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New method for inducing directional differentiation of human stem cells into hepatocytes

A technology of directed differentiation and cell differentiation, applied in the fields of biology and medicine, can solve the problem of unreported urea synthesis function and so on

Active Publication Date: 2017-04-05
TRANSCEND CYTOTHERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has not been reported that this "liver bud" tissue has the urea synthesis function of normal liver tissue or liver cells; and whether this method can be applied to human liver cell differentiation is still unknown, and there is still a certain distance from practical application; However, this research achievement provides a new idea for the clinical application research and development of ES / iPS cell differentiation liver cells

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  • New method for inducing directional differentiation of human stem cells into hepatocytes
  • New method for inducing directional differentiation of human stem cells into hepatocytes
  • New method for inducing directional differentiation of human stem cells into hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Embodiment 1, preparation of medium for inducing human embryonic stem cells or induced pluripotent stem cells to differentiate into hepatocytes

[0153] 1. Preparation of basic medium for cell differentiation

[0154] Cell differentiation basic medium was prepared according to conventional methods. That is: 0.5% N2, 1% B27, 1% Non-AA, 1% Sodium pyruvate, 1% penicillin and streptomycin mixed solution (100x) were added to DMEM / F12 (basic cell culture medium). Wherein, the percentages are all in v / v.

[0155] 2. Preparation of Hepatocyte Differentiation Medium

[0156] (1) Hepatocyte Differentiation Medium 1

[0157] Add the following components at the final concentrations to the above "basic cell differentiation medium":

[0158] GSK3β inhibitor CHIR-99021: 2uM;

[0159] TGFβ inhibitor SB431542: 5uM;

[0160] Retinoic acid (RA): 2uM.

[0161] (2) Hepatocyte Differentiation Medium 2

[0162] Add the following components at the final concentrations to the above "bas...

Embodiment 2

[0196] Example 2. Using Hepatocyte Differentiation Medium 1 and Hepatocyte Differentiation Enhanced Medium 2 for Human Hepatic Progenitor Cell Differentiation and Hepatic Mature Cell Culture

[0197] 1. Initiation of differentiation of human hepatic precursor cells

[0198] The culture plate was primed with matrigel for 12 hours, hepatocyte differentiation medium 1 was added to the culture plate, and then human embryonic stem cells (ES) (see figure 1 Left picture) Suspended in "Hepatocyte Differentiation Medium 1, plated; cultured at 37°C, 5% CO2, and the medium was changed every 72 hours.

[0199] Human embryonic stem cells are differentiated and cultured in hepatocyte differentiation medium 1 at 37°C and 5% CO2 for 10-15 days to obtain human hepatic precursor cells. The morphological comparison between human ES cells and the differentiated hepatic precursor cells is shown in figure 1 .

[0200] The hepatic precursor cells obtained by differentiation culture can be used for...

Embodiment 3

[0208] Example 3. Flow cytometric analysis of liver-specific marker staining for inducing human ES cells to differentiate into hepatic mature cells using hepatocyte differentiation medium 4

[0209] The method for inducing human ES cells to differentiate into hepatocytes is the same as in Example 2. The difference is that hepatocyte differentiation medium has been used for culture.

[0210] Flow cytometric analysis of liver-specific marker staining of human ES cells differentiated into hepatic mature cells. Using the conventional immunostaining method, the mature hepatic cells obtained after the above experimental steps induced the differentiation of human ES cells were immunostained for human hepatocyte-specific markers (AAT, ALB, Asgpr, CYP3A, HNF4a). The immunostaining method is:

[0211] (1) Discard the cell culture medium, rinse once with PBS,

[0212] (2) Digest with 0.05% trypsin at 37°C for 5 minutes, stop trypsin with a trypsin terminator, or cell culture medium co...

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Abstract

The invention relates to a new method for inducing directional differentiation of human stem cells, such as human embryonic stem cells (ES cells) or induced pluripotent stem cells, into hepatocytes. The invention discloses a medium and culture method for directional differentiation culture of the human stem cells into the hepatocytes. The new method realizes the directional differentiation of human stem cells into hepatocytes without introduction of exogenous genes to stem cells or growth factors, obtained differentiated human hepatocytes have the typical characteristics of human hepatocytes, and differentiated hepatic progenitor cells can be passaged for a long term; and differentiated hepatic mature cells are passaged in a limited manner. The culture method has the advantages of simple conditions, low cost, and good safety and stability.

Description

technical field [0001] The present invention belongs to the fields of biology and medicine; more specifically, the present invention relates to a new method for inducing directed differentiation of human stem cells, such as human embryonic stem cells or induced pluripotent stem cells, into hepatocytes using only small molecules, and a specific method applied to the new method. Differentiation medium. Background technique [0002] According to the statistics of the World Health Organization, millions of people die of liver disease every year in the world. China is a big country with liver disease, and there are 140 million hepatitis B and C virus carriers alone, accounting for 28% of the global total; acute and chronic liver failure caused by various reasons is critically ill, has a dangerous prognosis, and has a high mortality rate ( 70-80%). Liver cell transplantation and bioartificial liver replacement therapy can not only treat liver failure, but also treat genetic meta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N2501/15C12N2501/727C12N2506/45C12N5/067C12N2501/237C12N2501/39C12N2501/385C12N2501/12C12N2506/02C12N5/0663C12N5/0606C12N5/0696C12N2501/70
Inventor 张培霖陈立新
Owner TRANSCEND CYTOTHERAPY CO LTD
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