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Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications.

Human amniotic membrane (hAM) represents a tissue that is well established as biomaterial in the clinics with potential for new applications in regenerative medicine. For tissue engineering (TE) strategies, cells are usually combined with inductive factors and a carrier substrate. We have previously recognized that hAM represents a natural, preformed sheet including highly potent stem cells. In the present approach for cartilage regeneration we have induced chondrogenesis in hAM in vitro. For this, hAM biopsies were cultured for up to 56 days under chondrogenic conditions. The induced hAM was characterized for remaining viability, glycosaminoglycan (GAG) accumulation using histochemical analysis, and a quantitative assay. Collagen I, II and X was immunohistochemically determined and cartilage-specific mRNA expression of (sex determining region Y-) box 9, cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), versican (CSPG2), COL1A1, COL9A2, melanoma inhibitory activity (MIA), and cartilage-linking protein 1 (CRTL1) analyzed by quantitative real-time polymerase chain reaction. Human AM was successfully induced to accumulate GAG, as demonstrated by Alcianblue staining and a significant (p < 0.001) increase of GAG/viability under chondrogenic conditions peaking in a 29.9 ± 0.9-fold induction on day 56. Further, upon chondrogenic induction collagen II positive areas were identified within histological sections and cartilage-specific markers including COMP, AGC1, CSPG2, COL1A1, COL9A2, MIA, and CRTL1 were found upregulated at mRNA level. This is the first study, demonstrating that upon in vitro induction viable human amnion expresses cartilage-specific markers and accumulates GAGs within the biomatrix. This is a promising first step towards a potential use of living hAM for cartilage TE.

Pulmonary surfactant (PS) reduces surface tension at the air-liquid interface in the alveolar epithelium of the lung, which is required for breathing and for the pulmonary maturity of the developing foetus. However, the origin of PS had never been thoroughly investigated, although it was assumed to be secreted from the foetal developing lung. Human amniotic membrane (hAM), particularly its epithelial cell layer, composes the amniotic sac enclosing the amniotic fluid. In this study, we therefore aimed to investigate a potential contribution of the cellular components of the hAM to pulmonary surfactant found in amniotic fluid. We identified that cells within the native membrane contain lamellar bodies and express all four surfactant proteins as well as ABCA3. Lipidomic profiling by nanoESI – MS/MS revealed the presence of the essential lipid species as found in PS. Also, the biophysical activity of conditioned cell culture supernatant obtained from hAM was tested with captive bubble surfactometry. hAM supernatant showed the ability to reduce surface tension, similar to human PS obtained from bronchoalveolar lavage. This means that hAM produces the essential PS-associated components and can therefore contribute as second potential source of PS in amniotic fluid aside from the foetal lung.

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

Amniotic membrane is a highly promising cell source for tissue engineering. Being part of the placenta, this tissue is abundantly available. It can be processed easily to yield large amounts of epithelial and mesenchymal cells that have shown broad differentiation potential. For tissue-engineering purposes, cells may be applied either directly after isolation from the tissue or after a period of in vitro expansion to obtain higher cell numbers. In order to investigate the advantages and drawbacks of these strategies we compared freshly isolated and cultivated human amniotic epithelial cells (hAEC) regarding their surface antigen (Ag) expression profile and osteogenic differentiation capacity.Expression of surface Ag that are characteristic for mesenchymal stromal and embryonic stem cells was analyzed by flow cytometry. Different protocols for osteogenic and adipogenic differentiation were compared.We have demonstrated that expression of surface Ag changes dramatically during cultivation of hAEC. While not or only weakly expressed on primary isolates, the mesenchymal markers CD13, CD44, CD49e, CD54, CD90 and CD105 are strongly up-regulated during in vitro propagation. In contrast, expression of the embryonic markers TRA-1-60 and TRA-1-81, but not SSEA-4, rapidly decreases upon cultivation. This phenotypic shift is associated with a reduction in osteogenic differentiation.Our results suggest that phenotypic alterations of hAEC during in vitro cultivation might be responsible for a functional reduction of the differentiation potential, which has to be considered for the potential application of these cells in regenerative medicine.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been used for allogeneic application in tissue engineering but have certain drawbacks. Therefore, stem cells (SC)s derived from other adult tissue sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. The aim of our study was to compare the immunomodulatory potential of human amniotic mesenchymal and human amniotic epithelial cells with that of human adipose-derived SCs under identical experimental conditions. We have demonstrated a dose-dependent inhibition of peripheral blood mononuclear cell (PBMC) immune responses in mixed lymphocyte reactions (up to 66-93% inhibition) and phytohemagglutinin activation assays (up to 67-96% inhibition). The lowest SC-to-PBMC ratio able to inhibit PBMC proliferation significantly was 1:8. Subcultivation (passage 2-6) did not alter immunoinhibitory properties, whereas cryopreservation significantly reduced the immunomodulatory potential. Using transwell systems, we have demonstrated an inhibition mechanism that is dependent on cell contact. Additionally, in coculture with allogeneic PBMCs, SCs were well tolerated and at most provoked mild alloreactions in singular cases. This study demonstrates, for the first time, contact- and dose-dependent immunosuppression of mesenchymal and epithelial amniotic SC populations, as well as of adipose tissue-derived SCs. All three cell types may be considered as possible alternatives to BMSCs for allogeneic application in tissue engineering.

Objective: Development of cell therapy and advanced therapy medicinal products depends on in vitro expansion of human cells in fetal bovine serum (FBS) supplemented media. Human-derived supplements, such as human serum (huS) and human platelet lysate (hPL), represent suitable alternatives to FBS. Various studies demonstrated that the use of these human alternatives result in comparable or even improved proliferation and expansion ratios. Methods: Within this study three human supplement alternatives, huS, hPLP (plasma containing hPL) and hPLN (plasma replaced by saline), were compared by 2D gel electrophoresis, an important tool in proteomic analysis. 2D gel electrophoresis allows the determination of the protein number and the detection of protein changes (decreasing/increasing concentration). Results and Conclusion: The comparison of huS, hPLP, and hPLN gels resulted in clearly visible differences in protein pattern, protein number and concentration, particularly when comparing huS with hPL and hPLP with hPLN.

SummaryObjective: The need for an alternative to fetal bovine serum (FBS) is known to scientists and users involved in cell therapy or advanced therapy medicinal products. Human serum (huS) and platelet lysate (hPL) can be used as alternatives resulting in similar or even superior results concerning cell expansion. Methods: We developed protocols for the production of huS and two types of hPL and tested them in the expansion of human fibroblasts and adipose tissue-derived stem cells (ASC). Quality control included cell counts (platelets, red and white blood cells), sterility testing, pH levels, total protein concentrations and growth factor levels. ASC and fibroblasts were expanded for three passages in media supplemented with FBS, huS or hPL and evaluated microscopically. Proliferation in terms of population doubling times (PDT) was determined. In case of ASC, differentiation was performed as well. Results: All three alternatives demonstrated shorter PDT for fibroblasts and ASC compared to FBS. Furthermore, ASC maintained their differentiation potential. Conclusion: We conclude that hPL and huS can be used as alternatives to FBS for the cultivation and expansion of cells intended for human use.

Preserved amniotic membrane (AM) has been used in the field of ophthalmology and wound care due to its bacteriostatic, antiphlogistic, protease-inhibiting, re-epithelialization, wound-protecting and scar formation-reducing properties. Typically, AM is applied after banking in a glycerol-preserved or freeze-dried state. Cell viabilities in different forms of preparation vary substantially, which in consequence may also be reflected in the amount and type of growth factors released from the preserved material. Therefore, we characterized the angiogenic factor (AF) profile released from different AM preparations. For this, medium was conditioned with non-preserved, glycerol- and cryo-preserved AM for 48 h, which was screened for AFs using a protein array system. In parallel, the preparations were tested for cell viability. Non-preserved as well as cryo-preserved AM maintained viabilities at 106.5 ± 23.9% and 21.9 ± 23.3%, respectively, whereas glycerol-preserved AM was found to be non-viable. Of the 20 investigated factors, high levels of angiogenin, GRO, IL-6/8, TIMP-1/2 and MCP-1 and low levels of EGF, IFNγ, IGF-1, leptin, RANTES, TGFβ1 and thrombopoietin were identified to be secreted from non-preserved AM. Cryo-preserved AM secreted high levels of IL-8, intermediate levels of GRO and TIMP-1/2 but only low levels of angiogenin, IFNγ, IL-6 and MCP-1 and no detectable EGF, IGF-1, leptin, RANTES, TGFβ1 and thrombopoietin. After banking in glycerol, AM releases only minute amounts of TIMP-1/2. Along with viability, the AF profile of amniotic membrane largely depends on the preparation method applied for banking. This should be considered for selection of an AM product for a specific clinical application. Copyright © 2009 John Wiley & Sons, Ltd.

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23–24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.

Up to now freeze-dried, gamma-sterilised or glycerol-preserved amniotic membranes (AMs) have widely been used in the field of ophthalmology and wound care (e.g. leg ulcers, burns). After some preservation processes in use, like freeze-drying or glycerol-preserving, the cells in the AM are no longer viable. Within this study we evaluated the influence of different short-term and long-term storage conditions on cell viability in AM. Therefore AMs from cesarean section placentae were washed and biopsied to evaluate the microbiological status and to determine the viability of the tissue. Additionally, viability under various storage conditions was examined by assessment of mitochondrial activity. Preservation included temperatures above and below 0 degrees C as well as various media compositions. As expected, cell viability in amnion decreases during storage, in fact the effect was more pronounced when stored frozen, but the higher viability of amnion obtained by storage above 0 degrees C with medium is associated with the limitation to a short period of storage of about 28 days. The evaluated preservation methods are the basis for future non-clinical in-vivo studies in which the possible benefit of amnion as a viable biomaterial in wound healing will be investigated.

Background Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. Methods CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-γ) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. Results In CB, 59% of events double positive for Hb-γ and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. Conclusions Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible. Cytometry 48:87–92, 2002. © 2002 Wiley-Liss, Inc.