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1.
iScience ; 26(10): 108027, 2023 Oct 20.
Article in English | MEDLINE | ID: mdl-37860763

ABSTRACT

Invasive freshwater mussels, such as the zebra (Dreissena polymorpha), quagga (Dreissena rostriformis bugensis), and golden (Limnoperna fortunei) mussel have spread outside their native ranges throughout many regions of the North American, South American, and European continents in recent decades, damaging infrastructure and the environment. This review describes ongoing efforts by multiple groups to develop genetic biocontrol methods for invasive mussels. First, we provide an overview of genetic biocontrol strategies that have been applied in other invasive or pest species. Next, we summarize physical and chemical methods that are currently in use for invasive mussel control. We then describe the multidisciplinary approaches our groups are employing to develop genetic biocontrol tools for invasive mussels. Finally, we discuss the challenges and limitations of applying genetic biocontrol tools to invasive mussels. Collectively, we aim to openly share information and combine expertise to develop practical tools to enable the management of invasive freshwater mussels.

2.
Sci Rep ; 5: 15234, 2015 Oct 20.
Article in English | MEDLINE | ID: mdl-26482195

ABSTRACT

The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair, and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian, and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations.


Subject(s)
Ethnicity/genetics , Genetic Variation , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Animals , Biomarkers , Cell Differentiation/genetics , Cell Line , Cell Lineage/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Embryoid Bodies/cytology , Embryoid Bodies/metabolism , Endoderm/cytology , Fibroblasts/cytology , Gene Expression Profiling , Gene Order , Genetic Vectors/genetics , Humans , Karyotype , Mice , Teratoma/genetics , Teratoma/metabolism , Teratoma/pathology , Transgenes
3.
Exp Neurol ; 248: 491-503, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23891888

ABSTRACT

Neural progenitor cells (NPCs) have shown modest potential and some side effects (e.g. allodynia) for treatment of spinal cord injury (SCI). In only a few cases, however, have NPCs shown promise at the chronic stage. Given the 1.275 million people living with chronic paralysis, there is a significant need to rigorously evaluate the cell types and methods for safe and efficacious treatment of this devastating condition. For the first time, we examined the pre-clinical potential of NPCs derived from human induced pluripotent stem cells (hiPSCs) to repair chronic SCI. hiPSCs were differentiated into region-specific (i.e. caudal) NPCs, then transplanted into a new, clinically relevant model of early chronic cervical SCI. We established the conditions for successful transplantation of caudalized hiPSC-NPCs and demonstrate their remarkable ability to integrate and produce multiple neural lineages in the early chronic injury environment. In contrast to prior reports in acute and sub-acute injury models, survival and integration of hiPSC-derived neural cells in the early chronic cervical model did not lead to significant improvement in forelimb function or induce allodynia. These data indicate that while hiPSCs show promise, future work needs to focus on the specific hiPSC-derivatives or co-therapies that will restore function in the early chronic injury setting.


Subject(s)
Cell Differentiation/physiology , Neural Stem Cells/transplantation , Neurogenesis/physiology , Neuroglia/cytology , Neurons/cytology , Spinal Cord Injuries/therapy , Stem Cell Transplantation , Animals , Cell Survival/physiology , Disease Models, Animal , Female , Humans , Induced Pluripotent Stem Cells/transplantation , Motor Activity/physiology , Nerve Regeneration/physiology , Rats , Rats, Long-Evans , Recovery of Function/physiology , Spinal Cord Injuries/physiopathology
4.
Stem Cells Dev ; 22(19): 2641-54, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23672680

ABSTRACT

To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens-induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally "weak grip" on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines--even lines with similar origins--is likely the most direct means of improving reprogramming efficiency.


Subject(s)
Cell Differentiation/physiology , Induced Pluripotent Stem Cells/metabolism , Muscle, Skeletal/metabolism , Neurons/metabolism , Animals , Cell Line , Electrophysiology , Fibroblasts , Humans , Induced Pluripotent Stem Cells/cytology , Mice , Muscle, Skeletal/cytology , Neurons/cytology , Patch-Clamp Techniques , Phenotype , Skin/cytology
5.
Cell Reprogram ; 15(1): 24-34, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23276226

ABSTRACT

The production of healthy, live, cloned animals by somatic cell nuclear transfer (SCNT) has been hampered by low efficiencies. Significant epigenetic changes must take place to ensure proper chromatin remodeling in SCNT. We hypothesized that exogenous expression of OCT4 in donor fibroblasts prior to its fusion with enucleated oocytes would facilitate SCNT reprogramming. We infected bovine adult fibroblasts with retroviral vectors containing yellow fluorescent protein (YFP) only, or the OCT4 gene fused to YFP (YO). We found that development to the blastocyst stage was not different between NT-YFP and NT-YO groups. NT-YFP embryos had the fewest trophoblast cells, measured by numbers of CDX2-positive cells. Fibroblasts expressing OCT4 had reduced levels of histone 3 lysine 9 or 27 trimethylation (H3K9me3 and H3K27me3, respectively). NT-YO blastocysts displayed higher H3K9me3 levels than IVF and NT-YFP embryos; however, they did not have different H3K27me3 levels. Levels of XIST mRNA expression in NT-YO and NT-YF were higher when compared to in vitro-fertilized blastocysts. We observed no differences in the expression of SOX2, NANOG, and CDX2. Although overexpression of OCT4 in donor cells increased H3K9me3 and did not reduce XIST gene expression, we show that a single transcription factor can affect the number of trophectoderm cells in bovine SCNT embryos.


Subject(s)
Animals, Genetically Modified/embryology , Blastocyst/metabolism , Cell Nucleus/metabolism , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Nuclear Transfer Techniques , Octamer Transcription Factor-3/biosynthesis , Animals , Animals, Genetically Modified/genetics , Blastocyst/cytology , Cattle , Cell Nucleus/genetics , Cloning, Organism/methods , Fibroblasts/cytology , Histones/genetics , Histones/metabolism , Humans , Methylation , Octamer Transcription Factor-3/genetics , RNA, Long Noncoding/biosynthesis , RNA, Long Noncoding/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transfection
6.
Stem Cells Dev ; 22(5): 735-49, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23025754

ABSTRACT

Approximately 12.5% of all 9,920 extant bird species in the world are threatened with extinction, and yet conservation efforts through natural breeding of captive species continue to encounter difficulties. However, sperm cryopreservation and artificial insemination offer potential benefits over natural breeding, but their applicability is still limited in nondomestic species. In this study, we aimed to exploit the potential of germ cell xenotransplantation as an alternative tool for preserving germplasm of endangered birds. The study was designed to investigate whether transfer of either spermatogonia-enriched cell fraction (SEF) or crude testicular cell fraction (CTF) from adult Japanese quails (as a model for wild species) would result in recolonization of gamma-irradiated gonads of adult recipient chickens. One month after transplantation, 75% of recipients injected with SEF and 25% of recipients injected with CTF resumed spermatogenesis. However, it took more than 3 months for 33% of the negative controls to resume marginal production of sperm. Some SEF recipients produced more spermatozoa bearing head morphology compared with donor controls. DNA analysis using quail-specific primers did not detect donor's DNA in these recipients' semen. However, 6 months after xenotransplantation, presence of quail germ cells was demonstrated by polymerase chain reaction and by immunohistochemistry in 1 rooster injected with SEF. These findings indicate that spermatogonia from adult quails were capable of colonizing immunocompetent testis of adult chickens but failed to produce sufficient sperm. Despite this limitation, the present approach represents a potential conservation tool that may be used to rescue germ cells of endangered adult male birds.


Subject(s)
Chickens , Coturnix , Spermatogenesis , Spermatogonia/transplantation , Spermatozoa/transplantation , Testis/cytology , Transplantation, Heterologous/veterinary , Animals , Breeding , Chickens/physiology , Coturnix/physiology , Endangered Species , Female , Insemination, Artificial , Male , Spermatozoa/physiology
7.
Epigenomics ; 4(6): 665-74, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23244311

ABSTRACT

The epigenetic machinery plays a pivotal role in the control of many of the body's key cellular functions. It modulates an array of pliable mechanisms that are readily and durably modified by intracellular or extracellular factors. In the fast-moving field of neuroepigenetics, it is emerging that faulty epigenetic gene regulation can have dramatic consequences on the developing CNS that can last a lifetime and perhaps even affect future generations. Mounting evidence suggests that environmental factors can impact the developing brain through these epigenetic mechanisms and this report reviews and examines the epigenetic effects of one of the most common neurotoxic pollutants of our environment, which is believed to have no safe level of exposure during human development: lead.


Subject(s)
Brain/drug effects , Environmental Exposure , Epigenesis, Genetic , Lead Poisoning, Nervous System, Childhood/genetics , Brain/growth & development , Child , Humans
8.
Stem Cells Dev ; 20(10): 1669-78, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21495906

ABSTRACT

For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases.


Subject(s)
Aging/drug effects , Cell Culture Techniques/methods , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/cytology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Leukemia Inhibitory Factor/pharmacology , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dogs , Embryoid Bodies/cytology , Epigenesis, Genetic/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Induced Pluripotent Stem Cells/metabolism , Karyotyping , Kruppel-Like Factor 4 , Male , Mice , Microsatellite Repeats/genetics , Testis/cytology
9.
PLoS One ; 5(11): e14095, 2010 Nov 23.
Article in English | MEDLINE | ID: mdl-21124794

ABSTRACT

BACKGROUND: As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion "resets" some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal. CONCLUSIONS/SIGNIFICANCE: These results - coupled with earlier data from our laboratory - suggest that IPSC conversion not only resets the "biological clock", but can also rejuvenate the energetic capacity of derived cells.


Subject(s)
Cell Differentiation/physiology , Fibroblasts/physiology , Induced Pluripotent Stem Cells/physiology , Mitochondria/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Cell Line , Energy Metabolism/physiology , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Membrane Potential, Mitochondrial/physiology , Microscopy, Electron , Mitochondria/physiology , Mitochondria/ultrastructure
10.
PLoS One ; 4(12): e8124, 2009 Dec 02.
Article in English | MEDLINE | ID: mdl-19956585

ABSTRACT

BACKGROUND: Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell "rejuvenation." METHODOLOGY/PRINCIPAL FINDINGS: We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres. CONCLUSIONS/SIGNIFICANCE: While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.


Subject(s)
Cellular Reprogramming/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Telomere/metabolism , Aged , Cell Differentiation , Cell Line , Genetic Vectors/genetics , Humans , Lentivirus/genetics , Male , Microscopy, Phase-Contrast , Phenotype , Teratoma/pathology
11.
Gene Expr Patterns ; 9(1): 54-64, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18723125

ABSTRACT

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.


Subject(s)
Animals, Genetically Modified/genetics , Antigens, Viral/genetics , Cytomegalovirus/genetics , Gene Expression Regulation, Viral , Hot Temperature , Immediate-Early Proteins/genetics , Promoter Regions, Genetic/genetics , Zebrafish/genetics , Age Factors , Animals , Tissue Distribution , Transgenes/physiology
12.
J Cell Biochem ; 91(4): 821-39, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14991773

ABSTRACT

Skeletal muscle contractile activity has been implicated in many aspects of muscle cell differentiation and maturation. Much of the research in this area has depended upon costly and labor-intensive cultures of isolated primary muscle cells because widely available immortalized muscle cell lines often do not display a high level of either spontaneous or stimulated contractile activity. We sought to develop conditionally-immortalized skeletal muscle cell lines that would provide a source of myofibers that exhibit robust spontaneous contractile activity similar to primary muscle cultures. Using a tetracycline-regulated retroviral vector expressing a temperature-sensitive T-antigen to infect primary myoblasts, we isolated individual clonal muscle precursor cell lines that have characteristics of activated satellite cells during growth and rapidly differentiate into mature myotubes with spontaneous contractile activity after culture in non-transformation-permissive conditions. Comparison of these cell lines (known as rat myoblast-like tetracycline (RMT) cell lines) to primary cell cultures revealed that they share a wide variety of morphological, physiological, and biochemical characteristics. Most importantly, the time-course and extent of activity-dependent gene regulation observed in primary cell culture for all genes tested, including subunits of the nicotinic acetylcholine receptor (nAChR), muscle specific kinase (MuSK), and myogenin, is reproduced in RMT lines. These immortalized cell lines are a useful alternative to primary cultures for studying muscle differentiation and molecular and physiological aspects of electrical activity in muscle fibers.


Subject(s)
Gene Expression Regulation/drug effects , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , Stem Cells/drug effects , Tetracycline/pharmacology , Animals , Antigens, Polyomavirus Transforming/genetics , Antigens, Polyomavirus Transforming/metabolism , Cell Differentiation/drug effects , Cell Line, Transformed , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , DNA-Binding Proteins/metabolism , Electric Stimulation , Homeodomain Proteins/metabolism , Immunohistochemistry , Muscle Contraction , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/virology , MyoD Protein/metabolism , Myogenic Regulatory Factor 5 , Myogenic Regulatory Factors/metabolism , Myogenin/metabolism , PAX7 Transcription Factor , Rats , Receptors, Cholinergic/metabolism , Retinoblastoma Protein/metabolism , Retroviridae/genetics , Simian virus 40/genetics , Stem Cells/cytology , Stem Cells/metabolism , Stem Cells/virology , Temperature , Trans-Activators/metabolism
13.
Cell Signal ; 16(5): 551-63, 2004 May.
Article in English | MEDLINE | ID: mdl-14751541

ABSTRACT

During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whose products are destined for the synapse, is suppressed by muscle activity. One of the best-studied examples of activity-dependent gene regulation in muscle are those encoding nicotinic acetylcholine receptor (nAChR) subunits. We recently showed that nAChR gene expression is inhibited by calcium/calmodulin-dependent protein kinase II (CaMKII) and CaMKII inhibitors block activity-dependent suppression of these genes. Here we report results investigating the mechanism by which CaMKII suppresses nAChR gene expression. We show that the muscle helix-loop-helix transcription factor, myogenin, is necessary for activity-dependent control of nAChR gene expression in cultured rat myotubes and is a substrate for CaMKII both in vitro and in vivo. CaMKII phosphorylation of myogenin is induced by muscle activity and this phosphorylation influences DNA binding and transactivation. Thus we have identified a signaling mechanism by which muscle activity controls nAChR gene expression in developing muscle.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Muscle Development/physiology , Myogenin/metabolism , Neuromuscular Junction/embryology , Neuromuscular Junction/enzymology , Receptors, Nicotinic/genetics , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Cells, Cultured , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Luciferases/genetics , Luciferases/metabolism , Muscle Contraction/physiology , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/enzymology , Phosphorylation/drug effects , Promoter Regions, Genetic , Rats , Receptors, Nicotinic/metabolism , Recombinant Proteins/metabolism , Signal Transduction/drug effects
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