369 GMP Isolation and Quality Assesment on human Amnion Epithelial Cell for Clinical Therapies
Monday November 16, 2015 from 15:30 to 17:00
Room 111-112

Roberto Gramignoli, Sweden

Assistant Professor

Dept. of Laboratory Medicine

Karolinska Institute


GMP Isolation and Quality Assesment on human Amnion Epithelial Cell for Clinical Therapies

Roberto Gramignoli1, Kristina Kannisto1, Raghuraman C Srinivasan1, Carl Jorns2, Stephen C Strom1.

1Laboratory Medicine, Karolinska Institute, Stockholm, Sweden; 2Clinical Science, Intervention and Technology , Karolinska Institute, Stockholm, Sweden

Background: Placenta is a non-controversial and readily available source of stem cells that can be used in regenerative medicine. We previously reported that amnion epithelial cells (hAEC) from term human placenta express surface markers and genes characteristic of pluripotent stem cells.  hAECs are not tumorigenic, have immunomodulatory and anti-inflammatory properties and once transplanted differentiate to hepatocyte-like cells resulting in correction of acute liver failure and mouse models of metabolic liver diseases, such as Maple Syrup Urine Disease (MSUD) and Phenylketonuria (PKU). These successful results have motivated isolation and banking of hAEC at our Institute for clinical therapy.
During the last year we have standardized reagents and procedures in accordance with current Good Manufacturing Practice (cGMP). Release criteria have been designed as quality control, and biosafety distribution of infused cells has been evaluated in two different preclinical models.
Methods: A complete profile of mRNA levels of stem cell markers (Oct-4, Nanog, Sox-2, Activin-A) was performed by qRT-PCR. Flow cytometry analysis for surface and nuclear markers was performed using several antibodies, including pluripotency markers (Nanog, Sox2, Oct4), embryonic stem cell markers (SSEA-3, SSEA-4, TRA1-60, TRA1-81), adhesion molecules (integrin subunits and several CAMs), HLA (-ABC, -DR, -G), CD24, CD31, CD34, CD44, CD45, CD73, CD105, CD90, CD117, E- and N-cadherins.
Results: All preparations were negative for hematopoietic markers, CD45 and CD90, while stromal markers were observed in less than 5% of cells. Adhesion molecules such as integrin subunits (CD29 and CD49f) and EpCAM were highly expressed (above 85%). Stem cell markers, SSEA-3, TRA1-60 and 1-81 positive cells comprised 12-15% of the population. We measured high level of expression in both membrane-bound and soluble HLA-G isoforms.  Ongoing analyses are addressing the hypothesis if this peculiar high level of HLA-G expression in these fetal-derived cells is on the basis of lack of rejection after long-term (100 days) in immunocompetent animal without administration of immunosuppressive drugs.
As part of preclinical safety studies, we investigated the distribution of hAECs, previously labeled, and infused into the portal vein in mice. There was almost complete localization of infused cells into the liver at 3 and 20 hours after injection, without obvious side effects. Similar findings were obtained in a large animal model (pig).
Conclusions: Preclinical studies suggest that amnion-derived epithelial stem cells are a useful alternative to hepatocytes for the treatment of liver-based inborn errors of metabolism. Isolations according to cGMP procedures in combination with completed biosafety studies will allow the translation of amnion-derived stem cells for clinical applications.

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