Multipotent adult stem cells having an ability of Oct4 expression derived from umbilical cord blood and method for preparing the same

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

Inactive Publication Date: 2008-12-24
SEOUL NAT UNIV R&DB FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] However, because stem cells can differentiate into a limited variety of cell types, including mesenchymal cells into bone or skeletal muscle, cardiac stem cells into cardiac cells, and endothelial progenitor cells into vascular endothelial cells, it is difficult to define these cells as true pluripotent stem cells

Method used

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  • Multipotent adult stem cells having an ability of Oct4 expression derived from umbilical cord blood and method for preparing the same
  • Multipotent adult stem cells having an ability of Oct4 expression derived from umbilical cord blood and method for preparing the same
  • Multipotent adult stem cells having an ability of Oct4 expression derived from umbilical cord blood and method for preparing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Isolation of adult stem cells from umbilical cord blood

[0047] Cord blood was collected from full-term prenatal newborns at Seoul National University Hospital and Samsung Cheil Hospital according to the Institutional Review Board guidelines.

[0048] Dilute 70-100 ml of the collected cord blood sample in PBS at a ratio of 1:1 and stir. Then, the blood sample was separated on Ficoll at a ratio of 15:25, where the blood sample (diluted 1:1 in PBS) was slowly spilled onto 15 ml of Ficoll solution to cause layering, followed by centrifugation at 2500 rpm for 20 min . After centrifugation, three different layers were formed from the bottom. Among the three layers, the buffy coat (middle layer: mononuclear cell layer) was aspirated with a pipette and washed three times with HBSS, followed by centrifugation at 1800 rpm for 15 minutes. The upper layer was completely removed from the centrifugate and the pellet was stored on ice.

[0049] The precipitate was shak...

Embodiment 2

[0052] Example 2: Immune properties of pluripotent adult stem cells from umbilical cord blood

[0053] In order to examine the immune properties of the umbilical cord blood-derived pluripotent adult stem cells obtained in Example 1, the expression patterns of cell surface antigens were analyzed. For this purpose, the 2×10 cultured in Example 1 6 -10 7 The cells were washed with PBS and incubated with their corresponding antibodies at room temperature. Antigen expression and non-expression were analyzed by flow cytometry. Furthermore, PAS staining (Herperiodic acid staining method) was performed.

[0054] results, such as Figure 4 As shown, the pluripotent adult stem cells from umbilical cord blood of the present invention showed 63.38%, 96.54% and 63.99% positive responses to CD45, SH-2 and SH-3, respectively, and more than 90% negative responses to CD34. Moreover, the immunophenotype of other antigens was analyzed. As a result, the immune characteristics of pluripotent ...

Embodiment 3

[0056] Example 3: Differentiation of pluripotent adult stem cells from umbilical cord blood into osteoblasts

[0057] The pluripotent adult stem cell culture solution from umbilical cord blood obtained in Example 1 was mixed with 1 ml of osteoblast induction medium (0.1 μmol / L dexamethasone (Sigma, USA), 0.05 mmol / L ascorbic acid-2-phosphate (Sigma , USA), 10 mmol / L β-glycerol phosphate (Sigma), and 5-30% human serum or plasma), and the cells were counted. Then, the cells were cultured in flasks (5% CO 2 ; 37°C; the medium was replaced every 3-4 days) to induce pluripotent adult stem cells to differentiate into osteoblasts. Fourteen days after the start of culture, the differentiation of pluripotent adult stem cells from umbilical cord blood into osteoblasts was confirmed by Von-Kassa staining (see Figure 4 ).

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Abstract

The present invention relates to multipotent adult stem cells expressing Oct4, derived from umbilical cord blood (UCB) and also these cell are expressing CD29, CD31, CD44, simultaneously, a method for preparing the same, and more specifically to multipotent adult stem cells which are obtained by culturing umbilical cord blood-derived monocytes in a medium containing bFGF (basic fibroblast growth factor) and human serum or plasma. In addition, multipotent adult stem cells expressing Oct-4 from UCB are morphologically spindle or round shaped cells Although the stem cells according to the present invention are adult stem cells, they are multipotent and capable of differentiating into ectodermal-, messodermal-, emdodermal-originated tissue or cells including osteogenic cells or nerve cells etc, thus they can be effectively used in the treatment of intractable diseases and incurable diseases.

Description

technical field [0001] The present invention relates to pluripotent adult stem cells with Oct4 expression ability from umbilical cord blood and a preparation method thereof, more specifically, relates to a method of producing mononuclear cells derived from umbilical cord blood by containing bFGF (basic fibroblast growth factor) and human The isolated pluripotent adult stem cells are cultured in serum or plasma medium. Background technique [0002] Although many diseases in the past have been cured due to the development of life science and medicine in our modern society, there are still some deep-rooted diseases or incurable diseases such as avascular necrosis, cancer, dementia and severe burns. Furthermore, there are many problems in the field of organ transplantation. These cell therapies are currently at the center of attention as treatments to treat the root causes of these diseases. [0003] Cell therapy is a method of treating or preventing diseases by externally pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/02C12N5/06A61K35/30A61K35/32C12N5/074
CPCC12N5/0607C12N2501/115C12N5/0647
Inventor 姜庆顺
Owner SEOUL NAT UNIV R&DB FOUND
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