scholarly journals Изучение регенеративного потенциала стволовых клеток цельного костного мозга для лечения механических травм кожи в модельных экспериментах на мышах

2020 ◽  
Author(s):  
E.V. Bogdanenko ◽  
L.A. Sergievich ◽  
A.V. Karnaukhov ◽  
N.A. Karnaukhova ◽  
I.A. Lizunova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Marker Gene ◽  
Fluorescent Microscopy ◽  
Mechanical Trauma ◽  
Donor Cells ◽  
Study Results ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Stromal Stem Cells

Введение. Культивированные мезенхимальные стромальные стволовые клетки (МССК), выделенные из костного мозга (КМ), можно использовать для лечения обширных ран, однако это очень дорого, трудоёмко и возможно только через несколько дней после их получения. При этом цельный донорский КМ можно вводить системно непосредственно после травмы без выделения МССК, так как они содержатся в нём естественным образом. Это важно для предотвращения гибели и инвалидизации пострадавших. Цель исследования - изучение терапевтического потенциала цельного донорского КМ, пересаживаемого мышам-реципиентам после нанесения им механических травм. Методика. Эксперимент выполнен на 38 животных. На следующий день после облучения в дозе 6,5 Гр 18 мышам-реципиентам линии C57BL/6 наносили резаную рану в межлопаточной области спины, а затем вводили внутривенно 100 мкл суспензии клеток донорского цельного КМ, несущих маркерный ген зеленого флуоресцентного белка EGFP. Реципиентов забивали через 1, 2, 3, 7, 11, 14, 21, 28 и 35 сут после трансплантации. Под флуоресцентным микроскопом изучали участки кожи, прилегающие к ране, а также дно раны и струп. Скорость заселения этих зон сравнивали со скоростью заселения участков кожи без раны на пояснице данных реципиентов и в межлопаточной и поясничной областях (20) у облучённых животных-реципиентов без травмы. Результаты. Уже на следующий день после трансплантации в участках кожи, прилегающих к ране, и на дне раны обнаруживали донорские клетки. Через 7 сут наблюдалось массированное заселение раны флуоресцирующими клетками различных типов; в то же время в участках кожи без раны на пояснице данных реципиентов донорские клетки появились в существенных количествах только через 11 сут. Донорские клетки сохранялись в коже по меньшей мере 35 сут после трансплантации без всяких признаков элиминирования. У животных без травмы заселение кожи донорскими клетками происходило медленнее, чем у травмированных, с похожим типом заселения обеих изучаемых зон (межлопаточной и поясничной). Заключение. Полученные результаты позволяют предположить, что повреждённые ткани выделяют цитокины, обладающие способностью привлекать большинство донорских клеток именно к ране. МССК цельного КМ заращивали рану с очень большой скоростью, из чего можно предположить, что его трансплантация сразу после получения различных травм по эффективности может быть не хуже, чем лечение культивированными МССК, а по оперативности воздействия и экономичности намного его превосходить.Introduction. Cultured mesenchymal stromal stem cells (MSSC), isolated from the bone marrow (BM) may be used to treat extensive wounds, but this treatment is very expensive, time consuming and possible only in several days after the injury. However, donor whole BM can be systemically administered directly after an injury without isolating MSSC because they are naturally contained in the BM. This is important for preventing death and disability of accident victims. The aim of the work was to study the therapeutic potential of donor whole BM transplanted to recipient mice after inflicting a mechanical trauma. Methods. On the next day after irradiation at a dose of 6.5 Gy, recipient C57BL/6 mice were subjected to a cut wound in the interscapulum and then injected intravenously with 100 μl of cell suspension of the donor whole BM carrying a marker gene of the green fluorescent protein, EGFP. Recipients were sacrificed in 1, 2, 3, 7, 11, 14, 21, 28, and 35 days after transplantation. The bottom of the wound and the scab on it and also areas of the skin adjacent to the wound were examined by fluorescent microscopy. The rate of colonization of these zones was compared to the rate of colonization of non-injured lumbar skin areas of these recipients and interscapular and lumbar regions of irradiated recipients without traumas. Results. Already on the next day after transplantation, the donor cells were detected in skin areas adjacent to the wound and on the bottom of the wound. In 7 days, massive wound colonization with various types of fluorescent cells was observed; at the same time, substantial amount of donor cells appeared in the non-injured lumbar skin of these recipients only in 11 days. The donor cells remained in the skin for at least 35 days after transplantation without any signs of elimination. Colonization of skin with the donor cells was slower in animals without than with wounds with a similar type of colonization in both of the studied zones (interscapular and lumbar). Conclusions. The study results suggested that damaged tissues secrete cytokines that are capable of attracting the majority of donor cells specifically to the wound. MSSC of the whole BM healed the wound very fast, which indicated that the MSSC transplantation immediately after a trauma is not inferior in effectiveness to the treatment with cultured MSSC and may be much superior in both promptness of the effect and cost-efficiency.

scholarly journals Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein

2017 ◽  
Vol 107 (4) ◽  
pp. 483-490 ◽  
Author(s):  
Nisha Govender ◽  
Mui-Yun Wong
Keyword(s):  
Oil Palm ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Early Stage ◽  
Marker Gene ◽  
Induction Medium ◽  
Root Penetration ◽  
Ganoderma Boninense ◽  
Agrobacterium Strain ◽  
Green Fluorescent

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

1999 ◽  
Vol 112 (16) ◽  
pp. 2705-2714
Author(s):  
E.M. Burns ◽  
L. Christopoulou ◽  
P. Corish ◽  
C. Tyler-Smith
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Quantitative Measurement ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Chromosome Loss ◽  
Facs Analysis ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

6 Invitro validation of gene edited phenotypes using CRISPR-dCas9 transcriptional activators

2020 ◽  
Vol 32 (2) ◽  
pp. 127
Author(s):  
K. M. Polkoff ◽  
N. K. Gupta ◽  
J. A. Piedrahita
Keyword(s):  
Dna Sequences ◽  
Fluorescent Protein ◽  
Transcriptional Start Site ◽  
Fluorescent Microscopy ◽  
Large Animal ◽  
Transcriptional Activators ◽  
Gfp Expression ◽  
Time Investment ◽  
Donor Cells ◽  
Fetal Fibroblasts

Despite the extensive advantages of gene-edited large animals for agriculture and biomedical purposes, they represent a large monetary and time investment due to high husbandry costs, long gestation lengths, and few offspring; that is, 9 months for one calf and almost 4 months for pigs. Even with known DNA sequences before somatic cell nuclear transfer (SCNT), inserted transgenes are often not expressed as expected. Therefore, there is a need to phenotypically validate the gene modifications invitro before investing time and resources in the creation of a gene-edited large animal; however, many gene targets are tissue specific and not expressed in SCNT donor cells. In this work, we show that CRISPR-dCas9 transcriptional activators (TAs) can be used to validate functional transgene insertion in nonexpressing SCNT donor cells, in our case fetal fibroblasts. To demonstrate this concept, we first generated a DNA knockin of the H2B-GFP sequence into the porcine LGR5 locus. CRISPR/Cas9 nuclease was used to create a double-stranded break in the genomic DNA downstream of the LGR5 promoter. A homology-directed repair template plasmid containing H2B-GFP flanked by 1000bp homology arms flanking the cut site was co-transfected with the Cas9 and gRNA, and cells were seeded at low density for colony outgrowth. Colonies were genotyped by PCR and sequencing to verify successful targeted transgene integration. To test whether TAs allow for invitro validation of transgene expression, 5×105 wildtype or gene-edited fibroblasts were nucleofected (Lonza) with 500ng total of four gRNA plasmids (Addgene #43860) designed to target the 1-kb region upstream of the LGR5 transcriptional start site in combination with 500ng VP64-dCas9 (Addgene #47107). Detection of green fluorescent protein (GFP) was analysed by fluorescent microscopy followed by flow cytometry; at least 30 000 events were recorded for each treatment (Cytoflex). Our results show that GFP was detected in on average 28.6% of the gene-edited cells transfected with LGR5 TAs but not detected in gene-edited cells that were not transfected with LGR5 TAs (0%) or in wild-type cells transfected with the LGR5 TAs (0%). The experiment was repeated three times. Next, to prove that our invitro validation replicates the invivo phenotype, the gene-edited colonies heterozygous for the insertion were used for SCNT to generate piglets. Epidermal cells, which contain a population of LGR5+ stem cells, were isolated from the skin and sorted for GFP expression. The RT-qPCR results from GFP+ or GFP− cells showed the presence of LGR5 transcripts in the GFP+ cells but not GFP− cells. In conclusion, TAs were necessary and sufficient to detect LGR5-promoter driven H2B-GFP expression in gene-edited fibroblasts invitro, which faithfully recapitulates the invivo phenotype of the gene-edited animal. Further preliminary data from our laboratory suggest that our novel method can be used to detect successful gene knockouts in addition to transgene knockins and can be used to validate phenotypic outcomes of DNA modifications before the generation of gene-edited animals.

scholarly journals Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing

2019 ◽  
Vol 5 (8) ◽  
pp. eaax0801 ◽  
Author(s):  
Minh Khanh Nguyen ◽  
Cong Truc Huynh ◽  
Alex Gilewski ◽  
Samantha E. Wilner ◽  
Keith E. Maier ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Sirna Delivery ◽  
Hydrolytic Degradation ◽  
Nucleic Acid Delivery ◽  
Covalent Bonds ◽  
Disulfide Linkages ◽  
Green Fluorescent ◽  
Interfering Rna ◽  
Transfection Agent

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein–expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

scholarly journals Directed Fiber Outgrowth from Transplanted Embryonic Cortex-Derived Neurospheres in the Adult Mouse Brain

2009 ◽  
Vol 2009 ◽  
pp. 1-7
Author(s):  
Vesna Radojevic ◽  
Josef P. Kapfhammer
Keyword(s):  
Neural Progenitor Cells ◽  
Fluorescent Protein ◽  
Neuronal Progenitor ◽  
Axonal Projections ◽  
Axonal Connections ◽  
Donor Cells ◽  
The Central Nervous System ◽  
Green Fluorescent ◽  
Host Brain

Neural transplantation has emerged as an attractive strategy for the replacement of neurons that have been lost in the central nervous system. Multipotent neural progenitor cells are potentially useful as donor cells to repopulate the degenerated regions. One important aspect of a transplantation strategy is whether transplanted cells are capable of fiber outgrowth with the aim of rebuilding axonal connections within the host brain. To address this issue, we expanded neuronal progenitor from the cortex of embryonic day 15 ubiquitously green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the entorhinal cortex of 8-week-old mice immediately after a perforant pathway lesion. After transplantation into a host brain with a lesion of the entorhino-hippocampal projection, the neurosphere-derived cells extended long fiber projections directed towards the dentate gyrus. Our results indicate that transplantation of neurosphere-derived cells might be a promising strategy to replace lost or damaged axonal projections.

scholarly journals Development and Dynamics of Pseudomonassp. Biofilms

2000 ◽  
Vol 182 (22) ◽  
pp. 6482-6489 ◽  
Author(s):  
Tim Tolker-Nielsen ◽  
Ulla C. Brinch ◽  
Paula C. Ragas ◽  
Jens Bo Andersen ◽  
Carsten Suhr Jacobsen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Physiological State ◽  
Cell Aggregation ◽  
Flow Chamber ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Biofilm Bacteria ◽  
Fluorescent Cells ◽  
Confocal Scanning Laser ◽  
Green Fluorescent

ABSTRACT Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.

scholarly journals Isolation, Characterization, and Transduction of Endometrial Decidual Tissue Multipotent Mesenchymal Stromal/Stem Cells from Menstrual Blood

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Filippo Rossignoli ◽  
Anna Caselli ◽  
Giulia Grisendi ◽  
Serena Piccinno ◽  
Jorge S. Burns ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Menstrual Blood ◽  
Decidual Tissue ◽  
Promising Tool ◽  
Clonogenic Potential ◽  
Cell And Gene Therapy ◽  
Green Fluorescent ◽  
Stromal Stem Cells

Mesenchymal stromal/stem cells (MSCs) reveal progenitor cells-like features including proliferation and differentiation capacities. One of the most historically recognized sources of MSC has been the bone marrow, while other sources recently include adipose tissue, teeth, bone, muscle, placenta, liver, pancreas, umbilical cord, and cord blood. Frequently, progenitor isolation requires traumatic procedures that are poorly feasible and associated with patient discomfort. In the attempt to identify a more approachable MSC source, we focused on endometrial decidual tissue (EDT) found within menstrual blood. Based also on recent literature findings, we hypothesized that EDT may contain heterogeneous populations including some having MSC-like features. Thus, we here sought to isolate EDT-MSC processing menstrual samples from multiple donors. Cytofluorimetric analyses revealed that resulting adherent cells were expressing mesenchymal surface markers, including CD56, CD73, CD90, CD105 and CD146, and pluripotency markers such as SSEA-4. Moreover, EDT-MSC showed a robust clonogenic potential and could be largely expandedin vitroas fibroblastoid elements. In addition, differentiation assays drove these cells towards osteogenic, adipogenic, and chondrogenic lineages. Finally, for the first time, we were able to gene modify these progenitors by a retroviral vector carrying the green fluorescent protein. From these data, we suggest that EDT-MSC could represent a new promising tool having potential within cell and gene therapy applications.

scholarly journals Gonadotropes in medaka grow long extensions with varicosity-like swellings, projecting towards each other and blood vessels

2019 ◽  
Author(s):  
Heidi Kristine Grønlien ◽  
Romain Fontaine ◽  
Kjetil Hodne ◽  
Isabelle Tysseng ◽  
Eirill Ager-Wick ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Network Formation ◽  
Scientific Literature ◽  
Fluorescent Protein ◽  
Flash Photolysis ◽  
Close Contact ◽  
Primary Cultures ◽  
Fluorescent Microscopy ◽  
Cell Types ◽  
Green Fluorescent

AbstractAccumulating evidence in the scientific literature indicates that some pituitary cell types are organized in complex networks. Previous observations have indicated that this may also be the case in medaka (Oryzias latipes), where long cellular extensions with varicosity-like swellings are formed by luteinizing hormone (Lh)-producing gonadotropes expressing green fluorescent protein. In this study, immunofluorescence of intact pituitaries reveal that Lh beta polypeptides are mainly located in the varicosity-like swellings and at the extremity of the extensions. Some extensions approach nearby Lh-producing cells, and other extensions are in close contact with blood vessels. To investigate whether these extensions may contribute to network formation, we followed their development using confocal and fluorescent microscopy on primary cultures. During the first two days in culture, the extensions initiated the formation of homotypic cellular networks and clustering. The extensions were classified as either major or minor. Major extensions were several cell diameters long, dependent on microtubules, and displaying varicosity-like swellings at regular intervals. Minor extensions typically protruded from the major, were significantly shorter and thinner, and dependent on actin. The swellings were dependent on both microtubules and actin. Flash photolysis of caged Ca2+ showed that the signal was propagated along the major extensions, intensifying in each swelling, indicating a continuous structure. However, the Ca2+ signal did not transfer to the next cell in the network, but was transferred between cells merged at their somas. In summary, Lh-producing gonadotropes in medaka display a complex cellular structure of extensions, possibly linked to communication with blood vessels and/or other gonadotrope cells.

scholarly journals Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 431-440 ◽  
Author(s):  
Bruno Verhasselt ◽  
Magda De Smedt ◽  
Rita Verhelst ◽  
Evelien Naessens ◽  
Jean Plum
Keyword(s):  
Gene Therapy ◽  
Dendritic Cells ◽  
Green Fluorescent Protein ◽  
T Cell ◽  
Gene Transfer ◽  
Cord Blood ◽  
Fluorescent Protein ◽  
Marker Gene ◽  
Green Fluorescent

Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

2000 ◽  
Vol 12 (2) ◽  
pp. 1 ◽  
Author(s):  
Sangho Roh ◽  
Hosup Shim ◽  
Woo-suk Hwang ◽  
Jong-taek Yoon
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

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