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A method of converting human hematopoietic progenitor cells into hematopoietic stem cells

A technology of hematopoietic progenitor cells and hematopoietic stem cells, applied in the field of hematopoietic stem cells, can solve the problems of low number of hematopoietic stem cells, increase the fatality rate of opportunistic infections, etc., and achieve the effect of high number

Active Publication Date: 2019-01-15
NEWISH TECH (BEIJING) CO LTD
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Problems solved by technology

However, due to the small number of hematopoietic stem cells contained in a single umbilical cord blood, it is not enough to rebuild the immune system of adult patients in a short period of time, thereby increasing the fatality rate of opportunistic infections. There is an urgent need for a method to increase the number of hematopoietic stem cells in umbilical cord blood

Method used

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  • A method of converting human hematopoietic progenitor cells into hematopoietic stem cells
  • A method of converting human hematopoietic progenitor cells into hematopoietic stem cells
  • A method of converting human hematopoietic progenitor cells into hematopoietic stem cells

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Experimental program
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Embodiment 1

[0023] Embodiment 1: Using the special transformation medium for human umbilical cord blood hematopoietic stem cells to obtain a large number of umbilical cord blood hematopoietic stem cells from hematopoietic progenitor cells, including the following steps:

[0024] 1. Obtain peripheral blood mononuclear cells (PBMC).

[0025] (1) Collect 80-120ml of umbilical cord blood with a disposable blood bag (containing anticoagulants such as heparin sodium), transfer the umbilical cord blood from the blood bag to a 500ml culture bottle, add normal saline to dilute 2-3 times, mix well and gradually Add dropwise to 0.4 times the volume of lymphocyte separation medium, be careful not to damage the interface.

[0026] (2) Centrifuge at 1500-2000rpm / min for 20 minutes. Due to different densities, the centrifuge tube is divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white mononuclear cell layer (PBMC), the The thi...

Embodiment 2

[0041] Embodiment 2: The above-mentioned isolated human placental blood hematopoietic stem cells are subjected to phenotype identification, activity rate and purity detection and functional identification, including the following steps:

[0042] 1. Cell count

[0043] Count the number of CD34+CD90+ and CD34+CD45RA- hematopoietic stem cells after culture respectively.

[0044] Table 2 Statistics of cell number after culture of CD34+CD90- hematopoietic progenitor cells

[0045]

[0046] Table 3 Statistics of cell number after culture of CD34+CD45RA+ hematopoietic progenitor cells

[0047]

[0048] 2. Cell Flow Cytometry Analysis

[0049] Using BD’s FACS Verse flow detector, take 20 μl of cell suspension and add 0.2 μl of FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, and APC-labeled CD90 dissolved in 0.5% BSA. After vortexing each tube, incubate at room temperature in the dark for 15 minutes, add an appropriate amount of PBS, centrifuge at 1600 rpm for 5 mi...

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Abstract

The invention discloses a method for preparing human hematopoietic stem cells by using human hematopoietic progenitor cells, comprising the following steps: S1 preparing CD34+CD90- and CD34+CD45RA+ umbilical cord hematopoietic progenitor cells from human umbilical cord blood; S2) suspending the CD34+CD90- and CD34+CD45RA+ umbilical cord hematopoietic progenitor cells in a medium specially used forumbilical cord hematopoietic stem cells transformation and culturing the cells, the medium adopting a StemSpan SFEM II serum-free culture medium and 100ng / ml SCF, 100ng / ml FLT3 and 50ng / ml TPO beingadded into the medium, wherein the cell seeding density in 24-well plate is 0.55 *10<4> cells / well for CD34+CD90-cells, and 0.13 *10<4> cells / well CD34+CD45RA+ cells; 1[mu]M of MS-275 is added, and culture is performed at 37 DEG C in an incubator with the CO2 concnetration being 5%. The method has the beneficial effects that the hematopoietic stem cells prepared by using the human hematopoietic progenitor cells are high in quantity and have the differentiation potential of each lineage, and can provide a hematopoietic stem cell donor for clinical application.

Description

technical field [0001] The invention relates to the technical field of hematopoietic stem cells, in particular to a method for preparing human hematopoietic stem cells from human hematopoietic progenitor cells by using small chemical molecules. Background technique [0002] Hematopoietic stem cells are an extremely important type of stem cells in adults. Although they only account for less than one ten-thousandth of human blood cells, they have strong self-renewal and differentiation capabilities, and can rebuild the entire blood system and immune system of the body for a long time. Differentiation potential of blood and immune cells of various lineages. Therefore, hematopoietic stem cells are widely used in the clinical treatment of malignant blood diseases such as leukemia and lymphoma. Not only that, hematopoietic stem cell transplantation can also help treat metabolic diseases, congenital immunodeficiency, diabetes and other conditions. According to statistics, there a...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/999
Inventor 陈立功刘德芳杨欢郭潇马骏凡孙忠杰
Owner NEWISH TECH (BEIJING) CO LTD
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