Neural crest cell carrying specific head-tail axis marker gene and preparation method and application thereof

Abstract

The invention provides a neural crest cell carrying a specific head-tail axis marker gene and a preparation method and application thereof, and relates to the technical field of biology, and the preparation method comprises the following steps: culturing stem cells to form an embryoid body; performing nerve induced differentiation on the embryoid body under the gradient driving of morphogenetic factors to form neural crest cells carrying specific head-tail axis marker genes, and promoting the formation of a neural tube-like structure with the polarity of the head-tail axis and the dorsal-abdominal axis when the embryoid body enters the nerve induced differentiation beginning by the gradient driving of morphogenetic factors. And inducing to generate neural crest cells carrying position-specific marker genes. The technical problem that neural crest cells carrying specific head-tail axis marker genes cannot be formed by an induced differentiation method in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a neural crest cell carrying a specific head-tail axis marker gene and a preparation method and application thereof. BACKGROUND

[0002] At present, relevant literatures at home and abroad report that human stem cells are directly used to induce neural crest cells, but these neural crest cells do not have specificity. Some literatures also report that human stem cells are used to form neural tube head-tail axis and back-abdominal axis and scattered neural crest cells. However, a large number of animal experiments show that neural crests formed at different positions along the head-tail axis of the neural tube carry different marker genes, which represent the head-tail axis position information of the neural crest. These neural crest cells carrying different marker genes will form peripheral nerves, craniofacial bones and the like at different positions of the human body. The neural crest cells induced by directly using human stem cells do not have these characteristics. Although some literatures report that human stem cells are used to form neural tube head-tail axis and back-abdominal axis and scattered neural crest cells, there are not a large number of neural crest cells carrying specific marker genes.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] One of the purposes of the present application is to provide a preparation method of a neural crest cell carrying a specific head-tail axis marker gene, so as to solve the technical problem in the prior art that the induced differentiation method cannot form a neural crest cell carrying a specific head-tail axis marker gene.

[0005] The second purpose of the present application is to provide a neural crest cell carrying a specific head-tail axis marker gene.

[0006] The third purpose of the present application is to provide an application of the neural crest cell prepared by the above preparation method in preparing a cell preparation for treating peripheral nerve injury or craniofacial defect.

[0007] The fourth purpose of the present application is to provide an application of the neural crest cell prepared by the above preparation method in preparing a region-specific neural organoid or disease model.

[0008] In order to achieve the above purposes of the present application, the following technical solutions are adopted: In a first aspect, the present application provides a preparation method of a neural crest cell carrying a specific head-tail axis marker gene, comprising the following steps: A. A step of culturing pluripotent stem cells to form a blastoid; B. A step of performing neural induction differentiation on the blastoid cultured in step A under the gradient driving of morphogens to form a neural crest cell carrying a specific head-tail axis marker gene.

[0009] Further, the morphogen gradient comprises a head-tail axis direction diffusion gradient and a dorsal-ventral axis direction diffusion gradient, and the diffusion direction of the dorsal-ventral axis direction diffusion gradient is perpendicular to the diffusion direction of the head-tail axis direction diffusion gradient. The morphogen of the head-tail axis direction gradient comprises CHIR. The morphogen of the dorsal-ventral axis direction gradient comprises BMP4 and SHH.

[0010] Further, step B comprises: B1, inducing differentiation of the embryoid body into primitive neuroectoderm under the driving of the morphogen gradient; B2, inducing differentiation of the primitive neuroectoderm cultured in step B1 into early neural crest cells carrying specific head-tail axis marker genes under the driving of the morphogen gradient; B3, culturing the early neural crest cells to differentiate into neural crest cells carrying specific head-tail axis marker genes under the driving of the morphogen gradient.

[0011] Further, the primitive neuroectoderm has head-tail axis and dorsal-ventral axis polarity.

[0012] Further, the inducing differentiation in step B1 comprises using a three-dimensional neuroectoderm differentiation medium containing a dual SMAD inhibitor and a ROCK inhibitor to induce differentiation; Preferably, the three-dimensional neuroectoderm differentiation medium comprises KSR medium containing Matrigel. Preferably, the dual SMAD inhibitor comprises SB431542 and / or LDN193189. Preferably, the ROCK inhibitor comprises Y-27632.

[0013] Further, the inducing differentiation in step B2 comprises using a neural induction medium to gradually replace the three-dimensional neuroectoderm differentiation medium containing a dual SMAD inhibitor and a ROCK inhibitor, and the amount of the three-dimensional neuroectoderm induction differentiation system is gradually reduced from 100% to 25%; Preferably, the amount of the three-dimensional neuroectoderm induction differentiation system is reduced by 25% every 1-2 days. Preferably, the neural induction medium comprises NIM medium.

[0014] Further, the inducing differentiation in step B3 comprises using a neural induction medium containing an exogenous growth factor to induce differentiation; Preferably, the exogenous growth factor comprises FGF2 and / or EGF.

[0015] In a second aspect, the present application provides a neural crest cell carrying a specific head-tail axis marker gene, which is prepared by the preparation method described above. Preferably, the head-tail axis marker gene comprises HOX3-5.

[0016] In a third aspect, the present application provides use of the neural crest cell prepared by the preparation method described above in the preparation of a cell preparation for treating peripheral nerve injury or craniofacial defects.

[0017] In a fourth aspect, the present application provides use of the neural crest cell prepared by the preparation method described above in the preparation of a region-specific neural organoid or disease model.

[0018] The present application provides that the morphogen gradient drives the initiation of neural induction differentiation of the embryoid body, promotes the formation of a neural tube-like structure with head-tail axis and dorsal-ventral axis polarity, and induces the generation of neural crest cells carrying position-specific marker genes. The technical problem that the prior art induction differentiation method cannot form neural crest cells carrying specific head-tail axis marker genes is solved. BRIEF DESCRIPTION OF DRAWINGS In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0019] Figure 1 A morphogen diffusion gradient model structure is provided for example 1 of the present application; Figure 2 A mold for the three-dimensional craniofacial bone morphology is provided for example 2 of the present application. DETAILED DESCRIPTION

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of "or" means "and / or." Furthermore, the use of the term "including" as well as other forms such as "include," "includes," and "included" is non-limiting.

[0021] Unless otherwise indicated, the methods and techniques of the present application are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.

[0022] The technical solutions of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0023] In one aspect of the present application, a preparation method of neural crest cells carrying specific head-tail axis marker genes is provided, comprising the following steps: A, a step of culturing pluripotent stem cells to form a blastula; B, a step of inducing and differentiating the blastula cultured in step A into neural crest cells carrying specific head-tail axis marker genes under the driving of a morphogen gradient.

[0024] The morphogen gradient driving starts as soon as the blastula enters the neural induction differentiation stage, promotes the formation of a neural tube structure with head-tail axis and dorsal-ventral axis polarity, and induces the generation of neural crest cells carrying position-specific marker genes. The technical problem that the prior art induction differentiation method cannot form neural crest cells carrying specific head-tail axis marker genes is solved.

[0025] In some specific embodiments, the morphogen gradient comprises a head-tail axis direction diffusion gradient and a dorsal-ventral axis direction diffusion gradient, and the diffusion direction of the dorsal-ventral axis direction diffusion gradient is perpendicular to the diffusion direction of the head-tail axis direction diffusion gradient. The morphogen gradient is implemented based on a microfluidic chemotaxis chamber, and the morphogen is replaced once every 24 hours to ensure the concentration and gradient formed by the morphogen.

[0026] In some specific embodiments, the morphogen of the head-tail axis direction gradient comprises CHIR; and the morphogen of the dorsal-ventral axis direction gradient comprises BMP4 and SHH.

[0027] Among them, the CHIR factor promotes the formation of neural tube and neural crest head-tail axis, and the parallel diffusion direction of the BMP4 and SHH factors is perpendicular to CHIR, which promotes the formation of neural tube and neural crest dorsal-ventral axis, and the most important role of the BMP4 factor is to promote the closure of the neural tube.

[0028] In some specific embodiments, step B comprises: B1, inducing and differentiating the blastula into primitive neural ectoderm under the driving of the morphogen gradient; B2, inducing and differentiating the primitive neural ectoderm cultured in step B1 into early neural crest cells carrying specific head-tail axis marker genes under the driving of the morphogen gradient; and B3, culturing the early neural crest cells under the driving of the morphogen gradient to differentiate into neural crest cells carrying specific head-tail axis marker genes.

[0029] Step B1 is patterning culture, promoting formation of neural tube-like structures with clear head-tail axis and dorsal-ventral axis polarity. In some specific embodiments, the primitive neuroectoderm has head-tail axis and dorsal-ventral axis polarity.

[0030] In some specific embodiments, the inducing differentiation in step B1 comprises inducing differentiation using a three-dimensional neuroectoderm differentiation medium containing dual SMAD inhibitors and ROCK inhibitors. Dual SMAD inhibitors combined with ROCK inhibitors efficiently promote neuroectoderm formation. In some specific embodiments, the dual SMAD inhibitors comprise SB431542 and / or LDN193189; in some specific embodiments, the ROCK inhibitors comprise Y-27632.

[0031] In some specific embodiments, the three-dimensional neuroectoderm differentiation medium comprises KSR medium containing Matrigel. Matrigel is added to promote three-dimensional structure assembly and neural tube closure.

[0032] In some specific embodiments, the inducing differentiation in step B2 comprises inducing differentiation using a neural induction medium gradually replacing the three-dimensional neuroectoderm differentiation medium containing dual SMAD inhibitors and ROCK inhibitors, with the three-dimensional neuroectoderm induction differentiation system gradually reducing from 100% to 25%. Under the driving of morphogen gradient, neural crest cell formation is promoted by culture medium gradient conversion, and specific marker genes are expressed. In some specific embodiments, the three-dimensional neuroectoderm induction differentiation system is reduced by 25% every 1-2 days; in some specific embodiments, the neural induction medium comprises NIM medium.

[0033] In some specific embodiments, the inducing differentiation in step B3 comprises inducing differentiation using a neural induction medium containing exogenous growth factors. Exogenous growth factors promote neural precursor proliferation and survival and maintain their differentiation activity. In some specific embodiments, the exogenous growth factors comprise FGF2 and / or EGF.

[0034] According to another aspect of the present application, there is also provided a neural crest cell carrying specific head-tail axis marker genes, which is prepared by the above preparation method.

[0035] In some specific embodiments, the head-tail axis marker genes comprise HOX3-5.

[0036] According to another aspect of the present application, there is also provided use of the neural crest cell prepared by the above preparation method in preparation of a cell preparation for treating peripheral nerve injury or craniofacial defects.

[0037] According to another aspect of the present application, there is also provided use of the neural crest cells prepared by the method described above in the preparation of a region-specific neural organoid or disease model.

[0038] The application will be further described by way of examples. Unless otherwise stated, the materials in the examples were prepared according to existing methods or purchased directly from the market.

[0039] Equipment: microfluidic chemotaxis chamber (Ibiditreat m-slide chemotaxis chambers, Ibidi, Grafelfing, Germany).

[0040] Culture medium: mTeSR (STEMCELL Technologies, #100-0276); KSR medium: 15% KSR (Invitrogen), KO DMEM (Invitrogen), L-glutamine (Gibco), 100x non-essential amino acids (NEAA) (Gibco), 100x penicillin-streptomycin (Gibco), and 1000x β-mercaptoethanol (Gibco); Neural induction medium (NIM): DMEM / F12 (Invitrogen), 100x N2 supplement (Gibco), 50x B27 supplement without vitamin A (Life Technologies), 100x Glutamax (Gibco), 100x NEAA (Gibco), 100x penicillin-streptomycin (Gibco), 0.15% D-(+)-glucose solution (Sigma G8644).

[0041] Terminal neural differentiation medium (TD): Neurobasal (Gibco), 100x N2 supplement (Gibco), 50x B27 supplement without vitamin A (Life Technologies), 100x Glutamax (Gibco), 100x NEAA (Gibco), 100x penicillin-streptomycin (Gibco), 150x HEPES (Gibco), and 1000x β-mercaptoethanol (Gibco).

[0042] Example 1 A neural crest cell carrying a specific head-tail axis marker gene is differentiated from a human stem cell, and the operation is performed according to the following steps: 1. Preparation of embryoid bodies Human stem cell aggregates were dissociated into single cell suspension using Accutase (diluted 1 :2 with DPBS IX, SCR005). Four million cells were plated in a single well of a 6-well plate (Corning) with 4 mL of mTeSR medium containing Y-27632 (5 mM) and the culture plate was placed on an orbital shaker (Labstrong) at 95 rpm at 37°C, 5% CO2 to form embryoid bodies. Fresh medium was changed at 24 hours and 72 hours.

[0043] 2. Neural induction Neural induction was initiated at 96 hours after embryoid body formation (designated day 0) using KSR medium supplemented with SB431542 (10 mM), LDN193189 (1 mM) (referred to as dual SMAD inhibition) and Y-27632 (5 mM), Matrigel (4%, v / v) to induce differentiation into primitive neuroectoderm.

[0044] From day 5 to day 11, the embryoid bodies were subjected to a gradient adaptation from KSR medium to neural induction medium with continuous use of dual SMAD inhibitors: day 4 (100% KSR), day 7 (75% KSR medium + 25% NIM), day 9 (50% KSR medium + 50% NIM), day 11 (25% KSR medium + 75% NIM) to induce early neural crest cells carrying specific head-tail axis marker genes.

[0045] From day 13 to day 16, the culture system was maintained using 100% NIM supplemented with FGF2 (10 ng / ml) and EGF (10 ng / ml). Neural crest cells carrying specific head-tail axis marker genes were formed.

[0046] From day 17 onwards, the culture system was switched to TD medium supplemented with 10 nM BDNF, 10 nM GDNF and 200 mM ascorbic acid.

[0047] 3. Simultaneous use of construct morphogen diffusion gradient At day 0 (i.e. the first day of neural induction using dual SMAD inhibition in KSR medium in step 2), approximately 200 embryoid bodies (average diameter 230 pm) were collected into 1.5 ml centrifuge tubes, washed with DMEM / F12 and gently mixed with a p1000 pipette and custom hydrogel (1.5 mg / ml rat tail collagen I + 1% agarose) before injection through a needle into the culture area of each device (polymerisation occurs within approximately 2 minutes after mixing warm agarose and cold collagen). The pH of the hydrogel was tested and adjusted to 7.5 prior to the experiment.

[0048] AsFigure 1 As shown, 2 μΜ CHIR (CHIR99021, activator of WNT pathway) was added to reservoir one A, and 2 μg (100 nM) of 20 kDa protein SHH was added to reservoir two C. 5 ng / mL of BMP4 was added to reservoir three B. The medium in all reservoirs was changed every 24 hours to maintain the continuous concentration and gradient of BMP4, CHIR, and SHH. After the 5-day patterning, the organoids were cultured in the device for another 4 days and removed on day 9. CHIR promotes the formation of neural tube and neural crest head-tail axis, and the parallel diffusion direction of BMP4 and SHH is perpendicular to CHIR, which promotes the formation of neural tube and neural crest dorsal-ventral axis. The most important role of BMP4 is to promote the closure of the neural tube.

[0049] 4. Screening HOX-gene marked neural crest cells by single cell sequencing.

[0050] Example 2 HOX-gene marked neural crest cells prepared in Example 1 were selected, and the HOX-gene marked neural crest cells, which are formed from the neural tube of the head, can form craniofacial bone. The HOX-gene marked neural crest cells and the brain tissue from which they are generated were implanted together in a mold with a three-dimensional craniofacial bone shape, and the mold model is as shown in Figure 2 Then chondrogenesis was induced. The operation was performed according to the following steps: a. The HOX-gene marked neural crest cells and the brain tissue from which they are generated implanted in the mold were maintained in culture. In the first two weeks, the liquid was changed every other day: each time the old culture medium was discarded and 10 mL of fresh neural crest stem cell maintenance medium was added.

[0051] b. After two weeks, the liquid was changed three times a week (for example, on Monday, Wednesday, and Friday): on Monday and Wednesday, half the liquid was changed (50% of the old culture medium was discarded and an equal amount of fresh maintenance medium was added); and on Friday, the liquid was changed completely (all the old culture medium was discarded and 10 mL of fresh maintenance medium was added).

[0052] c. The above culture scheme needs to be continued for more than 90 days.

[0053] d. During this period, the cells will migrate, aggregate, and self-organize to form organoids, and this organoid will form a preliminary chondrocranial with a craniofacial bone shape according to the shape of the mold.

[0054] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing neural crest cells carrying specific head-tail axis marker genes, characterized in that, Includes the following steps: A. Steps for culturing stem cells to form embryoid bodies; B. The step of neurally inducing differentiation of embryoids cultured in step A into neural crest cells carrying specific head-tail axis marker genes under the drive of morphogen gradient.

2. The preparation method according to claim 1, characterized in that, The morphogenetic gradient includes a head-to-tail axis diffusion gradient and a dorsal-ventral axis diffusion gradient, wherein the diffusion direction of the dorsal-ventral axis diffusion gradient is perpendicular to the diffusion direction of the head-to-tail axis diffusion gradient. The morphogenetic elements of the head-to-tail axis diffusion gradient include CHIR; The morphogenetic elements of the dorsal-ventral axis diffusion gradient include BMP4 and SHH.

3. The preparation method according to claim 2, characterized in that, Step B includes: B1. Inducing embryoids to differentiate into primitive neuroectoderm under the drive of morphogen gradient; B2. The primitive neuroectoderm cultured in step B1 is induced to differentiate into early neural crest cells carrying specific head-tail axis marker genes under the drive of morphogenetic gradient. B3. Early neural crest cells are cultured under morphogen gradient-driven conditions to differentiate into neural crest cells carrying specific head-tail axis marker genes.

4. The preparation method according to claim 3, characterized in that, The primitive neuroectoderm has cephalo-tail axis and dorsal-ventral axis polarity.

5. The preparation method according to claim 3, characterized in that, The induction of differentiation in step B1 involves using a three-dimensional neuroectodermal differentiation medium containing both SMAD inhibitors and ROCK inhibitors to induce differentiation; Preferably, the three-dimensional neural ectoderm differentiation medium comprises KSR medium containing Matrigel; Preferably, the dual SMAD inhibitors comprise SB431542 and / or LDN193189; Preferably, the ROCK inhibitor includes Y-27632.

6. The preparation method according to claim 5, characterized in that, Step B2 involves gradually replacing the three-dimensional neuroectodermal differentiation medium containing dual SMAD inhibitors and ROCK inhibitors with a neural induction medium, with the amount of the three-dimensional neuroectodermal differentiation induction system gradually decreasing from 100% to 25%. Preferably, the dosage of the three-dimensional neuroectodermal differentiation induction system is reduced by 25% every 1-2 days; Preferably, the neural induction medium comprises NIM medium.

7. The preparation method according to claim 3, characterized in that, The induction of differentiation in step B3 includes inducing differentiation using a neural induction medium containing exogenous growth factors; Preferably, the exogenous growth factor includes FGF2 and / or EGF.

8. A neural crest cell carrying a specific head-tail axis marker gene, characterized in that, The neural crest cells are prepared by the preparation method according to any one of claims 1 to 7; Preferably, the head-tail axis marker genes include HOX3-5.

9. The use of neural crest cells prepared by the method according to any one of claims 1 to 7 in the preparation of cell preparations for treating peripheral nerve injury or craniofacial defects.

10. The use of neural crest cells prepared by the method according to any one of claims 1 to 7 in the preparation of region-specific neural organoids or disease models.

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