78 research outputs found

    Isolation and Characterization of Neural Crest Stem Cells Derived From In Vitro–Differentiated Human Embryonic Stem Cells

    Get PDF
    The neural crest is a transient structure of vertebrate embryos that initially generates neural crest stem cells (NCSCs) which then migrate throughout the body to produce a diverse array of mature tissue types. Due to the rarity of adult NCSCs as well as ethical and technical issues surrounding isolation of early embryonic tissues, biologic studies of human NCSCs are extremely challenging. Thus, much of what is known about human neural crest development has been inferred from model organisms. In this study, we report that functional NCSCs can be rapidly generated and isolated from in vitro—differentiated human embryonic stem cells (hESCs). Using the stromal-derived inducing activity (SDIA) of PA6 fibroblast co-culture we have induced hESCs to differentiate into neural crest. Within 1 week, migrating cells that express the early neural crest markers p75 and HNK1 as well as numerous other genes associated with neural crest induction such as SNAIL, SLUG, and SOX10 are detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the p75-positive population enriches for cells with genetic, phenotypic, and functional characteristics of NCSCs. These p75-enriched cells readily form neurospheres in suspension culture, self-renew to form secondary spheres, and give rise under differentiation conditions to multiple neural crest lineages including peripheral nerves, glial, and myofibroblastic cells. Importantly, these cells differentiate into neural crest derivatives when transplanted into developing chick embryos in vivo. Thus, this SDIA protocol can be used to successfully and efficiently isolate early human NCSCs from hESCs in vitro. This renewable source of NCSCs provides an invaluable source of cells for studies of both normal and disordered human neural crest development

    Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consor tium

    Get PDF
    The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community

    Molecular, phenotypic, and sample-associated data to describe pluripotent stem cell lines and derivatives

    Get PDF
    The use of induced pluripotent stem cells (iPSC) derived from independent patients and sources holds considerable promise to improve the understanding of development and disease. However, optimized use of iPSC depends on our ability to develop methods to efficiently qualify cell lines and protocols, monitor genetic stability, and evaluate self-renewal and differentiation potential. To accomplish these goals, 57 stem cell lines from 10 laboratories were differentiated to 7 different states, resulting in 248 analyzed samples. Cell lines were differentiated and characterized at a central laboratory using standardized cell culture methodologies, protocols, and metadata descriptors. Stem cell and derived differentiated lines were characterized using RNA-seq, miRNA-seq, copy number arrays, DNA methylation arrays, flow cytometry, and molecular histology. All materials, including raw data, metadata, analysis and processing code, and methodological and provenance documentation are publicly available for re-use and interactive exploration at https://www.synapse.org/pcbc. The goal is to provide data that can improve our ability to robustly and reproducibly use human pluripotent stem cells to understand development and disease

    Evaluation of short‐term safety of ultrasound‐guided foetal fluid sampling in the dog (Canis lupus familiaris)

    Get PDF
    Background: In humans, analysis of amniotic fluid is widely used for diagnostic and prognostic purposes. Amniocentesis has scarcely been used in veterinary medicine to date, despite a tremendous potential for clinical and research applications in dogs. Our study aimed to establish a safe method for foetal fluid sampling in female dogs. Methods: Two transabdominal ultrasound-guided methods were assessed: the "free hand" and the needle-guided bracket sampling. In addition, through a subsequent routinely scheduled ovariohysterectomy, fluid was directly collected. Samples from 98 conceptuses were collected at day 46.7 +/- 7.5 of pregnancy. Results: The amount of fluid retrieved varied between 0.5 and 5.0 ml per collection. Macroscopic examination of the uterus and conceptuses identified 53% of the puncture sites. Neither fluid leakage nor foetal injury was detected, and six hematomas (5.8%) were visible. Ultrasound-guided foetal fluid collection was found to be potentially safe, and it can be performed by using either transabdominal method. Conclusion: Foetal fluid collection is possible with relative ease and low short-term risk, and may open paths for diagnostic, therapeutic and research purposes in dogs. The procedure can provide new insights into prenatal clinical medicine, including diagnostics of foetal deaths, early identification of heritable diseases and so on

    Gene therapy for canine mucopolysaccharidosis type I

    No full text
    grantor: University of TorontoHematopoietic stem cell (HSC) gene transfer is being developed as a treatment for genetic disease. This thesis evaluated the therapeutic potential of HSC gene therapy in a canine model of MPS I which results from a deficiency of a -L-iduronidase. The first experiment demonstrated the maintenance of 5-10% (87/1220) of hematopoietic progenitors (CFU) and 1% of leukocytes provirus positive for 2 years post-infusion in normal recipients. Specific antibodies against culture media components were detected but did not abrogate engraftment with potentially therapeutic levels of provirally marked cells. To evaluate the therapeutic potential of HSC gene therapy, marrow from 5 MPS I dogs was transduced with iduronidase containing recombinant retroviruses. Following adoptive transfer into autologous unconditioned recipients, proviral DNA was detected in 2-10% CFU (51/1288) and 1% of leukocytes for 1-3 years post-infusion. However, proviral iduronidase expression was not detected in any dog and all dogs had continued disease progression. Humoral and cellular immune responses against iduronidase protein and autologous cells expressing iduronidase were identified, which possibly abrogated the therapeutic benefits of gene therapy. To evaluate gene therapy in the absence of confounding anti-transgene immune responses, iduronidase transduced MPS I marrow cells were adoptively transferred into fetal pups. 17 fetal pups were injected with iduronidase transduced MPS I marrow cells. Nine normal and 3 MPS I fetal recipients survived and proviral DNA was detected in 2-12% (76/1790) CFU and ~ 1% of leukocytes for l year. Proviral iduronidase activity was detected in 1.5% (6/404) CFU but not mature leukocytes from MPS I pups. Specific humoral immune responses against iduronidase were not detected in MPS recipients, even after boosting with iduronidase transduced marrow cells, consistent with the induction of tolerance to iduronidase in these pups. All MPS I dogs died at 8-10 months with no evidence for disease amelioration. These results show that iduronidase transduced progenitors can engraft in adult, juvenile and fetal recipients, and contribute to hematopoiesis in normal and MPS I dogs. However, the therapeutic potential of HSC gene transfer in canine MPS I appears to be limited by poor maintenance of proviral iduronidase expression and low levels of genetically corrected circulating leukocytes.Ph.D

    Постановка задачі оптимального синтезу автономної високоточної стійкої до збурень системи стабілізації та визначення курсу

    No full text
    <p><em> Проаналізовано підходи до постановки задач оптимального синтезу систем управління рухомими об’єктами широкого класу. Сформульовано формалізовану постановку задачі оптимального синтезу стійкої до збурень автономної високоточної системи стабілізації та визначення курсу. Визначено критерій оптимальності сформульованої задачі. Проаналізовано структуровані та неструктуровані невизначеності системи.</em><em></em></p

    Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes

    No full text
    In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (Eμ) with and without associated matrix attachment regions (EμMAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34(+) progenitors in vitro transduced with Eμ- and EμMAR-containing lentivectors. Lastly, we evaluated the expression from the EμMAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the Eμ and EμMAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the EμMAR-containing vector and not other cells types or vectors. Proviral genomes with the EμMAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the EμMAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies

    Recovery of Multipotent Progenitors from the Peripheral Blood of Patients Requiring Extracorporeal Membrane Oxygenation Support

    No full text
    Rationale: Studies have demonstrated that bone marrow-derived cells can be recruited to injured lungs through an unknown mechanism. We hypothesize that marrow progenitors are mobilized into the circulation of patients with cardiac and/or respiratory failure, and may then traffic to and incorporate into the sites of tissue injury. Objectives: To determine whether progenitor populations are increased in the blood of patients with severe acute cardiorespiratory failure placed on extracorporeal membrane oxygenation (ECMO). Methods: Mononuclear cells from ECMO, umbilical cord, and control blood samples were evaluated in colony-forming assays for hematopoietic, mesenchymal, and epithelial cells. Progenitors were identified by proliferative and differentiative capacities, and confirmed by the expression of lineage-specific markers. Measurements and Main Results: Significantly higher levels of hematopoietic progenitors were observed in ECMO (n 5 41) samples than neonatal intensive care unit (n 5 16) or pediatric intensive care unit controls (n 5 14). Hematopoietic progenitor mobilization increased with time on ECMO support. Mesenchymal progenitors (MSC) were recovered from 18/58 ECMO samples with rapid sample processing (, 4 h) critical to their recovery. MSC were not recovered from normal controls. ECMO-derived MSC had osteogenic, chondrogenic, and adipogenic differentiation potential. The recovery of MSC did not influence survival outcome (61%). Epithelial progenitors were observed in eight ECMO samples but not in control samples. Their presence was associated with a lower survival trend (38%). Conclusions: Hematopoietic, mesenchymal, and epithelial progenitors were mobilized into the circulation of patients on ECMO. This may reflect a response to severe cardiopulmonary injury, blood-foreign surface interactions with the ECMO circuit, and/or hemodilution
    corecore