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For more than 100 years, the human amniotic membrane (hAM) has been used in multiple tissue regeneration applications. The hAM consists of cells with stem cell characteristics and a rich layer of extracellular matrix. Undoubtedly, the hAM with viable cells has remarkable properties such as the differentiation potential into all three germ layers, immuno-modulatory, and anti-fibrotic properties. At first sight, the hAM seems to be one structural entity. However, by integrating its anatomical location, the hAM can be divided into placental, reflected, and umbilical amniotic membrane. Recent studies show that cells of these amniotic sub-regions differ considerably in their properties such as morphology, structure, and content/release of certain bioactive factors. The aim of this review is to summarize these findings and discuss the relevance of these different properties for tissue regeneration. In summary, reflected amnion seems to be more immuno-modulatory and could have a higher reprogramming efficiency, whereas placental amnion seems to be pro-inflammatory, pro-angiogenic, with higher proliferation and differentiation capacity (e.g., chondrogenic and osteogenic), and could be more suitable for certain graft constructions. Therefore, we suggest that the respective hAM sub-region should be selected in consideration of its desired outcome. This will help to optimize and fine-tune the clinical application of the hAM.

Background In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. Methods Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. Findings Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. Interpretation Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. Funding Austrian Research Promotion Agency FFG (“CartiScaff” #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08), Graphical abstract Image, graphical abstract

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application.

Peripheral nerve fibrosis and painful adhesions are common, recurring pathological sequelae following injury. In this study, vital human amnion (hAM), an increasingly interesting biomaterial for regenerative medicine, was investigated as a novel therapy. hAM was first analyzed in vitro regarding its anti-adhesive characteristics. Then, the reflected region of hAM which was identified as more suitable, was transplanted into female Sprague Dawley rats with recurring sciatic nerve scarring (n = 24) and compared with untreated controls (n = 30) at one, four and twelve weeks. Immune response and fibrosis were investigated by (immuno)histochemical analysis. Nerve structure was examined and function determined using electrophysiology and gait analysis. Here we identified strongly reduced adhesions in the hAM-treated rats, displaying a significant difference at four weeks post transplantation compared to untreated controls (p = .0052). This correlated with the in vitro cell attachment test on hAM explants, which demonstrated a distinctly limited ability of fibroblasts to adhere to amniotic epithelial cells. Upon hAM transplantation, significantly less intraneural fibrosis was identified at the later time points. Moreover, hAM-treated rats exhibited a significantly higher sciatic functional index (SFI) after four weeks compared to controls (p .05), which indicated a potentially pro-regenerative effect of hAM. As a possible explanation, an impact of hAM on the endogenous immune response, including T cell and macrophage subsets, was indicated. We conclude that hAM is strongly effective against recurring nerve scarring and induces an anti-fibrotic and pro-regenerative effect, making it highly promising for treating adhesion-related disorders.Abnormal fibrotic bonding of tissues, frequently involving peripheral nerves, affects millions of people worldwide. These so-called adhesions usually cause severe pain and drastically reduce quality of life. To date, no adequate treatment exists and none is routinely used in the clinical practice. In this study, vital human amnion, the innermost of the fetal membranes, was transplanted in a rat model of peripheral nerve scarring and recurring adhesions as novel therapeutic approach. Amniotic cells have already demonstrated to feature stem-cell like properties and produce pro-regenerative factors, which makes the amnion an increasingly promising biomaterial for regenerative medicine. We identified that its transplantation was very effective against peripheral nerve scarring and distinctly reduced recurring adhesions. Moreover, we identified a pro-regenerative effect. This study showed that the amnion is a highly promising novel therapeutic approach for adhesion-related disorders.

One of the mainstays of facial rejuvenation strategies is volume restoration, which can be achieved by autologous fat grafting. In our novel approach, we treated the adipose tissue harvest site with extracorporeal shock wave therapy (ESWT) in order to improve the quality of the regenerative cells in situ. The latter was demonstrated by characterizing the cells of the stromal vascular fraction (SVF) in the harvested liposuction material regarding cell yield, adenosine triphosphate (ATP) content, proliferative capacity, surface marker profile, differentiation potential and secretory protein profile. Although the SVF cell yield was only slightly enhanced, viability and ATP concentration of freshly isolated cells as well as proliferation doublings after 3 weeks in culture were significantly increased in the ESWT compared with the untreated group. Likewise, cells expressing mesenchymal and endothelial/pericytic markers were significantly elevated concomitant with an improved differentiation capacity towards the adipogenic lineage and enhancement in specific angiogenic proteins. Hence, in situ ESWT might be applied in the future to promote cell fitness, adipogenesis and angiogenesis within the fat graft for successful facial rejuvenation strategies with potential long-term graft survival.

Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and in vitro characterization and clinical application of PnD., Austrian Science Fund (FWF) DOC 31-B26, Medical University Graz, Universita Cattolica del Sacro Cuore, PRIN 2017 program of Italian Ministry of Research and University (MIUR) 2017RSAFK7, Ministry of Health, Italy GR-2018-12366992, Slovenian Research Agency - Slovenia P3-0108, MRIC UL IP-0510, Plan Estatal de Investigacion Cientifica y Tecnica y de Innovacion, ISCIII Subdireccion General de Evaluacion y Fomento de la Investigacion, Ministerio de Economia y Competitividad, Spain PI16/01642, European Union (EU), European Community (EC), German Research Foundation (DFG) GE-2223/2-1

Scaffold-free 3D cell cultures (e.g. pellet cultures) are widely used in medical science, including cartilage regeneration. Their drawbacks are high time/reagent consumption and lack of early readout parameters. While optimisation was achieved by automation or simplified spheroid generation, most culture systems remain expensive or require tedious procedures. The aim of this study was to establish a system for resource efficient spheroid generation with additional early readout parameters. This was achieved by a new approach for spheroid generation via self-assembly from monolayer via compartmentation of cell culture surfaces utilising laser engraving (grid plates). The compartmentation triggered contraction and rolling up of the cell monolayer, finishing in condensation into a spheroid in human adipose-derived stem cell (ASC/TERT1) and human articular chondrocytes (hACs)-ASC/TERT1 co-cultures, when cultivated on grid plates under chondrogenic conditions. Plates with 1 and 3 mm grid size yielded stable diameters (about 140 µm and 300 µm, respectively). ASC/TERT1 spheroids fully formed within 3 weeks while co-cultures took 1–2 weeks, forming significantly faster with increasing hAC ratio (p < 0.05 and 0.01 for 1:1 and 1:4 ASC/TERT1:hAC ratio, respectively). Co-cultures showed slightly lower spheroid diameters, due to earlier spheroid formation and incomplete monolayer formation. However, this was associated with a more homogeneous matrix distribution in the co-culture. Both showed differentiation capacity comparable to standard pellet culture in (immune-)histochemistry and RT-qPCR. To assess usability for cartilage repair, spheroids were embedded into a hydrogel (fibrin), yielding cellular outgrowth and matrix deposition, which was especially pronounced in co-cultures. The herein presented novel cell culture system is not only a promising tool for autonomous spheroid generation with the potential of experimental and clinical application in tissue engineering, but also for the generation of ‘building blocks’ for subsequential biofabrication strategies such as bioprinting.

Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.

Purpose Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. Results Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs’ transplanted vessels. Conclusion We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration.

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of “AuriScaff”, an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. Statement of Significance After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.

Natural extracellular matrix-derived biomaterials from decellularized allogenic tissues are of increasing interest for tissue engineering because their structure and composition provide a complexity that is not achievable with current manufacturing techniques. The prerequisite to bring allogenic tissue from bench to bedside as a functional biomaterial is the full removal of cells while preserving most of its native characteristics such as structure and composition. The exceptionally dense structure of articular cartilage, however, poses a special challenge for decellularization, scaffold preparation, and reseeding. Therefore, we tested 24 different protocols aiming to remove cells and glycosaminoglycans (GAG) while preserving the collagen backbone and ultrastructure. The resulting matrices were analyzed for cell removal (DNA quantification, haematoxylin and eosin staining), GAG content (dimethyl methylene blue assay, Alcian blue staining and micro-computed tomography), collagen integrity (immunohistochemistry and ultrastructure), and biomechanics (compression test). Furthermore, seeding tests were conducted to evaluate cell viability and attachment to the scaffolds. Sodium dodecyl sulfate-based protocols yielded satisfactory reduction of DNA content, yet had negative effects on cell viability and attachment. Hydrochloric acid efficiently decellularized the scaffold and pepsin emerged as best option for GAG depletion. Combining these two reagents led to our final protocol, most efficient in DNA and GAG depletion while preserving the collagen architecture. The compressive modulus decreased in the absence of GAG to ∼1/3 of native cartilage, which is significantly higher than that by commercially available scaffolds tested as a reference (ranging from 1/25 to 1/100 of native cartilage). Cytocompatibility tests showed that human adipose-derived stromal cells readily adhered to the scaffold. In this study, we established a protocol combining freeze-thaw cycles, osmotic shock, and treatment with hydrochloric acid followed by a pepsin digestion step, achieving successful decellularization and GAG depletion within 1 week, resulting in a cytocompatible material with intact collagen structure. The protocol provides a basis for the generation of allogeneic scaffolds, potentially substituting manufactured scaffolds currently used in clinical articular cartilage treatment.

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.

OBJECTIVES We tested whether resuscitation supplemented with rat adipose-derived stem cells (ASCs) or secretome (conditioned media) of ASCs can ameliorate inflammation, cell/organ injury, and/or improve outcome after hemorrhagic traumatic shock (HTS). INTERVENTIONS Rats were subjected to HTS and a resuscitation protocol that mimics prehospital restrictive reperfusion followed by an adequate reperfusion phase. Twenty minutes into the restrictive reperfusion, animals received an intravenous bolus of 2 × 10 cells (ASC group) or the secretome produced by 2 × 10 ASCs/24 h (ASC-Secretome group). Controls received the vehicle (Vehicle group). All rats were observed for 28-day survival. MEASUREMENTS AND MAIN RESULTS HTS-induced inflammation represented by IL-6 was inhibited in the ASC (80%, P

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.

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.

INTRODUCTION: The demand for new therapeutic approaches to treat bone defects and fractures is increasing in trauma surgery and orthopaedics because the number of patients with degenerative diseases is continuously growing. "Tissue Engineering" offers promising new technologies that combine the three components - cells, growth factors and matrix. Efforts are targeted at improving and accelerating recovery, especially for long bone fractures, and reducing the risk of delayed bone healing or pseudoarthrosis. Adult human adipose-derived stem cells (ASC) can differentiate into osteoblasts in an osteogenic surrounding. Bone morphogenetic protein-2 (BMP-2) accelerates and initiates this differentiation. Fibrin, a matrix that promotes wound healing, is a promising carrier for ASCs and BMP-2.MATERIALS AND METHODS: In this study, a 2mm transcortical drill hole in the femur of male rats served as a small non-critical size defect model for fracture simulation. In vivo bone healing was investigated upon administration of the growth factor BMP-2 embedded with ASCs in a locally applied fibrin matrix. Groups with the components alone were also investigated. After 2 and 4 weeks, μCT and histology were performed to determine the bone and callus volume.RESULTS AND DISCUSSION: After only a short period of time (2 and 4 weeks), this animal model discloses comparative information about the osteogenetic potential and bone regeneration with little effort (no osteosynthesis necessary). The most significant result found in this model is that the combination of ASCs and BMP-2 in a fibrin matrix significantly reduces callus formation after 2 weeks compared to BMP-2 alone. BMP-2 alone significantly increased callus formation. ASCs embedded alone in the fibrin matrix did not lead to increased bone regeneration.CONCLUSION: Transplantation of ASC modulated the callus induction by BMP-2 to a normal volume.

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Tissue engineering strategies usually require cell isolation and combination with a suitable biomaterial. Human amniotic membrane (AM) represents a natural two-layered sheet comprising cells with proven stem cell characteristics. In our approach, we evaluated the differentiation potential of AM in toto with its sessile stem cells as alternative to conventional approaches requiring cell isolation and combination with biomaterials. For this, AM-biopsies were differentiated in vitro using two osteogenic media compared with control medium (CM) for 28 days. Mineralization and osteocalcin expression was demonstrated by (immuno)histochemistry. Alkaline phosphatase (AP) activity, calcium contents and mRNA expression of RUNX2, AP, osteopontin, osteocalcin, BMP-2 (bone morphogenetic protein), and BMP-4 were quantified and AM viability was evaluated. Under osteogenic conditions, AM-biopsies mineralized successfully and by day 28 the majority of cells expressed osteocalcin. This was confirmed by a significant rise in calcium contents (up to 27.4 ± 6.8 mg/dl d28), increased AP activity, and induction of RUNX2, AP, BMP-2 and BMP-4 mRNA expression. Relatively high levels of viability were retained, especially in osteogenic media (up to 78.3 ± 19.0% d14; 62.9 ± 22.3% d28) compared to CM (42.2 ± 15.2% d14; 35.1 ± 8.6% d28). By this strategy, stem cells within human AM can successfully be driven along the osteogenic pathways while residing within their natural environment.

Large animals such as pigs are good models for skeletal tissue engineering, since they provide physical forces similar to those of humans. Porcine bone marrow mesenchymal stem cells (BMSCs) have shown regenerative capacity similar to those of human BMSCs and can therefore be preclinically applied in settings corresponding to autologous transplantation in patients. Aiming at a one-step procedure for cartilage regeneration with autologous BMSCs, three straightforward isolation methods for BMSCs of Göttingen minipigs were compared. For this purpose, the BMSC fraction was enriched by red blood cell (RBC) lysis, dextran sedimentation or density gradient centrifugation. Isolated BMSCs were evaluated with regard to cell yield, proliferation capacity, phenotype and ability to differentiate to the chondrogenic lineage. Highest cell yields determined at the time of subcultivation were obtained using RBC lysis. In comparison, dextran sedimentation was less efficient but superior to density gradient centrifugation, which yielded significantly lower cell numbers than RBC lysis. The evaluated isolation methods resulted in cultures with equal proliferative capacity, with constant population doubling times of 50-55 h for at least 100 days (approximating to 40 cumulative population doublings) in vitro. Chondrogenic differentiation in micromass pellet cultures was evaluated by glycosaminoglycan quantification, histological staining with Alcian blue and safranin O and immunohistochemical analysis for collagen type II. These evaluations demonstrated that all three isolation methods yielded cells capable of generating cartilaginous tissue in vitro. According to our data, RBC lysis can be used to efficiently isolate porcine BMSCs in a short time frame which would allow for intraoperative one-step procedures in preclinical cartilage regeneration studies.

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.

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca2+ dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Statement of Significance Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.

An approach of using RFP-transfected human foreskin fibroblasts (hFS-RFP) to support the growth of GFP expressing human embryonic stem cells (hES; HS181-GFP) is reported. The two-color system was applied to detect interactions between hFS and human embryonic stem cells (hES). After overnight culture, the hES cell colonies showed a behavior of "pushing away" the underlying feeder cells. This phenomenon occurred with both a low and high density of feeders. The density of the feeder cell layer, however, influenced the growth pattern of hES cell colonies. At a high feeder cell density, the hES colonies were more pointed and aligned with the direction of the fibroblasts, whereas less dense feeder layers allowed a more rounded and flat hES colony formation. Not surprisingly, a small fraction of mitotically inactivated feeder cells reattached after passage and remained viable in the cultures for up to four subsequent passages. The prospect of using the two-color system for detection of possible fusion events between hES cells and feeder cells was assessed by screening a large number of cell cultures for double RFP/EGFP expressing cells. The results indicate that fusion events are extremely rare (10(-6)), or alternatively that after fusion the dual expression of both EGFP and RFP is not easily detected for other reasons. In summary, a two-color system allows analysis of colony formation and also helps to identify and follow the differentiation of cells.

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), S100β, 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, S100β 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.

The aim of the present study was to investigate the changes in absolute myocardial blood flow (AMF) after intracoronary injections of mesenchymal SC (MSC) and compared to controls in closed-chest reperfused acute myocardial infarction (AMI) in pigs. Male MSCs, transiently transfected with Luciferase (Luc-MSC) were delivered $$ \left( {{9}.{7}\pm {1}.{2} \times {1}{0^{{6}}}} \right) $$ intracoronary in the open infarct-related artery one-week post-AMI in female pigs (group MSC), while saline was injected with the same injection rate in controls (group C). The AMF was measured immediately after, and 3, 12 and 24 h post-intracoronary Luc-MSC or saline injections. In vitro bioluminescence images and quantitative real-time TaqMan PCR measurements were performed to quantify the sex-mismatched MSCs. No difference between the groups was observed regarding the weight, heart rate, blood pressure and global ejection fraction 1-week post-AMI. The baseline AMF were similar in the groups (61.3 ± 15. vs 61.1 ± 12.0 ml/min). AMF was decreased significantly immediately after intracoronary MSC delivery (42.0 ± 12.4 vs 57.7 ± 15.7 ml/min p = 0.013), and remained low at 3 h (40.9 ± 13.4 vs 55.8 ± 4.9 ml/min, p = 0.004), 12 h (43.0 ± 3.7 vs 57.8 ± 5.4 ml/min, p = 0.001) with incomplete recovery at 24 h (47.2 ± 5.5 vs 62.1 ± 14.1 ml/min, p = 0.038) as compared to controls, respectively. In vitro bioluminescence displayed transfected Luc-MSCs along the proximal and mid part of the LAD, with limited number (295 ± 101 sry copied/million cardiac cells) of Y-chromosome-MSCs in the infarcted area. Intracoronary injection of SCs results in immediate decrease of AMF, with delayed recovery. The delivery of the SC into the injured myocardium might be hindered by the altered coronary pressure and flow conditions.

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.

SummarySomatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

Adipose tissue is easily available and contains high numbers of stem cells that are capable for chondrogenic differentiation. We hypothesize that a partial substitution of chondrocytes with autologous adipose-derived stem cells (ASC) might be a possible strategy to reduce the number of chondrocytes needed in matrix-associated autologous chondrocyte transplantation. To lay the ground, in vitro coculture experiments were performed using human chondrocytes and human ASC. Chondrocytes were obtained from donors undergoing matrix-associated autologous chondrocyte transplantation. ASC were isolated from liposuction material. Chondrocytes and ASC were seeded either in fibrin (Tisseel; Baxter, Vienna, Austria) or collagen matrix (Tissue Fleece; Baxter, Unterschleissheim, Germany). RNA for quantitative reverse transcriptase (RT)-polymerase chain reaction was isolated after 2 weeks of culture in chondrogenic medium, and after 4 weeks samples were processed for histology. Related to the number of chondrocytes used, coculture with ASC led to strong increase in collagen type IX mRNA expression, which is an indicator for long-term stability of cartilage. Moderate upregulation was shown for SOX9, aggrecan, melanoma inhibitory activity, cartilage link protein 1, and cartilage oligomeric matrix protein mRNA. However, expression of collagen I and collagen II indicates the synthesis of fibrous tissue, which might be due to the use of dedifferentiated chondrocytes. Tisseel provided slightly better chondrogenic conditions than Tissue Fleece. These data support the possibility to take advantage of ASC in cartilage regeneration in conjunction with autologous chondrocytes.

Fracture healing is a complicated process involving many growth factors, cells, and physical forces. In cases, where natural healing is not able, efforts have to be undertaken to improve healing. For this purpose, tissue engineering may be an option. In order to stimulate cells to form a bone tissue several factors are needed: cells, scaffold, and growth factors. Stem cells derived from bone marrow or adipose tissues are the most useful in this regard. The differentiation of the cells can be accelerated using mechanical stimulation. The first part of this chapter describes the influence of longitudinal strain application. The second part uses a sophisticated approach with stem cells on a newly developed biomaterial (Sponceram) in a rotating bed bioreactor with the administration of bone morphogenetic protein-2. It is shown that such an approach is able to produce bone tissue constructs. This may lead to production of larger constructs that can be used in clinical applications.

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.

In the past decade, adipose tissue became a highly interesting source of adult stem cells for plastic surgery and regenerative medicine. The isolated stromal vascular fraction (SVF) is a heterogeneous cell population including the adipose-derived stromal/stem cells (ASC), which showed regenerative potential in several clinical studies and trials. SVF should be provided in a safe and reproducible manner in accordance with current good manufacturing practices (cGMP). To ensure highest possible safety for patients, a precisely defined procedure with a high-quality control is required. Hence, an increasing number of adipose tissue-derived cell isolation systems have been developed. These systems aim for a closed, sterile, and safe isolation process limiting donor variations, risk for contaminations, and unpredictability of the cell material. To isolate SVF from adipose tissue, enzymes such as collagenase are used. Alternatively, in order to avoid enzymes, isolation systems using physical forces are available. Here, we provide an overview of known existing enzymatic and non-enzymatic adipose tissue-derived cell isolation systems, which are patented, published, or already on the market.