Human Placenta Laminin-111 as a Multifunctional Protein for Tissue Engineering and Regenerative Medicine.

Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.

Validation of an alternative microbiological method for tissue products.

According to the European Pharmacopoeia sterility testing of products includes an incubation time of 14 days in thioglycollate medium and soya-bean casein medium. In this case a large period of time is needed for product testing. So we designed a study to evaluate an alternative method for sterility testing. The aim of this study was to reduce the incubation time for the routinely produced products in our tissue bank (cornea and amnion grafts) by obtaining the same detection limit, accurateness and recovery rates as the reference method described in the European Pharmacopoeia. The study included two steps of validation. Primary validation compared the reference method with the alternative method. Therefore eight bacterial and two fungi test strains were tested at their preferred milieu. A geometric dilution series from 10 to 0.625 colony forming unit per 10 ml culture media was used. Subsequent to the evaluation the second part of the study started including the validation of the fertility of the culture media and the parallel testing of the two methods by investigating products. For this purpose two product batches were tested in three independent runs. Concerning the validation we could not find any aberration between the alternative and the reference method. In addition, the recovery rate of each microorganism was between 83.33 and 100 %. The alternative method showed non-inferiority regarding accuracy to the reference method. Due to this study we reduced the sterility testing for cornea and amniotic grafts to 9 days.

In toto differentiation of human amniotic membrane towards the Schwann cell lineage.

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100ß, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100ß was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.

Intact human amniotic membrane differentiated towards the chondrogenic lineage.

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.

Proteomic analysis of human-derived cell culture supplements.

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.

Human-derived alternatives to fetal bovine serum in cell culture.

OBJECTIVE: 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.

Anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model.

The human amniotic membrane (hAM), thanks to its favorable properties, including anti-inflammatory, anti-fibrotic and pro-regenerative effects, is a well-known surgical material for many clinical applications, when used both freshly after isolation and after preservation. We have shown previously that hAM patching is a potential approach to counteract liver fibrosis. Indeed, when fresh hAM was used to cover the liver surface of rats with liver fibrosis induced by the bile duct ligation (BDL) procedure, the progression and severity of fibrosis were significantly reduced. Since cryopreservation enables safety and long-term storage of hAM but may influence its functional properties, here we compared the anti-fibrotic effects of fresh and cryopreserved hAM in rats with BDL-induced liver fibrosis. After BDL, the rat liver was covered with a piece of fresh or cryopreserved hAM, or left untreated. Six weeks later, the degree of liver fibrosis was assessed histologically using the Knodell and the METAVIR scoring systems. Digital image analysis was used to quantify the percentage of the areas of each liver section displaying ductular reaction, extracellular matrix (ECM) deposition, activated myofibroblasts and hepatic stellate cells (HSCs). Liver collagen content was also determined by spectrophotometric technique. The degree of liver fibrosis, ductular reaction, ECM deposition, and the number of activated myofibroblasts and HSCs were all significantly reduced in hAM-treated rats compared to control animals. Fresh and cryopreserved hAM produced the same anti-fibrotic effects. These findings indicate that cryopreservation maintains the anti-fibrotic properties of hAM when used as a patch to reduce the severity of liver fibrosis.

Development and validation of a production process of platelet lysate for autologous use.

Growth factors (GF) contained in platelets are a potential source to improve wound healing by the stimulation and acceleration of soft tissue and bone healing. This resulted in the idea that autologous platelet-rich plasma or platelet lysate (PL) containing high levels of GF might improve healing processes. Today platelet products are already applied in bone and maxillofacial surgery. In recent years, cosmetic surgery and facial rejuvenation procedures are growing steadily. New methods including platelet products aiming to induce non-surgical reduction of wrinkles upon topical injection and to minimize surgical risks in general are developed. Several point-of-care devices are already available on the market. However, the amount of PL obtained by these kits is far too high for certain applications in cosmetic surgery and they offer no possibility of storing the remaining material in a sterile manner. Therefore we developed a procedure for the sterile production of smaller amounts of PL in a closed system that can also be split into several products for repeated administration. The closed system was determined to be a bag system designed for an autologous blood donation of 100 ml whole blood. We set a special focus on the validation of the production procedure, mainly regarding sterility and platelet recovery. For validation 22 healthy volunteers were asked for a blood donation, which was centrifuged twice to obtain concentrated platelets (CP). A freeze-thaw cycle caused lysis of the CP to get approximately 8.48 ± 1.36 ml PL. We yielded satisfying results of 100% sterility and a platelet recovery of 36.92% ± 18.71%. We therefore conclude that the PL obtained is ready for studies comparing it with traditional treatments.

Novel Human Placenta-Based Extract for Vascularization Strategies in Tissue Engineering.

There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.

Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.

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.

Cellular and Site-Specific Mitochondrial Characterization of Vital Human Amniotic Membrane.

Over a century ago, clinicians started to use the human amniotic membrane for coverage of wounds and burn injuries. To date, literally thousands of different clinical applications exist for this biomaterial almost exclusively in a decellularized or denuded form. Recent reconsiderations for the use of vital human amniotic membrane for clinical applications would take advantage of the versatile cells of embryonic origin including the entirety of their cell organelles. Recently, more and more evidence was found, showing mitochondria to be involved in most fundamental cellular processes, such as differentiation and cell death. In this study, we focused on specific properties of mitochondria of vital human amniotic membrane and characterized bioenergetical parameters of 2 subregions of the human amniotic membrane, the placental and reflected amnion. We found significantly different levels of adenosine triphosphate (ATP) and extracellular reactive oxygen species, concentrations of succinate dehydrogenase, and lactate upon inhibition of ATP synthase in placental and reflected amnion. We also found significantly different rates of mitochondrial respiration in isolated human amniotic epithelial cells and human amniotic mesenchymal stromal cells, according to the subregions. Differences in metabolic activities were inversely related to mitochondrial DNA copy numbers in isolated cells of placental and reflected amnion. Based on significant differences of several key parameters of energy metabolism in 2 subregions of vital amnion, we propose that these metabolic differences of vital placental and reflected amnion could have critical impact on therapeutic applications. Inclusion of region-specific metabolic properties could optimize and fine-tune the clinical application of the human amniotic membrane and improve the outcome significantly.

Oxygen Tension Strongly Influences Metabolic Parameters and the Release of Interleukin-6 of Human Amniotic Mesenchymal Stromal Cells In Vitro.

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. In vivo (in utero), cells of the hAM are exposed to low oxygen tension (1-4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.

Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: a comparison with human mesenchymal stem cells from adipose tissue.

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.

An Effective Method of Atelocollagen Type 1/3 Isolation from Human Placenta and Its In Vitro Characterization in Two-Dimesional and Three-Dimensional Cell Culture Applications.

Pepsin-solubilized atelocollagen can be used to form highly complex three-dimensional matrices for a broad spectrum of tissue engineering applications. Moreover, it has a long history as a favorable biomaterial in pharmaceutical and medical industries. So far, the main sources for these approaches are collagens from xenogenic sources. Yet, these nonhuman collagens carry a risk of provoking immune reactions in patients. Here we describe an effective method of isolating atelocollagen type 1/3 (COL1/3) from human placenta. By combining a single pepsin digestion step with tangential flow filtration and further precipitation steps, we could purify COL1/3 within only 4 days of processing. The resulting COL1/3 was biochemically characterized by determining residual DNA content, proving the absence of impurities by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis combined with total amino acid quantification, identifying the isolated collagen types by Western blot analysis, and analyzing the spontaneous formation of fibrous structures on freeze-drying via scanning electron microscopy. Finally, the cytocompatibility of the isolated collagen was demonstrated in two dimensional using primary rat hepatocytes and in three dimensional by a sprouting assay of human umbilical vein endothelial cell. The isolation method described not only fulfills demands for cost-efficient bioengineering using a human waste material but also potentially increases overall safety for patients by use of homologous products.

Detection and relocation of rare events. A comparative study using the laser scanning cytometer and the Metafer/RCDetect microscope scanning system.

We compared instrumental analysis of enriched cord blood nucleated red blood cells (CB-NRBC) out of in vitro contamination preparations of dilutions of minute volumes of male cord blood into peripheral blood from nonpregnant women. This was done using the laser scanning cytometer (LSC) and the Metafer/RCDetect microscope scanning system, both allowing for relocation of positive cells defined on the basis of fluorescence parameters. Both instruments were efficient in performing scanning and relocation; a difference in the recovery of CB-NRBC was not significant and can be explained by the method of preparation used.

Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites.

Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative.

Secondary correction of posttraumatic orbital wall adhesions by membranes laminated with amniotic membrane.

The objective of the study was to find out if human amniotic membrane could be used for corrective surgery after trauma to the orbital wall. Because of its proposed antiadhesive qualities, it seemed to be potentially suitable. We studied 8 men (mean age 37 (range 19-74) years) who had deficient ocular movement after fractures of the orbital floor. Five of them had already been operated on. Inclusion criteria were trauma dating back more than 4 months and a soft tissue stricture in the orbital floor diagnosed by magnetic resonance imaging. Patients were treated secondarily with lysis of adhesions and insertion of allogeneic human amniotic membrane laminated on to polyglactin 910/polydioxanone foil, which functioned as the carrier material. Patients were followed up for 3 months, by which time disorders of motility of the ocular bulb had disappeared completely in 5. Two patients had improved motility and a reduction in both their subjective and objective symptoms. One patient had no improvement. The considerable reduction in adhesions and scarring after insertion of the membrane confirms previous assumptions, according to which the epithelial side of the human amniotic membrane has an antiadhesive effect because of its smooth surface.

Mesenchymal stem or stromal cells from amnion and umbilical cord tissue and their potential for clinical applications.

Mesenchymal stem or stromal cells (MSC) have proven to offer great promise for cell-based therapies and tissue engineering applications, as these cells are capable of extensive self-renewal and display a multilineage differentiation potential. Furthermore, MSC were shown to exhibit immunomodulatory properties and display supportive functions through parakrine effects. Besides bone marrow (BM), still today the most common source of MSC, these cells were found to be present in a variety of postnatal and extraembryonic tissues and organs as well as in a large variety of fetal tissues. Over the last decade, the human umbilical cord and human amnion have been found to be a rich and valuable source of MSC that is bio-equivalent to BM-MSC. Since these tissues are discarded after birth, the cells are easily accessible without ethical concerns.

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table.

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.