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Very small embryonic-like (vsel) stem cells and methods of isolating and using the same

A technology of stem cells and endoderm cells, applied in non-embryonic pluripotent stem cells, biochemical equipment and methods, artificially induced pluripotent cells, etc.

Inactive Publication Date: 2009-11-04
UNIV OF LOUISVILLE RES FOUND INC
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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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  • Very small embryonic-like (vsel) stem cells and methods of isolating and using the same
  • Very small embryonic-like (vsel) stem cells and methods of isolating and using the same
  • Very small embryonic-like (vsel) stem cells and methods of isolating and using the same

Examples

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Embodiment 1

[0175] bone marrow cells

[0176] Murine mononuclei 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). cells (MNC). Red blood cells were removed with hypotonic solution (Lysing Buffer, BD Biosciences, San Jose, California, United States of America).

[0177] Alternatively, 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.

[0178] Obtain light-dense human BMMNC from four cadaveric BM donors (aged 52-65 years) and, if necessary, remove adherent cells and T-lymphocytes (A-T-MNC) as described in Ratajczak et ...

Embodiment 2

[0180] Classification of bone marrow-derived cells

[0181] 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-CD...

Embodiment 3

[0185] Side population (SP) cell isolation

[0186] 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 then 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 Hoechst red d...

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Abstract

The presently disclosed subject matter provides populations of stem cells that are purified 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 the benefit of US Provisional Patent Application Serial No. 60 / 748,685, filed December 8, 2005; the entire disclosure of which is hereby incorporated by reference. technical field [0003] The presently disclosed subject matter generally relates to the identification, isolation, and use of stem cell populations isolated from bone marrow, umbilical cord blood, and / or other sources, and referred to herein as very small embryonic-like (VSEL) stem cells . More specifically, the presently disclosed subject matter relates to isolating said VSEL stem cells and using them (optionally after in vitro treatment) to treat tissue and / or organ damage in a subject in need thereof. Background technique [0004] The use of stem cells and stem cell derivatives has gained increasing interest in medical research, especially in the field of providing reagents for treating tissue damage due to any of genetic defect...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/074
CPCC12N2501/115C12N2501/11C12N2501/23C12N2501/22C12N5/0607A61P9/10
Inventor M·拉塔查克M·库恰J·拉塔查克
Owner UNIV OF LOUISVILLE RES FOUND INC
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