Conditioned media derived from mesenchymal stem cell cultures: The next generation for regenerative medicine.

Recent studies suggest that the main driving force behind the therapeutic activity observed in mesenchymal stem cells (MSCs) are the paracrine factors secreted by these cells. These biomolecules also trigger antiapoptotic events to prevent further degeneration of the diseased organ through paracrine signalling mechanisms. In comparison with the normal physiological conditions, an increased paracrine gradient is observed within the peripheral system of diseased organs that enhances the migration of tissue-specific MSCs towards the site of infection or injury to promote healing. Thus, upon administration of conditioned media derived from mesenchymal stem cell cultures (MSC-CM) could contribute in maintaining the increased paracrine factor gradient between the diseased organ and the stem cell niche in order to speed up the process of recovery. Based on the principle of the paracrine signalling mechanism, MSC-CM, also referred as the secretome of the MSCs, is a rich source of the paracrine factors and are being studied extensively for a wide range of regenerative therapies such as myocardial infarction, stroke, bone regeneration, hair growth, and wound healing. This article highlights the current technological applications and advances of MSC-CM with the aim to appraise its future potential as a regenerative therapeutic agent.

[Effect of conditioned medium from mesenchymal stem cells on regeneration of endothelium at HCl-induced damage trachea in rats].

The purpose. Respiratory epithelium regeneration is studied in rats with tracheal damage induced by inhaling hydrochloric acid vapor. Method. Regeneration process after the chemical burn was activated by intratracheal administration of preparations obtained from the same-species mesenchymal stem cells (MSC). Results. Tracheal epithelium is shown to recover almost completely on day 3-7 after applying MSC compositions (MSCs). Closed structures containing ciliated cells similar to ciliated cells of the respiratory epithelium lining the trachea are formed in the submucosal epithelium during regeneration. These structures migrate towards epithelium and get incorporated into the damaged epithelium. This phenomenon is apparently indicative of the special mechanism of respiratory epithelium regeneration after HCl-induced injury. Conclusion. It is demonstrated in this study that cell-free MSCs instilled intratracheally promote the recovery of normal submucosal epithelium by either preventing or reducing necrosis and inflammation. Such topical MSCs administration significantly accelerates migration of ciliated cell towards the surface and de novo formation of the ciliary epithelium.

Conditioned medium from renal tubular epithelial cells stimulated by hypoxia influences rat bone marrow-derived endothelial progenitor cells.

BACKGROUND AND AIMS: It has been well documented that endothelial progenitor cells (EPCs) participate in neovascularization in adults and that rarefaction of peritubular capillaries (PTCs) is closely associated with progressive kidney disease. Therefore, we investigated whether EPCs were influenced by conditioned medium (CM) of renal tubular epithelial cells (RTECs) stimulated by hypoxia, to provide evidence for EPCs transplantation in vivo in the future. METHODS: Mononuclear cells of rat bone marrow were isolated by density gradient centrifugation and were cultured according to previously described techniques. RTECs were cultured primarily with routine tissue block adhering wall method. In addition, CM was harvested from RTECs cultivated for 48 h in 5% O(2). EPCs proliferation and migration were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and transwell. The protein and mRNA expression of stromal cell-derived factor (SDF-1), vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1), and C-X-C chemokine receptor 4 (CXCR4) was separately assessed by Western blot, enzyme-linked immunosorbent assay (ELISA), and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. RESULTS: We showed that hypoxia increased the expression of SDF-1 and VEGF in RTEC. In addition, hypoxic CM improved proliferation, migration, and expression of VEGF, Ang-1, and CXCR4 of EPCs. CONCLUSIONS: These results suggest that hypoxic CM improves neovascularization of EPCs and may also be considered as therapeutic agents to supply the potent origin of reconstituion of PTCs of progressive kidney disease.

Astrocyte and muscle-derived secreted factors differentially regulate motoneuron survival.

During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40-50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75(NTR) - and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.

Toxic effects of fumonisin in mouse liver are independent of the peroxisome proliferator-activated receptor alpha.

Fumonisin mycotoxins occur worldwide in corn and corn-based foods. Fumonisin B1 (FB1) is a rodent liver carcinogen and suspected human carcinogen. It inhibits ceramide synthase and increases tissue sphinganine (Sa) and sphingosine (So) concentrations. Events linking disruption of sphingolipid metabolism and fumonisin toxicity are not fully understood; however, Sa and So were shown to bind mouse recombinant peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the role of PPARalpha in fumonisin hepatotoxicity in vivo, wild-type (WT) and PPARalpha-null mice were fed control diets or diets containing 300 ppm FB1, Fusarium verticillioides culture material (CM) providing 300 ppm FB1, or 500 ppm of the peroxisome proliferator WY-14,643 (WY) for 1 week. WY-fed WT mice exhibited hepatomegaly, an effect not found in WY-fed PPARalpha-null mice, and WY did not change liver sphingoid base concentrations in either strain. Hepatotoxicity found in FB1- and CM-fed WT and PPARalpha-null mice was similar, qualitatively different from that found in WY-treated animals, and characterized by increased Sa concentration, apoptosis, and cell proliferation. Transcript profiling using oligonucleotide arrays showed that CM and FB1 elicited similar expression patterns of genes involved in cell proliferation, signal transduction, and glutathione metabolism that were different from that altered by WY. Real-time RT-PCR analysis of gene expression demonstrated PPARalpha-dependence of lipid metabolism gene expression in WY-treated mice, whereas PPARalpha-independent alterations of genes in lipid metabolism, and other categories, were found in CM- and FB1-fed mice. Together, these findings demonstrate that FB1- and CM-induced hepatotoxicity in mice does not require PPARalpha.

Stimulated endothelial cell adhesion and angiogenesis with laminin-5 modification of expanded polytetrafluoroethylene.

Biomedical implants often exhibit poor clinical performance due to the formation of a periimplant avascular fibrous capsule. Surface modification of synthetic materials has been evaluated to accelerate the formation of functional microcirculation in association with implants. The current study used a flow-mediated protein deposition system to modify expanded polytetrafluoroethylene (ePTFE) with a laminin-5-rich conditioned growth medium and with medium from which laminin-5 had been selectively removed. An in vitro model of endothelial cell adherence determined that laminin-5 modification resulted in significantly increased adhesion of human microvessel endothelial cells to ePTFE. In vivo studies evaluating the periimplant vascular response to laminin-5-treated samples indicated that absorption of laminin-5-rich conditioned medium supported accelerated neovascularization of ePTFE implants. A flow system designed to treat porous implant materials facilitates laminin-5 modification of commercially available ePTFE, resulting in increased endothelial cell adhesion in vitro and increased vascularization in vivo.

Isolation of a retinal pigment epithelial cell-derived fraction which promotes Müller cell proliferation.

Retinal pigment epithelial cells (RPE) secrete a factor(s) in vitro which promotes Müller cell proliferation. To begin the isolation of the active factor, medium conditioned by cultured RPE (RPE-CM) was fractionated by fast protein liquid chromatography. The fractions were tested in an assay for Müller cell proliferation and it was found that one fraction contained biological activity comparable to that of complete RPE-CM. Gel electrophoresis demonstrated that this fraction consisted of proteins with approximate molecular masses of 35-88 kDa. An affinity assay revealed that only an 88-kDa protein in the RPE-CM binds to Müller cells. This protein was also present in the active fraction; therefore, it is the most likely source of the mitogenic activity of RPE-CM.

Mitogenic factors released from smooth muscle cells are responsible for neointimal cell proliferation after balloon catheter deendothelialization.

Smooth muscle cell (SMC) proliferation results in neointimal formation in response to selective deendothelialization. The neointimal SMC are characteristically different from normal, medial SMC. The major difference is that neointimal SMC have a higher proliferation rate. The high proliferation rate has been observed 15 weeks after endothelial injury. In this study, it is noted that neointimal SMC release some mitogenic factor(s) which is(are) responsible for inducing persistent SMC proliferation in an autocrine manner. The SMC were cultured from the media of normal rabbit aorta as well as the neointimal tissue formed 15 weeks after an endothelial injury. From the culture of the neointimal SMC, the conditioned medium was collected and growth factors, including PDGF-AB, TGF-beta 1, TGF-beta 2, and bFGF, were assayed. The conditioned medium was used to culture the medial SMC from normal rabbit aorta. The mitogenic effect of the conditioned medium was evaluated by the incorporation of [3H]thymidine into the SMC. Results demonstrated that PDGF-AB and TGF-beta 1 were increased in neointimal SMC-conditioned medium. After incubation with the conditioned medium, medial SMC incorporated significantly higher [3H]thymidine, compared to incubation with control medium (P < 0.01). The data indicate that endothelial injury induces production of some growth factors, including PDGF and TGF-beta, by the neointimal SMC. These growth factors may act in an autocrine manner to stimulate SMC proliferation for a long time following a single deendothelialization.

Efecto de medio condicionado de placenta humana sobre linfocitos periféricos in vitro / Effects of human placenta conditioned media on peripheric lymphocytes in vitro

En este trabajo se investiga la acción del medio condicionado de placenta humana (MCPH) sobre linfocitos periféricos in vitro. Se cultivaron leucocitos periféricos de varones, mujeres con diferente número de partos, gestantes y nulíparas, en su propio plasma sin y con MCPH al 5, 10 y 20 por ciento. Luego de 120 h de cultivo se determinó la Transformación Blástica Linfocitaria (TBL, valores ò 0,5 indican estimulación) por observación de la morfología celular. Los linfocitos de varones y mujeres nulíparas no responden a ninguna de las concentraciones de MCPH ensayadas. En mujeres que han tenido hijos, la más alta transformación blástica linfocitaria se observa con MCPH al 10 por ciento, siendo esta respuesta correlativa con el número de partos (r = 0,73). En gestantes, si bien la máxima respuesta se obtiene con MCPH al 10 por ciento, el agregado de 5 por ciento del extracto es suficiente para producir estimulación linfocitaria (TBL x = 0,54 ñ 0,05). En conclusión, MCPH al 10 por ciento sigue siendo la concentración óptima para provocar blastogénesis linfocitaria. Además, en el plasma de las mujeres gestantes existirían sustancias que potencian la acción del extracto in vitro, permitiendo TBL > 0,5 con la adición de sólo 5 por ciento de MCPH

Efecto de medio condicionado de placenta humana sobre linfocitos periféricos in vitro / Effects of human placenta conditioned media on peripheric lymphocytes in vitro

En este trabajo se investiga la acción del medio condicionado de placenta humana (MCPH) sobre linfocitos periféricos in vitro. Se cultivaron leucocitos periféricos de varones, mujeres con diferente número de partos, gestantes y nulíparas, en su propio plasma sin y con MCPH al 5, 10 y 20 por ciento. Luego de 120 h de cultivo se determinó la Transformación Blástica Linfocitaria (TBL, valores ò 0,5 indican estimulación) por observación de la morfología celular. Los linfocitos de varones y mujeres nulíparas no responden a ninguna de las concentraciones de MCPH ensayadas. En mujeres que han tenido hijos, la más alta transformación blástica linfocitaria se observa con MCPH al 10 por ciento, siendo esta respuesta correlativa con el número de partos (r = 0,73). En gestantes, si bien la máxima respuesta se obtiene con MCPH al 10 por ciento, el agregado de 5 por ciento del extracto es suficiente para producir estimulación linfocitaria (TBL x = 0,54 ñ 0,05). En conclusión, MCPH al 10 por ciento sigue siendo la concentración óptima para provocar blastogénesis linfocitaria. Además, en el plasma de las mujeres gestantes existirían sustancias que potencian la acción del extracto in vitro, permitiendo TBL > 0,5 con la adición de sólo 5 por ciento de MCPH (AU)