CN112877364A - Reprogramming induction protocol for direct transformation of subchondral bone cells into articular chondrocytes - Google Patents
Reprogramming induction protocol for direct transformation of subchondral bone cells into articular chondrocytes Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses an induction scheme for direct transformation from reprogrammed subchondral osteoblasts to functional articular chondrocytes, which is suitable for regenerative repair treatment of severe articular cartilage defect. The induction protocol of the present invention comprises: the genes Sox9, Sox5 and Plagl1 or Sox8 and ZCchc5 were introduced into subchondral bone cells to obtain cartilage-like cells.
Description
Technical Field
The invention belongs to the technical field of biology, relates to cell and molecular biology, eukaryotic cell reprogramming, cell transformation and differentiation, and tissue regeneration and repair, and also relates to application of a technical scheme for cell reprogramming in articular cartilage injury treatment.
Background
Articular cartilage tissue (articular cartilage) has a fine layered structure and contains chondrocytes (chondrocytes) of different functions. The deep-medial structures lack vessels, nerves and lymphatic vessels, and the number of chondrocytes is rare. Although the matrix mainly contains collagen (collagen), proteoglycan (proteoglycan) and non-collagen components, and plays a role in buffering and lubrication, it lacks self-renewal ability (Bhosale and Richardson 2008). If the joint movement causes high-intensity impact and excessive abrasion, the cartilage is seriously damaged, and the cartilage tissue can not be self-repaired.
With the rapid progress of the aging of the population in China, the incidence of articular cartilage injury is gradually increased; the rapid development of the national fitness exercise also enables the cartilage patients to show a trend of youthfulness. Osteoarthritis (osteoarthritis), a high-incidence joint disease, currently accounts for over 10% of the total population, and most of them have pathological changes such as articular cartilage defects (Bhosale and Richardson 2008). Because clinical intervention is limited, the course of disease is not healed, and the wound surface and the periphery are further abraded, the defect gradually develops into full-thickness cartilage defect (full-thickness defect), so that a bone plate below the cartilage is exposed (Frobell, Wirth et al 2010), the joint movement is limited and aggravated, and finally the joint movement is disabled.
The patients with severe cartilage defects can only improve the quality of life and avoid disability by total joint replacement (Juni, Reichenbach et al 2006), but at the same time need to undergo what can be called the most difficult postoperative rehabilitation process, and also face a series of problems such as prosthesis maintenance and non-negligible complications, while for the majority of elderly patients, there is no doubt a need to bear more serious challenges. Therefore, the development of minimally invasive treatment methods with low risk and high safety is of great significance.
Currently, the regenerative repair treatment for cartilage defects mainly includes two modes of endogenous cell recruitment and exogenous cell transplantation. Microfracture (microfracture) promotes bone marrow extravasation containing mesenchymal stem cells through a bone surface drilling technology, fills defects, and repairs damaged cartilage. However, this treatment has the problem that fibrocartilage is easily formed after the operation, which is different from hyaline cartilage, and the joint function repair effect is weakened (Oussedik, Tsutskaris et al 2015). Although autologous chondrocyte transplantation (MACI) using cartilage tissue at a non-load bearing part has a good hyaline cartilage repair effect, there are also unavoidable problems, such as surgical material acquisition, limited autologous cartilage source, dedifferentiation phenomenon in vitro culture and postoperative complications. Due to the layered structure of articular cartilage, it is still difficult to achieve the level of repair in normal physiological state by simply transplanting differentiated and mature chondrocytes. Mesenchymal stem cells have attracted attention in recent years as seed cells, and have become a focus of basic and clinical research (Harrell, Markovic et al 2019). However, in practical application, there are many problems that in vitro operations such as cell acquisition, culture and preservation, and the like, and the directional differentiation of cells is accurately regulated to avoid ossification and calcification, and the technical bottleneck needs to be broken through.
For the regenerative repair of a full-thickness defect of cartilage, subchondral bone cells (subchondral bone cells) may be potential seed cells due to the following unique advantages: (1) the chondrocytes are adjacent to subchondral osteocytes, and the exposed subchondral osteocytes become wound surfaces (versions); (2) the subchondral bone plate carries subchondral bone cells, forming a natural cell-scaffold complex (cell-scaffold complex); (3) both are of mesodermal origin and belong to the same lineage (linkage); the chondrocytes are transdifferentiated into osteoblasts (chondrocyte-to-osteoblast transduction) via the endochondrial ossification (Yang, Tsang et al 2014); (4) subchondral bone cells are terminally differentiated by skeletal stem cells and are abundant in source. Therefore, the conversion of subchondral bone cells into chondrocytes with a repair function becomes the primary key for realizing in-situ repair of the cartilage full-thickness defect.
The exogenous over-expression transcription factor combination Oct4, Sox2, KLF4 and c-Myc can induce fibroblast reprogramming (cell reprogramming) to be transformed into Induced Pluripotent Stem Cells (iPSC) (Takahashi and Yamanaka 2006), and based on the technology, other adult cells can also be reprogrammed to be transformed into pluripotent stem cells, wherein the pluripotent stem cells comprise chondrocytes and synoviocytes which are self-derived from osteoarthritis patients and direct induced transformation from the synoviocytes to the iPSC (Kim, Son et al.2011, Wei, Zeng et al.2012), and then are transformed into chondrocytes. Chondrocytes differentiated from ipscs may show better cartilage matrix forming ability than mesenchymal stem cells (Ko, Kim et al.2014). In view of the limitation of the potential tumorigenic risk of the iPSC on the aspect of clinical application, a plurality of key transcription factors are introduced to induce one type of adult cells to be directly transformed (direct conversion) into another type of adult cells, so that the process of cell dedifferentiation and then directed differentiation can be passed over, and the potential safety hazard brought by the iPSC stage is avoided.
As early as 1987, MyoD transcription factors were reported to induce the direct conversion of fibroblasts into myocytes (Davis, Weintraub et al 1987). In recent years, fibroblasts were induced by Gata4, Mef2c and Tbx5 to be directly transformed into myocardial-like cells (Ieda, Fu et al 2010), and chondrocytes were also transformed by direct reprogramming (Hiramatsu, Sasagawa et al 2011, Ishii, Kami et al 2012, Takimoto, or o et al 2012). Therefore, the direct reprogramming technology can more directly utilize the adult cells to regenerate and repair.
Aiming at the repair of the cartilage full-layer defect, subchondral bone cells exposed at the bottom of a wound surface can be directly used for directionally inducing transformed seed cells to the chondrocytes. If in-situ induction is carried out, the operation can be realized only by injecting reprogramming factors into joints, which is equivalent to minimally invasive operation, the patients have nearly no pain, the defects of ACI/MACI therapy can be overcome, and a new mode of utilizing a natural stent transplantation technology is opened.
Disclosure of Invention
The purpose of the present invention is to provide a direct transformation pathway from subchondral bone cells to articular chondrocytes and a cell reprogramming induction scheme.
The specific bone tissue cells derived from the subchondral bone plate, including osteoblasts, osteocytes or osteoclasts, are distributed on the subchondral bone plate and are exposed on the joint surface due to damage or abrasion of the articular cartilage tissue above the specific bone tissue cells.
The bone joints containing the subchondral bone plate comprise knee joints, hip joints, vertebral joints, sacroiliac joints, costal joints, sternoclavicular joints, acromioclavicular joints, shoulder joints, elbow joints, wrist joints, foot joints and finger joints.
The direct transformation from the subchondral osteocyte to the articular chondrocyte comprises the action of single or multiple transcription factors on a specific target gene promoter region in a cell, so that specific osteocyte cells derived from a subchondral bone plate undergo cell direct reprogramming to lose the marker function and the phenotypic characteristic of the original specific cell type, and are directionally transformed into another specific type of articular cartilage-like cells with the marker function and the phenotypic characteristic of the articular chondrocyte.
The articular chondrocyte has the characteristic of marking function and phenotype, and comprises expressing high-level II type collagen, proteoglycan and appropriate expression level IX and XI type collagen, presents a polygonal paving stone-like shape, does not express or expresses low-level I type collagen, alkaline phosphatase and osteocalcin.
The direct transformation from subchondral bone cells to chondrocytes requires the introduction of at least one reprogramming factor, has a transient or stable expression level, can directly or indirectly induce the expression of at least one cell endogenous gene, and promotes the formation of stable and determined marker functions and phenotypic characteristics belonging to articular chondrocytes.
The reprogramming factors can promote at least one reprogramming factor to directly or indirectly induce the expression of at least one cell endogenous gene for promoting the formation of articular chondrocytes by participating in chromatin appearance modification.
At least one of the reprogramming factors can prevent the maintenance of the marker function and the phenotype characteristic of the original bone tissue cell or promote the formation of the marker function and the phenotype characteristic of the original bone tissue cell.
The reprogramming factor should not promote transformation of a third cell type, including fibroblasts, myocytes, neurons, epithelial cells, endothelial cells, hematopoietic cells, lymphocytes, into non-bone tissue cells or chondrocytes.
The reprogramming factors described in the present invention include Sox and one or more of Sox, Sox forming a composition, which in combination with one or more of Foxa, Id, Ndn, Peg, Plagl, Zcchc, Zfp354 and Zfp385 induces and promotes the directional transformation of subchondral bone cells into cartilage-like cells, and in combination with one or more of AEBP, ATF, CREB3L, Eid, Foxa, Foxc, Foxo, Foxp, Hoxa, Hoxc, KLF, Nr4a, Nfix, Nrep, Pitx, 3f, Prickle, RARG, Runx, Sfr, Shox, Snai, Sp and Trps, enhances the induction or promotion of direct transformation of subchondral bone cells into chondrocytes.
The reprogramming factor introduced into the subchondral bone cells can be transported by methods of viral vectors, plasmid transfection, nucleic acid forms, protein mediation, transposons and nanoparticles, and acts on a specific target gene promoter region in the form of nucleic acid, protein, polypeptide and small molecular compounds.
All or partial gene segments in some combinations are constructed on the same recombinant expression vector and transferred into cells for co-expression.
The reprogramming factor composition is induced for a period of 1-8 weeks and expresses at least one stable, defined marker functional and phenotypic characteristic of articular chondrocytes for at least 1-8 weeks.
The cartilage-like cells induced by direct reprogramming can be cultured in three dimensions in vitro to form cartilage microspheres and form cartilage tissues in vivo.
The method provided by the invention can take subchondral bone cells as in-situ seed cells to carry out effective direct reprogramming transformation into cartilage-like cells, and meets the treatment requirements of various joint diseases accompanied with severe cartilage injury, including osteoarthritis, rheumatoid arthritis, cartilage injury, traumatic rupture or shedding, achondroplasia and ankylosing spondylitis.
The invention has the advantages that:
subchondral osteocytes are part of cartilage defect wound surfaces, are locally obtained and have sufficient sources, are natural seed cells for in-situ regeneration and repair, and are reversely induced by the same lineage to contribute to improvement of induction efficiency and transformation reliability.
The subchondral bone cells and the bone plate form a natural cell scaffold complex, which is beneficial to repairing large-area defects and reducing the dependence on artificial scaffolds.
Drawings
FIG. 1 shows the immunofluorescence assay for the expression of collagen and proteoglycan type II, IX and XI markers specific to chondrocytes after transfection of Sox9, Sox5 in combination with Plagl1 factor.
FIG. 2 shows the immunofluorescence assay of chondrocyte-specific markers for the expression of collagen type II, IX and XI and proteoglycan after transfection of Sox9, Sox5, Sox8 in combination with ZCchc5 factor.
FIG. 3 shows the immunofluorescence assay of the expression of collagen type II, IX and XI and proteoglycan specific markers of chondrocytes after transfection of Sox9, Sox6 in combination with ZCchc5 factor.
Detailed Description
Embodiments of the invention are described below, in conjunction with the appended drawings, and all such specific compositions, materials, and methods described below are intended to be in whole or in part within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the invention but merely to illustrate specific embodiments within the scope of the invention. It will be appreciated that many variations to the described solution of the invention are possible while still remaining within the scope of the invention.
Example 1
First, experimental material
Mouse knee joint subchondral bone osteoblast, 293T cell
Lentiviral packaging plasmid psPAX2, envelope plasmid pMD2.G and shuttle plasmid pSIN4-CMV
Second, Experimental methods
Construction of a Lentiviral vector carrying a segment of a reprogramming factor Gene
Sox9, Sox5 and Plagl1 reprogramming factor genes amplified from cDNA were constructed on pSIN4 lentiviral vector backbone, the resulting plasmid was purified and obtained to a titer of 1-3X 10 by second generation lentiviral packaging system8Tu/ml of lentivirus particles.
TABLE 1 primer sequences
Reprogramming factor induction based on mouse knee joint subchondral bone osteoblasts
Subchondral bone osteoblasts at a rate of 5000/cm224 hours after inoculation, the reprogramming factors lentiviruses were treated with Sox9, Sox5, Sox6, Sox8, ZCchc5 or Plagl1 single factors and SThe ox9, Sox5 and Plagl1 were combined and transduced into a culture medium containing 8 μ g/ml polybrene at a multiplicity of infection equal to 100, and after 16h incubation the virus-containing medium was removed and DPBS washed 2 times and replaced with chondrocyte complete medium.
Immunofluorescence method for detecting cartilage specific protein expression
After 14 days of induction, the obtained induced cells were fixed with 4% paraformaldehyde for 15min, washed with DPBS for 5min, and repeated 2 times. 0.2% TritonX-100 permeates cell membrane for 10min, DPBS washes for 3min, repeats for 3 times. Blocking with 3% BSA at room temperature for 1h, adding primary antibody COL2A1(1:500), ACAN (1:750), COL9A1(1:1000) or COL11A1(1:1000), and incubating overnight at 4 ℃. The primary antibody was aspirated, washed with DPBS for 3min and repeated 4 times, a goat anti-mouse IgG2b secondary antibody labeled with AF568 (1:2000) and a donkey anti-rabbit secondary antibody labeled with AF647 (1:750) were added, incubation was continued for 1h at room temperature, DAPI (1:1000) was added for 5min, and washing with DPBS for 5min and repeated 4 times. And observing and recording the cell fluorescence expression by a high content imaging system.
Third, experimental results
The results of immunofluorescent staining and the statistical results of fluorescence expression intensity are shown in FIG. 1 and Table 2. Osteoblasts not induced by transcription factor combination, empty vector control and single-factor transduced groups of Sox9, Sox5, Sox6, Sox8, Zcchc5 or Plagl1 have no expression of collagen and proteoglycan of type II, IX and XI; sox9, Sox5 and Plagl1 combined induced cells to express cartilage cell markers functional proteins type II, IX and XI collagen and proteoglycan, and the morphology was transformed from fusiform zigzag to polygonal paving stone.
The above results indicate that single factor transduction of Sox9, Sox5, Sox6, Sox8, Zcchc5 or Plagl1 is not sufficient to induce the transformation of subchondral bone cells into chondrocytes, and the combination of Sox9, Sox5 and Plagl1 factors can effectively reprogram subchondral bone cells to directly transform them into cartilage-like cells expressing key functional proteins required for the construction of cartilage tissues.
TABLE 2 mean fluorescence intensity of cellular protein expression after Induction
Example 2
First, experimental material
Mouse knee joint subchondral bone osteoblast, 293T cell
Lentiviral packaging plasmid psPAX2, envelope plasmid pMD2.G and shuttle plasmid pSIN4-CMV
Second, Experimental methods
Construction of a Lentiviral vector carrying a segment of a reprogramming factor Gene
Constructing the reprogramming factor genes of Sox9, Sox5, Sox8 and Zcchc5 amplified from cDNA onto pSIN4 lentiviral vector backbone, purifying the obtained plasmid, obtaining the titer of 1-3 × 10 by using second generation lentiviral packaging system8Tu/ml of lentivirus particles.
TABLE 3 primer sequences
Reprogramming factor induction based on mouse knee joint subchondral bone osteoblasts
Subchondral bone osteoblasts at a rate of 5000/cm2After 24 hours of inoculation, the reprogramming factor lentiviruses were added to the culture broth containing 8 μ g/ml polybrene at a multiplicity of infection equal to 100 with a combination of Sox9, Sox5, Sox8 and Zcchc5, the virus-containing medium was removed after 16h incubation, and DPBS was washed 2 times and replaced with chondrocyte complete medium.
Immunofluorescence method for detecting cartilage specific protein expression
After 14 days of induction, the obtained induced cells were fixed with 4% paraformaldehyde for 15min, washed with DPBS for 5min, and repeated 2 times. 0.2% TritonX-100 permeates cell membrane for 10min, DPBS washes for 3min, repeats for 3 times. Blocking with 3% BSA at room temperature for 1h, adding primary antibody COL2A1(1:500), ACAN (1:750), COL9A1(1:1000) or COL11A1(1:1000), and incubating overnight at 4 ℃. The primary antibody was aspirated, washed with DPBS for 3min and repeated 4 times, a goat anti-mouse IgG2b secondary antibody labeled with AF568 (1:2000) and a donkey anti-rabbit secondary antibody labeled with AF647 (1:750) were added, incubation was continued for 1h at room temperature, DAPI (1:1000) was added for 5min, and washing with DPBS for 5min and repeated 4 times. And observing and recording the cell fluorescence expression by a high content imaging system.
Third, experimental results
The results of immunofluorescent staining and the statistical results of fluorescence expression intensity are shown in FIG. 2 and Table 4. Osteoblasts and empty vector control groups which are not induced by transcription factor combination have no expression of collagen proteins II, IX and XI and proteoglycan; the combination of Sox9, Sox5, Sox8 and Zcchc5 induces the cells to express cartilage cell marker functional proteins type II, IX and XI collagen and proteoglycan, and the morphology is converted from fusiform saw-tooth shape to polygonal paving stone shape.
The above results indicate that the combination of Sox9, Sox5, Sox8 and Zcchc5 factors has the effect of efficiently reprogramming subchondral bone cells to directly convert them into cartilage-like cells expressing key functional proteins required for constructing cartilage tissues.
TABLE 4 mean fluorescence intensity of cellular protein expression after Induction
Example 3
First, experimental material
Mouse knee joint subchondral bone osteoblast, 293T cell
Lentiviral packaging plasmid psPAX2, envelope plasmid pMD2.G and shuttle plasmid pSIN4-CMV
Second, Experimental methods
Construction of a Lentiviral vector carrying a segment of a reprogramming factor Gene
Constructing the reprogramming factor genes of Sox9, Sox6 and Zcchc5 amplified from cDNA onto pSIN4 lentiviral vector backbone, purifying the obtained plasmid, obtaining the titer of 1-3 × 10 by the second generation lentiviral packaging system8Tu/ml of lentivirus particles.
TABLE 5 primer sequences
Reprogramming factor induction based on mouse knee joint subchondral bone osteoblasts
Subchondral bone osteoblasts at a rate of 5000/cm2After 24 hours of inoculation, the reprogramming factor lentiviruses were added to the culture broth containing 8 μ g/ml polybrene at a multiplicity of infection equal to 100 with a combination of Sox9, Sox6 and Zcchc5, the virus-containing medium was removed after 16h incubation, and the DPBS was washed 2 times and replaced with chondrocyte complete medium.
C. immunofluorescence method for detecting cartilage specific protein expression
After 14 days of induction, the obtained induced cells were fixed with 4% paraformaldehyde for 15min, washed with DPBS for 5min, and repeated 2 times. 0.2% TritonX-100 permeates cell membrane for 10min, DPBS washes for 3min, repeats for 3 times. Blocking with 3% BSA at room temperature for 1h, adding primary antibody COL2A1(1:500), ACAN (1:750), COL9A1(1:1000) or COL11A1(1:1000), and incubating overnight at 4 ℃. The primary antibody was aspirated, washed with DPBS for 3min and repeated 4 times, a goat anti-mouse IgG2b secondary antibody labeled with AF568 (1:2000) and a donkey anti-rabbit secondary antibody labeled with AF647 (1:750) were added, incubation was continued for 1h at room temperature, DAPI (1:1000) was added for 5min, and washing with DPBS for 5min and repeated 4 times. And observing and recording the cell fluorescence expression by a high content imaging system.
Third, experimental results
The results of immunofluorescent staining and fluorescence expression intensity statistics are shown in FIG. 3 and Table 6. Osteoblasts and empty vector control groups which are not induced by transcription factor combination have no expression of collagen proteins II, IX and XI and proteoglycan; sox9, Sox6 and Zcchc5 in combination induce cells to express cartilage cell markers functional proteins type II, IX and XI collagen and proteoglycan, and the morphology is converted from fusiform zigzag to polygonal paving stone.
The above results indicate that Sox9, Sox6 and Zcchc5 are able to reprogram subchondral bone cells and directly convert them into cartilage-like cells expressing key functional proteins required for the construction of cartilage tissues.
TABLE 6 mean fluorescence intensity of cellular protein expression after reprogramming
Claims (10)
1.A reprogramming induction scheme for directional transformation of bone tissue cells to articular chondrocytes is characterized in that specific bone tissue cells derived from a subchondral bone plate undergo direct reprogramming of cells, lose the marker function and phenotypic characteristic of the original specific cell type, and are directionally transformed into another specific articular cartilage-like cell with the marker function and characteristic of the articular chondrocytes.
2. The reprogramming induction protocol of claim 1, wherein the specific bone tissue cells of the subchondral plate undergo direct cell reprogramming by transferring at least one reprogramming factor into the bone tissue cells of the subchondral plate, and acting on the specific target gene promoter region in the cells to induce the transformation of the bone tissue cells into the articular cartilage-like cells.
3. The reprogramming induction protocol of claim 2, wherein the reprogramming factors express at least one reprogramming factor in bone tissue cells, induce the expression of at least one cellular endogenous gene and its product, and form at least one stable, defined, articular chondrocyte that is insufficient to maintain the original bone tissue cells or induce and promote the marker functions and phenotypic characteristics of the third cell type.
4. The reprogramming induction protocol of claim 3, wherein the reprogramming factors are selected from Sox9 and Sox5, Sox6, Sox8 in combination with one or more factors to induce and promote the directional transformation of bone tissue cells into cartilage-like cells.
5. The reprogramming induction protocol of claim 3, wherein the reprogramming factors are selected from the group consisting of Sox9, Sox5 in combination with Plagl1, or Sox9, Sox5, Sox8 in combination with ZCchc 5; or the reprogramming factors are selected from Sox9 and one or more of Sox5, Sox6 and Sox8 which are combined and then combined with one or more of Foxa3, Id1, Id3, Ndn, Peg3, Zfp354c and Zfp385b to induce and promote the directional transformation of subchondral bone cells into cartilage-like cells.
6. The reprogramming induction protocol according to any one of claims 4 to 5, comprising the step of introducing the gene encoding the protein of interest into a somatic cell in vivo or in vitro.
7. The reprogramming induction protocol of claim 6, comprising the steps of: inserting the protein coding gene of the composition into an expression vector to obtain a recombinant vector.
8. The reprogramming induction protocol of claim 6, wherein the reprogramming factor is induced for a period of 1 to 8 weeks.
9. A reprogramming induction composition for the directional transformation of bone tissue cells into articular chondrocytes, which is characterized in that the composition is selected from Sox9 and one or more factors selected from Sox5, Sox6 and Sox8, or the combination of the factors and one or more factors selected from Foxa3, Id1, Id3, Ndn, Peg3, Plagl1, ZCchc5, Zfp354c and Zfp385b, so as to induce and promote the directional transformation of subchondral osteocytes into cartilage-like cells.
10. Use of the reprogramming induction composition according to claim 9 for the preparation of a medicament for treating severe cartilage defects.
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| CN115089614A (en) * | 2022-06-28 | 2022-09-23 | 中国人民解放军军事科学院军事医学研究院 | Method for enhancing performance of skeletal stem cells and application of method in treatment of osteoarthritis |
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