Gene Expression in Amnion-Derived Cells Cultured on Recombinant Laminin 332—A Preliminary Study

Human amniotic cells (hAC) exhibit characteristics of undifferentiated cells and immunomodulatory properties. Recognition of the relationship between amniotic cells and components of the extracellular matrix is an important condition for their ex vivo preparation and further successful clinical application in regenerative medicine and transplantology. Laminin 332 (LN-332), as a natural component of the basement membrane of amniotic epithelial cells and a ligand for integrin receptors, may strongly influence the phenotype and fate of amniotic cells. We investigated the impact of recombinant LN-332 on hAC viability and expression of markers for pluripotency, early differentiation, adhesion, and immunomodulatory properties. During 14 days of culture, hAC were quantified and qualified by light microscopy, immunohistochemistry, immunocytochemistry, and flow cytometry. Gene expression was assessed with real-time polymerase chain reaction (RT-PCR) arrays and compared with differentiated cells originated from the three germ layers. LN-332 caused an over 2-fold increase in the total number of hAC, accompanied by a 75% reduction of SSEA-4-positive cells and an increase in HLA-ABC-positive cells. In particular, we observed that the presence of laminin 332 in the medium of a short-time culture modifies the effect of culture duration on hAC, enhancing time-dependent inhibition of expression of certain genes, including pluripotency and differentiation markers, laminin 332 subunits (which may be part of self-regulation of LN-332 synthesis by amniotic cells), and integrins. The changes observed in hAC were more distinct with respect to differentiated mesenchymal cells, resulting in more comparable phenotypes than those represented by differentiated endo- and ectodermal cells. We concluded that laminin 332 present in the culture medium influences to a certain extent proliferation, adhesion, and differentiation of amniotic cells in culture.

Immunosuppressive Potential of Activated Human Amniotic Cells in an Experimental Murine Model of Skin Allo- and Xenotransplantation

Isolated human amniotic cells (hAC) could be used as a source of immunomodulatory factors in regenerative medicine and transplantation. However, in previous experimental studies, native hAC administered to skin graft recipients did not induce graft immunotolerance. To strengthen the immunomodulatory properties of hAC prior to administration to the recipient, we activated them ex vivo using pro-inflammatory cytokines. In this study, we compared the transplantation efficiency of skin allografts (mouse to mouse) and xnografts (rat to mouse) in recipient mice divided into three main groups receiving: 1. Placebo (control group); 2. Cyclosporine A (CsA) [10 or 50 mg/kg body weight (bw)]; 3. suspension of hAC activated ex vivo by IL-1β and INFγ, administered into a tail vein or subcutaneously. During 15 days of observation, hAC administered intravenously or subcutaneously after allotransplantation appeared to be as safe and efficient as CsA at the dose of 10 mg/kg bw in preventing rejection of skin allo- and xenografts. After xenotransplantation, however, only hAC administered intravenously prevented rejection to an extent comparable to CsA. Both CsA (10 mg/kg bw) and activated hAC reduced inflammatory infiltration in the skin (after intravenous injection) and did not increase the concentration of the inflammation marker SAP in serum or percentage of leukocytes in blood. Finally, we concluded that administration of activated hAC is safe and efficient in the presented animal model of skin allo- and xenotransplantation in a route-dependent manner. Activated hAC injected intravenously exhibit an immunosuppressive effect comparable to CsA administered at the dose of 10 mg/kg bw in both allo- and xenotransplantation.

Current Trends in Orthobiologics: An 11-Year Review of the Orthopaedic Literature

Background: The use of “orthobiologics” or regenerative therapies in orthopaedic surgery has grown in recent years. Particular interest has been raised with regard to platelet-rich plasma, bone marrow aspirate, adipose-derived cells, and amniotic cells. Although studies have analyzed outcomes after orthobiologic treatment, no study has analyzed how the literature as a whole has evolved. Purpose: To evaluate trends in platelet-rich plasma, bone marrow aspirate, adipose-derived cells, and amniotic cell publications and to assess how these might inform efforts to establish minimum reporting standards and forecast future use. Study Design: Systematic review; Level of evidence, 4. Methods: A database was compiled systematically using PubMed to identify articles published between 2009 and 2019 within 9 prominent orthopaedic journals and pertaining to the use of platelet-rich plasma, bone marrow aspirate, adipose-derived cells, and amniotic cells in the treatment of musculoskeletal conditions. Included articles were classified as clinical, nonclinical (translational or basic science), or review, and a variety of study parameters were recorded for each. Additional queries were performed to identify articles that utilized minimum reporting standards. Results: A total of 474 articles (132 clinical, 271 nonclinical, 71 review) were included, consisting of 244 (51.5%) platelet-rich plasma, 146 (30.8%) bone marrow aspirate, 72 (15.2%) adipose-derived cells, and 12 (2.5%) amniotic cells. The greatest annual increase in publications for each orthobiologic topic was from 2018 to 2019. The American Journal of Sports Medicine demonstrated the highest number of overall (34.2%) and clinical (50.0%) publications, and accounted for 44.3% of all platelet-rich plasma publications. The Journal of Orthopaedic Research accounted for the second highest overall number of publications (24.9%) and highest nonclinical publications (41.0%). Platelet-rich plasma accounted for 91.5% of all level 1 clinical studies, while much greater than half of bone marrow aspirate, adipose-derived cells, and amniotic cell publications were level 3 or lower. Out of the 207 articles that used some form of reporting protocol, 59 (28.5%) used an established algorithm and 125 (60.4%) used their own. Conclusion: Interest in orthobiologics continues to grow, as evidenced by an increasing trend in publications over an 11-year period. However, current reporting on orthobiologic formulations is largely heterogeneous, emphasizing the need for minimum reporting standards and higher-quality studies.

Reprogrammed iBlastoids contain amnion-like cells but not trophectoderm

Two recent papers in Nature show that human blastocyst-like structures (or blastoids) can be generated from human pluripotent stem cells (Yu et al 2021) or through reprogramming of fibroblasts (Liu et al 2021), respectively. Both papers perform extensive single cell transcriptional analysis and compare blastoid cells with the cells in preimplantation human embryos, leading to a conclusion that the blastoids contain cell lineages corresponding to the epiblast, primitive endoderm and trophectoderm in preimplantation human embryos. Transcriptional analysis is, however, critically dependent on having relevant reference samples, not only of targeted cell types but also of potential alternative cell lineages. For this reason, we have reevaluated the blastoid data with a more comprehensive cellular reference, including extended cultures of blastocysts, several stem cell-based embryo models and a gastrulation stage human specimen. From this reanalysis we resolve that reprogrammed blastoids by Liu et al. fail to generate cells with trophectoderm profiles. Instead, cells identified as trophectoderm lineages in reprogrammed blastoids possess a transcriptional profile more representative of amniotic cells in post-implantation human embryos. Our reanalysis also shows that stem cell-derived blastoids did contain trophectoderm-like cells, highlighting the potential of human blastoids to model blastocyst development.

Stem cells and COVID-19: are the human amniotic cells a new hope for therapies against the SARS-CoV-2 virus?

A new coronavirus respiratory disease (COVID-19) caused by the SARS-CoV-2 virus, surprised the entire world, producing social, economic, and health problems. The COVID-19 triggers a lung infection with a multiple proinflammatory cytokine storm in severe patients. Without effective and safe treatments, COVID-19 has killed thousands of people, becoming a pandemic. Stem cells have been suggested as a therapy for lung-related diseases. In particular, mesenchymal stem cells (MSCs) have been successfully tested in some clinical trials in patients with COVID-19. The encouraging results positioned MSCs as a possible cell therapy for COVID-19. The amniotic membrane from the human placenta at term is a valuable stem cell source, including human amniotic epithelial cells (hAECs) and human mesenchymal stromal cells (hAMSCs). Interestingly, amnion cells have immunoregulatory, regenerative, and anti-inflammatory properties. Moreover, hAECs and hAMSCs have been used both in preclinical studies and in clinical trials against respiratory diseases. They have reduced the inflammatory response and restored the pulmonary tissue architecture in lung injury in vivo models. Here, we review the existing data about the stem cells use for COVID-19 treatment, including the ongoing clinical trials. We also consider the non-cellular therapies that are being applied. Finally, we discuss the human amniotic membrane cells use in patients who suffer from immune/inflammatory lung diseases and hypothesize their possible use as a successful treatment against COVID-19.

Critical Impact of Human Amniotic Membrane Tension on Mitochondrial Function and Cell Viability In Vitro

Amniotic cells show exciting stem cell features, which has led to the idea of using living cells of human amniotic membranes (hAMs) in toto for clinical applications. However, under common cell culture conditions, viability of amniotic cells decreases rapidly, whereby reasons for this decrease are unknown so far. Recently, it has been suggested that loss of tissue tension in vivo leads to apoptosis. Therefore, the aim of this study was to investigate the effect of tissue distention on the viability of amniotic cells in vitro. Thereby, particular focus was put on vital mitochondria-linked parameters, such as respiration and ATP synthesis. Biopsies of hAMs were incubated for 7–21 days either non-distended or distended. We observed increased B-cell lymphoma 2-associated X protein (BAX)/B-cell lymphoma (BCL)-2 ratios in non-distended hAMs at day seven, followed by increased caspase 3 expression at day 14, and, consequently, loss of viability at day 21. In contrast, under distention, caspase 3 expression increased only slightly, and mitochondrial function and cellular viability were largely maintained. Our data suggest that a mechano-sensing pathway may control viability of hAM cells by triggering mitochondria-mediated apoptosis upon loss of tension in vitro. Further studies are required to elucidate the underlying molecular mechanisms between tissue distention and viability of hAM cells.

NPs-TiO2 and Lincomycin Coexposure Induces DNA Damage in Cultured Human Amniotic Cells

Titanium dioxide nanoparticles (NPs-TiO2 or TiO2-NPs) have been employed in many commercial products such as medicines, foods and cosmetics. TiO2-NPs are able to carry antibiotics to target cells enhancing the antimicrobial efficiency; so that these nanoparticles are generally used in antibiotic capsules, like lincomycin, added as a dye. Lincomycin is usually used to treat pregnancy bacterial vaginosis and its combination with TiO2-NPs arises questions on the potential effects on fetus health. This study investigated the potential impact of TiO2-NPs and lincomycin co-exposure on human amniocytes in vitro. Cytotoxicity was evaluated with trypan blue vitality test, while genotoxic damage was performed by Comet Test, Diffusion Assay and RAPD-PCR for 48 and 72 exposure hours. Lincomycin exposure produced no genotoxic effects on amniotic cells, instead, the TiO2-NPs exposure induced genotoxicity. TiO2-NPs and lincomycin co-exposure caused significant increase of DNA fragmentation, apoptosis and DNA damage in amniocytes starting from 48 exposure hours. These results contribute to monitor the use of TiO2-NPs combined with drugs in medical application. The potential impact of antibiotics with TiO2-NPs during pregnancy could be associated with adverse effects on embryo DNA. The use of nanomaterials in drugs formulation should be strictly controlled in order to minimize risks.