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Preparation method of hemopoietic progenitor cells and special medium for the same

A technology of hematopoietic progenitor cells and culture medium, applied in the field of preparation of hematopoietic progenitor cells, to achieve the effect of broad application prospects

Inactive Publication Date: 2011-10-05
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows humans with blood vessels or bone marrow to grow new stem cells called VEGFR-3+ BFGFLCs from their own vasculature. These cells were able to form placental tissue during fetal life but they lacked this ability when grown into adult animals due to inherited defects caused by gene mutations like X chromosome syndrome. By inducting these cells through specific stages of different types of reprogramming it was possible to create homogeneous populations of immune system precursors which could potentially replace some individuals' natural defenses against diseases such as autoimmunity.

Problems solved by technology

The technical problem addressed by this patented technology relates to improving the efficiency with which certain types of plasma cells develop towards various tissues like blood vessels, muscles, nerves, brain neuron systems, etc.. Current techniques involve culturing isolated populations of specific lineages but they lack accurate simulation over time due to factors like variations between individual linesage stages.

Method used

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  • Preparation method of hemopoietic progenitor cells and special medium for the same
  • Preparation method of hemopoietic progenitor cells and special medium for the same
  • Preparation method of hemopoietic progenitor cells and special medium for the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1. Inducing Human Embryonic Stem Cells to Differentiate into Hematopoietic Progenitor Cells

[0093] I. Embryonic stem cells are induced to differentiate into mesoderm cells

[0094] 1. Preparation method

[0095] 1. Preheat differentiation medium I-2a in a water bath at 37°C.

[0096] 2. Add differentiation medium I-2a to the human embryonic stem cell line H1 (human ES cell line H1), and transfer the clones to a non-attached six-well plate, and place them in an incubator at 37°C for mesoderm differentiation;

[0097] 3. After 36 hours, mesoderm differentiation was completed, and the cells of the combined treatment group of human BMP4 and human Wnt3a (BMP4+Wnt3a) were obtained, which were mesoderm cells (H1).

[0098] In the same way, the human ES cell line H1 was cultured in the first-stage differentiation medium and differentiation medium I-1 to obtain the cells in the control group without induction factors (No cytokine) and the cells in the human BMP4-trea...

Embodiment 2

[0203] Example 2, Drug Toxicity Detection of Hematopoietic Progenitor Cells

[0204] I. Enrichment and purification of hematopoietic progenitor cells

[0205] 1. Method

[0206] 1. Digest hematopoietic progenitor cells (H1) obtained from differentiation medium III-2a with trypsin, then add serum-containing medium to stop the trypsin digestion, and use a 40 μm cell mesh to filter cells that have not been digested into single cells Agglomerate, the cells were collected by centrifugation; the serum-containing medium was the aforementioned DMEM medium containing 10% (volume percentage) fetal bovine serum.

[0207] 2. The cells were resuspended in phosphate buffer containing 2% (volume percentage) fetal bovine serum, and the cells were labeled with an elutable CD34 magnetic bead antibody in a refrigerator at 4°C for 20 minutes;

[0208] 3. Collect the cells by centrifugation, and resuspend the cells in phosphate buffer containing 2% fetal bovine serum;

[0209] 4. Add the cells ...

Embodiment 3

[0261] Example 3, Inducing Human Embryonic Stem Cells (H9) to Differentiate into Hematopoietic Progenitor Cells

[0262] I. Embryonic stem cells are induced to differentiate into mesoderm cells

[0263] 1. Preparation method

[0264] Human embryonic stem cells (H9) were induced to differentiate into mesoderm cells (H9) in differentiation medium I-2a using the preparation method described in Example 1-I.

[0265] 2. Detection of differentiation results

[0266] The mesoderm cells (H9) obtained in the differentiation medium I-2a above were analyzed by flow cytometry. The experimental results showed that in the combined treatment group of human BMP4 and human Wnt3a (BMP4+Wnt3a) (differentiation medium I-2a was used), 93% of the cells differentiated into T-positive mesoderm cells.

[0267] II. Differentiation of mesoderm cells into hematopoietic-endothelial precursor cells

[0268] 1. Preparation

[0269] The above-mentioned mesoderm cells (H9) were induced to generate hemato...

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Abstract

The invention provides a preparation method of hemopoietic progenitor cells and a special medium for the same. The special medium consist of a differentiation medium I-1 or a differentiation medium I-2, a differentiation medium II-1 or a differentiation medium II-2, and a differentiation medium III-1 or a differentiation medium III-2. The differentiation medium I-2 is obtained by adding a human BMP4 and a human Wnt3a into a first phase differentiation medium; the differentiation medium II-2 is obtained by adding a human vascular endothelial growth factor and a human basic fibroblast growth factor into a second phase differentiation medium; the differentiation medium III-1 is a third phase differentiation medium; and the differentiation medium III-2 is obtained by adding an all-trans-RA (retinoic acid) into a third phase differentiation medium. The hemopoietic progenitor produced by the method of the invention is suitable for being used as a substitute of a human primary hemopoietic progenitor.

Description

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Claims

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Application Information

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Owner PEKING UNIV
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