Human amniotic epithelial cells

The human amnion is a tissue of fetal origin consisting of amniotic epithelial (AE) cells on a basement collagenous membrane, an acellular compact layer filled with reticular fibres, a fibroblast layer with Hofbauer cells/histiocytes and a highly hygroscopic spongy layer with fibrils between the chorion and the amniotic sac.

hAECs can be obtained from the amnion membrane with differential enzymatic digestion after it is separated from the underlying chorion . The ease of hAEC isolation, their ability to differentiate into all three germ layers, their low immunogenicity, anti-inflammatory properties and their non-controversial nature have contributed in their promising potential for regenerative medicine therapies . The amnion contains epithelial cells that express a variety of surface markers with positive expression of embryonic cell markers such as stage-specific antigens (SSEAs) 3 and 5, Tra-1-60, Tra-1-81 and mesenchymal and haematopoietic markers CD105, CD90, CD73, CD44, CD29, HLA-A, -B, -C, CD13, CD10, CD166 and CD117. hAEC pluripotency was hypothesized due to the fact that they originate from the amnion membrane, and they are formed from the epiblast 2 weeks after gastrulation occurs. hAECs can mature under specific culture conditions into neuronal cells that synthesize acetylcholine, norepinephrine and dopamine , and they can express Oct-4, SOX-2 and Nanog . hAECs also appear to be resistant to rejection after allotransplantation probably due to their immunosuppression properties (CD59 and human leukocyte antigen (HLA)-G ). In vivo, hAECs have been reported to lower the blood glucose levels of streptozotocin-induced diabetic mice several weeks after implantation, potentially by differentiation into beta cells .

For clinical application, hAECs have been considered for a broad variety of conditions including ophthalmic diseases, lung fibrosis, liver fibrosis, multiple sclerosis, congenital metabolic disorders such as ornithine transcarbamylase deficiency, familial hypercholesterolaemia, spinal cord injuries, Parkinson’s disease and for allogeneic cell transplantations . Recently, human amniotic membrane (hAM) has been tested as a biological template through decellularization to repair human tissue defects, and when compared with polypropylene mesh, it possessed higher biocompatibility after 10 months of implantation into a rabbit model . However, the optimal recellularization strategies with hAECs and their differentiated types remain to be investigated.

Cultured hAECs produce albumin and alpha-fetoprotein, and they show glycogen storage and hepatic differentiation potential in vitro . In vitro, hAECs had the capacity to metabolize ammonia, testosterone and 17a-hydroxyprogesterone caproate, whereas they expressed hepatocyte markers such as albumin, A1AT, CYP2A4, 3A7, 1A2, 2B6, ASGPR1 and inducible fetal cytochromes. After intrahepatic transplantation into immunodeficient (SCID)/beige mice, hAECs demonstrated functional hepatic characteristics , and following the pretreatment of SCID/beige mice with retrorsine (RS), hAECs expressed mature liver genes, plasma proteins and hepatic enzymes to a level equal to adult liver tissue. Recently, hAECs showed therapeutic efficacy after transplantation in a mouse model of cirrhosis . The relevant properties of hAECs with hepatic progenitors such as the expression of FoxA2 and GATA-4, a1-anti-trypsin and OTC suggest their potential in liver regeneration. However, further evidence is required on the expression of genes associated with the metabolism of cholesterol, fat urea and xenobiotics such as low-density lipoprotein (LDL) and intracerebral haemorrhage (ICH) as they are associated with functional properties of hepatocytes.

Human amniotic mesenchymal stromal cells (hAMSCs) and chorionic mesenchymal stromal cells (hCMSCs)

Mesenchymal stem cells are found in a variety of sites including the bone marrow, adipose tissue, tooth buds, umbilical cord blood, Wharton’s jelly, amniotic fluid, muscle, lungs and the placenta . Placental mesenchymal stromal cells are derived from the extraembryonic mesoderm. They show trilineage differentiation potential, maintain long telomeres, express markers associated with pluripotency such as Oct4 and SSEA-3, although the level of SSEA-4 expression is controversial , and they have immunoprivileged characteristics, augmented proliferative rates and high clonogenicity. Both hAMSCs and hCMSCs express limited levels of HLA-A,B,C suggesting an immunoprivileged profile , and their remarkable immunosuppressive properties contribute to their usage in a transplantation setting. For example, human placenta-derived MSCs can engraft in multiple organs in vivo, and they can actively migrate when transplanted intraperitoneally and intravenously with the expression of migration molecules P-selectin, L-selectin and CD29 .

Placenta-derived MSCs exhibit the same morphological features as adipose-derived MSCs, and they have a positive expression for CD29, CD44, CD105 and CD166 with similar osteogenic differentiation capacity . Zhu et al. studied the effect of adenovirus-mediated gene transfer of LIM mineralization protein-1 (LMP-1) on human placental-derived MSCs . LMP-1 plays an important role in osteoblast differentiation, maturation and bone formation. Using a proteomic approach, the authors identified changes in cytoskeletal proteins, cadmium-binding proteins and metabolic proteins in transduced cells that suggest a role in osteogenic differentiation. Chang et al. have shown the neurogenic potential of hAM- MSCs at early passages from term placental samples identified by neuronal-specific markers and their ability to secrete dopamine. Neural differentiation of human decidual placental stem cells can be mediated using the biomechanical properties of carbon nanotube–collagen substrates without the need for proneural media supplements .

The properties of the MSCs vary according to the developmental stage and gestation of the placental source and chorionic villi. Cells selected at lower gestational ages exhibit greater generation doubling times, proliferative potential, differentiation capacity and phenotypic stability than cells sourced from term placental tissue . In addition, the characteristics, function and therapeutic potential of placental MSCs differ according to their fetal and maternal origin . Fetal placental MSCs express high levels of hepatocyte growth factor and CD200, and they have higher immunosuppressive properties in vivo compared with maternal origin placental MSCs. hAMSCs and hCMSCs have a direct suppressive effect on the proliferation of T-lymphocytes from human adult peripheral blood and umbilical cord blood in vitro . In vivo studies in immune-competent rats and pigs show that hAMSCs can exhibit extended survival with no evidence of immunological rejection after xenogeneic transplantation . Wolbank et al. were able to establish human amnion-derived MSC (hAMC) lines through the ectopic expression of the catalytic subunit of human telomerase (hTERT) , which is known to extend the cellular lifespan of bone marrow and adipose tissue stem cells with a high differentiation capacity . However, this hTERT transduction methodology may compromise the immunomodulatory properties of the hAMC cells, thus rendering the cells incompatible with biobanking procedures and compromising their use in allogeneic cell therapies.

Bone marrow-derived MSCs and human placenta-derived MSCs appear to have similar patterns of adhesion molecules (β1 integrins CD29, CD44, CD54, E-cadherin, CD166, CD49, integrin a11 and chemokine receptor displays (CCR1, CCR3, CXCR3, CXCR4 and CXCR6)), including the absence of selectin and fucosyltransferase expression . On this basis, placenta-derived MSCs show promising biological properties that could be maintained under good manufacturing practice (GMP)-grade culture, and they can be widely used in translational medicine.

Clinically, placental mesenchymal stromal cells have been used in a variety of conditions. Ringden et al. used placenta-derived decidual stromal cells to treat steroid refractory acute graft-versus-host disease (GVHD), and they showed 1 year 73% survival rates in patients versus 6% in retrospective controls . Placenta-derived MSCs can accelerate wound healing, and they have been used as transplants to close cutaneous wounds and to treat epidermolysis bullosa , an inherited blistering skin condition. Fetal membrane MSCs are susceptible to human herpes virus infection ; thus, there is the potential to use modified herpes viruses for gene transfer.

Haematopoietic stem cells

For the past decade, the placenta has been investigated as a potential niche source of haematopoietic stem cells (HSCs), a type of stem cells that are formed during a limited time window of embryogenesis. The extraembryonic yolk sac is the pool of uni- or oligolineage progenitors that exhibit erythroid and myeloid potential, and it can give rise to HSCs under specific extracellular signalling pathways . The common origin of endothelial and HSCs is identified by the co-expression of the markers CD31/CD34, C-kit and Sca-1 as well as the role of Hedgehog (Hh) signalling pathway and the common requirement of Vegf and Notch . The placenta is an enabling haematopoietic microenvironment for growth and expansion of HSCs, but it is not certain whether HSCs are intrinsically or extrinsically generated. In mouse development, Runx1 is expressed in both endothelial cells within the blood vessels and haematopoietic and mesenchymal cells in the placenta, and it may be involved in HSC emergence . Portilho et al. have recently reported that only the labyrinth region in mouse placenta seemed to have a haematopoietic activity near trophoblast giant cell activity . The placenta niche encourages the proliferation and expansion of HSC without inducing myeloerythroid lineage differentiation, but further studies are needed to investigate the kinetics and pathways of HSC activity during placental maturation.

Clinically, Elmacken et al. reported in a pilot trial the administration of unrelated donor placenta-derived stem cells in conjunction with unrelated single or double umbilical cord blood transplantation in children and adults with malignant and non-malignant diseases giving high tolerance levels .