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Method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro

A technology of embryonic stem cells and erythroid cells, which is applied in the field of in vitro induction of embryonic stem cells to differentiate into erythroid cells, can solve the problems of poor stability of hemoglobin oxygen carriers, weak oxygen-carrying capacity of fluorocarbons, complex process, etc., and achieve effective cell sources , easy to obtain materials, and easy to amplify in vitro

Inactive Publication Date: 2015-06-10
奥思达干细胞有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, hemoglobin oxygen carriers have poor stability, difficult purification, complicated process and high cost, and fluorocarbons have weak oxygen-carrying capacity, slow metabolism, and great toxic and side effects. Therefore, these two types of erythroid cell substitutes cannot be mass-produced. applications, it is necessary to continue to search for new alternative sources of blood erythroid cells

Method used

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  • Method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro
  • Method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro
  • Method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro

Examples

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specific Embodiment 1

[0035] Specific example 1: in vitro induction of embryonic stem cells to differentiate into erythroid cells

[0036] (1) Routine culture of embryonic stem cells, the specific steps include:

[0037] Embryonic stem cells are routinely cultured for passage and expansion. The medium at this stage is: 85% DMEM medium as the basal medium, supplemented with 15% Knockout Serum Replacement, 1mmol / L essential amino acids, 100IU / mL penicillin, 50μg / mL streptomycin and 4ng / mL bFGF. Cell cultures were incubated at 37°C, 5% CO2, and passaged once a week.

[0038] (2) Pre-induction of embryonic stem cells, the specific steps include:

[0039] In order to induce the differentiation of human embryonic stem cells into hematopoietic cells, the embryonic stem cells were induced for 6-7 days in culture. Digest with 1 mg / mL type Ⅳ collagenase at 37°C for 3-5 minutes and pipette into small cell clusters, then use ultra-low adhesion 6-well culture plates to suspend for 7 days, use 100 IU / mL penic...

specific Embodiment 2

[0048] Specific embodiment 2: the mensuration of each index of hematopoietic stem cell

[0049] (1) Specific expression on the cell surface: detection of specific markers of hematopoietic stem cells

[0050] CD34 antigen is a glycoprotein with a molecular weight of 115kD on the cell surface. The gene encoding CD34 is located on the long arm of chromosome 1 and contains 8 exons. CD34 antigen is a differentiation group antigen present only on the surface of hematopoietic stem / progenitor cells. CD34 is strongly positively expressed on hematopoietic stem cells, but weakly positive on late-differentiated progenitor cells. Mature blood cells do not express the CD34 antigen. Therefore, CD34 can be used as a basic marker for detecting hematopoietic stem cells.

[0051] Take the well-growth cells from step (3) of Example 1 for detection: collect, centrifuge at 1500rpm for 10min, discard the supernatant, add PBS solution to resuspend, draw 100μL of cell suspension (about 3×10 5 cel...

specific Embodiment 3

[0056] Specific embodiment 3: the determination of various indexes of erythroid cells

[0057] 1. Erythroid cell smear test

[0058] (1) Collect the suspension-cultured cells at different time points in step (4) of Example 1, centrifuge at 2000 rpm for 5 minutes. Discard the supernatant, add 1ml PBS to wash the cells twice. Resuspend the cells to 100 μl;

[0059] (2) The slides were soaked in acid solution, rinsed repeatedly with distilled water, and coated with poly-lysine. Install it on the cell smear machine according to the operating instructions, and mark it with a pencil;

[0060] (3) Add the cell suspension, set the speed of the cytocentrifuge to 1000 rpm, and centrifuge for 2 minutes. After centrifugation, separate the middle filter paper layer from the slide in a vertical direction, carefully remove the slide, and dry the slide.

[0061] The results showed that the hematopoietic stem cells grew in suspension in the erythroid induction medium and expanded rapidly...

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Abstract

The invention discloses an effective method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro. The embryonic stem cells are pre-induced through an inducing system containing dexamethasone, subjected to induction culture through a serum-free medium system containing a serum-free culture additive N2 or B27, prostaglandin E2 and L-glutamine, and then induced to be differentiated into the erythroid cells through a serum-free medium system containing stem cell factors (SCF), interleukin 3 and hemopoietin. The method specifically includes the following steps of conventionally culturing the embryonic stem cells in vitro, pre-inducing the embryonic stem cells, directionally differentiating the pre-induced cells into hematopoietic stem cells, and directionally differentiating the hematopoietic stem cells into the erythroid cells. According to the method for inducing the embryonic stem cells to be differentiated into the erythroid cells in vitro, materials are convenient to obtain, expansion in vitro is facilitated, immunogenicity is low, and therefore the human embryonic stem cells are differentiated into the mature erythroid cells more safely and efficiently, and the technological application prospects are good.

Description

technical field [0001] The invention belongs to the field of regenerative medicine and biotechnology, and relates to a preparation method of erythroid cells, in particular to a method for inducing embryonic stem cells to differentiate into erythroid cells in vitro. Background technique [0002] With the rapid development of my country's medical technology and the increasing requirements of people's lives, component blood transfusion has been deepened in clinical medical practice, among which erythroid cells (also called blood cells) are the most used. Erythrocyte transfusion is a vital cell therapy method in modern medical technology. The main purpose is to improve the hypoxic state of patients. It is widely used in the treatment of postoperative and trauma, chronic anemia and other diseases. However, at present, clinical practice still relies on voluntary blood donation as the main source of blood. The blood supply is very tight, and reports of insufficient blood supply are...

Claims

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Application Information

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IPC IPC(8): C12N5/078
Inventor 周萱
Owner 奥思达干细胞有限公司
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