WO2023174447A1 - 间充质干细胞创面修复优势功能亚群及其鉴定和应用 - Google Patents

间充质干细胞创面修复优势功能亚群及其鉴定和应用 Download PDF

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WO2023174447A1
WO2023174447A1 PCT/CN2023/094202 CN2023094202W WO2023174447A1 WO 2023174447 A1 WO2023174447 A1 WO 2023174447A1 CN 2023094202 W CN2023094202 W CN 2023094202W WO 2023174447 A1 WO2023174447 A1 WO 2023174447A1
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cell
wound repair
stem cells
subpopulation
cells
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陈小松
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陈小松
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention belongs to the biological field, and specifically relates to a mesenchymal stem cell wound repair superior functional subgroup and its identification and application.
  • HUCMSC Human umbilical cord mesenchymal stem cells
  • Single-cell transcriptome sequencing technology can realize the reproduction of genes in a single cell, which is of great significance for discovering cell heterogeneity, revealing cell functions, elucidating the relationships between groups (across-omes), and establishing a complete map.
  • Single-cell multi-omics ATAC+GEX Assay for transposase-accessible chromatin and gene expression
  • ATAC+GEX Assay for transposase-accessible chromatin and gene expression
  • Single-cell multi-omics ATAC+GEX Assay for transposase-accessible chromatin and gene expression
  • ATAC+GEX Assay for transposase-accessible chromatin and gene expression
  • RNA and chromatin accessibility in a single cell, more deeply characterize cell types of different lineages, and capture cell heterogeneity , combining epigenetic expression profiles and gene expression profiles to infer the full picture of the molecular regulatory network and obtain more realistic and systematic molecular change information, which is a new direction for panoramic analysis of the spatiotemporal heterogeneity of umbilical cord mesenchymal stem cells.
  • Flow cytometry is a rapidly developing biomedical analysis technology that integrates laser technology, photoelectric measurement technology, computer technology, fluid mechanics, cell immunofluorescence chemistry technology, and monoclonal antibody technology. New high-tech cell analysis technology.
  • Flow cytometer is an instrument built using flow cytometry. It allows cells or particles to flow in a liquid flow, pass through an incident light beam one by one, and use a high-sensitivity detector to record scattered light and various fluorescence signals, thereby enabling multi-parameter analysis of particles, including particle shape and size, cell cycle, etc. , intracellular cytokines, bacterial surface antigens, cellular DNA contents, etc.
  • the particles detected by FCM are generally active cells.
  • FCM can also perform cell sorting, which can be sorted according to certain properties of the detected cells, and if the sorting process is carried out under sterile conditions, these cells can also be used for culture.
  • the technical problem to be solved by the present invention is to overcome the defects of inconsistent repair efficacy, poor repeatability and low implantation rate of mesenchymal stem cells used for wound repair in the prior art, and to provide a mesenchymal stem cell wound repair superior functional cell subtype. Groups and their identification and applications.
  • the advantageous functional cell subpopulation for wound repair of the present invention has the same functional characteristics and good reproducibility, and can be used to accelerate wound healing.
  • the inventors conducted heterogeneity exploration and experimental demonstration of mesenchymal stem cells, such as umbilical cord mesenchymal stem cells, using single-cell multi-omics ATAC+GEX and spatial transcriptome sequencing, and applied single-cell multi-modal omics multi-dimensional and comprehensive
  • the heterogeneity map of umbilical cord mesenchymal stem cells was drawn, and combined with the inventor's research results in clinical practice and wound repair, the dominant functional subpopulation related to wound healing was finally screened out through flow cell sorting technology, and the This subpopulation was expanded, characterized, and initially verified in animal models.
  • the present invention solves the above technical problems through the following technical solutions.
  • the first aspect of the present invention provides a biomarker combination of cells with dominant functions in wound repair, and the biomarker combination includes CD29, CD142 and CYR61.
  • the CD29 is also called ITGB1, and the CD142 is also called F3.
  • the second aspect of the present invention provides a biomarker combination of cells with dominant functions in wound repair, and the biomarker combination includes CD29, CD142 and S100A9.
  • the wound refers to the damage caused by external wounding factors on normal skin, which damages the skin function.
  • the wound includes loss of part of normal tissue, necrosis of part of tissue cells, and stress reaction of part of tissue.
  • the biomarker combination may also include one or more biomarkers selected from the following: DCN, TGFBl, COL3A1, COLIA2 and COLIA1.
  • the biomarker combination may also include the following markers: DCN, TGFBl, COL3A1, COLIA2 and COLIA1.
  • the biomarker combination may also include one or more biomarkers selected from the following: DCN, TGFBl, COL3A1, COLIA2, COLIA1, NEAT1, TMP1, GFBP4, SAT1, TGM2, LUM, SERPNF1, MXD4, FOS, ACTA2, HTRA3, HTRA1, PNRC1, APOE and CDKN1C.
  • the biomarker combination also includes the following markers: DCN, TGFB1, COL3A1, COLIA2, COLIA1, NEAT1, TMP1, GFBP4, SAT1, TGM2, LUM, SERPNF1, MXD4, FOS, ACTA2, HTRA3, HTRA1, PNRC1, APOE and CDKN1C.
  • the third aspect of the present invention provides a wound repair superior functional cell preparation, which includes CD29, CD142 and CYR61 positive stem cells.
  • the fourth aspect of the present invention provides a wound repair superior functional cell preparation, which includes CD29, CD142 and S100A9 positive stem cells.
  • the stem cells are preferably mesenchymal stem cells.
  • the stem cells preferably express one or more biomarkers selected from DCN, TGFB1, COL3A1, COLIA2 and COLIA1.
  • the stem cells more preferably express one or more biomarkers selected from NEAT1, TMP1, GFBP4, SAT1, TGM2, LUM, SERPNF1, MXD4, FOS, ACTA2, HTRA3, HTRA1, PNRCl, APOE and CDKN1C. .
  • a fifth aspect of the present invention provides a cell subpopulation, the cell subpopulation comprising CD29, CD142 and CYR61 positive stem cells; the proportion of the stem cells in the cell subpopulation is at least 90%.
  • a sixth aspect of the present invention provides a cell subpopulation, the cell subpopulation comprising CD29, CD142 and S100A9 positive stem cells; the proportion of the stem cells in the cell subpopulation is at least 90%.
  • the positive expression ratio of the biomarker in the cell subpopulation is preferably at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% .
  • the stem cells are preferably as described in the third or fourth aspect.
  • a seventh aspect of the present invention provides a pharmaceutical composition, which includes stem cells and pharmaceutically acceptable carriers and/or excipients; the stem cells are as described in the third or fourth aspect.
  • An eighth aspect of the present invention provides a pharmaceutical combination, which includes a first therapeutic agent and a second therapeutic agent; the first therapeutic agent includes the pharmaceutical composition according to the seventh aspect.
  • the second therapeutic agent is selected from one or more of immunosuppressants, analgesics and anti-infective agents.
  • the ninth aspect of the present invention provides a biomarker combination as described in the first or second aspect, a wound repair advantageous functional cell preparation as described in the third or fourth aspect, as described in the fifth or sixth aspect.
  • a tenth aspect of the present invention provides a method for promoting wound repair, which method includes administering to a subject in need a wound repair superior functional cell preparation as described in the third or fourth aspect, such as the fifth aspect or The cell subpopulation according to the sixth aspect, the pharmaceutical composition according to the seventh aspect, or the pharmaceutical combination according to the eighth aspect.
  • the eleventh aspect of the present invention provides a method for identifying a predominant functional cell subpopulation in wound repair for non-diagnostic purposes.
  • the method includes:
  • the cell subpopulation is the wound repair superior functional cell subpopulation
  • the characteristic gene expresses the biomarker combination described in the first aspect or the second aspect.
  • the non-diagnostic purpose means that the predominant functional cell subpopulation for wound repair obtained according to the identification method cannot be used as a single clinical indicator for wound repair; the wound repair still requires the experience and professional analysis of clinical medical personnel. It is judged that the superior functional cell subpopulation for wound repair of the present invention will be an important reference index.
  • the positive expression ratio of the biomarker combination as described in the first aspect or the second aspect in the cell subpopulation is at least 90%, for example, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100%.
  • the single-cell multimodal omics technology may be routine in the art, for example, including single-cell multimodal omics technology such as ATAC+GEX technology.
  • the cells are mesenchymal stem cells; for example, umbilical cord mesenchymal stem cells.
  • the mesenchymal stem cells are the wound repair superior functional subpopulation (Hr-MSC) of human umbilical cord mesenchymal stem cells. This concept was first proposed by the inventor.
  • (1) is followed by (1-1): obtaining positioning information of the target candidate cell subpopulation through spatial transcriptomics, where the positioning information includes enrichment status and positioning status.
  • a twelfth aspect of the present invention provides a system for identifying superior functional subpopulation cells for wound repair, the system including a typing module and a gene display module;
  • the typing module performs molecular typing on the cells to be tested through single-cell multimodal omics technology to obtain target candidate cell subpopulations;
  • the gene display module performs characteristic gene analysis on the target candidate cell subpopulations obtained by the typing module. Display and sort to obtain functional cells expressing characteristic genes, which are the superior functional subpopulation cells for wound repair;
  • the characteristic gene includes/expresses the biomarker combination as described in the first aspect or the second aspect.
  • the positive expression ratio of the marker combination as described in the first aspect or the second aspect in the functional cell subpopulation is at least 90%, for example, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100%.
  • a thirteenth aspect of the present invention provides a kit for identifying the biomarker combination as described in the first or second aspect, or the cell subpopulation as described in the fifth or sixth aspect.
  • the kit includes a reagent for detecting the expression level of the biomarker combination as described in the first aspect or the second aspect.
  • the reagents are, for example, reagents for detecting the protein expression level of the marker, or reagents for detecting the gene or transcription level of the marker.
  • the kit includes the marker combination as described in the first aspect or the second aspect.
  • the cell preparation of the present invention can be used to prepare materials for regenerative medicine.
  • the stem cells are cells with the ability to differentiate into various cells and the ability to self-renew, including embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, umbilical cord blood stem cells, adipose stem cells, pluripotent stem cells, and induced pluripotent stem cells. wait.
  • the term "positive” means that cells can be sorted as positive cells by FACS, or the expression of corresponding markers can be detected by RT-PCR.
  • the present invention selects the superior functional subpopulation Hr-MSC with the function of promoting wound healing.
  • a comprehensive preliminary screening was conducted based on the expression of Marker genes in each subgroup, the functional enrichment of each subgroup, the expression of transcription factors of each subgroup, and combined with Cellmarker, PanglaoDB, EBI and other databases.
  • the preliminary screening includes: positioning: subcellular positioning of subgroup Marker genes and tissue positioning of immunofluorescence; quantification: horizontal comparison of the proportions of subgroups in different generations, different individuals, and different genders.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the wound repair superior functional subpopulation (Hr-MSC) of the present invention can effectively promote the healing of wounds (including various types of acute wounds, chronic refractory wounds, etc.), and can be used to accelerate the healing of various clinical acute and chronic wounds. Intervene at each stage in order to achieve the goal of accelerating scar-free wound healing; and provide sufficient data support and technical support for the research and development of innovative stem cell drugs, accelerate the clinical application of innovative stem cell drugs, and facilitate customized development to shorten the course of the disease and improve the quality of wound healing. "Innovative Stem Cell Drugs" and finally establish a scientific and reasonable stem cell application standard system.
  • Figure 1 is a schematic diagram of the umbilical cord mesenchymal stem cell grouping results.
  • FIGS 2A to 2D are schematic diagrams of the heat map results of the Marker genes of Hr-MSCs.
  • Figure 3A and Figure 3B are schematic diagrams of the expression levels of Marker genes in Hr-MSCs.
  • Figure 4 shows the functional enrichment analysis and gene heat map display of 13 clusters.
  • Figure 5 shows the UMAP grouping diagram of four different WJ-MSCs subpopulations.
  • Figure 6 is a schematic diagram of the umbilical cord stem cell grouping results.
  • Figure 7 is a schematic diagram of the sorting results of CD29 + /CD142 + /S100A9 + Hr-MSC.
  • Figure 8 is a schematic diagram of the wound healing efficiency results of Example 6.
  • Figure 9 is a schematic diagram of the migration ability of CD29 + /CD142 + /S100A9 + Hr-MSC on HDF and HUCV.
  • Figure 10 is a schematic diagram of zebrafish skin wound healing.
  • Figure 11 is a system 21 for identifying superior functional cell subpopulations in wound repair, which includes a typing module 11 and a gene display module 21.
  • This example uses single-cell transcriptomics to sequence and analyze umbilical cord mesenchymal stem cells.
  • the umbilical cord stem cells are initially divided into different subpopulations at the single cell level.
  • Bioinformatics analysis is used to functionally annotate each subpopulation to determine the target candidate subpopulation. .
  • Sample human umbilical cord Huatong jelly tissue
  • the enrolled pregnant women gave birth at 37-40 weeks of gestation, and the umbilical cords of newborns were selected for experiments as the source of subsequent P0 and P3 generation umbilical cord mesenchymal stem cells, number: PUM-A1-000152;
  • Each run provides 8 channels, and each channel can collect 100 to 10,000 cells;
  • umbilical cord mesenchymal stem cells have limited heterogeneity. It is preliminarily determined that different individuals, different generations, and different genders have the same cell composition; different individuals, different generations, and different genders have different cell proportions; different Individuals, different generations, and cells of different genders express genes differently.
  • This example creates a gene expression heat map for the Marker genes of each subpopulation of umbilical cord stem cells obtained in Example 1, and the results are shown in Figures 2A to 2D.
  • the Marker genes in Figure 2A to Figure 2D include: UBE2C, TOP2A, CDK1, CENPF, KPNA2, CKS2, PLK1, HMGB2, AURKA, ARL6IP1, GTSE1, MKI67, ASPM, CENPE, NUSAP1, SGO2, AURKB, TPX2, UBE2S, SMC4 , NEAT1, ACTA2, MXD4, TIMP1, LMCD1, TAGLN, DCN, IGFBP4, TGM2, PAPPA, ANGPTL4, COL8A1, KCNE4, INHBA, MEG3, SAT1, TUBA1A, CDKN1A, SELENOM, SAA1, CDC20, CCNB1, NCL, PNN, HSP90AA1 , HSPA8, DDX21, SFPQ, SLBP, CCNB2, SRSF7, TUBB4B, ODC1, PTTG1, BIRC5, HIST1H4C, HIST1H1A, HIST1H
  • DCN is expressed in all subgroups, but is expressed in the 4th subgroup (Cluster 4). reached the highest level, and the remaining figures and gene analyzes were the same as before. It can be seen that the expression levels of Marker genes are different.
  • clusters 1, 4 and 11 have ACTA2, TGM2, FOS, TGFB1, FLNA and COL3A1 as characteristic genes.
  • the cluster mainly enriches biological processes such as glucocorticoid response, pericyte differentiation, putrescine catabolism, and extracellular matrix organization; cluster 10 highly expresses S100A10, PDLIM1, and CAV1; clusters 5, 8, and 12 are characterized by UBE2S, TAGLN, TPM1 and TMSB4X are highly expressed, and the significant genes in clusters 6 and 9 are mainly involved in immune regulation and wound repair-related pathways. These genes include B4GALT1, HEG1, CXCL3, CXCL1 and HMOX1.
  • proliferative stem cells proliferative_MSC; composed of clusters 0, 2, 3 and 7
  • microenvironment supporting stem cells niche-supporting_MSC; composed of clusters 1, 4 and 11
  • metabolic stem cells metabolic stem cells
  • biofunctional type_MSC functional stem cells; composed of clusters 6 and 9, namely CD29 of this application + /CD142 + /S100A9 + Hr-MSC
  • CD29, CD142 and CYR61 of biofunctional type MSC were used as candidate markers for Hr-MSC; and CD29, CD142 and S100A9 of biofunctional type MSC were used as candidate markers for Hr-MSC for follow-up respectively.
  • the positive expression ratio of the above-mentioned selected markers in each subpopulation of Hr-MSC is at least 90%.
  • this example uses single-cell transcriptomic sequencing analysis to obtain the Marker genes of a subpopulation with target therapeutic functions.
  • the target subpopulation is extracted from primary mixed umbilical cord interstitial cells. Mesenchymal stem cells were sorted. As shown in Figure 6, this subpopulation accounted for about 5.310% of the total cells.
  • This subgroup was identified by flow cytometry with positive markers: CD29+, CD142+, and CYR61+.
  • the target subpopulation that is all positive for these three indicators is P6, accounting for 5.3% of the total stem cells, and is the target subpopulation for subsequent expansion.
  • Example 5 Sorting of CD29 + /CD142 + /S100A9 + Hr-MSC
  • this example uses single-cell transcriptomic sequencing analysis to obtain the Marker genes of a subpopulation with target therapeutic functions.
  • the target subpopulation is extracted from primary mixed umbilical cord interstitial cells. mesenchymal stem cells. This subgroup was identified by flow cytometry with positive markers: CD29+, CD142+, and S100A9+. As shown in Figure 7, the target subpopulation that is positive for these three indicators accounts for 9.53% of the total stem cells, which is the target subpopulation for subsequent expansion.
  • This example creates a full-thickness skin defect in adult zebrafish, and evaluates the healing time, that is, the healing efficiency, of different administrations (i.e., the following groups) of the defect wound in adult zebrafish to verify the effectiveness (for model construction and effectiveness verification, please refer to Kennard Andrew S et al., Theriot Julie A.(2021).Wounding Zebrafish Larval Epidermis by Laceration.Bio Protoc,11(24),e4260; Mhlongo Fikile et al., Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.[J ] .J Ethnopharmacol,2022,286:114867).
  • This example creates a full-thickness skin defect in adult zebrafish, and evaluates the healing time, that is, the healing efficiency, of different administrations (i.e., the following groups) of the defect wound in adult zebrafish to verify the effectiveness (for model construction and effectiveness verification, please refer to Kennard Andrew S et al., Theriot Julie A.(2021).Wounding Zebrafish Larval Epidermis by Laceration.Bio Protoc,11(24),e4260; Mhlongo Fikile et al., Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.[J ] .J Ethnopharmacol,2022,286:114867). details as follows:
  • the wound healing efficiency of the positive sorting group reached 0.857 ⁇ 0.050
  • the wound healing efficiency of the control group, negative sorting MSC group and unsorted MSC group were 0.575 ⁇ 0.110, 0.648 ⁇ 0.129, 0.646 ⁇ 0.129, respectively.
  • the wound healing efficiency of the sorting group was statistically different, indicating that CD29 + /CD142 + /S100A9 + MSC could better promote wound healing.
  • Example 8 System for identifying dominant functional cell subpopulations in wound repair
  • This embodiment provides a system 21 for identifying superior functional cell subpopulations for wound repair, as shown in Figure 11 , which includes: a typing module 11 and a gene display module 21.
  • the typing module 11 performs molecular typing on the cells to be tested through single-cell multimodal omics technology to obtain target candidate cell subpopulations.
  • the gene display module 12 displays and sorts the characteristic genes of the target candidate cell subpopulations obtained by the typing module 11 to obtain a cell subpopulation containing the characteristic genes.
  • the resulting cell subpopulation is a subpopulation of cells with superior wound repair function.

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Abstract

本发明公开了一种间充质干细胞创面修复优势功能亚群(Hr-MSC)及其鉴定和应用。所述间充质干细胞创面修复优势功能亚群具有包括CD29、CD142和CYR61或S100A9的生物标志物组合。本发明的创面修复优势功能亚群能够高效促进创面(包括各类急性创面、慢性难愈性创面等)愈合，可用于加速临床各类急慢性创面的愈合，在创面愈合的各个阶段予以干预，以期达到加快创面无瘢痕愈合目标；并且为干细胞创新药物的研发提供充分的数据支撑及技术支持，加速干细胞创新药物的临床应用，有利于定制化开发缩短病程、提高创面愈合质量的"干细胞创新药物"，最终建立科学合理的干细胞应用标准体系。

Description

间充质干细胞创面修复优势功能亚群及其鉴定和应用
本申请要求申请日为2022年3月14日的中国专利申请CN202210249611.4的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物领域，具体涉及一种间充质干细胞创面修复优势功能亚群及其鉴定和应用。
背景技术
人脐带间充质干细胞(Human umbilical cord mesenchymal stem cell，HUCMSC)是理想的种子细胞之一，其在组织损伤修复领域已展现出良好的应用前景。然而，回顾诸多研究发现，干细胞治疗效果存在着不同程度的差异性，植入效率以及整合率均较低，究其根本是因为间充质干细胞(mesenchymal stem cell，MSC)功能变异和细胞异质性。
单细胞转录组测序技术可实现单个细胞内的基因重现，对于发现细胞异质性、揭示细胞功能、阐述各组间关系(across-omes)以及建立完整图谱等方面具有重大的意义。
单细胞多组学ATAC+GEX(Assay for transposase-accessible chromatin and gene expression)技术可在单个细胞内同时检测RNA和染色质可及性，更深入地表征不同谱系的细胞类型，捕获细胞异质性，结合表观遗传表达谱及基因表达谱推测分子调控网络的全貌，获得更真实、系统的分子变化信息，对于全景式解析脐带间充质干细胞时空异质性是一个全新方向。
流式细胞术(Flow cytometry，FCM)是一项发展迅速的生物医学分析技术，它是集激光技术、光电测量技术、计算机技术、流体力学以及细胞免疫荧光化学技术、单克隆抗体技术为一体的新型高科技细胞分析技术。流式细胞仪是应用流式细胞术建立起来的仪器装置。它使细胞或微粒在液流中流动，逐个通过一入射光束，并用高灵敏度检测器记录下散射光及各种荧光信号，从而得以对粒子进行多参数分析，包括诸如粒子形状和大小、细胞周期、细胞内细胞因子、细菌表面抗原、细胞DNA内含物等。FCM检测的粒子一般为活性细胞，通过激光光源激发细胞上所标记的荧光物质的强度和颜色以及散射光的强度可以得到细胞内部各种各样的生物信息。另外FCM还可进行细胞分选，可以根据所检测细胞的某种性质进行分选，并且如果分选的过程是在无菌条件下进行的，这些细胞还可以用来培养。
发明内容
本发明所要解决的技术问题是为了克服现有技术中用于创面修复的间充质干细胞修复效力不一致、重复性差、植入率低的缺陷，提供一种间充质干细胞创面修复优势功能细胞亚群及其鉴定和应用。本发明的创面修复优势功能细胞亚群具有相同的功能特性，重复性好，可用于加速创面的愈合。
发明人对间充质干细胞例如脐带间充质干细胞进行了异质性探讨和实验论证，采用单细胞多组学ATAC+GEX及空间转录组测序，应用单细胞多模态组学多维度全方位绘制出了脐带间充质干细胞异质性图谱，并结合发明人在临床实践以及创面修复中的研究成果，通过流式细胞分选技术最终筛选出与创面愈合相关的优势功能亚群，并对该亚群进行扩增、鉴定及在动物模型初步验证。
本发明通过以下技术方案解决上述技术问题。
本发明的第一方面提供一种创面修复优势功能细胞的生物标志物组合，所述生物标志物组合包括CD29、CD142和CYR61。
所述CD29也称ITGB1，所述CD142也称F3。
本发明的第二方面提供一种创面修复优势功能细胞的生物标志物组合，所述生物标志物组合包括CD29、CD142和S100A9。
本发明中，所述创面是指外界致伤因子在正常皮肤上所造成的损害，使皮肤功能受损。所述创面包括部分正常组织丢失、部分组织细胞坏死和部分组织发生应激反应。
本发明中，所述生物标志物组合还可包括选自以下一种或多种的生物标志物：DCN、TGFB1、COL3A1、COLIA2和COLIA1。
本发明中，所述生物标志物组合还可包括以下标志物：DCN、TGFB1、COL3A1、COLIA2和COLIA1。
本发明中，所述生物标志物组合还可包括选自以下一种或多种的生物标志物：DCN、TGFB1、COL3A1、COLIA2、COLIA1、NEAT1、TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE和CDKN1C。
本发明中，所述生物标志物组合还包括以下标志物：DCN、TGFB1、COL3A1、COLIA2、COLIA1、NEAT1、TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE和CDKN1C。
本发明的第三方面提供一种创面修复优势功能细胞制剂，所述创面修复优势功能细胞制剂包含CD29、CD142和CYR61阳性的干细胞。
本发明的第四方面提供一种创面修复优势功能细胞制剂，所述创面修复优势功能细胞制剂包含CD29、CD142和S100A9阳性的干细胞。
本发明中，所述干细胞优选为间充质干细胞。
本发明中，所述干细胞优选表达选自DCN、TGFB1、COL3A1、COLIA2和COLIA1中的一种或多种生物标志物。
本发明，所述干细胞更优选表达选自NEAT1、TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE和CDKN1C中的一种或多种生物标志物。
本发明的第五方面提供一种细胞亚群，所述细胞亚群包含CD29、CD142和CYR61阳性的干细胞；所述干细胞在所述细胞亚群的比例至少为90％。
本发明的第六方面提供一种细胞亚群，所述细胞亚群包含CD29、CD142和S100A9阳性的干细胞；所述干细胞在所述细胞亚群的比例至少为90％。
本发明中，所述细胞亚群中，所述生物标志物的阳性表达比例优选至少为92％、至少为95％、至少为97％、至少为98％、至少为99％或至少为100％。
本发明中，所述干细胞优选如第三方面或第四方面所述。
本发明的第七方面提供一种药物组合物，所述药物组合物包括干细胞以及药学上可接受的载体和/或辅料；所述干细胞如第三方面或第四方面所述。
本发明的第八方面提供一种药物组合，所述药物组合包括第一治疗剂和第二治疗剂；所述第一治疗剂包含如第七方面所述的药物组合物。
本发明一些实施方案中，所述第二治疗剂选自免疫抑制剂、止痛剂和抗感染剂中的一种或多种。
本发明的第九方面提供一种如第一方面或第二方面所述的生物标志物组合、如第三方面或第四方面所述的创面修复优势功能细胞制剂、如第五方面或第六方面所述的细胞亚群、如第七方面所述的药物组合物或者如第八方面所述的药物组合在制备促进创面修复的制剂中的应用。
本发明的第十方面提供一种促进创面修复的方法，所述方法包括向有需要的受试者施用如第三方面或第四方面所述的创面修复优势功能细胞制剂、如第五方面或第六方面所述的细胞亚群、如第七方面所述的药物组合物或者如第八方面所述的药物组合。
本发明的第十一方面提供一种非诊断目的的创面修复优势功能细胞亚群的鉴定方法，所述方法包括：
(1)基于单细胞多模态组学技术对待测细胞进行分子分型，获得目标候选细胞亚群；
(2)对(1)中获得的目标候选细胞亚群进行特征基因展示和分选，获得包含特征基因的细胞亚群，所述细胞亚群即为创面修复优势功能细胞亚群；
所述特征基因表达如第一方面或第二方面所述的生物标志物组合。
本发明中，所述非诊断目的是指：根据所述鉴定方法获得的创面修复优势功能细胞亚群并不能作为创面修复的单一临床指标；所述创面修复仍需要临床医疗人员的经验和专业分析判断，本发明的创面修复优势功能细胞亚群将是重要的参考指标。
本发明一些实施方案中，所述细胞亚群中，如第一方面或第二方面所述的生物标志物组合的阳性表达比例至少为90％，例如至少为92％、至少为95％、至少为97％、至少为98％、至少为99％或至少为100％。
本发明一些实施方案中，所述单细胞多模态组学技术可为本领域常规，例如包括单细胞多组学技术例如ATAC+GEX技术。
本发明一些实施方案中，所述细胞为间充质干细胞；例如为脐带间充质干细胞。
本发明中，所述间充质干细胞为人脐带间充质干细胞的创面修复优势功能亚群(Hr-MSC)，该概念为发明人所首次提出。
本发明一些实施方案中，所述(1)后包括(1-1)：通过空间转录组学获得所述目标候选细胞亚群的定位信息，所述定位信息包括富集情况、定位情况。
本发明的第十二方面提供一种鉴定创面修复优势功能亚群细胞的系统，所述系统包括分型模块和基因展示模块；
其中，所述分型模块通过单细胞多模态组学技术对待测细胞进行分子分型，获得目标候选细胞亚群；所述基因展示模块对分型模块获得的目标候选细胞亚群进行特征基因展示和分选，获得表达特征基因的功能细胞，所述功能细胞即为创面修复优势功能亚群细胞；
所述特征基因包括/表达如第一方面或第二方面所述的生物标志物组合。
本发明一些实施方案中，所述功能细胞亚群中，如第一方面或第二方面所述的标志物组合的阳性表达比例至少为90％，例如至少为92％、至少为95％、至少为97％、至少为98％、至少为99％或至少为100％。
本发明的第十三方面提供一种用于鉴定如第一方面或第二方面所述的生物标志物组合、或者如第五方面或第六方面所述的细胞亚群的试剂盒。
本发明的一些实施方案中，所述试剂盒包含检测如第一方面或第二方面所述的生物标志物组合的表达水平的试剂。所述试剂例如用于检测标志物的蛋白表达水平的试剂，或用于检测标志物的基因或转录水平的试剂。
本发明一些实施方案中，所述试剂盒包含如第一方面或第二方面所述的标志物组合。
本发明的细胞制剂可用于制备用于再生医疗的材料。
本发明中，所述干细胞为具有分化为各种细胞的能力以及自我更新能力的细胞，包括胚胎干细胞、间充质干细胞、脐带干细胞、脐带血干细胞、脂肪干细胞、多能干细胞、诱导多能干细胞等。
本发明中，所述阳性是指，细胞能够通过FACS分选为阳性细胞，或者能够通过RT-PCR检测到相应标志物的表达。
本发明基于单细胞多模态组学的分群及特征基因展示结果，筛选具有促进创面愈合功能的优势功能亚群Hr-MSC。依据Marker基因在各亚群中的表达情况、各亚群功能富集情况、各亚群转录因子表达情况以及结合Cellmarker、PanglaoDB、EBI等数据库进行综合的初步筛选。其中，将单细胞多组学ATAC+GEX技术及空间转录组学技术应用于脐带华通胶间充质干细胞空间异质性的研究为本发明首创。所述初步筛选包括：定位：亚群Marker基因亚细胞定位及免疫荧光的组织定位；定量：横向比较亚群在不同代次、不同个体、不同性别的比例。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的创面修复优势功能亚群(Hr-MSC)能够高效促进创面(包括各类急性创面、慢性难愈性创面等)愈合，可用于加速临床各类急慢性创面的愈合，在创面愈合的各个阶段予以干预，以期达到加快创面无瘢痕愈合目标；并且为干细胞创新药物的研发提供充分的数据支撑及技术支持，加速干细胞创新药物的临床应用，有利于定制化开发缩短病程、提高创面愈合质量的“干细胞创新药物”，最终建立科学合理的干细胞应用标准体系。
附图说明
图1为脐带间充质干细胞分群结果示意图。
图2A～图2D为Hr-MSC的Marker基因的热图结果示意图。
图3A和图3B为Hr-MSC的Marker基因的表达水平示意图。
图4为13个cluster的功能富集分析以及基因热图展示。
图5为四种不同WJ-MSCs亚群的UMAP分群图。
图6为脐带干细胞分群结果示意图。
图7为CD29+/CD142+/S100A9+Hr-MSC的分选结果示意图。
图8为实施例6伤口愈合效率结果示意图。
图9为CD29+/CD142+/S100A9+Hr-MSC在HDF和HUCV上的迁移能力示意图。
图10为斑马鱼皮肤伤口愈合的示意图。
图11为鉴定创面修复优势功能细胞亚群的系统21，其包括分型模块11与基因展示模块21。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1：脐带干细胞分群
本实施例利用单细胞转录组学对脐带间充质干细胞进行测序分析，初步在单细胞水平将脐带干细胞分成不同亚群，利用生物信息学分析对各亚群进行功能注释，确定目标候选亚群。
1、样本：人脐带华通胶组织；
入组孕妇在孕期37-40周分娩，选取新生儿脐带进行实验，作后续P0、P3代脐带间充质干细胞组织来源，编号：PUM-A1-000152；
选取6个样本(huc_1P0、huc_1P3、huc_2P0、huc_2P3、huc_3P0和huc_3P3)的细胞进行下游分析。
2、仪器：10×Chromium；
3、参数：
(1)每次运行提供8个通道，每个通道可以收集100～10000个细胞；
(2)细胞捕获效率～65％；
(3)测序结果中doublet比例为0.9％/1000个细胞；
(4)不对细胞大小设限。
4、利用Illumina NovaSeq测序平台进行测序并进行后续的数据分析：对原始测序数据提取barcode、UMI和RNA序列，选定人类参考基因组进行比对获取feature-barcode矩阵。后续分析如下：
①对数据进行基因数、UMI数及线粒体的过滤，使用sctransform构建基因表达正则化负二项模型进行数据归一化，然后进行PCA降维，根据碎石图确定PCA的个数；
②使用降维算法例如t-SNE、UMAP将测序方法得到的生物信息学结果转化为生物学结果，确定不同个体、不同区域中细胞类型的异质性，并进行可视化，确定不同个体细胞类型的异质性，得到分型结果，获得13个亚群(Cluster 0、Cluster 1、Cluster 2、Cluster3、Cluster 4、Cluster 5、Cluster 6、Cluster 7、Cluster 8、Cluster 9、Cluster 10、Cluster 11和Cluster 12)；
③使用FindClusters函数对分群结果进行基因的差异表达分析，并采用不同形式展现亚群的特征性Marker基因，根据结果筛选出可能具有相似功能的群Cluster 6和Cluster 9(即Hr-MCS)；
④对Hr-MCS中每个cluster的差异表达基因进行GO、KEGG富集、蛋白互作网络、转录因子注释等分析，得到Hr-MSC上调基因富集影响的信号通路，推测其可能的功能。
如图1的A、B所示，脐带间充质干细胞具有有限异质性，初步确定不同个体、不同代次、不同性别细胞组成一致；不同个体、不同代次、不同性别细胞比例不同；不同个体、不同代次、不同性别细胞表达基因不同。
实施例2：Hr-MSC的Marker基因分析
本实施例针对实施例1获得的脐带干细胞各亚群的Marker基因制作基因表达热图，结果如图2A～图2D所示。
图2A～图2D中的Marker基因包括：UBE2C、TOP2A、CDK1、CENPF、KPNA2、CKS2、PLK1、HMGB2、AURKA、ARL6IP1、GTSE1、MKI67、ASPM、CENPE、NUSAP1、SGO2、AURKB、TPX2、UBE2S、SMC4、NEAT1、ACTA2、MXD4、TIMP1、LMCD1、TAGLN、DCN、IGFBP4、TGM2、PAPPA、ANGPTL4、COL8A1、KCNE4、INHBA、MEG3、SAT1、TUBA1A、CDKN1A、SELENOM、SAA1、CDC20、CCNB1、NCL、PNN、HSP90AA1、HSPA8、DDX21、SFPQ、SLBP、CCNB2、SRSF7、TUBB4B、ODC1、PTTG1、BIRC5、HIST1H4C、HIST1H1A、HIST1H1D、HIST1H1B、ANKRD1、CLSPN、CYR61、GINS2、PTX3、FEN1、MCM7、ATAD2、FBXO5、GMNN、PCNA、HIST1H1C、MCM3、HIST1H1E、TYMS、LUM、SERPINF1、HTRA3、COL3A1、FOS、PNRC1、HTRA1、APOE、TGFBI、COL1A2、CDKN1C、COL1A1、KRT18、GYPC、TK1、CENPX、GNG11、LSM4、S100A16、SRM、SNRPB、CDKN3、MYL9、PFN1、RRM2、RPS27L、EXOSC8、LRRC75A、SET、GOLGA4、DST、KPNB1、B4GALT1、FLNA、HNRNPH1、ANKRD11、ZFP36L1、HEG1、 HDLBP、PEG10、ATP2B1、PAK2、CDV3、COL5A1、HBZ、NUDC、GAS6、SLC20A1、MALAT1、BOP1、SERBP1、LRRC59、HNRNPU、IL6ST、HNRNPA3、CAPN2、PFKP、EBNA1BP2、CTGF、HMGN2、TPM1、ACTB、CYCS、RBM8A、STMN1、TPM2、SMS、TMSB4X、ACTG1、FTL、SNRPG、MARCKSL1、RANBP1、HMOX1、SQSTM1、SLC3A2、GADD45A、NUPR1、DDIT3、FTH1、CXCL3、HLA-B、GDF15、TSC22D3、ZFAS1、CXCL1、S100A4、PPP1R15A、LGALS3、IL32、S100A10、KRT7、PDLIM1、CAV1、RTN4、TM4SF1、FAH、CRIP2、PDPN、CD9、SPON2、LMNA、PRSS23、S100A6、MAOA、CYBA、ABHD5、EZR、MT-ND3、FN1、MT-ND6、MT-ATP6、THBS1、MT-ND4、MT-ND1、MT-CYB、MT-ND2、SLC38A2、FBN1、COL4A1、COL5A2、RAB13、POSTN、LDHA、ACTG2、CTSC、COTL1、POLR3K、ANXA2、MT2A、PLAC9、NPW和TUBA1B。
各亚群分类及前4或前5的Marker基因如表1所示。
表1亚群分类及其Marker基因
实施例3：Hr-MSC的Marker基因的表达水平
对实施例2中所列的Hr-MSC的部分Marker基因测定表达水平，得到各亚群Marker基因的表达情况，如图3A和图3B所示。
以Marker基因DCN为例，DCN在各亚群中均表达，但在第4亚群(Cluster 4)表 达最高，其余各图、各基因分析同前。由此可知，Marker基因的表达水平具有差异。
如图4所示，在经过Seurat一系列标准质控及数据预处理，基于细胞的特征基因，我们得到13个细胞簇(Cluster 0-12)，我们进一步对各细胞簇的特征基因及功能富集进行分析，以明确细胞身份。结果显示，Cluster 0、2、3、7与其他细胞群相比，高表达UBE2C、TOP2A、HMGB2、CDC20、PCNA、HIF1A等已知的增殖相关基因，提示这些簇具有强的增殖能力。GO富集分析结果表明，这三个簇在细胞核分裂、DNA复制和干细胞增殖中也显著富集；cluster 1、4和11以ACTA2、TGM2、FOS、TGFB1、FLNA和COL3A1为特征基因，这些基因集主要富集糖皮质激素反应、周细胞分化、腐胺分解代谢、细胞外基质组织等生物学过程；cluster 10高表达S100A10、PDLIM1和CAV1；cluster 5、8和12的特征是UBE2S、TAGLN、TPM1和TMSB4X的高表达，而cluster 6和9显著性基因主要参与免疫调节及创面修复相关通路，这些基因包括B4GALT1、HEG1、CXCL3、CXCL1和HMOX1。
最终，基于基因表达谱、功能相似性及文献回顾，我们将相似的亚群进行合并，并命名为增殖型干细胞(proliferative_MSC；由cluster 0、2、3和7组成)，微环境支持型干细胞(niche-supporting_MSC；由cluster1、4和11组成)，代谢型干细胞(metabolism-related_MSC；由cluster 5、8和12组成)以及biofunctional type_MSC(功能型干细胞；由cluster 6和9组成，即本申请的CD29+/CD142+/S100A9+Hr-MSC)(如图5所示)。其中cluster10因占比小且GO富集无法很好归类于其他亚群，故不纳入后续分析。
根据以上结果，结合单细胞数据，将biofunctional type MSC的CD29、CD142和CYR61作为Hr-MSC的候选标志物；以及将biofunctional type MSC的CD29、CD142和S100A9作为Hr-MSC的候选标志物分别进行后续实验。上述修选标志物在Hr-MSC的各亚群中的阳性表达比例均至少为90％。
实施例4：CD29+/CD142+/CYR61+Hr-MSC的分选
参照实施例1，本实施例利用单细胞转录组学测序分析，得到具有目标治疗功能的亚群的Marker基因，通过与流式细胞分选技术结合，将目标亚群从原代混合的脐带间充质干细胞中分选出，如图6所示，该亚群占总细胞的5.310％左右。通过流式细胞术标记阳性Marker：CD29+、CD142+、CYR61+鉴定该亚群。此三个指标全阳性的目标亚群为P6，占总体干细胞比值为5.3％，为后续扩增目标亚群。
实施例5：CD29+/CD142+/S100A9+Hr-MSC的分选
参照实施例1，本实施例利用单细胞转录组学测序分析，得到具有目标治疗功能的亚群的Marker基因，通过与流式细胞分选技术结合，将目标亚群从原代混合的脐带间充质干细胞中分选出。通过流式细胞术标记阳性Marker：CD29+、CD142+、S100A9+鉴定该亚群。如图7所示，此三个指标全阳性的目标亚群占总体干细胞比值为9.53％，为后续扩增目标亚群。
实施例6：效果例
本实施例通过制造成年斑马鱼全层皮肤缺损，评估成年斑马鱼缺损创面不同给药(即下述分组)愈合时间即愈合效率进行有效性验证(模型构建和有效性验证参考Kennard Andrew S等，Theriot Julie A.(2021).Wounding Zebrafish Larval Epidermis by Laceration.Bio Protoc,11(24),e4260；Mhlongo Fikile等，Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.[J].J Ethnopharmacol,2022,286:114867)。
将成年斑马鱼在28℃的自动鱼类饲养系统中以14小时的光照/10小时的黑暗的周期饲养。每天喂食一次刚孵化的卤虫。将斑马鱼分为三组：对照组(PBS，n＝6)、阳性分选MSC组(CD29+/CD142+/CYR61+MSC，n＝6)和阴性分选MSC组(CD29-/CD142-/CYR61-MSC，n＝6)。
初步验证的结果如图8所示，阳性分选MSC组和阴性分选MSC组在伤口愈合效率上较对照组均有显著差异(p<0.001)，阳性分选MSC组的伤口愈合效率较阴性分选MSC组的伤口愈合效率有显著差异。
实施例7：效果例
本实施例通过制造成年斑马鱼全层皮肤缺损，评估成年斑马鱼缺损创面不同给药(即下述分组)愈合时间即愈合效率进行有效性验证(模型构建和有效性验证参考Kennard Andrew S等，Theriot Julie A.(2021).Wounding Zebrafish Larval Epidermis by Laceration.Bio Protoc,11(24),e4260；Mhlongo Fikile等，Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.[J].J Ethnopharmacol,2022,286:114867)。具体如下：
将成年斑马鱼在28℃的自动鱼类饲养系统中以14小时的光照/10小时的黑暗的周期饲养。每天喂食一次刚孵化的卤虫。将斑马鱼分为四组：对照组(PBS，n＝6)、未分选MSC组(华通胶组织MSC，n＝6)、阳性分选MSC组(CD29+/CD142+/S100A9+MSC，n＝6) 和阴性分选MSC组(CD29-/CD142-/S100A9-MSC，n＝6)。然后，使用0.03％三嗪(中国有限公司阿拉丁生化科技有限公司)对斑马鱼进行麻醉。麻醉后，用微型眼科剪刀在斑马鱼背部左侧造成全层伤口(直径～3mm)。然后，将配制好的10μL相应实验溶液(1×104个细胞)皮下注射到伤口周围区域，使用33号注射器(Hamilton)。注射后，将针头小心地保持在原位5分钟，然后将鱼放回循环系统中的水中。如果观察到任何注射液在注射部位泄漏，则对该鱼实施安乐死，并且不包括在随后的实验中。在创伤后0、5、15和30天(dpw)，将鱼麻醉并随后拍照。使用ImageJ软件测量伤口面积。
结果如图9和10所示，创伤后第5天，伤口表面可见少量上皮化组织，其随着时间的推移逐渐增加(图10)。创伤后第30天，对照组的伤口仍较为明显，上皮细胞组织较薄；在未分选组、阳性分选组和阴性分选组中均能观察到不同程度的伤口愈合。值得注意的是，阳性分选组的上皮化更为显著，银条纹重建良好，伤口周围黑色素细胞形成的黑色素斑点延伸至伤口区域。具体而言，阳性分选组的伤口愈合效率达到0.857±0.050，对照组、阴性分选MSC组和未分选MSC组的伤口愈合效率依次为0.575±0.110、0.648±0.129、0.646±0.129，阳性分选组的伤口愈合效率具有统计学差异，表明CD29+/CD142+/S100A9+MSC能够更好的促进伤口愈合。
实施例8：鉴定创面修复优势功能细胞亚群的系统
本实施例提供了一种鉴定创面修复优势功能细胞亚群的系统21，如图11所示，其包括：分型模块11与基因展示模块21。
其中，分型模块11通过单细胞多模态组学技术对待测细胞进行分子分型，获得目标候选细胞亚群。
基因展示模块12对分型模块11获得的目标候选细胞亚群进行特征基因展示和分选，获得包含特征基因的细胞亚群，所得细胞亚群即为创面修复优势功能亚群细胞。
特征基因表达以下标志物组合中的一种或多种：
(1)CD29、CD142和CYR61；
(2)DCN、TGFB1、COL3A1、COLIA2和COLIA1；
(3)NEAT1、TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE、和CDKN1C。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (18)

  1. 一种创面修复优势功能细胞的生物标志物组合，其特征在于，所述生物标志物组合包括CD29、CD142和CYR61。
  2. 一种创面修复优势功能细胞的生物标志物组合，其特征在于，所述生物标志物组合包括CD29、CD142和S100A9。
  3. 如权利要求1或2所述的生物标志物组合，其特征在于，所述生物标志物组合还包括选自DCN、TGFB1、COL3A1、COLIA2和COLIA1中的一种或多种；
    较佳地，所述生物标志物组合包括选自NEAT1、TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE和CDKN1C中的一种或多种。
  4. 一种创面修复优势功能细胞制剂，其特征在于，所述创面修复优势功能细胞制剂包含CD29、CD142和CYR61阳性的干细胞。
  5. 一种创面修复优势功能细胞制剂，其特征在于，所述创面修复优势功能细胞制剂包含CD29、CD142和S100A9阳性的干细胞。
  6. 如权利要求4或5所述的创面修复优势功能细胞制剂，其特征在于，所述干细胞为间充质干细胞；
    和/或，所述干细胞表达选自DCN、TGFB1、COL3A1、COLIA2和COLIA1中的一种或多种生物标志物。
  7. 如权利要求6所述的创面修复优势功能细胞制剂，其特征在于，所述干细胞表达选自NEAT1、TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE和CDKN1C中的一种或多种生物标志物。
  8. 一种细胞亚群，其特征在于，所述细胞亚群包含CD29、CD142和CYR61阳性的干细胞；
    所述干细胞在所述细胞亚群的比例至少为90％。
  9. 一种细胞亚群，其特征在于，所述细胞亚群包含CD29、CD142和S100A9阳性的干细胞；
    所述干细胞在所述细胞亚群的比例至少为90％。
  10. 如权利要求8或9所述的细胞亚群，其特征在于，所述干细胞为间充质干细胞；
    和/或，所述干细胞表达选自DCN、TGFB1、COL3A1、COLIA2和COLIA1中的一种或多种。
  11. 如权利要求10所述的细胞亚群，其特征在于，所述干细胞表达选自NEAT1、 TMP1、GFBP4、SAT1、TGM2、LUM、SERPNF1、MXD4、FOS、ACTA2、HTRA3、HTRA1、PNRC1、APOE和CDKN1C中的一种或多种。
  12. 一种药物组合物，其特征在于，所述药物组合物包括干细胞以及药学上可接受的载体和/或辅料；所述干细胞如权利要求4～7任一项所述。
  13. 一种药物组合，其特征在于，所述药物组合包括第一治疗剂和第二治疗剂；所述第一治疗剂包含如权利要求12所述的药物组合物；
    较佳地，所述第二治疗剂选自免疫抑制剂、止痛剂和抗感染剂中的一种或多种。
  14. 一种促进创面修复的方法，所述方法包括向有需要的受试者施用如权利要求4～7任一项所述的创面修复优势功能细胞制剂、如权利要求8～11任一项所述的细胞亚群、如权利要求12所述的药物组合物或者如权利要求13所述的药物组合。
  15. 一种鉴定创面修复优势功能细胞亚群的方法，其特征在于，所述方法包括：
    (1)基于单细胞多模态组学技术对待测样本中的细胞进行分子分型，获得目标候选细胞亚群；
    (2)对(1)中获得的目标候选细胞亚群进行特征基因展示和分选，获得包含特征基因的细胞亚群，所述细胞亚群即为创面修复优势功能细胞亚群；
    所述特征基因编码如权利要求1～3任一项所述的生物标志物组合。
  16. 如权利要求15所述的方法，其特征在于，(1)后包括(1-1)：通过空间转录组学获得所述目标候选细胞亚群的定位信息，所述定位信息包括富集情况和定位情况。
  17. 一种鉴定创面修复优势功能细胞亚群的系统，其特征在于，所述系统包括分型模块和基因展示模块；
    其中，所述分型模块通过单细胞多模态组学技术对待测细胞进行分子分型，获得目标候选细胞亚群；所述基因展示模块对分型模块获得的目标候选细胞亚群进行特征基因展示和分选，获得包含特征基因的细胞亚群，所述细胞亚群即为创面修复优势功能亚群细胞；
    所述特征基因编码如权利要求1～3任一项所述的生物标志物组合。
  18. 一种用于鉴定创面修复优势功能细胞亚群的试剂盒；
    所述试剂盒包含检测如权利要求1～3任一项所述的生物标志物组合的试剂。
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