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Background Mesenchymal stem/stromal cells (MSC) are commonly used in regenerative medicine. Among different tissues, iliac crest bone marrow (BM) represents the most exploited source, but its disadvantages are a painful aspiration procedure and low cell number. An alternative, readily available source of MSC for research would be beneficial for regenerative medicine development. This work aimed to propose a new source of bone marrow isolation in which the femoral shaft is taken during total hip arthroplasty (THA). Methods In preliminary experiments, three different gradient methods for cell separation (Ficoll-Paque 1.078 g/mL, 17% sucrose gradient, BM seeding fraction) were tested with regard to the time of primary culture, initial cell number, the phenotype, and morphology of MSC. Then human bone marrow MSC derived from two different sources, iliac crest aspirate (BM-MSCi) or femoral shaft (BM-MSCt), were analyzed in terms of cell number and colony-forming ability followed by differentiation potential of MSC into osteo-, chondro-, and adipogenic lineages as well as mRNA expression of a variety of cytokines and growth factors. Results Our studies showed that MSC isolated from the bone marrow of two different sources and cultured under appropriate conditions had similar characteristics and comparable propensity to differentiate into mesodermal cells. MSC derived from BM-MSCi or BM-MSCt expressed various growth factors. Interestingly, the expression of EGF, FGF, IGF, and PDGF-A was much higher in BM-MSCt than BM-MSCi. Conclusions The results of our study demonstrate that human MSC isolated from the BM of the femoral shaft have similar biological characteristics as MSC derived from the iliac crest, suggesting the femoral shaft as a possible alternative source for mesenchymal stem/stromal cells.

Cell-based therapies deliveredviaintrathecal injection are considered as one of the most promisingsolutions for the treatment of amyotrophic lateral sclerosis (ALS). Herein, injectable manganese-basedbiocompatible hydrogel blends were developed, that can allow image-guided cell delivery. Thehydrogels can also provide physical support for cells during injection, and at the intrathecal space aftertransplantation, while assuring cell survival. In this regard, different formulations of methacrylated gellangum/hyaluronic acid hydrogel blends (GG-MA/HA) were considered as a vehicle for cell delivery. Thehydrogels blends were supplemented with paramagnetic Mn2+to allow a real-time monitorization ofhydrogel depositionvia T1-weighted magnetic resonance imaging (MRI). The developed hydrogels wereeasily extruded and formed a stable fiber upon injection into the cerebrospinal fluid. Hydrogels preparedwith a 75 : 25 GG-MA to HA ratio supplemented with MnCl2at 0.1 mM showed controlled hydrogeldegradation, suitable permeability, and a distinct MRI signalin vitroandin vivo. Additionally, human-derived adipose stem cells encapsulated in 75 : 25 GG-MA/HA hydrogels remained viable for up to14 days of culturein vitro. Therefore, the engineered hydrogels can be an excellent tool for injectableimage-guided cell delivery approaches., Sı´lvia Vieira acknowledges the FCT PhD scholarship (SFRH/BD/102710/2014). The FCT distinction attributed to J. Miguel Oliveira under the Investigator FCT program (IF/01285/2015) and J. Silva-Correia (IF/00115/2015) are also greatly acknowledged. The authors also acknowledge the funds provided under the project NanoTech4ALS, funded under the EU FP7 M-ERA.NET program, and ESF (POWR.03.02.00-00-I028/17-00).

Background: Dysfunction of glia contributes to the deterioration of the central nervous system in a wide array of neurological disorders, thus global replacement of glia is very attractive. Human glial-restricted precursors (hGRPs) transplanted intraventricularly into neonatal mice extensively migrated and rescued the lifespan in half of the studied mice, whereas mouse GRPs (mGRPs) presented no therapeutic benefit. We studied in the same experimental setting canine GRPs (cGRP) to determine whether their therapeutic potential falls between hGRPs and mGRPs. Additional motivation for the selection of cGRPs was a potential for use in veterinary medicine. Methods: cGRPs were extracted from the brain of dog fetuses. The cells were transplanted into the anterior or posterior aspect of the lateral ventricle (LV) of neonatal, immunodeficient, dysmyelinated mice (Mbpshi, Rag2 KO, shiv/rag2). Outcome measures included early cell biodistribution, animal survival and myelination assessed with MRI, immunohistochemistry and electron microscopy. Results: Grafting of cGRP into posterior LV significantly extended animal survival, whereas no benefit was observed after anterior LV transplantation. In contrast, myelination of the corpus callosum was more prominent in anteriorly transplanted animals. Conclusions: The extended survival of animals after transplantation of cGRPs could be explained by the vicinity of the transplant near the brain stem.

Haemophilia A and B are rarely occurring X chromosome-linked congenital coagulation disorders dominated by spontaneous joint bleedings and chronic synovitis, leading to development of haemophilic arthropathy (HA). Progranulin (PGRN) is a growth factor with anti-inflammatory and immunomodulatory properties. PGRN is an important molecule in the pathogenesis of osteoarthritis (OA) and rheumatological disorders. This study was aimed at investigating the potential role of PGRN in the mechanisms underlying the pathogenesis of HA. The serum levels of PGRN were measured by enzyme-linked immunosorbent assay (ELISA) in patients with end-stage knee joint HA (n = 20) and end-stage primary knee joint OA (n = 20) who met the inclusion and exclusion criteria. The clinical and radiological assessment of disease severity was evaluated by the Knee Society Score (KSS) and Kellgren-Lawrence scale. Median PGRN levels in HA patients was 349.1 ng/mL (232.8-415.6 ng/mL) and in OA patients 148.3 ng/mL (112.1-275.3 ng/mL) with statistically significant differences between both groups (P < 0.015). Further analysis revealed no correlation between PGRN levels and any of the patient demographics and clinical parameters. This study demonstrates increased PGRN serum levels in patients with HA and provides new insights into the mechanisms underlying the pathogenesis of HA indicating a new potential target for therapeutic intervention.

Heterotopic ossification (HO) is an abnormal formation of mature lamellar bone within extraskeletal soft tissues, such as muscles, tendons, and ligaments. This process is thought to be induced by inflammation associated with tissue injuries. HO is classified using two subtypes: resulting from injury or genetically inherited. HO formation is associated with polytrauma patients with traumatic brain injuries and spinal cord injuries. Moreover, HO is also considered to be a post-operative risk factor in some orthopaedic procedures. In this review, we summarize our current understanding of the pathology of different types of HO and discuss its current and future therapies. Thus far, research has revealed cellular and molecular pathways leading to HO formation and proposed several possible mechanisms leading to HO and conserved signalling pathways common in the different HO subtypes. Non-steroidal anti-inflammatory drug treatment and localized low-dose irradiation are currently the only available prophylactic treatments for HO. However, they are not always effective and do not target the osteogenic processes directly. New therapeutic strategies targeting the pathological processes of HO, such as bone morphogenetic protein (BMP) inhibitors like noggin, BMP type 1 receptor inhibitor, and nuclear retinoid acid receptor-gamma (RARγ) agonists, are currently being investigated. In-depth understanding of the HO pathological process could help to develop effective therapeutic strategies.

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.

The most recent research concerning amyotrophic lateral sclerosis (ALS) emphasizes the role of glia in disease development. Thus, one can suspect that the effective therapeutic strategy in treatment of ALS would be replacement of defective glia. One of the basic problems with human glial progenitors (hGRPs) replacement strategies is the time needed for the cells to become fully functional in vivo. The lifespan of most popular high copy number SOD1 mutant mice might be too short to acknowledge benefits of transplanted cells. We focused on developing immunodeficient rag2−/− model of ALS with lower number of transgene copies and longer lifespan. The obtained hSOD1/rag2 double mutant mice have been characterized. QPCR analysis revealed that copy number of hSOD1 transgene varied in our colony (4–8 copies). The difference in transgene copy number may be translated to significant impact on the lifespan. The death of long- and short-living hSOD1/rag2 mice is preceded by muscular weakness as early as one month before death. Importantly, based on magnetic resonance imaging we identified that mutant mice demonstrated abnormalities within the medullar motor nuclei. To conclude, we developed long-living double mutant hSOD1/rag2 mice, which could be a promising model for testing therapeutic utility of human stem cells.

Stem cell therapy is being intensely investigated within the last years. Expectations are high regarding mesenchymal stem cell (MSC) treatment in translational medicine. However, many aspects concerning MSC therapy should be profoundly defined. Due to a variety of approaches that are investigated, potential effects of stem cell therapy are not transparent. On the other hand, most results of MSC administration in vivo have confirmed their safety and showed promising beneficial outcomes. However, the therapeutic effects of MSC-based treatment are still not spectacular and there is a potential risk related to MSC applications into specific cell niche that should be considered in long-term observations and follow-up outcomes. In this review, we intend to address some problems and critically discuss the complex nature of MSCs in the context of their effective and safe applications in regenerative medicine in different diseases including graft versus host disease (GvHD) and cardiac, neurological, and orthopedic disorders.

Mesenchymal stem cells (MSC) exhibit enormous heterogeneity which can modify their regenerative properties and therefore influence therapeutic effectiveness as well as safety of these cells transplantation. In addition the high phenotypic plasticity of MSC population makes it enormously sensitive to any changes in environmental properties including fluctuation in oxygen concentration. We have shown here that lowering oxygen level far below air atmosphere has a beneficial impact on various parameters characteristic for umbilical cord Wharton Jelly- (WJ-) MSC and adipose tissue- (AD-) derived MSC cultures. This includes their cellular composition, rate of proliferation, and maintenance of stemness properties together with commitment to cell differentiation toward mesodermal and neural lineages. In addition, the culture genomic stability increased significantly during long-term cell passaging and eventually protected cells against spontaneous transformation. Also by comparing of two routinely used methods of MSCs isolation (mechanical versus enzymatic) we have found substantial divergence arising between cell culture properties increasing along the time of cultivationin vitro. Thus, in this paper we highlight the urgent necessity to develop the more sensitive and selective methods for prediction and control cells fate and functioning during the time of growthin vitro.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Cell-based gene therapy holds a great promise for the treatment of human malignancy. Among different cells, mesenchymal stem cells (MSCs) are emerging as valuable anti-cancer agents that have the potential to be used to treat a number of different cancer types. They have inherent migratory properties, which allow them to serve as vehicles for delivering effective therapy to isolated tumors and metastases. MSCs have been engineered to express anti-proliferative, pro-apoptotic, and anti-angiogenic agents that specifically target different cancers. Another field of interest is to modify MSCs with the cytokines that activate pro-tumorigenic immunity or to use them as carriers for the traditional chemical compounds that possess the properties of anti-cancer drugs. Although there is still controversy about the exact function of MSCs in the tumor settings, the encouraging results from the preclinical studies of MSC-based gene therapy for a large number of tumors support the initiation of clinical trials.

Background aims As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly–derived MSCs (WJ-MSCs). Methods Primary cultures growing in 21% oxygen were either transferred into 5% O 2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α–3α were evaluated. Results Lowering O 2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O 2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration. Conclusions A physiologically relevant microenvironment of 5% O 2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α–mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin–laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Stroke is the leading cause of severe disability, and lacunar stroke is related to cognitive decline and hemiparesis. There is no effective treatment for the majority of patients with stroke. Thus, stem cell-based regenerative medicine has drawn a growing body of attention due to the capabilities for trophic factor expression and neurogenesis enhancement. Moreover, it was shown in an experimental autoimmune encephalomyelitis (EAE) model that even short-lived stem cells can be therapeutic, and we have previously observed that phenomenon indirectly. Here, in a rat model of lacunar stroke, we investigated the molecular mechanisms underlying the positive therapeutic effects of short-lived human umbilical cord-blood-derived neural stem cells (HUCB-NSCs) through the distinct measurement of exogenous human and endogenous rat trophic factors. We have also evaluated neurogenesis and metalloproteinase activity as cellular components of therapeutic activity. As expected, we observed an increased proliferation and migration of progenitors, as well as metalloproteinase activity up to 14 days post transplantation. These changes were most prominent at the 7-day time point when we observed 30 % increases in the number of bromodeoxyuridine (BrdU)-positive cells in HUCB-NSC transplanted animals. The expression of human trophic factors was present until 7 days post transplantation, which correlated well with the survival of the human graft. For these 7 days, the level of messenger RNA (mRNA) in the analyzed trophic factors was from 300-fold for CNTF to 10,000-fold for IGF, much higher compared to constitutive expression in HUCB-NSCs in vitro. What is interesting is that there was no increase in the expression of rat trophic factors during the human graft survival, compared to that in non-transplanted animals. However, there was a prolongation of a period of increased trophic expression until 14 days post transplantation, while, in non-transplanted animals, there was a significant drop in rat trophic expression at that time point. We conclude that the positive therapeutic effect of short-lived stem cells may be related to the net increase in the amount of trophic factors (rat + human) until graft death and to the prolonged increase in rat trophic factor expression subsequently.