Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.

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Divergent functions of endotrophin on different cell populations in adipose tissue

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00314.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. E952-E963 ◽

Cited By ~ 20

Author(s):

Yueshui Zhao ◽

Xue Gu ◽

Ningyan Zhang ◽

Mikhail G. Kolonin ◽

Zhiqiang An ◽

...

Keyword(s):

Adipose Tissue ◽

Lipid Accumulation ◽

Lysyl Oxidase ◽

Stromal Vascular Fraction ◽

Cell Types ◽

Marker Genes ◽

New Perspective ◽

Collagen Genes ◽

Different Cell Types

Endotrophin is a cleavage product of collagen 6 (Col6) in adipose tissue (AT). Previously, we demonstrated that endotrophin serves as a costimulator to trigger fibrosis and inflammation within the unhealthy AT milieu. However, how endotrophin affects lipid storage and breakdown in AT and how different cell types in AT respond to endotrophin stimulation remain unknown. In the current study, by using a doxycycline-inducible mouse model, we observed significant upregulation of adipogenic genes in the white AT (WAT) of endotrophin transgenic mice. We further showed that the mice exhibited inhibited lipolysis and accelerated hypertrophy and hyperplasia in WAT. To investigate the effects of endotrophin in vitro, we incubated different cell types from AT with conditioned medium from endotrophin-overexpressing 293T cells. We found that endotrophin activated multiple pathological pathways in different cell types. Particularly in 3T3-L1 adipocytes, endotrophin triggered a fibrotic program by upregulating collagen genes and promoted abnormal lipid accumulation by downregulating hormone-sensitive lipolysis gene and decreasing HSL phosphorylation levels. In macrophages isolated from WAT, endotrophin stimulated higher expression of the collagen-linking enzyme lysyl oxidase and M1 proinflammatory marker genes. In the stromal vascular fraction isolated from WAT, endotrophin induced upregulation of both profibrotic and proinflammatory genes. In conclusion, our study provides a new perspective on the effect of endotrophin in abnormal lipid accumulation and a mechanistic insight into the roles played by adipocytes and a variety of other cell types in AT in shaping the unhealthy microenvironment upon endotrophin treatment.

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Gigaspora margarita and Its Endobacterium Modulate Symbiotic Marker Genes in Tomato Roots under Combined Water and Nutrient Stress

Plants ◽

10.3390/plants9070886 ◽

2020 ◽

Vol 9 (7) ◽

pp. 886

Author(s):

Matteo Chialva ◽

Luisa Lanfranco ◽

Gianluca Guazzotti ◽

Veronica Santoro ◽

Mara Novero ◽

...

Keyword(s):

Mycorrhizal Fungi ◽

Marker Gene ◽

Arbuscular Mycorrhizal ◽

Crop Productivity ◽

Nutrient Stress ◽

Growth Effect ◽

Limiting Factors ◽

Marker Genes ◽

Gigaspora Margarita ◽

The Difference

As members of the plant microbiota, arbuscular mycorrhizal fungi (AMF) may be effective in enhancing plant resilience to drought, one of the major limiting factors threatening crop productivity. AMF host their own microbiota and previous data demonstrated that endobacteria thriving in Gigaspora margarita modulate fungal antioxidant responses. Here, we used the G. margarita–Candidatus Glomeribacter gigasporarum system to test whether the tripartite interaction between tomato, G. margarita and its endobacteria may improve plant resilience to combined water/nutrient stress. Tomato plants were inoculated with spores containing endobacteria (B+) or not (B-), and exposed to combined water/nutrient stress. Plants traits, AM colonization and expression of AM marker genes were measured. Results showed that mycorrhizal frequency was low and no growth effect was observed. Under control conditions, B+ inoculated plants were more responsive to the symbiosis, as they showed an up-regulation of three AM marker genes involved in phosphate and lipids metabolism compared with B− inoculated or not-inoculated plants. When combined stress was imposed, the difference between fungal strains was still evident for one marker gene. These results indicate that the fungal endobacteria finely modulate plant metabolism, even in the absence of growth response.

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Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

Journal of General Virology ◽

10.1099/vir.0.82436-0 ◽

2007 ◽

Vol 88 (4) ◽

pp. 1225-1230 ◽

Cited By ~ 41

Author(s):

Nele Tamberg ◽

Valeria Lulla ◽

Rennos Fragkoudis ◽

Aleksei Lulla ◽

John K. Fazakerley ◽

...

Keyword(s):

Fluorescent Protein ◽

Semliki Forest Virus ◽

Cultured Cells ◽

Marker Gene ◽

Virus Production ◽

Marker Genes ◽

Replicase Gene ◽

Marker Virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.

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Pilot investigation on the dose-dependent impact of irradiation on primary human alveolar osteoblasts in vitro

Scientific Reports ◽

10.1038/s41598-021-99323-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anna-Klara Amler ◽

Domenic Schlauch ◽

Selin Tüzüner ◽

Alexander Thomas ◽

Norbert Neckel ◽

...

Keyword(s):

Bone Cells ◽

Marker Gene ◽

Marker Genes ◽

Dependent Manner ◽

Current Standard ◽

Functional Changes ◽

Standard Procedures ◽

The Impact ◽

Dose Dependent

AbstractRadiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

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New strategies in regenerative medicine and surgery and their application to orthopedic surgery field: The bio-active composite therapies (BACTs)

IP International Journal of Orthopaedic Rheumatology ◽

10.18231/j.ijor.2021.014 ◽

2022 ◽

Vol 7 (2) ◽

pp. 58-64

Author(s):

Michele Zocchi

Keyword(s):

Regenerative Medicine ◽

Orthopedic Surgery ◽

New Technology ◽

Stromal Vascular Fraction ◽

Active Components ◽

Recipient Site ◽

The One ◽

Active Composite

Regenerative Medicine and Surgery is a rapidly expanding branch of translational research in tissue engineering, cellular and molecular biology. To date, the methods to improve cell intake, survival and isolation need to comply with a complex and still unclear regulatory frame, becoming everyday more restrictive and often limiting effectiveness and outcome of the therapeutic choices. Thus, the author developed a novel regenerative strategy, based on the synergic action of several bio-active components, called the Bio-Active Composite Therapies (BACTs) to improve grafted cells intake and survival in total compliance with the legal and ethical limits of the current regulatory frame. The rationale at the origin of this new technology is based on the evidence that cells need supportive substrate to survive in vitro and this observation, applying the concept of translational medicine, is true also in vivo. Many different sources have been used in the past for MSCs, molecules and growth factors (GF) isolation and extraction, but the Adipose Tissue and its Stromal Vascular Fraction (SVF) definitely remains the most valuable, abundant, safe and reliable. Bio-Active Composite Mixtures (BACMs) are tailor-made injectable “cocktails” containing several bio-active components to support cells survival and induce a strong regenerative response in vivo by stimulating the recipient site to act as an in-situ real Bioreactor. In this article, the author analyze the main causes of cell’s death and the strategies for preventing it, and outline all the technical steps for preparing the main components of BACMs and the different mixing modalities to obtain the most efficient regenerative action on different clinical and pathological conditions in several surgical specialties. Orthopedic Surgery is definitely the one that most can benefit of these new therapeutic strategies. The final part of this work is anticipating the logical and sequential evolution toward other fundamental technical steps for further supporting and enhancing the most efficient regenerative activity.

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Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

International Journal of Molecular Sciences ◽

10.3390/ijms21072627 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2627

Author(s):

Olivier Gröninger ◽

Samuel Hess ◽

Dirk Mohn ◽

Elia Schneider ◽

Wendelin Stark ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Calcium Phosphate ◽

Amorphous Calcium Phosphate ◽

Marker Gene ◽

Ion Concentration ◽

Marker Genes ◽

Calcium Phosphate Nanoparticles ◽

Cell Commitment

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

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Potential Application of Cord Blood-Derived Stromal Cells in Cellular Therapy and Regenerative Medicine

Journal of Blood Transfusion ◽

10.1155/2012/365182 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Simone Maria Kluth ◽

Teja Falk Radke ◽

Gesine Kogler

Keyword(s):

Regenerative Medicine ◽

Cord Blood ◽

Stromal Cells ◽

Cellular Therapy ◽

Adipogenic Differentiation ◽

Cell Populations ◽

Differentiation Potential ◽

Differentiation And Proliferation

Neonatal stromal cells from umbilical cord blood (CB) are promising alternatives to bone marrow- (BM-) derived multipotent stromal cells (MSCs). In comparison to BM-MSC, the less mature CB-derived stromal cells have been described as a cell population with higher differentiation and proliferation potential that might be of potential interest for clinical application in regenerative medicine. Recently, it has become clear that cord blood contains different stromal cell populations, and as of today, a clear distinction between unrestricted somatic stromal cells (USSCs) and CB-MSC has been established. This classification is based on the expression of DLK-1, HOX, and CD146, as well as functional examination of the adipogenic differentiation potential and the capacity to support haematopoiesis in vitro and in vivo. However, a marker enabling a prospective isolation of the rare cell populations directly out of cord blood is yet to be found. Further analysis may help to reveal even more subpopulations with different properties, which could be useful for the directed application of these cells in preclinical models.

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A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

BIO Web of Conferences ◽

10.1051/bioconf/20191201019 ◽

2019 ◽

Vol 12 ◽

pp. 01019 ◽

Cited By ~ 1

Author(s):

S. Sabbadini ◽

L. Capriotti ◽

C. Limera ◽

O. Navacchi ◽

G. Tempesta ◽

...

Keyword(s):

Marker Gene ◽

Marker Genes ◽

Wine Grape ◽

Thompson Seedless ◽

Multiple Virus ◽

Almost All ◽

New Breeding Techniques ◽

Breeding Techniques ◽

Selection Of

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants for Agrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to control B. cinerea infection.

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miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/β-catenin signaling

Journal of Molecular Endocrinology ◽

10.1530/jme-14-0013 ◽

2014 ◽

Vol 52 (3) ◽

pp. 311-320 ◽

Cited By ~ 31

Author(s):

Chen Chen ◽

Yongdong Peng ◽

Yinglin Peng ◽

Jian Peng ◽

Siwen Jiang

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Binding Protein ◽

Adipogenic Differentiation ◽

Transcriptional Regulators ◽

Marker Genes ◽

Preadipocyte Differentiation ◽

Fatty Acid Binding ◽

Canonical Wnt ◽

Adipogenic Marker

MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator-activated receptor γ (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/β-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of β-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/β-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/β-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue.

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Advances in mesenchymal stem cell exosomes: a review

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02138-7 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Yaya Tang ◽

Yan Zhou ◽

Hong-Jun Li

Keyword(s):

Stem Cells ◽

Regenerative Medicine ◽

Targeted Delivery ◽

Tissue Repair ◽

Medical Problems ◽

Medicine Research ◽

Paracrine Signalling ◽

Tissue Repair And Regeneration ◽

In Vitro Expansion

AbstractStem cells can be used for regenerative medicine and as treatments for disease. The application of tissue engineering-related transplantation, stem cells, and local changes in the microenvironment is expected to solve major medical problems. Currently, most studies focus on tissue repair and regeneration, and mesenchymal stem cells (MSCs) are among the most common research topics. MSCs are applicable as seed cells, and they represent one of the current hot topics in regenerative medicine research. However, due to storage limitations and because cell senescence occurs during in vitro expansion, their clinical application is challenging. Exosomes, which are secreted by MSCs through paracrine signalling, not only have the same effects as MSCs, but they also have the advantages of targeted delivery, low immunogenicity, and high repairability. This article reviews the acquisition methods, characteristics, biological functions, and clinical applications of exosomes.

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