Effect of gold nanoparticles on adipogenic differentiation of human mesenchymal stem cells

Gold nanoparticles are very attractive for biomedical products. However, there is a serious lack of information concerning the biological activity of nanosized gold in human tissue cells. An influence of nanoparticles on stem cells might lead to unforeseen consequences to organ and tissue functions as long as all cells arising from the initial stem cell might be subsequently damaged. Therefore the effect of negatively charged gold nanoparticles (9 and 95 nm), which are certified as reference material for preclinical biomedical research, on the adipogenic differentiation of human mesenchymal stem cells (hMSCs) is investigated here. Bone marrow hMSCs are chosen as differentiation model since bone marrow hMSCs are well characterized and their differentiation into the adipogenic lineage shows clear and easily detectable differentiation. In this study effects of gold nanoparticles on adipogenic differentiation are analyzed regarding fat storage and mitochondrial activity after different exposure times (4–21 days). Using time lapse microscopy the differentiation progress under chronically gold nanoparticle treatment is continuously investigated. In this preliminary study, chronically treatment of adipogenic differentiating hMSCs with gold nanoparticles resulted in a reduced number and size of lipid vacuoles and reduced mitochondrial activity depending on the applied concentration and the surface charge of the particles.     

Pluripotency of adult stem cells derived from human and rat pancreas

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases. PACS 87.17.-d; 87.18.Ed; 81.17.Ez; 87.18.-h     

In vitro labeling and MRI of mesenchymal stem cells from human umbilical cord blood

OBJECTIVE: The aim of this study was to label human umbilical cord blood mesenchymal stem cells (MSCs) with poly-l-lysine (PLL)-conjugated superparamagnetic iron oxide particles and to obtain magnetic resonance (MR) images of the labeled MSCs' suspension at 1.5 T. MATERIAL AND METHODS: PLL was conjugated with iron oxide to form superparamagnetic particles called Fe(2)O(3)-PLL. Human umbilical cord blood MSCs were isolated, purified, expanded and incubated with Fe(2)O(3)-PLL. Prussian blue stain was performed to show intracellular iron; spectrometry was used to quantify iron uptake within cells. Tetrazolium salt (MTT) assay was applied to evaluate toxicity and proliferation of MSCs labeled with various concentrations of Fe(2)O(3)-PLL. The cell apoptosis rate was determined by annexin V/propichium iodide (PI) double staining method. Vials containing cells underwent MR imaging (MRI) with T(1), T(2) and T(2)* weighted MRI. RESULTS: Iron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining in all samples except the unlabeled control. The iron content per cell determined by spectrometry was 64.51+/-10.32 pg. Among MSCs with and without labeling of various concentrations of Fe(2)O(3)-PLL, MTT values of light absorption had no statistically significant difference (Kruskal-Wallis test, chi(2)=10.35, P=.17). A concentration at 20 mug/ml of iron appeared most suitable for incubating cells. Of labeled and unlabeled MSCs, the early [annexin V-fluorescein isothiocyanate (FITC)-positive/PI-negative] and late (annexin V-FITC-positive/PI-positive) apoptotic cells were 10.34+/-0.43%/11.36+/-1.30% and 4.01+/-1.76%/2.98+/-1.37%, respectively, and there were no significant differences between them (P>.05). T(2) weighted image (WI) and T(2)*WI demonstrated significant decrease of signal intensity (SI) in vials containing 1 x 10(6) (1 day), 1x10(6) (8 days) and 5 x 10(5) labeled cells, in comparison with unlabeled cells (P<.05). The percentage change of SI (DeltaSI) was significantly higher in 10(6) labeled cells after 1-day culture than that in the same number of labeled cells after 8-day culture and that in 5 x 10(5) labeled cells, particularly on T(2)*WI (P<.05). Among pulse sequences, T(2)*WI demonstrated the highest DeltaSI (P<.05). CONCLUSION: The human umbilical cord blood MSCs can be labeled with Fe(2)O(3)-PLL without significant change in viability and apoptosis. The suspension of labeled MSCs can be imaged with standard 1.5-T MR equipment.     

In situ Raman spectroscopic monitoring of hydroxyapatite as human mesenchymal stem cells differentiate into osteoblasts

The differentiation of stem cells into specific cell types is playing an essential role in the development of stem cell therapy, tissue engineering, and regenerative medicine. In this research, Raman microspectroscopy was applied to monitor the development of hydroxyapatite [HA, Ca₅ (PO₄)₃ (OH)], which is associated with the differentiation of the human mesenchymal stem cells (hMSCs) into osteoblasts. Raman spectra exhibited dramatic changes in the HA region, 950–970 cm⁻¹, over the period of 7–21 days after the start of differentiation. This work demonstrates the successful application of Raman spectroscopy for monitoring and quantitatively evaluating hMSC differentiation into osteoblasts. Copyright © 2008 John Wiley & Sons, Ltd.     

Tumor necrosis factor α triggers proliferation of adult neural stem cells via IKK/NF-κB signaling

Background  

Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-α) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation.     

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) is crucial step for its clinical applications in cell transplantation therapy. In the cryopreservation of MSCs, dimethyl sulfoxide has been widely used as a cryoprotectant (CPA). However, it has been proved that DMSO has toxic side effects to human body. In this study, DMSO-free CPA solutions which contained ethylene glycol (EG), 1, 2-propylene glycol (PG) and sucrose as basic CPAs, supplemented with polyvinyl alcohol (PVA) as an additive, were developed for the cryopreservation of UCB-derived MSCs. The cryopreservation of UCB-derived MSCs was achieved by vitrification via plunging into liquid nitrogen and by programmed freezing via an optical-DSC system respectively. The viability of thawed UCB-derived MSCs was tested by trypan blue exclusion assay. Results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence of PVA for vitrification, but only < 10% to 45% of viability was found for programmed freezing. These results indicate that PVA exerts a beneficial effect on the cryopreservation of UCB-derived MSCs and suggest the vitrification in combination with the dimethyl sulfoxide free CPA solutions supplemented with PVA would be an efficient protocol for the cryopreservation of UCB-derived MSCs.     

Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled

Objective

Three different kinds of transfection reagents were used to mediate the transfection of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) into human umbilical-cord-derived mesenchymal stem cells (hUCMSCs). The efficacy of different transfection reagents and the feasibility of NMR tracer in vitro of magnetized stem cells were estimated.

Methods

After purification by tissue explants adherent method, the biological characteristics of hUCMSCs in vitro were identified by subculture and amplification. Calcium phosphate, Effectene and liposome2000 were used to transfect Gd-DTPA-labeled hUCMSCs respectively, and cell counting was used to mediate the transfection of Gd-DTPA into hUCMSCs, which were then induced to lipoblast and osteoblast in vitro. The determination of the transfection activities of the transfection reagents was conducted by measuring the magnetic resonance imaging (MRI) signal intensity of the Gd-DTPA-labeled cells and the concentration of gadolinium ion in the cells. Furthermore, the relationship between the signal intensity of Gd-DTPA-labeled hUCMSCsMRI, cell subculture and generations was studied.

Results

Primary cells were obtained by tissue explants adherent for two weeks. The cells displayed a long spindle form and grew in swirl. After two passage generations, the cellular morphology became more homogeneous. The result detected by the flow cytometer showed that CD29C, D44, CD90, and CD105 were highly expressed, while no CD45, CD40, and HLA-DR expression was detected in the third generation cells. Directional induction in vitro caused the differentiation into lipoblast and osteoblast. After transfected by calcium phosphate, Effectene and liposome 2000, the signal intensity of stem cells was 2281.2 ± 118.8, 2031.9 ± 59.7 and 1887.4 ± 40.8 measured by MRI. Differences between these three groups were statistically significant (P < 0.05). The concentrations of gadolinium ion in three groups of stem cells were 0.178 ± 0.009 mg/L, 0.158 ± 0.003 mg/L and 0.120 ± 0.002 mg/L respectively, examined by inductively coupled plasma atomic emission spectrometry. No significant differences were found among these three groups (P < 0.05). The proliferation and differentiation abilities of the Gd-DTPA-labeled stem cells were not affected. A minimum 5 × 10⁴ Gd-DTPA-labeled stem cells could be traced with MRI in vitro and presented in high signal. The trace duration time in vitro was about 12 days.

Conclusions

Tissue explants adherent method can be availably applied to purify hUCMSCs. The Effectene method was proved to have the best transfection effect. The proliferation ability and differentiation potency of Gd-DTPA-labeled hUCMSCs were not affected, and the NMR of labeled stem cells in vitro was proved to be feasible.     

Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4

Background  

Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies.     

Relations between reactive oxygen species and Raman spectral variations of human umbilical cord mesenchymal stem cells with different viability

Laser Physics - In this study, the Reactive Oxygen Species (ROS) levels in Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) with different cell viability were measured by ROS probe...     

MR tracking of magnetically labeled mesenchymal stem cells in rats with liver fibrosis

Purpose

In vivo magnetic resonance (MR) tracking of magnetically labeled bone marrow mesenchymal stem cells (BMSCs) administered via the mesenteric vein to rats with liver fibrosis.

Materials and Methods

Rat BMSCs were labeled with superparamagnetic iron oxide (SPIO) and the characteristics of the BMSCs after labeling were investigated. Eighteen rats with CCL4-induced liver fibrosis were randomized to three groups to receive SPIO-labeled BMSCs (BMSC-labeled group), cell-free SPIO (SPIO group), or unlabeled BMSCs (control group). MR imaging of the liver was performed at different time points, and signal-to-noise ratio (SNR) of the liver was measured. In vivo distribution of delivered BMSCs was assessed by histological analysis.

Results

Labeling of BMSCs with SPIO did not significantly alter cell viability and proliferation activity. In BMSC-labeled group, the liver SNR immediately decreased from 8.56±0.26 to 3.53±0.41 at 1 h post injection and remained at a significantly lower level till 12 days (P<.05 versus the level before). By contrast, the liver SNR of the SPIO group almost recovered to the preinjection level (P=.125) at 3 days after a transient decrease. In control group, the liver SNR demonstrated no significant difference at the tested time points. Additionally, Prussian blue-positive cells were mainly distributed in the liver parenchyma, especially in injured areas.

Conclusion

The magnetically labeled BMSCs infused through the mesenteric vein can be detected in the fibrotic liver of rats using in vivo MR imaging up to 12 days after injection.     

Visualization of exosomes from mesenchymal stem cells in vivo by magnetic resonance imaging

Background and purposeWe develop a method of imaging exosomes in vivo according to the vital role of exosomes in intercellular communication. This study aims to design a new label method that allows the visualization of labeled exosomes with magnetic resonance imaging (MRI).MethodsWe designed a fusion protein consisting of two parts, namely, ferritin heavy chain (FTH1) and a truncated lactadherin. FTH1 is used as an MRI reporter. Lactadherin is a trans-membrane protein. The lactadherin protein are mostly located on the outer surface of exosomes. We replaced the outer membrane part of lactadherin with FTH1, infected mesenchymal stem cells with lentivirus carrying the fusion protein, and isolated exosomes from the labeled cells by ultracentrifugation. Labeled exosomes were validated by transmission electron microscopy images, Western blot, nanosight particle tracking, and visualized in vitro and in vivo by MRI.ResultsFTH1 expression would suppress mesenchymal stem cell proliferation, whereas the characterization of labeled exosomes remains comparable with unlabeled exosomes. MR imaging shows that exosomes labeled with FTH1 can be visualized in vitro and in vivo.ConclusionThis innovative reporter–imaging approach to track and visualize exosomes with MRI can be utilized as a tool for the study of the role of exosomes under different conditions.     

Embedding methods for poly(l-lactic acid) microfiber mesh/human mesenchymal stem cell constructs

Fiber mesh scaffolds were recently investigated in tissue engineering as possible support for stem cell growth and differentiation, in order to repair lesion areas in clinical practice. In particular, the literature is focused on fiber mesh scaffolds constituted of biocompatible and resorbable polymeric structures, like poly(l-lactic acid) (PLLA). However, as regards the study of constructs constituted of PLLA microfibers and cells, only quantitative and SEM analyses were reported, lacking histological analysis. Histological evaluation of these constructs could give important information about cellular distribution in the scaffold, cell–scaffold interactions and extracellular matrix production. The purpose of our study was to find a valid method to analyze PLLA microfiber/cell constructs from both histological and histochemical angles. Biodegradable non-woven fiber meshes were prepared using hollow microfibers, based on PLLA.We first evaluated different embedding methods useable for histological analysis and the results showed that among the paraffin, Killik, and acrylic resin the only suitable medium was the latter. Then we employed the acrylic resin to embed the constructs made up of PLLA microfibers and bone marrow-derived human mesenchymal stromal cells, which we then analyzed with Toluidine Blue, PAS and Alcian Blue staining. These constructs, previously analyzed for cell viability by MTT and CCK-8 tests, showed viable/proliferating cells until 6 weeks of culture. The stainings performed on constructs confirmed viability data obtained with SEM and MTT/CCK-8 and supplied other information on the cell behaviors such as the distribution and organization onto the scaffold and the production of extracellular matrix molecules. In conclusion, this methodological study mainly suggests a suitable method to analyze PLLA microfiber/cell constructs, at the same time confirming and enriching the literature data on the compatibility between PLLA microfibers and hMSCs.     

Cerebrospinal fluid promotes survival and astroglial differentiation of adult human neural progenitor cells but inhibits proliferation and neuronal differentiation

Background  

Neural stem cells (NSCs) are a promising source for cell replacement therapies for neurological diseases. Growing evidence suggests an important role of cerebrospinal fluid (CSF) not only on neuroectodermal cells during brain development but also on the survival, proliferation and fate specification of NSCs in the adult brain. Existing in vitro studies focused on embryonic cell lines and embryonic CSF. We therefore studied the effects of adult human leptomeningeal CSF on the behaviour of adult human NSCs (ahNSCs).     

Directed neuronal differentiation of human embryonic stem cells

Background

We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4⁺/SSEA-4^- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).

Results

A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons.

Conclusion

This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4⁺ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

     

Enhanced homing of mesenchymal stem cells to the ischemic myocardium by ultrasound-targeted microbubble destruction

In recent years, ultrasound-targeted microbubble destruction (UTMD) has been utilised for the targeted delivery of stem cells. We tested the effects of the myocardial micro-environment changes induced by UTMD on promoting the homing of mesenchymal stem cells (MSCs) to the ischemic myocardium. Dogs were randomly divided into two groups and treated with or without UTMD after the establishment of myocardial infarction models. 4,6-diamino-2-phenyl indole (DAPI) labelled MSCs were transplanted via coronary injections 2 weeks after myocardial infarction in both groups. The results from real-time PCR and western blot analyses indicated that the expression of various cytokines in UTMD-treated dogs was much higher than that observed in non-treated dogs. Histopathological findings demonstrate that ultrasound at a frequency of 1 MHz and an intensity of 1.0 W/cm² provoked inflammatory reactions with mild myocardial damage. Myocardial microenvironment changes caused by UTMD may promote the homing of MSCs to the ischemic myocardium. This non-invasive technique may be a promising method for cardiac cell transplantation therapy.     

Micrometer-sized iron oxide particle labeling of mesenchymal stem cells for magnetic resonance imaging-based monitoring of cartilage tissue engineering

Purpose

To examine mesenchymal stem cell (MSC) labeling with micrometer-sized iron oxide particles (MPIOs) for magnetic resonance imaging (MRI)-based tracking and its application to monitoring articular cartilage regeneration.

Methods

Rabbit MSCs were labeled using commercial MPIOs. In vitro MRI was performed with gradient echo (GRE) and spin echo (SE) sequences at 3T and quantitatively characterized using line profile and region of interest analysis. Ex vivo MRI of hydrogel-encapsulated labeled MSCs implanted within a bovine knee was performed with spoiled GRE (SPGR) and T_1ρ sequences. Fluorescence microscopy, labeling efficiency, and chondrogenesis of MPIO-labeled cells were also examined.

Results

MPIO labeling results in efficient contrast uptake and signal loss that can be visualized and quantitatively characterized via MRI. SPGR imaging of implanted cells results in ex vivo detection within native tissue, and T_1ρ imaging is unaffected by the presence of labeled cells immediately following implantation. MPIO labeling does not affect quantitative glycosaminoglycan production during chondrogenesis, but iron aggregation hinders extracellular matrix visualization. This aggregation may result from excess unincorporated particles following labeling and is an issue that necessitates further investigation.

Conclusion

This study demonstrates the promise of MPIO labeling for monitoring cartilage regeneration and highlights its potential in the development of cell-based tissue engineering strategies.     

Effect of various freezing solutions on cryopreservation of mesenchymal stem cells from different animal species

The objective of this study is to compare the effects of different well defined freezing solutions with a reduced concentration of dimethylsulfoxide (DMSO) combined with polyethylene glycol (PEG) and/or trehalose on cryopreservation of mesenchymal stem cells (MSCs) from mice, rats and calves. Post-thaw cell viability, proliferation capacity and differentiation potential of MSCs from different species were assessed after cryopreservation with the conventional slow freezing method. Although the post-thaw viabilities and metabolic activities varied among the different species, satisfactory results were obtained with 5 percent (v/v) DMSO, 2 percent (w/v) PEG, 3 percent (w/v) trehalose and 2 percent (w/v) bovine serum albumin (BSA) as the freezing solution. Our results showed that mouse MSCs were more robust to cryopreservation compared with rat and bovine MSCs.     

Period 2 regulates neural stem/progenitor cell proliferation in the adult hippocampus

Background  

Newborn granule neurons are generated from proliferating neural stem/progenitor cells and integrated into mature synaptic networks in the adult dentate gyrus of the hippocampus. Since light/dark variations of the mitotic index and DNA synthesis occur in many tissues, we wanted to unravel the role of the clock-controlled Period2 gene (mPer2) in timing cell cycle kinetics and neurogenesis in the adult DG.     

Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

Background  

Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.     

Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

Background

Caffeine is the most commonly consumed psycho-stimulant in the world. The effects of caffeine on the body have been extensively studied; however, its effect on the structure of the brain has not been investigated to date.

Results

In the present study we found that the long-term consumption of caffeine can induce ventriculomegaly; this was observed in 40% of the study rats. In the caffeine-treated rats with ventriculomegaly, there was increased production of CSF, associated with the increased expression of Na⁺, K⁺-ATPase and increased cerebral blood flow (CBF). In contrast to the chronic effects, acute treatment with caffeine decreased the production of CSF, suggesting 'effect inversion' associated with caffeine, which was mediated by increased expression of the A₁ adenosine receptor, in the choroid plexus of rats chronically treated with caffeine. The involvement of the A₁ adenosine receptor in the effect inversion of caffeine was further supported by the induction of ventriculomegaly and Na⁺, K⁺-ATPase, in A₁ agonist-treated rats.

Conclusion

The results of this study show that long-term consumption of caffeine can induce ventriculomegaly, which is mediated in part by increased production of CSF. Moreover, we also showed that adenosine receptor signaling can regulate the production of CSF by controlling the expression of Na⁺, K⁺-ATPase and CBF.