CN112481364B - Method for screening new sub-population of periodontal ligament specific stem cells based on single cell sequencing and application thereof - Google Patents

Abstract

The invention discloses a method for screening a new sub-population of periodontal ligament specific stem cells based on single cell sequencing and application thereof, wherein the screened new sub-population specifically expresses FBLN2 and membrane protein CADM3, and simultaneously discloses application of the new sub-population of periodontal ligament specific stem cells in preparation of a medicament for treating periodontal diseases. According to the invention, periodontal ligament specific stem cell subsets are obtained through single cell sequencing, and in vitro flow sorting and cell subset performance verification are carried out through marker genes; the cell subset screened by the invention is beneficial to searching intervention targets of periodontal steady imbalance, and creates possibility for developing new drugs; the periodontal ligament specific stem cells are used as seed cells to form organoids, so that scientific basis is provided for the research and verification of new materials of medical instruments; further explores the feasibility of the autologous/allogenic transplantation of the periodontal ligament stem cells and provides a new idea for treating periodontal diseases.

Description

Method for screening new sub-population of periodontal ligament specific stem cells based on single cell sequencing and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a method for screening a new sub-population of periodontal ligament specific stem cells based on single cell sequencing and application thereof.

Background

In the complex microenvironment of the mouth, the periodontal tissue is highly dynamically balanced. Periodontal homeostasis is the biological foundation for achieving normal physiological functions of the oral cavity such as mastication and pronunciation, and clinical treatment modes such as orthodontics and implantation. Imbalance in periodontal homeostasis can lead to loss of periodontal attachment, destruction of alveolar bone, and initiation of periodontal disease. Periodontal disease has the characteristics of high morbidity, easy destruction and difficult recovery, is the most common reason for tooth loss of human, is closely related to various systemic diseases, and is one of chronic diseases which are harmful to human health and need to be mainly prevented and treated by the world health organization. Therefore, the exploration of the periodontal homeostasis maintenance mechanism and the tooth-supported tissue damage repair remodeling mechanism are key breakthrough points for the response of periodontal disease prevention and treatment.

Periodontal tissue is a three-layer tooth attachment device consisting of two mineralized tissues of alveolar bone cementum and a dense fibrous connective tissue periodontal ligament gum sandwiched therebetween. The periodontal ligament, a layer of unmineralized and tough tissue secretory fibers, is embedded in the cementum at one end and in the alveolar bone at the other end to anchor the teeth elastically and firmly in the alveolar fossa like a bridge. The elastic connection mode allows periodontal membrane to quickly respond and coordinate when the periodontal tissue is stimulated by the outside, and the specific processes comprise new bone deposition and old bone absorption, and the degradation and secretion formation of fibrous matrix. However, periodontal Ligament Stem Cells (HPDLSCs) play an important role in the process of stimulation response and physiological reconstruction due to their self-renewal and multi-directional differentiation potential. To better answer how periodontal disease is prevented and treated, we need to further study the role and related mechanisms that HPDLSCs play in. Several articles have been published to answer periodontal pathogenesis from HPDLSCs or regenerate periodontal tissue in vitro using HPDLSCs as seed cells. However, in the prior art, the tissue cell types are difficult to be accurately defined, most of the research can only be carried out by screening common mesenchymal stem cell surface markers, however, the stem cell markers are various, and the markers selected in different articles are different, so that the definition of the periodontal ligament stem cells seems to be rather lack of specificity, and on the other hand, the disordered differentiation regulation and control mechanism brought by the stem cell markers hinders the research of key mechanisms and the transformation of clinical treatment. Therefore, it is important to separate, screen and identify periodontal ligament-specific stem cells.

The single-cell RNA sequencing (scRNA-seq) breaks through the bottleneck of the prior art, and each cell in the tissue is sequenced to obtain the unique characteristics of the transcriptome, so that each cell is accurately grouped according to the transcriptome. With the support of single cell sequencing technology, it becomes possible to map tissue cells and to discover tissue-specific cell types. There have been reports of finding new subsets of tissue-specific stem cells in bone marrow, retina, and breast. Then for the periodontal ligament, is there such a population of tissue-specific stem cells? What, if any, play a role in the maintenance of periodontal tissue homeostasis and in the repair of tissue damage? How to promote the transformation of disease into the prognosis?

Disclosure of Invention

The technical problem is as follows: at present, periodontal scaling, root surface leveling and other methods are adopted in clinical tradition, only the lesion development can be controlled to a certain extent, and the damaged periodontal tissue regeneration and repair cannot be improved. The prior art is limited to stem cell based periodontal tissue engineering. But the tissue engineering results can not be clinically transformed due to the limitation of research technology-1 researchers can only screen periodontal ligament stem cells through common mesenchymal surface markers, and the cell populations obtained in this way are a group of mixed cell populations with different biological functions, different lineages and different differentiation tendencies, thereby having different prognosis on diseases. The prognosis of the disease cannot be really understood only by common surface markers, and the intervention is silent; (2) seed cells are the key to periodontal regenerative therapy. At present, embryonic stem cells and adult stem cells are used as sources of seed cells for periodontal tissue regeneration therapy. Some researchers select embryonic stem cells to carry out targeted gene editing to assist the formation of periodontal tissue fibers, but the materials are difficult to obtain, the ethical problem exists, and the universality is not high; BMSCs were also selected by researchers as subjects, but not considered for their allogenic antigen response and poor availability; (3) the tissue engineering uses the surface marker to screen cells, then carries out in vitro amplification culture, and transplants the cells into the dorsal subcutaneous tissues of nude mice to form periodontal tissue organoids. The in vitro simulation mode is different from the in vivo actual condition, and the in vivo condition cannot be truly reflected; in conclusion, none of the prior art has been able to isolate, screen and identify subpopulations of cells that maintain periodontal homeostasis from a heterogeneous population of cells.

The purpose of the invention is as follows: periodontal homeostasis is important for the development, maintenance, reconstruction, and development of a variety of oral treatment modalities for the oral and maxillo system. There is currently no clear determination as to the true cell population to maintain/restore periodontal homeostasis. According to the invention, the periodontal tissue is subjected to single cell sequencing, a specific periodontal ligament stem cell subset which can possibly maintain/recover periodontal homeostasis is screened through the transcriptome level, in vitro flow type sorting and multidirectional differentiation potential verification are carried out through marker genes, a CreER-loxP inducible reporter gene system is constructed by using model animals to carry out in vivo lineage tracing and verify the characteristics of the dryness and fate operation of the specific periodontal ligament stem cell subset and the core genome which maintains/recovers periodontal homeostasis, and therefore, the purposes of separating, screening and identifying the specific periodontal ligament stem cell subset are achieved.

In order to achieve the purpose, the invention provides the following technical scheme:

the method for screening the new sub-population of the periodontal ligament specific stem cells based on single cell sequencing comprises the following steps:

a. acquiring healthy premolar teeth which need to be extracted due to orthodontics;

b. preparing a single cell suspension of periodontal membrane tissue;

c. detecting the quality of the single cell suspension sample by cell staining;

d. sequencing and analyzing the single cell transcriptome;

e. biological information quasi-time sequence analysis finds possible new sub-groups of periodontal ligament stem cells, and high specificity and high sensitivity marker genes of the possible new sub-groups are determined;

f. in vitro selecting human periodontal ligament subgroup according to the marker gene, culturing in vitro, and verifying its dryness;

g. constructing a CreER mouse driven by a marker gene promoter of a candidate population for lineage tracing and cell elimination verification;

h. and g, carrying out in vivo verification by using the mouse model constructed in the step g.

Further, the new sub-population of periodontal ligament-specific stem cells obtained by the method of the present invention.

Furthermore, a new sub-population of periodontal ligament-specific stem cells specifically express FBLN2 and the membrane protein CADM3.

The periodontal ligament specific stem cell new subgroup is applied to preparing a medicament for treating periodontal diseases.

Has the beneficial effects that: the invention provides a method for screening a new sub-population of periodontal ligament specific stem cells based on single cell sequencing and application thereof, the invention obtains the sub-population of periodontal ligament specific stem cells by single cell sequencing, and carries out in vitro flow type sorting and verification of the performance of the cell sub-population by a marker gene; the cell subset screened by the invention is beneficial to searching intervention targets of periodontal steady imbalance, and creates possibility for developing new drugs; periodontal ligament specific stem cells are used as seed cells to form organoids, so that scientific basis is provided for research and verification of new materials of medical instruments; further explore the feasibility of the autologous/allogeneic transplantation of the periodontal ligament stem cells and provide a new idea for treating periodontal diseases.

Drawings

FIG. 1 is a schematic diagram of the preparation and single cell sequencing of a single cell suspension of human periodontal tissue.

FIG. 2 is a diagram of the analysis of the results of single cell transcriptome sequencing; a is a 2D-tSNE graph which shows the coloring result graph of the cluster analysis of 5562 periodontal ligament cell single-cell RNA-seq; b is the expression level and distribution diagram of known periodontal ligament cell types and representative marker genes shown by a single gene tSNE diagram and a Violin diagram; c is the expression level and profile of 5 typical MSC marker genes in periodontal ligament tissue shown in 2D-tSNE diagram; d is a cell type enriched gene heatmap; e is a GO analysis chart of a new sub-group of periodontal tissues; f is the expression level and profile of the novel subgroup marker gene shown by the tSNE diagram and the Violin diagram of the single gene.

FIG. 3 is a diagram of a periodontal tissue subdivision analysis; a is a result graph obtained by further subdividing original 3,5,6 and 8 cell populations to obtain O1-O9 cell subsets; b is a heat map of the gene enriched by the cell types of the sub-population; c is the expression level and profile of the 6 typical MSC marker genes in the cell population of the sub-population shown in the 2D-tSNE plot; d is a quasi-time sequence analysis display development locus diagram of each subgroup.

FIG. 4 is a graph of the results of in vitro validation of the properties of the new subpopulation; a is the expression level and the distribution diagram of the new subgroup marker genes shown by a single gene tSNE diagram and a Violin diagram; b is a graph showing the results of expressing FBLN2 by human and mouse periodontal membrane immunohistochemical IHC; c is a graph showing the results of expressing FBLN2 by mouse periodontal membrane immunofluorescence IF; d is a result graph obtained by selecting CD146 as a positive control and sorting a new subgroup in vitro by using CADM 3; e is CADM3+ cell in vitro monoclonal crystal violet staining, ALP, alizarin red and oil red O multidirectional differentiation staining pattern.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention. Example 1 method for screening a novel subpopulation of periodontal ligament-specific stem cells based on single cell sequencing

The invention provides a method for screening a new sub-population of periodontal ligament specific stem cells based on single cell sequencing, which comprises the following steps:

a. selecting healthy premolar teeth which need to be extracted due to orthodontics, requiring complete tooth shape, no caries, no tooth body, dental pulp, periodontal and periapical tissue diseases and no pollution during extraction (considering that teeth can be washed, gargled, disinfected and the like before tooth extraction), immediately placing the teeth into alpha-MEM culture solution containing 2% FBS and 2% double antibody after extraction, and quickly transferring the teeth into a biosafety cabinet at low temperature.

b. Single cell suspension preparation of periodontal Membrane tissue

(1) Clamping the crown of the tooth with the root of the tooth of a needle holder facing upwards in the biological safety cabinet sterilized by ultraviolet irradiation;

(2) repeatedly washing with 2% green/streptomycin double-antibody-containing PBS (washing root surface with PBS for 15-20 times) until the tooth surface is free of blood stain;

(3) scraping 1/3 of gingival tissues on the root by using a sterile surgical sharp blade, and keeping the root tissues wet;

(4) transferring the teeth with the gingival tissues removed into a 15ml centrifuge tube, adding 1.5ml of 3mg/ml of I-type collagenase and 1.5ml of 4mg/ml of II-type Dispase into each tooth, and digesting for 45min at 220rpm of a constant temperature shaker at 37 ℃;

(5) adding 10ml of alpha-MEM complete culture medium containing 10% FBS to terminate digestion, preliminarily removing impurities by passing through a 40 μm sieve, centrifuging at 500g for 7min, and removing the supernatant by pipetting;

(6) adding 6 times volume of erythrocyte lysate, incubating for 5min at 4 ℃, adding excessive PBS for washing for 1-2 times, 600g, centrifuging for 8min, removing supernatant, and adding 200ul BD buffer.

c. Single cell suspension sample quality detection

(1) Calcein and Draq7 fluorescence staining are used for detecting cell activity and cell quantity, and whether the requirements of single cell on-machine are met or not is judged.

d. Single cell transcriptome sequencing analysis

(1) Capturing single cells;

(2) CB/UMI label addition;

(3) RNA reverse transcription;

(4) constructing a library;

(5) high throughput sequencing.

A schematic diagram of the preparation of a single cell suspension of human periodontal tissue and single cell sequencing is shown in FIG. 1.

e. Biological information quasi-time sequence analysis finds possible new subgroup of periodontal ligament stem cells, and defines high specificity and high sensitivity marker gene of the possible new subgroup

And (3) data analysis: standardization (Cell Normalization), cell Clustering (Cell Cluster), rdsplot Analysis, cell regrouping (Cluster), pseudo-temporal Analysis (pseudo-temporal), gene function Analysis (GOanalysis), signaling Pathway Analysis (Pathway Analysis), SCENIC (Single Cell Regulation Network Inference and Cluster), quSAGE Analysis, cell communication Analysis (CellPhone Analysis), geneModule Analysis.

f. In vitro selecting human periodontal ligament subgroup according to marker gene, in vitro culturing, and verifying its dryness

(1) Human pericarp tissue was digested and enzymatically digested as described above to obtain a single cell suspension, cultured adherent with complete medium MEM (containing 10% fetal calf serum, 100U/ml penicillin, 100ug/ml streptomycin), changed at 3d for the first time, and the suspension cells were discarded, after which time every 3d was changed. After 80% confluence of cell growth, passage was performed with 0.25% pancreatin. And taking the cells with good growth in the 1 st to 4 th generations for subsequent flow sorting.

(2) Flow sorting

Digesting with 0.25% pancreatin to obtain periodontal ligament single cell suspension, sealing cell surface Fc receptor with CD16/CD32, and coupling with CADM3 of fluorescein ⁺ Antibody (marker gene encoding candidate cell subset) for cell surface marker staining, DAPI (0.05-0.2. Mu.g/mL) for dyingColor flow sorting the membrane protein high expression live cell population and simultaneously sorting MCAM ⁺ Cell subsets served as positive controls.

(3) Detecting stem cell characteristics

Osteogenic differentiation: replacement of osteogenic induction solution was performed when the cells reached 80% confluence, and every 3 days. The change of related protein and mRNA was detected at 7 days of induction, and alkaline phosphatase enzymatic chemical staining and alizarin red staining were performed at 7 days and 14 days, respectively.

Chondrogenic differentiation: 3-4X 10 ⁵ Resuspending the cells in 0.5ml complete chondrogenic differentiation medium of human bone marrow mesenchymal stem cells, placing at 37 ℃ and 5% CO ₂ Culturing in the incubator until the cell mass is gathered, replacing the cells with fresh chondroblast inducing and differentiating complete culture medium every 2-3 days until cartilage balls are formed, and carrying out Alixin blue staining identification.

Adipogenic differentiation: according to 2X 10 ⁴ cells/cm ² The cell density of (2) is inoculated in a six-hole plate, after 80% of cells are fused, 2ml of adipogenic induction differentiation culture medium A liquid is added into the six-hole plate, after 3 days of induction, the A liquid in the six-hole plate is sucked away, 2ml of adipogenic induction differentiation culture medium B liquid is added, after 24 hours, the B liquid is sucked away, the A liquid is changed back for induction, and after 3-5 times of alternate action of the A liquid and the B liquid (12-20 days), lipid drops are observed by a microscope.

Neurogenesis differentiation: the number of cells is 5000/cm ² Inoculating on polylysine coated glass slide, neurobasal medium, adding 1% ITS,100ng/ml basic fibroblast growth factor (bFGF) for 5 days; post-neurobasal medium supplemented with 100ng/ml bFGF,10ng/ml FGF8, 100ng/ml (SHH) for 5 days, post IF detection nestin, beta3-tubulin crystal violet staining: after fixation with 4% paraformaldehyde, washing with distilled water, the monoclonal colonies formed were observed microscopically after staining with crystal violet staining solution.

g. Construction of candidate population creER mice driven by marker gene promoter for lineage tracing and cell elimination verification

(1) Modifying candidate marker gene by CRISPR/Cas9 technology, and inserting P2A-Cre before stop codon ^ERT2 Obtaining the tamoxifen-induced conditional Cre expression mouse modelAnd constructing a CreER mouse driven by the marker gene promoter of the candidate population.

(2) Constructing an inducible conditional fluorescent expression mouse model: marker gene-Cre ^ERT2 Mouse and R26 ^tdTomato The mice are hybridized for the first generation to obtain the induction type conditional fluorescence expression mice.

(3) Constructing an inducible cell-knockout mouse model: marker gene-Cre ^ERT2 And R26 ^DTA (Rosa 26-loxP-STOP-loxP-DTR) mice were mated to obtain a knockout model mouse.

h. In vivo validation

(1) Induced conditional fluorescence expression mice were used to analyze the fate transition and functional effects of specific cell subsets by lineage tracing.

(2) The induced cell knockout mouse model is utilized to verify the function and function of the periodontal ligament specific stem cell subset in the processes of periodontal tissue growth reconstruction and tissue injury repair.

(3) Specific cell subsets are sorted in a flow mode, and after in vitro culture, the biological material is implanted into immunodeficient mice to verify whether periodontal tissue organoids can be formed or not.

Example 2 analysis of Experimental results

(1) Single cell transcriptome sequencing analysis to discover new subpopulations

Periodontal tissue maps were generated from single cell transcriptome sequencing analysis, and were divided into 14 subsets, fibroblasts (0-4), osteoblasts (5), new subpopulations (6), endothelial cells (7), pericytes (8), T cells (9), epithelial cells (10), macrophages (11), neutrophils (12), monocytes (13), and possibly schwann cells (14). As can be seen from FIG. 2, the sequencing of single-cell transcriptome revealed that there may be a new sub-population specific to periodontal ligament tissue (population 6), the 2D-tSNE graph of FIG. 2A shows the staining result of single-cell RNA-seq cluster analysis of 5562 periodontal ligament cells, and the new sub-population of periodontal ligament tissue is circled in dark blue color; 2B, single gene tSNE and Violin plots showing the expression levels and distribution of known periodontal ligament cell types and representative marker genes; 2C 2D-tSNE plot showing expression levels and distribution of 5 typical MSC marker genes in periodontal membrane tissue, with a gradual color from grey to red indicating low to high gene expression levels; 2D is a cell type enriched gene heatmap, each column representing a cell and each row representing a marker gene; 2E new sub-population of periodontal tissue (population 6) GO map for its possible functional effects; the single gene tSNE and Violin plots for 2F show the expression levels and distribution of the marker genes for the new subpopulation (population 6).

(2) Further sub-clustering of the known population and the new sub-population, by common MSC labeling and modeling timing analysis, it is speculated that the new sub-population (population 6) may be a sub-population of periodontal tissue-specific stem cells

To further understand the function of the new subpopulations, the original 3,5,6,8 cell population was further subdivided into O1-O9 cell subpopulations, in which O2 and O6 subpopulations were separated from the original new subpopulation (new transplantation), as shown in FIG. 3A; the cell type enriched gene heatmap of the population of cells is shown in FIG. 2B, with each column representing a cell and each row representing a marker gene; FIG. 3C is a 2D-tSNE plot showing the expression levels and distribution of 6 typical MSC marker genes in a cell population of a subpopulation, the gradual change in color from gray to red indicating a low to high gene expression level, indicating that common MSC markers are more centrally expressed in the new subpopulation; FIG. 3D is a graph showing the development trajectory of each subpopulation by pseudo-temporal analysis showing that the new subpopulation is located at the position of the origin upstream of development, thereby suggesting that O6 is a periodontal tissue-specific stem cell.

(3) In vitro verification that the subgroup has dryness and multidirectional differentiation potential

Analyzing the high expression gene of O6, selecting the marker genes of a new subgroup from specificity and sensitivity, namely FBLN2 and membrane protein CADM3, and displaying the expression level and distribution of the marker genes of the new subgroup in a graph of FIG. 4A; immunohistochemistry and immunofluorescent staining results confirmed the specific perivascular expression of FBLN2 in the periodontal ligament of human and mouse, see fig. 4B-C; further utilizing the subgroup specificity to express the membrane protein CADM3, carrying out flow sorting, in-vitro culture, observation and multidirectional differentiation to determine the dryness of the membrane protein CADM 3; the previous MSC marker CD146, commonly used by humans, was used as a positive control to sort out cell populations strongly positive for CADM3, see fig. 4D; in vitro monoclonal crystal violet staining, ALP, alizarin red, oil red O multi-directional differentiation staining of CADM3+ cells of fig. 4E show that CADM3+ cells have the ability to form monoclonal colonies in vitro and the multi-directional differentiation potential, indicating that they are dry.

Claims (1)

1. The method for screening the periodontal ligament specific stem cells based on single cell sequencing is characterized by comprising the following steps of:

a. acquiring healthy premolar teeth which need to be extracted due to orthodontics;

b. preparing a single cell suspension of periodontal membrane tissue;

c. detecting the quality of the single cell suspension sample by cell staining;

d. sequencing and analyzing the single cell transcriptome;

e. biological information pseudo-timing analysis finds high-sensitivity marker genes, wherein the marker genes are genes for specifically expressing FBLN2 and membrane protein CADM 3;

f. in vitro selecting specific stem cells of human periodontal ligament according to CADM3, culturing in vitro, and verifying the dryness;

g. constructing a CreER mouse driven by a marker gene promoter of a candidate population for lineage tracing and cell elimination verification;

h. and g, carrying out in vivo verification by using the mouse model constructed in the step g.

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