Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.
22 Related JoVE Articles!
Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set
In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1
The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2
Additionally, James Thomson et al.
reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.
Developmental Biology, Issue 34, iPS, reprogramming, lentivirus, stem cell, induced pluripotent cell, pluripotency, fibroblast, embryonic stem cells, ES cells, iPS cells
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Institutions: National Institute of Environmental Health Sciences.
Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3
. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5
. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7
. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5
, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8
. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.
Stem Cell Biology, Issue 63, Molecular Biology, Genetics, Embryonic stem cell, ESC, self-renewal, differentiation, Oct4, GFP, reporter assay, RNAi
Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells
Institutions: Vanderbilt University.
Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1
. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4
. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+
mice are cancer stem cells, which are capable of initiating and propogating tumors 10
. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11
, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.
Medicine, Issue 43, medulloblastoma, stem cells, isolation, in vitro culture
Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System
Institutions: Stemgent, MIT - Massachusetts Institute of Technology.
Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1
Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6
iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.
Developmental Biology, Issue 33, reprogramming, Doxycycline, DOX, iPS, induced pluripotent stem cells, lentivirus, pluripotency, transduction, stem cells
Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP
Institutions: Yale School of Medicine.
Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro
disease modeling and regenerative medicine1
. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4
) and become induced pluripotent stem cells (iPSCs)2-4
. Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4
. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.
Stem Cell Biology, Issue 62, Human iPS cells, iPSCs, Reprogramming, Retroviral vectors and Pluripotency
Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts
Institutions: University of Texas M.D. Anderson Cancer Center, Poznan University of Medical Sciences, The Scripps Research Institute.
Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3
. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5
. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog.
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6
, human newborn fibroblasts, as well as human keratinocytes.
Developmental Biology, Issue 60, stem cells, induced pluripotent stem cells, iPSC, somatic cell reprogramming, pluripotency, retroviral transduction
Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres
Institutions: George Mason University, Virginia Surgery Associates.
Breast ductal carcinoma in situ
(DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2
incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.
Cancer Biology, Issue 93, Breast, ductal carcinoma in situ, epidermal growth factor, mammosphere, organoid, pre-invasive, primary cell culture, serum-free, spheroid
Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells
Institutions: University of Michigan Comprehensive Cancer Center, University of Michigan Comprehensive Cancer Center.
Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1
. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo
. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4
. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3
. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7
. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.
Medicine, Issue 86, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
Institutions: University of Ulm.
Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo
. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls.
DTI data analysis is performed in a variate fashion, i.e.
voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e.
differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.
Medicine, Issue 77, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF
Institutions: Stemgent, Stemgent.
Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1
However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2
The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3
Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.
Cellular Biology, Issue 33, SC1(Pluripotin), LIF, mESC, mouse ESC, mouse ES cells, pluripotency, self-renewal, small molecule
Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development
Institutions: The George Washington University, The George Washington University.
Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus
embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.
Developmental Biology, Issue 71, Cellular Biology, Molecular Biology, Anatomy, Physiology, Biochemistry, Xenopus laevis, fate mapping, lineage tracing, cell-cell signaling, cell fate, blastomere, embryo, in situ hybridization, animal model
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
Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation
Institutions: Vanderbilt University Medical Center, Vanderbilt University Medical Center, Veteran Affairs TVHS.
Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.
Medicine, Issue 83, Glioma, Malignant glioma, primary orthotopic xenograft, isocitrate dehydrogenase
Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons
Institutions: Ludwig Maximilians University Munich, Ludwig-Maximilians University Munich, Friedrich-Alexander-Universität Erlangen-Nürnberg, Johannes Gutenberg University Mainz.
Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro
modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ
within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro
expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-β and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro
lineage conversion of brain-resident pericytes into functional human iNs.
Neuroscience, Issue 87, Pericytes, lineage-reprogramming, induced neurons, cerebral cortex
Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors
Institutions: Fred Hutchinson Cancer Research Center, The Children's Hospital of Philadelphia, The Children's Hospital of Philadelphia.
This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4
, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1
) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.
Cellular Biology, Issue 92, Induced pluripotent stem cells, iPSC, iPSC generation, human, HDAC inhibitors, histone deacetylase inhibitors, reprogramming, episomes, integration-free
Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens
Institutions: The Ohio State University Medical Center, The Ohio State University Medical Center.
The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro
culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.
We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.
Medicine, Issue 53, Glioblastoma multiforme, glioma, temozolomide, therapeutics, drug design
In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors
Institutions: University College London, University of Manchester.
Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo
by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo
, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo
reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo
may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro
performed protocols and can be applied to different tissue types in the future.
Stem Cell Biology, Issue 82, Pluripotent Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Transcription Factors, General, Gene Therapy, Gene Expression, iPS, OKSM, regenerative medicine
Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain
Institutions: University of Dresden, Center for Regerative Therapies Dresden.
Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as "niches" that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo
behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro
. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.
Neuroscience, Issue 81, adult neural stem cells, proliferation, differentiation, cell culture, growth factors
Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2
. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5
. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6
. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7
. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9
. Bevacizumab however failed to prolong overall survival in a recent phase III trial26
. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12
, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Institutions: McMaster University .
Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo
. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.
Cancer Biology, Issue 67, Stem Cell Biology, Medicine, Cellular Biology, Molecular Biology, BTIC (brain tumor initiating cells), tumorspheres, self-renewal, flow cytometry, single cell RT-PCR
A Zebrafish Model of Diabetes Mellitus and Metabolic Memory
Institutions: Rosalind Franklin University of Medicine and Science, Rosalind Franklin University of Medicine and Science.
Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo
system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.
Medicine, Issue 72, Genetics, Genomics, Physiology, Anatomy, Biomedical Engineering, Metabolomics, Zebrafish, diabetes, metabolic memory, tissue regeneration, streptozocin, epigenetics, Danio rerio, animal model, diabetes mellitus, diabetes, drug discovery, hyperglycemia
Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4
Institutions: Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology.
Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.
Cell Biology, Issue 14, Reprogramming, inducible lentiviruses, iPS cells, MEFs, ES cells, virus transduction, doxycycline