The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140]     

The effects of preservation procedures on amniotic membrane's ability to serve as a substrate for cultivation of endothelial cells

Amniotic membrane (AM) has been used as a scaffold for the ex vivo expansion of different types of cells and a cell delivery matrix in regenerative medicine. Since the preservation procedures can influence the AM properties for experimental and clinical purposes, this study was established to investigate the feasibility of using the AM after different preservation methods to serve as substrates for endothelial cell expansion ex vivo. The effects of cryopreservation and lyophilization were evaluated on mechanical and histological characteristics of the AM, and the results were compared with the fresh AM. The ECM components of the basement membrane were well conserved in all groups. Although lyophilization resulted in more histological changes and lower level of physical variables including thickness, F_max, elongation at break and suture retention than the fresh and cryopreserved AM, endothelial cells grown on the lyophilized AM were better attached to the basement membrane. Cytotoxicity assay by MTT showed that the lyophilized AM is a compatible substrate for endothelial cells cultivation. The findings of this study suggest that the lyophilized AM is a suitable matrix for cultivation of endothelial cells due to this fact that lyophilization led to exposure of basement membrane of the AM.     

Homocysteine modulates the proteolytic potential of human vascular endothelial cells

Pathological levels of homocysteine induce a metalloproteinase-dependent degradation of the elastic structures in arterial wall. This elastolytic process is preferentially localized toward the internal elastic laminae and in the first layers of the media, suggesting endothelium could participate in extracellular matrix degradation induced by homocysteine. Therefore, we studied the effects of homocysteine on proteolytic potential of endothelial cells. Human umbilical vein endothelial cells were cultured with concentrations of homocysteine matching human physiological (10 microM) and pathological (50, 100, and 250 microM) plasma homocysteine levels. Pathological levels of homocysteine increased the secretion of elastolytic metalloproteinase-2 and -9, but not of metalloproteinase-3 and -7. Homocysteine also increased the expression of human tissue kallikrein, a potential activator of matrix metalloproteinase-2 and -9, while the expression of urokinase plasminogen activator was not altered. These results suggest vascular endothelial cells could participate in the subendothelial degradation of the arterial elastic structures occurring in hyperhomocysteinemia.     

Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.     

Caution regarding the combined effects of extracellular matrices and nutrient media on cultured endothelial cells

To study the influence of smooth muscle cells (SMC) on endothelial cells (EC), different co-culture designs are available, including EC seeding on SMC extracellular matrix (ECM). We explored human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on either in situ or coated ECM, elaborated by HUVECs or human arterial smooth muscle cells (HUASMCs), in the presence of different nutrient media containing varying amounts of fetal calf serum. Coating wells with HUVEC or HUASMC ECMs did not improve HUVEC adhesion 1 h after cell seeding, compared with uncoated wells. HUVEC adhesion on in situ HUVEC-ECM and HUASMC-ECM was significantly increased compared with uncoated wells. The substratum upon which cells are maintained was found to play a crucial role, in conjunction with the medium to which HUVECs are exposed for their proliferative response. These results stress the importance of selecting media in relation to the particular substratum, in order to avoid misinterpretation of data.     

Compartmentalized coculture of porcine arterial endothelial and smooth muscle cells on a microporous membrane

Summary Endothelial and smooth muscle cells were harvested from porcine pulmonary arteries and grown to two passages from primary culture in serum-containing medium. Thereafter, the cells were plated on the opposite sides of a microporous poly-(ethylene terephthalate) membrane and cultivated in a chemically defined, serum-free medium. The membrane with pores of 1 μm diameter allowed the passage of molecules and the extension of cell processes, while maintaining separate homogeneous cell populations. Pores of 3 μm diameter permitted the crossing of smooth muscle cells through the membrane. The coating of the polymer with constituents of the extracellular matrix optimized cell adhesion. Morphological analysis of the model showed typical cobblestone pattern and ultrastructure of endothelial cells, which lost rapidly the expression of von Willebrand factor but kept that of angiotensin-converting enzyme. Smooth muscle cells were spindle shaped and specific α-actin was revealed by immunochemistry and quantitated by enzyme-linked immunosorbent assay (ELISA). Their ultrastructure featured an intermediate contractile-synthetic phenotype. Permeability studies to different molecules showed a marked reduction of the albumin clearance. Finally, in coculture in the presence of endothelial cells, the smooth muscle cells proliferation was increased, whereas it was not the case in autologous cocultures. In conclusion, such a coculture model may help to a better understanding of the interactions between endothelial and smooth muscle cells that may be important in the pathogenesis of vascular diseases.     

Human amniotic membrane as an alternative source of stem cells for regenerative medicine

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.     

Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

C-reactive protein induced expression of adhesion molecules in cultured cerebral microvascular endothelial cells

Ischemic stroke can trigger an acute phase response resulting in a rise of plasma concentration of C-reactive protein (CRP). Clinical data about the relationship between CRP and prognosis suggest that CRP might be involved in the pathogenesis of cerebral ischemia. In the present work, a significant increase of circulating level of CRP was observed in an vivo rat brain ischemia model of middle cerebral artery occlusion. To determine the possible effects of CRP on brain microvessel endothelium, we performed a dose-dependent experiment in mouse brain microvascular endothelial cells (bEnd.3 cells) with emphasis on its relation to cell adhesions molecules. Incubation with CRP (1-75 mg/L) for 24 h significantly increased Lactate dehydrogenase (LDH) leakage from bEnd.3 cells (P<0.01) in a dose-dependent manner, and induced significant up-regulations of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions analyzed by Western blotting (P<0.01). In contrast to earlier report, CRP also induced significant increase in ICAM-1 expression in the absence of serum (P<0.01). In conclusion, the present results suggest that CRP may be involved directly in the development of inflammation in response to cerebral ischemia.     

The influence of various storage conditions on cell viability in amniotic membrane

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°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°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. A part of this work was presented at the World Congress on Tissue Banking in Rio in May 2005 and was honoured by the Poster Award Commission.     

Methylmercury-injury effect on tube formation by cultured human vascular endothelial cells

The effect of methylmercury chloride (MeHg) on growth and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were collected by enzymatic digestion with collagenase. Precultivation of HUVECs with MeHg at concentrations of 1.0–50.0 mol/L exerted negligible effects on the viable cell number, while the viable cell number was slightly reduced at 100 mol/L and fell to zero at concentrations exceeding 500.0 mol/L MeHg. The viable cell number was depressed in a concentration-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 mol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal bovine serum (FBS) increased tube formation in a dose-dependent manner, with half-maximum stimulation of tube formation at approximately 3.4% FBS. The length of tube formation decreased time-dependently at concentrations of 0.1 and 1.0 mol/L MeHg. Pretreatment of Matrigel with 1 mol/L MeHg before the cell seeding reduced the tube formation by HUVECs. These results suggest that the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function.Abbreviations MeHg methylmercury chloride - HUVECs human umbilical vein endothelial cells     

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.     

Comparison of characteristics of cultured limbal cells on denuded amniotic membrane and fresh conjunctival, limbal and corneal tissues

This study aimed to evaluate proposed molecular markers related to eye limbal stem cells (SC) and to identify novel associated genes. The expression of a set of genes potentially involved in stemness was assessed in freshly prepared limbal, corneal and conjunctival tissues. PAX6, AC133, K12 and OCT4 were detected in all the tissues and p63(+)/K3(-)/K12(+)/Nodal(+)/Cx43(+) were expressed in conjunctival, p63(-)/K3(+)/K12(+)/Nodal(-)/Cx43(+) in corneal, and p63(+)/K3(-)/K12(-)/Nodal(-)/Cx43(-) in limbal tissues. Limbal explants were cultured on human amniotic membrane for 21 days. The cells expressed p63 but not K3, K12, Nodal and Cx43, however, the expression of K3, K12 and Cx43 was detected, and p63 and the high BrdU-labeling index decreased with more culture. Ultrastructure analysis of the cultured cells showed typically immature organization of intracellular organelles and architecture. Our data suggest that limbal, corneal and conjunctival tissues are heterogeneous with some progenitors. Also, the expression of traditional SC markers may not be a reliable indicator of limbal SC and there is an increasing need to determine factor(s) involved in their stemness.     

Silica gel dissication of amniotic membrane with related epithelium cells for ocular surface reconstruction

Cornea reparative regeneration when in various pathological states needs creating certain conditions to intensify the potential of regional stem cells mitotic activity. Aims of the Research. To find out the degree of the epithelium AM preservation after preliminary processing and conservation by means of dehydration over silica gel with further sterilization; to study the effectiveness of clinical treatment of AM conserved in the surroundings with vital ability epithelium and AM dried over silica gel. Materials and methods. There was carried out an investigation of 18 samples of native amnion treated with antibiotics and 18 total surface amnion samples conserved by drying over silica gel and then sterilized by gamma rays. Clinical experiments were carried out on patients with severe chemical and thermal burns – 18 people (21 eyes). After the burn trauma all the patients underwent the standard procedure of necrectomy, the covering of the eyeball with amniotic membrane dried over silica gel. Conclusion. The drying out of the amniotic membrane over silica gel on frames without being fixed on nitrocellulose paper makes the process of the amniotic membrane conservation simpler and makes it possible to preserve its unique biological qualities. The effectiveness of the regeneration of epithelium tissue of the eyeball surface with amniotic membrane dried over silica gel without the vital capacity cells of the epithelium layer is analogous to the regeneration of epithelium cells with amniotic membrane with vital capacity cells. With eye burns AM coverage hinders the formation of rough conjunctiva cicatrix, provides a favorable out-of-cell matrix substrate for epithelium migration and leads to quicker regeneration of one's own epithelium, makes further visual rehabilitation simpler. This revised version was published online in July 2006 with corrections to the Cover Date.     

“The preadipocyte factor” DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential

Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as “the” preadipocyte marker, yet the phenotype of DLK1⁺ cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1⁺ cells encompass around 1-2% of the adult mouse adipose stromal vascular fraction (SVF). Unexpectedly, the DLK1⁺SVF population was enriched for cells expressing genes generally ascribed to the vascular lineage and did not possess any adipogenic differentiation potential in vitro. Instead, DLK1⁺ cells comprised an immediate ability for cobblestone formation, generation of tube-like structures on matrigel, and uptake of Acetylated Low Density-Lipoprotein, all characteristics of endothelial cells. We therefore suggest that DLK1⁺SVF cells are of a vascular origin and not them-selves committed preadipocytes as assumed hitherto.     

Differential regulation of biglycan and decorin synthesis by connective tissue growth factor in cultured vascular endothelial cells

It is possible that connective tissue growth factor (CTGF) serves as either an independent regulator or a downstream effector of transforming growth factor-beta (TGF-beta) on the proteoglycan synthesis in vascular endothelial cells. Since TGF-beta regulates endothelial proteoglycan synthesis in a cell density-dependent manner, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(35)S]sulfate or (35)S-labeled amino acids in the presence of CTGF, and the labeled proteoglycans were characterized by biochemical techniques. The results indicate that CTGF suppresses the synthesis of biglycan but newly induced that of decorin in the cells when the cell density is low; in addition, no change was observed in the hydrodynamic size and the glycosaminoglycan chain length of these two small chondroitin/dermatan sulfate proteoglycans. The regulation of endothelial proteoglycan synthesis by CTGF is completely different from that by TGF-beta, suggesting that CTGF is not a downstream effector of TGF-beta but an independent regulator in vascular endothelial cells with respect to the proteoglycan synthesis.     

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the alpha(v)-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.     

In vitro studies on the expansion of endothelial cell monolayers on components of the basement membrane

The purpose of the present study was to observe the expansion of a monolayer of endothelial cells over specific components of the basement membrane. This was performed in vitro in a monolayer expansion assay over 5 days. The control surface was uncoated glass in the form of coverslips. Test substances were coated at a concentration of 10 μg/ml. The highest expansion was obtained with a high molecular weight fragment mixture of collagen type IV (IV-F, consisting of 75, 120 and 140 KD fragments), followed by fibronectin. Collagens type I, III and IV tetramer gave similar results, less than fibronectin or collagen type IV-F, although all of the above basement membrane coatings promoted expansion significantly above that of the control (P<0.01). The poorest expansion was obtained with laminin, which was significantly less than the control. The pentapeptide GRGDS, related to the fibronectin cell binding region, gave expansion significantly below that of the intact fibronectin molecule, as did the intact collagen type IV molecule compared with type IV-F (P<0.025). This indicates that sequences of the fibronectin molecule other than the cell binding sequence may be involved in promoting endothelial cell expansion. In addition, the integrity of the collagen type IV molecule does not appear necessary for this effect. On the contrary, the higher movement on IV-F may represent an inherent repair mechanism in damaged endothelium. Autoradiographic studies show that endothelial cell proliferation at the expanding front is involved in the migration assay.     

Anti-apoptotic and anti-senescence effects of Klotho on vascular endothelial cells

Klotho-mutated mice manifest multiple age-related disorders that are observed in humans. A recent study suggested that Klotho protein might function as an anti-aging hormone in mammals. Because it has been reported that apoptosis and senescence in vascular endothelial cells are closely related to the progression of atherosclerosis, we investigated Klotho's ability to interfere with apoptosis and cellular senescence in human umbilical vascular endothelial cells (HUVEC). Klotho overexpression decreased H(2)O(2)-induced apoptosis in COS-1 cells and Jurkat cells. Klotho protein also reduced H(2)O(2)- and etoposide-induced apoptosis in HUVEC. Caspase-3 and caspase-9 activity was lower in Klotho-treated HUVEC than in control cells. Senescence-associated beta-gal staining showed that Klotho protein interferes with H(2)O(2)-induced premature cellular senescence. The expression of p53 and p21 was lower in Klotho-treated cells. Our study suggests that Klotho acts as a humoral factor to reduce H(2)O(2)-induced apoptosis and cellular senescence in vascular cells.