Research method based on regulation effect of extraembryonic tissue on generation of protointestinal motility
By establishing a culture system suitable for extraembryonic mesodermal stem cells, optimizing the culture conditions and signaling pathways, and building a co-culture system, the problem of extraembryonic mesodermal cell culture is solved, and the regulatory role of extraembryonic cells in the occurrence of gastrulation is realized, providing an efficient in vitro model.
Patent Information
- Application Number
- CN202510485220.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-17
- Publication Date
- 2025-08-05
AI Technical Summary
The lack of an effective extraembryonic mesoderm cell culture system in the prior art leads to insufficient understanding of the role of extraembryonic tissue in the early stages of embryonic development, especially in the process of gastrulation.
A culture system suitable for extraembryonic mesoderm stem cells was established. By optimizing the culture conditions and signaling pathways, a co-culture system without serum, growth factor, and feeder layer was constructed. Type I collagen and Matrigel matric gel were used, combined with the WNT activator CHIR, to study the regulatory effect of extraembryonic cells on gastrulation movement.
The stable culture and purification of extraembryonic mesoderm stem cells was realized, revealing the regulatory role of extraembryonic cells in early human embryonic development, providing a controllable in vitro research platform for the occurrence of gastrulation, and clarifying the regulatory differences between different extraembryonic lineages.
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Figure CN120424855A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of stem cell culture and human early development research, and relates to the establishment of an extraembryonic mesodermal stem cell culture system and a research method based on the regulatory effect of extraembryonic tissue on gastrulation. Background Art
[0002] Mammalian embryonic development is an intricately coordinated process involving synchronized lineage diversification and morphogenetic tissue reorganization. In humans, the basic body plan is established during the third week of development. During this critical period, known as gastrulation, the embryo forms its three germ layers—the precursors of all organs—and establishes the body axis, laying the foundation for precise development. However, during the second week, prior to gastrulation, the embryo undergoes its most significant changes: the emergence and differentiation of various extraembryonic cell types, including the trophoblast, amnion, and extraembryonic mesoderm. These extraembryonic cells, along with embryonic cells, establish the embryo's basic architecture. Logically, these extraembryonic tissues must be highly involved in embryonic development. Despite this, our understanding of their specific roles and regulatory mechanisms, particularly with regard to the initiation of gastrulation in humans, remains largely unknown. While stable trophoblast stem cell culture protocols have been established for extraembryonic cell lines, including the rapid and stable induction of amnion-like cells, efficient extraembryonic mesoderm cell culture systems remain lacking. Summary of the Invention
[0003] To address these issues, this study optimized culture conditions and established a stable and efficient in vitro culture system for extraembryonic mesoderm stem cells (ExMSCs). Using a fully defined co-culture system (serum-free, growth factor-free, chemical inhibitor-free, and feeder-free, i.e., APEL medium), we determined the impact of different extraembryonic lineages on gastrulation. This demonstrated the important role of extraembryonic cells in the development of the human primitive streak, enabling us to analyze in vitro the initiation of gastrulation during the black box period of human embryonic development.
[0004] In order to achieve the above objectives, this application provides the following technical solutions:
[0005] The present invention aims to provide a research method based on the regulatory effect of extraembryonic tissue on gastrulation, which comprises the following steps:
[0006] Step 1: using an established extraembryonic mesodermal stem cell culture system and inducing extraembryonic mesodermal stem cells in vitro through the extraembryonic mesodermal stem cell culture system;
[0007] Step 2: Use a co-culture system to study extraembryonic mesoderm stem cells, trophoblast stem cells (TSCs), and amnion-like cells (AMLCs) to explore the regulatory role of extraembryonic lineage cells on gastrulation.
[0008] Specifically, the present invention establishes an extraembryonic mesodermal stem cell culture system using a matrix gel with the addition of cytokines and chemical molecules. Previous reports have shown that extraembryonic mesodermal stem cells can be differentiated from naive human pluripotent stem cells by adapting them to trophoblast stem cell culture medium (ASECRiAV). However, these extraembryonic mesodermal stem cells exhibit high cellular heterogeneity, intermix with trophoblast stem cells, and cannot be cultured for extended periods of time.
[0009] The first step is to screen the extracellular matrix suitable for extraembryonic mesodermal stem cells. Therefore, the inventors conducted a variety of tests and optimizations on the conditions for in vitro extraembryonic mesoderm derivation. The results showed that the use of type I collagen (Collagen I) as a culture substrate can significantly enrich extraembryonic mesoderm cells. Secondly, the important signaling pathways for regulating extraembryonic lineage development, such as TGFβ, WNT, and FGF, were tested. The original culture system was optimized by adding corresponding protein factors and small molecule inhibitors. Finally, the establishment of the extraembryonic mesodermal stem cell culture system was confirmed by the stable in vitro culture of cells, as well as vitality detection indicators such as CCK8, and the comprehensive expression of characteristic genes of extraembryonic mesoderm cells. The extraembryonic mesoderm stem cell culture system is: ASECRiAV culture medium + 20ng ml -1 Activin A+2ng ml -1 TGFβ1+0.5μM FGFR1 inhibitor, FGFR1 inhibitor is PD173074.
[0010] In particular, the present invention establishes an extraembryonic and embryonic stem cell co-culture system to study the regulation of extraembryonic lineages, specifically in the following manner:
[0011] In order to explore the regulation of embryonic development in a controllable environment, the inventors conducted a series of co-culture condition tests in vitro, including tests of 0.1% to 5% concentrations of two types of matrix gels, Collagen I and Matrigel, the inoculation ratio of extraembryonic cells to embryonic stem cells of 1:1 to 3:1, a basic culture medium without exogenous components, and culture time. Finally, a fully definable co-culture system was established. The culture medium only uses APEL medium, which is an animal component-free, multifunctional, growth factor-free formula. The culture plates are coated with a defined extracellular matrix, 0.5% Matrigel, to promote cell adhesion and growth, and then embryonic stem cells and extraembryonic cells are inoculated at a density of 1:3 and cultured continuously for 72 hours. The co-culture system is maintained under standard conditions (37°C, 5% CO2), and the culture medium is replaced every 24 hours to ensure a stable environment. The specific inoculation conditions are as follows:
[0012] In order to clarify the occurrence of gastrulation, a small molecule triggering system was constructed. We screened the activation of WNT and FGF, the key pathways of gastrulation, by adding 3μM and 5μM concentrations of CHIR and 8ng.ml - 1 Finally, the applicant found that adding 3 μM CHIR to APEL can effectively induce the emergence of gastrulation cell populations in embryonic stem cells, and the most significant state is reached after 48 hours of culture.
[0013] Based on this, we conducted a study to evaluate the extraembryonic lineage regulation of gastrulation. When embryonic stem cells (ESCs) reached 80% confluence in mTeSR PLUS medium, the cells were cultured with TrypLE TM Express dissociation into single cells or ReleSR dissociation into small cell clumps. Resuspend the cell clumps in mTeSRPLUS and then seed them into 12-well plates pre-coated with 0.5% Matrigel at a density of 1:20. Incubate the clumps at 37°C for 24 hours. Then use TrypLE TMExpress dissociated amnion-like cells, extraembryonic mesoderm stem cells, and trophoblast stem cells, respectively. These cells were seeded into pre-seeded embryonic stem cell culture wells at a 3:1 ratio of extraembryonic cells (extraembryonic mesoderm stem cells, trophoblast stem cells, and amnion-like cells) to intraembryonic cells. Extraembryonic cells were distributed around the embryonic stem cells for co-culture. Co-cultures were maintained in APEL medium (fully defined conditions) for 1-3 days with or without CHIR, using WNT activation. The CHIR-free group was used to determine whether gastrulation was promoted, while the CHIR-containing group was used to determine whether gastrulation was inhibited. Immunostaining and flow cytometric analysis of TBXT-positive populations in embryonic stem cells were performed at different culture days to compare the effects of different extraembryonic lineage cells.
[0014] Definition of noun:
[0015] The extraembryonic mesoderm cells referred to in the present invention are an important cell lineage during gastrulation.
[0016] Compared with the prior art, the present invention has the following advantages:
[0017] (1) The present invention establishes an extraembryonic mesodermal stem cell culture system suitable for culturing extraembryonic mesodermal stem cells. This system is capable of effectively inducing and purifying extraembryonic mesodermal stem cells. The induced extraembryonic mesodermal cells can be stably cultured in vitro (to date, for over 100 days), have higher cell viability, uniformly express extraembryonic mesodermal marker genes, and exhibit stem cell self-renewal characteristics. Using the established extraembryonic mesodermal stem cell culture system, uniform extraembryonic mesodermal stem cells representing the extraembryonic mesoderm in early human embryos were successfully obtained.
[0018] (2) Through the co-culture system, the present invention clarifies the differences in the regulation of embryonic cells by various extraembryonic lineage cells (such as trophoblast stem cells, amniotic cells, and extraembryonic mesoderm stem cells), reveals the key role of extraembryonic cells in the formation of the human primitive streak, and provides an effective in vitro model and research method for the study of the mechanism of the "black box" stage of early human embryonic development. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1A The efficiency of obtaining extraembryonic mesodermal stem cells induced by different matrix gels is demonstrated exemplarily.
[0020] Figure 1B The optimization results of the extraembryonic mesodermal stem cell culture system for TGFβ, WNT, and FGF signaling pathways are exemplified.
[0021] Figure 2A The schematic diagram of the optimization process of extraembryonic mesodermal stem cells is shown as an example.
[0022] Figure 2B The bright field morphology and staining identification images of extraembryonic mesoderm stem cells are shown as examples.
[0023] Figure 3 The bright field morphology and staining identification images of characteristic markers of embryonic stem cells and extraembryonic lineage cells are exemplified, and there is a schematic diagram of the distribution of different lineage cells in the embryo.
[0024] Figure 4A The results of co-culture of amnion-like cells, trophoblast stem cells and embryonic stem cells are exemplified, in which amnion-like cells promote the expression of gastrulation markers in embryonic stem cells, while trophoblasts do not have this characteristic.
[0025] Figure 4B The flow cytometry analysis results show that amniotic-like cells, trophoblast stem cells and embryonic stem cells have a certain inhibitory effect on the expression of gastrulation characteristic marker T. The figure below shows that amniotic-like cells and trophoblast stem cells can regulate the occurrence of gastrulation through secretion.
[0026] Figure 5A The results of co-culture of extraembryonic mesoderm stem cells and embryonic stem cells were exemplified, showing that it had no significant effect on the expression of gastrulation characteristic genes, but there was a phenotype of promoting cell migration under the regulation of CHIR treatment.
[0027] Figure 5B The results exemplify the dynamic process of promoting cell migration between extraembryonic mesodermal stem cells and embryonic stem cells under the regulation of CHIR treatment.
[0028] Figure 6 The results exemplify the dynamic process of promoting cell migration of extraembryonic mesodermal stem cells and embryonic stem cells under the regulation of CHIR treatment, and that these cells have the characteristics of epithelial-mesenchymal transition. DETAILED DESCRIPTION
[0029] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.
[0030] This example mainly introduces a method for studying the regulation of gastrulation by extraembryonic lineages based on stably cultured extraembryonic mesodermal stem cells, including the following parts:
[0031] 1. Obtaining extraembryonic mesoderm cells by in vitro induction
[0032] Using TrypLE TM Express human pluripotent stem cells (PXGL or 5iLAF medium) were dissociated into single cells and then cultured at 2×10 5 cells / cm 2 Extraembryonic mesoderm cells were obtained by seeding cells at a density of 100 μg / mL on culture dishes coated with collagen IV and culturing them in ASECRiAV medium for 13-18 days. The cells were cultured at 37°C in 5% CO2.
[0033] For passaging, use TrypLE TM Express cells were dissociated into single cells and passaged to 5 μg ml at a split ratio of 1:3 to 1:6 every 3–5 days. -1 Collagen IV coated plates.
[0034] 2. Obtaining a stable culture system for extraembryonic mesodermal stem cells by adjusting the culture system
[0035] like Figure 1A 、 1B The present invention uses flow cytometry to sort BST2-positive cells, a characteristic gene of the extraembryonic mesoderm, and maintain them in a culture medium for extraembryonic mesoderm stem cells. The cells are then inoculated into Collagen I-coated cell dishes. Activin A and TGF-β1, which are the main signaling pathways for regulating extraembryonic lineage development, are added to the ASECRiAV system. The protein factors SB43 and A83, which are the corresponding factors in the basic culture system, are removed. WNT activator CHIR, inhibitors IM2 and IWR1 are added, and small molecule inhibitors PD03 and PD17 corresponding to FGF are added. Finally, the applicant screened out the addition of 20 ng ml -1 Activin A, 2ngml -1 Extraembryonic mesodermal stem cell culture system (e.g., TGF-β1 and 0.5 μM PD173074) Figure 2A 、 2B ). Cells were cultured at 37°C in 5% O2. For passaging, TrypLE TM Express cells were dissociated into single cells and passaged onto Collagen I-coated dishes at a split ratio of 1:3 to 1:6 every 3-5 days. 10 μM Y-27632 was added 1 day before each passage.
[0036] 3. Construction of a co-culture system of different extraembryonic lineages and embryonic stem cells
[0037] The extraembryonic mesodermal stem cells, trophoblast stem cells, and amnion-like cells were grouped into three groups: extraembryonic mesodermal stem cell group, trophoblast stem cell group, and amnion-like cell group. The extraembryonic mesodermal stem cell group, trophoblast stem cell group, and amnion-like cell group were co-cultured with human embryonic stem cells using a co-culture system to analyze the effects of extraembryonic mesodermal stem cells, trophoblast stem cells, and amnion-like cells on the formation of primitive streaks.
[0038] For example, the specific process of co-culturing the extraembryonic mesodermal stem cell group and human embryonic stem cells is as follows:
[0039] Human embryonic stem cells were cultured in mTeSR PLUS medium until approximately 80% confluence, dissociated into single cells or small clumps using ReleSR, and plated at a density of 1:20 on culture plates pre-coated with 0.5% Matrigel and cultured for 24 h to form adherent structures;
[0040] TrypLE TM After Express dissociation into single cells, the cells were seeded around human embryonic stem cells at a ratio of 3:1 between trophoblast stem cells and human embryonic stem cells.
[0041] The cells were co-cultured in APEL medium containing WNT activators for 1-3 days, and controls with or without CHIR were set up to evaluate the induction or inhibition of gastrulation.
[0042] Among them, the treatment method of extraembryonic mesoderm cells and amniotic-like cells is the same as above.
[0043] like Figure 3 Here, by constructing a co-culture system, the induction effect was systematically evaluated by first inoculating embryonic stem cells to form a sheet structure and then inoculating different numbers of amniotic-like cells. The inventors collected a variety of reported methods for inducing human amniotic-like cells in vitro, and induced and verified them. Amniotic-like cells can be derived from primed human naive pluripotent stem cells with TGFβ inhibition SB43 and addition of BMP4, and human naive pluripotent stem cells can also be induced into amniotic-like cells. All of these induced amniotic-like cells co-express amniotic-specific genes, such as ISL1, GABRP, and WNT6. Previous studies have shown that amniotic cells can act as a signal transduction hub for gastrulation.
[0044] Next, a co-culture system was used to study the effect of these amniotic-like cells on the occurrence of primitive streak movement. The results showed that no matter which pluripotent stem cells the amniotic-like cells came from, they were able to induce the occurrence of gastrulation cells in the absence of any exogenous factors. In addition, we observed that, unlike the gastrulation cells induced by the WNT pathway agonist CHIR, these amniotic-like cell-induced gastrulation cells maintained the compactness of the colony, appeared from the 24-hour co-culture and persisted for up to 72 hours, and were mainly located at the edge of the embryonic stem cell colony in contact with the gastrulation cells (such as Figure 4A ).
[0045] In contrast, unlike gastrulation cells, another type of extraembryonic cell, human blastocyst-derived trophoblast stem cells, did not exhibit the same inductive effect on embryonic stem cells. Even at high concentrations of CHIR, trophoblast stem cells not only failed to induce gastrulation cells but also hindered gastrulation cell induction (e.g., Figure 4A As a result, gastrulation cells were almost undetectable, demonstrating the unexpected inhibitory effect of trophoblast stem cells on embryonic stem cells. This inhibitory effect of trophoblast stem cells on gastrulation cells does not require direct cell-cell contact. Using a non-contact Transwell culture system, trophoblast stem cells were still able to inhibit the emergence of gastrulation cells under CHIR treatment (e.g., Figure 4B This suggests that trophoblast stem cells may regulate gastrulation induction by secreting certain factors. In addition, trophoblast stem cells and amnion-like cells do not affect the proliferation of embryonic stem cells.
[0046] Purified extraembryonic mesoderm stem cells were used to study the interaction between extraembryonic mesoderm stem cells and embryonic stem cells by co-culture. In the absence of CHIR, embryonic stem cells co-cultured with extraembryonic mesoderm stem cells did not show the expression of TBXT, a marker characteristic of gastrulation, or morphological changes (such as Figure 5A ). When CHIR was added to the co-culture system to induce TBXT expression in embryonic stem cells, the embryonic stem cells underwent significant cell migration. This occurred without changing the cell proliferation rate. Morphological changes of embryonic stem cells within 48 hours when co-cultured with extraembryonic mesoderm stem cells in the presence of CHIR. Cell migration began to appear at 3 hours, and the empty area gradually increased and expanded rapidly between 37-45 hours (as shown in Figure 2). Figure 5B). Over time, this migration led to the formation of blank areas within the embryonic stem cells. In addition, 3D assembly assays of embryonic stem cells and extraembryonic mesoderm stem cells showed that embryonic stem cells migrated into extraembryonic mesoderm stem cell spheres in the presence of CHIR, compared to the non-CHIR treated group, and the contact area and length increased since 12 hours after the cell aggregates were put together. Epithelial-mesenchymal transition (EMT) characterization was detected, and in the presence of CHIR, embryonic stem cells migrated to extraembryonic mesoderm stem cells faster than in the control group. Time-course immunostaining results showed that after 24 hours, gastrulation cells began to appear evenly within the embryonic stem cell colonies. Over time, these TBXT cells began to migrate out of the cell colony and move toward the extraembryonic mesoderm cells. By 72 hours, these gastrulation cells were mainly located at the leading edge of the migrating cell population. In addition, these migrating cells exhibited EMT characteristics and began to express N-cadherin (such as Figure 6 ).
[0047] It can be understood that the above specific description of the present invention is only used to illustrate the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents to achieve the same technical effects; as long as the use requirements are met, they are within the scope of protection of the present invention.
Claims
1. A research method based on the regulatory effect of extraembryonic tissues on gastrulation, characterized in that: The research methodology includes the following steps: Step 1: Establish an extraembryonic mesodermal stem cell culture system, and induce extraembryonic mesodermal stem cells in vitro through the extraembryonic mesodermal stem cell culture system; Step 2: Explore the regulatory effects of extraembryonic mesoderm stem cells, trophoblast stem cells, and amniotic-like cells on gastrulation.
2. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 1, characterized in that: Step 1: Extraembryonic mesodermal stem cell culture system includes: ASECRiAV medium + 20ng ml -1 Activin A+2ng ml -1 TGFβ1+0.5μM FGFR1 inhibitor.
3. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 2, characterized in that: The FGFR1 inhibitor is PD173074.
4. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 3, characterized in that: The specific operations of step 1 are as follows: Using TrypLE TM Human embryonic stem cells were dissociated into single cells by Express and then cultured at 2×10 5 cells / cm 2 The cells were seeded at a density of 100 μg / cm2 on a culture dish coated with collagen IV and cultured in an extraembryonic mesodermal stem cell culture system at 37°C and 5% CO2 for 13-18 days. Subculture using TrypLE TM Express extraembryonic mesodermal stem cells were dissociated into single cells and passaged onto Collagen I-coated plates at a split ratio of 1:3 to 1:6 every 3-5 days.
5. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 1, characterized in that: In step 2, a co-culture system is used to explore the regulatory effects of extraembryonic mesoderm stem cells, trophoblast stem cells, and amniotic-like cells on gastrulation. The co-culture system is an APEL medium that does not contain animal components or growth factors.
6. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 5, characterized in that: In step 2, the extraembryonic mesoderm stem cell group, trophoblast stem cell group, and amnion-like cell group were designed. The extraembryonic mesoderm stem cell group, trophoblast stem cell group, and amnion-like cell group were co-cultured with human embryonic stem cells using a co-culture system to analyze the effects of the extraembryonic mesoderm stem cells, trophoblast stem cells, and amnion-like cells on the formation of primitive streaks.
7. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 6, characterized in that: The specific cultivation process of step 2 is as follows: Human embryonic stem cells were cultured in mTeSR PLUS medium until approximately 80% confluence, dissociated into single cells or small clumps using ReleSR, and plated at a density of 1:20 on culture plates pre-coated with 0.5% Matrigel and cultured for 24 h to form adherent structures; Trophoblast stem cells, extraembryonic mesoderm stem cells, or amniotic-like cells were cultured with TrypLE TM After express dissociation into single cells, trophoblast stem cells, extraembryonic mesoderm stem cells, or amnion-like cells are seeded around human embryonic stem cells at a ratio of 3:1; The cells were co-cultured in APEL medium containing WNT activators for 1-3 days, and controls with or without CHIR were set up to evaluate the induction or inhibition of gastrulation.
8. The research method based on the regulatory effect of extraembryonic tissue on gastrulation according to claim 6, characterized in that: The expression levels of gastrulation marker genes in human embryonic stem cells were detected by immunofluorescence and flow cytometry, and the regulatory effects of extraembryonic mesoderm stem cells, trophoblast stem cells, and amniotic-like cells on gastrulation were evaluated.