Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.
23 Related JoVE Articles!
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Institutions: Northeastern University.
More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1
. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression.
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5
. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention
. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7
. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9
. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10
. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.
Bioengineering, Issue 59, Gelatin Nanoparticle, Gene Therapy, Targeted Delivery, Pancreatic Cancer, Epidermal Growth Factor Receptor, EGFR
A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Waterloo.
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H
]-thymidine incorporation, protein abundance, and mRNA expression.
Physiology, Issue 88, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
Generation and Recovery of β-cell Spheroids From Step-growth PEG-peptide Hydrogels
Institutions: Indiana University - Purdue University at Indianapolis.
Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (C
, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 β-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.
Biomedical Engineering, Issue 70, Bioengineering, Tissue Engineering, Cellular Biology, Molecular Biology, Biomaterials, beta cells, β-cell, PEG, PEG-peptide hydrogels, hydrogel, MIN6, poylmers, peptides, spheroids, pancreas
Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells
Institutions: University of Colorado Denver, University of Colorado Denver, University of Colorado Denver, University of Colorado Denver.
Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo
pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.
Developmental Biology, Issue 56, mesenchymal stem cell, MSC, lung stem cell, resident lung mesenchymal stem cells, stem cells
Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging
Institutions: Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco.
In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.
Cell Biology, Issue 13, cell labeling, stem cell, MR imaging, cell tracking, iron oxide, contrast agents, mesenchymal stem cells
Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures
Institutions: Medical University of Graz, Austria.
Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2
and in vitro
. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5
or lysed by freeze/thaw cycles6-14
before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17
Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19
and a current lack of standardized protocols for platelet preparation.15,16
This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.
Cellular Biology, Issue 32, Pooled human platelet lysate (pHPL), platelet derived growth factors (PDGFs), cell culture, stem cells
Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)
Institutions: University Medicine Göttingen, Göttingen, Germany.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.
Medicine, Issue 61, neuroscience, amyotrophic lateral sclerosis, ALS, spinal cord, mouse, rotarod, hanging wire
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System
Institutions: Washington University in St. Louis School of Medicine.
Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1
as well as shift isoform ratios of specific genes 2
. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.
Neurobiology, Issue 75, Neuroscience, Medicine, Biomedical Engineering, Genetics, Anatomy, Physiology, Surgery, Pharmacology, Cerebrospinal Fluid, Rodentia, Oligonucleotides, Antisense, Drug Administration Routes, Injections, Intraventricular, Drug Delivery Systems, mouse, rat, brain, antisense oligonucleotide, osmotic pump, Bolus, Ventricle, Neurosciences, Translational, Cerebrospinal fluid, CNS, cannula, catheter, animal model, surgical techniques
Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters
Institutions: University of California, San Diego.
For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9
. However in vitro,
genesis of insulin producing cells from human fetal ICCs is low10
; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro
genesis of insulin producing cells from hESC is much less robust11-17
. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22
, far fewer exist for ICCs10,23,24
. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6
. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.
Medicine, Issue 87, human fetal pancreas, islet cell cluster (ICC), transplantation, immunofluorescence, endocrine cell proliferation, differentiation, C-peptide
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via
functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v
.) or intracerebroventricular (i.c.v.
) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.
) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo
Here we describe the methods that we have developed for the i.v
. and i.c.v.
delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo
preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
Intramyocardial Cell Delivery: Observations in Murine Hearts
Institutions: Imperial College London, Imperial College London, Monash University.
Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.
Medicine, Issue 83, intramyocardial cell injection, heart, grafting, cell therapy, stem cells, fibrotic tissue
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle
Institutions: University of Pittsburgh, University of Pittsburgh, Nazarbayev University, University of California at Los Angeles, Erasmus MC Stem Cell Institute, Oregon Health & Science University, Queen's Medical Research Institute and University of Edinburgh, University of California at Los Angeles, University of Pittsburgh.
Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs
have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.
Cellular Biology, Issue 90, Blood Vessel; Pericyte; Adventitial Cell; Myogenic Endothelial Cell; Multipotent Precursor
Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies
Institutions: University of California, San Francisco - UCSF.
Neuroscience, Issue 6, neurogenesis, cortex, electroporation, injection, stem cells, brain, Translational Research
Propagation of Human Embryonic Stem (ES) Cells
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 1, ES, embryonic stem cells, tissue culture
ES Cell-derived Neuroepithelial Cell Cultures
Institutions: Harvard Medical School.
ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).
Cellular Biology, issue 1, embryonic stem (ES) cells, rosettes, neuroepithelial precursors, stromal cells, differentiation
Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
Institutions: AntiCancer, Inc..
There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.
Cellular Biology, Issue 13, Springer Protocols, hair follicles, liposomes, adenovirus, genes, stem cells
ALS - Motor Neuron Disease: Mechanism and Development of New Therapies
Institutions: Johns Hopkins University.
Medicine, Issue 6, Translational Research, Neuroscience, ALS, stem cells, brain, neuron, upper motor neuron, transplantation
Transplantation of Cells Directly into the Kidney of Adult Zebrafish
Institutions: Massachusetts General Hospital.
Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1
. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2
. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3
. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.
Cellular Biology, Issue 51, zebrafish, kidney, regeneration, transplantation