CN118460478B - Multi-osteochondroma induced pluripotent stem cell line and application thereof - Google Patents

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a multiple osteochondroma induced pluripotent stem cell line and its application. The cell line is a multiple osteochondroma induced pluripotent stem cell line with EXT1 gene breakage caused by balanced chromosome translocation. It has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 8, 2024, with a deposit number of CGMCC No.45839. It has been verified by experiments that it is not only similar to embryonic stem cells in cell morphology, gene expression, proliferation and differentiation potential, but also can effectively differentiate into three germ layers, thereby providing important resources for the study of pathological mechanisms related to multiple osteochondromas and personalized drug screening and treatment, and has good practical application value.

Description

A multiple osteochondroma induced pluripotent stem cell line and its application

Technical Field

The invention belongs to the technical field of biomedicine, and specifically relates to a multiple osteochondroma induced pluripotent stem cell system and application thereof.

Background Art

The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

Hereditary multiple exostoses (HME) is an autosomal dominant genetic disease characterized by impaired cartilaginous bone formation and multiple osteochondromas. It is characterized by multiple bony protrusions covered with cartilage, most commonly in the metaphysis of long bones, but can also occur in the shafts of long bones. Flat bones, vertebrae, and ribs may also be affected, but the skull is usually not affected. Deformities of the legs, forearms (similar to Madelung deformity), and hands are common. The disease is often accompanied by pain, asymmetrical short stature, and a variety of skeletal malformations, such as bowing deformities of the forearms and lower legs, unequal limb lengths, and genu varum. Domestic and foreign studies have found that the disease is associated with mutations in the tumor suppressor genes EXT1 and EXT2, but its pathogenesis is not yet fully understood. Current mechanism studies largely use functional cell lines or transgenic mice. EXT1 mutations lead to systemic heparan sulfate deficiency. Functional cell line studies have shown that local heparan sulfate levels drop sharply, disrupting the normal homeostatic signaling pathway that maintains the perichondrial stroma; leading to excessive BMP signaling; and causing ectopic cartilage formation and osteochondroma formation. Data from heparan sulfate-deficient mouse models show that systemic treatment with BMP signaling antagonists can significantly reduce the formation of osteochondromas. Although these animal models have provided important research results for the development of multiple osteochondromas, they also have some limitations. Due to racial differences, animals sometimes cannot fully reflect the characteristics of human diseases. In short, the early theory proves that osteochondroma formation is a druggable process, which provides a preliminary theoretical basis for the creation of clinically relevant treatments. However, due to the limitations of animal models, drug research is still very difficult.

Human induced pluripotent stem cells (iPS) can be reprogrammed from the patient's own peripheral blood, fibroblasts, etc. to form the patient's own specific iPS, which has greater application value in the study of the biological mechanism of the disease, drug development, and disease treatment research. However, the inventors found that there are few studies and reports on induced pluripotent stem cell lines for multiple osteochondromas.

Summary of the invention

In view of the deficiencies in the prior art, the present invention aims to provide a multiple osteochondroma induced pluripotent stem cell line and its application. Specifically, in a multiple osteochondroma family, two family members were diagnosed with multiple osteochondroma. The proband had protrusions at the ends of long bones and exostoses at the epiphysis. The clinical manifestations were consistent with multiple osteochondroma, and his mother had similar clinical manifestations. The proband's full exogenous test results were negative, and the karyotype analysis showed 46,XX,t(1:8)(p13;q24.1). Further whole-genome optical mapping technology and long-fragment PCR sequencing results showed that one of the breakpoints of the proband's balanced translocation occurred within the EXT1 gene on chromosome 8, which is a common pathogenic gene for multiple osteochondroma. The long-fragment PCR and second-generation sequencing results clearly showed that the breakpoint of chromosome 8 was located at intron 1 of the EXT1 gene [hg19]chr8:118921743, and there is currently no report on this mutation. Therefore, the present invention collects peripheral blood from the proband and induces specific iPS cells by electroporation of plasmids, thereby obtaining a multiple osteochondroma induced pluripotent stem cell line with EXT1 gene disruption caused by balanced chromosome translocation. Based on the above research results, the present invention is completed.

In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

The first aspect of the present invention provides a human induced pluripotent stem cell EXT1-PHYW-iPS, which is an induced pluripotent stem cell line for multiple osteochondromas in which the EXT1 gene is broken due to balanced chromosomal translocation. It has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on April 8, 2024, with the deposit number CGMCC No.45839.

The second aspect of the present invention provides a method for preparing the above-mentioned human induced pluripotent stem cell EXT1-PHYW-iPS, which comprises: introducing reprogramming factors into somatic cells of patients with multiple osteochondromas who have balanced chromosome 8 translocation resulting in EXT1 gene breakage, thereby reprogramming the cells.

The third aspect of the present invention provides a composition, wherein the composition comprises as an active ingredient at least one of the above-mentioned human induced pluripotent stem cells EXT1-PHYW-iPS and a culture of the human induced pluripotent stem cells EXT1-PHYW-iPS.

The fourth aspect of the present invention provides the use of the above-mentioned human induced pluripotent stem cell EXT1-PHYW-iPS and/or the above-mentioned composition in any one or more of the following:

(a) Establishment of a multiple osteochondroma disease model;

(b) Study on the disease mechanism of multiple osteochondromas.

Beneficial technical effects of one or more of the above technical solutions:

The above technical solution is the first to establish a patient-specific induced pluripotent stem cell line with multiple osteochondromas caused by a balanced chromosome translocation breakpoint located within the EXT1 gene, providing a cell model for patients with multiple osteochondromas to find the cause of the disease at the genetic level; at the same time, the establishment of this cell line provides a cell model that is closer to the real microenvironment in the patient's body for the mechanism research and drug development of multiple osteochondromas; in addition, the above technical solution provides new ideas for genetic disease diagnosis and consultation: for cases with clear phenotypes but negative results of whole exome sequencing, and families with abnormal chromosome karyotype analysis, whole genome optical mapping technology and long fragment sequencing technology can be used to improve the positive detection rate. At the same time, it preserves human single-gene genetic disease cell resources, enriches the patient-derived specific iPS cell bank, and provides a practical research model for subsequent research.

In summary, the above technical scheme provides a pluripotent stem cell line with stable propagation and multiple osteochondroma-specific genetic information (balanced translocation of chromosome 8 leads to the breakage of EXT1 gene), which is not only similar to embryonic stem cells in cell morphology, gene expression, proliferation and differentiation potential, but also can effectively differentiate into three germ layers, thus providing an important resource for the study of pathological mechanisms related to multiple osteochondroma and personalized drug screening and treatment, and has good practical application value.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

FIG. 1 is the morphological observation of iPS cells of the present invention.

FIG. 2 is a diagram showing the stemness markers of iPS cell lines labeled by immunofluorescence chemistry experiments of the present invention, with a scale bar of 20 μm.

FIG3 is a diagram showing the results of the iPS cell line of the present invention differentiating into three germ layers: inner (A), middle (B), and outer (C).

FIG. 4 is a pedigree diagram of a family with multiple osteochondromas according to the present invention.

FIG. 5 is a karyotype diagram of multiple osteochondroma cells of the present invention.

FIG. 6 is a diagram showing the results of OGM detection using the whole genome optical mapping technology of multiple osteochondroma cells of the present invention.

FIG. 7 is the long fragment sequencing result of multiple osteochondroma cells of the present invention.

DETAILED DESCRIPTION

It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and/or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

The present invention provides a multiple osteochondroma-specific induced pluripotent stem cell line EXT1-PHYW-iPS, which shows 46,XX,t(1:8)(p13;q24.1) in karyotype analysis and one breakpoint of the balanced translocation occurs within the EXT1 gene of chromosome 8. Specifically, peripheral blood is collected from the proband, and peripheral blood PBMC is separated by density gradient centrifugation. CD34 ⁺ cells are cultured for 5-7 days until the cell amount reaches 1×10^6, and the cells are reprogrammed into iPS cells by electroporation of plasmids. After electroporation, the cells are cultured with Stemcell E7 culture medium until the iPS cell morphology is formed, and the mTeSR1 culture medium is replaced to maintain the culture. The induced iPS cells are passaged to P10 respectively, and the mutation is detected by OGM and long fragment amplification. The stem cell markers OCT4 and Nanog are marked by immunocytochemistry to prove that the three cell lines have the differentiation potential of embryonic stem cells, and the real-time fluorescence quantitative PCR method is used to confirm that the three cell lines have the potential to differentiate into the endoderm, middle and outer germ layers.

Therefore, in a typical embodiment of the present invention, a human induced pluripotent stem cell EXT1-PHYW-iPS is provided, which is an induced pluripotent stem cell line for multiple osteochondromas in which the EXT1 gene is broken due to balanced chromosomal translocation, and which has been deposited in the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China) on April 8, 2024, with the deposit number CGMCC No.45839.

In another specific embodiment of the present invention, a method for preparing the above-mentioned human induced pluripotent stem cell EXT1-PHYW-iPS is provided, and the preparation method comprises: introducing reprogramming factors into somatic cells of patients with multiple osteochondromas who have balanced translocation of chromosome 8 resulting in EXT1 gene breakage, thereby reprogramming the cells.

More specifically, the preparation method comprises: collecting somatic cells from patients with multiple osteochondromas who have balanced chromosome 8 translocation leading to EXT1 gene disruption, and culturing the cells;

The plasmid containing the reprogramming factors was introduced into the cultured somatic cells, and the cells were cultured in a reprogramming medium. The successfully reprogrammed cells were screened to obtain the above-mentioned human induced pluripotent stem cells EXT1-PHYW-iPS.

Furthermore, the somatic cells are specifically peripheral blood mononuclear cells PBMC in peripheral blood.

Furthermore, the plasmid containing the reprogramming factor may be pCE-hOCT4, pCE-hSK, pCE-hUL, pCE-mp53DD and pCXB-EBNA1 plasmids; the above plasmids can be obtained commercially.

In another specific embodiment of the present invention, a composition is provided, wherein the active ingredient of the composition comprises at least one of the above-mentioned human induced pluripotent stem cells EXT1-PHYW-iPS and the culture of human induced pluripotent stem cells EXT1-PHYW-iPS.

The culture of human induced pluripotent stem cells EXT1-PHYW-iPS includes a purified population of human induced pluripotent stem cells EXT1-PHYW-iPS, and cells differentiated from human induced pluripotent stem cells EXT1-PHYW-iPS (such as a population of purified differentiated cells).

In another specific embodiment of the present invention, the use of the above human induced pluripotent stem cell EXT1-PHYW-iPS and/or the above composition in any one or more of the following is provided:

(a) Establishment of a multiple osteochondroma disease model;

(b) Study on the disease mechanism of multiple osteochondromas.

Wherein, in (a), the multiple osteochondroma disease model may specifically be a multiple osteochondroma cell model.

The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1: Isolation and in vitro expansion of single cells from umbilical cord blood and peripheral blood

(1) In accordance with the Declaration of Helsinki and reviewed by the Ethics Committee, each participant signed a written informed consent form; medical history and clinical examination data were collected at the same time, and the family tree is shown in Figure 4; 2 ml of venous blood was collected from the research subjects for storage. The whole exome sequencing results of a patient with multiple osteochondromas were negative and the karyotype was abnormal. The whole genome optical mapping technology and long fragment sequencing technology confirmed that the breakpoint of the balanced translocation was located in intron 1 of the EXT1 gene. The results of the patient's whole genome optical mapping technology are shown in Figure 6.

(2) Reagents and consumables are as follows:

A. Human peripheral blood lymphocyte separation medium (Tianjin Haoyang Biological Products Co., Ltd., catalog number: LTS1077-1);

B. PBS buffer (BI catalog number: 02-024-1ACS);

C Pasteur pipette;

D. 15ml centrifuge tube; Corning: #339650;

E. Heparin sodium blood collection tube;

(3) Instruments used:

A. Centrifuge;

B. Carbon dioxide incubator;

C. Inverted microscope;

(4) Specific steps

A. Take 2 ml of umbilical cord blood or peripheral blood and add PBS at a 1:1 ratio. Transfer to a new 15 ml centrifuge tube and mix well using a Pasteur pipette.

B. Take another 2 ml of lymphocyte separation solution and place it in a new 15 ml centrifuge tube. Slowly add the umbilical cord blood or peripheral blood mixed in step A.

C. Centrifuge at 500g for 25min, with the centrifuge's vertical and horizontal accelerations set to level 0.

D. After centrifugation, remove the centrifuge tube, aspirate the buffy coat with a Pasteur pipette, add it to a new 15ml centrifuge tube, add PBS to 10ml, and centrifuge at 450g for 10 minutes.

E. Remove the supernatant, keep the cell pellet, resuspend the cells in expansion medium, and culture and expand. The culture medium is Gibco StemPro-34 SFM (1×).

Example 2: Cell reprogramming and passaging

(1) Cells: CD34+ cells expanded in Example 1

(2) Reagents and consumables:

A. ReproTeSRrm Medium for Reprogramming (stemcell Catalog# 05926);

B. Maintenance medium: mTeSR1 basal medium (stemcell catalog # 85850);

C. Electroporation culture medium;

D. pCE-hOCT4, pCE-hSK, pCE-hUL, pCE-mp53DD, pCXB-EBNA1 plasmids;

E. Matrigel, BD Sciences: #354277;

F. DMEM/F12, GIBCO:#11330;

(3) Instruments and equipment

A. Electroporator;

B. Centrifuge;

C. Inverted microscope;

D. Carbon dioxide incubator;

(4) Specific steps

A. One day before electroporation, take a 6-well cell culture plate and spread it with Matrigel;

B. On the day of electroporation, remove the culture plate, remove the Matrigel and add 2 ml Gibco StemPro-34 SFM medium;

C. Pipette the expanded cells into a 15 ml centrifuge tube, add PBS to 10 ml, centrifuge at 300 g for 5 min, and remove the supernatant;

D. Add 2 ml of electroporation medium to resuspend and wash the cells, centrifuge at 300 g for 5 min, and discard the supernatant;

E. Add 90ul electroporation medium and relevant electroporation plasmids to a final volume of 100ul;

F. Transfer to an electroporation cup, and after electroporation, transfer to a cell culture well on a Matrigel plate supplemented with Gibco StemPro-34 SFM medium, and place in a carbon dioxide incubator for culture;

G. On the second day after electroporation, add 1 ml of Gibco StemPro-34 SFM medium;

H. On the third day, add 1 ml of ReproTeSRrm Medium for Reprogramming (stemcell Catalog # 05926);

I. On the fifth day, add 1 ml of ReproTeSRrm Medium for Reprogramming (stemcell Catalog # 05926);

J. On the seventh day, completely replace the medium and add 2 ml of new ReproTeSRrm Medium for Reprogramming (stemcell Catalog # 05926);

K. Continue culturing until iPS-like cell clusters are formed as shown in Figure 1, and then perform clone selection and subculture.

Example 3: Identification of stem cell characteristics by immunofluorescence cytometry

Cell: EXT1 gene mutation induces pluripotent stem cells

(1) Reagents and consumables

A. Antibodies: primary antibody, secondary antibody;

B. DAPI;

C. Millicell EZ SLIDE (Catalog #PEZGS0416);

D. PBST;

E. BSA;

F. PBS;

(2) Instruments used

A. Confocal microscopy;

B. Pipette;

C. Shaking bed;

(3) Specific steps

A. Take out the cell slides that have been laid in advance, add PBST (0.1% Triton x100) to permeabilize the cells for 10 minutes, and wash twice with PBS, each time for 10 minutes;

B. Block with 5% BSA at room temperature for 1 hour (prepared in PBS, Hyclone #SH30256.01B);

C. Primary antibody at 4℃ overnight. Note: 5% BSA dilution, no need to wash off BSA with PBS before applying antibody, 100ul/circle;

D. Remove the primary antibody incubation solution and wash with PBS for 10 min x 3 (can be placed on a shaker);

E. Incubate with secondary antibody at RT for 1h. Protect from light from this step.

F. Wash with PBS 3 times, 10 min each time;

G. Add a mountant (one drop is enough, VECTOR cat# H-1200) to the slides in a dark place. Store at 4°C. It is best to take photos within 3 days after completion. The results show that the iPS cells described in Example 2 can express the stemness factor OCT4 and Nanog markers, as shown in Figure 2.

Example 4: Differentiation of the three germ layers: endoderm, mesoderm, and ectoderm

Cell: EXT1 gene mutation induces pluripotent stem cells

(1) Reagents and consumables

A. DPBS

B. Culture Media

(2) Instruments used

A. Microscope

B. Incubator

(3) Specific steps

A. Remove the iPSCs culture medium, add 1 mL of DPBS to wash the cells once and then discard;

B. Add 1mL Accutase and digest for 7-8min at room temperature;

C. Gently shake the plate to remove the cells from the bottom of the dish, and add 2mL DMEM/F12 to terminate digestion;

D. Mix the cells by gently pipetting and add them to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes at room temperature.

E. Discard the supernatant and add 1 mL of mTeSR1 medium supplemented with 10 nM Y27632 to resuspend the cells;

F. After counting the cells, add 80×10 ⁴ , 20×10 ⁴ , and 80×10 ⁴ cells to the Matrigel-coated 12-well plates, respectively, and culture them in a 5% CO ₂ , 37°C constant temperature incubator;

G. The next day, discard the original culture medium, add 1 mL of DPBS to each well to wash the cells and then discard;

H. Add STEMdiff™ Trilineage Ectoderm Medium, STEMdiff™ Trilineage Mesoderm Medium, and STEMdiff™ Trilineage Endoderm Medium culture media to induce iPSCs to differentiate into the three germ layers;

I. The medium was changed every day. After 5 days, the mesoderm and endoderm cells were collected to extract RNA. After 7 days, the ectoderm cells were collected and photographed under a microscope. As shown in Figure 3, the cells differentiated into endoderm (A), mesoderm (B), and ectoderm (C) morphological cells.

Example 5: G-banded chromosome karyotype analysis

Cell: EXT1 gene mutation induces pluripotent stem cells

(1) Reagents and consumables

A. Colchicine

B. Fixative

(2) Instruments used

A. Microscope

B. Incubator

(3) Specific steps

A. The cells are cultured normally and their growth status is observed. They are inoculated into culture plates and cultured to the logarithmic growth phase.

B. Colchicine treatment: Add colchicine to the cell culture medium and continue culturing to block spindle formation and obtain cells in the metaphase phase.

C. Hypotonic treatment disperses the chromosomes in the cells and makes them easier to observe.

D. Use a specific fixative to fix the cells, then prepare the slides and use optical or electrochemical colorimetric equipment to observe the specific morphological structure of the chromosomes.

E. Result analysis: Karyotype analysis showed 46,XX,t(1:8)(p13;q24.1) as shown in Figure 5.

Example 6: Whole-genome optical mapping technology

(1) Reagents and consumables

A. Bionano DNA Extraction Reagent

B. Chip

(2) Instruments used

A. Qubit 3.0

B. Sequencer

(3) Specific steps

A. Use the Bionano kit to extract high-quality, ultra-long fragment gDNA.

B. Fluorescent labeling and staining of ultra-long DNA fragments using the Bionano Prep DLS Labeling Kit.

C. DNA was straightened by electrophoresis and transported to the nanopore channel, and single molecules were photographed by fluorescence, and a digital representation of motif-specific labeling was generated (optical map, non-base sequence). The results showed that one of the breakpoints of the proband's balanced translocation occurred within the EXT1 gene on chromosome 8, as shown in Figure 6.

Example 7: Long fragment amplification based on Bionano whole genome optical map breakpoint interval

(1) Reagents and consumables

A. Primers

B. Amplification Reagents

(2) Instruments used

A. PCR instrument

B. Sequencer

(3) Specific steps

A. Use KP0131-F and KP0131-R primers to amplify long fragments. The fragment size is about 15778 bp. The primer sequences are shown in Table 1.

B. The amplified long fragment was subjected to Sanger sequencing using sequencing primers KP0150-F and KP0150-R. The primer sequences are shown in Table 2. The sequencing results showed that the breakpoint of chromosome 8 was located in intron 1 of the EXT1 gene [hg19]chr8:118921743, as shown in Figure 7.

Table 1. LR-PCR amplification primers

Table 2. Sanger sequencing primers

Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (4)

1. A human induced pluripotent stem cell EXT1-PHYW-iPS cell line is an induced pluripotent stem cell line for multiple osteochondromas in which the EXT1 gene is disrupted due to balanced chromosomal translocation. It has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 8, 2024, with the deposit number CGMCC No.45839. 2. A composition, characterized in that the active ingredient of the composition comprises the human induced pluripotent stem cell EXT1-PHYW-iPS according to claim 1. 3. Use of the human induced pluripotent stem cell EXT1-PHYW-iPS described in claim 1 and/or the composition described in claim 2 in constructing a multiple osteochondroma disease model. 4. The use according to claim 3, characterized in that the multiple osteochondroma disease model is specifically a multiple osteochondroma cell model.