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Phase contrast image of human epithelial cells isolated from full-term amnion membrane and seeded in a collagen-coated vessel.

What? The placenta is a temporary organ that preserves and supports embryos and fetus(es) to grow and mature during pregnancy. It is the sole tissue in the human body generated by two distinct individuals: the mother and the (semi)allogenic fetus. The complexity of placenta is second only to the vast number of critical functions and properties that characterize it. In humans, such an organ lasts for approximately nine months, but not all the parts of the placenta are originated and formed simultaneously and immediately after fertilization. While several parts of the placenta originate from the hypoblast, the amnion membrane arises from the pluripotent epiblast (as later all the fetal tissues and organs do). Indeed, the very first group of cells that the embryo generates, before implantation and gastrulation, are cells that encapsulate the embryonic body to protect it from maternal immune rejection and support implantation to the uterus. These cells that extrude out on day 8 post-fertilization are called amnion or amniotic epithelial cells (AEC), and they pave the surface of the amnion membrane, a thin layer in the human placenta surrounding the fetus. The amnion membrane is an avascular tissue (contains no blood vessels), but it receives nourishment via diffusion of substances from the underlying connective tissue, through the basement membrane. The amnion membrane represents the internal surface of the placenta, and dresses the amniotic sac up, surrounding the embryo first, and later the fetus, for all months of pregnancy. The amnion membrane contains a fibrous compact layer with few mesenchymal stromal cells dispersed and physically separated from the AEC layer by the basement membrane, the thickest of the human body. The amniotic sac wraps the fetus but also prevents leakage of the amniotic fluid (initially generated by the same AEC at early phase of pregnancy), to insulate and protect the baby. The amnion membrane and sac cushion the fetus by virtue of a large number of extracellular proteins, including elastin, that AECs secrete during the first weeks of gestation.

Who? The epithelial cells covering the amnion membrane form a thin, continuous, protective layer of compactly cuboidal cells, strongly attached to a basement membrane. Like several other epithelial cells, human AECs are characterized by polygonal morphology, a large presence of mitochondria, and functional polarization leading to extensive microvilli on the cell surface and tight junctions in the basolateral membrane. Human AECs constitute approximately 80-90% of the cells present in the amnion membrane, and it has been estimated that a few hundreds of millions AEC can be found in amnionic tissue at full term. The amnion epithelial cells have been described as secreting several soluble proteins and proteases, but also releasing a great amount of vesicles, both micro-vesicles, and exosomes (1). Both intact amnion tissue (exposed to different decellularization processes) and intact, viable cells have been proposed and tested for clinical use. Notably, the amnion membrane has been used as a biomedical tool for more than one century, e.g. to cover burned and ulcerated epidermis and replacement of normal mucosa in periodontal surgery (2). Nowadays, the most common application for amnion membranes is in ophthalmology, where the membrane is spread onto the ocular surface to treat ulcers or limbal pathologies (2). In all the aforementioned applications, a decellularized product is sufficient to reverse chronic inflammation and enhance regeneration. Indeed, despite being deprived of viable cells, decellularized amnion maintains several extra-cellular proteins and growth factors, critical mediators to reverse inflammation and fibrosis, but also serving as anti-microbial and angiogenic factors (2). Decellularized products have recently been combined with, or even replaced by, intact viable cells isolated from full-term amnion membranes. The release of intact AEC has been associated with ameliorated and long-term effects in congenital disorders (3). During the past 15 years, several preclinical studies and transplants in human subjects attested to AEC immune privilege and drew attention to a plethora of additional cellular properties and characteristics that may dramatically change the way we currently treat congenital or chronic disorders, opening the gate to new interventional approaches and potential cures. Several organs and tissues have benefitted from the implantation or systemic infusion of human AEC, including the liver, lungs, pancreas, kidneys, joints, and bone, but also the brain and spinal cord (4).

When? In 2005, a group in Pittsburgh (Pennsylvania), firstly reported the stemness of AEC and attested the constitutive expression of surface markers and genes commonly observed on pluripotent stem cells (such as embryonic or induced pluripotent stem cells) (5). The same group reported human AEC capable of differentiation into all three germ layers, in response to their origin long before the fetal germ layers originate. Importantly, unlike pluripotent cells, AECs have been reported as not expressing telomerase, described as not immortal, and, more important, as non-tumorigenic. Such important features and safety profile have encouraged studies and preclinical analyses in support of AEC use in regenerative medicine or oncological treatments. Placental AEC multipotency and immunomodulatory (rather than immunosuppressive) properties may produce a paradigm shift in cell transplantation. Conversely to other current allogenic cell-based therapy, although not the patient’s own cells, AECs do not require any immunosuppressant for long-term acceptance, nor lead to diminished immune protection in the host. On the contrary, these perinatal products have been implied to play a critical role in reversing autoimmune disorders in mothers affected by such a life-threatening disease. Previous reports have highlighted clinical effects in uncomplicated pregnancies, where afflicted mothers were relieved from symptoms during the nine months of pregnancy, to later experience disorder flare once the baby (and the placenta) were expelled.

Where? The first documented use of isolated amnion cells in clinical settings has been described in 1992, when a group in Trieste (Italy) implanted subcutaneously human amnion cells to treat patients with Niemann-Pick disease (6), an inherited disease that affects the metabolism of fat (cholesterol and lipids), leading to cell death in the brain, nerves, liver, spleen, bone marrow and lungs. Repeated implantations of amnion cells have been described as an exogenous source of sphingomyelinase, the missing enzyme in this inherited lysosomal storage disease. Full-term human placentae were collected after successful caesarean section procedures, and the amnion membrane was immersed in a mixture of collagenase and trypsin to dissolve the avascular matrix and release all amnion cells. Following a short selection of cells seeded in culture for 48 hours, amnion cell products were lately implanted subcutaneously in the middle band of the chest. Such a procedure was repeated every 2-4 months, leading to described remission for all the patients. More recently, a registered phase 1 clinical trial has been initiated at Monash Children’s Hospital (Australia), where approximately one million allogeneic human AEC have been injected in premature babies (7). Six premature babies received a suspension of cryopreserved human AEC, isolated according to current Good Manufacturing Practice (GMP) pharmaceutical quality management regulations and injected in the umbilical vein. Such treatment, a first-in-human study where the injection of amnion cells was proposed as instrumental for the treatment of bronchopulmonary dysplasia, supports the safety and tolerability of AECs in allogeneic compartments without the use of any immunosuppressive drugs.

How? The release of viable and functional human AEC requires a precise and accurate methodology based on selected, clinical-grade reagents and procedures. The first description of a mechano-enzymatic procedure to selectively isolate AEC was reported in 2010 (8). Continuous refinements and procedure revisions have led to the optimization of a clinical-grade procedure, in accordance with GMP requirements (9). This advanced technology allows for the complete release of AEC from every full-term placenta, with a range recovery between 100 and 400 million cells. The cell viability has been reported as excellent by almost every group with experience in epithelial cell isolation (95% or above). Finally, human AECs have been reported to successfully withstand cryogenic procedures, allowing long-term storage and cell banking similar to current activities with cord blood products (10). The same group that detailed AEC isolation, also established criteria for the characterization and release of the final product (9). As part of safety studies, the authors described validated routes of administration for AEC suspensions to efficiently and safely implant cells in different target organs and tissues (11).

Did you know that... the amnion membrane has been recognized as an important adjunct to current medicinal treatments since ancient times. Dated as early as 400 years BC, scripts found in China and Greece describe the use of placental material and amnion membrane as healing agents for bodily injuries. The Taoists, during the Jinn Dynasty, paid great respect to material donated by Nature and believed that perinatal tissues would be instrumental to preserve health and have characteristic indications for human disease treatment, while the great tyrant of the Qin dynasty relied on extra-embryonic tissues to extract supplements for longevity. However, it was not until the first years of the 20th century that the use of amnion membrane was first documented in medical practice. Before the Great War rose in Europe, Dr. Davis at the John Hopkins Hospital, first described the use of amnion membrane as a natural bandage for burned skin (12), capable to maintain the damaged tissue moist, and supporting the natural regeneration of the skin. Since then, exponential growth in clinical reports and biomedical descriptions has been experienced, leading to a new millennium where cell-based therapies and perinatal derivatives will clearly show the way to new, important treatments and cures.

References

1. Morandi F, Marimpietri D, et al. Human Amnion Epithelial Cells Impair T Cell Proliferation: The Role of HLA-G and HLA-E Molecules. Cells 2020; 9(9):2123; doi:10.3390/cells9092123
2. Gramignoli R. Therapeutic Use of Human Amnion-Derived Products: Cell-Based Therapy for Liver Disease. Curr Pathobiol Rep 2016; 4:157–167; doi:10.1007/s40139-016-0112-8
3. Skvorak KJ, Dorko K, et al. Placental stem cell correction of murine intermediate maple syrup urine disease. Hepatology. 2013;57(3):1017; doi: 10.1002/hep.26150
4. Miki T. Stem cell characteristics and the therapeutic potential of amniotic epithelial cells. Am J Reprod Immunol. 2018;e13003; doi: 10.1111/aji.13003
5. Miki, T., Strom, S.C. Amnion-derived pluripotent/multipotent stem cells. Stem Cell Rev 2006; 2, 133; doi: 10.1007/s12015-006-0020-0
6. Bembi B, Comelli M, et al. Treatment of sphingomyelinase deficiency by repeated implantations of amniotic epithelial cells. Am J Med Genet 1992;44(4):527; doi: 1002/ajmg.1320440430
7. Lim R, Malhotra A, et al. First-In-Human Administration of Allogeneic Amnion Cells in Premature Infants With Bronchopulmonary Dysplasia: A Safety Study. Stem Cells Transl Med 2018;7(9):628; doi: 1002/sctm.18-0079
8. Miki T, Marongiu F, et al. Isolation of Amniotic Epithelial Cells. Current protocols in stem cell biology. 2007; 12:1E.3.1; doi: 10.1002/9780470151808.sc01e03s3
9. Gramignoli R, Srinivasan RC, et al. Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures. Curr Protoc Stem Cell Biol 2016;12;37:1E.10.1; doi: 10.1002/cpsc.2
10. Srinivasan R, Strom S, Gramignoli R. Effects of Cryogenic Storage on Human Amnion Epithelial Cells. Cells 2020; 9(7):1696; doi: 10.3390/cells9071696
11. Srinivasan RC, Kannisto K, et al. Evaluation of different routes of administration and bio-distribution of human amnion epithelial cells in mice. Cytotherapy 2019;21(1):113; doi: 1016/j.jcyt.2018.10.007
12. Davis J. Skin transplantation with a review of 550 cases at the John Hopkins Hospital. John Hopkins Medical Journal 1910;15:307

 
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