CN113846063B - Universal human stem cell suitable for allogeneic transplantation and construction method thereof - Google Patents

Abstract

The invention provides a universal human stem cell suitable for allogeneic transplantation and a construction method thereof, wherein the enhancer gene sequence of a B2M gene in the stem cell is partially knocked down or silenced, so that the stem cell can maintain low immunogenicity in an inflammatory environment, desensitizes the stimulation of inflammatory factors IFN-gamma, does not cause immune response of allogeneic peripheral blood mononuclear cells, and can escape allogeneic CD8 cells ⁺ Killing of T cells and NK cells. The stem cell transplantation method is expected to transplant into the body of a patient without considering HLA matching, can avoid the attack of the immune system of the patient, plays a role in treatment, and solves the problem of immunological rejection of the immune system of the human body to allogeneic source cells in the regenerative therapy based on stem cell transplantation.

Description

Universal human stem cell suitable for allogeneic transplantation and construction method thereof

Technical Field

The invention belongs to the medical field firstly, in particular to a universal human stem cell which can be used for allogeneic transplantation without causing rejection reaction; and belongs to the field of biotechnology, in particular to a method for constructing universal human stem cells.

Background

The description of the background art is intended to be merely a general description of the invention in order to facilitate an understanding of the invention and is not intended to limit the invention in any way.

Whether transplant rejection occurs or not depends mainly on the histocompatibility between donor and recipient, and the degree of matching of major histocompatibility antigens, i.e., MHC molecules, is particularly important. Human MHC molecules, also known as Human Leukocyte Antigens (HLA), are highly polymorphic, and HLA matching has been a key bottleneck problem in clinical treatment of human cells and organ transplantation, since the HLA molecular patterns are often not identical between the donor and recipient.

HLA is classified into class I antigen (HLA-I), class II antigen (HLA-II) and class III antigen (HLA-III) according to its distribution and function. Beta 2microglobulin is also called B2M, and in a human body, the beta 2microglobulin is coded by a B2M gene and is an important component in HLA-I molecules. HLA-I is used as a membrane protein, the expression level of the HLA-I directly determines the immunogenicity of cells, and B2M (Beta 2Microglobulin, also called Beta 2M) is used as a component of HLA-I and plays an important role in fixing the HLA-I protein on the surface of a cell membrane. In the absence of B2M expression, few HLA-class I proteins can be detected on the cell surface [1].

Despite the low immunogenicity of stem cells currently widely accepted, a large body of experimental data demonstrates that transplantation of native stem cells directly still cannot avoid the immunological rejection of Eryun [2-4 ]]. The low immunogenicity of stem cells compared to adult cells is manifested by low expression of HLA class I antigens and no expression of HLA class II antigens. However, our preliminary studies of the subject groups showed that the immunogenicity of Mesenchymal Stem Cells (MSCs) was inducible. IFN-gamma is one of the most common proinflammatory factors in immune rejection, and the HLA expression level of MSC is increased under the action of the inflammatory factor IFN-gamma, and the immunogenicity is increased along with the increase [5 ]](ii) a Compared with the unstimulated group, the total HLA-I expression level and the membrane HLA-I expression level of the MSC after IFN-gamma stimulation are both obviously up-regulated and are more easily rejected by organisms [6]. After IFN-gamma stimulation, the expression level of HLA-I molecules on the surfaces of ESCs and iPSCs is increased and is enough to cause CD8 in vitro and in vivo ⁺ T cell mediated immune rejection [7]. However, the mechanism of how IFN- γ regulates HLA-I gene expression is still unclear.

Researchers have created a variety of donor stem cells (i.e., "universal stem cells") that escape immune rejection and are compatible with any patient. Existing studies have mainly adapted stem cells to develop immune tolerance by the following strategies: (1) ESC constitutively expresses immunosuppressive molecules CTLA4-Ig and PD-L1[8] by knock-in strategy; (2) Knocking out the ESC B2M gene by CRISPR technology, and introducing a synthetic protein construct to rebuild HLA-E expression to inhibit attack from NK cells [9]; (3) Using CRISPR technology to knock out HLA-A, HLA-B, HLA-C and CIITA one by one, and using knock-in strategy to make ESC express immune regulatory factors PD-L1, CD47 and HLA-G10. However, the existing scheme for constructing the universal stem cells still needs to introduce exogenous DNA, so that potential safety hazards such as unstable cell karyotypes are caused.

This requires a new approach to construct stem cells, thereby avoiding some of the disadvantages of conventional techniques.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a universal human stem cell suitable for allogeneic transplantation, wherein the enhancer gene sequence of the B2M gene in the stem cell is partially knocked down or silenced or a part of the enhancer gene sequence is knocked down, the low immunogenicity can be maintained in an inflammatory environment, the desensitization is realized on the stimulation of inflammatory factors IFN-gamma, the immune response of allogeneic Peripheral Blood Mononuclear Cell (PBMC) cells is not caused, and the stem cell can simultaneously escape allogeneic CD8 ⁺ Killing of T cells and NK cells. Is hopeful to transplant into the body of a patient without considering HLA matching, can avoid the attack of the immune system of the patient, plays a role in treatment and solves the problem of immunological rejection of the immune system of the human body to foreign cells in regenerative therapy based on stem cell transplantation.

The existing method for constructing the universal stem cell generally depends on high expression of various vector-mediated immune suppressor molecules, so that exogenous DNA needs to be introduced into the genome of the stem cell, and unknown safety hazards exist. Moreover, the method usually involves gene editing for several times, and the obtained multiple engineering stem cells even have the defects of unstable karyotype and the like, and the safety and the quality control of the cells are unstable. In addition, genome editing strategies of some protocols are cumbersome and multiple genetic engineering manipulations can affect the integrity of the stem cell genome. The invention is intended to explore and develop more safe and reliable universal human stem cells from the perspective of the Super Enhancer (SE) we have found.

The invention is based on epigenetics, compares the signal change condition of the modified region of the enhancer marker H3K27ac before and after IFN-gamma stimulation through ChIP-Seq technology, finds an enhancer sequence on the B2M gene for the first time, and the enhancer has the characteristic of SE after IFN-gamma stimulation and obviously improves the expression of the B2M gene. By knocking down or silencing the enhancer sequence, the expression of the B2M gene and the membrane HLA-class I protein can be significantly reduced.

The enhancer sequences found in the present invention are located: 45002906-45022119, namely the initial position of the enhancer is 45002906 site of 15 chromosome, the termination position is 45022119 site of 15 chromosome, the total length is 19214bp, as shown in SEQ ID NO:7, or a fragment thereof. The enhancer has the characteristic of a super enhancer after IFN-gamma stimulation, and the expression of the B2M gene is obviously improved. Conversely, if the sequence can be modified in appearance or silenced by other techniques, such as by cutting or cleaving nucleotides in the sequence using current gene editing techniques to disable or reduce enhancement, and to reduce the level of transcription or translation of the B2M gene, expression of the membrane HLA class I protein will be reduced, thereby reducing or eliminating rejection. This is because, in the absence or reduced expression of B2M, few HLA class I proteins can be detected on the cell surface. The enhancer is one of the key factors influencing the expression of B2M.

The invention proves that the enhancer belongs to SE through a bioinformatics approach, and finds that an SE inhibitor JQ1 can obviously reverse the effect of IFN-gamma on up-regulating B2M expression. Further, the activity of a certain sequence is reduced by CRISPR/Cas9 gene editing technology (knocking-down is the activity reduction of the modified gene editing technology, and is also called knocking-down, and is different from knocking-out caused by cutting the sequence), the SE causes the B2M gene transcription to be obviously inhibited, and the stimulation desensitization to IFN-gamma can be realized.

On the basis, the invention constructs the universal human stem cell suitable for allogeneic transplantation, wherein the core enhancer gene sequence of the B2M gene in the stem cell is partially knocked down or silenced, or the partial sequence is silenced or the partial sequence is knocked down.

The existing method for constructing the universal stem cell generally depends on high expression of various vector-mediated immune suppression molecules, so that exogenous DNA needs to be introduced into the genome of the stem cell, and unknown safety hazards exist. Moreover, the method usually involves gene editing for several times, and the obtained multiple engineering stem cells even have the defects of unstable karyotype and the like, and the safety and quality control of the cells are unstable. In addition, certain protocols are cumbersome in genome editing strategies, and multiple genetic engineering manipulations can affect the integrity of the stem cell genome. The present invention intends to explore and develop a safer and more reliable general stem cell from the perspective of the SE we have found.

According to the invention, SE is knocked down or silenced by a CRISPR gene editing technology, so that B2M gene transcription is obviously inhibited, and IFN-gamma stimulation desensitization can be realized, thereby constructing an MSC (hereinafter referred to as hypo-MSC) capable of maintaining low immunogenicity in an inflammatory environment. hypo-MSC low expression membrane HLA-I protein, desensitization to inflammatory factor IFN-gamma stimulation, no immunoreaction of allogeneic PBMC cell, and capability of avoiding allogeneic CD8 ⁺ Killing of T cells and NK cells. The novel hypo-MSC is expected to be prevented from being attacked by the immune system of a patient after being transplanted into the body of the patient without considering HLA matching, plays a role in treatment and solves the problem of immunological rejection of the immune system of a human body to allogeneic cells. Meanwhile, the feasibility and repeatability of the construction method are verified in ADSC, and the result shows that the method has universality.

In one aspect, the invention provides an enhancer, which is located in proximity to the B2M gene and is capable of forming SE upon stimulation by IFN- γ.

The invention provides a nucleic acid sequence, which is an enhancer and can enhance the expression of B2M and membrane HLA-I protein; conversely, if the sequence is silenced or knocked down, the expression of B2M and membrane HLA-class I proteins is significantly reduced or attenuated, thereby reducing the immunogenicity and immune rejection of the cell.

In some embodiments, the invention provides nucleic acid sequences that enhance the expression level of HLA class I genes of MSCs by the action of the inflammatory factor IFN- γ.

Enhancement as used herein describes enhancement in that the above-mentioned nucleic acid sequence does not directly transcribe and translate the HLA-I class gene, but enhances transcription of the HLA-I class gene. Therefore, also called enhancer sequences, when an enhancer is present, it can enhance the expression ability of a target gene sequence, but does not directly express the target gene itself. For example, a gene directly expressing HLA-class I is the gene, i.e., the DNA sequence (including exons and introns) of the gene is transcribed into RNA, and the RNA of the exon part is translated into a protein sequence, whereas the enhancer sequence provided by the present invention can significantly enhance the expression of the gene, i.e., regulate the expression of the gene (the so-called regulation may be either enhanced or reduced, or even leave it unexpressed).

Further, the enhancer region is located in chr15:45002906-45022119, and the chr15:45002906-45022119 has the amino acid sequence shown in SEQ ID NO:7, or a fragment thereof.

The enhancer sequence provided by the present invention is located near the B2M gene, and the vicinity of the B2M gene means that the enhancer sequence is located upstream or downstream of the B2M gene, and some of the enhancer sequence are introns of the B2M gene (fig. 1). The enhancer region sequences found in the present invention are located: 45002906-45022119, namely the initial position of the enhancer is 45002906 site of 15 chromosome, the termination position is 45022119 site of 15 chromosome, the total length is 19214bp, as shown in SEQ ID NO:7, or a sequence shown in the figure.

Further, the enhancer region has 6 active sequences, namely E1-E6, and the E1-E6 have the sequences shown in SEQ ID NO:1 to SEQ ID NO: 6.

Enhancers are DNA sequences that significantly increase the transcription frequency of a gene, and are mainly present in eukaryotic genes, and may be present upstream, downstream, or as introns in the gene, and thus have multiple active sequences in the region of the enhancer, which may be introns in the gene, upstream or downstream sequences, and the like.

Introns are non-coding sequences in the gene and are spliced out during mRNA processing, so mature mRNA has no intron coding sequences. Introns are not significant to the structure of the translated product and do not affect gene expression.

According to the invention, a large number of researches show that the first intron of B2M is also part of an enhancer, E2 and E3 are both positioned in the first intron of B2M, and when E2 or E3 is knocked down, knocked out or silenced, the expression of the B2M gene is remarkably reduced.

The enhancer region and the positional relationship between the active sequence and the B2M gene are shown in FIG. 1.

The E1-E6 are respectively positioned in chr15:45002980-45003650, chr15:45003850-45005400, chr15:45005530-45007575, chr15:45010414-45015507, chr15:45017960-45021156 and chr15:45021762-45023541, and respectively have the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO: 6. Knockdown, knock-out, or silencing of any of the active regions reduces the expression of the B2M gene, but to a different extent.

Furthermore, the core enhancer fragment of the enhancer region is E2, and the core enhancer is the enhancer region which affects the expression of the B2M gene to the highest extent.

In another aspect, the invention provides the use of an enhancer as described above for enhancing expression of the B2M gene.

Further, the enhancer is capable of forming SE under IFN- γ stimulation, which SE further promotes the expression of the B2M gene.

In yet another aspect, the present invention provides a method for the low expression of a B2M gene, wherein all or part of the enhancer sequence of the B2M gene is knocked down or silenced, and wherein the enhancer region has the sequence as set forth in SEQ ID NO:7, or a sequence shown in the figure.

In still another aspect, the present invention providesbase:Sub>A method for producing low-expression membrane HLA-class I protein, wherein the membrane HLA-class I protein comprises one of HLA-A, HLA-B, HLA-C, and B2M protein; the whole or partial sequence of the enhancer of the B2M gene is knocked down or silenced, and the region of the enhancer has the sequence shown as SEQ ID NO:7, or a sequence shown in the figure.

In yet another aspect, the invention provides a method of reducing expression of a membrane HLA-class I protein, primarily by knocking down or silencing all or part of the sequence of the enhancer as described above; the membrane HLA-I protein comprises one of HLA-A, HLA-B and HLA-C and B2M protein.

Further, the method comprises the steps of:

(1) Knocking down all or part of the sequence of the enhancer by a CRISPR gene editing method;

(2) Cells with the full or partial sequence of the enhancer knocked down are selected.

According to the invention, the B2M gene expression is obviously inhibited by knocking down (the knocking down is to adopt a modified gene editing technology to reduce the activity of a certain sequence but not to cause the deletion of the sequence) or silencing the enhancer through a CRISPR gene editing technology.

Further, the enhancer is knocked down by lentivirus-mediated CRISPRi, whose guide nucleotide sequences are sgRNA F and sgRNA R, respectively, whose sequences are as set forth in SEQ ID NO:16 and SEQ ID NO:17 is shown; the knocked down enhancer sequence is located at chr15:45,004,417-45,004,436.

Theoretically, any active sequence of the enhancer can obviously promote the expression of the B2M gene, so that the expression of the B2M gene can be reduced by knocking down any active sequence from E1 to E6.

The invention discovers that the core enhancer fragment of the SE region is E2, and the effect of reducing the expression of the B2M gene is most obvious after the E2 region is knocked down.

The B2M gene and the membrane HLA-I protein have universality for stem cells, and are theoretically suitable for any human cells.

Further, the stem cells are MSCs or ADSCs.

In yet another aspect, the invention provides a human stem cell having all or part of the sequence of the core enhancer of the B2M gene knocked-down or silenced.

In some embodiments, the sequence of the core enhancer is partially knocked down, or silenced, or a partial sequence is silenced or a partial sequence is knocked down; or a partial sequence is silenced while a partial sequence is knocked down.

Further, the enhancer region is located at chr15:45002906-45022119, and the chr15:45002906-45022119 has the amino acid sequence shown as SEQ ID NO:7, or a sequence shown in the figure.

Further, the enhancer region has 6 active sequences, namely E1-E6, and the E1-E6 have the sequences shown in SEQ ID NO:1 to SEQ ID NO: 6.

Further, the core enhancer fragment of the enhancer region is E2; the stem cells are MSC or ADSC cells. Theoretically, all nucleated human cells, not limited to stem cells, can be used to construct the low immunogenicity universal cells provided by the present invention. The stem cell type of MSC or ADSC is not therefore a limitation of the present invention.

In some embodiments, a portion of the enhancer sequence is knocked down, and the sequence knocked down and thus deleted is located at chr15:45,004,417-45,004,436.

In yet another aspect, the invention provides a method of producing a stem cell, primarily by knocking down or silencing all or part of the core enhancer sequence of the B2M gene.

Further, the method comprises the steps of:

(1) Knockdown of all or part of a fragment on an enhancer sequence located at chr15:45002906-45022119 by CRISPRi;

(2) Cells are selected for which all or part of the sequence segment of the enhancer is knocked down.

Further, the guide nucleotide sequences of the lentivirus-mediated CRISPRi are sgRNA F and sgRNA R, respectively, and the sequences of the sgRNA F and the sgRNA R are respectively shown in SEQ ID NO:16 and SEQ ID NO: shown at 17.

Further, the cells in the step (2) are MSC or ADSC cells; the knocked down sequence is located at chr15:45,004,417-45,004,436.

Theoretically, all nucleated human cells, not limited to stem cells, can be used to construct the low immunogenicity universal cells provided by the present invention. The stem cell type of MSC or ADSC is not therefore a limitation of the present invention.

In some aspects, the method comprises the steps of: providing a cell; the core plasmid (pHR-SFFV-KRAB-dCas 9-P2A-mChery or pLV-U6-gRNA-UbC-eGFP-P2A-Bsr; packaging plasmid psPAX2 and envelope plasmid pMD2.G are co-cultured with the cells to obtain the screened double-positive cells as the cells with the enhancer knocked down.

In yet another aspect, the present invention provides a stem cell having a sequence of all or part of the core enhancer region of the B2M gene knocked-down or silenced for suppressing an allogeneic immune rejection.

In some embodiments, the enhancer gene of the B2M gene in the stem cell is silenced or knocked down, or part of the sequence is silenced, or part of the sequence is knocked down; or a partial sequence is silenced while a partial sequence is knocked down.

Further, the enhancer region is located at chr15:45002906-45022119, and the chr15:45002906-45022119 has the amino acid sequence shown as SEQ ID NO: 7; the stem cells are MSC or ADSC cells.

The gene knockdown, silencing and cleavage described in the present invention are all intended to reduce, lose or prevent the function of the target gene as an enhancer.

The invention has the beneficial effects that:

1. the enhancer sequence on the B2M gene responding to IFN-gamma stimulation is explained for the first time;

2. the sequence is found to have the characteristic of enhancer before IFN-gamma stimulation for the first time, and has the characteristic of SE after stimulation, thereby enhancing the expression of B2M gene;

3. the sequence is found to promote the expression of B2M gene under the stimulation of IFN-gamma for the first time, so as to promote the expression of HLA-I protein of the membrane;

4. the novel epigenetic mechanism that the inflammatory factor IFN-gamma regulates the expression of the B2M gene by inducing SE is elucidated for the first time;

5. providing a new target for constructing human cells with low immunogenicity;

6. by applying CRISPI technology, the SE is genetically edited in the stem cells to construct the universal stem cells, which can realize low immunogenicity and escape from allogeneic immune reaction;

7. when the universal stem cell is constructed, foreign DNA such as immunosuppressive molecules and the like is not required to be introduced, and multiple gene editing is not required, so that the universal stem cell is simpler and more convenient to implement;

8. the invention uses reversible appearance modification without changing DNA sequence, so the constructed general stem cell (such as hypo-MSC) is safer and more reliable;

9. the method provided by the invention has universality, is not specific to MSC (mesenchymal stem cell) cells, can be realized in ADSC (all-dielectric dependent cell) cells, and can be theoretically suitable for constructing universal cells by any human stem cells and other nucleated human cells.

Drawings

FIG. 1 is a schematic diagram showing the region where the enhancer is located, and the positional relationship between the active sequence and the B2M gene.

FIG. 2 isbase:Sub>A diagram showing the results of RNA extraction and real-time fluorescent quantitative PCR detection of HLA-A, HLA-B, HLA-C and B2M expression levels of three groups of MSCs in example 1.

FIG. 3 is the results of the expression levels of HLA-A, HLA-B, HLA-C and B2M by the five groups of MSCs in example 2 using q-PCR and flow cytometry, respectively, wherein 3A is the result of q-PCR detection and 3B is the result of flow cytometry detection.

FIG. 4 is a graph showing the results of H3K27ac ChIP-Seq analysis and bioinformatics analysis in example 3, wherein 4A is a graph showing the results of H3K27ac ChIP-Seq analysis and 4B is a graph showing the results of bioinformatics analysis.

FIG. 5 is a schematic diagram of the experiment process of knocking down large fragments of super enhancer and the result of detecting the expression level of HLA-I in example 4, in which 5A is a schematic diagram of the experiment process of knocking down a part of DNA sequence of super enhancer, and 5B is a schematic diagram of the result of detecting the expression level of HLA-I.

FIG. 6 is a schematic diagram of the results of the modified hypo-MSC cell obtained by the lentivirus-mediated CRISPR technique of example 5, wherein 6A is the hypo-MSC cell flow fluorescence sorting result; 6B is a schematic diagram of a lentivirus-mediated CRISPR technical process; 6C is a schematic diagram comparing the B2M expression levels of MSC and ADSC before and after SE modification.

FIG. 7 is a graph showing the results of co-culture of PBMCs with hypo-MSCs or MSCs in example 6, wherein 7A and 7B are the results of flow-sampling and proliferation assay of PBMCs with hypo-MSCs or MSCs; 7C is a schematic representation of the comparison of B2M expression levels in human interferon gamma (IFN-. Gamma.) enzyme-linked immunosorbent assay hypo-MSC or MSC.

FIG. 8 is a graph showing the co-culture results of NK cells with hypo-MSC or MSC in example 7, wherein 8A and 8B are graphs showing the results of detecting NK cell activation marker molecule (CD 107 a); 8C is a schematic diagram of the results of measuring NK cell cytotoxicity (LDH).

FIG. 9 shows CD8 of example 8 ⁺ T cells andschematic representation of hypo-MSC or MSC co-culture results, wherein 9A is for detecting CD8 ⁺ Activation of T cells marker (CD 69) results; 9B is the detection of CD8 ⁺ Schematic representation of cytotoxicity (LDH) results for T cells; 9C is the detection of CD8 ⁺ The result of the secretion of the inflammatory factor IFN-gamma of the T cell is shown schematically; 9D and 9E are for detecting CD8 ⁺ The results of the T cells and hypo-MSC or MSC co-culture flow-type collection detection proliferation condition are shown in the figure.

Detailed Description

The specific embodiments of the present invention are set forth merely to illustrate how the invention may be practiced and are not to be construed as limiting the invention, which is defined by the claims.

English abbreviation of noun and Chinese meaning comparison table: MSC: mesechymal Stem Cells, mesenchymal Stem Cells; ADSC: adipose-derived mesensical Stem Cell, adipose-derived Mesenchymal Stem Cell; B2M: beta 2Microglobulin, beta 2 Microglobulin; ESC: embryonic Stem Cells, embryonic Stem Cells; and (2) iPSC: induced Pluripotent Stem Cells; and SE: super Enhancer; TE: typical Enhance, canonical enhancer; KD: knockdown.

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example 1: IFN-gamma upregulating HLA-I gene expression of MSCs

This example demonstrates, through specific experiments, that IFN- γ can up-regulate the expression of HLA-I gene of MSC.

1.1 cell culture and treatment

MSC culture conditions: L-DMEM medium (containing 10% fetal bovine serum), 5% CO2, and 37 ℃ constant temperature culture. 24 hours before treatment, MSCs were treated at 5X 10 ⁵ And inoculating the cells into a 6-well plate for culture, wherein the cell fusion degree reaches 60-70% the next day.

Dividing the cultured MSC into three groups, wherein the first group is a blank control, the second group adds IFN-gamma into the culture medium according to the concentration of 100ng/ml, and continuously culturing for 24 hours; in the third group, IFN-gamma is added into the culture medium according to the concentration of 100ng/ml, and the culture is continued for 48 hours; collecting cells for subsequent RNA extraction and other treatments.

1.2 RNA extraction and fluorescent quantitative PCR (qRT-PCR) of MSC

(1) RNA extraction:

1) Respectively adding 1ml of Trizol cell lysis solution into the three groups of collected MSC cells;

2) Adding 200 mul (1/5 volume) of chloroform, carrying out vortex oscillation, and standing for 10min;

3) And a precooling centrifuge, centrifuging: 12000g,4 ℃,15min;

4) The supernatant was transferred to a new 1.5ml RNase-free EP tube;

5) Adding equivalent isopropanol, turning upside down, mixing well, and standing at room temperature for 10min;

6) And centrifuging: 12000g,4 ℃,15min;

7) Removing the supernatant, adding 1ml of 75% ethanol, and slightly reversing the mixture to wash the mixture;

8) And centrifuging: 12000 Xg, 4 ℃,10min;

9) Removing the supernatant, and air-drying for 10min;

10 According to the size of the precipitate, 20. Mu.l of DEPC water was added, RNA was dissolved at 4 ℃ and the mixture was allowed to stand for 30min, and the concentration was measured.

(2) Real-time fluorescent quantitative PCR (qRT-PCR)

1) Using a TOYOBO reverse transcription kit (cat No.: FSQ-201) and carrying out reverse transcription on the RNA extracted in the last step to obtain cDNA, wherein the system comprises the following steps:

2) According to Takara corporation (cat No.: RR 420A) for real-time fluorescent quantitative PCR of cDNA using the system on SYBR mix kit instructions:

the PCR reaction cycle conditions of the real-time fluorescent quantitative PCR vii 7 instrument are as follows:

the primer sequences used are listed in the following table:

the contents of HLA-A, HLA-B, HLA-C and B2M in the three groups of MSCs are respectively detected by real-time fluorescent quantitative PCR, and the results of RNA extraction and real-time fluorescent quantitative PCR of the three groups of MSCs are shown in FIG. 2.

As can be seen from the q-PCR results (FIG. 2), IFN-gamma withbase:Sub>A concentration of 100ng/ml can significantly increase the transcriptional level (i.e., the content of mRNA) of HLA-I genes (HLA-A, HLA-B, HLA-C (also called HLA-ABC for short) and B2M) of MSC; and with the prolonged action time of IFN-gamma, the transcription level of HLA-I molecules of MSC is increased, and the transcription peak can be reached within 48 hours (h).

The experiment proves that IFN-gamma can obviously or very obviously improve the transcription level of several genes related to HLA-I family, the expression of the antigen can be obviously or very obviously improved, and the higher the expression of the antigen protein is, the higher the possibility of causing rejection reaction is (the transcription level of the direct gene) is.

Example 2: experiments involving enhancers in the stimulation of HLA-I expression by IFN-gamma

In this example, after MSC is pretreated with JQ1 (purchased from abcam, cat # ab146612, lot # APN15092-1-1; an enhancer inhibitor) with three concentrations of 125, 250 and 500nmol/ml for 2 hours, 100ng/ml IFN-gamma is added to treat MSC, the cells are collected after 48 hours of continuous culture, RNA is extracted to perform qPCR to detect HLA-I related gene expression, corresponding MSC blank control is set, and IFN-gamma treatment is performed on the MSC blank control, five groups of MSCs are compared, and q-PCR and flow cytometry are respectively adopted for detection, wherein the results are shown in FIG. 3, wherein the specific process and sequence of q-PCR refer to the process and conditions of example 1, the q-PCR result refers to FIG. 3A, and the expression levels of HLA-A, HLA-B, HLA-C and B2M of the five groups of MSCs are shown; the flow cytometry results are shown in figure 3B.

1. q-PCR results

As can be seen from the q-PCR results (FIG. 3A), IFN- γ can significantly increase the transcriptional level of HLA-I genes (HLA-A, HLA-B, HLA-C and B2M) of MSC cells. However, this stimulation can be inhibited by small molecule JQ1, an enhancer inhibitor, and there is a dose response relationship, with increased inhibition as the concentration of JQ1 increases.

2. Flow cytometry results:

the specific process is as follows:

(1) Collecting the above cultured five groups of MSC cells, counting, and collecting 1 × 10 cells ⁶ Mixing cells/100 μ L with 10 μ L diluted HLA-ABC antibody (NOVUS, catalog # NB 100-64775), and incubating on ice for 30min;

(2) 1mL of PBS is added to resuspend and wash the cells;

(3) Centrifuging at 200 Xg for 5min, and removing supernatant;

(4) Resuspend the mixed cells with 40. Mu.L of diluted Anti-Mouse 488 secondary antibody (Invitrogen, catalog # A-21202), incubate for 15min on ice in the dark;

(5) 1mL of PBS was added to resuspend the washed cells;

(6) Centrifuging at 200 Xg for 5min, and discarding the supernatant;

(7) Cells were resuspended in 300. Mu.L PBS, transferred to a flow tube, and flow-loaded.

In this example, the mRNA level of HLA-I was measured by q-PCR, and the expression of HLA-I protein on the cell membrane was further measured by flow cytometry. The flow results (FIG. 3B) are consistent with the q-PCR results, when the MSC is stimulated by IFN-gamma, the membrane HLA-I expression level is increased (the peak value is shifted to the right), when the MSC pretreated by JQ1 is stimulated by IFN-gamma, although the membrane HLA-I expression level is increased to a certain degree, the peak value right shift degree is not obvious in the IFN-gamma stimulation group, namely IFN-gamma can up-regulate the HLA-I expression level on the surface of the MSC cell membrane, but JQ1 can obviously inhibit the effect of IFN-gamma, which means that when the cell enhancer is inhibited by JQ1, the effect of the IFN-gamma stimulating the up-regulation of HLA-I is blocked. From these results, it can be speculated that there may be an enhancer involved in the stimulation of HLA-I expression by IFN- γ, but how this enhancer is involved in particular is demonstrated in the examples below.

Example 3 IFN-. Gamma.has an effect of promoting the opening and activation of an enhancer in the vicinity of the B2M gene

To further understand whether enhancers are involved in the vicinity of the HLA-I associated gene following IFN- γ stimulation and are directly involved in expression of that gene, we performed H3K27ac ChIP-Seq analysis and bioinformatics analysis.

1. The procedure for the H3K27ac ChIP-Seq analysis was as follows:

1X 10 at room temperature ⁷ The non-IFN- γ -stimulated MSCs and IFN- γ -stimulated MSCs of (a) were fixed with 1% formaldehyde for 10 minutes, respectively, to obtain DNA-protein cross-links. After 1.5 hours of sonication to obtain chromatin fragments of 200-300bp in length, the chromatin fragments were incubated overnight at 4 ℃ with an H3K27ac antibody (H3K 27ac is a putative marker Protein for active enhancers, and all enhancer DNA binding to H3K27ac Protein was captured using the H3K27ac antibody), and then incubated with Dynabeads Protein G at 4 ℃ for 4 hours to obtain antibody-chromatin complexes. Then, RNase A and proteinase K are used for crosslinking, and a PCR purification kit is used for extracting and purifying the DNA of the immune precipitation, and finally, a DNA library is prepared for sequencing.

The results of the H3K27ac ChIP-Seq analysis revealed that the H3K27ac signal was significantly enhanced near the B2M gene after the stimulation of MSC with IFN-gamma, and a new H3K27ac signal was generated (FIG. 4A). Here, it was demonstrated that the B2M gene has an enhancer sequence and is a novel sequence. In FIG. 4A, the case of the enhancer near the B2M gene before IFN- γ stimulation is shown at the top, and the case of the enhancer near the B2M gene after IFN- γ stimulation is shown at the bottom, and it can be seen that the peak signal of H3K27ac is significantly increased after IFN- γ stimulation, indicating that these enhancers are activated after IFN- γ stimulation.

2. Bioinformatic analysis of SE identification:

1) H3K27ac ChIP-Seq data preprocessing

The quality of the original sequencing data is firstly evaluated by using FastQC (v0.11.9), then low-quality bases and linker sequences existing in the sequencing data are removed by using trimmatic (v 0.38, default parameters) software, finally, only sequences with the length of more than 50 bases are reserved for subsequent analysis, and the quality of the filtered sequencing data is re-evaluated by using FastQC. Then, using a second generation sequencing data alignment software bowtie2 (v2.4.1- — very-sensitive), the filtered qualified sequences were aligned to the reference genes. To avoid the effect of PCR duplication on subsequent analyses, markDuplicates (Picard) was used to label and remove redundant sequences resulting from PCR.

2) H3K27ac ChIP-Seq result visualization

To more intuitively observe the abundance distribution of H3K27ac ChIP-Seq sequencing reads on the reference Genome, the aligned BAM formatted file was converted into bedGraph format using bamCoverage (deepTools, v3.5.1, - - -binSize 200- -smoothLength 600), and then the abundance distribution characteristics of H3K27 ChIP-Seq reads on the reference gene were displayed using UCSC Genome Browser (UCSC Genome Browser).

3) Finding enhancers and super enhancers

Based on the aligned BAM format files, genomic regions significantly enriched in H3K27ac ChIP-Seq reads were identified using the callpeak (MACS 2, v 2.2.6) algorithm, which were considered to be the presence of H3K27ac modifications. We next used ROSE (RANK ORDERING OF SUPER-enhancer) software developed by Richard a young laboratory, the institute OF technology, ma, to find ENHANCERS and SUPER-ENHANCERS based on the aligned BAM files and MACS 2-recognized H3K27ac modified region files, which first identified enhancer regions, then merged adjacent ENHANCERS less than 15kb apart, and finally compared the abundance distribution OF H3K27ac reads within the merged enhancer regions to identify SUPER-ENHANCERS. Based on the H3K27ac data of hMSC before and after IFN- γ stimulation, a emerging super enhancer in hMSC after IFN- γ stimulation can be identified.

Bioinformatic analysis revealed that these regions were typical enhancers in MSC cells that were not stimulated by IFN- γ and were characterized by a super enhancer after IFN- γ stimulation (fig. 4B). FIG. 4B isbase:Sub>A bioinformatic analysis of the sequencing results of H3K27ac, on the left side, the enhancer signals of all the genes of MSC before IFN- γ stimulation, and it can be seen that HLA-I related genes (HLA-A, HLA-B, HLA-C and B2M) are all located below the intersection of two dotted lines, i.e. the enhancers of HLA-I related genes before IFN- γ stimulation do not reach the threshold of super enhancer and do not belong to the super enhancer category; in the right side of the enhancer signal of all the genes of MSC after IFN-gamma stimulation, it can be seen that only the enhancer of B2M reaches the threshold of super enhancer in HLA-I related genes (HLA-A, HLA-B, HLA-C and B2M), namely, the super enhancer belongs to the category of super enhancers. This also indicates that the enhancer sequence is located in the vicinity of the B2M gene and not in the vicinity of other genes HLA-A, HLA-B, and HLA-C.

Example 4 reduction of Membrane HLA-I expression levels following super enhancer Large fragment knockout

By utilizing the characteristic that paired sgRNAs in a paired sgRNA CRISPR gene editing method (paired-gRNA CRISPR-Cas 9) can cause two breaks in the same gene, a large fragment knockout is carried out on a partial DNA sequence (a red arrow in a figure 5A shows that the knockout position is: chr15:45004785-45006364 and the length of the knockout sequence is 1579bp, and a knockout cell (KO) is sorted out by using a flow fluorescence cell sorting mode, wherein the large fragment spans partial regions of E2 and E3.

The sequences of the paired sgrnas are as follows, sg-KO1-F: ATTCTCCAGAGCAAAACTGGG (SEQ ID NO: 19); sg-KO1-R: CCCAGTTTTGCTCTGGAGAAT (SEQ ID NO: 20); sg-KO2-F: TAGTTTACAGCAATCACCTG (SEQ ID NO: 21); sg-KO2-R: CAGGTGATTGCTGTAACTA (SEQ ID NO: 22).

SE core sequence knockout experiments show that when the sequence is knocked out, the expression level of HLA-I of a membrane of a KO cell is obviously reduced compared with that of an MSC control group, and even can not be detected; and the cell is desensitized to IFN-gamma stimulation, and the expression level of membrane HLA-I of KO cells is obviously reduced compared with that of MSC control group after IFN-gamma stimulation.

Fig. 5A is a schematic diagram of an experiment, that is, two sgrnas are designed at positions before and after peak 1, and DNA fragmentation is caused at the positions of the two sgrnas by using CRISPR technology, so that large fragment DNA sequence knockout of a part of an enhancer is realized. The flow cytometry analysis showed that after the enhancer was knocked out (KO group), the expression level of membrane HLA-I was significantly decreased (left shift of peak) compared to the non-knocked out group (MSC group) and was almost at the level of completely non-expressed B2M gene compared to blank group, as shown in fig. 5B; and when the KO group is stimulated by IFN-gamma (KO + IFN-gamma group), the rising degree of the expression quantity of the membrane HLA-I is not obvious in the undepressed group (MSC + IFN-gamma group), and compared with the blank group, the rising degree is almost at the level of completely not expressing the B2M gene, which indicates that the MSC after the enhancer is knocked out is completely desensitized to IFN-gamma stimulation. It was also demonstrated that deletion of the large fragment sequence of this enhancer severely affected the expression of the B2M gene and thereby also reduced the amount of membrane HLA-I expression.

Therefore, large fragment knockout easily leads to almost no expression of the B2M gene, which increases the immunogenicity of MSCs (because deletion of B2M causes immune rejection of natural killer cells), and cannot be used to construct universal stem cells, further suggesting that the knockdown method is more suitable for constructing universal stem cells than knockout.

Example 5: lentivirus-mediated CRISPR technology for obtaining modified hypo-MSC cells

In order to construct the universal stem cell hypo-MSC, this example employs a small-segment knockdown method for a partial sequence in the core enhancer segment E2 of the enhancer, so as to successfully construct the hypo-MSC cell.

The hypo-MSC cells provided in this example were prepared by the following steps:

(1) sgRNA design and recombinant plasmid construction:

sgRNA was designed against the enhancer sequence (SEQ NO: 1) (Benchling website:https:// www.benchling.com/crispr/). sgRNA sequences are shown in the table below:

enzyme cutting sites are respectively added at two ends of the sgRNA, CACC is added at the 5 'end of a sense strand of a sgRNA sequence, and AAAC is added at the 5' end of an antisense strand, so that a cohesive end which is complementary to pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid (Addge: # 83925) after being cut by Fast Digest Bbs I is formed. If the first base at the 5' end of the sense strand is not a G, a G is added after the CACC at the 5' end, and a C is added at the 3' end of the corresponding antisense strand. pLV-U6-gRNA-UbC-eGFP-P2A-Bsr is an sgRNA framework expression vector containing a U6 promoter, and is provided with a GFP green fluorescent protein gene and ampicillin resistance.

(1) pLV-U6-gRNA-UbC-eGFP-P2A-Bsr is cut by Fast Digest Bbs I, and a linearized vector is recovered after DNA gel electrophoresis.

(2) Phosphorylating and annealing the sgRNA sequence with T4 PNK; and respectively connecting the linear pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid vector with the annealed sgRNA double-stranded sequence for 1h at room temperature by using T4 ligase. The ligation product was transformed into competent bacteria, trans 109, on ice at 30min, 45s at 42 ℃ for 2min on ice. Clones were selected on ampicillin resistant LB plates. And (4) picking positive clone shake bacteria and sequencing. The sequencing primer is the forward primer sequence of the U6 promoter, 5 'GAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO: 18). The clone with correct sequencing is the recombinant plasmid.

(2) HEK293FT cells were plated on 10cm cell culture dishes at 2X 106/dish one day in advance to ensure that the degree of cell fusion reached 60% effect the next day.

(3) The optimal transfection time is 12-18 hours after plating. HEK293FT cells were co-transfected with the core plasmid (pHR-SFFV-KRAB-dCas 9-P2A-mCherry (Addgene: # 60954) or the recombinant plasmid from step 1, the packaging plasmid psPAX2 (Addgene: # 12260), the envelope plasmid pMD2.G (Addgene: # 12259) according to the ViaFect transfection reagent instructions, in the proportions of 4.

(4) The virus stock was collected 3 times consecutively at 24 hours, 48 hours and 72 hours after transfection, and each collected virus solution was filtered through a 0.45 μm filter to remove cell debris and other impurities.

(5) 1 × 10 a day in advance ⁵ MSC cells were seeded in 6-well plates per well, preferably to ensure 50% cell confluence the next day of infection.

(6) Before infection, virus stock solution is mixed with fresh culture medium in equal volume (step 3), 2 mul of polybrene with 10mg/ml (final concentration is 10 mug/ml) is added, mixed evenly and added into a six-well plate. This was repeated three times.

(7) And (3) performing flow fluorescence sorting 48 hours after the cell fusion degree reaches 80-90%, sorting the cells with double positive of mCherry (red fluorescence) and GFP (green fluorescence), and performing amplification culture. Theoretically, the surviving MSC is a cell line that stably knockdown the enhancer gene.

Based on the discovery of a super enhancer SE on the B2M gene in response to IFN- γ stimulation, this example uses the approach of lentivirus CRISPR to apparently modify this SE to knock down a partial fragment of the enhancer's E2 active sequence, located at chr15:45,004,417-45,004,436.

The results show that the apparent inhibition of SE can significantly reduce B2M gene expression and can greatly suppress IFN-gamma stimulated B2M gene expression. We further verified the feasibility and reproducibility of this approach in primary ADSC cells, and the results obtained in ADSC were consistent with MSC results (see fig. 6A and 6B in particular). This shows that our method for constructing low immunogenicity cells is not specific to MSC and has universality. We refer to the less immunogenic MSCs constructed by this method as hypo-MSCs.

It is understood that in any other cell, modification of the SE sequence, partial gene sequence knock-down, or reverse gene silencing found in the present invention can be used to inhibit SE, and after inhibition, the expression of B2M gene is reduced, thereby reducing the activity of other similar genes, reducing immunogenicity, and thus reducing rejection. It will also be appreciated that, in addition to the gene of the invention which knockdown the sequence may alter or inhibit the SE gene, at other locations, the activity or function of the gene may be significantly inhibited, thereby reducing B2M gene expression.

As can be seen in fig. 6A, BF (bright field) was used to control the intensity of the fluorescent signal (red or green fluorescence), and the gene knockdown cells had mCherry (red fluorescence) and GFP (green fluorescence) appearance, indicating the presence of stable SE-knocked-down cells.

FIG. 6C is a graph showing the comparison of B2M expression levels before and after SE modification between MSC and ADSC, wherein MSC-KRAB represents MSC stably expressing dCas9-KRAB, and MSC-sgRNA represents MSC stably expressing dCas9-KRAB and sgRNA simultaneously. Only under the guidance of sgRNA does dCas9-KRAB function. This example designs a pair of sgrnas (plus and minus two) for enhancer, which can direct dCas9-KRAB to the enhancer region to play the role of knocking down enhancer. MSC-KRAB cells, lacking the pair of sgRNAs, failed to exert enhancer knockdown effect and were used as control cells.

From the expression level of B2M of fig. 6C, the MSC expression levels were significantly different for MSC cells, cells without SE modification and cells with SE sequence modification. Similarly, the two showed significant differences in IFN-. Gamma.stimulated B2M gene expression. This fully demonstrates that the methods of the invention can inhibit SE activity, thereby significantly reducing the expression level of the B2M gene. The same conclusion is true for ADSC cells.

However, compared with the large fragment knockout in example 4, the normal expression of B2M is not completely inhibited by the small fragment knock-down of the core enhancer fragment, so that a universal stem cell hypo-MSC can be constructed.

Example 6 hypo-MSCs do not elicit an immune response in allogeneic PBMCs

In this example, allogeneic PBMC labeled with CFSE dye was cultured with hypo-MSC (cells prepared in example 5) and MSC (control), PBMC cell proliferation (PBMC proliferation reflecting the intensity of immune rejection) was detected by flow cytometry, and secretion of inflammatory factors in immune response was detected by human interferon-gamma (IFN-. Gamma.) ELISA, as follows:

1. PBMC mixed culture

(1) Blood was diluted with PBS;

(2) Taking a proper amount of Ficoll in a 15mL centrifuge tube, and slowly adding diluted blood to the upper layer of the Ficoll;

(3) Centrifuging at 300 Xg for 30min, putting the middle flocculent layer in a new centrifuge tube, adding PBS for cleaning, and counting;

(4) Isolated PBMCs (allogenic cells) from the reacted cells were stained at 37 ℃ for 10min according to the CFDA-SE cell proliferation and tracer detection kit instructions. Then washing for 2 times by using complete cell culture solution, centrifuging, and counting the number of the cells which are re-suspended by using the complete cell culture solution to serve as reaction cells;

(9) After MSC and hypo-MSC adhere to the wall, incubating the wall with 5ng/mL mitomycin at 37 ℃ for 15min, then washing the wall for 2 times by using complete cell culture solution, centrifuging, and counting the number of the cells which are re-suspended by using the complete cell culture solution to serve as stimulated cells;

(10) At 1 × 10 ⁴ cells/cell density of 100 μ L stimulated cells (i.e., both MSC and hypo-MSC stimulated cells, MSC used as a control for hypo-MSC) were seeded in U-type 96-well plates;

(11) At 1 × 10 ⁵ cells/cell density of 100. Mu.L the reaction cells were seeded in U-shaped 96-well plates;

(12) And (3) putting the U-shaped 96-well plate back to the cell culture box for culturing for 3-5 days respectively, and then collecting samples in a flow mode to detect the proliferation condition of the reaction cells.

2. Human interferon gamma (IFN- γ) enzyme-linked immunosorbent assay:

(1) Pre-calculating the required number of battens, taking out the kit 30min before the experiment, and recovering to the room temperature;

(2) Adding 100 mul of standard working solution and a detection sample into each reaction hole (if the concentration of the sample is higher than the detection range, the sample needs to be diluted by the standard and sample diluent and then sampled), making a plurality of holes for the standard, and incubating in an incubator at 37 ℃ for 90min after closing the plates;

(3) Discarding the liquid, spin-drying, adding 100 μ l biotin-labeled gamma interferon antibody working solution into each reaction well (for two treatment of co-culture, PBMC + hypo-MSC; PBMC + MSC), sealing, and incubating at 37 deg.C for 60min;

(4) Washing: discarding the liquid, spin-drying, adding 350 μ l of washing solution into each reaction hole, soaking for 1-2min, spin-drying the washing solution, and repeating for 4 times;

(5) Adding 100 mu l of HRP marked streptavidin working solution into each reaction hole, and incubating in an incubator at 37 ℃ for 30min after closing a plate;

(6) Washing: 300 mul of washing solution was added to each reaction well, and the washing solution was spun down at intervals of 30 seconds. Repeating for 4 times;

(7) Adding 90 μ l of color developing agent (in dark) into each reaction hole, sealing the reaction holes, and developing in dark at 37 deg.C for about 15min;

(8) Adding 50 μ l of stop solution into each reaction hole, and immediately measuring OD value (within 5 min) with a microplate reader at a wavelength of 450 nm;

(9) Measuring OD value with 450nm wavelength of a microplate reader;

(10) Calculating the average OD value of the standard substance and the sample: the OD value of zero wells should be subtracted from the OD value of each standard and sample;

(11) The concentration of the standard substance is used as the abscissa and the OD value of the absorbance is used as the ordinate, and a standard curve is drawn by software (values of blank groups are removed in drawing).

(12) If the OD value of the sample is higher than the upper limit of the standard curve, the sample is diluted properly and then detected again, and the concentration is multiplied by the dilution times when being calculated.

Allogeneic PBMCs were labeled with CFSE dye and co-cultured with hypo-MSCs (cells of example 5), MSCs (control), and FIGS. 7A and 7B are schematic graphs showing the results of flow-through assays for proliferation of PBMCs with hypo-MSCs or MSCs, where E/T is the efficiency: target ratio (ratio of Effector cells (here PBMCs) to Target cells (here MSCs or hypo-MSC cells)), indicating that the MSC group elicited a strong immune response, that PBMCs proliferated extensively, and that PBMCs of the hypo-MSC group were almost non-proliferating (FIGS. 7A and 7B). The positive control is PBMC mixed culture from two different sources, and the source of the PBMC is different, so that immune rejection reaction is caused certainly, and the PBMC is called as the positive control; the negative control is PBMC cultured alone and not mixed with other cells, so that immune rejection reaction does not occur; the positive and negative controls were used together to demonstrate the success of the experiment without false positives and false negatives.

The immunogenicity change conditions of hypo-MSC and MSC before and after co-culture are detected in one step, and the result shows that the B2M gene of MSC is obviously up-regulated after co-culture with PBMC, and hypo-MSC can maintain low-level B2M gene expression before and after co-culture. Indicating hypo-MSC had low immunogenicity and could maintain low immunogenicity in inflammatory environments (fig. 7C).

Example 7 hypo-MSC is able to escape killing of NK cells

Separating NK cells (natural killer cells) in the PBMC, continuously co-culturing the NK cells with hypo-MSC and MSC according to the proportion relation of 1.

The results show that there was no difference in the proportion of hypo-MSC and MSC-activated NK cells in the case of NK cells co-cultured with hypo-MSC or MSC in three mixing ratios of 1, 3, 1 and 10, respectively (fig. 8A and 8B), nor was there any statistical difference in LDH (fig. 8C), indicating that hypo-MSC was not sufficiently hypoimmunogenic to activate allogeneic NK cells.

Example 8 hypo-MSCs are able to escape killing of CD8+ T cells

The mixed culture is carried out for five days by using MSC and PBMC in advance, CD8+ T cells are separated out and are continuously cultured together with hypo-MSC and MSC, and the secretion of activation marker (CD 69), cytotoxicity (LDH) and inflammatory factor IFN-gamma of the CD8+ T cells is detected.

The results indicate that hypo-MSC showed lower CD8+ T activation (fig. 9A), lower T cell killing (fig. 9B) and lower IFN- γ secretion (fig. 9C) than MSC group; CD8+ T cell proliferation experiments also demonstrated that hypo-MSC was barely able to induce CD8+ T cell proliferation (fig. 9D and 9E). Indicating that the low immunogenicity of hypo-MSC is able to escape killing of allogeneic CD8+ T cells.

The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

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aacccaggag gcggaggttg cagtgagctg agatctcacc actgcacact gcactccagc 1380

ctgggcaatg gaatgagatt ccatcccaaa aaataaaaaa ataaaaaaat aaagaacata 1440

ccttgggttg atccacttag gaacctcaga taataacatc tgccacgtat agagcaattg 1500

ctatgtccca ggcactctac tagacacttc atacagttta gaaaatcaga tgggtgtaga 1560

tcaaggcagg agcaggaacc aaaaagaaag gcataaacat aagaaaaaaa atggaagggg 1620

tggaaacaga gtacaataac atgagtaatt tgatgggggc tattatgaac tgagaaatga 1680

actttgaaaa gtatcttggg gccaaatcat gtagactctt gagtgatgtg ttaaggaatg 1740

ctatgagtgc tgagagggca tcagaagtcc ttgagagcct ccagagaaag gctcttaaaa 1800

atgcagcgca atctccagtg acagaagata ctgctagaaa tctgctagaa aaaaaacaaa 1860

aaaggcatgt atagaggaat tatgagggaa agataccaag tcacggttta ttcttcaaaa 1920

tggaggtggc ttgttgggaa ggtggaagct catttggcca gagtggaaat ggaattggga 1980

gaaatcgatg accaaatgta aacacttggt gcctgatata gcttgacacc aagttagccc 2040

caagtg 2046

<210> 4

<211> 5094

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccacttggta aaaaatgtga accccttgta tatagagttt ggctcacagt gtaaagggcc 60

tcagtgattc acattttcca gattaggaat ctgatgctca aagaagttaa atggcatagt 120

tggggtgaca cagctgtcta gtgggaggcc agccttctat attttagcca gcgttctttc 180

ctgcgggcca ggtcatgagg agtatgcaga ctctaagagg gagcaaaagt atctgaagga 240

tttaatattt tagcaaggaa tagatataca atcatccctt ggtctccctg ggggattggt 300

ttcaggaccc cttcttggac accaaatcta tggatattta agtcccttct ataaaatggt 360

atagtatttg catataacct atccacatcc tcctgtatac tttaaatcat ttctagatta 420

cttgtaatac ctaatacaat gtaaatgcta tgcaaatagt tgttattgtt taaggaataa 480

tgacaagaaa aaaaagtctg tacatgctca gtaaagacac aaccatccct ttttttcccc 540

agtgtttttg atccatggtt tgctgaatcc acagatgtgg agcccctgga tacggaaggc 600

ccgctgtact ttgaatgaca aataacagat ttaaaatttt caaggcatag ttttatacct 660

gatggccagc tttgtttatt tgaccaagaa tctgagttag ctagttctag gtactgacag 720

gataaataaa acacaacact gctcccgatc ttctcagttt agcagaggga cagatatgca 780

ctcaaataat taaaatatat cctgataaga atatagcata ggtacgcgcg aagaacttgg 840

caatcgaaat tttgttgttc aggctgggcg aggtgtctca tgtctgtaat cccagcactt 900

tgggaggcca tggtaggatg atcgcttgag cccaggagtc cgagaccagc ctgggcaaca 960

taacaagacc ctgtctcaat tcaaagaatt gaattaaaaa aaacaaaaaa taattttttt 1020

aaaaaagaaa tgttgttgtt caaggaacaa caacaaaaat ctagggaggt gttagagaag 1080

ccatttgcct gagctgagag taagttgcta gtggttctct tgattggtag gtggggcctg 1140

ggtttccagg catggtagcc aggaaggaca gccacatggc aggtttgggt aattccaaac 1200

agtggaggaa gggtgtctgg gggaagactt gtaggaactc agctgaaaaa attgggggat 1260

gatactctga aagaaaaaca aagttttaaa atttctactc ttacacttaa cacataatgc 1320

ttctgtgacc ggatatttag gggttttccc cccacactct gttaggagaa aaattttaga 1380

cagattaaat ttaacagagt ttaactgagc aaaaatgatt ctcgaaccag gcagctgccg 1440

gagccagaat aggttcaaaa tgactctggg ggtgccacat ggttggatga catttaggga 1500

cagaaaaagg aaagtgatgt gcagaaaatg gaagtcaggg gcagaagcag ccagattggt 1560

tgcagttcag catttgcctc atttaaacag ggtttgaaga gttggccacc tgtgattggc 1620

tgagactctg tggtataaga gtaagttaca gtctgtttac acatccagtt aggttacagt 1680

tcactatgca gagagaaatc tttagcctga acttacacag ggaggcagtt ttatttattt 1740

atttaatttt ttttttttga gacaaggtct cactctgtca cccgggctgg agttcagtgg 1800

tatgatcatg gcttattgca gcctcgactt cctggcctca agcaatcctt ccgcctcagg 1860

ctctagagta gctgggacta caggcacatg tcagcatgcc tggctaattt tgttttttaa 1920

tttttagtag agatgaactc ctggccttgc acaattcttt cgcctcagcc tccgaaaatg 1980

ctgggattac aggtgtgagc cactgtgccc agctaaggca actttaggct aaaccttttt 2040

ttgagacaga gtttctctct tgttgtctag gctggagtgc agttgcacca tcttggctca 2100

ctgcaacctc cacctccagg gttcaagtga ttctcgttcc tcagcctccc gagtaactgg 2160

gattacaggc atgcgccacc acgcctggct aattttgtgt ttttagtaga gatggtgttt 2220

caccatgttg tccaggctgg tctcaaactc ctggcctcaa gtgatcctct ggcctcagct 2280

tcccagagta ctgagattac aggcatgagc cactgtgccc tgcctaggct aaacttaatt 2340

taacaacacc aaacaatctc cagcagacac caactgggta tcccataatt caattcgatt 2400

ttgattggat ctacctggag atggtgtcag atcccgctgg ttgagggttc agtcccacaa 2460

gactgccctc cacttcagat gccaatcaca cattgtaggt tgttacctct acttctgact 2520

gaccagctgg aaaccagaac tcccatgact gcctccttga ctttggttaa tttgctagga 2580

cagttcatat ttaccaatct attataaaag attaaaggct acagacgaat aactagatga 2640

aaagatgaat agggctatat gtagggggtt gtggtggtga cagtccatgc cctctccagg 2700

tgtatgccac cctcccagca cctccacaca ttcagcaaac aggaagctca tcgttcaaga 2760

gtttttatag agcttgatct ccagctcccc ttcaccttcc cagaggtgga tgggtggggg 2820

tggaagttcc aacacactaa tcttctcatc acttggtctt tctggtgact agcaccatcc 2880

tgaggctatc taggggccca aacctatgag tataacctca ttagcatata ctcaagggtt 2940

accaaagagg cttattatta ataataaaag acactcctat cactcaaaat tcaaaggatt 3000

ttaggaactt ctgacaggaa ctggggacaa agaccaaatg tgtttcatat tataccacac 3060

ttacctaggc cattcattaa ctcttcacct ggctgcaaac taaaattaca tgtagagttt 3120

gtaagaacaa aacaaacaca cacacagaca cacacacaca cacacacaca cacacacaca 3180

cacacacaca aaacaacaag gcaggcacgg tggctcacgc ctgtaagccc aacactttgg 3240

aaagtcaagg tgagcagatt acgaggagtt caagaccagc ctggctaaca tggtgaaacc 3300

ctgtctctac taagaataca aaaattagcc aggcatggtg gcacgtgcct ataatcccag 3360

ctactcagga ggctgaggca agagaatcgc ttgaacctcg gaggcagagg ttgcagtgag 3420

ccgagatcat gccattgcac tccagcctga gtgacagagt gagactccat ctcaaaaaca 3480

gcaacaacaa caacaacaac aacaacaaca acaacaacaa ctcaccagct tcccagactc 3540

ccatccaggc caattaaacc ataatctctc ggtatggagc ccaggtatag tgttattttt 3600

taaaaaacct ctagatagtt ttaatgtaca tccaagattg acagacccat tgagttcagt 3660

cataaagaac tctctctcca ttccatatca ttcaacagag ctattttgtg aggggctgct 3720

ctgctggctg atattacaag gtaatactat gtcaaagtca aatgaaatat agagttgaat 3780

ctctgaaatt aaaatgtttt atttgggagg aaagaattgc aactcagggc atacacacag 3840

accagctggt ctttggcatg tctgaagtac aaaaaggttt tataaaatgg gaaaatgtta 3900

cttatcgctc tgagaaaatt tcactggtgc tagtaaagtt ttgaggagct ggcaagtttt 3960

gattggcaaa tgatgacaat aaacagaact agtctcagag ttgtagcagg tcatttcagt 4020

agccattagt taaacctggt ttcagatgat agcaggcagt ttcagcttcc aggcttgcag 4080

aaaatcgcat ttttggaaca atgttttgtg ccctgagtgc ttctccctgg cttcttgact 4140

ctgttttagt tgagtaagac aaaaatgacc cagtttgatg accaactttc acagttcctt 4200

tggagcatgc cttagtttct ctttttagaa gagagcaagc aaagaggagg aatacaggaa 4260

gcttaattaa ccttattaaa tataattgtg caatgcaaat taaactgaaa agtcccaaca 4320

tcagtaatag agaagtagat cctacatact gacatggaag catacccata ctatattgct 4380

gaaacaaaaa ttagtttcta caaaatatgt gtaagaagcc actttaaaaa aacaaactag 4440

gtggggcatg gtggctcaca cctgtaatcc cagcattttt ggaggccaag gtgggaggac 4500

tgcgtgagct aagaagtttg agacaagcct gggcaacata gtgagaccca gtctctctct 4560

ctatttatgt atttatttat gtatgtacat atttattgat ttatttattt gagacggagt 4620

cttgccctgt cacccaggct ggagtgcaat gtcgtgatct cagctcactg caacctccac 4680

ctcccaggtt caaacgattc tcctgcctca gcctcctgag tagctggaat tacaggcacc 4740

cgccaccata cccagctaat tattttttgt atttttagta gagacgggat ttcatcatgt 4800

tggtcaggct ggtctcaaac tcctgacctc atgatccacc cgcctcggcc tcccaaagtg 4860

ctgggattac aggtgtgagc caccgcgccc ggccgagacc cagtctcttt aaaaaaaaat 4920

ggctaacatg gtgaaacccc atctctacta aaaatacaaa aattagctgg gtgtggtggt 4980

acatacctgt aatcccagct acttgggagg ctgaggcagg agaatcactt gaacccagga 5040

ggcggaggtt gcagtgagcg gagattgcat caccgcacac tagcctgggt gaca 5094

<210> 5

<211> 3197

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagatggagt cttgctaggt cacccaggct ggagtgcagt ggtgcgatct cggctcactg 60

caacctctgc ctcctgggtt cacaccattc tcctgcctca gcctcccaag tagctgggac 120

tacaggtgcc tgccaccacg ccaggttaat tttttgtatt tttagtagag acagggtttc 180

accgtgttag ccaggatggt ctcgatctcc tgacctcatg atccacccat cttggcctcc 240

caaagtgctg ggattacagg catgaaccac tgcgcccggc cgcatcgcta gtttttaaaa 300

actttttgta gagacagatt cttactatgt tgccaaggct ggtctcaaac tcctggcctc 360

aagagatcct ccagtcttcg gcctcccaaa aagatgggat tacaggcatg agccacctca 420

cctggcctct tttttttgta tattacctga tctcaggtat tctgctatag caacagaaag 480

acgaagacag aatccttagc tgtctgcaag tgtgcatgcc attttcatca tctgaagagt 540

cagcgagtgt cttaggtgga gtcttgcaaa agcaggccct gagccaaaga tttggatgca 600

aatgacttgt taagaaaggg ctcttcgaga ccgtgccatt gcactccagc ccgggcaaga 660

agagtgaaac tctgtttcaa aaaaaaaaaa gtggggggct cctaggaaaa gaacagtaaa 720

ggagtggggg atgaaggaca gggaatggga agaagccaag cgagagcatg atttccgaag 780

tcctacactc agcctgatca cacgggaagc tttagaacaa agaacacacc tcagagtttt 840

tcctgcctca acacaaagga gctgggcttt ggtgctcttc atcagcctgt ctttggctat 900

ccagggttgt ggaacgaggt gaaacataaa actccgaggt acttccggct ccctccagtg 960

tctgagggta atctgcagga ctgagggtaa ttgcagatgc tagctgttag cagcaaacta 1020

tgcaaaagct gaggactggc ttataaagcc agatctgggt gagtcatgtt tcctgtcaac 1080

atcctctgct gggcccataa cacatgcaac cccaaacttc cattacaagt tcaaagtttc 1140

taaggggata gcattacagt gtgtatgata ttggactcag acctgagttt gaatcctaat 1200

tccacaaaag aaattggaaa agagtcatat tgctgacttg accctttgtc accatatcca 1260

taaaatggga taattattcc tatatcataa atttacttat ttattcactt agtcatttgt 1320

taaataaata tggagtgtct actttgtgcc gggcactctt tttagggtgg ttctgagaag 1380

gggatggcaa tgagaagggc tctctaagat gcaagactcc aggcaactgc ttttacttcc 1440

agtggttctt tattttcaca gctcattaga gcaaattacc acagcaggga gatacaggtt 1500

gagtatccct tatccgaaaa gcctggcacc agaagtgttt taaattttgg attttttttt 1560

gatttttgga atatttgtta attatcagtt gagcatctct aatgtgaaaa tctaaaatcc 1620

aaaatgcccc agtgaccctt tcctttgagc atcatgttgg tgctcaaaaa gtttgagatg 1680

ttggagcatt taggatttca gatttttgga ttaaggatac tcattttgta caatggaaaa 1740

cttcttggca ctaatctgat gaaagaagaa actacgggag aatccctttc cctaaaaggc 1800

tttcagtaac aagatggccc tgactcattc tggacagttc ctccaaaggt aggcctacag 1860

gtagaactct tttctggcag cataggggtt ttccagaatg aaggtgtatt ttttttttac 1920

tgaggccagg aaaggtgaag agaatgaagg gttcctagtg acgatgcagc aaccaagtca 1980

tttatgtaat ggagccttat ctgacagatt tctaggatag aaactcagta tccaagctca 2040

ggcttggcag actgaggtgg gcaccgtccc caggattgca gtgtggatta gaggtttcaa 2100

ccccaatcat attcaacagc tccctttccc ccaacaaccc ctttttataa caattgtttt 2160

gtggtgattc ttttactaat atgatcatga aaattaagta atttagcttg tgaatgctct 2220

agcttggctg cactgctggt ataaagaagt gccagacact tgcaactgcc taggatctct 2280

gtgaaccagc ggcatggaag actgagaggc acgctggatt gatggctcag attcaagtat 2340

ggtattgcag gttctggtgt aattttctaa aatggcgaaa atctctaggt aaaactccaa 2400

aaataaaaaa ccaacataca tcttcctttg agttacctgg tagttatatt cctggaaaat 2460

tcagtgtata tgagcctgag gtagtagatg gtcaataaat actcaagaaa taaaaaaata 2520

aaaaaatata aaaatataaa acacatttct tacatgtcct tggtgcttgt aagcccagat 2580

tatatgcagg cttttttttg gacggagtct ctgttgccca ggctggagtg aagtggcgag 2640

atctgggctc actgcaacct ccacctcctg ggttcaagcg atcctcctgc ctcagcctcc 2700

tgagtagctg ggattacagg cacgcgccac caccctcagc tgatctttgt atttttagta 2760

gagaaggggt ttcaccatgt tggccaggct gatgtagaac tcctgacctc aagtgatccg 2820

cccacctcgg cctcccaaag tgttgggatt acaggcgtga gtcacagcgc ccggctgctt 2880

ttttatacat taagtgtgct tgcagaggac tgcgacctct ggccctcggc ctcttaataa 2940

atactccaag tgacttcatt ggaacaacca ctgagaatca cttatctaga gagtgggaag 3000

ttgctgatct catcactgca tggggtgggg tgagggaagc aaccctgccc ttcccctttc 3060

tcggaaagca gctggctttg aagaaagaga aaccaggacg ggaaagtcct gatttctaat 3120

ctgaaacagc gctttttgtc cagagaccgg tgacgagcga ccctgggctc gggttttgat 3180

tgggcagttc ggaaact 3197

<210> 6

<211> 1780

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtgcatgca ggggagcgca gagccctaag cccttctctg gggtccgccc gttttcctgc 60

tgggcttctc atttcctcac taggttctac ggtttgccga tctaaatcca gttcgtgtcc 120

tattgtattg tgtaacattt ttggcagctt acatctttgt ttattgagat atagttcaca 180

aaccatacaa ttaacatatt taagtgtaca tttcagtggg ttttagtata ttcacagggt 240

tgtgtaacca cctcaatttt aggacatttt cgtcactccc aaaagaaatt ttgtaccagt 300

tcggtgtcac tcccatttct cccaaacccc tagtcctagg aaaccaccaa tctttctgtc 360

tctatgggtt tgcctattag tatttcatag aaacagaaac atataatatg tggtcttttg 420

taactggctt ctgttttcaa aggtcgtcat attgtagcaa ggatcagttc ttcattccta 480

tttattgatg aatattactc tactgtatag atatactaag ttttgtttat ccatctatag 540

ttaatgggca tttggattgt ttccactctt cggctactga gaataaaatg tggttatcaa 600

tattcatgta taaatttttg gtgtggacat agattttcaa ttctcttgag tatatatgca 660

ggagtggaat tgctgggtga tatggtaact ctatgtttaa tcttttaagg aactactagg 720

ctgttctcca aagctgaatg taccattgtg tatgagagtt ccaatttttc tacatcctca 780

ccaatacttt taatcttttt ttattataac cattctagtg gatacgaaat ggtatctctt 840

tatggttttg atttgtattt ccctaatgac tatgcattat tttaaaatta taagctaatt 900

aatttttaca aagatttaca tgctgtgatt ttgacaactt aacttctagg tcacatgctc 960

aaaagtcaca gagcctagac ccaaaccaag cctttctatg gctaatgctc gaactccatc 1020

ctctctttcc tagaaggcaa tctgggataa atttttcaga actccatttg ttgcctctgt 1080

tagaaagaaa acactgggca agatgaacct gcaagtagag taaatggttt atttcttatt 1140

tgcttttagg agtatgtaag gagatttttt ggtctcccgt ttttacttta taaaaaatga 1200

gtgagtgggc caagcgctgt ggctcatgcc tgtgatccca gcactttggg agaccgaggc 1260

aggcagatca cctgaggtca ggagtttgat atcagcctga ccaacatggc aaaaccccat 1320

ctctgctaaa aatacaaaaa ttagccgggt gtagtgccat gcacctgtaa ttccagctat 1380

atgggaggct gaggcatgaa cattgcttga accccgggga gacggaggtt gcagtgagcc 1440

aagattgcac cactgcactc cagcctgggt gacagagtga ggctctttct caaaaaaaaa 1500

aaaaaggaaa gagaaagctc ttaattgaag tagtaaccct ttcttgggtg ttcaatgata 1560

ttgtgcttct cttcatgtaa gtgcccctta gattcctcct cctcactacc atcaggtcat 1620

agaataggga ggggcactca gtttcaacag cagtggcctc caaattttgt tcaaatgcat 1680

aactataaaa agttttttgt atacattctc tatatatagc tatacatttt atacatacac 1740

tactatcagt cttgatttag acattactaa agcttaattt 1780

<210> 7

<211> 19214

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acctactatg tgccagcccc tgttctaggg tggaaactaa gagaatgatg tacctagagg 60

gcgctggaag ctctaaagcc ctagcagtta ctgcttttac tattagtggt cgtttttttc 120

tcccccccgc cccccgacaa atcaacagaa caaagaaaat tacctaaaca gcaaggacat 180

agggaggaac ttcttggcac agaactttcc aaacactttt tcctgaaggg atacaagaag 240

caagaaaggt actctttcac taggaccttc tctgagctgt cctcaggatg cttttgggac 300

tatttttctt acccagagaa tggagaaacc ctgcagggaa ttcccaagct gtagttataa 360

acagaagttc tccttctgct aggtagcatt caaagatctt aatcttctgg gtttccgttt 420

tctcgaatga aaaatgcagg tccgagcagt taactggctg gggcaccatt agcaagtcac 480

ttagcatctc tggggccagt ctgcaaagcg agggggcagc cttaatgtgc ctccagcctg 540

aagtcctaga atgagcgccc ggtgtcccaa gctggggcgc gcaccccaga tcggagggcg 600

ccgatgtaca gacagcaaac tcacccagtc tagtgcatgc cttcttaaac atcacgagac 660

tctaagaaaa ggaaactgaa aacgggaaag tccctctctc taacctggca ctgcgtcgct 720

ggcttggaga caggtgacgg tccctgcggg ccttgtcctg attggctggg cacgcgttta 780

atataagtgg aggcgtcgcg ctggcgggca ttcctgaagc tgacagcatt cgggccgaga 840

tgtctcgctc cgtggcctta gctgtgctcg cgctactctc tctttctggc ctggaggcta 900

tccagcgtga gtctctccta ccctcccgct ctggtccttc ctctcccgct ctgcaccctc 960

tgtggccctc gctgtgctct ctcgctccgt gacttccctt ctccaagttc tccttggtgg 1020

cccgccgtgg ggctagtcca gggctggatc tcggggaagc ggcggggtgg cctgggagtg 1080

gggaaggggg tgcgcacccg ggacgcgcgc tacttgcccc tttcggcggg gagcagggga 1140

gacctttggc ctacggcgac gggagggtcg ggacaaagtt tagggcgtcg ataagcgtca 1200

gagcgccgag gttgggggag ggtttctctt ccgctctttc gcggggcctc tggctccccc 1260

agcgcagctg gagtggggga cgggtaggct cgtcccaaag gcgcggcgct gaggtttgtg 1320

aacgcgtgga ggggcgcttg gggtctgggg gaggcgtcgc ccgggtaagc ctgtctgctg 1380

cggctctgct tcccttagac tggagagctg tggacttcgt ctaggcgccc gctaagttcg 1440

catgtcctag cacctctggg tctatgtggg gccacaccgt ggggaggaaa cagcacgcga 1500

cgtttgtaga atgcttggct gtgatacaaa gcggtttcga ataattaact tatttgttcc 1560

catcacatgt cacttttaaa aaattataag aactacccgt tattgacatc tttctgtgtg 1620

ccaaggactt tatgtgcttt gcgtcattta attttgaaaa cagttatctt ccgccataga 1680

taactactat ggttatcttc tgcctctcac agatgaagaa actaaggcac cgagatttta 1740

agaaacttaa ttacacaggg gataaatggc agcaatcgag attgaagtca agcctaacca 1800

gggcttttgc gggagcgcat gccttttggc tgtaattcgt gcattttttt ttaagaaaaa 1860

cgcctgcctt ctgcgtgaga ttctccagag caaactgggc ggcatgggcc ctgtggtctt 1920

ttcgtacaga gggcttcctc tttggctctt tgcctggttg tttccaagat gtactgtgcc 1980

tcttactttc ggttttgaaa acatgagggg gttgggcgtg gtagcttacg cctgtaatcc 2040

cagcacttag ggaggccgag gcgggaggat ggcttgaggt ccgtagttga gaccagcctg 2100

gccaacatgg tgaagcctgg tctctacaaa aaataataac aaaaattagc cgggtgtggt 2160

ggctcgtgcc tgtggtccca gctgctccgg tggctgaggc gggaggatct cttgagctta 2220

ggcttttgag ctatcatggc gccagtgcac tccagcgtgg gcaacagagc gagaccctgt 2280

ctctcaaaaa agaaaaaaaa aaaaaaagaa agagaaaaga aaagaaagaa agaagtgaag 2340

gtttgtcagt caggggagct gtaaaaccat taataaagat aatccaagat ggttaccaag 2400

actgttgagg acgccagaga tcttgagcac tttctaagta cctggcaata cactaagcgc 2460

gctcaccttt tcctctggca aaacatgatc gaaagcagaa tgttttgatc atgagaaaat 2520

tgcatttaat ttgaatacaa tttatttaca acataaagga taatgtatat atcaccacca 2580

ttactggtat ttgctggtta tgttagatgt cattttaaaa aataacaatc tgatatttaa 2640

aaaaaaatct tattttgaaa atttccaaag taatacatgc catgcataga ccatttctgg 2700

aagataccac aagaaacatg taatgatgat tgcctctgaa ggtctatttt cctcctctga 2760

cctgtgtgtg ggttttgttt ttgttttact gtgggcataa attaattttt cagttaagtt 2820

ttggaagctt aaataactct ccaaaagtca taaagccagt aactggttga gcccaaattc 2880

aaacccagcc tgtctgatac ttgtcctctt cttagaaaag attacagtga tgctctcaca 2940

aaatcttgcc gccttccctc aaacagagag ttccaggcag gatgaatctg tgctctgatc 3000

cctgaggcat ttaatatgtt cttattatta gaagctcaga tgcaaagagc tctcttagct 3060

tttaatgtta tgaaaaaaat caggtcttca ttagattccc caatccacct cttgatgggg 3120

ctagtagcct ttccttaatg atagggtgtt tctagagaga tatatctggt caaggtggcc 3180

tggtactcct ccttctcccc acagcctccc agacaaggag gagtagctgc cttttagtga 3240

tcatgtaccc tgaatataag tgtatttaaa agaattttat acacatatat ttagtgtcaa 3300

tctgtatatt tagtagcact aacacttctc ttcattttca atgaaaaata tagagtttat 3360

aatattttct tcccacttcc ccatggatgg tctagtcatg cctctcattt tggaaagtac 3420

tgtttctgaa acattaggca atatattccc aacctggcta gtttacagca atcacctgtg 3480

gatgctaatt aaaacgcaaa tcccactgtc acatgcatta ctccatttga tcataatgga 3540

aagtatgttc tgtcccattt gccatagtcc tcacctatcc ctgttgtatt ttatcgggtc 3600

caactcaacc atttaaggta tttgccagct cttgtatgca tttaggtttt gtttctttgt 3660

tttttagctc atgaaattag gtacaaagtc agagaggggt ctggcatata aaacctcagc 3720

agaaataaag aggttttgtt gtttggtaag aacatacctt gggttggttg ggcacggtgg 3780

ctcgtgcctg taatcccaac actttgggag gccaaggcag gctgatcact tgaagttggg 3840

agttcaagac cagcctggcc aacatggtga aatcccgtct ctactgaaaa tacaaaaatt 3900

aaccaggcat ggtggtgtgt gcctgtagtc ccaggaatca cttgaaccca ggaggcggag 3960

gttgcagtga gctgagatct caccactgca cactgcactc cagcctgggc aatggaatga 4020

gattccatcc caaaaaataa aaaaataaaa aaataaagaa cataccttgg gttgatccac 4080

ttaggaacct cagataataa catctgccac gtatagagca attgctatgt cccaggcact 4140

ctactagaca cttcatacag tttagaaaat cagatgggtg tagatcaagg caggagcagg 4200

aaccaaaaag aaaggcataa acataagaaa aaaaatggaa ggggtggaaa cagagtacaa 4260

taacatgagt aatttgatgg gggctattat gaactgagaa atgaactttg aaaagtatct 4320

tggggccaaa tcatgtagac tcttgagtga tgtgttaagg aatgctatga gtgctgagag 4380

ggcatcagaa gtccttgaga gcctccagag aaaggctctt aaaaatgcag cgcaatctcc 4440

agtgacagaa gatactgcta gaaatctgct agaaaaaaaa caaaaaaggc atgtatagag 4500

gaattatgag ggaaagatac caagtcacgg tttattcttc aaaatggagg tggcttgttg 4560

ggaaggtgga agctcatttg gccagagtgg aaatggaatt gggagaaatc gatgaccaaa 4620

tgtaaacact tggtgcctga tatagcttga caccaagtta gccccaagtg aaataccctg 4680

gcaatattaa tgtgtctttt cccgatattc ctcaggtact ccaaagattc aggtttactc 4740

acgtcatcca gcagagaatg gaaagtcaaa tttcctgaat tgctatgtgt ctgggtttca 4800

tccatccgac attgaagttg acttactgaa gaatggagag agaattgaaa aagtggagca 4860

ttcagacttg tctttcagca aggactggtc tttctatctc ttgtactaca ctgaattcac 4920

ccccactgaa aaagatgagt atgcctgccg tgtgaaccat gtgactttgt cacagcccaa 4980

gatagttaag tggggtaagt cttacattct tttgtaagct gctgaaagtt gtgtatgagt 5040

agtcatatca taaagctgct ttgatataaa aaaggtctat ggccatacta ccctgaatga 5100

gtcccatccc atctgatata aacaatctgc atattgggat tgtcagggaa tgttcttaaa 5160

gatcagatta gtggcacctg ctgagatact gatgcacagc atggtttctg aaccagtagt 5220

ttccctgcag ttgagcaggg agcagcagca gcacttgcac aaatacatat acactcttaa 5280

cacttcttac ctactggctt cctctagctt ttgtggcagc ttcaggtata tttagcactg 5340

aacgaacatc tcaagaaggt ataggccttt gtttgtaagt cctgctgtcc tagcatccta 5400

taatcctgga cttctccagt actttctggc tggattggta tctgaggcta gtaggaaggg 5460

cttgttcctg ctgggtagct ctaaacaatg tattcatggg taggaacagc agcctattct 5520

gccagcctta tttctaacca ttttagacat ttgttagtac atggtatttt aaaagtaaaa 5580

cttaatgtct tccttttttt tctccactgt ctttttcata gatcgagaca tgtaagcagc 5640

atcatggagg taagtttttg accttgagaa aatgtttttg tttcactgtc ctgaggacta 5700

tttatagaca gctctaacat gataaccctc actatgtgga gaacattgac agagtaacat 5760

tttagcaggg aaagaagaat cctacagggt catgttccct tctcctgtgg agtggcatga 5820

agaaggtgta tggccccagg tatggccata ttactgaccc tctacagaga gggcaaagga 5880

actgccagta tggtattgca ggataaaggc aggtggttac ccacattacc tgcaaggctt 5940

tgatctttct tctgccattt ccacattgga catctctgct gaggagagaa aatgaaccac 6000

tcttttcctt tgtataatgt tgttttattc ttcagacaga agagaggagt tatacagctc 6060

tgcagacatc ccattcctgt atggggactg tgtttgcctc ttagaggttc ccaggccact 6120

agaggagata aagggaaaca gattgttata acttgatata atgatactat aatagatgta 6180

actacaagga gctccagaag caagagagag ggaggaactt ggacttctct gcatctttag 6240

ttggagtcca aaggcttttc aatgaaattc tactgcccag ggtacattga tgctgaaacc 6300

ccattcaaat ctcctgttat attctagaac agggaattga tttgggagag catcaggaag 6360

gtggatgatc tgcccagtca cactgttagt aaattgtaga gccaggacct gaactctaat 6420

atagtcatgt gttacttaat gacggggaca tgttctgaga aatgcttaca caaacctagg 6480

tgttgtagcc tactacacgc ataggctaca tggtatagcc tattgctcct agactacaaa 6540

cctgtacagc ctgttactgt actgaatact gtgggcagtt gtaacacaat ggtaagtatt 6600

tgtgtatcta aacatagaag ttgcagtaaa aatatgctat tttaatctta tgagaccact 6660

gtcatatata cagtccatca ttgaccaaaa catcatatca gcattttttc ttctaagatt 6720

ttgggagcac caaagggata cactaacagg atatactctt tataatgggt ttggagaact 6780

gtctgcagct acttctttta aaaaggtgat ctacacagta gaaattagac aagtttggta 6840

atgagatctg caatccaaat aaaataaatt cattgctaac ctttttcttt tcttttcagg 6900

tttgaagatg ccgcatttgg attggatgaa ttccaaattc tgcttgcttg ctttttaata 6960

ttgatatgct tatacactta cactttatgc acaaaatgta gggttataat aatgttaaca 7020

tggacatgat cttctttata attctacttt gagtgctgtc tccatgtttg atgtatctga 7080

gcaggttgct ccacaggtag ctctaggagg gctggcaact tagaggtggg gagcagagaa 7140

ttctcttatc caacatcaac atcttggtca gatttgaact cttcaatctc ttgcactcaa 7200

agcttgttaa gatagttaag cgtgcataag ttaacttcca atttacatac tctgcttaga 7260

atttggggga aaatttagaa atataattga caggattatt ggaaatttgt tataatgaat 7320

gaaacatttt gtcatataag attcatattt acttcttata catttgataa agtaaggcat 7380

ggttgtggtt aatctggttt atttttgttc cacaagttaa ataaatcata aaacttgatg 7440

tgttatctct tatatctcac tcccactatt acccctttat tttcaaacag ggaaacagtc 7500

ttcaagttcc acttggtaaa aaatgtgaac cccttgtata tagagtttgg ctcacagtgt 7560

aaagggcctc agtgattcac attttccaga ttaggaatct gatgctcaaa gaagttaaat 7620

ggcatagttg gggtgacaca gctgtctagt gggaggccag ccttctatat tttagccagc 7680

gttctttcct gcgggccagg tcatgaggag tatgcagact ctaagaggga gcaaaagtat 7740

ctgaaggatt taatatttta gcaaggaata gatatacaat catcccttgg tctccctggg 7800

ggattggttt caggacccct tcttggacac caaatctatg gatatttaag tcccttctat 7860

aaaatggtat agtatttgca tataacctat ccacatcctc ctgtatactt taaatcattt 7920

ctagattact tgtaatacct aatacaatgt aaatgctatg caaatagttg ttattgttta 7980

aggaataatg acaagaaaaa aaagtctgta catgctcagt aaagacacaa ccatcccttt 8040

ttttccccag tgtttttgat ccatggtttg ctgaatccac agatgtggag cccctggata 8100

cggaaggccc gctgtacttt gaatgacaaa taacagattt aaaattttca aggcatagtt 8160

ttatacctga tggccagctt tgtttatttg accaagaatc tgagttagct agttctaggt 8220

actgacagga taaataaaac acaacactgc tcccgatctt ctcagtttag cagagggaca 8280

gatatgcact caaataatta aaatatatcc tgataagaat atagcatagg tacgcgcgaa 8340

gaacttggca atcgaaattt tgttgttcag gctgggcgag gtgtctcatg tctgtaatcc 8400

cagcactttg ggaggccatg gtaggatgat cgcttgagcc caggagtccg agaccagcct 8460

gggcaacata acaagaccct gtctcaattc aaagaattga attaaaaaaa acaaaaaata 8520

atttttttaa aaaagaaatg ttgttgttca aggaacaaca acaaaaatct agggaggtgt 8580

tagagaagcc atttgcctga gctgagagta agttgctagt ggttctcttg attggtaggt 8640

ggggcctggg tttccaggca tggtagccag gaaggacagc cacatggcag gtttgggtaa 8700

ttccaaacag tggaggaagg gtgtctgggg gaagacttgt aggaactcag ctgaaaaaat 8760

tgggggatga tactctgaaa gaaaaacaaa gttttaaaat ttctactctt acacttaaca 8820

cataatgctt ctgtgaccgg atatttaggg gttttccccc cacactctgt taggagaaaa 8880

attttagaca gattaaattt aacagagttt aactgagcaa aaatgattct cgaaccaggc 8940

agctgccgga gccagaatag gttcaaaatg actctggggg tgccacatgg ttggatgaca 9000

tttagggaca gaaaaaggaa agtgatgtgc agaaaatgga agtcaggggc agaagcagcc 9060

agattggttg cagttcagca tttgcctcat ttaaacaggg tttgaagagt tggccacctg 9120

tgattggctg agactctgtg gtataagagt aagttacagt ctgtttacac atccagttag 9180

gttacagttc actatgcaga gagaaatctt tagcctgaac ttacacaggg aggcagtttt 9240

atttatttat ttaatttttt ttttttgaga caaggtctca ctctgtcacc cgggctggag 9300

ttcagtggta tgatcatggc ttattgcagc ctcgacttcc tggcctcaag caatccttcc 9360

gcctcaggct ctagagtagc tgggactaca ggcacatgtc agcatgcctg gctaattttg 9420

ttttttaatt tttagtagag atgaactcct ggccttgcac aattctttcg cctcagcctc 9480

cgaaaatgct gggattacag gtgtgagcca ctgtgcccag ctaaggcaac tttaggctaa 9540

accttttttt gagacagagt ttctctcttg ttgtctaggc tggagtgcag ttgcaccatc 9600

ttggctcact gcaacctcca cctccagggt tcaagtgatt ctcgttcctc agcctcccga 9660

gtaactggga ttacaggcat gcgccaccac gcctggctaa ttttgtgttt ttagtagaga 9720

tggtgtttca ccatgttgtc caggctggtc tcaaactcct ggcctcaagt gatcctctgg 9780

cctcagcttc ccagagtact gagattacag gcatgagcca ctgtgccctg cctaggctaa 9840

acttaattta acaacaccaa acaatctcca gcagacacca actgggtatc ccataattca 9900

attcgatttt gattggatct acctggagat ggtgtcagat cccgctggtt gagggttcag 9960

tcccacaaga ctgccctcca cttcagatgc caatcacaca ttgtaggttg ttacctctac 10020

ttctgactga ccagctggaa accagaactc ccatgactgc ctccttgact ttggttaatt 10080

tgctaggaca gttcatattt accaatctat tataaaagat taaaggctac agacgaataa 10140

ctagatgaaa agatgaatag ggctatatgt agggggttgt ggtggtgaca gtccatgccc 10200

tctccaggtg tatgccaccc tcccagcacc tccacacatt cagcaaacag gaagctcatc 10260

gttcaagagt ttttatagag cttgatctcc agctcccctt caccttccca gaggtggatg 10320

ggtgggggtg gaagttccaa cacactaatc ttctcatcac ttggtctttc tggtgactag 10380

caccatcctg aggctatcta ggggcccaaa cctatgagta taacctcatt agcatatact 10440

caagggttac caaagaggct tattattaat aataaaagac actcctatca ctcaaaattc 10500

aaaggatttt aggaacttct gacaggaact ggggacaaag accaaatgtg tttcatatta 10560

taccacactt acctaggcca ttcattaact cttcacctgg ctgcaaacta aaattacatg 10620

tagagtttgt aagaacaaaa caaacacaca cacagacaca cacacacaca cacacacaca 10680

cacacacaca cacacacaaa acaacaaggc aggcacggtg gctcacgcct gtaagcccaa 10740

cactttggaa agtcaaggtg agcagattac gaggagttca agaccagcct ggctaacatg 10800

gtgaaaccct gtctctacta agaatacaaa aattagccag gcatggtggc acgtgcctat 10860

aatcccagct actcaggagg ctgaggcaag agaatcgctt gaacctcgga ggcagaggtt 10920

gcagtgagcc gagatcatgc cattgcactc cagcctgagt gacagagtga gactccatct 10980

caaaaacagc aacaacaaca acaacaacaa caacaacaac aacaacaact caccagcttc 11040

ccagactccc atccaggcca attaaaccat aatctctcgg tatggagccc aggtatagtg 11100

ttatttttta aaaaacctct agatagtttt aatgtacatc caagattgac agacccattg 11160

agttcagtca taaagaactc tctctccatt ccatatcatt caacagagct attttgtgag 11220

gggctgctct gctggctgat attacaaggt aatactatgt caaagtcaaa tgaaatatag 11280

agttgaatct ctgaaattaa aatgttttat ttgggaggaa agaattgcaa ctcagggcat 11340

acacacagac cagctggtct ttggcatgtc tgaagtacaa aaaggtttta taaaatggga 11400

aaatgttact tatcgctctg agaaaatttc actggtgcta gtaaagtttt gaggagctgg 11460

caagttttga ttggcaaatg atgacaataa acagaactag tctcagagtt gtagcaggtc 11520

atttcagtag ccattagtta aacctggttt cagatgatag caggcagttt cagcttccag 11580

gcttgcagaa aatcgcattt ttggaacaat gttttgtgcc ctgagtgctt ctccctggct 11640

tcttgactct gttttagttg agtaagacaa aaatgaccca gtttgatgac caactttcac 11700

agttcctttg gagcatgcct tagtttctct ttttagaaga gagcaagcaa agaggaggaa 11760

tacaggaagc ttaattaacc ttattaaata taattgtgca atgcaaatta aactgaaaag 11820

tcccaacatc agtaatagag aagtagatcc tacatactga catggaagca tacccatact 11880

atattgctga aacaaaaatt agtttctaca aaatatgtgt aagaagccac tttaaaaaaa 11940

caaactaggt ggggcatggt ggctcacacc tgtaatccca gcatttttgg aggccaaggt 12000

gggaggactg cgtgagctaa gaagtttgag acaagcctgg gcaacatagt gagacccagt 12060

ctctctctct atttatgtat ttatttatgt atgtacatat ttattgattt atttatttga 12120

gacggagtct tgccctgtca cccaggctgg agtgcaatgt cgtgatctca gctcactgca 12180

acctccacct cccaggttca aacgattctc ctgcctcagc ctcctgagta gctggaatta 12240

caggcacccg ccaccatacc cagctaatta ttttttgtat ttttagtaga gacgggattt 12300

catcatgttg gtcaggctgg tctcaaactc ctgacctcat gatccacccg cctcggcctc 12360

ccaaagtgct gggattacag gtgtgagcca ccgcgcccgg ccgagaccca gtctctttaa 12420

aaaaaaatgg ctaacatggt gaaaccccat ctctactaaa aatacaaaaa ttagctgggt 12480

gtggtggtac atacctgtaa tcccagctac ttgggaggct gaggcaggag aatcacttga 12540

acccaggagg cggaggttgc agtgagcgga gattgcatca ccgcacacta gcctgggtga 12600

cagagcaaga ctttgtcaca cacacacaca caaaaaagtg gacgcttgca tcacttgagc 12660

ctgggaggtt gaggctgcag tgagcagaga tcaggacact acactccagc ctgggtgatg 12720

aagcaagtcc ctcaaaaagg aaaaaaaaaa aaagtagacc cttgcacccc aaatctgtat 12780

tgttcaaagg ttaactgtat cctcctttaa aaaaggggtt actgttaaat aataattttt 12840

atatcttact ttttttcatg atccctaagg aaaacatgtc acaaatggat atgtctttct 12900

agttttgtca acaaccactt tcacggtatt tttgggctgt tgctttttac ttgtcatttt 12960

tgtgttgtaa acccttgaag caaaactcaa ggtcttttct ttttttcttt tcttttcttt 13020

tctgagacag ggtattaccc tgtcacccag gcttgtgtgc agaggcacaa tcttggctca 13080

ctgcagcctc agcctcttgg actcaagcaa tcctcccatc tcagcctccc aagtagctga 13140

gaccacaggt gtgcaccacc cattcctggc taattttttt tgtgtatttt tttgtagaga 13200

tgggggtctc actatgtttc cctggctggt cttgaactct tgggttcaag cgatcctcca 13260

gtctcgcctc tcaaagtgct gggattacag agccaccaca ctcagccatt tcctataata 13320

aaaaaatatt ttgtctgtga tgtagccatc aaagttttag ggggaaaaat tggcattgtc 13380

tttccctgga gtctagacct ataggaaaaa aattgaaggc taatatttgg gttctagata 13440

acgcaaaata aataataaaa catctgtgat taccttgatt gaaaagtttg tgcatagaaa 13500

ttattctggt actttggaga gctacaagaa ggcctaacag aaaaaaaaac tgataaatac 13560

aattaaaata acagtcgagc atatattaaa gcacttacta tgcactaaac tctatatatc 13620

tcctgtcatt agttggatgc aattattaga catctttggt cctccacaac ataggtgatt 13680

aaaaagccta agaaactgaa gcgttatcta tgtctttggt aagacacaca ggcccaagtg 13740

aagcctgcaa tgatgtgcag gcttcccctc ccccacggac cacatggacc acttcctctt 13800

tttttttttt tttttttttc tttagacaga gtctcactgt atcgcctagg ctggagtgca 13860

gtggcgcaat ctcggctcac tgcaacctct gcttcctggg ttcaagtgat tctcctgcct 13920

cagcctcccg agtagctggg aatacaggca tgcaccacca tgcctgacta atttttttat 13980

gtgtgtgttt ttggtagaga tggggtttca ccatgctggc caggctggtc tcaaactcct 14040

gacctcgtga tctgctcgcc ttggcctccc aaagtgctgg gattacaggc acttcctctt 14100

tttactgttt tcacttagaa aaactgagag gacttttgct gccactgaat gacacagcat 14160

agttgaagtg gttttctcgt catttattat ggctcgaatg tttgtccctt ctgaaattca 14220

tgttgaaact taatcctggc tgggcacagt ggctcacact tctaatccca gcattttggg 14280

aggctgagac tggaggatcg catgagccca ggagtttgag accagcctgg gcaacatagt 14340

gagtcctcct ctctacaaaa tcaaacaaac aaaaattagc tgggtgtggt agcacacacc 14400

tgtggtccca gctacttgga aggcggaggc aggaggattg tttgaaccca ggaggagttt 14460

aagagcagtc tgggcaatat agtgagacct catatctgtc tatctatgta tctatgtatc 14520

tatgtatcta tgtatctatc tatctatcta tctatctaaa ataaattaaa ttaaaaaata 14580

aaaaataatg aggcaaagaa aagaaagtta atcctcaata taacagtatt aagaagtagg 14640

gcatttaaga ggtgactggg tcatgagggc tccttcatga atggattaat ggattaatgg 14700

gttatcctga gaatgggtct gttataaaag ccagtttcgc tctttctctt gtgcccctct 14760

aaatctttat ctttagccat gttatgatgc ctctctgata tctttaccca tgttatgatg 14820

cggaacaaag ccctcaccca aagcaaacca gatacagccc ctcaatgttg aacttctcag 14880

cctcttccat acatttttct tcctttcttt tttttgagat aggatcttgc tctgccattg 14940

tgccattgca gcctcaaact cttgggctca agtgatcctg ctgcttcagc atcctgagta 15000

gcggggctta cagatgcaca cccccatgca tcactaattt tttttttttt ttttgagatg 15060

gagtcttgct aggtcaccca ggctggagtg cagtggtgcg atctcggctc actgcaacct 15120

ctgcctcctg ggttcacacc attctcctgc ctcagcctcc caagtagctg ggactacagg 15180

tgcctgccac cacgccaggt taattttttg tatttttagt agagacaggg tttcaccgtg 15240

ttagccagga tggtctcgat ctcctgacct catgatccac ccatcttggc ctcccaaagt 15300

gctgggatta caggcatgaa ccactgcgcc cggccgcatc gctagttttt aaaaactttt 15360

tgtagagaca gattcttact atgttgccaa ggctggtctc aaactcctgg cctcaagaga 15420

tcctccagtc ttcggcctcc caaaaagatg ggattacagg catgagccac ctcacctggc 15480

ctcttttttt tgtatattac ctgatctcag gtattctgct atagcaacag aaagacgaag 15540

acagaatcct tagctgtctg caagtgtgca tgccattttc atcatctgaa gagtcagcga 15600

gtgtcttagg tggagtcttg caaaagcagg ccctgagcca aagatttgga tgcaaatgac 15660

ttgttaagaa agggctcttc gagaccgtgc cattgcactc cagcccgggc aagaagagtg 15720

aaactctgtt tcaaaaaaaa aaaagtgggg ggctcctagg aaaagaacag taaaggagtg 15780

ggggatgaag gacagggaat gggaagaagc caagcgagag catgatttcc gaagtcctac 15840

actcagcctg atcacacggg aagctttaga acaaagaaca cacctcagag tttttcctgc 15900

ctcaacacaa aggagctggg ctttggtgct cttcatcagc ctgtctttgg ctatccaggg 15960

ttgtggaacg aggtgaaaca taaaactccg aggtacttcc ggctccctcc agtgtctgag 16020

ggtaatctgc aggactgagg gtaattgcag atgctagctg ttagcagcaa actatgcaaa 16080

agctgaggac tggcttataa agccagatct gggtgagtca tgtttcctgt caacatcctc 16140

tgctgggccc ataacacatg caaccccaaa cttccattac aagttcaaag tttctaaggg 16200

gatagcatta cagtgtgtat gatattggac tcagacctga gtttgaatcc taattccaca 16260

aaagaaattg gaaaagagtc atattgctga cttgaccctt tgtcaccata tccataaaat 16320

gggataatta ttcctatatc ataaatttac ttatttattc acttagtcat ttgttaaata 16380

aatatggagt gtctactttg tgccgggcac tctttttagg gtggttctga gaaggggatg 16440

gcaatgagaa gggctctcta agatgcaaga ctccaggcaa ctgcttttac ttccagtggt 16500

tctttatttt cacagctcat tagagcaaat taccacagca gggagataca ggttgagtat 16560

cccttatccg aaaagcctgg caccagaagt gttttaaatt ttggattttt ttttgatttt 16620

tggaatattt gttaattatc agttgagcat ctctaatgtg aaaatctaaa atccaaaatg 16680

ccccagtgac cctttccttt gagcatcatg ttggtgctca aaaagtttga gatgttggag 16740

catttaggat ttcagatttt tggattaagg atactcattt tgtacaatgg aaaacttctt 16800

ggcactaatc tgatgaaaga agaaactacg ggagaatccc tttccctaaa aggctttcag 16860

taacaagatg gccctgactc attctggaca gttcctccaa aggtaggcct acaggtagaa 16920

ctcttttctg gcagcatagg ggttttccag aatgaaggtg tatttttttt ttactgaggc 16980

caggaaaggt gaagagaatg aagggttcct agtgacgatg cagcaaccaa gtcatttatg 17040

taatggagcc ttatctgaca gatttctagg atagaaactc agtatccaag ctcaggcttg 17100

gcagactgag gtgggcaccg tccccaggat tgcagtgtgg attagaggtt tcaaccccaa 17160

tcatattcaa cagctccctt tcccccaaca accccttttt ataacaattg ttttgtggtg 17220

attcttttac taatatgatc atgaaaatta agtaatttag cttgtgaatg ctctagcttg 17280

gctgcactgc tggtataaag aagtgccaga cacttgcaac tgcctaggat ctctgtgaac 17340

cagcggcatg gaagactgag aggcacgctg gattgatggc tcagattcaa gtatggtatt 17400

gcaggttctg gtgtaatttt ctaaaatggc gaaaatctct aggtaaaact ccaaaaataa 17460

aaaaccaaca tacatcttcc tttgagttac ctggtagtta tattcctgga aaattcagtg 17520

tatatgagcc tgaggtagta gatggtcaat aaatactcaa gaaataaaaa aataaaaaaa 17580

tataaaaata taaaacacat ttcttacatg tccttggtgc ttgtaagccc agattatatg 17640

caggcttttt tttggacgga gtctctgttg cccaggctgg agtgaagtgg cgagatctgg 17700

gctcactgca acctccacct cctgggttca agcgatcctc ctgcctcagc ctcctgagta 17760

gctgggatta caggcacgcg ccaccaccct cagctgatct ttgtattttt agtagagaag 17820

gggtttcacc atgttggcca ggctgatgta gaactcctga cctcaagtga tccgcccacc 17880

tcggcctccc aaagtgttgg gattacaggc gtgagtcaca gcgcccggct gcttttttat 17940

acattaagtg tgcttgcaga ggactgcgac ctctggccct cggcctctta ataaatactc 18000

caagtgactt cattggaaca accactgaga atcacttatc tagagagtgg gaagttgctg 18060

atctcatcac tgcatggggt ggggtgaggg aagcaaccct gcccttcccc tttctcggaa 18120

agcagctggc tttgaagaaa gagaaaccag gacgggaaag tcctgatttc taatctgaaa 18180

cagcgctttt tgtccagaga ccggtgacga gcgaccctgg gctcgggttt tgattgggca 18240

gttcggaaac tgtaaaagcg aattaaaagg gtgacaagct agtgttttag cctatccagt 18300

tccgggagtt tgcacgcaga cgctctgctt cgtgaccttg gctctgctct gtgggcgccg 18360

cccccagcct gggcgcgtcc atcgtcgagt accttctcct ctgcctcccc ctccctctgc 18420

ttctatctct ctccaattgc cctccctggc ctgcggccgc ccggtcctcc ttcccagccc 18480

agtgcagcca ggcacccggg ttcggcttgc tcaggtctct gtccgggact gggaagccac 18540

ggagggccgg gaaagtggca cactcctgga gctcaagctt cctactctct catcgctgaa 18600

taactaccgg gcaggactgg gtggaacaga cagcatattt aggtcattga tgggctgctg 18660

ggtggatgga gctggaaagt gatgggcact gctttctggg gctgcccagg ttccttccga 18720

gggctcgctt ttcctgggca gagcgggaaa gaggaggggc aggccggtcg cgaaacgcaa 18780

atagtcgaga atagcgatcc ggggagaagc aggtgtctgt ggggcccaag agaagtacca 18840

tctcggtaag taggccggtg catgcagggg agcgcagagc cctaagccct tctctggggt 18900

ccgcccgttt tcctgctggg cttctcattt cctcactagg ttctacggtt tgccgatcta 18960

aatccagttc gtgtcctatt gtattgtgta acatttttgg cagcttacat ctttgtttat 19020

tgagatatag ttcacaaacc atacaattaa catatttaag tgtacatttc agtgggtttt 19080

agtatattca cagggttgtg taaccacctc aattttagga cattttcgtc actcccaaaa 19140

gaaattttgt accagttcgg tgtcactccc atttctccca aacccctagt cctaggaaac 19200

caccaatctt tctg 19214

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tccttggagc tgtgatcact 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aagggcagga acaactcttg 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cctagcagtt gtggtcatcg g 21

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctccctcctt ttccacctga ac 22

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tcctggttgt cctagctgtc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caggctttac aagtgatgag 20

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gagtgctgtc tccatgtttg atg 23

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aagttgccag ccctcctaga 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgcttggctg tgatacaaag 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ctttgtatca cagccaagca 20

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gagggcctat ttcccatgat tcc 23

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

attctccaga gcaaactggg 20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cccagtttgc tctggagaat 20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tagtttacag caatcacctg 20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

caggtgattg ctgtaaacta 20

Claims (6)

1. A human-derived stem cell, wherein a partial sequence of an enhancer near a B2M gene is knocked down, and a region of the enhancer is located near the B2M gene; the enhancer region is positioned in chr15:45002906-45022119, and the sequence of the chr15:45002906-45022119 is shown as SEQ ID NO:7 is shown in the specification; the enhancer region has 6 active sequences which are respectively E1-E6, and the sequences of E1-E6 are respectively shown in SEQ ID NO: 1-SEQ ID NO:6 is shown in the specification; the enhancer can form a super enhancer under IFN-gamma stimulation; the core enhancer fragment of the enhancer region is E2, and the knockdown sequence is located at chr15:45,004,417-45,004,436.

2. The human stem cell of claim 1, wherein the stem cell is an MSC or an ADSC cell.

3. A method for producing human stem cells, characterized by knocking down a partial sequence of an enhancer near the B2M gene, comprising the steps of:

(1) Knocking down a partial fragment on an enhancer sequence by CRISPR, wherein the enhancer region is positioned at chr15:45002906-45022119;

(2) Sorting out the cells with partial sequence segment of the knocked-down enhancer;

the enhancer can form a super enhancer under IFN-gamma stimulation; the knocked down sequence is located at chr15:45,004,417-45,004,436.

4. The preparation method of claim 3, wherein the CRISPR guide nucleotide sequences are sgRNA F and sgRNA R, respectively, and the sequences of the sgRNA F and the sgRNA R are shown in SEQ ID NO:16 and SEQ ID NO: shown at 17.

5. The method according to any one of claims 3 to 4, wherein the cells in step (2) are human mesenchymal stem cells or human adipose-derived stem cells.

6. Use of human stem cells for the preparation of stem cells capable of suppressing alloimmune rejection, said use being non-disease therapeutic use; partial sequence knockdown of an enhancer region near the B2M gene of the stem cell; the enhancer can form a super enhancer under IFN-gamma stimulation; the enhancer region is positioned in chr15:45002906-45022119, and the chr15:45002906-45022119 has the nucleotide sequence shown in SEQ ID NO: 7; the enhancer region has 6 active sequences respectively from E1 to E6, and the E1 to E6 have the sequences shown in SEQ ID NO: 1-SEQ ID NO: 6; the core enhancer fragment of the enhancer region is E2, and the knockdown sequence is located at chr15:45,004,417-45,004,436.

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