Aging is considered the main risk factor for IPF [7– 11]. Along with others, we have demonstrated that there is an increase in markers of cell senescence in lung fibroblasts from IPF patients [12–15]. Additionally, we have shown that, in animal models of lung injury, aged bonemarrow-derivedmesenchymalstemcells (B-MSCs) have decreased protective activity . This is in contrast to what we had previously described in young animal models of pulmonary fibrosis, where infusion of B-MSCs isolated from normal young donors in the ini- tial stages of the injury results in a decrease in collagen deposition in the lung after bleomycin instillation [17, 18]. Therefore, we aimed to determine the differences in the biological and functional characteristics of B-MSCs from healthy individuals and IPF patients within the same age range. Characterization of IPF B-MSCs shows an increase in cell senescence linked to an upsurge of senescence-associated secretory phenotypes (SASPs) promoting a proinflammatory milieu and increasing de- position of components from the extracellular matrix. Our data suggest that extrapulmonary alterations in B-MSCs from IPF patients might contribute to the pathogenesis of the disease. To our knowledge, this is the first report describing amelioration in functional and reparative capacities of the endogenous nonpulmonary MSCs from patients who have developed IPF.
Antioxidant supplementation has been suggested as a means to reduce the DNA dam- age and relieve oxidative stress during the expansion and proliferation of bonemarrow-derivedmesenchymalstemcells (bmMSCs) . This oxidative stress is a re- sult of the imbalance between ROS production and diminished endogenous antioxi- dant protection. Accumulative ROS (especially • OH with a half-life of 10 − 9 s) not only have the potential to damage all types of biomolecules (such as DNA, proteins, lipids and carbohydrates), but can also inhibit MSC immunomodulation, thus increasing sen- escence and reducing ex vivo expansion, which is critical for clinical application off the cells . Effective antioxidants that could protect MSCs from oxidative stress are a de- sirable focus of research.
Mammal Bonemarrow is an invaluable source of Mesenchymalstemcells. BoneMarrowderivedMesenchymalStemCells (BM-MSCs) are multipotent, self-renewing cells found in almost all postnatal organs / tissues and are used in the treatment of various disease conditions. Diabetes mellitus is a metabolic syndrome characterized by increased levels of blood glucose leading to various complications like Diabetic Foot, Diabetic Neuropathy, Diabetic Retinopathy, Diabetic Cardiomyopathy and Diabetic Nephropathy. BM-MSCs are able to differentiate into many cell types, and to proliferate ex vivo. These attributes makes them a potential therapeutic tool for cell replacement therapy in diabetes and other diseases. The present review discusses the isolation and culturing of BM-MSCs along with their potential as new therapeutic agent in the treatment of Diabetes related complications and its limitations.
We have previously reported on both the os- teogenic potential of hydroxyapatite (HA) com- bined with bonemarrow-derivedmesenchymalstemcells (BMSCs) and a method involving os- teogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMS- Cs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphat- ase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. How- ever, P3 cell/HA constructs which had incre- ased cell-loading showed abundant bone for- mation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The pre- sent study indicates that the osteogenic poten- tial of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require
Purpose: Mesenchymalstemcells (MSCs) are multipotent and give rise to distinct- ly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechani- cally made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). Materials and Methods: BMSCs grown confluent were mechanically separated with cell scrapers and masses of sepa- rated cells were cultured to form cluster BMSCs. As described here cluster of BM- SCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differenti- ated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immu- nocytochemistry to identify the expression of neural specific markers. Results: For the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Par- ticularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neu- ral cell phenotype, expressing several neuro-glial specific markers. Conclusion: bonemarrow-derivedmesenchymalstemcells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could dif- ferentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neu- ronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.
Bonemarrow-derivedmesenchymalstemcells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional defi- cits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of ␤ -galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migra- tion of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.
Bonemarrow–derivedstemcells contribute to cell turnover and repair in various tissue types, including the kidneys [35,36]. MSCs are attractive candidates for renal repair, because nephrons are of mesenchymal origin and because stromal cells are of crucial importance for sig- naling, leading to differentiation of both nephrons and collecting ducts . In the present study, bonemarrowderivedmesenchymalstemcells were isolated from male rats, grown and characterized by their adhesiveness and fusiform shape and by detection of CD 29; one of the surface markers of rat mesenchymalstemcells, and were used to detect their possible anti-inflammatory, anti- apoptotic and vascular role in amelioration of renal function in experimental DN model. These cells were actually insinuating themselves into the renal tissue as detected by fluorescent microscope. Similar results have been reported by Morigi et al. , who. injected labeled human bonemarrow MSCs with PKH 26 dye into mice with induced acute renal failure. The red fluorescence of the MSC was clearly detected in renal tissues.
Background: Stem cell-fate is highly regulated by stem cell niche, which is composed of a distinct microenvironment, including neighboring cells, signals and extracellular matrix. Bonemarrow-derivedmesenchymalstemcells (BM-MSCs) are multipotent stemcells and are potentially applicable in wide variety of pathological conditions. However, the niche microenvironment for BM-MSCs maintenance has not been clearly characterized. Accumulating evidence indicated that heparan sulfate glycosaminoglycans (HS-GAGs) modulate the self-renewal and differentiation of BM-MSCs, while overexpression of heparanase (HPSE1) resulted in the change of histological profile of bonemarrow. Here, we inhibited the enzymatic activity of cell-autonomous HPSE1 in BM-MSCs to clarify the physiological role of HPSE1 in BM-MSCs. Results: Isolated mouse BM-MSCs express HPSE1 as indicated by the existence of its mRNA and protein, which includes latent form and enzymatically active HPSE1. During in vitro osteo-differentiations, although the expression levels of Hpse1 fluctuated, enzymatic inhibition did not affect osteogenic differentiation, which might due to increased expression level of matrix metalloproteinase 9 (Mmp9). However, cell proliferation and colony formation efficiency were decreased when HPSE1 was enzymatically inhibited. HPSE1 inhibition potentiated SDF-1/CXCR4 signaling axis and in turn augmented the migratory/anchoring behavior of BM-MSCs. We further demonstrated that inhibition of HPSE1 decreased the accumulation of acetylation marks on histone H4 lysine residues suggesting that HPSE1 also modulates the chromatin remodeling.
Aim: This study was designed to evaluate the therapeutic potential of adipose tissue-derivedmesenchymalstemcells (AT- MSCs) compared to bonemarrow-derivedmesenchymalstemcells (BM-MSCs) on the regeneration of surgically created cleft alveolus in dogs. Methods: Split mouth experimental study was performed on twelve healthy mongrel dogs. The dogs were divided into two groups (A and B): Ingroup A, the surgically created alveolus was transplanted with AT-MSCs, scaffold and growth factors at the experimental side (right side of the maxilla). Ingroup B, the surgically created alveolus was transplanted with BM-MSCs, scaffold and growth factors at the experimental side (right side of the maxilla). In the control side (left side of the maxilla), the surgically created alveolus was transplanted with scaffold and growth factors only. The flaps were replaced and sutured with resorbable sutures. Bone regeneration was evaluated clinically and radiographically after 1.5 and 3 months following dogs’ scarification. The data were evaluated with descriptive and t-test methods (p=0.05). Results: Stemcells whether AT-MSCs or BM-MSCs accelerate the healing and regeneration of the defected area by increasing the bone width and surface area; providing the bone quantity and quality as early as 1.5 and 3 months. Conclusions: AT-MSCs and BM-MSCs are attractive tools in bone regeneration. AT-MSCs in experimental studies showed that their effectiveness is comparable to BM-MSCs, in addition to its low cost, ease of harvesting and safer procedure to obtain stemcells as well as less risk of infection.
One successful approach is to isolate cells that express specific molecules on their cell surfaces using monoclo- nal antibodies and cell sorting technologies. Enriched populations of BMMSCs have been isolated from human bonemarrow aspirates using a STRO-1 mono- clonal antibody in conjunction with antibodies against VCAM-1/CD106 , CD146 , low affinity nerve growth factor receptor/CD271, PDGR-R, EGF-R and IGF-1-R , fibroblast cell marker/D7-Fib  and integrin alpha 1/CD49a . A more recent study has also identified molecules co-expressed by a CD271 + mesenchymalstem cell population including platelet derived growth factor receptor-b (CD140b), human epi- dermal growth factor 2/ErbB2 (CD340) and frizzled-9 (CD349) . Further cell separation based upon multi- parameter FACS identified a population of proposed mouse mesenchymal precursors with the composite phenotype Lin - CD45 - CD31 - Sca-1 + . Another recent study also identified and characterized an alternate population of primitive mesenchymalcellsderived from adult mouse bonemarrow, based upon their expression of the SSEA-1 . All approaches used for BMMSC purification and isolation will undergo ex vivo expansion to enrich cell numbers for tissue regeneration or sys- temic therapies by plastic adherent assay. In addition to identifying a novel sub-population of BMMSCs that pos- sess enhanced immunomodulatory properties when compared to regular BMMSCs, we showed that CD34
Unlike other sources of stemcells, bonemarrow is easy to obtain requiring minimal donor site morbidity, invasiveness and anaesthetic and these properties make BM-MSCs an appropriate choice for musculoskeletal injuries (Kennard et al., 2011; Malik and Khan, 2011). For example, to obtain ACL fibroblasts an arthroscopy is required in an already injured knee, whereas BM-MSCs can be aspirated from the iliac crest. BM-MSCs can be isolated with relative ease due to their superior ability to bind to tissue culture plastic relative to other bonemarrowcells (Petite et al., 2000). Once isolated, BM-MSCs can be easily proliferated in vitro without losing their capacity for differentiation. Due to their multi-lineage potential, repair of complex injuries is possible. For example, when injected into a knee joint with injuries to the ACL, medial meniscus and cartilage of the femoral condyles, BM-MSCs mobilize to affected areas and contribute to regeneration (Agung et al., 2006).
C. A half amount of the medium was changed 48 hours after plating and once every 3 to 4 days thereafter. The cell morphology and growth were observed under an Axiovert 200 inverted microscope (Carl Zeiss Meditec AG; Jena, Germany), when cells were de ﬁ ned as passage 0. Following culture for 10 to 12 days when cell con ﬂ uence reached 80% to 90%, each ﬂ ask was added with 1 mL pancreatin containing 0.25% EDTA (GIBCO; Rockville, MD, USA) for incubation of 30 s, and then the pancreatin was removed. Then, cells were digested with pancreatin again at 37°C for 1 min, immediately added with 2 to 3 mL L-DMEM supplemented with 10% FBS, and gently pipetted. The digested cells were passaged at a ratio of 1:2 or 1:3 (numbering: 1.25 to 1.5 × 10 5 cells/mL), when cells were de ﬁ ned as passage 1, and all non-digested cells were dis- carded. Subsequently, cells were passaged once every 3 to 7 days, which were de ﬁ ned as passages 2, 3, etc., and cells at
Six male patients aged between 25 and 45 years, scheduled for coronary artery bypass graftingwere recruited for the study after informed consent. The study received institutional Ethical committee approval as well. Bonemarrow was aspirated from the posterior iliac crest after induction of anesthesia. 20 ml of bone-marrow aspirate was collected in heparinized centrifuge tube and transferred to the laboratory immediately under aseptic condition. The sample was processed within one hour to isolate mononuclear cells (MNCs) using Ficoll-paque density gradient separation method and the other mature blood cell lineages were removed. Mononuclear cells were then washed in PBS and resuspended in DMEM F-12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic (Gibco), plated at a density of 1×10 6 cells/cm 2 . The cells were incubated at 37°C with 5% CO 2 and 95% humidity. This was followed by
elastin-expressing BMSCs cultured with bFGF-loaded PLGA NPs at an equivalent concentration of 20 ng/ml. Cells in all four groups were cultured in proliferation medium for 7 days and then changed to differentiation medium for a further 7 days. At the end of the experi- ment, cells from each group were harvested for evalu- ation of intracellular elastin and collagen expression using reverse transcription (RT)-PCR and western blot- ting, and supernatants of cell culture were also collected to measure the secretion of elastin and collagen into cul- ture media using ELISA. In both RT-PCR and western blotting analysis, significantly increased expressions of elastin and collagen were observed in both groups of elastin-expressing BMSCs, compared with those from BMSCs not expressing elastin, regardless of incubation with either free bFGF or bFGF-loaded PLGA (Fig. 4a, b). In addition, administration of bFGF-loaded PLGA NPs to the elastin-expressing BMSCs further upregulated the expression of elastin and collagen, compared with the same cells incubated with free bFGF. Consistently, co- culture of elastin-expressing BMSCs with bFGF-loaded PLGA exhibited a higher extent of stimulation on the se- cretion of elastin and collagen, compared with the same cells cocultured with free bFGF (Fig. 4c). These results indicated that overexpressing elastin in the BMSCs not only upregulated the expression of elastin itself, but also stimulated the intracellular production of collagen as well as extracellular secretion of both elastin and collagen.
Abstract: Liver transplantation is the most effective treatment for advanced liver disease, but its application is restricted. Cell therapy provides a fresh approach for treating liver cirrhosis. The immunological superiority and stem cell characteristics of mesenchymalstemcells (MSCs) from bonemarrow, umbilical cord, adipose tissue, or dental tissues might allow them to perform well as alternative cell therapy treatments. Many previous animal experi- ments and clinical studies of liver disease have been reported and have demonstrated the efficacy and safety of MSCs therapy; no serious side effects or complications were noted, but not all trials have shown efficacy. Potential mechanisms of action, for example, secreting cytokines and growth factors, promoting liver regeneration, inhibiting inflammation in the liver, facilitating the degradation of excessive extracellular matrix (ECM) and differentiating into multi-lineage cells, could account for how MSCs therapy helps improve liver function. However, exploring the mecha- nism of action still requires further study, and a multitude of problems still await solutions in clinical practice. The use of MSCs in treating liver disease shows broad potential as well as undoubted challenges. In this paper, we will focus on the current possible mechanisms by which bonemarrowmesenchymalstemcells (BM-MSCs) improve liver function, show the relationship between the application of MSCs and the occurrence of liver tumors and hepatic fibrosis, and present recent controversies and prospects for the treatment of liver cirrhosis.
Result: Histochemical and immunohistochemistry studies revealed that ECM components such as Glycosaminoglycans (GAGs), neutral carbohydrate, elastic fibers, collagen I & IV, laminin, and fibronectin were well preserved, and the cells were completely removed after decellularization. Scanning electron microscopy (SEM) showed that 3D ultrastructure of the testis remained intact. In vivo and in vitro studies point out that decellularized scaffold was non-toxic and performed a good platform for cell division. In vivo implant of the scaffolds with or without mesenchymalstemcells (MSCs) showed that appropriate positions for transplantation were the mesentery and liver and the scaffolds could induce donor-loaded MSCs or host migrating cells to differentiate to the cells with phenotype of the sertoli- and leydig-like cells. The scaffolds also provide a good niche for migrating DAZL-positive cells; however, they could not differentiate into post meiotic-cell lineages.
For immunofluorescence staining, the frozen samples were cut into 5-μm sections using a cryostat microtome (Leica, Wetzlar, Hesse, Germany). The slides were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 minutes at room temperature and washed with cold PBS. After treatment with PBS containing 0.25% Triton X-100 for 10 minutes for permeabilization, the samples were incu- bated with 5% normal goat serum/1% BSA/PBS for 30 minutes to block unspecific binding of the antibodies. Subsequently the slides were incubated with primary antibodies specific to CD31 (Abcam, Cambridge, UK) at 1:50, alpha smooth muscle actin (α-SMA; Abcam) at 1:100 and Ki67 (Abcam) at 1:200 overnight at 4°C, followed by PBS washing three times. Alexa Fluor 488- conjugated secondary antibody or Alexa Fluor 594- conjugated secondary antibody was incubated for 1 hour at room temperature (Invitrogen). ProLong Gold antifade reagent with DAPI (Invitrogen) was applied to mount and counterstain the slides. All of the fluorescence pictures were captured and analyzed under a fluorescent micro- scope (Zeiss-spot; Carl Zeiss MicroImaging GmbH, Jena, Thuringia, Germany). For quantification of the microvessel area, 10 fields at 200× were randomly selected from sections stained with CD31 antibody and the vessel area was measured using ImageJ software . For assess- ment of cell proliferation in vivo, the percentage of Ki-67 -positive cells was calculated using the ImageJ software.
However, an important aspect to be noted is the senes- cence associated with these stemcells, which will later lead to their death. Alessio and colleagues have shown that low dose of radiation can lead to senescence . Senescence of dendrimer-labeled MSCs needs further in- vestigation. Since the dendrimers do not leak out of the cells, they are retained within the MSCs and do not label surrounding host cells, thereby avoiding false positives that would occur if the label was transferred to host cells. Given that MSCs can differentiate into different lineages including osteoblasts, adipocytes, and chondrocytes, we assessed whether this differentiation might be affected by the dendrimer label. Previous studies have shown that introduction of certain nanoparticles to MSCs affects their differentiation. For example, nanophase hydroxyapatite, HA-PLGA nanocomposites, and iron oxide nanoparticles (IONP) have been shown to induce osteogenic differenti- ation from human MSCs . PLLA-hydroxyapatite nanocomposites also induce chondrogenic differentiation in MSCs, while graphene quantum dots (GQD) have been shown to enhance differentiation of MSCs into osteoblasts and adipocytes [24, 25]. Though this can be useful when treatments require for the differentiation into a specific cell line, these nanoparticles cannot be used for labeling if it is important for the MSCs to retain their “stemness.” Our results clearly show that our mixed-surface dendri- mers label MSCs without altering their inherent proper- ties of differentiation. Our experiments showed that MSCs were able to differentiate, with and without dendri- mers in them. This is critical if this label is used in MSCs that requires retention of their regenerative properties. Our findings also clearly show that dendrimer-labeled MSCs can be used for differentiation into any cell line that is needed for the purpose of regenerative medicine.
The treatments focus on retaining the function of the remaining cardiomyocytes, but myocardial infarction (MI) still remains a major cause of morbidity and mor- tality worldwide [1, 2]. Recently, mesenchymalstem cell (MSC) transplantation has held great promise for mul- tiple diseases, such as MI [3–6]. The illustrated under- lying therapeutic mechanisms include engraftment , paracrine , and other effects, like exosomes . These mechanisms play a role on the premise that the engrafted MSCs survive for a certain period. However, many studies have shown that only a small number of transplanted MSCs eventually survive to play protective effects [9–11]. Also, evidences indicated that under myo- cardial ischemia conditions, hypoxic stress caused in- creased apoptosis of MSCs and reduced its therapeutic effects [1, 12]. Therefore, it is reasonable to assume that promoting the low engrafted MSC survival in ischemia myocardial tissue may be the key to improving stem cell therapeutic efficacy after MI.