Method for isolating and culturing multipotent progenitor/stem cells from umbilical cord blood and method for inducing differentiation thereof

A technology for stem cell differentiation and umbilical cord blood, which is applied in the fields of blood/immune system cells, biochemical equipment and methods, animal cells, etc.

Inactive Publication Date: 2007-02-21
生命科得公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there are still problems in isolating and culturing MSCs from cord blood (Erices A, et al., Br. J. Haematol. 109: 235-242, 2000; Lee OK, et al., Blood 103: 1669-1675, 2004; Wexler SA, et al., Br. J. Haematol. 121:368-74, 2003)
There is also no report of a reliable method for isolating and culturing stem cells capable of differentiating into various types of cells such as neurons, osteoblasts, myoblasts, adipocytes, etc. from umbilical cord blood

Method used

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  • Method for isolating and culturing multipotent progenitor/stem cells from umbilical cord blood and method for inducing differentiation thereof
  • Method for isolating and culturing multipotent progenitor/stem cells from umbilical cord blood and method for inducing differentiation thereof
  • Method for isolating and culturing multipotent progenitor/stem cells from umbilical cord blood and method for inducing differentiation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Example 1: Isolation and culture of multipotent progenitor / stem cells from mononuclear cells derived from umbilical cord blood

[0157] Preparation of mononuclear cells derived from umbilical cord blood

[0158] Before the placenta is separated from the uterus after delivery, 60-150 ml of cord blood is taken from the umbilical cord vein using a cord blood sampling bag (capacity: 175 ml) containing 23 ml of the anticoagulant CPDA-1 or a 50 ml syringe containing 5 ml of heparin. All instruments used for cord blood sampling were sterile prior to use, and the sampling site was sterilized with alcohol or betadin.

[0159]For isolation of mononuclear cells from cord blood, 15 ml of cord blood samples were dispensed into 50 ml conical tubes within 24 hours of sampling. 15 ml of phosphate buffered saline (Dulbecco's PBS; Hyclone, SH300028.03) was added to the tube and mixed with the cord blood. Gently cover the bottom of the conical tube with 15ml of Ficoll-Hypaque solution ...

Embodiment 2

[0166] Example 2: Characterization of Mononuclear Cell Multipotent Progenitor / Stem Cells Isolated and Cultured from Umbilical Cord Blood

[0167] In order to perform immunophenotyping analysis on the multipotent progenitor / stem cells prepared from the mononuclear cells derived from umbilical cord blood in Example 1, the cultured cells were treated with 0.05% trypsin solution to detach the cells from the culture flask, and washed twice with PBS Separate cells. Cells at 5 x 10 5 Cells / 200 μl were suspended in PBS, 10 μl of various antibodies were added thereto, and the culture flask was placed in a dark room for 15 minutes. Cells were then washed twice with PBS for flow cytometry (Becton Dickinson), mixed with 500 [mu]l of the same PBS, and immunophenotyped using flow cytometry (FACScan, Becton Dickinson). At this time, CD14, CD31, CD34, CD44, CD45, CD49a, CD54, CD62E, CD73, CD90, CD104 (mentioned above, BD Sciences), CD105 (Ancell Co.), CD133 ( Miltenyi Biotec) and CD166 (An...

Embodiment 3

[0172] Example 3: Differentiation of multipotent progenitor / stem cells derived from umbilical cord blood into neurons

[0173] Induction of differentiation into neurons

[0174] In order to confirm whether the multipotent progenitor / stem cells obtained in Example 1 can differentiate into various types of cells, they were treated to induce differentiation into neurons as follows. Multipotent progenitor / stem cells at 4 x 10 4 cells / cm 2 The concentration was inoculated into the animal cell medium induced to differentiate into neurons, and cultured for 1-2 weeks, wherein the differentiation induction medium was supplemented with 1% FBS, 2mM L-glutamine, 10μM retinoic acid, 10μM folskolin , 100 ng / ml NGF, 1×N2 supplement (Gibco BRL), 0.00001% β-mercaptoethanol and 100 U / ml penicillin-100 μg / ml streptomycin in HG-DMEM.

[0175] Confirm differentiation into neurons

[0176] Using an optical microscope to observe whether the multipotent progenitor / stem cells derived from umbili...

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Abstract

Since the multipotent progenitor / stem cells isolated and cultured from the cord blood-derived mononuclear cells according to the method of the present invention are capable of differentiating into several types of cells including neurons, osteoblasts, myoblasts, endothelial cells, hepatocytes and dendritic cells, they can be effectively used for a cell therapy, a cell replacement therapy, an organ restoration technique or an organ production.

Description

technical field [0001] The present invention relates to a method for isolating and culturing multipotent progenitor / stem cells from mononuclear cells derived from umbilical cord blood and a method for inducing differentiation of the multipotent progenitor / stem cells to produce various types of cells. Background technique [0002] Stem cells have multiple differentiation potentials through specific differentiation induction to stimulate differentiation into cells of various tissues, as well as self-renewal abilities at the undifferentiated stage. Stem cells are classified into embryonic stem cells (ES cells) and adult stem cells according to their differentiation potential and generation time. ES cells are isolated from the inner cell mass (ICM) of blastocyst stage embryos. ES cells include three types of germ layers, ie, endoderm, mesoderm, and ectoderm, and ES cells are pluripotent cells capable of differentiating into almost every type of cell found in a living body. How...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/00C12N5/02C12N5/0789
CPCH04B1/40H04R3/00
Inventor 李明雨金映真崔祯恩梁末淑文荣俊金仙庆金孝哲朴峻成张寅根
Owner 生命科得公司
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