse models of human inborn errors of metabolism. We will also establish a clinically applicable small-scale preparatory Bio-banking system to provide immunotype-matched cells to patients affected by these diseases. These immunotype-matched cells can replace the missing enzyme function in patients who suffer from congenital liver metabolic disorders, and potentially will be cure the condition. Although this proposal focuses on the congenital liver metabolic disorders, success may lead to the use of these cells in other liver diseases.
We propose to develop a technology to isolate and derive functional hepatic cells from discarded human placentae. The therapeutic cells will be utilized to treat congenital metabolic disorders. Current therapy for congenital metabolic disorders requires life-long treatment. It is easy to imagine how the economical burden afflicts the patients' families and society. If successful, immuotype matched hAEC-derived cell replacement therapy may completely cure some of the congenital metabolic disorders. The benefit of this new regenerative medicine will be tremendous not only for the patients' quality of life but also for our society. Although this proposal focuses on the congenital liver metabolic disorders, the target disease can potentially be extended to other liver diseases. This cell therapy would be the first cell therapy for liver disease and could benefit thousands of patients in California who suffer various liver diseases.
Furthermore, once this therapeutic potential is demonstrated, a placenta collection system, placental stem cell banking system, and a stem cell-derived hepatic cell distribution system might be a novel industry or industries that could provide job opportunities to the citizens of California.
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.