CN111304148B

CN111304148B - Application of thiamine in promoting differentiation of human pluripotent stem cells to definitive endoderm cells

Info

Publication number: CN111304148B
Application number: CN201911353445.7A
Authority: CN
Inventors: 佟曼; 韩伟方; 马静; 辛文
Original assignee: BEIJING TRANSGEN BIOTECH CO LTD
Current assignee: Beijing quanshijin Biotechnology Co.,Ltd.
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-04-13
Anticipated expiration: 2039-12-25
Also published as: CN111304148A

Abstract

The invention discloses application of thiamine in promoting differentiation of human pluripotent stem cells to definitive endoderm cells. Further disclosed is a culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising basal medium, thiamine, a growth factor and a small molecule compound. The invention also provides a method for promoting the differentiation of the human pluripotent stem cells to definitive endoderm cells. The invention discovers for the first time that thiamine can greatly improve the efficiency and purity of inducing differentiation of human pluripotent stem cells to obtain definitive endoderm cells, improves the differentiation efficiency from 55-79% to 94-98%, and provides good cell resources for downstream differentiation of the definitive endoderm cells.

Description

Application of thiamine in promoting differentiation of human pluripotent stem cells to definitive endoderm cells

Technical Field

The invention relates to the technical field of cell culture. More particularly, it relates to the use of thiamine in promoting differentiation of human pluripotent stem cells to definitive endoderm cells.

Background

Endoderm is one of the three germ layers, and develops together with mesoderm and ectoderm to produce all the organs of an adult. In the early embryonic stage, endodermal cells are initially produced during gastrulation and develop into the digestive tract including the intestine, liver and pancreas, the respiratory tract including the lungs and trachea, and the thyroid gland. All these organs play an important role in the body, and the loss of function may cause fatal diseases. However, most of the functional cells constituting these organs are difficult to culture in vitro, and obtaining transplantable healthy organs by organ donation also has a limitation in the number of donors. Therefore, the lack of high quality endoderm cells severely limits this area of developmental biological basic research, disease modeling, and regenerative medicine applications.

With the rapid development of the stem cell field, the acquisition of definitive endoderm cells and cells derived therefrom from human pluripotent stem cells is becoming an increasingly hot point of regenerative medicine. Based on the rationale for mesendoderm formation in developmental biology, several research groups have been able to successfully differentiate human pluripotent stem cells to obtain relatively uniform definitive endoderm cells. The extracellular matrix for inducing the differentiation of definitive endoderm cells is selected from Fibronectin Fibronectin and Vitronectin, and a key signal path needing to be activated is an Nodal/Activin signal path, and the formation of endoderm is regulated and controlled by a downstream effector protein SMAD 2/3. Other signaling pathways such as WNT and FGF are essential for the formation of primitive streak and the expression of the endodermal key regulator SOX 17. In order to maintain cell viability during cell proliferation and differentiation, it is necessary to use nutritional supplements, which are commonly used and include serum, serum replacement or B27 supplement. The use of these nutritional additives introduces animal-derived components into the differentiation system, which on the one hand affects batch-to-batch stability, and on the other hand limits the use of definitive endoderm cells and derived cells in the field of cell replacement therapy due to the risk of animal viral infection. In order to establish a differentiation system with definite chemical components and no animal origin, the use of nutritional additives such as serum, serum substitutes, B27 additives and the like needs to be avoided. In addition, in the existing differentiation system, the purity of definitive endoderm cells is limited, and is only 60% -80%, and 20-40% of the cells are other types of cells. The low purity of definitive endoderm cells leads to increased difficulty in downstream differentiation to obtain high quality hepatocyte, islet beta cells, and other derived cells.

Disclosure of Invention

One object of the present invention is to provide the use of thiamine in promoting differentiation of human pluripotent stem cells into definitive endoderm cells.

The second purpose of the invention is to provide a culture medium for differentiating the human pluripotent stem cells to the definitive endoderm cells.

The third purpose of the invention is to provide a method for promoting the differentiation of human pluripotent stem cells to definitive endoderm cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

in accordance with the first object described above, the present invention provides the use of thiamine in promoting differentiation of human pluripotent stem cells into definitive endoderm cells.

Optionally, the concentration of thiamine is 40-50 mg/L.

According to the second object, the present invention provides a culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, comprising a basal medium, thiamine, a growth factor and a small molecule compound.

Optionally, the concentration of thiamine is 40-50 mg/L.

Optionally, the basic culture medium comprises one or more of DMEM/F12 culture medium and RPMI1640 culture medium.

Optionally, the growth factor comprises one or more of Actvina, TGF beta, Wnt3 a; preferably, the concentration of Actvina is 10-300 mug/L, the concentration of Wnt3a is 10-300 mug/L, and the concentration of TGF beta is 0.1-10 mug/L.

Optionally, the small molecule compound includes one or more of a PI3K signal pathway inhibitor and a trace element.

Optionally, the PI3K signal pathway inhibitor comprises one or more of LY294002 and Wortmannin; the microelements comprise one or more of sodium selenite and sodium selenate; preferably, the concentration of LY294002 is 0.1-50 μ M, and the concentration of Wortmannin is 0.1-5 μ M; the concentration of the sodium selenite is 1-40 mug/L, and the concentration of the sodium selenate is 0.01-1 mug M.

In accordance with the third object above, the present invention also provides a method for promoting differentiation of human pluripotent stem cells into definitive endoderm cells, the method comprising at least two stages: in the first stage, induced differentiation culture is carried out for 0-24 hours; and a second stage, inducing differentiation culture for 24-72 hours;

wherein, after the induction differentiation culture for 24-72 hours, the culture medium is used for inducing the differentiation of the human pluripotent stem cells to obtain the definitive endoderm cells.

Optionally, the method further comprises the step of culturing and expanding the human pluripotent stem cells in a human pluripotent stem cell culture medium containing a Vitronectin or Matrigel matrix before inducing differentiation.

Optionally, in the method of the present invention, in the first stage, induced differentiation culture is performed for 0 to 24 hours, and the used differentiation culture medium formula includes a basal culture medium, a growth factor and a small molecule compound; preferably, in the formula of the differentiation medium, the basic medium comprises one or more of DMEM/F12 medium and RPMI1640 medium; the growth factor comprises one or more of Actvina, TGF beta and Wnt3 a; the small molecule compound comprises one or more of a PI3K signal pathway inhibitor and a GSK3 beta signal pathway inhibitor.

In addition, the invention also claims the human definitive endoderm cells obtained by the method and the application of the human definitive endoderm cells in the differentiation of downstream hepatic progenitor cells, liver cells, bile duct cells and islet cells.

Because the human definitive endoderm obtained by the invention has high purity, the hepatic progenitor cells, the hepatic cells, the cholangiocytes and the islet cells obtained by differentiation also have good application prospects in the treatment of acute and chronic liver injury, decompensated liver cirrhosis and diabetes.

The invention has the following beneficial effects:

the invention discovers for the first time that thiamine can promote differentiation of human pluripotent stem cells to definitive endoderm cells. Especially, 40-50 mg/L thiamine has much higher induction effect (94-98%) on differentiation from human pluripotent stem cells to definitive endoderm cells than that of other concentrations (47-76%), such as 1mg/L and 100 mg/L. Compared with other definitive endoderm cell differentiation culture media which do not contain animal-derived components and only use ActivinA and Wnt3a to activate related signal paths, the culture medium greatly improves the efficiency and purity of inducing human pluripotent stem cells to differentiate to obtain definitive endoderm cells, improves the differentiation efficiency from 55-79% to 94-98%, and provides good cell resources for the downstream differentiation of the definitive endoderm cells.

The culture medium for differentiating the human pluripotent stem cells into the definitive endoderm cells has the advantages of definite chemical components, stable batch, no animal-derived components, no protein extract or hydrolysate, simple formula and low cost. The culture medium is safer in clinical application because the risk of introducing animal-derived and human-derived pathogenic microorganisms does not exist.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the morphology of definitive endoderm cells (Experimental group one, control group, scale: 100 μm).

FIGS. 2 a-2 f show the flow charts of the definitive endoderm marker gene SOX17 (experiment group one, experiment group two, experiment group three, low concentration group, high concentration group, control group).

Fig. 3 shows statistical graphs of differentiation efficiency of definitive endoderm cells (experiment group one, experiment group two, experiment group three, low concentration group, high concentration group, control group).

FIG. 4 shows the morphology of differentiation of definitive endoderm cells downstream into hepatic progenitors (Experimental group I, control group, scale: 100 μm).

FIG. 5 shows an AFP flow diagram of the hepatic progenitor marker gene (Experimental group one, control group).

Detailed Description

In order to make the technical solutions and advantages of the present invention clearer, the following will describe embodiments of the present invention in further detail with reference to the accompanying drawings.

Aiming at the problems in the prior art, such as the use risk of serum, serum substitutes or animal-derived components, the limited purity of differentiated definitive endoderm cells and the like, the invention provides the application of thiamine in promoting the differentiation of human pluripotent stem cells to definitive endoderm cells.

Thiamine, also known as vitamin B1, is involved in a variety of cellular functions, including energy metabolism, nerve conduction-related enzymatic reactions, etc., and plays a role in brain development and immune system regulation. In the in vitro culture of cells, thiamine with a certain concentration range can play a role in maintaining the energy metabolism of cells and promoting the growth, but the effect on the culture and the directional differentiation of pluripotent stem cells is not clearly concluded. The invention discovers the promoting effect of thiamine on the differentiation of pluripotent stem cells to definitive endoderm cells, and the effect is particularly obvious under specific concentration. For example, the concentration of thiamine is 40-50 mg/L, and specifically may be 40mg/L, 42mg/L, 45mg/L, 48mg/L, 50mg/L, and the like.

The invention provides a culture medium for differentiating human pluripotent stem cells to definitive endoderm cells on the basis of the culture medium. The culture medium has definite chemical components, no animal-derived components, no protein extract/hydrolysate, and can stably realize high-efficiency differentiation and obtain high-purity definitive endoderm cells. The differentiation rate of the definitive endoderm obtained by differentiating the human pluripotent stem cells by using the culture medium is higher than 94 percent and is far higher than the differentiation efficiency of 60 to 80 percent of a conventional differentiation system, and the differentiation efficiency is stable among batches. The culture medium comprises a basic culture medium, thiamine, a growth factor and a small molecule compound.

In a specific embodiment of the present invention, the concentration of thiamine in the culture medium is 40-50 mg/L, for example, 40mg/L, 42mg/L, 45mg/L, 48mg/L, 50mg/L, etc.

In a specific embodiment of the invention, the basic culture medium comprises one or more of DMEM/F12 culture medium and RPMI1640 culture medium.

In a specific embodiment of the invention, the growth factor comprises one or more of ActvinA, TGF β, Wnt3 a; preferably, the Actvina is at a concentration of 10 to 300. mu.g/L (e.g., 20 to 300. mu.g/L, 30 to 200. mu.g/L, 20 to 100. mu.g/L, 50 to 100. mu.g/L, etc.), the Wnt3a is at a concentration of 10 to 300. mu.g/L (e.g., 20 to 300. mu.g/L, 30 to 200. mu.g/L, 20 to 100. mu.g/L, 50 to 100. mu.g/L, etc.), and the TGF β is at a concentration of 0.1 to 10. mu.g/L (e.g., 0.5 to 5. mu.g/L, 1 to 3. mu.g/L, 1 to 5. mu.g/L, 0.5 to 8. mu.g/L, etc.).

In a specific embodiment of the invention, the small molecule compound comprises one or more of a PI3K signal pathway inhibitor and a trace element.

Optionally, the PI3K signal pathway inhibitor comprises one or more of LY294002 and Wortmannin; the microelements comprise one or more of sodium selenite and sodium selenate; preferably, the concentration of LY294002 is 0.1-50 μ M (e.g., 0.5-30 μ M, 1-20 μ M, 1-10 μ M, etc.), and the concentration of Wortmannin is 0.1-5 μ M (e.g., 0.2-4 μ M, 0.5-2 μ M); the concentration of sodium selenite is 1-40 μ g/L (e.g., 5-30 μ g/L, 10-20 μ g/L, etc.), and the concentration of sodium selenate is 0.01-1 μ M (e.g., 0.05-0.5 μ M, 0.1-0.2 μ M).

In addition, the present invention provides a method of promoting differentiation of human pluripotent stem cells into definitive endoderm cells, the method comprising at least 2 stages:

in the first stage, induced differentiation culture is carried out for 0-24 hours; at this stage, the culture medium may be used to culture human pluripotent stem cells without adding thiamine, and a differentiation medium known in the art may be used, for example, the differentiation medium formulation may include a basal medium, growth factors and small molecule compounds, and the invention is not limited to the medium formulation used in this step.

For example, in the first stage 0-24 hours, the differentiation medium formulation used includes, but is not limited to; the basic culture medium comprises one or more of DMEM/F12 culture medium and RPMI1640 culture medium; the growth factor comprises one or more of Actvina, TGF beta and Wnt3 a; the small molecule compound comprises one or more of a PI3K signal pathway inhibitor and a GSK3 beta signal pathway inhibitor.

In the second stage, induced differentiation culture is carried out for 24-72 hours; at this stage, the culture medium of the present invention is used to induce differentiation of human pluripotent stem cells to obtain definitive endoderm cells, and the addition of thiamine can significantly improve the efficiency of differentiation of human pluripotent stem cells to definitive endoderm cells.

It is conceivable for those skilled in the art that a step of culturing and expanding the human pluripotent stem cells in a culture medium of the human pluripotent stem cells containing a Vitronectin or Matrigel matrix may be further included before inducing differentiation in order to achieve a more stable differentiation effect.

The definitive endoderm prepared by the method has high purity, so that the differentiation efficiency of the definitive endoderm in the differentiation of downstream hepatic progenitor cells, hepatic cells, bile duct cells and islet cells is improved.

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention. Any range recited herein includes the endpoints and any values between the endpoints and any subranges subsumed therein or between the endpoints if not specifically stated.

The test methods used in the following examples are all conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The basal Medium RPMI1640Medium, chemical refined Xeno-free Human Pluripotent Stem Cell Medium used in the examples of the present invention was obtained from Beijing Quanyu gold Biotechnology Co., Ltd, the growth factor and the small molecule compound were obtained from R & D System, Selleck, and other materials were commonly used in the art.

Example 1

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 10. mu.g/L, LY 2940020.1. mu.M, thiamine 40mg/L, sodium selenite 1. mu.g/L.

Example 2

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 300. mu.g/L, LY 29400250. mu.M, thiamine 50mg/L, and sodium selenite 40. mu.g/L.

Example 3

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu.M, thiamine 45mg/L, and sodium selenite 20. mu.g/L.

Example 4

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, thiamine 45mg/L, sodium selenite 20. mu.g/L.

Example 5

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu.M, thiamine 45 mg/L.

Example 6

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, Wortmannin 1. mu.M, thiamine 45mg/L, sodium selenite 20. mu.g/L.

Example 7

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu.M, thiamine 45mg/L, sodium selenate 0.1. mu.M.

Comparative example 1

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu.M, thiamine 1mg/L, sodium selenite 20. mu.g/L.

Comparative example 2

A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, the culture medium comprising: RPMI1640Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu.M, thiamine 100mg/L, and sodium selenite 20. mu.g/L.

Test example 1

Comparative experiment for inducing differentiation of human pluripotent stem cells into definitive endoderm cells

D-2 digesting the human pluripotent stem cells with good growth state into single cells according to the formula 10⁵cells/mL were plated at a density of 10. mu. M Y-27632 in a Chemically Defined Xeno-free Human Pluripotent Stem Cell Medium in vitro or in Matrigel coated Petri dishes;

d0 changing the culture solution into a differentiation culture Medium A, namely a first stage of induced differentiation, wherein the treatment time is 0-24 hours, and the first experimental group (RPMI1640 Medium (1 x), ActivinA 100 μ g/L, LY 29400220 μ M, CHIR 990215 μ M), the second experimental group (RPMI1640 Medium (1 x), ActivinA 100 μ g/L, CHIR 990215 μ M), the third experimental group (RPMI1640 Medium (1 x), ActivinA 100 μ g/L, LY 29400220 μ M, CHIR 990215 μ M) and the third experimental group (RPMI1640 Medium (1 x), ActivinA 100 μ g/L, LY 29400220 μ M, CHIR 990215 μ M) are adopted; low concentration group (RPMI1640 Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu. M, CHIR 990215. mu.M), high concentration group (RPMI1640 Medium (1X), ActivinA 100. mu.g/L, LY 29400220. mu. M, CHIR 990215. mu.M); the control group is RPMI1640Medium (1X), ActivinA 100. mu.g/L, Wnt3a 50. mu.g/L;

d1 and D2, the culture solution is changed into a differentiation culture medium B, namely a second stage of induced differentiation, the treatment time is 24-72 hours, and an experiment group I (as a formula in an embodiment 3) is obtained; experimental group two (formulation as example 4); experimental group three (formulation as example 5); low concentration group (formulation of comparative example 1), high concentration group (formulation of comparative example 2), control group RPMI1640Medium (1X), ActivinA 100 μ g/L;

starting with D3, we obtained SOX17 positive definitive endoderm cells.

The differentiation results are shown in FIGS. 1 to 5.

FIG. 1 is a morphological diagram showing the induced differentiation of human pluripotent stem cells into definitive endoderm cells in differentiation media described in the experimental group I and the control group, respectively, for 3 days. As can be seen from the figure, the growth state of the cells during the induced differentiation was not different between the experimental group and the control group.

Fig. 2 shows the flow cytometric analysis results of the expression of the human definitive endoderm marker gene SOX17 induced by differentiating the human pluripotent stem cells into definitive endoderm cells induced for 3 days in the differentiation medium described in the experimental group one, the experimental group two, the experimental group three, the low concentration group, the high concentration group and the control group, respectively, which show that the differentiation efficiency of the experimental group is 96.29-97.67%, the differentiation efficiency of the experimental group is 94.14-95.74%, the differentiation efficiency of the experimental group is 94.48-95.65%, the differentiation efficiency of the low concentration group is 58.77-76.15%, the differentiation efficiency of the high concentration group is 46.75-72.58% and the differentiation efficiency of the control group is 55.28-78.63%.

Fig. 3 shows statistics of differentiation efficiencies of the experimental group and the control group, wherein a differentiation efficiency of the experimental group is 96.93% ± 0.74% (n ═ 4), the differentiation efficiency is high, and the batch-to-batch difference is small; the dichotomization efficiency of the experimental component is 95.09% +/-0.70% (n is 4), the differentiation efficiency is high, and the batch-to-batch difference is small; the differentiation efficiency of the experimental group is 95.04% + -0.49% (n is 4), the differentiation efficiency is high, and the batch-to-batch difference is small; the low-concentration component has the crystallization efficiency of 67.45% + -8.39% (n is 4), the crystallization efficiency is low and the batch-to-batch difference is large; the differentiation efficiency of the high concentration group is 63.57% ± 11.50% (n ═ 4), the differentiation efficiency is low and the batch-to-batch difference is large; the differentiation efficiency of the control group was 67.72% ± 10.27% (n ═ 4), the differentiation efficiency was low and the lot-to-lot variation was large.

Fig. 4 shows that in the differentiation media described in the experimental group i and the control group, human pluripotent stem cells were induced to differentiate into definitive endoderm cells, and the morphological pattern of downstream differentiation into hepatic progenitor cells is polygonal endothelial-like, and as a result, it can be seen that the efficiency and purity level of definitive endoderm cells obtained by the induction of the experimental group i can support smooth completion of downstream differentiation, and the differentiation stage of low-purity definitive endoderm cells of the control group into hepatic progenitor cells is poor and the purity is low.

Fig. 5 shows the flow cytometric analysis results of inducing differentiation of human pluripotent stem cells into definitive endoderm cells and downstream differentiation into hepatic progenitor cells in the differentiation medium described in the experimental group i and the control group i, and detecting the expression of the hepatic progenitor marker gene AFP, which shows that the efficiency of differentiation of definitive endoderm cells with high purity in the experimental group i to downstream differentiation to obtain hepatic progenitor cells is 88.61%, which is far higher than the differentiation efficiency of definitive endoderm cells with low purity in the control group 57.84%.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Claims

1. A culture medium for differentiation of human pluripotent stem cells into definitive endoderm cells, comprising a basal medium, thiamine, a growth factor, and a small molecule compound, wherein,

the basic culture medium is RPMI1640 culture medium;

the concentration of the thiamine is 40-50 mg/L;

the growth factor is ActvinnA, and the concentration is 100 mug/L;

the small molecular compound comprises one or more of PI3K signal channel inhibitor and trace elements.

2. The culture medium of claim 1, wherein the PI3K signaling pathway inhibitor comprises one or more of LY294002 and Wortmannin; the microelements comprise one or more of sodium selenite and sodium selenate.

3. The culture medium according to claim 2, wherein the concentration of LY294002 is 0.1 to 50 μ M and the concentration of Wortmannin is 0.1 to 5 μ M; the concentration of the sodium selenite is 1-40 mu g/L, and the concentration of the sodium selenate is 0.01-1 mu M.

4. A method of promoting differentiation of human pluripotent stem cells to definitive endoderm cells, the method comprising at least two stages:

in the first stage, induced differentiation culture is carried out for 0-24 hours; and a second stage, inducing differentiation culture for 24-72 hours;

wherein the culture medium for differentiating human pluripotent stem cells into definitive endoderm cells according to any one of claims 1 to 3 is used for 24 to 72 hours of induced differentiation culture.

5. The method according to claim 4, wherein the differentiation culture is induced for 0-24 hours, and the differentiation culture medium formula comprises a basal medium, growth factors and small molecule compounds; the basic culture medium comprises one or more of DMEM/F12 culture medium and RPMI1640 culture medium; the growth factor comprises one or more of Actvina, TGF beta and Wnt3 a; the small molecule compound comprises one or more of a PI3K signal pathway inhibitor and a GSK3 beta signal pathway inhibitor.