CN112011501B - HCM specificity induced pluripotent stem cell line carrying c.3369-3370 insC mutation - Google Patents
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Abstract
The invention provides an HCM specificity induced pluripotent stem cell line carrying c.3369-3370 insC mutation, which is preserved in China general microbiological culture Collection center (CGMCC) at 7, 9 and 2020, with the preservation number of CGMCC No.19956. The establishment of the cell line can provide a cell model for the new drug screening and drug treatment of clinical hypertrophic cardiomyopathy and provide an effective tool for the pathogenic mechanism research and the gene modification research of hypertrophic cardiomyopathy; the cell resource of the hereditary disease is preserved, the disease specificity iPSC cell bank is enriched, and the reserve is reserved for the subsequent research.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to an HCM (human chorionic gonadotropin) specificity induced pluripotent stem cell line carrying c.3369-3370 insC mutation.
Background
Familial Hypertrophic Cardiomyopathy (HCM) is an autosomal dominant hereditary heart disease of the myocardial sarcomere and is considered to be the most common hereditary heart disease in the world (Maron et al, 1995. HCM patients do not experience increased hemodynamic load, and the Left Ventricular (LV) myocardium becomes abnormally thickened, leading to an increased risk of developing clinical complications such as progressive heart failure, arrhythmias, and Sudden Cardiac Death (SCD) (Maron, 2002 Maron et al, 2003. Molecular genetic studies over the past 20 years have shown that HCM is caused by mutations in the gene encoding the sarcomeric myocardial protein (Geisterfer-Lowrance et al, 1990; seidman and Seidman, 2001). However, the mechanism by which sarcomere mutations produce the clinical phenotype of HCM is not clear. Studies on HCM are largely based on the design of transgenic animals carrying a human HCM mutant mouse α -myosin backbone (α -myosin backbone), e.g., transgenic mice carrying a human HCM mutant gene on the mouse α -myosin backbone. Although these animal models provide important research results for the development of HCM, they also have limitations in that animals sometimes do not fully characterize human disease due to ethnic differences, e.g., α -myosin rich mice lack β -myosin characteristic of the human cardiac sarcomere, the physiological properties of these two myosins are completely different and they cause functional contraction of completely different myosins (Fatkin et al, 1999 lomprep et al, 1981), which is particularly important in HCM, β -myosin being the most common disease of genetic mutations (Seidman and Seidman, 2001). A recent study highlighted that α -and β -myostatin of different biophysical properties were obtained by cloning the same mutations (R403Q) of the human HCM, α -myostatin backbone and β -myostatin backbone (Lowey et al, 2008). Notably, the effect of the same R403Q mutation on the contractile function of myosin resulting in a completely different one depends on whether it is represented in the alpha-or beta-myostatin subtype. This is probably the reason why animal models lead to contradictory results (Arad et al, 2002, marian et al, 1997.
Induced Pluripotent Stem Cells (iPSCs) introduced four transcription factors (c-Myc, oct3/4, SOX2, and Klf 4) into mouse fibroblasts by Takahashi and Yamanaka (Takahashi et al, 2006) in 2006 to reprogram them into ES-like cells; human induced pluripotent stem cells (hlipscs) were established in 2007 and were reprogrammed to form ES-like cells by introducing the same four transcription factors (c-Myc, oct3/4, SOX2, and Klf 4) and another set of transcription factors (Oct 3/4, SOX2, nanog, lin 28) into human fibroblasts (Takahashi et al, 2007, yu et al, 2007). The reprogramming technology has more application value if the biology and the treatment of diseases can be researched on the iPSCs of patients. By using the reprogramming technology, the skin fibroblasts of the patient or the T cells separated from the peripheral blood can be used as a cell source for reprogramming to generate the patient-specific iPSCs.
In the research of HCM and other genetic heart diseases, because it is almost impossible to obtain human heart pathological tissue specimens for in vitro research, and myocardial cells can not be cultured in vitro for a long time, the solution of the differences of animals and human beings is hindered. The generation of patient-specific ipscs circumvents the limitations of many transgenic animal models in disease studies, such as dilated cardiomyopathy, LEOPARD, and long QT syndrome (Carvajal-Vergara et al, 2010 itzhaki et al, 2011 moretti et al, 2010 narsinh et al, sun et al, 2012, yazawa et al, 2011), which have all successfully established disease-specific ipscs and were used to mimic a variety of inherited cardiovascular diseases in vitro.
We found a predecessor of hypertrophic cardiomyopathy in the clinic, 3 family members of which were diagnosed as HCM (FIG. 1) in the family survey, and used exon capture technology to target HCM-related 16 genes, including ACTC1, LAMP2, MYBPC3, MYH6, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPMI, PRKAG2, CSRP3, GLA, VCL, TNNC1, TTR and other common pathogenic genes encoding sarcomere proteins to screen the HCM family members for the pathogenic mutant genes by using target gene-targeted capture high-throughput sequencing technology, and found 2 cases of MYBPC3 c.3369-3370 insC mutations, and the patient DNA sequencing analysis electrophoresis shows that MYBPC3 gene has frame-shift mutation at C3369-3370 insC position, and cysteine is converted into leucine (p.Cys11225; EX 31/EX 31; het 2), and the transient heterozygous mutation is not reported in the transient map (the transient heterozygous site). One example was reprogrammed to patient-specific ipscs using the episomal method, and no international patent application was found for a hypertrophic cardiomyopathy-specific induced multifunctional stem cell line carrying the myosin binding protein C (MYBPC 3) c.3369-3370 insC mutation.
Disclosure of Invention
The invention aims to provide an HCM specific induced pluripotent stem cell line carrying a c.3369-3370 insC mutation.
In order to achieve the purpose, the invention adopts the following technical scheme:
an HCM specificity induced pluripotent stem cell line HCM-iPSC carrying a c.3369-3370 insC mutation is a Hypertrophic Cardiomyopathy (HCM) specificity induced pluripotent stem cell line FJMA0001i-HCM carrying a myosin binding protein C (MYBPC 3) c.3369-3370 insC mutation, which is preserved in China general microbiological culture Collection center (CGMCC) at 7, 9 and 2020, and the preservation number is CGMCC No.19956. The address is No. 3 of Xilu No.1 of Beijing, chaoyang, the institute of microbiology of Chinese academy of sciences.
The collected PBMNC was resuspended in 4ml PBMNC amplification medium (Nuwacell Ltd), and inoculated into 2 wells of a 6-well plate for culture, and 2ml PBMNC amplification medium (Nuwacell Ltd) was supplemented every 4 days during the culture.
Frozen PBMNC were thawed and plated onto fibronectin coated plates for expansion, and cells on day 7 in expansion culture were reprogrammed by adding EX-CYTE growth enhancement medium supplement (1.
The reprogramming method comprises the following steps: episomalt reprogramming vectors contain human OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT genes. Amplification ofThe cultured PBMNC cells were subjected to nuclear transfection by mixing Human CD34+ Cell Nuclear effector Kit (VPA-1003, amaxa) and 15-20ug of plasmid DNA, and the number of cells per nuclear transfection was 1X 10 6 The plasmid DNA proportion is 3.0 mu g pEP4EO2SEN2K,3.2 mu g pEP4EO2SET2K,2.4 mu g pCEP4-M2L. The nuclear transfection procedure was T-16. Cells immediately after nuclear transfection were seeded into 6-well plates previously plated with fibronectin/matrigel, and fibronectin was used with the matrigel to facilitate recovery of cells from nuclear transfection. After transfection, the cells were cloned at the third Day of passage 6 (P6, day 3), microscopically seen to grow in a typical clone shape, the clones were in a colony shape, a raised circular shape or an elliptical shape, the boundaries were clear, the cells within the cell mass were arranged closely, and the boundaries and morphology were not easily distinguished, by replacing the cells with DMEM/F12 culture solution containing 1X N-2, (1X B-27, 100 ng/ml bFGF, 0.5 uM PD0325901, 3 uM CHIR99021, 0.5 uM A-83-01, 1000U/ml hLIF, 10uM HA-100, and then carrying out iPSC amplification using hPCE culture solution (NuwaCell Ltd).
The invention has the advantages that:
1. a multifunctional stem cell line specifically induced by Hypertrophic Cardiomyopathy (HCM) carrying myosin binding protein C (MYBPC 3) c.3369-3370 insC mutation is established, and a cell model is provided for familial hereditary HCM caused by gene mutation;
2. the establishment of the cell line can provide a cell model for the new drug screening and drug treatment of clinical hypertrophic cardiomyopathy and provide an effective tool for the pathogenic mechanism research and the gene modification research of hypertrophic cardiomyopathy;
3. the cell resource of the hereditary disease is preserved, the disease specificity iPSC cell bank is enriched, and the reserve is reserved for the subsequent research.
Drawings
FIG. 1 is a family diagram of HCM, wherein ■ is male patient, ■ is male died from the disease, □ is normal male, and good is normal female.
FIG. 2. Electrophoresis picture of patient DNA sequencing analysis shows that MYBPC3 gene has frame-shift heterozygous mutation at C.3369-3370 insC position.
FIG. 3. Episomalt reprogramming vectors comprising human OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT genes.
FIG. 4 is a cell clone result chart of the cell line HCM-iPSC which is passaged to the third Day of 6 generation culture (P6 Day 3). Under the microscope, the cells grow in typical clone shapes, the clones are in colony shapes, raised round shapes or oval shapes, the boundaries are clear (figure 4A, 40X), the cells in the cell clusters are closely arranged, and the boundaries and the shapes are not easily distinguished (figure 4B, 200X).
FIG. 5. Real-time PCR amplification results, POU5F1 (blue) and NANOG (orange) gene expression was increased in HCM-iPSC (20180803 CQ-C2-P6) and hESC-H1 compared to MSC-P3. The internal reference is RPL13A (Ribosal protein L13A). P < 0.01, significant differences were considered.
FIG. 6 is a graph showing the results of simultaneous expression of SSEA4 and tra1-81 by HCM-iPSC.
FIG. 7. Results of GTG analysis of the cell line, which showed a normal male karyotype (46, XY).
FIG. 8 staining results of HCM-iPSC in vivo differentiation experiments, teratomas in grafts comprising all three germ layers, A: neural tissue (ectoderm), B: cartilage (mesoderm), C: primitive gut (endoderm), scale: 100 mm.
FIG. 9 detection result of loss of exogenous reprogramming genes of HCM-iPSC, and the detection result of exogenous reprogramming genes of HCM-iPSC cell samples (20180803 CQH-C1, 20180803CQH-C2, 20180803 CQH-C3) indicates the real gEBNA1 value of the HCM-iPSC sample. 1.0896 Y = x + 0.7276. The red dots in the red shaded areas of the figure are the locations of samples 20180803cqm-c1, 20180803cqm-c2, 20180803cqm-c 3.
FIG. 10 HCM-iPSC mutation patient DNA sequencing analysis and verification shows that MYBPC3 gene has heterozygous mutation at c.3369-3370 locus. The upper panel shows the sequencing result of the gene mutation of the patient, and the lower panel shows the verification of the same site gene mutation by HCM-iPSC.
FIG. 11.21 STR site electrophoretograms. One single peak (A) represents a homozygote and two peaks (B) of similar size represent a heterozygote.
FIG. 12 resuscitated HCM-iPSC passage 8 (40X).
FIG. 13 resuscitated HCM-iPSC passage 8 (200X).
Detailed Description
Example 1
HCM family cohort recruitment and disease mutation gene screening.
The diagnosis of HCM was based on the guidelines for hypertrophic cardiomyopathy and expert consensus, following the declaration of helsinki and reviewed by the ethical committee, each participant signed a written informed consent; meanwhile, collecting medical history data and clinical examination data, and determining the family of the familial hereditary hypertrophic cardiomyopathy; venous blood was collected from the subjects and stored at 10 ml. And the Shenzhen Huada clinical testing center utilizes a target gene targeted capture high-throughput sequencing technology to screen pathogenic mutant genes. HCM family members are subjected to high-throughput sequencing on 16 genes related to HCM by utilizing exon capture technology, including ACTC1, LAMP2, MYBPC3, MYH6, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPMI, PRKAG2, CSRP3, GLA, VCL, TNNC1, TTR and other common pathogenic genes encoding sarcomere proteins. We screened 3 families (I-1, II-2, II-3) of which 2 MYBPC3 c.3369-3370 insC mutations (II-2, II-3) were found, and electrophorograms of patient DNA sequencing analysis showed that the MYBPC3 gene was heterozygous for a frameshift at the C.3369-3370 insC position, converting cysteine to leucine (p.Cys1124Leufs 25; EX31/CDS31; het; (FIG. 2). There is no relevant literature report on the pathogenicity of the site.
II) isolation and culture of human peripheral blood mononuclear cells (PBMNC)
Selecting II-2 (65 years old, male, proband) patients in a family, collecting 5ml of venous blood (5 ml) of the patients in a heparin sodium anticoagulation tube, adding the anticoagulation into a lymphocyte separation tube (Nuwacell Ltd) for centrifugal separation of PBMNC, resuspending the collected PBMNC in 4ml of PBMNC amplification culture medium (Nuwacell Ltd), respectively inoculating the PBMNC in2 holes of a 6-hole plate for culture, and supplementing 2ml of PBMNC amplification culture medium (Nuwacell Ltd) every 4 days during the culture.
(III) reprogramming Process
Frozen PBMNC were thawed and plated onto fibronectin coated plates for expansion culture, and cells on day 7 in expansion culture were reprogrammed by addition of EX-CYTE growth enhancement medium supplement (1.
Epismal reprogramming vectors contained the human OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT genes (FIG. 3, gifted by Zhongsheng dating from Source). The cultured PBMNC cells were expanded and subjected to nuclear transfection by mixing Human CD34+ Cell Nuclear effector Kit (VPA-1003, amaxa) and 15-20ug of plasmid DNA, the number of cells per nuclear transfection was 1X 10 6 The plasmid DNA proportion is 3.0 μ g of pEP4EO2SEN2K,3.2 μ g of pEP4EO2SET2K and 2.4 μ g of pCEP4-M2L. The nuclear transfection procedure was T-16. Cells immediately after nuclear transfection were seeded into 6-well plates pre-plated with fibronectin/matrix gel, and fibronectin was used with the matrix gel to facilitate recovery of cells from nuclear transfection. From day 2 to day 11 after transfection, the culture medium DMEM/F12 containing N-2 (1X, thermoFisher Scientific), B-27 (1X, thermoFisher Scientific), bFGF (100 ng/ml, peprotech), PD0325901 (0.5 uM Stemgent), CHIR99021 (3 uM, stemgent), A-83-01 (0.5 uM, stemgent), hLIF (1000U/ml, millipore), HA-100 (10 uM, santa Cruz Biotechnology) was changed, and then the iPSC amplification was performed using hPSC culture medium (NuwatdLcell). FIG. 4 shows that the cell clone of the third Day of passage 6 (P6, day 3) shows that the cells grow in a typical clone shape under a microscope, the clone is in a colony shape, a raised circle or an ellipse, the boundary is clear (FIG. 4A, 40X), the cells in the cell mass are closely arranged, the boundary and the shape are not easily distinguished (FIG. 4B, 200X), and finally the cell line HCM-iPSC is obtained, namely the Hypertrophic Cardiomyopathy (HCM) specific induced pluripotent stem cell line FJMA0001i-HCM carrying the myosin binding protein C (MYBPC 3) c.3369-3370 insC mutation.
(IV) RNA extraction and qRT-PCR
Total RNA extraction of HCM-iPSC obtained in the previous step was performed by lysis of cells with Trizol Reagent (10296010, invitrogen, thermo Fisher Scientific) according to the kit instructions to isolate RNA, reverse transcription into cDNA using 1ug of RNA as template as described in the instructions (Prime Script Reagent kit, DRR037A, takara Bio), incubation at 37 ℃ for 15 min and 85 ℃ for 5 sec. qPCR analysis of transgene expression was performed using SYBR Premix Ex Taq (RR 820A, takara Bio) on the 7500 Real-Time PCR System (Applied Biosystems, thermo Fisher Scientific).
The PCR reaction conditions were 94 ℃ for 30 s,1 cycle; 94 ℃ for 5 sec,60 ℃ for 15 sec, requested for 45 cycles, 72 ℃ for 10s,1 cycle. RPL13 alpha is used as a reference gene.
Table 1 shows the upstream and downstream primers used. To assess the differential expression of pluripotency genes in HCM-iPSC (i.e., 20180803CQ-C2-P6 in the figure), MSC-P3 (referring to cells passaged to third generation MSC) and hESC-H1, we analyzed qRT-PCR results using f (x) =2- Δ CT. Real-time PCR showed increased expression of POU5F1 (blue) and NANOG (orange) genes in HCM-iPSC (20180803 CQ-C2-P6) and hESC-H1 compared to MSC-P3 (FIG. 5).
TABLE 1 qRT-PCR upstream and downstream primers
(V) flow cytometry analysis of pluripotency markers
The HCM-iPSC was digested with the digestion solution for 5 minutes to form a single cell suspension, centrifuged at 1 r/min for 5 min, and resuspended in 20% goat serum in FACS buffer. HCM-iPSC was incubated with PE-labeled anti-TRA1-81 antibody (12-8883-82, thermo) or anti-SSEA-4 antibody (MA 1-021-PE, thermo) (Table 2) for 30 min at 4 ℃ with Isotype as Isotype control and analyzed by flow cytometer (Beckman) and FlowJo (FlowJo Enterprise) software. The results showed that HCM-iPSCs expressed SSEA4 and tra1-81 simultaneously (FIG. 6), indicating that they were in an undifferentiated state.
TABLE 2 flow cytometry antibodies
(VI) karyotyping
When the confluency of the 6-well plate cells is 60%, 50ng/ml colchicine is added and incubated at 37 ℃ for 2h. The cells were made into a single cell suspension and mixed with a fixative (methanol: glacial acetic acid, 3:1) overnight at 4 ℃. The precipitated cells were dropped onto a pre-cooled slide and then dried at 75 ℃ for 1-2 hours, and the slide was stained with jimsa (5 ml jimsa +45ml PBS) and then dried at room temperature for karyotyping. The GTG analysis suggested that the cell line showed a normal male karyotype (46, XY) (FIG. 7).
(VII) in vivo differentiation test-teratoma formation test
2-4 x 10 6 Individual HCM-ipscs (P6) were injected into hind limb muscle of 6-week-old immunodeficient SCID beige mice. After 2 months, the grafts were dissected and fixed with 4% PFA for 24h at 4 ℃ and then paraffin embedded. Specimens were cut to 4mm thickness and stained with HE. The results are shown in FIG. 8, and the teratomas in the graft comprising all three germ layers A: nervous tissue (ectoderm), B: cartilage (mesoderm), C: primitive gut (endoderm) and scale: 100 mm.
(VIII) detection of loss of HCM-iPSC exogenous reprogramming genes
Extracting the total DNA of the HCM-iPSC cell sample, determining the Ct values of OCT4 and gEBNA1 by adopting real-time fluorescence quantitative PCR (the primers are shown in table 1), and detecting the reprogramming deletion condition of the HCM-iPSC exogenous gene. The true gEBNA1 value X (n) of the HCM-iPSC sample was calculated using the standard curve Y (n) (Y =1.0896X + 0.7276). In the exogenous gene standard curve (Y = 0.1), when Y (n) is smaller than the source, the exogenous gene is considered to be completely lost.
As shown by the exogenous gene standard curve, the red dots are the positions of the sample HCM-iPSC (20180803 CQH-C1, 20180803CQH-C2 and 20180803 CQH-C3) in the standard curve graph and are located in red shaded areas, namely the Y (n) value of the sample is less than 0.1 copy of Y (0.1), which indicates that the exogenous gene is lost.
MYBPC3 gene mutation verification of (nine) HCM-iPSC
Extracting DNA of the HCM-iPSC for PCR amplification and sequencing analysis. The results suggest that the HCM-iPSC has gene mutation at the same site (FIG. 10). The primer sequences are described below:
(ten) STR analysis
HCM-iPSC (20180803 CQ) and patient peripheral blood genomic DNA were extracted by the chelex method, and 21 STR sites (PowerPlex 21 System produced by Promega) were amplified in a 9700 amplification apparatus (Applied Biosystems inc., USA)) according to the instructions. The primer sequence of each STR locus is shown in the product instruction book. The size of the PCR product obtained was determined by capillary electrophoresis on a 3130 Gene analyzer (Applied Biosystems, inc) using Gene Mapper ID v3.2 (AB SCIEX, framingham, MA, USA) software (Table 3), and in 21 STR site electropherograms, a single peak (A) indicating homozygote and two peaks (B) of similar size indicating heterozygote (FIG. 11). The results showed that the genotypes of the HCM-iPSC and the patient peripheral blood genomic DNA at 21 STR sites are completely identical, indicating that they are from the same individual.
TABLE 3 iPSC and patient peripheral blood STR sites
Example 2
Resuscitation and amplification of HCM-iPSC (FJMA 0001 i-HCM): taking out the HCM-iPSC which is frozen and preserved for one year from a liquid nitrogen tank, quickly thawing in a water bath at 37 ℃, transferring to a centrifuge tube containing an hPSC culture solution, carrying out heavy suspension by using the hPSC culture solution (NuwaCell Ltd) after centrifugation, and inoculating to a six-well plate for HCM-iPSC amplification. FIG. 12 shows the cell clones at passage 8 (P8), and microscopic observation shows the typical clonal growth of the cells (FIG. 12, 40X), with the cells arranged closely (FIG. 13, 200X). Therefore, the cell line of the invention can still stably grow in a clone shape in vitro after being frozen for one year.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
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Claims (1)
1. An HCM-specific induced pluripotent stem cell line carrying the c.3369-3370 insC mutation, wherein: the cell line is a Hypertrophic Cardiomyopathy (HCM) specific induced pluripotent stem cell line FJMA0001i-HCM carrying myosin binding protein C (MYBPC 3) c.3369-3370 insC mutation, which is preserved in China general microbiological culture Collection center (CGMCC) at 7, 9 and 2020 years with the preservation number of CGMCC No.19956.
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| MYBPC3 基因p．Ile852Val 突变与家族性肥厚型心肌病的相关性研究;朱汀等;《心肺血管病杂志》;20200331;第39卷(第3期);第328-331页 * |
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