ALPL + PDGFD + periosteum stem cell as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a periosteum stem cell with positive expression of ALPL and PDGFD genes. The invention also relates to a method for identifying or screening the periosteum stem cells and application of the periosteum stem cells in bone injury repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a periosteal stem cell with positive expression of ALPL and PDGFD genes. The present invention also relates to a method for screening or identifying the periosteal stem cell and the use of the periosteal stem cell for bone damage repair. Background Art

[0002] Clinicians have recognized for centuries the critical role of the periosteum in bone growth and repair. [1] However, the identification and comprehensive exploration of periosteal stem cells (PSCs) is still in its infancy. The lack of periostin in the periosteum does not allow the regeneration of the periosteal cell pool after injury, pointing to the existence of a PSC population responsible for repairing bone damage. Periostin plays a crucial role in maintaining the homeostasis of the PSC pool. [2] Zhao et al. showed that Gli1 marks a major stem cell population in the adult craniofacial suture mesenchyme that is able to generate the periosteum, dura mater, and osteogenic front [3] Furthermore, Gli1-labeled PSCs in adult mice primarily promote the healing of bicortical fractures [4] Axin2 was also identified in the cranial sutures as a stem cell population with the capacity for self-renewal and differentiation [5] Recent studies have revealed the presence of two different types of stem cells in the cranial sutures. The first is the CTSK+ stem cell that specializes in intramembranous bone formation. [6] The second is a DDR2+ stem cell that uniquely mediates endochondral ossification without contributing to bone marrow formation [7] PSCs marked by Mx1+ αSMA+ exhibited remarkable self-renewal ability, rapidly migrated to the site of injury, differentiated into osteoblasts and chondrocytes, and generated new periosteum [8] Lineage tracing reveals that αSMA marks slowly cycling, self-renewing progenitor cells in the adult mouse periosteum, demonstrating their efficient role in processing bone injury [9] . These seminal studies provided valuable insights into the properties of PSCs that are critical for bone regeneration. Although various Cre models have pinpointed PSCs with similar features, the extent to which their identities and functions overlap or differ remains uncertain. These delineations provide an initial framework for the continued refinement of stem cell identity in the skull. Furthermore, Cre models may not be specific for a single cell population, as most periosteal markers are also expressed in bone marrow, skeletal muscle, or other stromal tissues. [1, 10] Currently, the classification of cell populations in PSCs needs to be further refined, and new markers are needed to identify the stem cells responsible for bone growth and repair.

[0003] Compared to mice, pigs are highly suitable candidates for preclinical studies due to their anatomical and physiological similarities to humans. [11, 12] Rodents have only one set of teeth that cannot be replaced, in contrast to humans who have two sets of teeth: deciduous and permanent. [13, 14] Minipigs exhibit a double set of dentition that mirrors the growth and development patterns of human teeth. In addition, the salivary glands of minipigs are very similar to those of humans in weight, size, length, diameter, and anatomical morphology. [11, 13, 14] Pigs, which have facial bones similar to those of humans, are an ideal animal model for studying craniofacial bone regeneration.

[0004] Alkaline phosphatase (ALPL) is expressed in a variety of tissue cells, mainly in the cytoplasm, but in different subtypes, it is also expressed in the cell membrane. Its function is to metabolize various phosphate compounds and plays a key role in the adaptive role of bone mineralization and function. [15, 16] . It has broad substrate specificity and can hydrolyze a wide range of compounds.

[17] Plays an important role in bone and tooth mineralization by hydrolyzing extracellular diphosphate, a potent inhibitor of mineralization, to phosphate, thereby promoting hydroxyapatite crystal formation and increasing inorganic phosphate concentrations [15, 16] ALPL is a hydrolase closely related to biomineralization. In addition to the above functions, it also plays an important role in the binding and metabolism of metal ions such as calcium, magnesium, and zinc.

[0005] Platelet derived growth factor D (PDGFD (also known as IEGF; MSTP036; or SCDGF-B)) is expressed in a variety of tissues, most abundantly in smooth muscle cells. This protein plays an important role in embryonic development, cell proliferation, cell migration, survival and chemotaxis.

[18] This factor plays an important role in wound healing. Induces macrophage recruitment, increases interstitial pressure, and vascular maturation during angiogenesis

[19] . Initiates the events leading to mesangial proliferative glomerulonephritis, including the influx of monocytes and macrophages and the production of extracellular matrix

[20] The protein encoded by this gene is a member of the platelet-derived growth factor family.

[0006] There is still a need to develop new periosteal stem cells. Summary of the invention

[0007] After in-depth research, the inventors used a pig model combined with single-cell sequencing technology to characterize adult stem cells in the skull periosteum for the first time, and identified ALPL+PDGFD+ (AP+) cells as unique skull PSCs that play a key role in craniofacial bone development and injury-induced bone regeneration. The following invention is provided.

[0008] In one aspect, the present invention provides a periosteal stem cell, wherein, in the periosteal stem cell, ALPL gene and PDGFD gene are expressed positively.

[0009] In certain embodiments, the periosteal stem cells are cranial periosteal stem cells or long bone periosteal stem cells.

[0010] In certain embodiments, the periosteal stem cells are derived from a mammal.

[0011] In certain embodiments, the periosteal stem cells are derived from a non-human mammal.

[0012] In certain embodiments, the periosteal stem cells are derived from a mammal in an embryonic development stage, or a mammal with bone damage (eg, skull damage, long bone damage, and / or cartilage damage).

[0013] In certain embodiments, the mammal is a pig (eg, a minipig, a mini-pig) or a mouse.

[0014] In certain embodiments, the mammal is a human.

[0015] In certain embodiments, the periosteal stem cells are derived from pigs (e.g., miniature pigs, mini pigs) at embryonic development stage E15 day (d)-E145d (e.g., E15d-E140d, E20d-E140d, E20d-E135d, E25d-E135d, E25d-E130d, E30d-E130d, E30d-E125d, E35d-E120d, E40d-E120d, E40d-E115d, E45d-E115d, E45d-E110d, E50d-E110d, E50d-E105d, E60d-E100d, E70d-E100d, or E80d-E100d).

[0016] In certain embodiments, the periosteal stem cells are derived from mice at embryonic development stage E5d-E20d (eg, E5d-E18d, E7d-E18d, E5d-E15d, E7d-E15d, or E7d-E10d).

[0017] In certain embodiments, the periosteal stem cells are derived from humans at embryonic development stage E8 weeks (w)-E40w (e.g., E8w-E36d, E8w-E32w, E12w-E32w, E12w-E28w, E12w-E24w, E16w-E24w, or E16w-E20w).

[0018] In the context of the present invention, the “periosteal stem cells positive for ALPL and PDGFD gene expression” have the same meaning as “ALPL+PDGFD+periosteal stem cells (AP+periosteal stem cells)”, “ALPL+PDGFD+ stem cells (AP+ stem cells)”, and “ALPL+PDGFD+ cells (AP+ cells)” and can be used interchangeably.

[0019] In certain embodiments, the periosteal stem cells have one or more of the following characteristics:

[0020] (1) capable of producing at least one daughter stem cell through cell division, wherein the daughter stem cell retains the same cell division activity and cell differentiation activity as the parent stem cell from which it is derived;

[0021] (2) having the ability to differentiate into other cells, for example, into osteoblasts or chondrocytes; preferably, the osteoblasts are pre-osteoblasts or mature osteoblasts;

[0022] (3) In a mammal, when the bone tissue in which the periosteal stem cells are located is defective or damaged, the periosteal stem cells: (a) proliferate and increase in number; and / or (b) further differentiate into other cells, for example, osteoblasts or chondrocytes; preferably, the osteoblasts are pre-osteoblasts or mature osteoblasts.

[0023] In certain embodiments, the cell division is symmetric division or asymmetric division.

[0024] In certain embodiments, the periosteal stem cells are present in the periosteum of the skull and / or the periosteum of the long bones.

[0025] On the other hand, the present invention also provides a method for preparing the periosteal stem cells of the present invention, which comprises the following steps:

[0026] (1) Collect cells from the periosteum of mammals;

[0027] (2) Among the collected cells, label the cells expressing ALPL and PDGFD;

[0028] (3) Sorting out the cells expressing ALPL and PDGFD in (2) to obtain the periosteal stem cells.

[0029] In certain embodiments, in step (1), cells are collected from the periosteum of a mammal in an embryonic development stage, or cells are collected from the periosteum of damaged bone tissue of a mammal.

[0030] In some embodiments, before step (2) and after step (1), the method further comprises the step of marking cells expressing CD45 and CD31 in the cells and removing the cells expressing CD45 and CD31 from the cells. In some embodiments, cells expressing CD45 and CD31 are removed by fluorescence-activated cell sorting (FACS).

[0031] In certain embodiments, in step (3), the cells are sorted by fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), density gradient centrifugation, or microfluidic cell sorting.

[0032] In certain embodiments, sorting is performed by FACS.

[0033] In certain embodiments, the mammal is a non-human mammal.

[0034] In certain embodiments, the mammal is a pig (eg, a minipig, a mini-pig) or a mouse.

[0035] In certain embodiments, the mammal is a human.

[0036] In certain embodiments, the periosteal stem cells are derived from pigs (e.g., miniature pigs, mini pigs) at embryonic development stage E15 day (d)-E145d (e.g., E15d-E140d, E20d-E140d, E20d-E135d, E25d-E135d, E25d-E130d, E30d-E130d, E30d-E125d, E35d-E120d, E40d-E120d, E40d-E115d, E45d-E115d, E45d-E110d, E50d-E110d, E50d-E105d, E60d-E100d, E70d-E100d, or E80d-E100d).

[0037] In certain embodiments, the periosteal stem cells are derived from mice at embryonic development stage E5d-E20d (eg, E5d-E18d, E7d-E18d, E5d-E15d, E7d-E15d, or E7d-E10d).

[0038] In certain embodiments, the periosteal stem cells are derived from humans at embryonic development stage E8 weeks (w)-E40w (e.g., E8w-E36d, E8w-E32w, E12w-E32w, E12w-E28w, E12w-E24w, E16w-E24w, or E16w-E20w).

[0039] In certain embodiments, the preparation method further comprises the steps of:

[0040] (4) Cultivating and sorting the obtained cells, namely the periosteal stem cells.

[0041] In certain embodiments, the preparation method further comprises the steps of:

[0042] (5) Subculturing the periosteal stem cells to obtain second-generation periosteal stem cells and third-generation periosteal stem cells.

[0043] In certain embodiments, culturing is performed by single cell cloning.

[0044] In certain embodiments, the preparation method further comprises the steps of:

[0045] (6) Detect the expression of ALPL and PDGFD in the periosteal stem cells obtained in step (3), step (4), the second-generation periosteal stem cells obtained in step (5), and the third-generation periosteal stem cells, and remove cells that do not express ALPL and PDGFD at the same time.

[0046] In certain embodiments, the expression of ALPL and PDGFD in the periosteal stem cells, the second-generation periosteal stem cells and the third-generation periosteal stem cells is detected by protein blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, mass spectrometry or immunohistochemistry.

[0047] In certain embodiments, cells that do not express both ALPL and PDGFD are removed by fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), density gradient centrifugation, or microfluidic cell sorting.

[0048] In certain embodiments, the periosteum is the skull periosteum or the long bone periosteum.

[0049] On the other hand, the present invention also provides a pharmaceutical composition comprising the periosteal stem cells of the present invention and one or more pharmaceutically acceptable excipients.

[0050] In certain embodiments, the pharmaceutical composition further comprises an additional bone repair drug.

[0051] In certain embodiments, the bone repair drug is selected from one or more of glucosamine, chondroitin sulfate, calcium, sodium hyaluronate, bone morphogenetic protein and alendronate sodium.

[0052] In certain embodiments, in the pharmaceutical composition, the periosteal stem cells of the present invention and the additional bone repair drug can be provided as separate components or as mixed components. Therefore, the periosteal stem cells of the present invention and the additional bone repair drug can be administered simultaneously, separately or sequentially.

[0053] In certain embodiments, the pharmaceutically acceptable excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0054] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" of the periosteum stem cells of the present invention. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0055] On the other hand, the present invention also provides a method for repairing bone damage in a subject, or treating a disease caused by bone damage in a subject, comprising administering an effective amount of the periosteal stem cells or pharmaceutical composition of the present invention to a subject in need thereof. The present invention also relates to the use of the periosteal stem cells or pharmaceutical composition for preparing a drug, the drug being used to repair bone damage in a subject, or being used to treat a disease caused by bone damage in a subject.

[0056] In certain embodiments, the bone injury is a skull injury, a long bone injury, and / or a cartilage injury.

[0057] In certain embodiments, the bone injury is a bone fracture, bone crack, and / or bone contusion.

[0058] In certain embodiments, the disease caused by bone damage is osteoporosis, bone tumor, osteomyelitis, bone spurs (bone hyperplasia), osteoarthritis, osteomalacia, osteopenia, bone defect, osteolysis or osteonecrosis.

[0059] In certain embodiments, the subject is a mammal.

[0060] In certain embodiments, the subject is a non-human mammal.

[0061] In certain embodiments, the mammal is a pig (eg, a minipig, a mini-pig) or a mouse.

[0062] In certain embodiments, the mammal is a human.

[0063] In certain embodiments, the periosteal stem cells are derived from pigs (e.g., miniature pigs, mini pigs) at embryonic development stage E15 day (d)-E145d (e.g., E15d-E140d, E20d-E140d, E20d-E135d, E25d-E135d, E25d-E130d, E30d-E130d, E30d-E125d, E35d-E120d, E40d-E120d, E40d-E115d, E45d-E115d, E45d-E110d, E50d-E110d, E50d-E105d, E60d-E100d, E70d-E100d, or E80d-E100d).

[0064] In certain embodiments, the periosteal stem cells are derived from mice at embryonic development stage E5d-E20d (eg, E5d-E18d, E7d-E18d, E5d-E15d, E7d-E15d, or E7d-E10d).

[0065] In certain embodiments, the periosteal stem cells are derived from humans at embryonic development stage E8 weeks (w)-E40w (e.g., E8w-E36d, E8w-E32w, E12w-E32w, E12w-E28w, E12w-E24w, E16w-E24w, or E16w-E20w).

[0066] In certain embodiments, the periosteal stem cells or pharmaceutical compositions are used alone or in combination with other bone repair drugs.

[0067] The periosteum stem cells or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0068] A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: incorporating the necessary dose of the periosteum stem cells or pharmaceutical compositions of the present invention into an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in suitable carriers before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffered solutions), Ringer's solutions, and any combination thereof.

[0069] In certain embodiments, the periosteal stem cells or pharmaceutical composition are further administered in combination with physical therapy. In certain embodiments, the physical therapy includes, but is not limited to, surgical treatment (e.g., minimally invasive surgery, open surgery), acupuncture, physical therapy, massage, etc.

[0070] In certain embodiments, the periosteal stem cells are from a mammal in an embryonic development stage, or a mammal with bone damage.

[0071] In certain embodiments, the periosteal stem cells are from a mammal 0 to 7 days (e.g., 0 to 6 days, 0 to 5 days, 0 to 4 days, 0 to 3 days, 0 to 2 days, 0 to 45 hours, 0 to 40 hours, 0 to 35 hours, 0 to 30 hours, 0 to 25 hours, 0 to 24 hours, 0 to 20 hours, 0 to 15 hours, or 0 to 10 hours) after bone injury.

[0072] In certain embodiments, the periosteal stem cells are from a mammal with bone damage, and the periosteal stem cells are located in the damaged bone tissue.

[0073] In certain embodiments, the mammal is a non-human mammal.

[0074] In certain embodiments, the mammal is a pig (eg, a minipig, a mini-pig) or a mouse.

[0075] In certain embodiments, the mammal is a human.

[0076] In certain embodiments, the periosteal stem cells are derived from pigs (e.g., miniature pigs, mini pigs) at embryonic development stage E15 day (d)-E145d (e.g., E15d-E140d, E20d-E140d, E20d-E135d, E25d-E135d, E25d-E130d, E30d-E130d, E30d-E125d, E35d-E120d, E40d-E120d, E40d-E115d, E45d-E115d, E45d-E110d, E50d-E110d, E50d-E105d, E60d-E100d, E70d-E100d, or E80d-E100d).

[0077] In certain embodiments, the periosteal stem cells are derived from mice at embryonic development stage E5d-E20d (eg, E5d-E18d, E7d-E18d, E5d-E15d, E7d-E15d, or E7d-E10d).

[0078] In certain embodiments, the periosteal stem cells are derived from humans at embryonic development stage E8 weeks (w)-E40w (e.g., E8w-E36d, E8w-E32w, E12w-E32w, E12w-E28w, E12w-E24w, E16w-E24w, or E16w-E20w).

[0079] In certain embodiments, the bone injury is a skull injury, a long bone injury, and / or a cartilage injury.

[0080] In certain embodiments, the mammal and the subject are the same individual of the same species or different individuals of the same species.

[0081] In certain embodiments, the periosteal stem cells are cranial periosteal stem cells.

[0082] In another aspect, the present invention also provides use of the periosteal stem cells of the present invention for forming bone tissue.

[0083] In certain embodiments, the bone tissue is skull bone tissue, long bone tissue, and / or cartilage tissue.

[0084] On the other hand, the present invention also provides use of the periosteal stem cells of the present invention for repairing bone damage.

[0085] In certain embodiments, the bone injury is a skull injury, a long bone injury, and / or a cartilage injury.

[0086] In certain embodiments, the bone injury is a bone fracture, bone crack, and / or bone contusion.

[0087] In certain embodiments, the periosteal stem cells are derived from a mammal in an embryonic development stage, or a mammal with bone damage.

[0088] In certain embodiments, the periosteal stem cells are derived from pigs (e.g., miniature pigs, mini pigs) at embryonic development stage E15 day (d)-E145d (e.g., E15d-E140d, E20d-E140d, E20d-E135d, E25d-E135d, E25d-E130d, E30d-E130d, E30d-E125d, E35d-E120d, E40d-E120d, E40d-E115d, E45d-E115d, E45d-E110d, E50d-E110d, E50d-E105d, E60d-E100d, E70d-E100d, or E80d-E100d).

[0089] In certain embodiments, the periosteal stem cells are derived from mice at embryonic development stage E5d-E20d (eg, E5d-E18d, E7d-E18d, E5d-E15d, E7d-E15d, or E7d-E10d).

[0090] In certain embodiments, the periosteal stem cells are derived from humans at embryonic development stage E8 weeks (w)-E40w (e.g., E8w-E36d, E8w-E32w, E12w-E32w, E12w-E28w, E12w-E24w, E16w-E24w, or E16w-E20w).

[0091] In certain embodiments, the periosteal stem cells are from a mammal 0 to 7 days (e.g., 0 to 6 days, 0 to 5 days, 0 to 4 days, 0 to 3 days, 0 to 2 days, 0 to 45 hours, 0 to 40 hours, 0 to 35 hours, 0 to 30 hours, 0 to 25 hours, 0 to 24 hours, 0 to 20 hours, 0 to 15 hours, or 0 to 10 hours) after bone injury.

[0092] In certain embodiments, the periosteal stem cells are from a mammal with bone damage, and the periosteal stem cells are located in the damaged bone tissue.

[0093] In certain embodiments, the bone injury is a skull injury, a long bone injury, and / or a cartilage injury.

[0094] In certain embodiments, the mammal and the subject are the same individual of the same species or different individuals of the same species.

[0095] In certain embodiments, the periosteal stem cells are cranial periosteal stem cells.

[0096] Definition of terms

[0097] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0098] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0099] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0100] As used herein, the term "ALPL (alkaline phosphatase, biomineralization associated)" refers to alkaline phosphatase, which is a membrane-bound glycosylase widely present in a variety of cells and tissues. The sequence of ALPL is well known to those skilled in the art (see, for example, Gene database accession number Gene ID: 249).

[0101] As used herein, the term "PDGFD (platelet derived growth factor D)" refers to platelet-derived growth factor D, which is an important growth factor protein that plays an important role in many biological processes, such as embryonic development, angiogenesis, and tissue repair. The sequence of PDGFD is well known to those skilled in the art (see, for example, Gene database accession number Gene ID: 80310).

[0102] As used herein, the term "embryo" refers to a mammalian organism from the unicellular stage. The embryo used in the present application can be obtained by separation from a mammalian body, or can be generated by mammalian stem cells through in vitro culture under appropriate conditions, and the embryo generated through in vitro culture is similar to the natural embryo produced in the body of the corresponding stage, such as having similar morphology, length, weight, cell type composition and expression of developmental marker genes, etc.

[0103] As used herein, the term "embryo development" refers to the biological process of growth and development of mammalian embryos during prenatal (e.g., before birth) development. Typically, embryo development is carried out in successive stages, wherein the process begins with fertilization of an egg cell (i.e., ovum) with sperm (i.e., sperm) to produce a diploid fertilized egg. After a series of mitotic cell divisions, the fertilized egg forms a multicellular embryo. After cell division, a blastocyst is formed, which then becomes a blastocyst. The further development of the blastocyst forms germ layers in a process called gastrulation. The subsequent stages of embryo development include the formation of a nervous system, organs, and somites.

[0104] The developmental stage of an embryo can be defined according to its gestational age. As used herein, the term "gestational age (embryonic day, E)" in the context of a mammalian embryo (e.g., a mouse embryo) refers to an embryo having the developmental characteristics of a mammalian embryo counterpart in vivo (in the fallopian tube or in the uterus) on a specific date after fertilization, wherein E0 day (d) is considered as a fertilized egg.

[0105] As used herein, the term "scRNA-seq" refers to single-cell RNA sequencing technology, which is a high-throughput sequencing method for analyzing cell-specific transcriptomes at the single-cell level. It includes single-cell capture, mRNA reverse transcription, cDNA library preparation, high-throughput sequencing, and data analysis. It is mainly used to study transcriptome heterogeneity, cell type identification, cell state transitions, etc. of individual cells.

[0106] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.

[0107] As used herein, the term "bone injury" refers to a condition in which bone tissue is defective due to trauma or disease, including but not limited to bone fractures, bone cracks, bone bruises, and the like.

[0108] As used herein, the term "bone repair drug" refers to a drug used to repair bone damage in a subject (eg, a human).

[0109] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0110] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease; an effective amount for treating a disease (e.g., a disease caused by bone damage) refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0111] Advantageous Effects of the Invention

[0112] The present invention combines single-cell sequencing technology and a miniature pig model to identify stem cells in the skull periosteum for the first time, and further identifies ALPL+PDGFD+ (AP+) cells as unique skull PSCs with self-renewal ability and differentiation potential, which has promising significance for the clinical application of bone injury repair and bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figures 1A-1D These are the results of an experiment to collect periosteum tissue from miniature pig skulls. Figure 1A This is the surgical flow chart. Figure 1B This is a drawing of the material collection mode. Figure 1C This is the H&E staining structure of the periosteum tissue. Figure 1D These are photos taken during the operation of collecting periosteal tissue from miniature pigs before and after birth.

[0114] Figure 2 This is a strategy for sorting 7-AAD-negative cells from single cell suspensions of periosteal tissue of prenatal and postnatal miniature pigs.

[0115] Figures 3A-3E This is the scRNA-seq experimental result of 7AAD negative periosteal cells, where: Figure 3A This is the tSNE diagram of the clustering of periosteal cells in miniature pigs before and after birth (combined sample analysis). Figure 3B This is the tSNE diagram of the clustering of periosteal cells in miniature pigs before and after birth (individual sample analysis). Figure 3C is the expression pattern of representative marker genes in each group of miniature pig periosteal cells, Figure 3D The specific expression pattern of ALPL and PDGFD in osteoblast progenitor cell clusters. Figure 3E The expression levels and expression pattern distribution of the selection marker genes (ALPL and PDGFD) in 14 minipig periosteal cell populations.

[0116] Figure 4This is a gating strategy for flow cytometric sorting of ALPL+ and PDGFD+ (AP+) periosteal stem cells.

[0117] Figure 5 H&E staining of subcutaneous transplants in nude mice of AP+ and non-AP+ cells in embryonic stage (90 days) samples.

[0118] Figure 6 H&E staining of AP+ and non-AP+ cells in postnatal (day 0) samples of subcutaneous transplants in nude mice.

[0119] Figure 7 It is a distribution pattern of 5 regions of interest (ROIs) of the skull periosteum and a schematic diagram of morphological staining.

[0120] Figure 8 The immunofluorescence chemical detection of AP+ cell localization was performed on the periosteum tissue of 5 ROIs areas.

[0121] Fig. 9 It is the t-SNE diagram of the target cell population (osteoblast progenitor cells) in the entire periosteal cell population.

[0122] Fig.10 Three distinct subpopulations within the target cell population (osteoblast progenitor cells) were identified by unsupervised clustering.

[0123] Figures 11A-11D is the expression level distribution of different genes in the three subgroups, where Fig.11A is the distribution of expression levels of LEPR and RUNX2 in the three subgroups, Fig. 11B The expression levels of CD200 and SPP1 in the three subgroups are distributed. Fig. 11C The expression level distribution of BGLAP and COLA1 in the three subgroups. Fig.11D is the distribution of expression levels of ALPL and PDGFD in the three subgroups.

[0124] Fig.12 Pseudo-time series analysis shows the differentiation trajectories of the three cell subsets.

[0125] Fig.13 It is a FACS flow sorting strategy for primary cells derived from single AP+ cells and cells after second and third generation clonal culture.

[0126] Fig.14 Microscope images (100 μm) of primary cells derived from single AP+ cells and second- and third-generation clones, as well as immunofluorescence chemical detection of osteogenic lineage marker genes (OCN) and chondrogenic lineage marker genes (COL2α1) in third-generation clones.

[0127] Fig.15This is the H&E staining image of the subcutaneous transplantation of the second-generation AP+ cell clone in nude mice.

[0128] Fig.16 is a flow chart of secondary transplantation of grafts in mice.

[0129] Fig.17 This is the in vivo fluorescence imaging of nude mice subcutaneously transplanted with ZsGreen fluorescently labeled AP+ cells.

[0130] Fig.18 This is the FACS flow sorting strategy for ZsGreen+ cells (AP+ cells) in the grafts of 10 primary transplanted nude mice.

[0131] Fig.19 This is a microscope image (25 μm) of a single clone of sorted ZsGreen+ cells (AP+ cells).

[0132] Fig. 20 It is the H&E staining image of the subcutaneous graft in nude mice after secondary transplantation of ZsGreen+ / AP+ cells.

[0133] Fig.21 ALP / ARS / Oil Red O and Alecin Blue staining were used to detect the in vitro differentiation activity of AP+ cells labeled with ZsGreen fluorescence.

[0134] Fig. 22 This is a flow cytometry gating strategy for isolating AP+ cell populations in miniature pig tibial periosteum tissue.

[0135] Fig.23 This is the H&E staining of AP+ and non-AP+ cells in the tibial periosteum tissue of embryonic minipigs at E90d in subcutaneous transplantation in nude mice.

[0136] Fig.24 This is the H&E staining of AP+ and non-AP+ cells in the tibial periosteum tissue of neonatal miniature pigs on P0d and subcutaneous transplantation in nude mice.

[0137] Fig.25 This is the H&E / Masson / Safranin Fast Green staining of the embryonic miniature pig tibia periosteum, and the distribution area of ​​AP+ cells was identified by immunofluorescence.

[0138] Fig.26 This is a flow chart for the construction of a miniature pig skull injury model and the preparation of periosteum single cell suspension.

[0139] Fig. 27 It is the statistical difference of CFU-F number of periosteal cells at different time points after miniature pig skull injury.

[0140] Fig.28It is a display of the CFU-F monoclonal level of periosteal cells at different time points after miniature pig skull injury.

[0141] Fig.29 These are H&E staining images of subcutaneous transplants of periosteal cells in nude mice at different time points after skull injury in newborn miniature pigs.

[0142] Fig.30 It is a FACS flow cytometry sorting strategy for periosteum AP+ cells at different time points after miniature pig skull injury.

[0143] Fig.31 These are H&E staining images of subcutaneous transplants of periosteal AP+ cells in nude mice at different time points after miniature pig skull injury.

[0144] Fig.32 These are H&E staining images of subcutaneous transplants of periosteal non-AP+ cells in nude mice at different time points after miniature pig skull injury.

[0145] Fig.33 This is a flow chart of the construction of the mandibular defect model in C57BL / 6 mice and the cell transplantation in each group.

[0146] Figures 34A-34C The results are the results of the bone healing test after different groups of cells were transplanted into the mandibular critical bone defect model of mice. Fig.34A This is the imaging test result of the bone healing level of the mouse mandibular defect model after receiving different groups of AP+ cell transplantation. Fig.34B The results of imaging tests on the bone healing level of the mandibular defect model in mice after transplantation of non-AP+ cells in different groups. Fig.34C These are the results of histomorphological examination of the bone healing level in the mouse mandibular defect model after receiving cell transplantation from different groups.

[0147] Fig.35 This is a flow chart of the construction of the C57BL / 6 mouse skull defect model and the cell transplantation of each group.

[0148] Figures 36A-36B The results of the bone healing test in the mouse skull defect model after different cell transplantation. Fig.36A These are the imaging and histomorphological test results of the bone healing level after different groups of AP+ cells were transplanted into the mouse skull defect model. Fig.36B These are the imaging and histomorphological test results of the bone healing level in the mouse skull defect model after receiving different groups of non-AP+ cell transplantation.

[0149] Fig.37 This is a histomorphometric pattern of the human periosteal tissue sample extraction and preparation area.

[0150] Figures 38A-38Fis the result of genomic analysis of human skull periosteal cells, Fig.38A Seurat cluster analysis shows 12 clusters of human periosteal cells. Fig.38B The 10X Genomics scRNA-seq technique was used to perform t-SNE clustering of 3 embryonic and 3 postnatal human periosteum 7AAD-negative cells. Fig.38C The distribution of 12 identified cell populations in each human periosteal cell sample, Fig.38D This is the expression pattern of key marker genes in 12 human cranial periosteal cell groups. Fig.38E The t-SNE diagram shows the specific expression pattern of the ALPL and PDGFD gene combination. Fig.38F tSNE distribution diagram of the expression pattern of key marker genes (ALPL and PDGFD) in human periosteal tissue cells.

[0151] Fig.39 These are H&E staining images of subcutaneous transplants of human periosteal cell monoclonal clones in nude mice in the P1.5y and E18w groups.

[0152] Fig.40 are the CFU-F counts of human periosteum AP+ and non-AP+ cells in the P1.5y and E18w groups.

[0153] Fig.41 The expression levels of EGR1 in AP+ and Non-AP+ periosteal cells of E18w and P1.5y human samples were detected by Western blot.

[0154] Fig.42 This is an H&E staining of a subcutaneous transplant of a human periosteal AP+ cell monoclone from E13w in nude mice.

[0155] Fig.43 It is a flow cytometry gating strategy for the isolation of AP+ cells in periosteal tissue cells at different developmental stages (E18w and P1.5y) in humans.

[0156] Fig.44 H&E staining of subcutaneous transplants in nude mice of monoclonal non-ap+ cells from E13w and P1.5y human periosteal tissue. DETAILED DESCRIPTION

[0157] The invention is now described in the following non-limiting examples.

[0158] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0159] The experimental animals used in the embodiments of the present invention include Bama miniature pigs (newborn piglets) purchased from the Chinese Academy of Sciences (Institute of Zoology, Animal Department), pregnant pigs and embryonic piglets purchased from Beijing Genesis Miniature Pig and Large Animal Cultivation and Breeding Center, and nude mice (BALB / c nude mice (female)) purchased from Charles River Laboratory Animal Technology.

[0160] Example 1: Preparation of ALPL+PDGFD+ stem cells

[0161] 1.1 Collection of periosteal cells

[0162] To study the biological characteristics of porcine calvarial PSCs, we initially collected Figures 1A-1D The skull periosteum tissue is shown. The whole skull parietal bone-periosteum hard and soft tissue composite flap was taken out at two developmental stages before and after birth (embryonic day 90 (E90d) and postnatal day 1 (P0d)). The periosteal stripper was pressed against the inner and outer skull bone walls to carefully peel off the whole periosteum interstitial tissue, and the interstitial tissue at the bone suture was also removed. The residual muscle and skin tissue connected to it were removed, and the tissue was rinsed with physiological saline three times and placed in a sterile 50ml centrifuge tube. Add collagenase (II+IV) at 2 times volume fraction (v / v) to the 50ml centrifuge tube where the periosteum tissue is collected. After thorough oscillation for 15 seconds, use sterile surgical scissors to reach into the centrifuge tube and fully cut the periosteum tissue into 1mm tissue fragments in a paste-like state. Digest the tissue suspension at a constant temperature (1200r / min, 37℃) for 20 minutes. After digestion, let it stand for 2 minutes and transfer the tissue supernatant to a new sterile 50ml centrifuge tube (pre-place 10-15ml PBS buffer in the centrifuge tube). Repeat the above steps 3 times (digest 3 times, and continue to add an equal volume of collagenase after each supernatant transfer to continue digestion. The isolated periosteum tissue was enzymatically dissociated into single periosteal cells, and then 2-3 ml of pre-cooled sterile water was quickly added to the centrifuged cell pellet (no more than 6 seconds), slowly pipetted several times, and then 3 volumes of PBS buffer were added to neutralize to destroy red blood cells. Subsequently, live cells were screened by FACS analysis using 7AAD antibody (Miltenyi Biotec, Cat#130-111-568) ( Figure 2 ).

[0163] 1.2 Classification of periosteal cells

[0164] Single-cell transcriptome analysis of 7AAD-negative periosteal cells was performed using 10X Genomics single-cell RNA sequencing (scRNA-seq) technology. 7826 and 11358 single cells that passed quality control were obtained from two developmental stages, E90d and P0d, respectively. The sequencing results are shown in Figures 3A-3E Overall, 14 clusters were identified in Harmony’s batch effect correction and Seurat’s unsupervised clustering ( Figure 3A-3B Based on representative marker genes, these clusters were annotated as follows: epithelial cells (expressing CDH1), cyclin (TOP2A), NK cells (GNLY), T cells (CD4), smooth muscle cells (ACTA2), osteoblast precursor cells (OPCs) (PRRX1, ACAN, CD200, SP7), erythroid precursors (AHSP), endothelial cells (VWF), macrophages (CD68), monocytes (CD14, CD68), mesenchymal cells (PRRX1), chondrocytes (PRRX1, ACAN), and neutrophils (S100A12) ( Figure 3C ).like Figure 3D and Figure 3E As shown, clusters of osteogenic precursor cells were detected that exclusively expressed the surface markers ALPL and PDGFD.

[0165] 1.3 Isolation of ALPL+PDGFD+periosteal stem cells

[0166] Next, the periosteal cells obtained in Example 1.1 were sorted by FACS. After the single cell suspension of periosteal cells was prepared in the above experiment, ALPL+PDGFD+ / CD45-CD31- (AP+) (where anti-human MSCA-1 antibody (REAlease® Miltenyi Biotec, Cat#130-119-917, RRID: AB_2751919), anti-PDGF-D / SCDGFB antibody (OTI1C1) (Alexa Fluor® 488 labeled) (Novus Biologicals, Cat: NBP2-73305AF488, RRID: AB_3083799)) cells were collected by flow sorting. CD45 and CD31 were used to exclude hematopoietic and endothelial cells (anti-human CD45 antibody (BioLegend, Cat#368540, RRID: AB_2716030) and anti-human CD31 antibody (BioLegend, Cat#303116) were used). The proportion of AP+ cells in E90d and P0d samples was 0.43±0.16% and 0.34±0.17%, respectively ( Figure 4 ). During FACS sorting, monoclonal experiments were performed simultaneously. Complete culture medium was added to the 96-well plate in advance. When FACS sorted AP+ cells, single cells were directly punched into the 96-well plate, with 1 single cell per well. The cells were cultured for 7-10 days and the clone growth was observed. The monoclonal clones amplified from single E90d AP+ cells and non-AP+ cells were fully mixed with β-TCP bone powder, and then transplanted subcutaneously into the dorsal side of nude mice in the form of spheres by centrifugation. The results were then tested by H&E. Figure 5 As shown in Figure 2, the monoclonal clone derived from a single E90d AP+ cell can efficiently form bones in vivo. In contrast, except for AP+ cells, the remaining cells (E90d non-AP+ cells) lack osteogenic potential in vivo. P0d AP+ cells and non-AP+ cells were also expanded and transplanted subcutaneously on the dorsal flank of nude mice for H&E analysis. The results are shown in Figure 2. Figure 6 As shown, neither AP+ nor non-AP+ cells were able to form new bone tissue.

[0167] 1.4 Distribution of ALPL+PDGFD+ cells in vivo

[0168] The presence of AP+ cells in vivo was verified by immunofluorescence. The periosteum tissue of E90d pig skull (including the central sagittal suture) was divided into five regions of interest (ROI) according to the anatomical structure ( Figure 7 ), and collect each part separately for cryosection staining. Figure 7 As shown in the figure, the five ROI regions of interest were peeled off, and 2 mm tissue blocks were cut and fixed, then frozen in liquid nitrogen. After tissue sections were cut, immunofluorescence staining of ALPL and PDGFD was performed (ALPL antibody, Abcam, Cat#ab126820; PDGFRD antibody, Abcam, Cat#ab234666). Figure 8 As shown, most AP+ cells were localized within the crevice (ROI 3) and in the periosteum extending from the crevice (ROI 2 and ROI 4). Very few AP+ cells were observed in the dura mater (ROI 5).

[0169] 1.5 Differentiation of ALPL+PDGFD+periosteal stem cells

[0170] Subpopulations of cells within the osteoblast precursor cell (OPCs) cluster were identified and further subdivided by transcriptome analysis using 10x single-cell sequencing. This group of cells was further classified into three secondary subpopulations based on their differential gene expression patterns: Fig. 9 , Fig.10 These subpopulations were identified as follows: OPCs (subpopulation 1), characterized by the expression of LEPR and RUNX2 ( Fig.11A); preosteoblasts (subpopulation 2), showing high expression of CD200 and SPP1 ( Fig. 11B ); and mature osteoblasts (subpopulation 3), expressing the maturation markers BGLAP and COL1A1 ( Fig. 11C ). In addition, ALPL was expressed at high levels in subpopulation 1 (OPCs) and subpopulation 3 (mature osteoblasts), while PDGFD was expressed in subpopulation 1 (OPCs) and subpopulation 2 (preosteoblasts). Notably, these two genes showed significant co-expression in subpopulation 1 ( Fig.11D To explore the relationship between these subgroups, a pseudo-time series analysis was performed ( Fig.12 ), with OPCs (subpopulation 1) as the starting point of cell differentiation, progressing to pre-osteoblasts (subpopulation 2) and mature osteoblasts (subpopulation 3). The results showed that OPCs subpopulation 1, which concentratedly expressed ALPL and PDGFD, was located at the starting point of OPCs differentiation and had the cell stemness to differentiate into pre-osteoblasts and mature osteoblasts. For OPCs subpopulation 1, in the context of the present invention, it is also called ALPL+PDGFD+ stem cells (or AP+ stem cells, AP+ periosteal stem cells, AP+ cells).

[0171] Example 2: Verification of the self-renewal and differentiation abilities of ALPL+PDGFD+ stem cells

[0172] In order to test the self-renewal ability of AP+ cells, the ability of AP+ cells to form secondary and tertiary clones was then tested. The primary AP+ cells obtained by FACS flow sorting in Example 1.3 were cultured. After the primary clones were formed, they were flow sorted for AP+ cells again. After the AP+ single cells were sorted, they were cultured again. The clones formed at this time were the second-generation clones. Subsequently, the second-generation clones formed by culture were flow sorted again, and the AP+ single cells obtained were cultured again. The clones formed after the second-generation clones were cultured again were the third-generation clones. Fig.13 The results showed that the primary clones derived from single AP+ cells, as well as the second and third generation clones, can stably retain the AP+ marker, which means that E90d AP+ cells have strong self-renewal ability. The third generation clone AP+ cells after culture were fixed and immunofluorescence cytochemistry was performed to detect osteoblast and chondrogenic lineage marker genes. Fig.14 The results showed that AP+ cells expressed osteocalcin (OCN, an osteoblast lineage marker gene) and type II collagen α1 chain (COL2α1, a chondrocyte lineage marker gene), indicating that the cell population from the third-generation cloned AP+ cells contained both osteoblastic and chondrogenic lineages, and that AP+ cells had the stemness to differentiate into osteoblasts and chondrocytes. In addition, the second-generation AP+ cell clones were transplanted subcutaneously on the dorsal side of nude mice and subjected to H&E testing. The second-generation AP+ cell clones demonstrated the ability to effectively form new bone in vivo ( Fig.15 ).

[0173] To further test whether the transplanted AP+ cells maintained their stem cell properties, Fig.16 The monoclonal AP+ cells after amplification and culture were transduced with lentivirus (pLV3-[Exp]-Ctrl-zsGreen-puromycin, GenePharma, Cat#NB-LV001). After maintaining stability, puromycin was used to select the stable transfected cell lines, and the AP+ cells marked with zsGreen fluorescent protein were selected and transplanted subcutaneously on the dorsal side of nude mice ( Fig.17 Six weeks after transplantation, the grafts were removed, enzymatically hydrolyzed into single-cell suspensions, and subjected to FACS flow cytometry sorting. Fig.18 The results show that zsGreen positive cells (AP+ cells) were obtained from 10 nude mice transplanted with AP+ cells by FACS sorting. The sorted single zsGreen+ cells were seeded individually into 96-well plates for monoclonal culture and secondary transplantation ( Fig.19 ). After the grafts were digested twice to form a single cell suspension, they were cultured for monoclonal growth. The expanded cells were fully mixed with β-TCP bone powder, centrifuged into spheres, and then transplanted into the subcutaneous tissue of the nude mice again. After 6 weeks, the grafts were harvested for H&E testing. H&E testing showed that these zsGreen+ colonies retained the ability to form bone nodules in vivo ( Fig. 20 ). The harvested grafts were stained with ALP / ARS / Oil Red O and Alecian Blue, and the results showed the multi-series differentiation potential of AP+ cells in vitro. Among them, ALP and ARS staining showed that these cells had good osteogenic potential; Oil Red O staining showed that these cells had good adipogenic differentiation potential; Alecian Blue staining results showed that these cells had chondrogenic potential ( Fig.21 These data further strongly demonstrate the self-renewal and differentiation capabilities of periosteal AP+ cells.

[0174] Subsequently, the periosteum tissue of the tibia of E90d and P0d miniature pigs was taken, and after the residual fat tissue and muscle tissue were fully removed, the ligaments of the cartilage at both ends were removed, and the periosteum tissue of the diaphysis and epiphysis was taken. After careful peeling, mechanical dissociation and enzyme digestion (the method is the same as that of Example 1.1) were used to prepare a single cell suspension of the tibial periosteum, and FACS sorting was performed to separate AP+ cells from the periosteum of the tibia (the method is the same as that of Examples 1.2 and 1.3) ( Fig. 22 The AP+ and non-AP+ cells of the tibial periosteum were expanded and then transplanted subcutaneously into the back of nude mice and stained with H&E. Fig.23 , Fig.24As shown, both AP+ and non-AP+ cells efficiently formed new bone. Unlike the skull periosteal cells, the in vivo osteogenic capacity of the tibia (long bone) periosteal cells of postnatal (P0d) miniature pigs was significantly stronger than that of the embryonic (E90d) miniature pigs ( Fig.24 ). The long bone periosteum tissue was frozen sectioned and immunofluorescence stained (the method is the same as that of Example 1.4). Fig.25 The results revealed the presence of AP+ cells in both the periosteum and bone marrow of long bones. In summary, although AP+ stem cells can also be found in the periosteum of long bones, the experimental results clearly demonstrate the significant biological differences in properties between cranial PSCs and long bone PSCs.

[0175] Example 3: ALPL+PDGFD+stem cells promote the repair of bone damage

[0176] Next, a craniofacial fracture model was established in an immature pig model at P0d ( Fig.26 ). The model was constructed by opening a window in the skull of a miniature pig after general anesthesia. Make a precise T-shaped incision at the top of the skull, extending along the anterior occipital and parietal sutures and the midsagittal suture, through the skin and subcutaneous tissue to expose the underlying bone. Each tissue layer is carefully separated to expose the bone. A 5mm diameter drill bit (Vincent Medical) is used to open a full-thickness bone window on both sides of the midsagittal suture (occipital-parietal) at about 1cm, and the internal periosteum is carefully preserved. Finally, the outer periosteal tissue and skin tissue are sutured in layers. At 12 hours and 24 hours after trauma (Tra-12h and Tra-24h), the experimental animals were killed, and after dissociating the entire skull, the inner and outer periosteal tissues and the periosteal tissues at the sutures were fully peeled off aseptically, and single-cell suspensions were obtained by mechanical dissociation and enzymatic digestion, and 7AAD-negative cells were sorted (the method is the same as in Example 1.1). After 7AAD flow sorting, the sorted single cells were placed in a 96-well plate and cultured for 7-10 days. The clone formation was observed and crystal violet staining was performed. The number of single clones formed was counted and compared with 96. The obtained value was the CFU-F activity. Compared with the control group P0d (P0d, non-injured) cells, Tra-24h cells showed the highest CFU-F activity, while the control group cells showed the lowest CFU-F number ( Fig. 27 , Fig.28 In addition, the expanded cells were fully mixed with β-TCP bone powder, centrifuged into spheres, and transplanted into the subcutaneous tissue of the back of nude mice. After 6 weeks, the transplants were harvested for H&E analysis. Compared with the control group cells and Tra-12h cells, Tra-24h cells showed the greatest in vivo differentiation potential ( Fig.29 ).

[0177] In order to further explore the potential role of periosteal AP+ stem cells in bone repair, AP+ cells were isolated from the periosteum of the control group (non-traumatic at P0d), Tra-12h, and Tra-24h (the process of mechanical dissociation and enzyme digestion to prepare single cell suspension was the same as in Example 1.1). Fig.30 ). FACS analysis showed that approximately 0.26±0.07% of periosteal cells in the control group were AP+ cells. In contrast, the abundance of AP+ cells increased significantly after bone injury, reaching 0.44±0.12% at Tra-12h and 0.71±0.14% at Tra-24h ( Fig.30 The expanded cells were fully mixed with β-TCP bone powder, centrifuged into spheres, and transplanted into the subcutaneous tissue of nude mice. After 6 weeks, the transplants were harvested for H&E testing. The experimental results showed that AP+ cells from Tra-12h and Tra-24h effectively regenerated bone tissue ( Fig.31 , Fig.32 ). These findings demonstrate activation of AP+ cells from postnatal day 0 minipigs in response to bone injury.

[0178] A mandibular defect model was constructed using C57BL / 6 mice (see Vajgel, A. et al., A Systematic Review on the Critical Size Defect Model., Clin Oral Implants Res25, no. 8 (2014): 879-93.), and the regenerative potential of activated PSCs was further studied. The expanded Tra-24h and control group AP+ cells from the craniofacial fracture model miniature pigs were fully mixed with HAMA hydrogel, centrifuged into spherical cells, and implanted into the bone defect site during the construction of the bone defect model ( Fig.33 After 6 weeks, micro-CT and morphometric analysis showed that recipients implanted with activated AP+ cells had significantly increased bone formation compared with controls ( Figures 34A-34C ). In addition, the contribution of AP+ cells to skull repair was evaluated. A skull defect model was constructed using C57BL / 6 mice. The expanded Tra-24h and control AP+ cells from the minipigs with craniofacial fractures, as well as the AP+ cells from the E90d minipigs, were fully mixed with HAMA hydrogel, centrifuged into spheres, and implanted into the bone defect site during the construction of the bone defect model ( Fig.35 ). After 6 weeks, micro-CT and morphological analysis were performed, and the results were as follows Figures 36A-36B As shown, activated AP+ cells displayed a remarkable ability to promote skull bone regeneration.

[0179] Example 4: Human skull periosteum contains ALPL+PDGFD+ stem cells

[0180] In order to explore the translational significance of the present invention, skull tissue bone flaps of human fetuses and adults (Skull tissue bone flap, Human beings, E13w~P30y, Department of Obstetrics and Gynecology, Peking University Third Hospital) were collected. Fig.37 Single periosteal cells were used for 10X Genomics analysis, and 14,248 and 15,569 quality-controlled single cells were obtained from two developmental stages, embryonic and postnatal, respectively ( Figures 38A-38B In total, 12 clusters were identified in Harmony’s batch effect correction and Seurat’s unsupervised clustering ( Figures 38C-38D ).like Figures 38E-38F As shown, it is clear that AP+ cells constitute a subset of human OPC clusters and neutrophil clusters, which is different from the AP+ cells in the pig dataset.

[0181] The PCs collected at the embryonic and postnatal stages were fully mixed with β-TCP bone powder, centrifuged into spheres, and transplanted subcutaneously into the back of nude mice. The grafts were harvested 6 weeks later for H&E analysis. PCs at the embryonic stage showed superior in vivo osteogenic ability compared with those at the postnatal stage ( Fig.39 ), which is consistent with the observations of porcine PCs. To better understand human skull AP+ cells, FACS sorting of embryonic week 18 (E18W) and postnatal month 1.5 (P1.5y) AP+ cells was performed ( Fig.43 ), Western blot detection of EGR1 expression levels in AP+ and non-AP+ periosteal cells of E18w and P1.5y human samples ( Fig.41 ), and the monoclonal clones of the selected AP+ and non-AP+ periosteal cells were cultured and transplanted subcutaneously on the back of nude mice. The grafts were harvested 6 weeks later for CFU-F activity detection ( Fig.40 At the same time, AP+ and non-AP+ periosteum cells from embryonic week 13 (E13W) and P1.5y were transplanted subcutaneously on the back of nude mice, and the grafts were harvested 6 weeks later for H&E examination ( Fig.42 The results showed that there were significant differences between embryonic and postnatal AP+ cells in CFU-F activity, EGR1 expression, and in vivo differentiation potential, similar to the observations of porcine skull PSCs, with embryonic AP+ cells having higher CFU-F activity, higher EGR1 expression, and better in vivo differentiation potential than postnatal AP+ cells.

[0182] In addition, non-AP+ cells from embryonic week 13 (E13W) and postnatal month 1.5 (P1.5y) were sorted and transplanted subcutaneously on the back of nude mice for H&E analysis. The experimental results showed that non-AP+ cells from the embryonic stage have limited ability to generate new bone in vivo ( Fig.44 This provides additional evidence that AP+ can be used as a reliable marker for isolating human calvarial PSCs.

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[0204] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. Periosteal stem cells, which express ALPL gene and PDGFD gene positively.

2. The periosteal stem cell according to claim 1, wherein The periosteal stem cells are skull periosteal stem cells or long bone periosteal stem cells; Preferably, the periosteal stem cells are derived from mammals; Preferably, the periosteal stem cells are derived from non-human mammals; Preferably, the periosteal stem cells are derived from mammals in the embryonic development stage, or mammals with bone damage; Preferably, the mammal is a pig (e.g., a minipig) or a mouse; Preferably, the mammal is a human.

3. The periosteal stem cell according to claim 1 or 2, having one or more of the following characteristics: (1) capable of producing at least one daughter stem cell through cell division, wherein the daughter stem cell retains the same cell division activity and cell differentiation activity as the parent stem cell from which it is derived; (2) having the ability to differentiate into other cells, for example, into osteoblasts or chondrocytes; preferably, the osteoblasts are pre-osteoblasts or mature osteoblasts; (3) In a mammal, when the bone tissue in which the periosteal stem cells are located is defective or damaged, the periosteal stem cells: (a) proliferate and increase in number; and / or (b) further differentiate into other cells, for example, osteoblasts or chondrocytes; preferably, the osteoblasts are pre-osteoblasts or mature osteoblasts.

4. A method for preparing the periosteal stem cells according to any one of claims 1 to 3, comprising the following steps: (1) Collect cells from the periosteum of mammals; (2) Among the collected cells, label the cells expressing ALPL and PDGFD; (3) sorting out the cells expressing ALPL and PDGFD in (2) to obtain the periosteal stem cells; Preferably, in step (1), cells are collected from the periosteum of a mammal in an embryonic development stage, or cells are collected from the periosteum of damaged bone tissue of a mammal; Preferably, before step (2) and after step (1), the method further comprises the step of marking cells expressing CD45 and CD31 in the cells and removing the cells expressing CD45 and CD31 from the cells; preferably, the cells expressing CD45 and CD31 are removed by fluorescence activated cell sorting (FACS); Preferably, in step (3), the cells are sorted by fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), density gradient centrifugation or microfluidic cell sorting; Preferably, sorting is performed by FACS; Preferably, the mammal is a non-human mammal; Preferably, the mammal is a pig (e.g., a minipig, a mini-pig) or a mouse; Preferably, the mammal is a human.

5. The preparation method according to claim 4, further comprising the steps of: (4) culturing and sorting the obtained cells, namely the periosteal stem cells; Preferably, it also includes the following steps: (5) subculturing the periosteal stem cells to obtain second-generation periosteal stem cells and third-generation periosteal stem cells; Preferably, the culture is performed by single cell cloning.

6. The preparation method according to claim 5, further comprising the steps of: (6) detecting the expression of ALPL and PDGFD in the periosteal stem cells obtained in step (3) or step (4), or the second-generation periosteal stem cells and the third-generation periosteal stem cells obtained in step (5), and removing cells that do not express ALPL and PDGFD at the same time; Preferably, the expression of ALPL and PDGFD in the periosteal stem cells, the second-generation periosteal stem cells and the third-generation periosteal stem cells is detected by protein blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, mass spectrometry or immunohistochemistry; Preferably, cells that do not express both ALPL and PDGFD are removed by fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), density gradient centrifugation or microfluidic cell sorting.

7. The preparation method according to any one of claims 4 to 6, wherein The periosteum is the skull periosteum or the long bone periosteum.

8. A pharmaceutical composition comprising the periosteum stem cells according to any one of claims 1 to 3, and one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further comprises an additional bone repair drug; Preferably, the bone repair drug is selected from one or more of glucosamine, chondroitin sulfate, calcium, sodium hyaluronate, bone morphogenetic protein and alendronate sodium.

9. Use of the periosteal stem cells according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a drug for repairing bone damage in a subject and / or for treating a disease caused by bone damage in a subject; Preferably, the bone lesion is a skull lesion, a long bone lesion, and / or a cartilage lesion; Preferably, the bone injury is a bone fracture, a bone crack, and / or a bone contusion; Preferably, the disease caused by bone damage is osteoporosis, bone tumor, osteomyelitis, bone spur (bone hyperplasia), osteoarthritis, osteomalacia, osteopenia, bone defect, osteolysis or osteonecrosis; Preferably, the subject is a mammal (eg, a human); Preferably, the periosteal stem cells or the pharmaceutical composition are used alone or in combination with another bone repair drug.

10. The use according to claim 9, wherein The periosteal stem cells are derived from mammals in the embryonic development stage, or mammals with bone damage; Preferably, the mammal and the subject are the same individual of the same species or different individuals of the same species; Preferably, the periosteal stem cells are cranial periosteal stem cells.

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