Concise review on clinical applications of conditioned medium derived from human umbilical cord-mesenchymal stem cells (UC-MSCS)

In recent years, mesenchymal stem cells have provoked much attentiveness in the field of regenerative medicine because of their differentiation potential and the capability to facilitate tissue repair via the emancipation of biologically active molecules. They have gained interest because of their distinctive curative properties. Mesenchymal stem cells are isolated from the Wharton\u2019s jelly part of umbilical cord possessing higher proliferation capacity, immunomodulatory activity, plasticity, as well as self-renewal capacity than the mesenchymal stem cells from various origins, and it is considered to be the best resource for allogeneic transplantation. The isolated umbilical cord-derived mesenchymal stem cells are cultured in the Dulbecco\u2019s Modified Eagle\u2019s Medium, and thereby it begins to release soluble factors into the medium during the period of culture which is termed as conditioned medium. This conditioned media has both differentiation capacity and therapeutic functions. Thus, it can be able to differentiate the cells into different lineages and the paracrine effect of these cells helps in replacement of the damaged cells. This medium may accord to optimization of diagnostic and prognostic systems as well as the generation of novel and targeted therapeutic perspectives

Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures

Background: In the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor \u3baB (NF-\u3baB), TNFa release, and IL10 expression. Methods: To investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out. Results: Flow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS. Conclusions: The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS

role of heparin sodium salt in the modulation of human umbilical cord derived mesenchymal stem cell differentiation

Introduction: The present study aims to investigate the role of low molecular weight compound heparin sodium salt (HSS) to control the differentiation of the human umbilical cord (UC) derived mesenchymal stem cells (MSCs) through possible interaction with WWTR1 protein. Materials and Methods: In order to carry out this study, the human UC-derived stem cells were isolated and characterized by stem cell specific markers and the effect of HSS was studied by altering the phenotypes of MSCs. An Insilco approach was employed to reveal the structural determination of the ligand, the WWTR1 protein binding site and to predict the strength of the interaction. After HSS treatment, WWTR1, Oct4, nanog, SOX9 gene expressions were studied using real-time polymerase chain reaction (PCR). Cell staining was performed using alizarin red to confirm the formation of osteocytes. Results: Mineralization indicated by osteocytes was confirmed using alizarin red after the treatment of HSS. Post, HSS treatment, OCT4, Nanog, RUNX2, COL1A1 and WWTR1 gene expressions were positively modulated. Heparin treatment of MSCs lead to the up regulation of WWTR1 along with the down regulation of stemness markers Oct4 and Nanog expression. In silico studies also predicted the possible interaction of WWTR1 with HS. Results indicated that Amino acid residues ASP57, GLN83, GLN109, THR135, and TYR141 came up as a prominent interaction centre; ASP57, GLN83 and THR135 recorded the highest interaction energy – while ASP57 mostly participated in an electrostatic interaction. Conclusions: To conclude, it can be stated that heparin can possibly interact with WWTR1 along with having the capability to direct cells towards osteogenic lineages

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality

Commitment of Autologous Human Multipotent Stem Cells on Biomimetic Poly-L-lactic Acid-Based Scaffolds Is Strongly Influenced by Structure and Concentration of Carbon Nanomaterial

Nanocomposite scaffolds combining carbon nanomaterials (CNMs) with a biocompatible matrix are able to favor the neuronal differentiation and growth of a number of cell types, because they mimic neural-tissue nanotopography and/or conductivity. We performed comparative analysis of biomimetic scaffolds with poly-L-lactic acid (PLLA) matrix and three different p-methoxyphenyl functionalized carbon nanofillers, namely, carbon nanotubes (CNTs), carbon nanohorns (CNHs), and reduced graphene oxide (RGO), dispersed at varying concentrations. qRT-PCR analysis of the modulation of neuronal markers in human circulating multipotent cells cultured on nanocomposite scaffolds showed high variability in their expression patterns depending on the scaffolds\u2019 inhomogeneities. Local stimuli variation could result in a multi- to oligopotency shift and commitment towards multiple cell lineages, which was assessed by the qRT-PCR profiling of markers for neural, adipogenic, and myogenic cell lineages. Less conductive scaffolds, i.e., bare poly-L-lactic acid (PLLA)-, CNH-, and RGO-based nanocomposites, appeared to boost the expression of myogenic-lineage marker genes. Moreover, scaffolds are much more effective on early commitment than in subsequent differentiation. This work suggests that biomimetic PLLA carbon-nanomaterial (PLLA-CNM) scaffolds combined with multipotent autologous cells can represent a powerful tool in the regenerative medicine of multiple tissue types, opening the route to next analyses with specific and standardized scaffold features

Preliminary investigation of blood vessel-derived acellular matrix for vascular graft application

Although autologous vascular grafts and artificial materials have been used for reconstruction of small diameter (<5mm) blood vessels, the poor availability of vessels and the occurrence of intimal hyperplasia and progressive atherosclerotic degeneration represent shortcomings of these vascular prostheses. Therefore, this preliminary study aimed to develop acellular matrix (AM)-based vascular grafts. Rat thoracic aortas were decellularized by means of a detergent-enzymatic treatment [1], whereas endothelial cells (ECs) were obtained through enzymatic digestion of rat skin followed by immunomagnetic separation of CD31-positive cells. Twenty male Lewis rats (8 week old) received either only AM and previously in vitro reendothelized AM as abdominal aorta Interposition grafts (about 2 cm). After 1 (n=10) and 3 (n=10) months from surgery, grafts were explanted and morphologically examined by scanning electron microscopy and Movat staining. The detergent enzymatic treatment completely removed the cellular part of vessels and both MHC class I and class II antigens. After 1 month from surgery, the luminal surface of implanted AMs was partially covered by ECs and several platelets adhered in the areas lacking cell coverage. Intimal hyperplasia, already detected after 1 month, increased at 3 months. On the contrary, all the grafts composed by AM and ECs were completely covered at 1 month and their structure was similar to that of native vessels at 3 months. Taken together, our findings show that prostheses composed of AM pre-seeded with ECs could be a promising approach for the replacement of blood vessels

Effects of surface topography on growth and osteogenic differentiation of human mesenchymal stem cells

The clinical success of an endosseous artificial implant is related to the quality of its osseointegration with the surrounding living bone. To achieve a stable anchorage, mesenchymal cells, migrating to the implant surface from the surrounding tissue, must differentiate towards mature osteoblasts rather than connective tissue cell types. It is well known that the cell response is affected by the physicochemical parameters of the biomaterial surface, such as surface energy, surface charges or chemical composition. Topography seems to be one of the most crucial physical cues for cells (1). In particular, interactions between mesenchymal stem cells (MSCs) and surfaces with specific micro and nano patterns can stimulate MSCs to produce bone mineral in vitro (2). Herein, stamps reporting different micro and nano features were fabricated in order to obtain several corresponding replicas in a short time through microinjec- tion molding. Then, the effects of the substrate topography on human bone marrowderived MSC adhesion, proliferation, and osteogenic differentiation were investigated in the absence of inductive growth factors. Collectively, our data show that both micro- and nano-structured surfaces possess osteoinductive properties. A relationship between dimensional feature of surface topography and differentiative potential was noted. On the contrary, cell adhesion and proliferation seemed to be unaffected. Further in vivo studies will be carried out to confirm the osteoinductive properties of selected surface geometries

Umbilical cord mesenchymal stem cells modulate dextran sulphate sodium induced acute colitis in immunodeficient mice.

Inflammatory bowel diseases (IBD) are complex multi-factorial diseases with increasing incidence worldwide but their treatment is far from satisfactory. Unconventional strategies have consequently been investigated, proposing the use of stem cells as an effective alternative approach to IBD. In the present study we examined the protective potential of exogenously administered human umbilical cord derived mesenchymal stem cells (UCMSCs) against Dextran Sulphate Sodium (DSS) induced acute colitis in immunodeficient NOD.CB17-Prkdc scid/J mice with particular attention to endoplasmic reticulum (ER) stress. METHODS: UCMSCs were injected in NOD.CB17-Prkdc scid/J via the tail vein at day 1 and 4 after DSS administration. To verify attenuation of DSS induced damage by UCMSCs, Disease Activity Index (DAI) and body weight changes was monitored daily. Moreover, colon length, histological changes, myeloperoxidase and catalase activities, metalloproteinase (MMP) 2 and 9 expression and endoplasmic reticulum (ER) stress related proteins were evaluated on day 7. RESULTS: UCMSCs administration to immunodeficient NOD.CB17-Prkdc scid/J mice after DSS damage significantly reduced DAI (1.45\u2009\ub1\u20090.16 vs 2.08\u2009\ub1\u20090.18, p\u20093-fold), which were significantly reduced in mice receiving UCMSCs. Moreover, positive modulation in ER stress related proteins was observed after UCMSC administration. CONCLUSIONS: Our results demonstrated that UCMSCs are able to prevent DSS-induced colitis in immunodeficient mice. Using these mice we demonstrated that our UCMSCs have a direct preventive effect other than the T-cell immunomodulatory properties which are already known. Moreover we demonstrated a key function of MMPs and ER stress in the establishment of colitis suggesting them to be potential therapeutic targets in IBD treatment

Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation