A method to reprogram fibroblasts into autonomic ganglia organoids

Abstract

The present invention discloses a method for reprogramming fibroblasts into autonomic ganglion organoids. The method uses a TetO-controlled lentiviral system to establish a method for reprogramming mouse somatic cells into autonomic ganglion organoids (induced autonomic ganglion, iAG) using a combination of transcription factors; the experimental results show that the combination of three transcription factors, Ascl1, Phox2a / 2b and Hand2, can induce mouse embryonic fibroblasts to generate self-organized, network-connected neural clusters. The method shows that the three factors Ascl1, Phox2a / 2b and Hand2 are effective means for reprogramming mouse fibroblasts into iAG organoids, and the induced iAG organoids may be used as a model to decipher the pathogenesis of autonomic neurological diseases and screen effective sources of drugs and cell replacement therapy.

Description

Technical Field

[0001] The present invention belongs to the field of cell reprogramming and cell replacement therapy biotechnology, and specifically relates to a method for reprogramming fibroblasts into autonomic ganglion organoids. Background Art

[0002] A ganglion is a cluster or group of nerve cells found in the peripheral nervous system (PNS) or central nervous system (CNS). They are often connected to each other and to other structures of the PNS and CNS to form a complex neural network. The autonomic nervous system (ANS) controls involuntary functions such as circulation, respiration, heart rate, body temperature, sweating, digestion, and metabolism that are regulated by smooth muscle fibers, cardiac muscle fibers, and glandular activity. It cannot be controlled by consciousness and is considered an "automatic" nervous system (Cannon, 1963; Goldstein, 2013). The ANS is composed of the sympathetic, parasympathetic, and enteric nervous systems, which contain a variety of different neuronal subtypes that innervate different tissues and organs. They are controlled by preganglionic neurons that input from the hypothalamus to the brainstem and spinal cord, which in turn determine the activity of primary visceral motor neurons in the autonomic ganglia. Autonomic ganglia are composed of different types of functional neurons (such as regulating vasoconstriction, secretory motility, and hair movement). The neuronal subpopulations that innervate each target tissue can express different neurotransmitters, neuropeptides, ion channels, and receptors, which together constitute the chemical phenotype of neurons (Cane and Anderson, 2009).

[0003] The specificity and differentiation of peripheral autonomic ganglion cells depend on the interaction of cell-extrinsic and cell-intrinsic factors. The intrinsic factors in its development process are mainly controlled by the regulatory network formed by transcription factors. Studies have shown that Ascl1 / Mash1 (Achaete-scute homolog 1) controls the development of neuronal progenitor cells in different nervous systems. Mutant Ascl1 mice die at birth, and the olfactory epithelium, sympathetic nerves, parasympathetic nerves and enteric nerves are severely damaged (Guillemot et al., 1993; Pattyn et al., 2006). In the absence of exogenous BMP2, Mash1 can induce the expression of Phox2a (Paired-like homeobox 2a) and c-RET and promote neuronal differentiation and the expression of pan-neuronal genes. In vivo, the expression of Phox2a in autonomic ganglia is decreased in Mash1. - / -Phox2a expression in chick embryos significantly decreased (Lo et al., 1998). Overexpression of Phox2a in cultured neural crests caused a strong increase in the number of sympathetic adrenergic cells. Phox2a expression in chick embryos promoted the expression of noradrenergic marker genes DBH (dopamine beta-hydroxylase) and TH (tyrosinehydroxylase), pan-neuronal genes SCG10 and NF160, and cholinergic genes ChAT (cholineacetyltransferase) and VAChT (vesicular acetylcholine transporter) (Stanke et al., 1999). The knockout results of Phox2b identified Phox2b as an important gene for the development of autonomic neurons (Pattyn et al., 1999). Studies have shown that Phox2 is sufficient to promote the generation of sympathetic neurons and directly or indirectly controls the expression of a large number of sympathetic neuron-specific genes. In fact. Ascl1 and Phox2 are both essential for the development of sympathetic neurons (Lo et al., 1998; Stanke et al., 2004). In addition, Hand2 (heart and neural crest derivatives expressed 2) is necessary and selective for the differentiation of noradrenergic sympathetic neurons. Neural crest-specific knockout of Hand2 leads to progressive neuronal loss, a significant reduction in tyrosine hydroxylase TH and dopamine hydroxylase DBH, and a block in initial noradrenergic differentiation (Hendershot et al., 2008; Lucas et al., 2006; Morikawa et al., 2007).

[0004] In recent years, great progress has been made in direct neuronal reprogramming in vitro. Chemical small molecules or transcription factors are used to transform fibroblasts or glial cells into dopaminergic, GABAergic, motor neurons, etc. The induced neurons can be used for cell therapy and disease models (Caiazzo et al., 2011; Liu et al., 2013; Son et al., 2011; Yang et al., 2017). Although studies have used chemical molecules to induce sympathetic neurons from human pluripotent stem cells (hPSCs) (Oh et al., 2016), the use of neurons from stem cells may increase the risk of tumor formation. In addition, there are studies that use transcription factors to transform fibroblasts into norepinephrine neurons (Li et al., 2019), but the neurons induced are dispersed and of a single type, which is different from the clustered ganglia in vivo and has multiple cell types. In a published study, fibroblasts were successfully reprogrammed to generate sensory ganglion organoids using a specific combination of transcription factors (Xiao et al., 2020), but it is still unknown whether it is possible to induce sympathetic ganglion organoids in vitro.

[0005] References

[0006] Caiazzo,M.,Dell'Anno,MT,Dvoretskova,E.,Lazarevic,D.,Taverna,S.,Leo,D.,Sotnikova,TD,Menegon,A.,Roncaglia,P.,Colciago,G.,et al.(2011).Directgeneration of functional dopaminergic neurons from mouse and humanfibroblasts.Nature 476,224-227.

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[0008] Cannon,W.B.(1963).The Wisdom of the Body(New York.:Norton Library).

[0009] Goldstein,D.S.(2013).Differential responses of components of theautonomic nervous system.

[0010] Guillemot,F.,Lo,L.C.,Johnson,J.E.,Auerbach,A.,Anderson,D.J.,andJoyner,A.L.(1993).Mammalian achaete-scute homolog 1is required for the earlydevelopment of olfactory and autonomic neurons.Cell 75,463-476.

[0011] Hendershot,T.J.,Liu,H.,Clouthier,D.E.,Shepherd,I.T.,Coppola,E.,Studer,M.,Firulli,A.B.,Pittman,D.L.,and Howard,M.J.(2008).Conditionaldeletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derivednoradrenergic sympathetic ganglion neurons.Dev Biol 319,179-191.

[0012] Li,S.,Shi,Y.,Yao,X.,Wang,X.,Shen,L.,Rao,Z.,Yuan,J.,Liu,Y.,Zhou,Z.,Zhang,Z.,et al.(2019).Conversion of Astrocytes and Fibroblasts intoFunctional Noradrenergic Neurons.Cell Rep28,682-697 e687.

[0013] Liu,M.L.,Zang,T.,Zou,Y.,Chang,J.C.,Gibson,J.R.,Huber,K.M.,and Zhang,C.L.(2013).Small molecules enable neurogenin 2 to efficiently convert humanfibroblasts into cholinergic neurons.Nat Commun 4,2183.

[0014] Lo,L.C.,Tiveron,M.-C.,and Anderson,D.(1998).MASH1 activatesexpression of the paired homeodomain transcription factor Phox2a,and couplespan-neuronal and subtype-specific components of autonomic neuronalidentity.Development 125,609-620.

[0015] Lucas,M.E.,Müller,F.,Rüdiger,R.,Henion,P.D.,and Rohrer,H.(2006).ThebHLH transcription factor<;em>;hand2<; / em>;is essential fornoradrenergic differentiation of sympathetic neurons.Development 133,4015.

[0016] Morikawa,Y.,D'Autréaux,F.,Gershon,M.D.,and Cserjesi,P.(2007).Hand2determines the noradrenergic phenotype in the mouse sympathetic nervoussystem.Dev Biol 307,114-126.

[0017] Oh,Y.,Cho,G.S.,Li,Z.,Hong,I.,Zhu,R.,Kim,M.J.,Kim,Y.J.,Tampakakis,E.,Tung,L.,Huganir,R.,et al.(2016).Functional Coupling with Cardiac MusclePromotes Maturation of hPSC-Derived Sympathetic Neurons.Cell Stem Cell 19,95-106.

[0018] Pattyn,A.,Guillemot,F.,and Brunet,J.-F.(2006).Delays in neuronaldifferentiation in Mash1 / Ascl1 mutants.Dev Biol 295,67-75.

[0019] Pattyn,A.,Morin,X.,Cremer,H.,Goridis,C.,and Brunet,J.F.(1999).Thehomeobox gene Phox2b is essential for the development of autonomic neuralcrest derivatives.Nature 399,366-370.

[0020] Son,E.Y.,Ichida,J.K.,Wainger,B.J.,Toma,J.S.,Rafuse,V.F.,Woolf,C.J.,and Eggan,K.(2011).Conversion of mouse and human fibroblasts into functionalspinal motor neurons.Cell Stem Cell 9,205-218.

[0021] Stanke,M.,Junghans,D.,Geissen,M.,Goridis,C.,Ernsberger,U.,and Rohrer,H.(1999).The Phox2homeodomain proteins are sufficient to promote the development of sympathetic neurons.Development 126,4087-4094.

[0022] Stanke,M.,Stubbusch,J.,and Rohrer,H.(2004).Interaction of Mash1 andPhox2b in sympathetic neuron development.Mol Cell Neurosci 25,374-382.

[0023] Xiao, D., Deng, Q., Guo, Y., Huang,

[0024] Yang,N.,Chanda,S.,Marro,S.,Ng,YH,Janas,JA,Haag,D.,Ang,CE,Tang,Y.,Flores,Q.,Mall,M.,et al.(2017).Generation of pure GABAergic neurons bytranscription factor programming.Nat Methods 14,621-628. Summary of the invention

[0025] The object of the present invention is to provide a method for reprogramming fibroblasts into autonomic ganglion organoids.

[0026] The autonomic nervous system plays an important role in maintaining physiological homeostasis. Its extensive connections make it vulnerable to damage, resulting in autonomic dysfunction, including primary causes such as Parkinson's disease, multiple system atrophy, pure autonomic failure and secondary causes such as diabetes, amyloidosis and immune-mediated diseases, which ultimately lead to multi-organ dysfunction such as cardiovascular, gastrointestinal, thermoregulatory and urogenital systems. In recent years, great progress has been made in direct neuronal reprogramming in vitro, using chemical small molecules or transcription factors to convert fibroblasts or glial cells into dopaminergic, GABAergic, motor neurons, etc. The induced neurons can be used for cell therapy and disease models.

[0027] In a published study, fibroblasts were successfully reprogrammed to generate sensory ganglion organoids using a specific combination of transcription factors, but it is unknown whether it is possible to induce sympathetic ganglion organoids in vitro. Although studies have used chemical molecules to induce sympathetic neurons from hPSCs, the use of neurons from stem cells may increase the risk of tumor formation. In addition, there are studies that use transcription factors to convert fibroblasts into norepinephrine neurons, but the neurons induced are scattered and of a single type, which is different from the clustered ganglia in the body and has a variety of cell types.

[0028] The present invention uses a TetO-controlled lentiviral system to overexpress key transcription factors (Ascl1, Phox2a / 2b and Hand2) in the development of autonomic ganglia in fibroblasts, and uses cell immunostaining, qRT-PCR, patch clamp, cell co-culture and other techniques to find that the three-factor combination APH can reprogram mouse fibroblasts into self-organized and networked iAG organoids, and the induced iAG neurons show various molecular characteristics, multiple cell types and electrophysiological characteristics of autonomic nerve cells, and can survive in vivo and regulate the beating of co-cultured cardiomyocytes. The iAG organoids generated by APH reprogramming somatic cells are similar to endogenous autonomic neurons in characteristics and functions, and can provide new possibilities for establishing autonomic neurological disease models, studying their pathogenesis, screening antagonistic drugs and developing cell replacement therapies.

[0029] Therefore, the first object of the present invention is to provide use of transcription factors Ascl1, Phox2a and Hand2 or transcription factors Ascl1, Phox2b and Hand2 in inducing the generation of autonomic ganglion organoids.

[0030] Preferably, the transcription factor is in the form of DNA, RNA or protein.

[0031] The second object of the present invention is to provide a method for obtaining autonomic ganglion organoids through reprogramming, comprising the following steps:

[0032] S1. Overexpressing or expressing transcription factors Ascl1, Phox2a and Hand2 or transcription factors Ascl1, Phox2b and Hand2 in mammalian somatic cells in vitro to obtain transgenic cells;

[0033] S2. The transgenic cells are induced and cultured in a neuronal induction medium to obtain autonomic ganglion organoids.

[0034] Preferably, the step S1 comprises the following steps: co-introducing transcription factors Ascl1, Phox2a and Hand2 or co-introducing transcription factors Ascl1, Phox2b and Hand2 into in vitro mammalian somatic cells to obtain transgenic cells.

[0035] Preferably, the introduction is carried out by separately constructing lentiviral vectors expressing each transcription factor, and then introducing them together into the mammalian somatic cells.

[0036] Preferably, the nucleotide sequence of the transcription factor Ascl1 is shown in SEQ ID NO.1, the nucleotide sequence of the transcription factor Phox2a is shown in SEQ ID NO.2, the nucleotide sequence of the transcription factor Hand2 is shown in SEQ ID NO.4, and the nucleotide sequence of the transcription factor Phox2b is shown in SEQ ID NO.3.

[0037] Preferably, the somatic cells are fibroblasts. More preferably, the somatic cells are embryonic skin fibroblasts.

[0038] Preferably, the mammal is a human, mouse, rat, monkey, rabbit, cow, horse, pig, dog or cat.

[0039] Preferably, in step S2, inducing and culturing the transgenic cells in a neuron induction medium is to culture the transgenic cells in neuron induction medium I for 7 days, then replace it with neuron induction medium II and continue culturing for 7-13 days, and during the induction culture, replace the medium every 2 days; the neuron induction medium I contains 1×B27, 1×Pen / Strep, 2ng / ml Dox, 10ng / ml bFGF, 100ng / ml IGF-1, 10ng / ml BDNF and 10ng / ml GDNF, and the balance is a 1:1 DMEM / F12 basal medium; the neuron induction medium II contains 10μM forskolin and 10ng / mlBMP4, and the balance is the neuron induction medium I.

[0040] The present invention also provides an autonomic ganglion organoid obtained according to the method.

[0041] The autonomic ganglion organoids obtained by the present invention can be used for autonomic nervous system disease models or cell therapy models, screening effective antagonistic drugs and developing cell replacement therapies.

[0042] The present invention establishes a method for reprogramming animal somatic cells into autonomic ganglion organoids, providing a new way to obtain induced autonomic ganglion cells, which is simpler, safer and more efficient than other methods, and has great application value in basic research on autonomic nervous diseases, drug screening and potential cell replacement therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 MEFs were directly reprogrammed into iAG for Ascl1, Phox2a / 2b and Hand2; Figure A, schematic diagram of reprogramming, mouse embryonic fibroblasts MEFs were derived from E13.5 Tau-GFP or C57BL6 / J mice, infected with lentivirus expressing transcription factors Ascl1, Phox2a / 2b and Hand2 controlled by tet-on elements, and induced using neuronal induction medium containing doxycycline (dox); Figure B, morphological changes of MEFs infected with GFP and APH (Ascl1, Phox2b and Hand2) viruses at different times, It can be seen that MEFs infected with APH for 8 days began to gather into clusters, and a large number of tightly connected iAG cell clusters were formed at 18 days; Figures CK, morphological changes of Tau-GFP-derived MEFs infected with different combinations of viruses for 18 days (A: Ascl1; P: Phox2a / 2b; H: Hand2), tightly connected cell clusters were observed in MEFs infected with AP and APH combinations, and the efficiency of the APH three-factor combination was better; Figure LO, Tuj1 and DAPI staining of neurons induced by different transcription factor groups; Figure P, statistical diagram of iAG induced by a single transcription factor and different combinations; 6×10 4 MEFs were plated in 12-well culture plates and GFP-positive cell clusters were counted under a fluorescence microscope 18 days after infection with different groups of viruses; Scale bar: 80 μm (BK); 40 μm (LO), * P<0.0001.

[0044] Figure 2Figure AV shows that the iAG induced by APH contains most types of autonomic ganglion neurons; Figure AV shows that the iAG induced by the combination of Ascl1, Phox2b and Hand2 expresses common neuronal marker molecules such as Tuj1, Map2, NF200, Synapsin1, NeuN, Peripherin, Vamp, Gad65, GABA, vGlut2, NPY, TH, DBH, Vmat2, SOM, VIP, ChAT and VAChT, as well as autonomic neuronal marker molecules, among which Figure AE shows MEFs derived from Tau-GFP mice, and Figure FV shows MEFs derived from C57BL6 / J mice; Figures W and X show the qRT-PCR results of the expression of the specified genes by iAG induced by APH combination. Compared with GFP, APH-induced iAG expressed most neuronal molecules and autonomic ganglion neuron-specific molecules; Figure Y, qRT-PCR results of the expression of designated genes in C57BL6 endogenous sympathetic ganglia compared with GFP virus-infected MEFs; Figure Z, qRT-PCR analysis of the expression of Tubb3 (Tuj1), Map2, TH and DBH in GFP, A, AP(2a), AP(2b), AH, AP(2a)H, AP(2b)H virus-infected MEFs, among which Tuj1 and Map2 are genes commonly expressed in neurons, and TH and DBH are genes expressed in autonomic ganglia neurons; Scalebar: 40μm (Figures AD, F, G, I, N, P, Q), 20μm (Figures E, H, J, KM, O, RT), * p<0.05, & p<0.01, # p<0.001.

[0045] Figure 3Figure 1 shows that iAG neurons induced by APH have autonomous neuronal electrophysiological characteristics; Figure A, iAG neurons induced by the combination of Ascl1, Phox2b and Hand2 clamped under a high-power water-immersion objective (40×); Figure B, rapidly activated and inactivated inward sodium currents and outward potassium currents of iAG neurons recorded by voltage clamp in neurons induced for 13 days; Figure C, spontaneous action potentials (sAPs) recorded in neurons induced for 10 days; Figures D and E, current clamp recordings under current injection show that iAG neurons have single-peak (Phasic, type I, D) and multi-peak (Tonic, type II, E) action potentials; Figure F, the proportion of recorded iAG neurons with single-peak (Phasic), multi-peak (Tonic) action potentials and no action potentials; Figure G, afterhyperpolarization (after sAPs) clamped at I = 0pA (n = 8 / 10) Figure H, resting membrane potential (RMP) of iAG neurons induced for 14-22 days; Figure I, action potential of neurons (RMP-40mV) was completely blocked after treatment with TTX (1μM, n=6 / 20); Figure J, Cd 2+ After the action of 300 μM (n=2 / 20), the action potential of neurons (RMP-34 mV) was completely blocked; Figure K, Cd 2+ (300μM) alone can reduce the action potential of neurons (RMP-49mV), while Cd 2+The combined action of 10 μM and 20 μM of CNQX and picrotoxin can completely block the action potential. After 30 minutes of washing, the action potential was partially restored (n=12 / 20); Figure L, the ratio of sAP or sPSC of synaptic activity recorded in iAG neurons; Figure M, the spontaneous postsynaptic current sPSC recorded in iAG neurons can be partially blocked by AMPA receptor antagonist CNQX (10 μM) or GABA receptor antagonist picrotoxin (50 μM), and the simultaneous administration of CNQX and picrotoxin can completely block the recorded postsynaptic current; Figure N, the action potential recorded in iAG neurons induced by dopamine (100 μM); Figure O, the application of noradrenaline (20 μM) The recorded postsynaptic current; Figure P, the action potential recorded from the iAG neurons induced by acetylcholine (Ach, 1μM) stimulation, which can be partially washed back; Figure Q, the action potential recorded from the iAG neurons induced by Ach stimulation can be partially blocked by the acetylcholine M receptor antagonist atropine (ATR, 10μM), while the simultaneous application of ATR and acetylcholine N receptor antagonist mecamylamine (MEC, 10μM) can completely block this effect; Figure R, the action potential recorded from the iAG neurons induced by 5-hydroxytryptamine (5-HT, 1μM) stimulation.

[0046] Figure 4 Survival of iAG neurons in the superior cervical ganglion in vivo; Figure A, schematic diagram of APH-transfected Tau-GFP-labeled cells digested and transplanted into the superior cervical ganglion of adult rats; Figure B-M, SCG was isolated 2 weeks after cell transplantation and injected and immunostained with GFP and TH antibody (BE), ChAT antibody (FI) or VAChT antibody (JM); arrows point to representative double-positive cells, scale bar: 20μm.

[0047] Figure 5Figure 1 shows iAG neurons innervating co-cultured cardiomyocytes; Figure A, schematic diagram of co-culture of iAG neurons and neonatal mouse cardiomyocytes (NMVMs); Figure B, representative images of co-staining of Tuj1 and NMVMs marker cTnT after 7-10 days of co-culture; the upper and right images are frontal views of Z-stack images (see Video S1 for 3D reconstructed images), the upper XY section is the XZ section image, marked with a green border, and the right side of the XY section is the image of the YZ section, marked with a red border, the projection of the XZ plane on the XY and YZ planes is represented by a red line, and the projection of the YZ plane on the other two planes is marked with a green line; Figures C and D, representative intensity images of increased cardiomyocyte beating, the cardiomyocyte beating speed increased after the addition of nicotine (1 μM); Figure E, statistical results of increased cardiomyocyte beating after nicotine treatment (n=13), ** p<0.0001; Figures F and G, representative intensity graphs of slowed cardiomyocyte beating, slowed cardiomyocyte beating after the addition of nicotine (1 μM); Figure H, statistical results of slowed cardiomyocyte beating after nicotine treatment (n=5), * p<0.05. DETAILED DESCRIPTION

[0048] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0049] The present invention utilizes the overexpression of three transcription factors (APH) Ascl1, Phox2a / 2b and Hand2 in animal somatic cells to reprogram somatic cells into autonomic ganglion organoids. The scheme adopted can be divided into four steps:

[0050] 1. Collection and expansion of animal somatic cells: The somatic cells used in this method are mouse embryonic fibroblasts.

[0051] 2. Overexpression of transcription factors in somatic cells: This method clones the transcription factor coding sequence into a lentivirus vector to overexpress the transcription factor.

[0052] 3. The transfected somatic cells are cultured in a specific culture medium to induce the formation of neural clusters.

[0053] 4. Take samples of the induced neural clusters and conduct identification, including morphological observation, immunostaining, qRT-PCR, electrophysiological recording, etc.

[0054] The specific experimental process is as follows:

[0055] Example 1

[0056] 1. Methods

[0057] 1. Preparation of Mouse Embryonic Fibroblasts (MEFs)

[0058] (1) Rapidly remove Tau-GFP or C57BL / 6J mouse E13.5 embryos and place them in a culture dish containing pre-cooled HBSS buffer (without calcium and magnesium, Gibco). After rinsing, transfer them to a culture dish containing new HBSS buffer, remove the capsule, umbilical cord, etc., and separate the embryos. Remove the head, spine, limbs and internal organs under a microscope, carefully separate the skin tissue with forceps and curved scissors, and place them in a new culture dish.

[0059] (2) For every 8 embryo skin tissues, add 1 mL of 0.25% trypsin-EDTA, cut the tissue into pieces with curved scissors, place in the incubator for digestion for 5 minutes, and continue to cut with curved scissors for digestion for 3 times (15 minutes). Add appropriate amount of MEF culture medium (containing DMEM high glucose medium, 10% FBS (Gibco), 1× Pen / Strep (Gibco), 1× MEM-NEAA (Gibco)) to terminate digestion, use a pipette to continuously blow until a single cell suspension is obtained, inoculate cells in a 10 cm culture dish according to 1.5-2 embryos, and place in the incubator.

[0060] (3) When the cells are fully grown, remove the culture medium, wash once with HBSS, add 1 mL of 0.25% trypsin-EDTA and incubate in an incubator for 3 min, add MEF culture medium to terminate digestion, sieve the dispersed cells with a 100 μm filter, and collect the cells by centrifugation at 1000 rpm for 5 min.

[0061] (4) First, resuspend the cells in 1 mL of cell freezing solution (DMEM high-glucose medium containing 10% DMSO and 30% FBS), then add the remaining freezing solution at a ratio of 1 dish:4 tubes, divide into cryopreservation tubes, place in a cryopreservation box, store at -80°C, and transfer to a liquid nitrogen tank the next day. The revived cells are passaged and used for subsequent induction.

[0062] 2. Lentivirus Preparation and MEFs Reprogramming

[0063] (1) Construction of lentiviral vector: The coding sequence of mouse transcription factor Ascl1 (its nucleotide sequence is shown in SEQ ID NO.1), transcription factor Phox2a (its nucleotide sequence is shown in SEQ ID NO.2), transcription factor Phox2b (its nucleotide sequence is shown in SEQ ID NO.3), and transcription factor Hand2 (its nucleotide sequence is shown in SEQ ID NO.4) were cloned into the EcoRI site of a lentiviral vector containing a TetO regulatory sequence (FUW-TetO-MCS, addgene #84008). The purpose is to co-transfect with RTTA virus in the future, and to add Dox (Doxycyclin) to control the expression of these transcription factors in somatic cells. After the correct cloning was verified by DNA sequencing, the plasmid for transfection was prepared.

[0064] (2) Lentivirus preparation: 24 hours before plasmid transfection, subculture confluent HEK293T cells into 10 cm culture dishes at a ratio of 1:4. About 1 hour before transfection, replace new culture medium (add 9 mL). Prepare the required amount of plasmid and transfection reagent for one 10 cm culture dish according to the following standards: ① Add target plasmids FUW-TetO-GFP / Ascl1 / Phox2a / Phox2b / Hand2, FUW-M2rtTA (10 μg), etc., as well as packaging plasmids pMDLg / pRRE (5 μg), pRSV-rev (2.5 μg), and pMD2.G (2.5 μg) into Opti-MEM (500 μl) and mix thoroughly; ② Add Opti-MEM (500 μL) and 40 μL lipofectamin 2000 into another centrifuge tube, mix gently, and incubate at room temperature for 5 minutes.

[0065] (3) Slowly add ② prepared in step (2) to ①, gently mix, and incubate at room temperature for 20-30 minutes. Then gently and evenly add the mixture (1 mL) dropwise to the cell culture dish. After about 48 hours, collect the culture medium, filter it with a 0.45 μm sterile filter, transfer it to an ultracentrifuge tube, and centrifuge it at 21000 rpm in an ultracentrifuge at 4°C for 2 hours.

[0066] (4) After centrifugation, carefully remove the cannula. A thin layer of white film-like substance can be seen at the bottom of the centrifuge tube. Gently pour out the supernatant in one direction (do not reflux), leaving about 1 mL. Shake overnight at 4°C. The next day, store the aliquots at -80°C and measure the virus titer. Avoid repeated freezing and thawing during use.

[0067] 3. MEFs reprogramming to produce iAG

[0068] (1) One day before virus infection, MEFs were plated at 6×10 4The number of cells per well was plated into a 12-well plate pre-coated with matrigel (more than 2 hours). On the day of infection, the target virus was mixed with M2rtTA at a ratio of 1:1, and 1 mL of MEF medium containing polybrene (10 μg / mL) was added. The cell supernatant was discarded and the virus-containing medium was added.

[0069] (2) 16-24 hours after infection, the supernatant was discarded and replaced with neuronal induction medium I (containing DMEM / F12 (1:1) (Life Technologies), 1×B27 (Gibco), 1×Pen / Strep (Gibco), 2ng / ml Dox, 10ng / ml bFGF (R&D Systems), 100ng / ml IGF-1 (R&D Systems), 10ng / ml BDNF (R&D Systems) and 10ng / ml GDNF (R&D Systems)). After 7 days of culture, the medium was replaced with neuronal induction medium II (neuronal induction medium I + 10μM forskolin (Selleck) + 10ng / ml BMP4 (R&D Systems)) and cultured for another 7-13 days. During the induction period, the medium was replaced every 2 days. After 2-3 weeks of induction, the neuronal cells were used for subsequent experiments.

[0070] 4. iAG identification

[0071] (1) Immunocytochemistry (ICC)

[0072] a. Take out the neuron culture plate that has been induced for 2-3 weeks, discard the culture medium, wash with PBS 3 times, 3 minutes each time, add appropriate amount of 4% PFA to fix for 15 minutes (not more than 20 minutes). Discard PFA, wash with PBS 3 times, 3 minutes each time. Then add ICC blocking solution and block at room temperature for 1.5 hours.

[0073] b. Prepare the antibody using antibody diluent (5% FBS, 0.07% Triton-100 in PBS) and incubate at 4°C overnight. The primary antibodies used in this experiment include: mouse anti-Tuj1(Millipore,MAB5564,1:500), rabbit anti-Tuj1(Abcam,ab18207,1:2000), mouse anti-Map2(Sigma,M1406,1:2000), rabbit anti-synapsin1(Calbiochem,574778,1:500), rabbit anti-peripherin(Millipore,ab1530,1:1000), NeuN(Millipore,ABN78,1:2000), rabbit anti-vGLUT2(abcam,ab79157,1:500), rabbit anti-GABA(Sigma,A-2052,1:1000), mouse anti-NF200(Millipore,MAB5266,1:500), rabbit anti-TH(Protos Biotech,CA-101bTHrab,1:1000),rabbit anti-DBH(Protos Biotech,CA-301bDBHrab,1:1000),NPY(Sigma,N9528,1:2000),Gad65(BDBiosciences,559931,1:500),Vmat2(Synaptic Systems, 138302, 1:200), ChAT (Millipore, AB144P, 1:500), VAChT (Synaptic Systems, 139103, 1:200), VIP (Immunostar, 20077, 1:200), goat anti-GFP (Abcam, ab6673, 1:2000).

[0074] c. The next day, remove the culture plate, rinse the slides with PBS, add the corresponding secondary antibody and DAPI reaction solution, and incubate at room temperature in the dark for 1.5 hours. Discard the liquid, rinse with PBS, seal the slides, observe under a fluorescence microscope, or temporarily store at 4°C.

[0075] (2) Fluorescence quantitative PCR (qRT-PCR)

[0076] Cells from different groups were taken and total RNA was extracted using TRIzol reagent (Invitrogen). Genomic DNA was removed from 1 μg RNA using HiScript IIQ RT SuperMix for qPCR (Vazyme Biotech) kit and reverse transcribed to synthesize cDNA. Kapa SYBR fast qPCR master mix (Kapa) dye was then used to prepare the reaction system. 3 G Real-Time PCR system (Analytikjena) was used for qRT-PCR reaction. The data obtained were used -ΔΔct The primers used for qRT-PCR are shown in Table 1.

[0077] Table 1 Genes and primers detected by qRT-PCR

[0078]

[0079]

[0080] (3) Electrophysiological analysis

[0081] MEFs were plated on 12 mm round glass slides coated with Matrigel in 24-well culture plates, and neurons were induced for 2-3 weeks for recording. The cell slides were placed in a chamber mounted on the stage of an upright microscope (BX51W1, Olympus, Japan) equipped with a 40× immersion objective and DIC optical system. The cells and recording electrodes were observed on the monitor of a CCD camera (evolve, Photometrics, Tucson, USA) mounted on the microscope. Whole-cell patch clamp recordings were performed using an EPC-10 USB amplifier (HEKA electronics, Lambrecht, Germany) and Patchmaster data acquisition software (HEKA). Borosilicate glass recording electrodes were stretched to 4-7 MΩ using a Sutter P-97 horizontal straightener. Oxygenated (95% O2 and 5% CO2) external fluid was continuously flowed into the chamber at a rate of 1.5-2 mL / min by a peristaltic pump (Lead-2, Longer Pump, Hebei, China). The composition of the external solution was (in mM): 140 NaCl, 2.5 KCl, 10 HEPES, 10 glucose, 2 CaCl2, and 2 MgCl2. The composition of the electrode internal solution was (in mM): 105 K-gluconate, 5 KCl, 5 NaOH, 15 KOH, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 2 adenosine 5'-triphosphate, 0.5 guanosine 5'-triphosphate, 10 HEPES, and 2 ascorbate (pH 7.2). Whole-cell currents were recorded in voltage clamp mode, with the base potential held at -60 mV and the voltage increased in 10 mV increments from -90 to +100 mV. For spontaneous synaptic currents, the cell membrane potential was held at -60 mV, and step current-evoked action potentials were recorded in current clamp mode with a current step interval of 5 pA. Spontaneous action potentials were recorded by current injection to maintain the initial holding potential at -60 mV. Data were digitized at 10 kHz with a 3 kHz low pass filter and analyzed using Patchmaster (HEKA).The antagonists and agonists used were: tetrodotoxin (TTX, Hamu-Shanghai, 1μM), 6cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris, 10μM), picrotoxin (Tocris, 50μM), norepinephrine (NE, Selleck, 20μM), dopamine (DA, Selleck, 100μM), acetylcholine (Ach, Selleck, 1μM), atropine (ATR, Selleck, 10μM), mecamylamine (MEC, Tocris, 10μM), 5-hydroxytryptamine (5-HT, Selleck, 1μM) and CdCl2 (Sigma, 300μM).

[0082] (4) Statistical analysis

[0083] In this study, cell cluster counting was performed under a 100X fluorescence microscope. qRT-PCR data were expressed as mean ± standard deviation (mean ± SD) and analyzed using GraphPad Prism 7 (GraphPad software, Inc.). The two groups were compared using two independent samples unpaired two-tailed student's t-test, and P < 0.05 was considered to be significantly different.

[0084] 2. Results

[0085] Using mouse embryonic fibroblasts (MEFs) derived from Tau-GFP heterozygous embryos, different combinations of Ascl1, Phox2b / 2a and Hand2 were used to infect MEFs, and the reprogramming ability of different combinations on MEFs was observed under doxycycline (dox)-containing neuronal differentiation medium. The results showed that GFP-positive cell clusters ( Figure 1 .H, I). This phenomenon was not observed in Ascl1 alone or in combination with other transcription factors ( Figure 1 .CG). When Ascl1, Phox2a / 2b and Hand2 (APH) were combined, the morphological changes of MEFs were also observed. The morphology began to change on the 8th day, and obvious cell formation was seen after 14 days, and more neural clusters were generated ( Figure 1.B, J, K, P). This suggests that Hand2 can enhance the ability of AP to reprogram MEFs into neural clusters. As can be seen from the figure, the neural clusters induced by two or three factors (AP, APH) are interconnected by relatively thick bundles of nerve fibers, which are morphologically similar to the autonomic ganglion plexus in vivo. Further immunostaining results showed that the neural clusters and bundles of nerve fibers induced by APH can be marked by the neuronal marker molecule Tuj1, while the Tuj1 markers in Ascl1 are all scattered neurons ( Figure 1 .LO). Tuj1 labeling reaction showed that neurons induced by Ascl1 alone were mainly in an immature neuronal state. We defined the APH-induced neural clusters as iAG (induced autonomic ganglia) organoids.

[0086] By immunofluorescence staining and qRT-PCR, we detected a variety of neuronal marker molecules, including neuronal universal molecules and cell type-specific molecules, to determine the characteristics of iAGs produced by APH reprogramming. The results showed that they highly expressed neuronal universal molecules Tuj1 and Map2 ( Figure 2 .A, B, F), also expresses synapsin and synaptobrevin (Vamp1) ( Figure 2 .D, I, J), indicating that iAG neurons can form synaptic connections and release synaptic vesicles. In addition, APH-induced iAG neurons also expressed NF200, NeuN, and Peripherin ( Figure 2 .C, G, H). Inhibitory neurotransmitter GABA, glutamate decarboxylase Gad65 and vesicular glutamate transporter vGlut2 were also detected ( Figure 2 .KM), which is consistent with the characteristics of autonomic ganglion neurons in vivo. Autonomic ganglion neurons contain multiple cell types, mainly noradrenergic and acetylcholinergic neurons. We detected the expression of specific molecules of different cell types. Immunostaining results showed that induced iAG expressed noradrenergic neuron marker molecules DBH, TH, NPY and Vmat2 ( Figure 2 .N, PR) and acetylcholinergic neuronal molecules ChAT, VAChT, VIP and SOM ( Figure 2 .E, S, T, O). qRT-PCR assay showed that the expression of genes such as Tuj1, Map2, NF200, p75NTR, vGlut2, GABARγ3, Vmat2, TH, DBH, SOM, VIP, ChAT, VAChT, and RET were significantly upregulated in APH-induced iAGs compared with GFP-infected MEFs, further confirming the above immunostaining results ( Figure 2.W, X). The expression of the specified genes in the endogenous sympathetic ganglia of C57BL6 was further confirmed by analyzing the expression of the autonomic ganglion marker molecules used in the above immunostaining and qRT-PCR experiments ( Figure 2 .Y). qRT-PCR was performed on the combination of Ascl1, Phox2a / 2b, and Hand2 ( Figure 2 .Z). Although Tubb3, Map2, and TH were significantly increased in AP- and APH-induced neurons compared with the GFP group, the TH expression in the APH group was significantly higher than that in the AP group, while DBH was not expressed in the AP group. This suggests that Hand2 can further promote the maturation of iAG neurons.

[0087] Subsequently, the electrophysiological properties of iAG neurons with neuronal morphology were evaluated by whole-cell patch clamp recording ( Figure 3 .A). The vast majority of neurons recorded had typical sodium and potassium currents and exhibited action potentials ( Figure 3 .B and C). 25.8% of neurons showed single-spiking action potentials ( Figure 3 .D and F), 65.2% of neurons showed multiple-spiking action potentials ( Figure 3 .E and F). When clamped at I = 0 pA (n = 8 / 10), iAG neurons showed significant after hyperpolarization (AHP) ( Figure 3 .G), and the resting membrane potential (RMP) of -41.7±8.8mV (n=65) was recorded at the same time. Figure 3 .H). By perfusing TTX (voltage-dependent Na + channel blockers) and / or Cd 2+ (nonspecific Ca 2+ The ion dependence of the action potential in these neurons was determined by TTX ( Figure 3 .I), 2 neurons were Cd 2+ Block( Figure 3 .J), 12 neurons require TTX and Cd 2+ There is also blocking ( Figure 3 .K). These data suggest that iAG neurons have electrophysiological properties similar to those of developing rodent autonomic neurons. At the same time, most neurons acquired spontaneous postsynaptic current (sPSC) responses ( Figure 3.L and M), sPSCs can be partially blocked by the AMPA receptor antagonist CNQX or the GABAA receptor antagonist picrotoxin, and are completely inhibited by both ( Figure 3 .M). These results are consistent with the immunoreactivities of synapsin, GABA, Gad65, and vGlut2 detected in iAG neurons ( Figure 2 .D, I, KM), suggesting that functional synapses have formed between iAG neurons. By detecting the responses of iAG neurons to several common neurotransmitters in the autonomic ganglia, we found that exogenous application of dopamine ( Figure 3 .N,n=5 / 30)、norepinephrine( Figure 3 .O,n=9 / 38)、acetylcholine( Figure 3 .P, Ach, n = 14 / 34) or 5-HT ( Figure 3 .R, 5-HT, n = 3 / 26), the induced sAPs or sPSCs in some iAG neurons were enhanced. The responses induced by dopamine, norepinephrine or 5-HT could be washed out ( Figure 3 .N,O,R), acetylcholine-induced secretin can be partially inhibited by atropine (ATR, an antagonist of the acetylcholine M receptor) and completely blocked by ATR and methylamine (MEC, an antagonist of the acetylcholine M receptor) ( Figure 3 .Q). These results further demonstrate that iAG neurons have the pharmacological and physiological properties of mature autonomic neurons.

[0088] APH-reprogrammed iAG neurons were injected into the superior cervical ganglion (SCG) of adult rats ( Figure 4 .A), we found that GFP + iAG neurons survive and proliferate in the SCG, expressing the autonomic neuronal markers TH, ChAT, and VAChT ( Figure 4 .BM), indicating that iAG neurons can survive in vivo and maintain somatic cell type specificity. At the same time, when these neural clusters were co-cultured with neonatal mouse ventricular myocytes (NMVM) ( Figure 5 .A), immunostaining showed that Tuj1-positive iAG neurons and co-cultured cTnT-positive cardiomyocytes were in physical contact ( Figure 5 .B). Among the 18 cardiomyocytes observed, the beating speed of 13 cells increased significantly ( Figure 5 .CE), the beating rate of 5 cells decreased ( Figure 5 .FH). Thus, iAG neurons were able to bind to mouse cardiomyocytes and regulate their beating rate depending on the neuronal subtype.

Claims

1. Application of transcription factors Ascl1, Phox2a and Hand2 or transcription factors Ascl1, Phox2b and Hand2 in inducing the production of autonomic ganglion organoids from mouse embryonic fibroblasts.

2. The use according to claim 1, characterized in that: The transcription factor is in the form of DNA, RNA or protein.

3. A method for obtaining autonomic ganglion organoids through reprogramming, characterized in that: The following steps are involved: S1. Overexpressing or expressing transcription factors Ascl1, Phox2a and Hand2 or transcription factors Ascl1, Phox2b and Hand2 in mouse embryonic fibroblasts to obtain transgenic cells; S2. The transgenic cells are induced and cultured in a neuronal induction medium to obtain autonomic ganglion organoids.

4. The method according to claim 3, characterized in that: The step S1 comprises the following steps: co-introducing transcription factors Ascl1, Phox2a and Hand2 or co-introducing transcription factors Ascl1, Phox2b and Hand2 into mouse embryonic fibroblasts to obtain transgenic cells.

5. The method according to claim 4, characterized in that The introduction is carried out by constructing lentiviral vectors expressing each transcription factor respectively, and then introducing them together into the mouse embryonic fibroblasts.

6. The method according to claim 4, characterized in that The nucleotide sequence of the transcription factor Ascl1 is shown in SEQ ID NO.1, the nucleotide sequence of the transcription factor Phox2a is shown in SEQ ID NO.2, the nucleotide sequence of the transcription factor Hand2 is shown in SEQ ID NO.4, and the nucleotide sequence of the transcription factor Phox2b is shown in SEQ ID NO.

3.

7. The method according to claim 3, characterized in that The step S2 of inducing and culturing the transgenic cells in a neuron induction medium comprises culturing the transgenic cells in neuron induction medium I for 7 days, then replacing the culture medium with neuron induction medium II for further culturing for 7-13 days, during which the culture medium is replaced every 2 days; the neuron induction medium I contains 1×B27, 1×Pen / Strep, 2ng / ml Dox, 10ng / ml bFGF, 100ng / ml IGF-1, 10ng / ml BDNF and 10ng / ml GDNF, and the remainder is a 1:1 DMEM / F12 basal medium; the neuron induction medium II contains 10μM forskolin and 10ng / ml BMP4, and the remainder is the neuron induction medium I.

8. Autonomic ganglion organoids obtained according to the method according to any one of claims 3-7.

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