Preparation method and application of dorsal root ganglion organoid

Abstract

The invention relates to a preparation method and application of dorsal root ganglion organoid. On one hand, the invention provides the preparation method of the dorsal root ganglion organoid, which comprises the following steps: (1) inducing stem cells in a neural stem cell induction culture medium to obtain neural stem cells; (2) inducing the neural stem cells in a neural crest cell induction culture medium to obtain neural crest cells; (3) inducing the neural crest cells in a sensory nerve progenitor cell induction culture medium to obtain sensory nerve progenitor cells; and (4) inducing the sensory nerve progenitor cells in the organoid induction culture medium to obtain the dorsal root ganglion organoid. Moreover, the invention also provides application of the dorsal root ganglion organoid obtained by the method, for example, the dorsal root ganglion organoid is used for drug screening or disease research.

Description

Technical Field

[0001] This invention relates to a method for culturing organoid models, specifically a method for inducing stem cells to differentiate in vitro into three-dimensional dorsal root ganglion organoids, wherein the dorsal root ganglion organoids include proprioceptors, mechanoreceptors, various nociceptor subtypes and Schwann glial cells, and have specific physiological functions. Background Technology

[0002] Embryonic stem cells are self-replicating, pluripotent cells. In vivo, embryonic stem cells respond to external signals, forming ectoderm, mesoderm, and endoderm, subsequently differentiating into more than 220 cell types performing specific functions. By mimicking the activation or inhibition of in vivo signals, stem cells can also be induced in vitro into cell types with specific functions in different tissues. In recent years, in vitro 3D cultured organoid models have become a hot topic in the field of stem cell applications, widely used to simulate various tissue structures and physiological functions of the body, such as brain-like organs and spinal cord organoids, demonstrating significant value. Organoid models overcome the shortcomings of traditional animal and cell experiments in simulating real human tissues. Their three-dimensional structure allows for further simulation of cell interactions under changes in the microenvironment, greatly restoring the cellular composition and molecular characteristics of real tissues. Organoid models are widely used in disease pathogenesis, preclinical drug screening, and cell transplantation therapy.

[0003] The differentiation of sensory neurons is strongly dependent on external cues, allowing us to induce stem cells into specific sensory neurons by sequentially adding signaling molecules. Current dorsal root ganglion (DRG) organoid culture protocols (see, for example, Mazzara PG, et al., Nat Commun., 2020; Chambers SM, et al., Nat Biotechnol., 2012) are mainly derived from molecular induction protocols for 2D nociceptors. After creating stem cell aggregates, human stem cells are induced to generate sensory neurons using the same combination of small-molecule inhibitors as in 2D culture, and neurotrophic factors supporting sensory neuron survival are added later. DRG organoids obtained using this culture protocol show a certain degree of similarity to the dorsal root ganglion at the transcriptomic level, and immunostaining results show the expression of sensory neuron type marker proteins and spontaneous excitatory postsynaptic currents. However, compared to 2D culture protocols, the differentiation efficiency of sensory neurons in 3D organoids obtained using existing techniques is reduced.

[0004] Current dorsal root ganglion organoid culture protocols cannot efficiently and stably generate sensory neurons, lacking the external signals necessary for the development of proprioceptors and mechanoreceptors. Immunostaining results show a lack of clear morphology of sensory neurons in the organoids, and functional experiments are lacking to assess their sensory neuron identity and physiological specificity. Summary of the Invention

[0005] Technical problems to be solved

[0006] This invention provides a simpler and more efficient method for preparing dorsal root ganglia. By adding different inducing agents, the differentiation signaling pathway is precisely regulated, thereby simulating the in vivo development of dorsal root ganglia through in vitro 3D stem cell culture. This results in the stable generation of dorsal root ganglion organoids containing various types of sensory neurons and glial cells. The sensory neurons possess the classic large cell body characteristics of sensory neurons while exhibiting the diversity of somatic sensory neurons, including multiple cell types such as proprioceptors, mechanoreceptors, and nociceptors, and expressing various function-related genes as the molecular basis for sensory function. Furthermore, these sensory neurons possess specific physiological characteristics and respond physiologically to sensory stimuli. The glial cells express myelin proteins that surround the neuronal axons, serving as the structural basis for auxiliary sensory signal transmission.

[0007] Technical solution

[0008] The dorsal root ganglia, located within the vertebral foramina on the dorsal side of the vertebrae, develop from neural crest cells and are the site of aggregation of sensory neuron cell bodies. Sensory neurons respond to a range of environmental and internal somatic stimuli, forming the biological basis for the precise processing of somatic sensations. Neural crest cells, controlled by cascade signals, form cell types with a strict developmental sequence, forming the basis of neuronal diversity. First neurogenesis produces proprioceptors (NTRK3+) and mechanoreceptors (NTRK2+), while second neurogenesis primarily produces nociceptors (NTRK1+). The axonal terminals of proprioceptors connect to muscle spindles and Golgi tendon organs, aiding in proprioception, while the axonal terminals of mechanoreceptors extend to sensory receptors in the skin or deeper to detect mechanical stimuli. Nociceptors respond to temperature and chemical stimuli, forming the essential cellular basis for sensations of heat, cold, pain, and itching. Degeneration or dysfunction of sensory neurons in the dorsal root ganglia often leads to severe sensory neurological disorders.

[0009] Based on signaling pathways enriched in the neural crest, sensory progenitor cells, and unspecialized neurons obtained from single-cell transcriptome sequencing data of human embryonic dorsal root ganglia, this invention designs a novel preparation scheme for dorsal root ganglia organoids, as detailed below.

[0010] Pretreatment steps for stem cells: Culture the starting cells to obtain stem cell aggregates.

[0011] The starting cells are stem cells, specifically embryonic stem cells or induced pluripotent stem cells.

[0012] In this document, the embryonic stem cells are commercially obtained embryonic stem cells, preferably cells from any of the following NIH-numbered cell lines: BG01, BG02, BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ​​ES06, TE03, TE32, TE33, TE04, TE06, TE62, TE07, TE72, UC01, UC06, WA01 (or H1), WA07, WA09 (or H9), WA13, and WA14; or, the embryonic stem cells may be human embryonic stem cells, which may be stem cells isolated or obtained from human embryos that have not undergone in vivo development and are within 14 days of fertilization.

[0013] In this article, induced pluripotent stem cells (iPS cells) refer to pluripotent stem cells reprogrammed from terminally differentiated somatic cells through the introduction of specific transcription factors. In 2006, Shinya Yamanaka of Kyoto University in Japan first reported research on induced pluripotent stem cells in *Cell*. They cloned the genes of four transcription factors—Oct3 / 4, Sox2, c-Myc, and Klf4—into a viral vector and introduced it into mouse fibroblasts. They found that this induced transformation, and the resulting iPS cells were similar to embryonic stem cells in morphology, gene and protein expression, epigenetic modification status, cell proliferation capacity, ability to form germ-like bodies and teratomas, and differentiation capacity.

[0014] The preparation process of dorsal root neural crest organoids includes the following steps:

[0015] (1) The stem cell aggregates were induced in a neural stem cell induction medium to obtain neural stem cells, wherein the neural stem cells were SOX2+ / PAX6+ cells.

[0016] The neural stem cell induction culture medium contains ROCK inhibitors, TGF-β inhibitors, WNT agonists, and FGF pathway activators.

[0017] In a specific embodiment of the present invention, the ROCK inhibitor refers to a small molecule inhibitor that blocks or reduces the expression or function of the ROCK protein. The ROCK protein is a serine-threonine kinase that interacts with Rho GTPase. Exemplary small molecule ROCK inhibitors include Y-27632 (US Patent No. 4,997,834) and fasudil (also known as HA 1077; Asano et al., J. Pharmacol. Exp. Ther. 241:1033-1040, 1987), as well as RKI-1447, GSK429286A, H-1152, SLx-2119, TC-S 7001, etc. These inhibitors bind to the kinase domain to inhibit ROCK enzyme activity. Other small molecules that have been reported to specifically inhibit ROCK include N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, GSK269962A, and fasudil hydrochloride (Tocris Bioscience). Other small molecule ROCK inhibitors include those described in PCT publications WO 03 / 059913, WO 03 / 064397, WO05 / 003101, WO 04 / 112719, WO 03 / 062225, and WO 03 / 062227; U.S. Patents 7,217,722 and 7,199,147; and U.S. Patent Application Publications 2003 / 0220357, 2006 / 0241127, 2005 / 0182040, and 2005 / 0197328.

[0018] In a preferred embodiment of the invention, the ROCK inhibitor is Y-27632. Y-27632, also known as (+ / -)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, is a small molecule inhibitor that selectively inhibits the activity of Rho-related kinases. Y-27632 is disclosed in U.S. Patent No. 4,997,834 and PCT Publication No. WO 98 / 06433. In some embodiments, when the ROCK inhibitor is Y-27632, the effective amount of the ROCK inhibitor is 5-15 μM, or about 5-10 μM, or about 10 μM.

[0019] In a specific embodiment of the present invention, the TGF-β inhibitor can be any inhibitor as long as it inhibits the function of the transforming growth factor (TGF)-β receptor and includes inhibitors of TGF-β / Smad signaling, such as small molecules, antibodies, antisense compounds, and negative regulators of TGF-β / Smad signaling molecules.

[0020] Exemplary small molecule inhibitors of TGF-β / Smad signaling include, but are not limited to, A83-01, SB431542, LDN-193189, Galuniserib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox (E-616452), LDN-193189 2HCl, K02288, BIBF-0775, TP0427736 HCl, LDN-21 4117, SD-20 8, Vactose rti b (TEW-71 97), ML347, and LDN-21. 2854, DMH1, Dorsomorphin (compound C), 2HCl, Pirfenidone (S-7701), Sulfasalazine (NSC667219), AUDA, PD 169316, TA-02, ITD-1, LY 3200882, Alantolactone, Halofuginone, SIS3 HCl, Dorsomorphin (compound C), and Hesperetin.

[0021] In a preferred embodiment of the present invention, the TGF-β inhibitor is SB431542, and its effective amount is 5-15 μM, or 5-10 μM, or 10 μM.

[0022] In a specific embodiment of the present invention, the WNT agonist is preferably a small molecule substance that promotes the activation of the classical Wnt signaling pathway, including CHIR99021 and 6-bromoindirubin-3′-oxime(6BIO) (CAS No. 667463-62-9), preferably CHIR99021; its effective amount is 2-4 μM, preferably 3 μM.

[0023] In a specific embodiment of the present invention, the FGF pathway activator is selected from one or more of the group consisting of FGF2, FGF7, FGF8 and FGF10, preferably FGF2; its effective amount is 10-25 ng / ml, 10-30 ng / ml, 15-25 ng / ml or 10-20 ng / ml, preferably 20 ng / ml.

[0024] (2) Neural stem cells are induced in a neural crest cell induction medium to obtain neural crest cells, wherein the neural crest cells are SOX10+ cells;

[0025] The neural crest cell induction culture medium contains a TGF-β inhibitor and retinoic acid;

[0026] The definition and dosage of the TGF-β inhibitor are as described above;

[0027] The CAS number of the retinic acid is 302-79-4, and its dosage in this article is 50-150 nM, 50-120 nM or 50-100 nM, preferably 100 nM.

[0028] (3) The neural crest cells were induced in a sensory nerve progenitor cell induction medium to obtain sensory nerve progenitor cells, wherein the sensory nerve progenitor cells were SOX10+ISL1+ cells.

[0029] The sensory nerve progenitor cell induction culture medium contains retinoic acid;

[0030] The amount of retinoic acid used is 50-150 nM, 50-120 nM or 50-100 nM, preferably 100 nM;

[0031] (4) Sensory nerve progenitor cells were induced in organoid induction medium to obtain dorsal root ganglion organoids.

[0032] The organoid induction culture medium contains BDNF, GDNF, β-NGF and NT-3;

[0033] In a specific embodiment of the present invention, the dosage of BDNF (also known as brain-derived neurotrophic factor) (purchased from PeproTech, catalog number 450-02) is 10-20 ng / ml, 5-20 ng / ml, 8-20 ng / ml or 8-15 ng / ml, preferably 10 ng / ml.

[0034] In a specific embodiment of the present invention, the dosage of GDNF (also known as glial cellline-derived neurotrophic factor) (purchased from PeproTech, catalog number 450-10) is 10-20 ng / ml, 10-30 ng / ml, 15-25 ng / ml or 15-22 ng / ml, preferably 20 ng / ml;

[0035] In a specific embodiment of the present invention, the dosage of β-NGF (also known as β-Nerve Growth Factor) (purchased from PeproTech, catalog number 450-01) is 5-20 ng / ml, 10-20 ng / ml, 5-15 ng / ml or 5-12 ng / ml, preferably 10 ng / ml.

[0036] In a specific embodiment of the present invention, the dosage of NT-3 (also known as neurotrophin-3) (purchased from PeproTech, catalog number 450-03) is 10-20 ng / ml, 5-20 ng / ml, or 5-15 ng / ml, preferably 10 ng / ml.

[0037] In summary, the applicant induced stem cells into pluripotent neural crest cells by adding ROCK inhibitors such as Y27632, TGF-β inhibitors such as SB431542, WNT agonists such as CHIR99021, FGF pathway activators such as FGF2, and RA (retinoic acid) in the early stages. Then, the continuous addition of RA guided the neural crest cells to transition to the sensory nerve fate, generating sensory nerve progenitor cells. Further addition of NT-3, BDNF, GDNF, and βNGF supported the differentiation, survival, and maturation of sensory neurons, thus obtaining dorsal root ganglion organoids. During the induction process, the inventors detected the expression of characteristic genes of neural crest cells, sensory neuron progenitor cells, and sensory neurons at multiple time points and compared them with the actual developmental process to ensure the accuracy of the induction protocol. Immunofluorescence staining results demonstrated that the culture protocol of this invention can reproduce the developmental process of the dorsal root ganglion, simultaneously obtaining proprioceptor, mechanoreceptor, and multiple nociceptor subtypes of cells. Furthermore, a human-specific enriched nociceptor and Schwann glial cells supporting sensory signal transmission were captured in long-term cultured organoids. Finally, we examined the expression of functional genes and performed functional validation based on calcium imaging. Capsaicin stimulation experiments were used to assess the physiological characteristics of sensory neurons. The results indicate that the culture protocol of this invention can obtain organoid models with composition and physiological function approximating human dorsal root ganglion cells. The organoid culture protocol of this invention can be found in [reference needed]. Figure 1 The dorsal root ganglion organoids obtained by this invention have the following advantages compared with organoids obtained in the prior art:

[0038]

[0039]

[0040] By simulating the cascade signals during the development of real dorsal root ganglia, stem cell aggregates are progressively induced into functional dorsal root ganglion organoids containing various sensory neurons. The dorsal root ganglion organoid culture protocol has broad application value. Our model effectively replicates the dorsal root ganglion differentiation process, allowing for integration with gene editing technology to comprehensively analyze the association between sensory nerve diseases and the genome. Based on the obtained multiple sensory neuron subtypes, we can conduct drug screening experiments and regenerative medicine research. Simultaneously, we can customize organoid models for patients to analyze the causes of disease, formulate treatment plans, and achieve precision medicine. For example, the dorsal root ganglion organoids of this invention can be used for in vitro studies simulating the development of human dorsal root ganglia and the function of sensory neurons, for screening targets for sensory nervous system-related diseases, for screening drugs to prevent or treat sensory nervous system-related diseases, or as in vitro models for constructing sensory circuits for scientific research or basic clinical research.

[0041] Specifically, the present invention provides the following technical solutions:

[0042] 1. A method for preparing dorsal root ganglion organoids, comprising the following steps:

[0043] (1) The stem cells were induced in a neural stem cell induction medium to obtain neural stem cells, wherein the neural stem cells were SOX2+ / PAX6+ cells.

[0044] The neural stem cell induction culture medium contains ROCK inhibitors, TGF-β inhibitors, WNT agonists, and FGF pathway activators.

[0045] (2) Neural stem cells are induced in a neural crest cell induction medium to obtain neural crest cells, wherein the neural crest cells are SOX10+ cells;

[0046] The neural crest cell induction culture medium contains a TGF-β inhibitor and retinoic acid;

[0047] (3) The neural crest cells were induced in a sensory nerve progenitor cell induction medium to obtain sensory nerve progenitor cells, wherein the sensory nerve progenitor cells were SOX10+ISL1+ cells.

[0048] The sensory nerve progenitor cell induction culture medium contains retinoic acid;

[0049] (4) Sensory nerve progenitor cells were induced in organoid induction medium to obtain dorsal root ganglion organoids.

[0050] The organoid induction culture medium contains BDNF, GDNF, β-NGF and NT-3.

[0051] 2. The preparation method according to Project 1 or 2, wherein the stem cells are embryonic stem cells or induced pluripotent stem cells.

[0052] 3. According to the preparation method described in Project 1 or 2, wherein the neural stem cell induction medium, the neural crest cell induction medium and the sensory nerve progenitor cell induction medium each contain a basal medium, wherein the basal medium is N2B27+10%KSR basal medium.

[0053] 4. The preparation method according to Project 1 or 2, wherein the organoid induction culture medium comprises a basal culture medium, and the basal culture medium is N2B27 basal culture medium.

[0054] 5. The preparation method according to item 1 or 2, wherein, in step (1),

[0055] The ROCK inhibitors include Y27632, fasudil, RKI-1447, GSK429286A, H-1152, SLx-2119, or TC-S 7001;

[0056] The TGF-β inhibitors include SB431542, SB505124, SB525334, LY364947, or A8301;

[0057] The WNT agonist includes CHIR99021 or 6-bromoindirubin-3′-oxime (6BIO);

[0058] The FGF pathway activators include FGF2, FGF7, FGF8, or FGF10;

[0059] Preferably, the concentration of the ROCK inhibitor is 5-15 μM;

[0060] Preferably, the concentration of the TGF-β inhibitor is 5-15 μM;

[0061] Preferably, the concentration of the WNT agonist is 2-4 μM;

[0062] Preferably, the concentration of the FGF pathway activator is 10-30 ng / ml.

[0063] 6. The preparation method according to item 1 or 2, wherein, in step (2), the TGF-β inhibitor includes SB431542, SB505124, SB525334, LY364947, and A8301;

[0064] Preferably, the concentration of the TGF-β inhibitor is 5-15 μM;

[0065] Preferably, the concentration of retinoic acid is 50-150 nM;

[0066] 7. The preparation method according to item 1 or 2, wherein, in step (3), the concentration of retinoic acid is 50-150 nM;

[0067] 8. The preparation method according to item 1 or 2, wherein in step (4), the concentration of BDNF is 5-20 ng / ml, the concentration of GDNF is 10-30 ng / ml, the concentration of β-NGF is 5-20 ng / ml, and the concentration of NT-3 is 5-20 ng / ml.

[0068] 9. The dorsal root ganglion organoid obtained by any one of the preparation methods in items 1-8.

[0069] 10. Uses of the dorsal root ganglion organoids described in Project 9, for example, for drug screening or disease research.

[0070] 11. A kit, preferably for differentiating stem cells into dorsal root ganglion organoids, comprising neural stem cell induction medium, neural crest cell induction medium, sensory nerve progenitor cell induction medium, and organoid induction medium;

[0071] Preferably, the neural stem cell induction culture medium contains a ROCK inhibitor, a TGF-β inhibitor, a WNT agonist, and an FGF pathway activator;

[0072] The neural crest cell induction culture medium contains a TGF-β inhibitor and retinoic acid;

[0073] The sensory nerve progenitor cell induction culture medium contains retinoic acid;

[0074] The organoid induction culture medium contains BDNF, GDNF, β-NGF and NT-3.

[0075] Beneficial effects

[0076] This invention provides a concise and stable method for preparing dorsal root ganglion organoids containing multiple sensory neuron types. Our method references signaling pathways enriched at different cellular developmental stages obtained from sequencing real human embryonic dorsal root ganglia. By simulating the signal cascades during real development in vitro, it induces the generation of neural crest cells and their differentiation into sensory neurons and glial cells. Immunohistochemical staining reveals that the dorsal root ganglion organoids of this invention possess cellular composition and morphology similar to those in real tissues, including proprioceptors, mechanoreceptors, multiple nociceptor subtypes, and glial cells, expressing various sensory function-related genes. Furthermore, calcium imaging demonstrates that the organoid model of this invention exhibits spontaneous calcium activity and capsaicin activation, proving its physiological function similar to that of real sensory neurons.

[0077] The preparation method of this invention generates dorsal root ganglion organoids containing multiple sensory neuron types more efficiently and stably, realizing the in vitro simulation of the development process of dorsal root ganglia in real human embryos, inducing the acquisition of neural crest cells and inducing them to differentiate into sensory neurons and glial cells, producing sensory neurons with similar composition and morphology to those in real tissues, including proprioceptors, mechanoreceptors, and multiple nociceptor subtypes. These neurons express their own specific sensory function genes and have the physiological characteristics of capsaicin activation. Attached Figure Description

[0078] Figure 1 A flowchart of the culture protocol for dorsal root ganglion organoids is shown, in which the addition of corresponding signaling molecules at different time points can induce stem cell aggregates into dorsal root ganglion organoids containing various sensory neurons and glial cells.

[0079] Figure 2The development of dorsal root ganglion organoids and the cell types they contain are shown. A shows the emergence of abundant neural stem cells (SOX2+ / PAX6+) in the organoids two days after induction; B shows the timeline of the emergence of major cell types during dorsal root ganglion organoid development, with abundant neural crest cells (SOX10+) induced on day 5 of culture, sensory progenitor cells (SOX10+ / ISL1+) induced on day 14, and sensory neurons (ISL1+) induced on day 30; C shows the types of sensory neurons (PRPH+) emerging on day 30 of culture, including... Nociceptors (NTRK1+), proprioceptors (NTRK3+), and mechanoreceptors (NTRK2+); D shows the functional genes expressed by sensory neurons (SCN10A+, SCN11A+) in dorsal root ganglion organoids cultured for 50 days; E shows the functional genes expressed by sensory neurons (RET+, P2RX3+) in dorsal root ganglion organoids cultured for 90 days, as well as a human-specific enriched nociceptor subtype (DCC+ / NTRK1+ / NTRK3+); F shows glial cells with MBP+ in dorsal root ganglion organoids cultured for 150 days.

[0080] Figure 3 The images show calcium activity signals in neurons of the dorsal root ganglion. A shows the spontaneous electrical activity of sensory neurons cultured for 40-50 days; B shows the activation properties of sensory neurons cultured for 40-50 days by capsaicin, indicating that these neurons are nociceptive neurons.

[0081] Figure 4 The study revealed that cell types and gene expression during the development of dorsal root ganglion organoids are highly similar to those in human dorsal root ganglia. Specifically, A and C show the cell types and sensory neuron subtypes in the DRG organoids; B shows the changes in the proportions of sensory progenitor cells, neurons, and glial cells in the organoids at days 30 and 60, with a decrease in progenitor cells and an increase in glial cells compared to day 30, indicating further cell differentiation; D shows the characteristic gene expression of cells at different developmental stages in the organoids; E shows that the sensory neuron subtypes contained in the organoids express multiple sensory function-related genes; F shows the integration results of organoid single-cell sequencing and human embryonic DRG sequencing data, demonstrating that our culture protocol can induce stem cells into neural crest cells in vitro and further differentiate into multiple sensory neuron subtypes; G and H show the transcriptomic correlations between organoids and human embryonic DRG at four cellular developmental windows, indicating a high correlation between the organoid transcriptome and the 7-15 week human embryonic DRG.

[0082] Figure 5This demonstrates that the organoids of the present invention can be used to investigate the effects of multiple genes on the differentiation of sensory neuron subtypes. Among them, Figure 5 The left figure of C and Figure 5 A shows that knocking down the expression of transcription factors MEIS2 and SKOR2 can reduce the number of mechanoreceptor cells and proprioceptor cells, respectively. Figure 5 Figures C and 5B show that knocking down the transcription factor FOXO1 reduces the number of nociceptors; Figures 5D and 5E show that knocking down MEIS2 and SKOR2 does not affect the number of nociceptors, while knocking down FOXO1 does not affect the number of mechanoreceptors and proprioceptors; Figures 5F and 5G show that adding MEISi-2 reduces the number of mechanoreceptors and proprioceptors, but does not affect the number of nociceptors. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0084] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0085] Example 1: Stem cell 3D in vitro culture and differentiation to form dorsal root ganglion organoids

[0086] Stem cell pretreatment steps:

[0087] H9 human embryonic stem cells (purchased from WiCell) were selected as the starting cells and cultured in stem cell culture medium. When the stem cell confluence reached 70%-80%, the cells underwent pretreatment for organoid preparation, as detailed below. 10 μM Y27632 was added to the stem cell culture medium to reduce apoptosis. After one hour, the culture medium was removed, and residual medium was washed away using DMEM / F12. 1 ml Accutase was added, and the cells were digested at 37°C for 6-8 min. When the cell clones were digested into single-cell states, 5 ml DMEM / F12 was added to terminate the reaction. The cells were collected in 15 ml centrifuge tubes, centrifuged at 800 rpm for 5 min, the supernatant was removed, and E6 culture medium was added to mix the cells. The single-cell suspension was transferred to 96-well low-adsorption culture plates, with 9 × 10⁶ cells per well. 3 Individual cells were placed in a 37°C, 5% CO2 incubator, and stem cell aggregates formed after 24 hours.

[0088] Preparation process of dorsal root ganglion organoids

[0089] (1) The stem cell aggregates obtained above are induced in neural stem cell induction medium. The first day of induction is Day 0. Neural stem cells are obtained by adding neural stem cell induction medium, wherein the neural stem cells are SOX2+ / PAX6+ cells.

[0090] The neural stem cell induction culture medium contains a ROCK inhibitor (Y27632, 10 μM, is used in this embodiment as an example), a TGF-β inhibitor (SB431542, 10 μM, is used in this embodiment as an example), a WNT agonist (CHIR99021, 3 μM, is used in this embodiment as an example), and an FGF pathway activator (FGF2, also known as human fibroblast growth factor 2, 20 ng / ml, is used in this embodiment as an example).

[0091] The neural stem cell induction medium includes a basal medium, which is N2B27+10%KSR basal medium.

[0092] The N2B27+10%KSR basal medium contains 45% Neurobasal Medium + 45% DMEM / F-12 + B27 without VA + N2 + 10% serum substitute KSR + Glutamax + non-essential amino acids (NEAA) + β-mercaptoethanol (β-ME) + Penicillin-Streptomycin Solution.

[0093] In this embodiment, the time for this step is induction culture Day 0-Day 2;

[0094] (2) Neural stem cells are induced in a neural crest cell induction medium to obtain neural crest cells, wherein the neural crest cells are SOX10+ cells;

[0095] The neural crest cell induction culture medium contains a TGF-β inhibitor (SB431542, 10 μM, is used exemplary in this embodiment) and Retinoic Acid (100 nM).

[0096] The neural crest cell induction culture medium includes a basal culture medium, which is N2B27+10% KSR basal culture medium.

[0097] The N2B27+10%KSR basal medium contains 45% Neurobasal Medium + 45% DMEM / F-12 + B27 without VA + N2 + 10% serum substitute KSR + Glutamax + non-essential amino acids (NEAA) + β-mercaptoethanol (β-ME) + Penicillin-Streptomycin Solution.

[0098] In this embodiment, the time for this step is Day 3-Day 5 of the induction culture;

[0099] (3) The neural crest cells obtained in step (2) are induced in a sensory nerve progenitor cell induction medium to obtain sensory nerve progenitor cells, wherein the sensory nerve progenitor cells are SOX10+ISL1+ cells.

[0100] The sensory nerve progenitor cell induction culture medium contains retinoic acid (RA, concentration 100 nM).

[0101] The sensory nerve progenitor cell induction medium includes a basal medium, which is N2B27+10% KSR basal medium.

[0102] The N2B27+10%KSR basal medium contains 45% Neurobasal Medium + 45% DMEM / F-12 + B27 without VA + N2 + 10% serum substitute KSR + Glutamax + non-essential amino acids (NEAA) + β-mercaptoethanol (β-ME) + Penicillin-Streptomycin Solution.

[0103] In this embodiment, the time for this step is Day 6-Day 14 of the induction culture;

[0104] (4) Sensory nerve progenitor cells were transferred into 24-well plates and induced in organoid induction medium to obtain dorsal root ganglion organoids.

[0105] The organoid induction culture medium contains BDNF (brain-derived neurotrophic factor, concentration 10 ng / ml), GDNF (glial cell line-derived neurotrophic factor, concentration 20 ng / ml), β-NGF (beta-Nerve Growth Factor, concentration 10 ng / ml), and NT-3 (neurotrophin-3, concentration 10 ng / ml).

[0106] The organoid induction culture medium includes a basal culture medium, which is N2B27 basal culture medium.

[0107] The N2B27 basal medium contains 50% Neurobasal medium + 50% DMEM / F-12 medium + B27 without VA + N2 + Glutamax + NEAA + β-ME + Penicillin-Streptomycin Solution;

[0108] Step (4) can be divided into two processes:

[0109] First, the sensory progenitor cells are induced to produce sensory neurons, i.e., a large number of different types of sensory neurons (ISL1+ / PRPH+), including nociceptive cells (ISL1+ / PRPH+NTRK1+), proprioceptive cells (ISL1+ / PRPH+NTRK3+), and mechanoreceptor cells (ISL1+ / PRPH+NTRK2+). This process usually occurs during Day 15-Day 30 of the induction culture.

[0110] Subsequently, the sensory neurons further differentiate into organoids including various sensory neuron subtypes. For example, on Day 50, genes related to nociception function (SCN10A+ / SCN11A+) are expressed; on Day 90, nociceptor subtype cells (P2RX3+ / RET+) and a human-specific enriched nociceptor (DCC+ / NTRK3+ / NTRK1+) are generated; and on Day 150 or Day 100, glial cells (MBP+) are generated. This process usually occurs after Day 30 of the induction culture, for example, Day 30-Day 90 or Day 30-Day 100. That is, obtaining glial cells (MBP+) marks the end of this stage, thus obtaining the dorsal root ganglion organoid of the present invention.

[0111] In the above-mentioned induction culture steps (1)-(3), the culture medium is changed every 24 hours. At each time point, the appearance of the target cell type is identified. If it appears, the next stage of induction is initiated, and the culture time is appropriately extended or shortened depending on the cell line.

[0112] In step (4) of the above-mentioned induction culture, the induction conditions are conventional conditions, such as culturing in a cell culture incubator at 37°C, 5% CO2, and 40% O2, and changing the culture medium every 2-3 days.

[0113] Furthermore, after obtaining glial cells (MBP+), the dorsal root ganglion organoids prepared as described above can still be maintained in the culture medium and retain the effects of the dorsal root ganglion organoids of the present invention for Day 150.

[0114] Example 2: Immunofluorescence staining to identify the DRG organoid differentiation process

[0115] (1) Preparation of frozen sections: Cells obtained at different times during the induction process in Example 1 (usually in the form of cell aggregates) were collected, fixed with 4% paraformaldehyde (PFA) at 4°C for 4 hours, washed with 1×PBS, and then incubated with 30% sucrose at 4°C overnight. The fluid around the organoids was aspirated, embedded in the embedding medium OCT (Tissue-Tek, 4583), flash-frozen on dry ice, and stored at -80°C for long-term storage. Tissue sections of 20 μm were prepared using a cryostat, attached to anti-detachment slides, and stored at -20°C for long-term storage.

[0116] (2) Immunofluorescence staining: Wash away the embedding agent OCT with 1×PBS, perform antigen retrieval using antigen retrieval solution (0.01M sodium citrate solution, pH=6.0), add 0.1% Triton X-100 in 1×PBS to increase cell membrane permeability for staining for 15 min. Add 10% serum (Jackson IR, 017-000-121) for blocking for 1 h, remove the blocking solution, add primary antibody (the sources of the primary antibodies NTRK1, NTRK3, NTRK2, ISL1, MBP, PRPH, RET, DCC, and SOX10 are shown in Table 1), and incubate overnight at 4℃. The next day, remove the primary antibody, wash three times with 1×PBS for 10 min each time, then add secondary antibody (all secondary antibodies were purchased from Invitrogen, their specific catalog numbers are shown in Table 1) and DAPI, incubate at room temperature for 2 h, and wash three times with 1×PBS for 10 min each time. Add the mounting medium and cover with a coverslip.

[0117] Table 1: Antibodies involved in immunofluorescence staining

[0118]

[0119]

[0120] (3) Use laser confocal imaging. The results showed that Day 2 generated a large number of neural stem cells (SOX2+ / PAX6+); Day 5 generated a large number of neural crest cells (SOX10+); Day 14 generated sensory progenitor cells (SOX10+ and ISL1+); Day 30 differentiated into a large number of different types of sensory neurons (ISL1+ / PRPH+), including nociceptors (ISL1+ / PRPH+NTRK1+), proprioceptors (ISL1+ / PRPH+NTRK3+), and mechanoreceptors (ISL1+ / PRPH+NTRK2+); Day 50 expressed genes related to nociceptive function (SCN10A+ / SCN11A+); Day 90 generated nociceptor subtypes (P2RX3+ / RET+) and a human-specific enriched nociceptor (DCC+ / NTRK3+ / NTRK1+); and Day 100 and Day 150 had glial cells (MBP+).

[0121] Figure 2 This study demonstrates key time points and characteristic gene expression during organoid development. Specifically, A shows the emergence of abundant neural stem cells (SOX2+ / PAX6+) in organoids two days after induction; B shows the time points of major cell types emerging during dorsal root ganglion organoid development: abundant neural crest cells (SOX10+) are induced in organoids on day 5 of culture, sensory progenitor cells (SOX10+ / ISL1+) are induced on day 14, and sensory neurons (ISL1+) are induced on day 30; C shows the sensory neuron (PRPH+) type emerging on day 30 of culture. The images show nociceptors (NTRK1+), proprioceptors (NTRK3+), and mechanoreceptors (NTRK2+); D shows the functional genes expressed by sensory neurons (SCN10A+, SCN11A+) in dorsal root ganglion organoids cultured for 50 days; E shows the functional genes expressed by sensory neurons (RET+, P2RX3+) in dorsal root ganglion organoids cultured for 90 days, as well as a human-specific enriched nociceptor subtype (DCC+ / NTRK1+ / NTRK3+); F shows glial cells with MBP+ in dorsal root ganglion organoids cultured for 150 days.

[0122] Example 3: Calcium Imaging to Identify Sensory Neuron Function

[0123] (1) Spontaneous calcium signal: Two weeks prior to imaging, organoids obtained in Example 1 were injected with a calcium virus (BrainCase, BC-0077) for labeling. Before imaging, the organoids were placed in culture dishes and perfused for 30 min with oxygenated artificial cerebrospinal fluid aCSF (125.0 mM NaCl, 26.0 mM NaHCO3, 2.5 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 1.25 mM NaH2PO4 at pH 7.4). Calcium imaging views were acquired at 5 frames per second using a two-photon microscope (LSM980NLO, ZEISS) and a 20 x 1.0 NA water objective (2.4 mm WD). The raw recorded data were analyzed using Fiji, ROIs were manually plotted to obtain fluorescence intensity, and the fluorescence change rate ΔF / F was calculated. base =(FF base ) / (F base -F background The response to the stimulus was defined as an increase of ΔF / Fbase > 5 SD from baseline. The procedures for calcium imaging and fluorescence intensity analysis can be found in existing techniques, such as Yang D, et al. (DOI:10.1016 / j.cell.2022.09.024). Calcium signal analysis results are shown below. Figure 3 A shows the spontaneous calcium activity of sensory neurons in organoids.

[0124] (2) Capsaicin activation of sensory neurons: For the capsaicin stimulation experiment, organoids obtained in Example 1 were first used, and baseline data were recorded for 4 minutes. Then, 1 μM capsaicin was added for 5 minutes to stimulate neurons, followed by elution with aCSF. After elution for 10 minutes, 40 mM KCl was added for 30 seconds, and then eluted with aCSF again. For the control group experiment, capsaicin stimulation was replaced with carrier (DMSO) stimulation. Changes in calcium signal in neurons during capsaicin stimulation, carrier stimulation, KCl stimulation, and elution were recorded. The results are shown in […]. Figure 3 B.

[0125] In conclusion, Figure 3 The results of calcium imaging show that sensory neurons in DRG organoids have spontaneous calcium activity and can be specifically activated by capsaicin, exhibiting physiological characteristics similar to those in the human body.

[0126] Example 4: Single-cell transcriptome analysis showed the similarity between in vitro differentiation and in vivo development of dorsal root ganglion organoids.

[0127] To investigate the similarity between the in vitro differentiation process and gene expression of dorsal root ganglion organoids and the development of real DRGs, we performed single-cell transcriptome sequencing analysis on dorsal root ganglion organoids cultured for 30 and 60 days. Through comparison of transcriptome data, we further clarified the expression of various characteristic and functional genes, including neural crest cells, neural progenitor cells, Schwann precursor cells, and various sensory neurons. By integrating the single-cell transcriptome data of human embryonic DRG development (GSE245310) in the publicly available Gene Expression Omnibus database (https: / / www.ncbi.nlm.nih.gov / geo / ), we found that DRG organoids can simulate the developmental process of human DRGs in vitro, demonstrating the feasibility of our constructed DRG organoid culture protocol.

[0128] Figure 4 Single-cell sequencing data of organoids cultured for 30 and 60 days were presented, showcasing cell types and gene expression profiles in DRG organoids, demonstrating the high similarity between RG organoids and human DRGs in cellular composition and gene expression characteristics. Among these, in Figure 4 In the diagram, A and C show the cell types and sensory neuron subtypes in DRG organoids; B shows the changes in the proportions of sensory progenitor cells, neurons, and glial cells in Day 30 and Day 60 organoids; compared to Day 30 organoids, Day 60 organoids show a decrease in sensory progenitor cells and an increase in glial cells, indicating further differentiation of cells in organoids; D shows the characteristic gene expression of cells at different developmental stages in organoids; E shows the expression of multiple sensory function-related genes by sensory neuron subtypes contained in organoids; F shows the integration results of organoid single-cell sequencing and human embryonic DRG sequencing data, which demonstrate that our culture protocol can induce stem cells into neural crest cells in vitro and further differentiate into multiple sensory neuron subtypes; G and H show the transcriptomic correlations of organoids and human embryonic DRG at four cell development windows, indicating a high correlation between the organoid transcriptome and GW7-GW15 human embryonic DRG.

[0129] Example 5: Application of dorsal root ganglion organoids to study gene function in dorsal root ganglion development

[0130] Because different transcription factors contribute differently to sensory neuron subtypes during human embryonic sensory neuron differentiation (see DOI:10.1016 / S0959-4388(99)00015-X), this invention, through single-cell sequencing and pedigree analysis of human embryonic DRGs, found that MEIS2 and SKOR2 may specifically participate in the differentiation of proprioceptors and mechanoreceptors (NTRK3 / NTRK2), while FOXO1 may specifically participate in the differentiation of nociceptors. Based on this, this embodiment uses dorsal root ganglion organoids obtained by this invention to verify the roles of the above three genes FOXO1, MEIS2, and SKOR2 in dorsal root ganglion development.

[0131] Specifically, lentiviruses were constructed using the pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE plasmid (wherein, the pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE plasmid and the lentivirus construction method are conventional techniques in the prior art, for example, see DOI:10.1073 / pnas.0407976101), to knock down target transcription factors TFs (FOXO1, MEIS2, and SKOR2, respectively). The shRNA sequences used to knock down FOXO1, MEIS2, and SKOR2 are as follows: 5'-CGTGCCCTACTTCAAGGATAA-3' (SEQ ID NO:1, which is the shRNA sequence corresponding to FOXO1), 5'-GAGCCAAGGAGCAGCATATAG-3' (SEQ ID NO:2, which is the shRNA sequence corresponding to MEIS2), and 5'-TGATGCTTCTGGAGGAGATTT-3' (SEQ ID NO:1). NO:3 (the shRNA sequence corresponding to SKOR2). H9 cells (purchased from WiCell) with 50% confluence were infected with the lentivirus containing shRNA constructed above, incubated at 37°C for 96 h, and then placed in selective medium containing 1 μg / ml puromycin. Puromycin-resistant clones were cultured from single cells. Total RNA was isolated from the cells using Trizol (Ambion, 15596018). Reverse transcription was performed using HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, R333-01). Quantitative real-time PCR was performed on a Quantstudio 6Flex Real-Time PCR system using a ChamQ SYBR Color qPCR MasterMix (High ROX Premixed) (Vazyme, Q441-02). The primer sequences for FOXO1, MEIS2, and SKOR2 in the PCR are shown in Table 2.

[0132] Table 2: qPCR primers

[0133]

[0134] In this embodiment, the function of these transcription factors was verified using organoids obtained by the present invention. Lentiviral viruses were constructed for transcription factor knockdown, and the organoids were infected with the lentivirus on day 15. The differentiation results of sensory neuron subtypes were statistically analyzed on day 30. The results showed that knockdown of MEIS2 and SKOR2 reduced the number of proprioceptors and mechanoreceptors, while knockdown of FOXO1 reduced the number of nociceptors, indicating that these transcription factors play a crucial role in the differentiation of different sensory neuron subtypes. In addition, an inhibitor of MEIS2 (MEISi-2, CAS No. 2250156-71-7) was added for further functional verification. The results showed that the number of proprioceptors and mechanoreceptors was reduced in organoids with added MEISi-2, further verifying that MEIS2 participates in the differentiation of proprioceptor and mechanoreceptor cells (NTRK3 / NTRK2).

[0135] Figure 5 This study demonstrates the validation of transcription factors that influence the production of sensory neuron subtypes in DRG organoids, serving as an application example of DRG organoids in exploring the transcriptional regulation of DRG development.

[0136] In summary, this invention provides a DRG organoid and its preparation method. The organoid, by mimicking the DRG development process, forms a 3D tissue similar to DRG cells, including various sensory neurons and glial cell subtypes, exhibiting sensory physiological characteristics. This provides an important research tool for exploring the development and function of the dorsal root ganglion. DRG organoids can replicate tissue development in vitro, allowing for the investigation of cell lineage canonical processes and transcriptional regulation mechanisms. The various functional sensory neurons contained within can be used for disease mechanism research, potential drug target analysis, and clinical drug development. Furthermore, constructing DRG organoids from the patient's own tissue allows for precise simulation of pathogenic characteristics, enabling precision medicine, organ regeneration, and the repair of functional impairments.

[0137] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a dorsal root ganglion organoid, comprising the following steps: (1) inducing stem cells in a neural stem cell induction medium to obtain neural stem cells, wherein the neural stem cells are SOX2+ / PAX6+ cells; wherein the neural stem cell induction medium comprises a ROCK inhibitor, a TGF-β inhibitor, a WNT agonist, and an FGF pathway activator; (2) inducing the neural stem cells in a neural crest cell induction medium to obtain neural crest cells, wherein the neural crest cells are SOX10+ cells; wherein the neural crest cell induction medium comprises a TGF-β inhibitor and retinoic acid; (3) inducing the neural crest cells in a sensory neural progenitor cell induction medium to obtain sensory neural progenitor cells, wherein the sensory neural progenitor cells are SOX10+ISL1+ cells; wherein the sensory neural progenitor cell induction medium comprises retinoic acid; (4) inducing the sensory neural progenitor cells in an organoid induction medium to obtain a dorsal root ganglion organoid; wherein the organoid induction medium comprises BDNF, GDNF, β-NGF, and NT-3.

2. The production method according to claim 1 or 2, wherein, The stem cells are embryonic stem cells or induced pluripotent stem cells.

3. The production method according to claim 1 or 2, wherein The neural stem cell induction medium, the neural crest cell induction medium, and the sensory neural progenitor cell induction medium each comprise a basal medium, and the basal medium is an N2B27+10% KSR basal medium.

4. The production method according to claim 1 or 2, wherein The organoid induction medium comprises a basal medium, and the basal medium is an N2B27 basal medium.

5. The production method according to claim 1 or 2, wherein In step (1), the ROCK inhibitor comprises Y27632, fasudil, RKI-1447, GSK429286A, H-1152, SLx-2119, or TC-S 7001; the TGF-β inhibitor comprises SB431542, SB505124, SB525334, LY364947, or A8301; the WNT agonist comprises CHIR99021 or 6-bromoindirubin-3'-oxime (6BIO); the FGF pathway activator comprises FGF2, FGF7, FGF8, or FGF10; Preferably, the concentration of the ROCK inhibitor is 5-15 μM; Preferably, the concentration of the TGF-β inhibitor is 5-15 μM; Preferably, the concentration of the WNT agonist is 2-4 μM; Preferably, the concentration of the FGF pathway activator is 10-30 ng / ml.

6. The production method according to claim 1 or 2, wherein In step (2), the TGF-β inhibitor comprises SB431542, SB505124, SB525334, LY364947, A8301; Preferably, the concentration of the TGF-β inhibitor is 5-15 μM; Preferably, the concentration of the retinoic acid is 50-150 nM.

7. The production method according to claim 1 or 2, wherein In step (3), the concentration of the retinoic acid is 50-150 nM.

8. The production method according to claim 1 or 2, wherein In step (4), the concentration of BDNF is 5-20 ng / ml, the concentration of GDNF is 10-30 ng / ml, the concentration of β-NGF is 5-20 ng / ml, and the concentration of NT-3 is 5-20 ng / ml.

9. The dorsal root ganglion organoid obtained by the preparation method of any one of claims 1-8.

10. Use of the dorsal root ganglion organoid of claim 9, for example, for drug screening or disease research.

11. A kit for differentiating stem cells into dorsal root ganglion organoids, comprising a neural stem cell induction medium, a neural crest cell induction medium, a sensory neural progenitor cell induction medium, and an organoid induction medium; Preferably, the neural stem cell induction medium comprises a ROCK inhibitor, a TGF-β inhibitor, a WNT agonist, and an FGF pathway activator; the neural crest cell induction medium comprises a TGF-β inhibitor and retinoic acid; the sensory neural progenitor cell induction medium comprises retinoic acid; the organoid induction medium comprises BDNF, GDNF, β-NGF, and NT-3.

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