Characterization of Dental Pulp Pluripotent-like Stem Cells (DPPSC) and their mesodermal differentiation potential.

Dental pulp represents an easily accessible autologous source of adult stem cell populations. Among them, a population named dental pulp pluripotent-like stem cells (DPPSC) has been isolated from the dental pulp of human third molars and express pluripotency markers such as OCT3/4 and SOX2. DPPSC show pluripotent-like behaviour differentiating in vitro into tissues of the three embryonic layers and being able to form teratoma-like structures. This population also represents a source of adult stem cells without the ethical controversy or safety issues that are associated with the use of embryonic stem cells or induced pluripotent stem cells. In this project, we studied the growth rate and genetic stability of several populations of DPPSC from different donors, comparing them to other stem cell populations obtained from the dental pulp (DPSC). We observed that DPPSC divide faster than DPSC and present no chromosomal abnormalities, in contrast to DPSC. We also confirmed the pluripotency markers expression of these DPPSC populations at mRNA and protein level. We then analyzed how the expression of these pluripotency markers varies when the cells are cultured in vitro for several passages (at least until passage 15), when they are seeded at higher densities or split at higher confluence, and when they are cultured using a GMP-approved maintaining medium. Our results showed that DPPSC expression of pluripotency markers is higher at passage 10, and that the cells up-regulate some pluripotency markers and down-regulate some others when they are cultured at higher densities or using GMP-approved medium. In this project, we also evaluated the in vitro and in vivo DPPSC differentiation potential to different mesodermal-derived lineages. We demonstrated that DPPSC can differentiate in vitro into the endothelial lineage, as well as into the smooth and skeletal muscle lineage, using DPPSC from different donors and passages. Regarding their in vivo differentiation, we performed a wound healing assay in nude mice. We found that DPPSC are able to accelerate wound closure, revascularization and matrix organization in the regenerating wound area, as well as being able to differentiate in vivo to the smooth muscle lineage. In another experiment, we injected DPPSC in the skeletal muscle of two immunodeficient dystrophic mouse models, Scid/mdx and Sgcb-null Rag2-null γc-null. DPPSC engrafted in the skeletal muscle of both mouse models and differentiated into the endothelial, smooth muscle and skeletal muscle lineage, since the cells showed integration in muscular fibres and vessels. Taken together, our results showed that DPPSC are a genetically stable population that can be expanded in vitro in GMP conditions and that own mesodermal differentiation potential in vitro and in vivo. They represent a potential source of stem cells for translational therapies to enhance the wound healing process and slow down dystrophic muscle degeneration

Interactions between microRNAs and long non-coding RNAs in cardiac development and repair

Non-coding RNAs (ncRNAs) are emerging players in muscle regulation. Based on their length and differences in molecular structure, ncRNAs are subdivided into several categories including small interfering RNAs, stable non-coding RNAs, microRNAs (miRs), long non-coding RNAs (lncRNAs), and circular RNAs. miRs and lncRNAs are able to post-transcriptionally regulate many genes and bring into play several traits simultaneously due to a myriad of different targets. Recent studies have emphasized their importance in cardiac regeneration and repair. As their altered expression affects cardiac function, miRs and lncRNAs could be potential targets for therapeutic intervention. In this context, miR- and lncRNA-based gene therapies are an interesting field for harnessing the complexity of ncRNA-based therapeutic approaches in cardiac diseases. In this review we will focus on lncRNA- and miR-driven regulations of cardiac development and repair. Finally, we will summarize miRs and lncRNAs as promising candidates for the treatment of heart diseases.status: publishe

Interactions between microRNAs and long non-coding RNAs in cardiac development and repair

Non-coding RNAs (ncRNAs) are emerging players in muscle regulation. Based on their length and differences in molecular structure, ncRNAs are subdivided into several categories including small interfering RNAs, stable non-coding RNAs, microRNAs (miRs), long non-coding RNAs (lncRNAs), and circular RNAs. miRs and lncRNAs are able to post-transcriptionally regulate many genes and bring into play several traits simultaneously due to a myriad of different targets. Recent studies have emphasized their importance in cardiac regeneration and repair. As their altered expression affects cardiac function, miRs and lncRNAs could be potential targets for therapeutic intervention. In this context, miR- and lncRNA-based gene therapies are an interesting field for harnessing the complexity of ncRNA-based therapeutic approaches in cardiac diseases. In this review we will focus on lncRNA- and miR-driven regulations of cardiac development and repair. Finally, we will summarize miRs and lncRNAs as promising candidates for the treatment of heart diseases

Direct contribution of skeletal muscle mesenchymal progenitors to bone repair

International audienceAbstract Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma

Aging affects the in vivo regenerative potential of human mesoangioblasts

Sarcopenia is the age-related loss of muscle mass, strength, and function. Although the role of human satellite cells (SCs) as adult skeletal muscle stem cells has been deeply investigated, little is known about the impact of aging on muscle interstitial stem cells. Here, we isolated the non-SC CD56- fraction from human muscle biopsies of young and elderly subjects. The elderly interstitial cell population contained a higher number of CD15+ and PDGFRα+ cells when compared to young samples. In addition, we found that the CD56- /ALP+ cells were well represented as a multipotent stem cell population inside the CD56- fraction. CD56- /ALP+ /CD15- cells were clonogenic, and since they were myogenic and expressed NG2, α-SMA and PDGFRβ can be considered mesoangioblasts (MABs). Interestingly, elderly MABs displayed a dramatic impairment in the myogenic differentiation ability in vitro and when transplanted in dystrophic immunodeficient Sgcb-null Rag2-null γc-null mice. In addition, elderly MABs proliferated less, but yet retained other multilineage capabilities. Overall, our results indicate that aging negatively impacted on the regenerative potential of MABs and this should be carefully considered for potential therapeutic applications of MABs

Additional file 1: Figure S1. of Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation

Characterization of undifferentiated DPPSC. a Cell morphology of DPPSC from passage 10 observed with optic microscopy. DPPSC are characterized as small-sized cells with large nuclei and low cytoplasm content. b Immunofluorescence analysis of OCT3/4-FITC, SSEA4-PE, and Merge. Hoechst (HT) as a nucleus control. DPPSC were positive for these embryonic markers, and both were located in the nucleus. c FACS analysis of DPPSC. c1 FACS analysis of membrane markers: CD105 (92,15%), CD29 (99,63%), CD146 (15,54%) and CD45 (0.04%). c2 FACS analysis of pluripotency nuclear markers: OCT3/4 (76,72%) and NANOG (30,18%). d RT-PCR of OCT3/4, NANOG and SOX2 expresions in DPPSC and DPMSC. e Western Blot analysis of OCT3/4 in DPPSC and DPMSC at different time points (5, 10 and 15 passages). GAPDH as a housekeeping. (TIF 1031 kb

Making physics fun: key concepts, classroom activities, and everyday examples, grades K-8

In easy-to-understand language, this resource presents engaging, ready-to-use learning experiences that address the "big ideas" in K-8 science education and help students make larger, real-world connections

Additional file 2: Table S2. of Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration

List of antibodies used for protein detection in immunofluorescence analyses. (DOCX 16 kb

Additional file 1: Table S1. of Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration

List of primers used for cDNA amplification in qRT-PCR analyses. (DOCX 11 kb

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions

: Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells