Blister-derived blastocyst induction culture medium and methods for inducing blastocyst culture in mice / humans

Abstract

This invention discloses a blastocyst induction culture medium derived from mouse and human embryonic stem cells (ESCs) and a method for inducing and culturing blastocysts in mice and humans. Using a chemically defined blastocyst induction culture medium, mouse / human ESCs differentiate into three different cell lineages found in natural blastocysts. Further, using the same blastocyst induction culture medium, the mixed state of mouse / human cells containing the three different cell lineages obtained in the previous step is induced to produce mouse and human blastocyst structures under suspension culture conditions. This invention utilizes a specific blastocyst induction culture medium and induction culture method to enable mouse / human ESCs to self-assemble into blastocyst structures. Experimental methods have verified that mouse and human blastocysts are similar to natural blastocysts in morphology, size, cell number, cell distribution, and gene expression characteristics, providing a new research model for in vitro tissue morphogenesis, organoid modeling, and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of embryonic stem cell culture technology, and specifically relates to a blastocyst induction culture medium for mice and humans, and particularly to a method for inducing and culturing blastocysts derived from mouse and human embryonic stem cells. Background Technology

[0002] Embryonic stem cells (ESCs) originate from the inner cell mass of the blastocyst and can contribute to all fetal tissues, but do not participate in placental development. Under suitable in vitro culture conditions, mouse and human ESCs can differentiate into a three-dimensional aggregate called embryoids (EBs), which contain three different lineage cell types and can be used to simulate early embryogenesis studies. The formation of EBs is an important feature of ESC self-assembly and lineage differentiation; however, EBs cannot completely recreate the initiation of embryonic development.

[0003] In recent years, breakthroughs have been made in embryonic stem cell-based embryonic-like research, with some studies reporting the in vitro reconstruction of blastocysts or gastrulation-stage embryonic developmental models using mouse or human embryonic stem cells. However, to date, the in vitro induction of mouse blastocysts still requires the co-culture of two or three different types of stem cells, and the culture conditions are very complex. Whether it is possible to obtain blastocyst-like structures using only mouse or human embryonic stem cells under chemically defined induction culture conditions has not been reported.

[0004] Therefore, obtaining a simple and efficient blastocyst induction culture medium for mice and humans, especially a blastocyst induction culture method derived solely from mouse and human embryonic stem cells, is key to overcoming the limitations of in vitro blastocyst induction methods. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a blastocyst induction culture medium derived from mouse and human embryonic stem cells, and to establish a blastocyst induction culture method, thereby obtaining a large number of mouse and human blastocyst structures for the first time using a single type of embryonic stem cell line for induction culture.

[0006] To address the aforementioned technical problems, this invention provides a blastocyst induction culture medium derived from mouse / human embryonic stem cells, characterized by comprising the following components: each 500 ml of blastocyst induction culture medium contains CHIR99021 with the following composition:

[0007]

[0008] The remainder is replenished by the basal culture medium.

[0009] The basal culture medium consists of basal culture medium DMEM / F12 and basal culture medium Neurobasal, with a volume ratio of 1:1.

[0010] Preferably, each 500 ml of blastocyst induction culture medium contains CHIR99021 with the following composition:

[0011]

[0012] 3. A method for embryonic-like induction culture of mouse / human embryonic stem cells, characterized by comprising the following steps:

[0013] (1) Prepare mouse embryonic stem cells and mTeSR cultured in 2i / L medium containing PD0325901+CHIR99021+LIF. TM Human embryonic stem cells were cultured under the following conditions: 1. Cultured at 37°C and 5% CO2 concentration.

[0014] (2) Take the 1×10 prepared in step (1) 5 Mouse / human ESCs were cultured in blastocyst-like induction medium at 37°C and 5% CO2 concentration for 5-7 days to obtain mixed cells containing three types of blastocyst cells from mice / humans.

[0015] (3) Take 1×10 of the mixed cells obtained in step (2). 5 The cells were cultured in suspension using blastocyst induction medium at 37°C and 5% CO2 concentration. The induction medium was changed daily. Mouse mixed cells began to form mouse and human blastocyst structures after 3-4 days of in vitro culture, or human mixed cells after 6-7 days of in vitro culture.

[0016] The beneficial effects of this invention are:

[0017] (1) A simple blastocyst induction culture medium with well-defined chemical composition was developed. Only one factor, CHIR99021, was used to achieve in vitro induction of mouse / human blastocysts. The culture conditions were very simple.

[0018] (2) Using this culture medium, mouse and human ESCs can be self-assembled in vitro to obtain blastocyst-like structures.

[0019] (3) Experimental methods were used to verify that mouse and human blastocysts are similar to natural blastocysts in terms of morphology, size, cell number, cell distribution and gene expression patterns.

[0020] (4) This invention provides an ideal research model for current research fields such as stem cell fate determination, early embryonic development, clinical application of stem cells, regenerative medicine and drug screening. Attached Figure Description

[0021] Figure 1 These are the early morphological characteristics of mouse ESCs cultured using blastocyst-like induction culture medium in this invention.

[0022] Figure 2 This invention describes the early morphological characteristics of human ESCs cultured using blastocyst-like induction culture medium.

[0023] Figure 3 This is the blastocyst induction and culture process derived from mouse embryonic stem cells in this invention.

[0024] Figure 4 This is the blastocyst induction and culture process derived from human embryonic stem cells in this invention.

[0025] Figure 5 This is a comparison of the diameter, total number of cells, and number of inner cell mass cells of mouse blastocysts in this invention with those of natural blastocysts.

[0026] Figure 6 This is an immunofluorescence image of the mouse blastocyst structure obtained in this invention, showing that the mouse blastocyst contains the expression of marker proteins of the three cell lineages contained in the natural blastocyst.

[0027] Figure 7 This is an immunofluorescence image of the human blastocyst structure obtained in this invention, showing that the human blastocyst contains the expression of the marker proteins of the three cell lineages contained in the natural blastocyst. Detailed Implementation

[0028] Example 1: Preparation method of mouse / human blastocyst induction culture medium

[0029] A blastocyst induction culture medium derived from mouse / human embryonic stem cells, wherein each 500 ml of blastocyst induction culture medium contains CHIR99021 (WNT pathway activator) and comprises: 2-3 ml N2 cell culture additive, 4-6 ml B27 cell culture additive, 20-30 mg bovine serum albumin, 4-6 ml non-essential amino acids, 4-6 ml L-glutamine, 0.5-2 ml β-mercaptoethanol, 4-5 ml penicillin-streptomycin, 3.0 μM CHIR99021, with the remainder supplemented by basal culture medium; wherein the basal culture medium is a mixture of DMEM / F12 and Neurobasal, with a volume ratio of 1:1.

[0030] Preferably, each 500 ml of blastocyst induction culture medium contains CHIR99021 (WNT pathway activator) with the following composition: 238.0 ml of basal culture medium DMEM / F12, 238.0 ml of basal culture medium Neurobasal, 2.5 ml of N2 cell culture additive, 5.0 ml of B27 cell culture additive, 25.0 mg of bovine serum albumin, 5.0 ml of non-essential amino acids, 5.0 ml of L-glutamine, 1.0 ml of β-mercaptoethanol, 5.0 ml of penicillin and streptomycin, and 3.0 μM CHIR99021.

[0031] Example 2: Method for inducing the culture of blastocysts derived from mouse / human embryonic stem cells

[0032] 1. Prepare mouse embryonic stem cells and mTeSRs cultured in 2i / L medium containing PD0325901+CHIR99021+LIF at 37℃ and 5% CO2 concentration. TM Human embryonic stem cells were cultured in a culture medium at 37°C and 5% CO2 concentration. The culture conditions for both cell lines are known.

[0033] 2. Differentiating mouse / human ESCs into three cell lineages of blastocysts

[0034] 1 × 10⁻⁶ ESCs were obtained from the mice or humans cultured in step 1. 5 Cells were cultured in blastocyst induction medium at 37°C and 5% CO2 concentration. After 5-7 days of culture, GOF / GFP-labeled mouse ESCs showed signs of differentiation, with some green fluorescent protein gradually disappearing. Cells were digested with Acctase and seeded into fibronectin-treated cell culture dishes. After 5-7 days of culture, human ESCs also began to differentiate. Cells were digested with EDTA (0.5 μM) and seeded into VTN (Thermo, catalog number: A14700) pretreated cell culture dishes to obtain mixed cell populations containing three types of blastocyst cells from both mice and humans. See details... Figure 1 and Figure 2 .

[0035] 3. Induction to obtain blastocyst-like structures derived from mouse / human embryonic stem cells

[0036] Cell counting was performed using 1 × 10⁻⁶ mouse / human mixed cells obtained in step 2. 5Cells were cultured in suspension at 37°C and 5% CO2 using blastocyst induction medium. This step required pretreatment of cell culture dishes with an anti-adhesion solution (purchased from STEMCELL, catalog number: 07010). The culture medium needed to be changed daily during suspension culture. Cells were observed to form tight aggregates within 24 hours, and a small cavity formed within each aggregate within 48-72 hours. Blastocyst structures derived from mouse and human embryonic stem cells were obtained after 3-4 days of culture (mixed mouse cells) or 6-7 days (mixed human cells). See details... Figure 3 and Figure 4 .

[0037] Example 3: Identification of the Morphology and Structure of Mouse Blastocysts

[0038] Measurements of blastocyst diameter revealed that it was quite similar to that of natural blastocysts, both hovering around 100 μm. Furthermore, counting the total cell number and inner cell mass number of blastocysts showed similarities to those of natural blastocysts. (See details...) Figure 5 .

[0039] Example 4: Identification of blastocyst cell distribution in mice and humans

[0040] Immunofluorescence assays were used to identify the cell distribution of the three germ layers in mouse and human blastocysts. Analysis revealed that mouse and human blastocysts exhibit similar spatiotemporal cell arrangement and expression characteristics of different cell-type specific proteins to natural blastocysts. (See details...) Figure 6 and Figure 7 The embodiments described above are only used to illustrate the technical ideas and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The patent scope of the present invention should not be limited by these embodiments alone. That is, all equivalent changes or modifications made in accordance with the spirit disclosed in the present invention still fall within the patent scope of the present invention.

Claims

1. A blastocyst induction culture medium derived from mouse / human embryonic stem cells, characterized in that: The composition of each 500ml of the blastocyst induction culture medium is as follows: Basic culture medium DMEM / F12 238.0ml Neurobasal basal culture medium 238.0 ml N2 cell culture additive 2.5ml B27 cell culture additive 5.0ml Bovine serum albumin 25.0 mg 5.0ml of non-essential amino acids L-glutamine 5.0ml 1.0 ml of β-mercaptoethanol Penicillin and streptomycin 5.0ml CHIR99021 3.0μM It does not contain PD0325901, leukemia inhibitory factor LIF, or retinoic acid RA.

2. An induction method using the culture medium of claim 1, characterized in that: Includes the following steps: (1) Mouse ESCs were cultured in 2i / L culture medium containing PD0325901+CHIR99021+LIF at 37℃ and 5% CO2 concentration, or human ESCs were cultured in mTeSR1 culture medium. (2) Take 1×10 from step (1) 5 One cell, cultured in the culture medium of claim 1 at 37°C and 5% CO2 concentration for 5-7 days, yields a mixed cell lineage containing three blastocyst cell lines; (3) Take the mixed cells obtained in step (2) and suspend them in the culture medium described in claim 1 in a culture dish pretreated with anti-adhesion solution: mouse cells form blastocysts in 3-4 days and human cells form blastocysts in 6-7 days.

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