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2.
Exp Neurol ; 263: 190-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25448007

ABSTRACT

OBJECTIVE: Charcot-Marie-Tooth (CMT) disease is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here, we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. METHODS: iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. RESULTS: iPSC-derived motor neurons exhibited gene and protein expression, ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient, but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities, including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. INTERPRETATION: Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations, including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies.


Subject(s)
Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/physiopathology , Motor Neurons/pathology , Adult , Animals , Cell Separation , Charcot-Marie-Tooth Disease/pathology , Child , Electrophysiological Phenomena , Female , GTP Phosphohydrolases/genetics , Gene Knock-In Techniques , Humans , Induced Pluripotent Stem Cells , Intermediate Filaments/pathology , Male , Mice , Mitochondria/pathology , Mitochondrial Proteins/genetics , Neurofilament Proteins/genetics , Patch-Clamp Techniques , Phenotype , Point Mutation , Real-Time Polymerase Chain Reaction
3.
Hum Mol Genet ; 22(2): 398-407, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23077215

ABSTRACT

Functional loss of SMN1 causes proximal spinal muscular atrophy (SMA), the most common genetic condition accounting for infant lethality. Hence, the hypomorphic copy gene SMN2 is the only resource of functional SMN protein in SMA patients and influences SMA severity in a dose-dependent manner. Consequently, current therapeutic approaches focus on SMN2. Histone deacetylase inhibitors (HDACi), such as the short chain fatty acid VPA (valproic acid), ameliorate the SMA phenotype by activating the SMN2 expression. By analyzing blood SMN2 expression in 16 VPA-treated SMA patients, about one-third of individuals were identified as positive responders presenting increased SMN2 transcript levels. In 66% of enrolled patients, a concordant response was detected in the respective fibroblasts. Most importantly, by taking the detour of reprograming SMA patients' fibroblasts, we showed that the VPA response was maintained even in GABAergic neurons derived from induced pluripotent stem cells (iPS) cells. Differential expression microarray analysis revealed a complete lack of response to VPA in non-responders, which was associated with an increased expression of the fatty acid translocase CD36. The pivotal role of CD36 as the cause of non-responsiveness was proven in various in vitro approaches. Most importantly, knockdown of CD36 in SMA fibroblasts converted non- into pos-responders. In summary, the concordant response from blood to the central nervous system (CNS) to VPA may allow selection of pos-responders prior to therapy. Increased CD36 expression accounts for VPA non-responsiveness. These findings may be essential not only for SMA but also for other diseases such as epilepsy or migraine frequently treated with VPA.


Subject(s)
CD36 Antigens/metabolism , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/metabolism , Valproic Acid/therapeutic use , CD36 Antigens/genetics , Cell Line , Fibroblasts/drug effects , Fibroblasts/metabolism , GABAergic Neurons/drug effects , GABAergic Neurons/metabolism , Gene Expression Profiling , Humans , Muscular Atrophy, Spinal/genetics , Valproic Acid/pharmacology
4.
Environ Health ; 11: 57, 2012 Aug 29.
Article in English | MEDLINE | ID: mdl-22931295

ABSTRACT

BACKGROUND: Studies have shown associations of diabetes and endogenous hormones with exposure to a wide variety of organochlorines. We have previously reported positive associations of polychlorinated biphenyls (PCBs) and inverse associations of selected steroid hormones with diabetes in postmenopausal women previously employed in a capacitor manufacturing plant. METHODS: This paper examines associations of PCBs with diabetes and endogenous hormones in 63 men previously employed at the same plant who in 1996 underwent surveys of their exposure and medical history and collection of bloods and urine for measurements of PCBs, lipids, liver function, hematologic markers and endogenous hormones. RESULTS: PCB exposure was positively associated with diabetes and age and inversely associated with thyroid stimulating hormone and triiodothyronine-uptake. History of diabetes was significantly related to total PCBs and all PCB functional groupings, but not to quarters worked and job score, after control for potential confounders. None of the exposures were related to insulin resistance (HOMA-IR) in non-diabetic men. CONCLUSIONS: Associations of PCBs with specific endogenous hormones differ in some respects from previous findings in postmenopausal women employed at the capacitor plant. Results from this study, however, do confirm previous reports relating PCB exposure to diabetes and suggest that these associations are not mediated by measured endogenous hormones.


Subject(s)
Diabetes Mellitus/chemically induced , Diabetes Mellitus/epidemiology , Environmental Exposure , Environmental Pollutants/toxicity , Hormones/blood , Polychlorinated Biphenyls/toxicity , Age Factors , Cross-Sectional Studies , Environmental Monitoring , Environmental Pollutants/blood , Follow-Up Studies , Humans , Illinois/epidemiology , Male , Middle Aged , Occupational Exposure , Polychlorinated Biphenyls/blood , Thyrotropin/blood , Triiodothyronine/blood
5.
Cell Stem Cell ; 10(5): 610-9, 2012 May 04.
Article in English | MEDLINE | ID: mdl-22560081

ABSTRACT

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies.


Subject(s)
Dystrophin/metabolism , Embryonic Stem Cells/physiology , Induced Pluripotent Stem Cells/physiology , Muscular Dystrophies/therapy , Satellite Cells, Skeletal Muscle/physiology , Animals , Cell Proliferation , Feasibility Studies , Humans , Mice , Mice, Inbred mdx , Muscle Contraction , PAX7 Transcription Factor/genetics , PAX7 Transcription Factor/metabolism , Stem Cell Transplantation , Transplantation, Heterologous
6.
Stem Cell Res Ther ; 2(5): 37, 2011 Sep 21.
Article in English | MEDLINE | ID: mdl-21936964

ABSTRACT

Five years after their initial derivation from mouse somatic cells, induced pluripotent stem (iPS) cells are an important tool for the study of neurological diseases. By offering an unlimited source of patient-specific disease-relevant neuronal and glial cells, iPS cell-based disease models hold enormous promise for identification of disease mechanisms, discovery of molecular targets and development of phenotypic screens for drug discovery. The present review focuses on the recent advancements in modeling neurological disorders, including the demonstration of disease-specific phenotypes in iPS cell-derived neurons generated from patients with spinal muscular atrophy, familial dysautonomia, Rett syndrome, schizophrenia and Parkinson disease. The ability of this approach to detect treatment effects from known therapeutic compounds has also been demonstrated, providing proof of principle for the use of iPS cell-derived cells in drug discovery.


Subject(s)
Induced Pluripotent Stem Cells/metabolism , Nervous System Diseases/therapy , Cell Differentiation , Cell Lineage , Drug Evaluation, Preclinical , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/transplantation , Neuroglia/cytology , Neuroglia/metabolism , Neurons/cytology , Neurons/metabolism
7.
Environ Res ; 111(6): 817-24, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21684538

ABSTRACT

There is an increasing body of literature showing associations of organochlorine exposure with risk of diabetes and insulin resistance. Some studies suggest that associations differ by gender and that diabetes risk, in turn, may be affected by endogenous steroid hormones. This report examines the relationships of serum PCBs and endogenous hormones with history of diabetes in a cohort of persons previously employed at a capacitor manufacturing plant. A total of 118 women were post-menopausal with complete data, of whom 93 were not using steroid hormones in 1996, at the time of examination, which included a survey of exposure and medical history, height, weight and collection of blood and urine for measurements of lipids, liver function, hematologic markers and endogenous hormones. This analysis examines relationships of serum polychlorinated biphenyls (PCBs), work exposure and endogenous hormones with self-reported history of diabetes after control for potential confounders. All PCB exposure groups were significantly related to history of diabetes, but not to insulin resistance as measured by the homeostatic model assessment of insulin resistance (HOMA-IR) in non-diabetics. Diabetes was also independently and inversely associated with follicle stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS) and triiodothyronine (T3) uptake. HOMA-IR was positively associated with body mass index (BMI) and C-reactive protein (CRP) and inversely associated with sex hormone binding globulin (SHBG) and T3 uptake after control for PCB exposure. Possible biologic mechanisms are discussed. This study confirms previous reports relating PCB exposure to diabetes and suggests possible hormonal pathways deserving further exploration.


Subject(s)
Diabetes Mellitus/chemically induced , Diabetes Mellitus/epidemiology , Environmental Pollutants/toxicity , Occupational Exposure , Polychlorinated Biphenyls/toxicity , Postmenopause , Aged , Body Mass Index , C-Reactive Protein/analysis , Cholesterol/blood , Cohort Studies , Dehydroepiandrosterone Sulfate/blood , Electric Capacitance , Environmental Pollutants/blood , Estradiol/blood , Estrone/metabolism , Estrone/urine , Female , Follicle Stimulating Hormone/blood , Humans , Insulin Resistance , Middle Aged , Polychlorinated Biphenyls/blood , Sex Hormone-Binding Globulin/analysis , Triglycerides/blood , Triiodothyronine/blood , Triiodothyronine/metabolism , United States/epidemiology
8.
Nat Biotechnol ; 29(3): 279-86, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21293464

ABSTRACT

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.


Subject(s)
Cell Culture Techniques/methods , Fibroblasts/cytology , Pluripotent Stem Cells/cytology , Skin/cytology , Tissue Engineering/methods , Cell Differentiation , Cells, Cultured , Humans
10.
Genes Dev ; 24(3): 312-26, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20123909

ABSTRACT

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.


Subject(s)
Cell Differentiation , Cell Lineage/genetics , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , HMGB Proteins/metabolism , SOXF Transcription Factors/metabolism , Animals , Embryonic Stem Cells/metabolism , Gene Regulatory Networks , HMGB Proteins/genetics , Mice , SOXF Transcription Factors/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
11.
Genes Dev ; 23(5): 561-74, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19270157

ABSTRACT

Neural stem cells (NSCs) persist throughout life in two forebrain areas: the subventricular zone (SVZ) and the hippocampus. Why forebrain NSCs self-renew more extensively than those from other regions remains unclear. Prior studies have shown that the polycomb factor Bmi-1 is necessary for NSC self-renewal and that it represses the cell cycle inhibitors p16, p19, and p21. Here we show that overexpression of Bmi-1 enhances self-renewal of forebrain NSCs significantly more than those derived from spinal cord, demonstrating a regional difference in responsiveness. We show that forebrain NSCs require the forebrain-specific transcription factor Foxg1 for Bmi-1-dependent self-renewal, and that repression of p21 is a focus of this interaction. Bmi-1 enhancement of NSC self-renewal is significantly greater with increasing age and passage. Importantly, when Bmi-1 is overexpressed in cultured adult forebrain NSCs, they expand dramatically and continue to make neurons even after multiple passages, when control NSCs have become restricted to glial differentiation. Together these findings demonstrate the importance of Bmi-1 and Foxg1 cooperation to maintenance of NSC multipotency and self-renewal, and establish a useful method for generating abundant forebrain neurons ex vivo, outside the neurogenic niche.


Subject(s)
Forkhead Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Prosencephalon/cytology , Prosencephalon/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Stem Cells/cytology , Animals , Cell Proliferation , Cell Survival , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Female , Gene Expression , Mice , Polycomb Repressive Complex 1 , Pregnancy , Prosencephalon/embryology , Stem Cells/metabolism
12.
Science ; 321(5893): 1218-21, 2008 Aug 29.
Article in English | MEDLINE | ID: mdl-18669821

ABSTRACT

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Cell Differentiation , Cellular Reprogramming , Fibroblasts/cytology , Motor Neurons/cytology , Pluripotent Stem Cells/cytology , Aged, 80 and over , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Cell Line , Embryonic Stem Cells/cytology , Female , Gene Expression , Humans , Motor Neurons/metabolism , Neuroglia/cytology , Retroviridae/genetics , Spinal Cord/cytology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Transcription Factors/genetics , Transcription Factors/metabolism , Transduction, Genetic
13.
Chemosphere ; 69(3): 435-43, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17583774

ABSTRACT

The composition of polychlorinated biphenyl (PCB) congeners in serum samples is compared between a cohort previously exposed to PCBs from working at a capacitor plant (n=180) and a cohort of Great Lakes sport-caught fish eaters (n=217). Fourteen congeners were measured in both samples. A multiple logistic regression model differentiating the two groups as a function of relative proportions amongst congeners 74, 138, 153, 180, and 201 correctly classifies more than 99% of the people (395/397); higher proportions of congeners 74, 153, and 201 characterize capacitor plant workers, while higher proportions of congeners 138 and 180 characterize fish eaters. The pattern is driven by the relative amounts of 74+153+201 compared to 138+180; all of the fish eaters, but only 5% of the capacitor plant workers, have a greater percent of 138+180 than 74+153+201. Consideration of combinations of congener levels and their relative proportions is relevant to tracking route of exposure and may also be relevant to modeling effects on health outcomes.


Subject(s)
Environmental Exposure , Fishes/metabolism , Occupational Exposure , Polychlorinated Biphenyls/analysis , Animals , Cohort Studies , Humans , Polychlorinated Biphenyls/pharmacokinetics
14.
Cell Stem Cell ; 1(1): 87-99, 2007 Jun 07.
Article in English | MEDLINE | ID: mdl-18371338

ABSTRACT

Knockout studies have shown that the polycomb gene Bmi-1 is important for postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is also important for NSC self-renewal in the embryo. We found that neural progenitors depend increasingly on Bmi-1 for proliferation as development proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1 reduction causes defects in embryonic and adult NSC proliferation and self-renewal that, unexpectedly, are mediated by a different cell-cycle inhibitor, p21. Gene array analyses revealed developmental differences in Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs, potentially with stage-related differences. Understanding stage-related mechanisms underlying NSC self-renewal has important implications for development of stem cell-based therapies.


Subject(s)
Cell Division , Cyclin-Dependent Kinase Inhibitor p21/physiology , Nervous System/cytology , Nuclear Proteins/physiology , Proto-Oncogene Proteins/physiology , RNA/genetics , Repressor Proteins/physiology , Retinoblastoma Protein/physiology , Stem Cells/cytology , Animals , Cell Proliferation , Electroporation , Female , Mice , Nuclear Proteins/genetics , Polycomb Repressive Complex 1 , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics
15.
Nat Neurosci ; 9(6): 743-51, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16680166

ABSTRACT

In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.


Subject(s)
Cell Differentiation/physiology , Cell Lineage/genetics , Cell Proliferation , Cerebral Cortex/embryology , Neurons/metabolism , Stem Cells/metabolism , Animals , Cell Adhesion Molecules, Neuronal/genetics , Cell Division/genetics , Cell Movement/genetics , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Down-Regulation/genetics , Extracellular Matrix Proteins/genetics , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Developmental/genetics , Mice , Microscopy, Video , Nerve Tissue Proteins/genetics , Neuronal Plasticity/genetics , Neurons/cytology , RNA Interference/physiology , Reelin Protein , Serine Endopeptidases/genetics , Stem Cells/cytology , Time Factors
16.
J Vis Exp ; (1): 86, 2006 Nov 09.
Article in English | MEDLINE | ID: mdl-18704174

ABSTRACT

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.


Subject(s)
Cell Culture Techniques , Embryonic Stem Cells/cytology , Freezing , Animals , Cells, Cultured , Culture Media , Embryo, Mammalian/cytology , Fibroblasts , Humans , Mice
17.
J Vis Exp ; (1): 49, 2006 Oct 12.
Article in English | MEDLINE | ID: mdl-18704181

ABSTRACT

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings. Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture. First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA. Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette. The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation. The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media. An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells. The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter. It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.


Subject(s)
Cell Culture Techniques , Cell Line , Embryonic Stem Cells/cytology , Trypsin , Animals , Culture Media , Embryo, Mammalian/cytology , Humans , Mice
18.
J Vis Exp ; (1): 50, 2006 Oct 12.
Article in English | MEDLINE | ID: mdl-18704182

ABSTRACT

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines. A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction. Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature. During this time cells can be observed rounding, and colonies lifting off the plate surface. Gentle repeated pipetting will remove cells and colonies from the plate surface. Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun. Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10 cm plate. After cells are adequately resuspended, ice cold freezing media is added at equal volume. Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80 C over 24 hours. Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.


Subject(s)
Embryonic Stem Cells , Freezing , Culture Media , Humans
19.
J Occup Environ Med ; 46(6): 565-76, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15213519

ABSTRACT

A mortality study of workers employed between 1944 and 1977 at an electrical capacitor manufacturing plant where polychlorinated biphenyls (PCBs), chlorinated naphthalenes, and other chemicals were used was undertaken. Age, gender, and calendar year-adjusted standardized mortality ratios (SMRs) were calculated for 2885 white workers. Total mortality and all-cancer mortality were similar to expected in both males and females. Females employed 10 or more years had a significantly elevated SMR of 6.2 for liver/biliary cancer. Intestinal cancer was significantly elevated in females employed 5 or more years after PCBs were introduced (SMR = 2.2). In males, stomach cancer (SMR = 2.2) and thyroid cancer (SMR = 15.2) were significantly elevated. Although individual exposure assessment was limited, PCBs alone or in combination with other chemicals could be associated with increased risks for liver/biliary, stomach, intestinal, and thyroid cancer.


Subject(s)
Naphthalenes/toxicity , Neoplasms/chemically induced , Neoplasms/mortality , Occupational Exposure/adverse effects , Polychlorinated Biphenyls/toxicity , Adult , Aged , Aged, 80 and over , Cause of Death , Cohort Studies , Female , Humans , Illinois/epidemiology , Industry/classification , Male , Middle Aged , Neoplasms/classification , Neoplasms/ethnology , Registries , Risk Assessment , Sex Distribution , Time Factors , White People/statistics & numerical data
20.
Science ; 298(5593): 601-4, 2002 Oct 18.
Article in English | MEDLINE | ID: mdl-12228721

ABSTRACT

Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells.


Subject(s)
Gene Expression Profiling , Gene Expression , Hematopoietic Stem Cells/physiology , Stem Cells/physiology , Totipotent Stem Cells/physiology , Adult , Animals , Cell Communication , Cell Cycle , Cell Differentiation , Cell Line , Cell Lineage , Cell Separation , Cells, Cultured , Computational Biology , Embryo, Mammalian/cytology , Expressed Sequence Tags , Genes, Homeobox , Hematopoiesis , Hematopoietic Stem Cell Transplantation , Humans , Mice , Neurons/cytology , Oligonucleotide Array Sequence Analysis , Signal Transduction , Transcription, Genetic
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