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Methods for isolating very small embryonic-like (vsel) stem cells

An embryo-like stem cell and cell technology, which is applied in non-embryonic pluripotent stem cells, animal cells, vertebrate cells, etc.

Inactive Publication Date: 2012-01-25
UNIV OF LOUISVILLE RES FOUND INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each growth factor has unique effects on differentiation pathways, but no growth factor exclusively directs differentiation into one cell type

Method used

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  • Methods for isolating very small embryonic-like (vsel) stem cells
  • Methods for isolating very small embryonic-like (vsel) stem cells
  • Methods for isolating very small embryonic-like (vsel) stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0289] bone marrow cells

[0290] Mouse singlets were isolated from BM obtained from femurs of pathogen-free, 3-week-, 1-month-, and 1-year-old female C57BL / 6 or DBA / 2J mice (obtained from The Jackson Laboratory, Bar Harbor, Maine, United States of America). Nucleated cells (MNC). Red blood cells were removed with hypotonic solution (Lysing Buffer, BD Biosciences, San Jose, California, United States of America).

[0291] Optionally, MNCs were isolated from murine BM obtained from femurs of pathogen-free, 4-6 week old female Balb / C mice (Jackson Laboratory) and subjected to Ficoll-Paque centrifugation to obtain light density MNCs. Sca-1 was isolated by using paramagnetic mini beads (Miltenyi Biotec, Auburn, California, United States of America) according to the manufacturer's protocol + cell.

[0292] Light-dense human BMMNC were obtained from four cadaveric BM donors (aged 52-65 years) and, if necessary, adherent cells and T-lymphocytes (A-T-MNC) were removed as described...

Embodiment 2

[0294] Classification of bone marrow-derived cells

[0295] For murine BM cells, using FACSVANTAGE TM SE (Becton Dickinson, Mountain View, California, United States of America), Isolation of Sca-1 from a Suspension of Murine BMMNC by Multiparametric, Viable Axenic Cell Sorting + / lin - / CD45 - and Sca-1 + / lin - / CD45 + cell. Briefly, BMMNC (100×10 6 cells / ml) were resuspended in cell sorting medium (CSM) containing 1× Hank's Balanced Salt Solution (GIBCO, Grand Island, New York, United States of America) without phenol red, 2% heat-inactivated Fetal calf serum (FCS; GIBCO), 10mM HEPES buffer (GIBCO) and 30U / ml gentamicin (GIBCO). The following monoclonal antibodies (mAbs) were used to stain these cells: biotin-conjugated rat anti-mouse Ly-6A / E (Sca-1; clone E13-161.7), streptavidin-PE- Cy5 conjugate, anti-CD45-APCCy7 (clone 30-F11), anti-CD45R / B220-PE (clone RA3-6B2), anti-Gr-1-PE (clone RB6-8C5), anti-TCRαβPE (clone H57 -597), anti-TCRγδ PE (clone GL3), anti-CD1...

Embodiment 3

[0299] Side population (SP) cell isolation

[0300] SP cells were isolated from bone marrow according to the method of Goodell et al. (2005) Methods Mol Biol 343-352. Briefly, BMMNC with 10 6 cells / ml were resuspended in pre-warmed DMEM / 2% FBS and pre-incubated at 37°C for 30 min. Cells were then labeled with 5 μg / ml Hoechst 33342 (Sigma Aldrich, St. Louis, Missouri, United States of America) in DMEM / 2% FBS and incubated at 37°C for 90 minutes. After staining, cells were pelleted, resuspended in ice-cold cell sorting medium, and subsequently maintained on ice until their sorting. Utilize FACSVANTAGE TM (Becton Dickinson, Mountain View, California, United States of America) for analysis and classification. The Hoechst dye was excited at 350 nm and its fluorescence emission collected with a 424 / 44 bandpass (BP) filter (Hoechst blue) and a 675 / 20 BP filter (Hoechst red). All parameters were collected using linear zoom in list mode and displayed as Hoechst blue versus Hoech...

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Abstract

The presently disclosed subject matter provides methods of isolating populations of stem cells that from bone marrow, peripheral blood, and / or other sources. Also provided are methods of using the stem cells for treating tissue and / or organ damage in a subject.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 61 / 194,719 filed September 30, 2008, U.S. Provisional Application No. 61 / 198,920 filed November 12, 2008, and US Provisional Application No. 61 / 245,650, the disclosure of each application is incorporated herein by reference in its entirety. [0003] Government interests [0004] This invention was made with government support under Grant Nos. R01 CA106281, R01 DK074720, R01 HL072410, HL055757, HL068088, and HL070897 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] The presently disclosed subject matter generally relates to the identification, isolation, and identification of populations of stem cells (referred to herein as very small embryonic-like (VSEL) stem cells) isolated from bone marrow, umbilical cord blood, and / or other sources and applications. More specifically, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0607
Inventor E·K·祖巴-叙尔马M·拉塔查克J·拉塔查克M·库恰
Owner UNIV OF LOUISVILLE RES FOUND INC