Size-tunable lipid vectors for controlled local delivery of siRNA from gene activated matrix.

Tissue engineering aims to restore or replace different types of biological tissues through the association of cells, biologic factors and biomaterials. Currently, stem cells arise as a major cell source for many therapeutic indications, and their association with 3D scaffolds allow increasing regenerative medicine efficiency. In this context, the use of RNA interference to enhance or control stem cell differentiation into the desired phenotype appears as a promising strategy. However, achieving high transfection efficiency of cells in a 3D structure requires the use of a vector allowing for the spatiotemporally controlled release of the genetic material from these scaffolds. In this study, we report a new siRNA nanovector, called solvent exchange lipoplexe formulation (SELF), which has a tunable size, is stable over time in cell culture conditions and possess a high efficiency to transfect primary human mesenchymal stromal cells (hMSC). We associated SELFs with porous 3D collagen microspheres and demonstrated that the loading capacity and release kinetics were different depending on the size of the associated SELF. Interestingly, these different release profiles resulted in differences in the transfection kinetics of hMSCs. This original and unique type of gene activated matrix, with adaptable release kinetics, could be of interest for long-term and/or sequential transfection profiles of stem cells in 3D culture. STATEMENT OF SIGNIFICANCE: This work combines the use of human mesenchymal stromal cell (hMSC) and gene therapy for tissue engineering. Here, a gene-activated matrix was elaborated with collagen microspheres supporting hMSCs and acting as a reservoir for transfection vectors. This injectable GAM allows for the local and sustained delivery of nucleic acids, hence long-lasting transfection of the supported cells. With the original synthesis protocol presented herein, the size of the nanocarriers can be easily adapted, resulting in different siRNA release profiles from the microspheres. Most interestingly, different siRNA release profiles gave rise to different cell transfection profiles as assessed by the downregulation of a target gene. This highlights the versatility of the system and its suitability for various pathophysiological needs in regenerative medicine.

Extracellular Vesicles Are More Potent Than Adipose Mesenchymal Stromal Cells to Exert an Anti-Fibrotic Effect in an In Vitro Model of Systemic Sclerosis.

Systemic sclerosis (SSc) is a complex disorder resulting from dysregulated interactions between the three main pathophysiological axes: fibrosis, immune dysfunction, and vasculopathy, with no specific treatment available to date. Adipose tissue-derived mesenchymal stromal cells (ASCs) and their extracellular vesicles (EVs) have proved efficacy in pre-clinical murine models of SSc. However, their precise action mechanism is still not fully understood. Because of the lack of availability of fibroblasts isolated from SSc patients (SSc-Fb), our aim was to determine whether a TGFß1-induced model of human myofibroblasts (Tß-Fb) could reproduce the characteristics of SSc-Fb and be used to evaluate the anti-fibrotic function of ASCs and their EVs. We found out that Tß-Fb displayed the main morphological and molecular features of SSc-Fb, including the enlarged hypertrophic morphology and expression of several markers associated with the myofibroblastic phenotype. Using this model, we showed that ASCs were able to regulate the expression of most myofibroblastic markers on Tß-Fb and SSc-Fb, but only when pre-stimulated with TGFß1. Of interest, ASC-derived EVs were more effective than parental cells for improving the myofibroblastic phenotype. In conclusion, we provided evidence that Tß-Fb are a relevant model to mimic the main characteristics of SSc fibroblasts and investigate the mechanism of action of ASCs. We further reported that ASC-EVs are more effective than parental cells suggesting that the TGFß1-induced pro-fibrotic environment may alter the function of ASCs.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Regulate the Mitochondrial Metabolism via Transfer of miRNAs.

Mesenchymal stromal cells (MSCs) are the most commonly tested adult progenitor cells in regenerative medicine. They stimulate tissue repair primarily through the secretion of immune-regulatory and pro-regenerative factors. There is increasing evidence that most of these factors are carried on extracellular vesicles (EVs) that are released by MSCs, either spontaneously or after activation. Exosomes and microvesicles are the most investigated types of EVs that act through uptake by target cells and cargo release inside the cytoplasm or through interactions with receptors expressed on target cells to stimulate downstream intracellular pathways. They convey different types of molecules, including proteins, lipids and acid nucleics among which, miRNAs are the most widely studied. The cargo of EVs can be impacted by the culture or environmental conditions that MSCs encounter and by changes in the energy metabolism that regulate the functional properties of MSCs. On the other hand, MSC-derived EVs are also reported to impact the metabolism of target cells. In the present review, we discuss the role of MSC-EVs in the regulation of the energy metabolism and oxidative stress of target cells and tissues with a focus on the role of miRNAs.

miR-155 Contributes to the Immunoregulatory Function of Human Mesenchymal Stem Cells.

Objectives: Mesenchymal stem/stromal cells (MSCs) are widely investigated in regenerative medicine thanks to their immunomodulatory properties. They exert their anti-inflammatory function thanks to the secretion of a number of mediators, including proteins and miRNAs, which can be released in the extracellular environment or in the cargo of extracellular vesicles (EVs). However, the role of miRNAs in the suppressive function of MSCs is controversial. The aim of the study was to identify miRNAs that contribute to the immunomodulatory function of human bone marrow-derived MSCs (BM-MSCs). Methods: Human BM-MSCs were primed by coculture with activated peripheral blood mononuclear cells (aPBMCs). High throughput miRNA transcriptomic analysis was performed using Human MicroRNA TaqMan® Array Cards. The immunosuppressive function of miRNAs was investigated in mixed lymphocyte reactions and the delayed type hypersensitivity (DTH) murine model. Results: Upon priming, 21 out of 377 tested miRNAs were significantly modulated in primed MSCs. We validated the up-regulation of miR-29a, miR-146a, miR-155 and the down-regulation of miR-149, miR-221 and miR-361 in additional samples of primed MSCs. We showed that miR-155 significantly reduced the proliferation of aPBMCs in vitro and inflammation in vivo, using the DTH model. Analysis of miRNA-mRNA interactions revealed miR-221 as a potential target gene that is down-regulated by miR-155 both in primed MSCs and in aPBMCs. Conclusion: Here, we present evidence that miR-155 participates to the immunosuppressive function of human BM-MSCs and down-regulates the expression of miR-221 as a possible inflammatory mediator.

Correction to: Characterization of immortalized human islet stromal cells reveals a MSC-like profile with pancreatic features.

An amendment to this paper has been published and can be accessed via the original article.

Characterization of immortalized human islet stromal cells reveals a MSC-like profile with pancreatic features.

BACKGROUND: Mesenchymal stromal cells (MSCs) represent an interesting tool to improve pancreatic islet transplantation. They have immunomodulatory properties and secrete supportive proteins. However, the functional properties of MSCs vary according to many factors such as donor characteristics, tissue origin, or isolation methods. To counteract this heterogeneity, we aimed to immortalize and characterize adherent cells derived from human pancreatic islets (hISCs), using phenotypic, transcriptomic, and functional analysis. METHODS: Adherent cells derived from human islets in culture were infected with a hTERT retrovirus vector and then characterized by microarray hybridization, flow cytometry analysis, and immunofluorescence assays. Osteogenic, adipogenic, and chondrogenic differentiation as well as PBMC proliferation suppression assays were used to compare the functional abilities of hISCs and MSCs. Extracellular matrix (ECM) gene expression profile analysis was performed using the SAM (Significance Analysis of Microarrays) software, and protein expression was confirmed by western blotting. RESULTS: hISCs kept an unlimited proliferative potential. They exhibited several properties of MSCs such as CD73, CD90, and CD105 expression and differentiation capacity. From a functional point of view, hISCs inhibited the proliferation of activated peripheral blood mononuclear cells. The transcriptomic profile of hISCs highly clusterized with bone marrow (BM)-MSCs and revealed a differential enrichment of genes involved in the organization of the ECM. Indeed, the expression and secretion profiles of ECM proteins including collagens I, IV, and VI, fibronectin, and laminins, known to be expressed in abundance around and within the islets, were different between hISCs and BM-MSCs. CONCLUSION: We generated a new human cell line from pancreatic islets, with MSCs properties and retaining some pancreatic specificities related to the production of ECM proteins. hISCs appear as a very promising tool in islet transplantation by their availability (as a source of inexhaustible source of cells) and ability to secrete a supportive "pancreatic" microenvironment.

Contribution of microRNAs to the immunosuppressive function of mesenchymal stem cells.

Mesenchymal stem/stromal cells (MSCs) are progenitor cells identified in many adult tissues, in particular in bone marrow or adipose tissue and, in placental tissues. MSCs exert pleiotropic functions that render them attractive for many clinical applications, both in degenerative and inflammatory diseases. Their main mode of action is through the secretion of trophic factors that can be released in the extracellular milieu or packaged within extracellular vesicles. These factors can be proteins, lipids, mRNAs or miRNAs that possess diverse effects, notably pro-angiogenic, anti-fibrotic, anti-apoptotic or anti-inflammatory. The anti-inflammatory role of a number of miRNAs has been demonstrated with different types of immune cells but few have been associated with the immunomodulatory function of MSCs. In this review, we summarize the data on the miRNAs that have been validated as participating to the anti-inflammatory role of MSCs.

Mesenchymal stem cells seeded on a human amniotic membrane improve liver regeneration and mouse survival after extended hepatectomy.

Liver failure remains the leading cause of post-operative mortality after hepatectomy. This study investigated the effect of treatment with allogenic mesenchymal stem cells (MSCs) on survival and liver regeneration 48 hr and 7 days after 80% hepatectomy in C57Bl/6 mice. To optimize their biodistribution, MSCs were grown on acellular human amniotic membranes (HAM) and applied as a patch on the remnant liver. This approach was compared with MSC infusion and HAM patch alone. Hepatectomized mice without any treatment were used as control group. Survival rate was calculated and biological and histopathological parameters were analysed to monitor liver function and regeneration. MSCs grown on HAM retained their ability to proliferate, to differentiate into osteoblasts and adipocytes and to respond to pro-inflammatory stimuli. Extended hepatectomy (80%) led to liver failure that resulted in death within 72 hr in 76% of mice. MSC infusion showed an early but transitory positive effect on survival. MSC/HAM patches stimulated regeneration and significantly improved survival rate (54% vs. 24% in the control group at 7 days). They also decreased the severity of hepatectomy-induced steatosis, suggesting a modulation of lipid metabolism in hepatocytes. MSCs were still present on HAM at Days 2 and 7 posthepatectomy. In conclusion, engineered tissue constructs that combine MSCs and HAM improve survival and liver regeneration after 80% hepatectomy in mice. These encouraging results pave the way to potential clinical application.

Serum-Mediated Oxidative Stress from Systemic Sclerosis Patients Affects Mesenchymal Stem Cell Function.

OBJECTIVES: Properties of mesenchymal stromal/stem cells (MSCs) from systemic sclerosis (SSc) patients have been reported to be altered. MSC-based therapy may therefore rely on the use of allogeneic MSCs from healthy subjects. Here, we investigated whether heterologous MSCs could exhibit altered properties following exposure to oxidative environment of SSc sera. METHODS: Human bone marrow-derived MSCs were cultured in the presence of various sera: control human serum AB (SAB), SAB with HOCl-induced AOPPs at 400 or 1,000 µmol/L (SAB400 or SAB1000, respectively), or H2O2-induced AOPPs or SSc patient serum (PS). Proliferation, apoptosis, and senescence rates of MSCs were evaluated after 3, 6, and 10 days in culture. Reactive oxygen species and nitric oxide production were quantified at 24 h. Trilineage potential of differentiation was tested after 21 days in specific culture conditions and immunosuppressive function measured in a T lymphocyte proliferative assay. RESULTS: In the presence of oxidative environment of PS, MSCs retained their proliferative potential and survived for at least the first 3 days of exposure, while the number of senescent MSCs increased at day 6 and apoptosis rate at day 10. Exposure to PS enhanced the antioxidant capacity of MSCs, notably the expression of SOD2 antioxidant gene. By contrast, the osteoblastic/adipogenic potential of MSCs was increased, whereas their immunosuppressive function was slightly reduced. DISCUSSION: Although some functional properties of MSCs were affected upon culture with PS, evidence from preclinical studies and the present one suggested that MSCs can adapt to the oxidative environment and exert their therapeutic effect.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Comparison between Stromal Vascular Fraction and Adipose Mesenchymal Stem Cells in Remodeling Hypertrophic Scars.

Hypertrophic scars (HTS) are characterized by excessive amount of collagen deposition and principally occur following burn injuries or surgeries. In absence of effective treatments, the use of mesenchymal stem/stromal cells, which have been shown to attenuate fibrosis in various applications, seems of interest. The objectives of the present study were therefore to evaluate the effect of human adipose tissue-derived mesenchymal stem cells (hASC) on a pre-existing HTS in a humanized skin graft model in Nude mice and to compare the efficacy of hASCs versus stromal vascular fraction (SVF). We found that injection of SVF or hASCs resulted in an attenuation of HTS as noticed after clinical evaluation of skin thickness, which was associated with lower total collagen contents in the skins of treated mice and a reduced dermis thickness after histological analysis. Although both SVF and hASCs were able to significantly reduce the clinical and histological parameters of HTS, hASCs appeared to be more efficient than SVF. The therapeutic effect of hASCs was attributed to higher expression of TGFß3 and HGF, which are important anti-fibrotic mediators, and to higher levels of MMP-2 and MMP-2/TIMP-2 ratio, which reflect the remodelling activity responsible for fibrosis resorption. These results demonstrated the therapeutic potential of hASCs for clinical applications of hypertrophic scarring.

Antifibrotic, Antioxidant, and Immunomodulatory Effects of Mesenchymal Stem Cells in HOCl-Induced Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a rare intractable disease with unmet medical need and fibrosis-related mortality. Absence of efficient treatments has prompted the development of novel therapeutic strategies, among which mesenchymal stem cells/stromal cells (MSCs) or progenitor stromal cells appear to be one of the most attractive options. The purpose of this study was to use the murine model of hypochlorite-induced SSc to investigate the systemic effects of MSCs on the main features of the diffuse form of the disease: skin and lung fibrosis, autoimmunity, and oxidative status. METHODS: We compared the effects of different doses of MSCs (2.5 × 10(5) , 5 × 10(5) , and 10(6) ) infused at different time points. Skin thickness was assessed during the experiment. At the time of euthanasia, biologic parameters were quantified in blood and tissues (by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, assessment of collagen content). Assessments of histology and immunostaining were also performed. RESULTS: A lower expression of markers of fibrosis (Col1, Col3, Tgfb1, and aSma) was observed in both skin and lung following MSC infusion, which was consistent with histologic improvement and was inversely proportional to the injected dose. Importantly, sera from treated mice exhibited lower levels of anti-Scl-70 autoantibodies and enhanced antioxidant capacity, confirming the systemic effect of MSCs. Of interest, MSC administration was efficient in both the preventive and the curative approach. We further provide evidence that MSCs exerted an antifibrotic role by normalizing extracellular matrix remodeling parameters as well as reducing proinflammatory cytokine levels and increasing antioxidant defenses. CONCLUSION: The results of this study demonstrate the beneficial and systemic effects of MSC administration in the HOCl murine model of diffuse SSc, which is a promising finding from a clinical perspective.

Mesenchymal Stem Cell-Derived Interleukin 1 Receptor Antagonist Promotes Macrophage Polarization and Inhibits B Cell Differentiation.

The role of interleukin 1 receptor antagonist (IL1RA) in mediating the immunosuppressive effect of mesenchymal stem/stromal cells (MSCs) has been reported in several studies. However, how MSC-derived IL1RA influences the host response has not been clearly investigated. We therefore derived MSCs from the bone marrow of IL1RA knockout mice and evaluated their immunosuppressive effect on different immune cell subsets. IL1RA deficient (IL1RA(-/-) ) or wild type (wt) MSCs inhibited to the same extend the proliferation of T lymphocytes. On the contrary, IL1RA(-/-) MSCs were less effective than wt MSCs to induce in vitro the macrophage polarization from M1 to M2 phenotype secreting IL10 and exerting a suppressive effect on CD4(+) T cells. Moreover compared with wt MSCs, IL1RA(-/-) MSCs did not efficiently support the survival of quiescent B lymphocytes and block their differentiation toward CD19(+) CD138(+) plasmablasts secreting IgG antibodies. The effectiveness of IL1RA secreted by MSCs in controlling inflammation was further shown in vivo using the collagen-induced arthritis murine model. MSCs lacking IL1RA expression were unable to protect mice from arthritic progression and even worsened clinical signs, as shown by higher arthritic score and incidence than control arthritic mice. IL1RA(-/-) MSCs were not able to decrease the percentage of Th17 lymphocytes and increase the percentage of Treg cells as well as decreasing the differentiation of B cells toward plasmablasts. Altogether, our results provide evidence of the key role of IL1RA secreted by MSCs to both control the polarization of macrophages toward a M2 phenotype and inhibit B cell differentiation in vivo.

Management of adolescents with very poorly controlled type 1 diabetes by nurses: a parallel group randomized controlled trial.

BACKGROUNDS: Fluctuation in glycemia due to hormonal changes, growth periods, physical activity, and emotions make diabetes management difficult during adolescence. Our objective was to show that a close control of patients' self-management of diabetes by nurse-counseling could probably improve metabolic control in adolescents with type 1 diabetes. METHODS: We designed a multicenter, randomized controlled, parallel group, clinical trial. Seventy seven adolescents aged 12-17 years with A1C >8% were assigned to either an intervention group (pediatrician visit every 3 months + nurse visit and phone calls) or to the control group (pediatrician visit every 3 months). The primary outcome was the evolution of the rate of A1C during the 12 months of follow-up. Secondary outcomes include patient's acceptance of the disease (evaluated by visual analog scale), the number of hypoglycemic or ketoacidosis episodes requiring hospitalization, and evaluation of A1C rate over time in each group. RESULTS: Seventy-seven patients were enrolled by 10 clinical centers. Seventy (89.6%) completed the study, the evolution of A1C and participants satisfaction over the follow-up period was not significantly influenced by the nurse intervention. CONCLUSION: Nurse-led intervention to improve A1C did not show a significant benefit in adolescents with type 1 diabetes because of lack of power. Only psychological management and continuous glucose monitoring have shown, so far, a slight but significant benefit on A1C. We did not show improvements in A1C control in teenagers by nurse-led intervention. TRIAL REGISTRATION: Clinical Trials.gov registration number: NCT00308256, 28 March 2006.

PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation.

The treatment of anterior cruciate ligament (ACL) failures remains a current clinical challenge. The present study aims at providing suitable degradable scaffolds for ligament tissue engineering. First, we focus on the design and the evaluation of poly(lactide)/poloxamer or poly(lactide)/poloxamine multiblock copolymers selected and developed to have suitable degradation and mechanical properties to match ACL repair. In the second part, it is shown that the copolymers can be processed in the form of microfibers and scaffolds consisting of a combination of twisted/braided fibers to further modulate the mechanical properties and prepare scaffold prototypes suitable for ligament application. Finally, after assessment of their cytocompatibility, the polymer scaffolds are associated with mesenchymal stem cells (MSCs). MSC differentiation toward a ligament fibroblast phenotype is promoted by a dual stimulation including an inductive culture medium and cyclic mechanical loads. RT-qPCR analyses confirm the potential of our scaffolds and MSCs for ACL regeneration with upregulation of some differentiation markers including Scleraxis, Tenascin-C and Tenomodulin.

Adipose Mesenchymal Stem Cells Isolated after Manual or Water-jet-Assisted Liposuction Display Similar Properties.

Mesenchymal stem or stromal cells (MSC) are under investigation in many clinical trials for their therapeutic potential in a variety of diseases, including autoimmune and inflammatory disorders. One of the main sources of MSCs is the adipose tissue, which is mainly obtained by manual liposuction using a cannula linked to a syringe. However, in the past years, a number of devices for fat liposuction intended for clinical use have been commercialized but few papers have compared these procedures in terms of stromal vascular fraction (SVF) or adipose mesenchymal stromal cells (ASC). The objective of the present study was to compare and qualify for clinical use the ASC obtained from fat isolated with the manual or the Bodyjet(®) water-jet-assisted procedure. Although the initial number of cells obtained after collagenase digestion was higher with the manual procedure, the percentage of dead cells, the number of colony forming unit-fibroblast and the phenotype of cells were identical in the SVF at isolation (day 0) and in the ASC populations at day 14. We also showed that the osteogenic and adipogenic differentiation potentials of ASCs were identical between preparations while a slight but significant higher in vitro immunosuppressive effect was observed with ASCs isolated from fat removed with a cannula. The difference in the immunomodulatory effect between ASC populations was, however, not observed in vivo using the delayed-type hypersensitivity (DTH) model. Our data, therefore, indicate that the procedure for fat liposuction does not impact the characteristics or the therapeutic function of ASCs.

Promyelocytic leukemia zinc-finger induction signs mesenchymal stem cell commitment: identification of a key marker for stemness maintenance?

INTRODUCTION: Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and bone tissue engineering given their ability to differentiate into chondrocytes and osteoblasts. However, the common origin of these two specialized cell types raised the question about the identification of regulatory pathways determining the differentiation fate of MSCs into chondrocyte or osteoblast. METHODS: Chondrogenesis, osteoblastogenesis, and adipogenesis of human and mouse MSC were induced by using specific inductive culture conditions. Expression of promyelocytic leukemia zinc-finger (PLZF) or differentiation markers in MSCs was determined by RT-qPCR. PLZF-expressing MSC were implanted in a mouse osteochondral defect model and the neotissue was analyzed by routine histology and microcomputed tomography. RESULTS: We found out that PLZF is not expressed in MSCs and its expression at early stages of MSC differentiation is the mark of their commitment toward the three main lineages. PLZF acts as an upstream regulator of both Sox9 and Runx2, and its overexpression in MSC enhances chondrogenesis and osteogenesis while it inhibits adipogenesis. In vivo, implantation of PLZF-expressing MSC in mice with full-thickness osteochondral defects resulted in the formation of a reparative tissue resembling cartilage and bone. CONCLUSIONS: Our findings demonstrate that absence of PLZF is required for stemness maintenance and its expression is an early event at the onset of MSC commitment during the differentiation processes of the three main lineages.

FOXO3A regulation by miRNA-29a Controls chondrogenic differentiation of mesenchymal stem cells and cartilage formation.

Skeletal development and cartilage formation require stringent regulation of gene expression for mesenchymal stem cells (MSCs) to progress through stages of differentiation. Since microRNAs (miRNAs) regulate biological processes, the objective of the present study was to identify novel miRNAs involved in the modulation of chondrogenesis. We performed miRNA profiling and identify miR-29a as being one of the most down-regulated miRNAs during the chondrogenesis. Using chromatin immunoprecipitation, we showed that SOX9 down-regulates its transcription. Moreover, the over-expression of miR-29a strongly inhibited the expression of chondrocyte-specific markers during in vitro chondrogenic differentiation of MSCs. We identified FOXO3A as a direct target of miR-29a and showed a down- and up-regulation of FOXO3a protein levels after transfection of, respectively, premiR- and antagomiR-29a oligonucleotides. Finally, we showed that using the siRNA or premiR approach, chondrogenic differentiation was inhibited to a similar extent. Together, we demonstrate that the down-regulation of miR-29a, concomitantly with FOXO3A up-regulation, is essential for the differentiation of MSCs into chondrocytes and in vivo cartilage/bone formation. The delivery of miRNAs that modulate MSC chondrogenesis may be applicable for cartilage regeneration and deserves further investigation.

Investigation on the properties of linear PLA-poloxamer and star PLA-poloxamine copolymers for temporary biomedical applications.

The objective of this work was to develop and study new biodegradable thermoplastics with improved mechanical properties for potential use as temporary implantable biomaterials. Linear poloxamer and star-shaped poloxamine have been used as macroinitiators for the ring-opening polymerization (ROP) of lactide to yield high molecular weight PLA-based thermoplastic block copolymers. The influence of the nature of the macroinitiator, PLA crystallinity and initial molecular weight on the copolymers properties was investigated by performing a 7-week degradation test in PBS. The evaluation of water uptakes and molecular weights during the degradation pointed out an early hydrolytic degradation of the 100-kg∙mol(-1) copolymers compared to the 200-kg∙mol(-1) ones (molecular weight decrease of ca. 40% and 20%, respectively). A dramatic loss of tensile mechanical properties was also observed for the 100-kg∙mol(-1) copolymers, whereas the 200-kg∙mol(-1) copolymers showed stable or even slightly improved properties with Young's moduli around 500 MPa and yield strains around 3% to 4%. Finally, the cytocompatibility of the more stable 200 kg∙mol(-1) copolymers was confirmed by murine mesenchymal stem cells (MSCs) culture.

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord.

BACKGROUND: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). RESULTS: Here we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. CONCLUSION: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.