CN113195724A - Low-immunogenicity engineered human mesenchymal stromal cells, preparation method and kit - Google Patents

Abstract

Methods and kits for preparing human mesenchymal stromal cells of reduced immunogenicity comprising one or more plasmids capable of mutating both alleles of the β 2-microglobulin gene in human pluripotent stem cells, which human mesenchymal stromal cells can be differentiated into genetically modified mesenchymal stromal cells that do not express β 2-microglobulin and that express reduced or do not express HLA-class I molecules located on the cell surface.

Description

Low-immunogenicity engineered human mesenchymal stromal cells, preparation method and kit

Cross Reference to Related Applications

This application claims priority from singapore patent application No. 10201808423W filed on 26.9.2018, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to a method and kit for preparing human mesenchymal stromal cells, in particular to human mesenchymal stromal cells with low immunogenicity and genetically modified human mesenchymal stromal cells.

Background

Mesenchymal stem cells have great potential in regenerative medicine applications due to their plasticity, immunoregulatory, and anti-inflammatory properties. It has many advantages in clinical applications, including high plasticity, ability to modulate inflammation, and ability to promote cell proliferation, cell differentiation, tissue repair through immunomodulation and immunosuppression.

Naturally occurring human Mesenchymal Stem Cells (MSCs) are a rare subset of non-hematopoietic stem cells, located around the vessels and trabeculae of the Bone Marrow (BM), accounting for 0.01-0.001% of all BM cells, rare and difficult to obtain. In recent years, technology has been developed to prepare Mesenchymal Stromal Cells (MSCs) exhibiting stem cell characteristics and immunoregulatory functions using human iPS cells. These iPS cell-derived MSCs and adult primary mesenchymal stromal cells have equal efficacy in treating various diseases ranging from tissue injury to immune disorders.

Although MSCs have been reported to achieve immune-sparing without triggering an damaging immune response, suitable for allogeneic transplantation, autoimmunity remains a worrying problem in awakening host rejection, such as humoral and cellular immune responses in vivo. Generally, both naturally occurring MSCs and iPS cell-derived MSCs are at risk of inducing host rejection by anti-graft antibodies or cellular immune memory against infused cells. Most cell types express HLA-I genes (HLA-A, HLA-B, and HLA-C) and function to present "non-self" antigen processed polypeptides to cytolytic CD8+ T cells to modulate immune rejection.

Disclosure of Invention

It is an object of the present invention to alleviate some of the above difficulties by preparing mesenchymal stromal cells of low immunogenicity derived from B2M knockout human pluripotent stem cells using a reliable, unlimited and standardized starting cell source, such as Induced Pluripotent Stem Cells (iPSCs), in human pluripotent stem cells.

Accordingly, one aspect of the present invention includes a method for preparing human mesenchymal stromal cells with reduced immunogenicity, the method comprising:

(a) mutating two alleles of a human pluripotent stem cell beta 2-microglobulin gene;

(b) differentiating the mutated human pluripotent stem cell into a derived human mesenchymal stromal cell having a biallelic mutation on a beta 2-microglobulin gene;

wherein the derived human mesenchymal stromal cells do not express beta 2-microglobulin and express attenuated or do not express HLA class I molecules located on the cell surface.

Another aspect of the invention includes genetically modified human mesenchymal stromal cells, including cells that do not express β 2-microglobulin and express attenuated or do not express HLA class I molecules located on the cell surface.

In another aspect, the present invention also includes a kit for preparing mesenchymal stromal cells with reduced immunogenicity, the kit comprising:

(a) one or more plasmids capable of mutating both alleles of the human pluripotent stem cell β 2-microglobulin gene;

(b) a culture medium of mesenchymal stromal cells, which is used for differentiating human pluripotent stem cells into the mesenchymal stromal cells.

Other aspects and features of the disclosed embodiments will become apparent to those skilled in the art upon review of the following detailed description of the disclosed embodiments taken in conjunction with the accompanying drawings.

Drawings

The technical solution of the invention is described, by way of example only, with reference to the accompanying drawings.

FIG. 1: and (3) preparing the hP-iPS cell with the B2M gene knockout by using CRISPR/Cas9 and a double-color selection method. (A) Schematic of CRISPR/Cas9 system targeting B2M first exon (EX1) with two selective donor templates for homologous recombination. The system is used for genetic modification of hP-iPS cells by electroporation. The arrows show the PCR primer binding sites used for genotyping. The restriction enzyme site HindIII is shown as H. (B) Representative diagram of B2M bi-allelic knock-out monoclonal hP-iPS cells. The scale bar is 200 μm and the exposure time for fluorescence is 1 second. (C) B2M single allele and double allele knockout hP-iPS single cell clones were analyzed by PCR for Wild Type (WT) and Homologous Direct Recombination (HDR) alleles of the B2M gene. WT hP-iPS cells were included as controls. (D) B2M single-and double-allele knock-out hP-iPS single cell clones were analyzed for B2M expression by western blot. Prior to analysis, the sample cells were treated with IFN-. gamma.for 48 hours. WT clones were control and β -actin was detected as an expression product of housekeeping gene. (E) Typical flow cytogram of hP-iPS single cell clone surface B2M and HLA-A, B, C expression of WT and B2M single allele and double allele knockout.

FIG. 2: selection of 18 single-allele clones for analysis (a) western blot analysis of EGFP expression in B2M single-allele knock-out hP-iPS single-cell clones. (B) The expression of B2M in B2M single allele knock-out hP-iPS single cell clones was analyzed by Western blotting. (C) Sequencing analysis of the target region.

FIG. 3: (A) southern blot hybridization of HindIII-cleaved EGFP probe with gDNA. (B) RT-PCR analysis of expression of pluripotent markers Oct4, Sox2 and Nanog of hP-iPS single cell clones knocked out by WT and B2M single allele and double allele. Expression of β -actin was included as a control for housekeeping genes. (C) Representative images of Embryoid Bodies (EBs) derived from WT, B2M single and double allele knock-out, hP-iPS single cell clones. The scale bar is 200 μm and the exposure time for fluorescence is 1 second. (D) RT-PCR analysis of marker expression in three germ layers of EBs derived from WT, B2M single and double allele knock-out hP-iPS cells. Pax6 is an ectodermal marker; MHC- α is a mesodermal marker and AFP is an endodermal marker. Expression of β -actin was included as a control for housekeeping genes.

FIG. 4: hP-iPS cells for preparing B2M gene knockout were selected by CRISPR/Cas9 and puromycin. (A) Schematic of CRISPR/Cas9 system targeting B2M first exon (EX 1). The system is used for genetic modification of hP-iPS cells by electroporation. The arrows show the PCR primer binding sites used for genotyping. (B) Typical flow cytogram of hP-iPS single cell clone surface B2M expression of B2M gene knock-out (B2 MKO).

FIG. 5: hP-iPS cells with B2M gene knockout are prepared by one step of CRISPR/Cas9 technology. (A) Typical flow cytograms of WT, B2M knock-out (B2MKO) #3 and #8hP-iPS single cell clone surface B2M and HLA-A, B, C expression. (B) Sanger sequencing analysis was performed on the B2M first exon of B2MKO #3 and #8hP-iPS single cell clones. The CRISPR target sequence is shown orange and the target site of the B2M gene is shown green. The insertion mutation was marked in red, and the deletion mutation was marked as "-". The genotypes of B2MKO #3 and #8hP-iPS single cell clones were summarized. (C) Chromosome karyotyping of clone #8 from the B2M knock-out.

FIG. 6: the B2M gene knockout hP-iPS cell pluripotency. (A) RT-PCR analysis of expression of the pluripotency markers Oct4, Sox2 and Nanog in WT, B2M single-allele and double-allele knock-out hP-iPS single-cell clones. Expression of β -actin was included as a control for housekeeping genes. (B) Representative plots of Embryoid Bodies (EBs) in hP-iPS single cell clones derived from WT, B2M single and double allele knockouts. The scale bar is 200 μm and the exposure time for fluorescence is 1 second. (C) RT-PCR analysis of marker expression in three germ layers of EBs derived from WT, B2M single and double allele knock-out hP-iPS cells. Pax6 is an ectodermal marker; MHC- α is a mesodermal marker and AFP is an endodermal marker. Expression of β -actin was included as a control for housekeeping genes.

FIG. 7: preparing mesenchymal stromal cells from hP-iPS cells with the B2M gene knocked out. (A) Representative images of WT and B2MKO hP-iPSC-derived mesenchymal stromal cells (iMSCs). The scale bar is 200 μm. (B) Typical flow cytograms of WT and B2MKO iMSCs surface B2M and HLA-A, B, C expression. (C) WT and B2MKO iMSCs were phenotyped with cell surface markers. Expression of MSC negative markers (CD14, CD24, CD34, CD45 and HLA-DR) and MSC positive markers (CD29, CD44, CD73, CD90, CD105 and CD166) are shown with typical flow cytograms.

FIG. 8: pluripotency of hP-iPS cell-derived mesenchymal stromal cells knocked out by B2M gene. Expression of specific markers for WT, B2MKO iMSCs and differentiated cells thereof was examined by RT-PCR. (A) Lipoprotein lipase (LPL) is used to indicate adipogenesis; (C) bone alkaline phosphatase (ALP) is used to indicate bone formation and (E) type ii collagen fiber α 1(COL2a) is used to indicate cartilage formation. B2MKO ismscs (B) adipogenesis, (D) bone formation and (F) cartilage formation. The lipid fraction of adipocytes is stained red with oil red O; calcium deposition by osteocytes was stained red with alizarin red S and acidic polysaccharides by chondrocytes were stained blue with Alcian blue 8GX (Alcian blue 8 GX). The scale bar for all images was 80 μm.

FIG. 9: low immunogenicity of hP-iPS cell-derived mesenchymal stromal cells knocked out by B2M gene. (A, B) immunogenicity of WT and B2MKO iMSCs when challenged with (iMSC-primed) Peripheral Blood Mononuclear Cells (PBMCs) activated by iMSC. As shown in the flow cytogram, phenotyping of PBMCs activated by iMSCs was shown to express CD3, CD56, and CD 8. DELFIA EuTDA cytotoxicity assay (2 hour release of Eu ligand) was used to test the immunogenicity of WT and B2MKO iMSCs as target cells when challenged with PBMCs activated with iMSCs. (C) Sensitivity of WT and B2MKO iMSC to NK lysis. In the DELFIA EuTDA cytotoxicity assay (Eu ligand release at 2 hours), primary NK cells target WT and B2MKO iMSCs as effector cells. The cytotoxicity assay was performed as three independent assays from three separate donors. As shown, the percent lysis of target cells (mean. + -. standard deviation of triplicate samples) for different E: T ratios in a representative experiment. P < 0.001.

FIG. 10: b2MKO iMSCs had higher immunosuppressive properties than WT and PBMC iMSCs. (B) OKT-3 induced hPBMC proliferation in the presence of MSCs hPBMC proliferation was assessed on day three and expressed as percentage of CFSE/far red stained cells. Data are expressed as percentage of hPBMC proliferation in the absence of MSCs and presented as mean ± standard deviation of three independent experiments. P <0.05, P < 0.01.

Detailed Description

The invention discloses a method for preparing Human Leukocyte Antigen (HLA) class I negative Mesenchymal Stromal Cells (MSCs). Human PBMC-derived iPS cells (hP-iPSCs) were first prepared and genetically modified by knock-out of the B2M gene. The selected clones were then differentiated into iPS-derived MSCs (imscs) with B2M gene knockout, which express the corresponding MSC marker without expressing HLA-class I antigen and showed pluripotency to differentiate into osteoblasts, chondrocytes and adipocytes. Importantly, for allogeneic immune cells, the iMSCs exhibit less immunogenicity as compared to wild-type iMSCs. Therefore, hP-iPSCs with the B2M gene knockout can be used as a cell resource of 'off-the-shelf' to prepare MSCs with low immunogenicity. The prepared iMSCs have great potential in the field of regenerative medicine. However, h iPSCs are notoriously difficult to transfect and considerations to optimize experimental design are often necessary.

The present technology provides a novel mesenchymal stromal cells (mesenchyme stromal cells) with low or reduced immunogenicity derived from induced pluripotent stem cells (hP-iPSCs) derived from human PBMCs with a B2M gene knockout. The B2M knock-out iMSCs are HLA-class I negative and exhibit low immunogenicity, thereby reducing the risk of allograft rejection as described above and providing extended survival and therapeutic functions following allograft transplantation. In this case, B2M knockout iMSCs have the potential to be a universal therapeutic cellular resource for MSC-based cell therapy due to their reduced immunogenicity.

Accordingly, a first aspect of the invention includes a method of making mesenchymal stromal cells with reduced immunogenicity, the method comprising:

(a) mutating both alleles of human pluripotent stem cell β 2-microglobulin;

(b) differentiating the mutated human pluripotent stem cells into derived human mesenchymal stromal cells having a biallelic mutation in the beta 2-microglobulin gene;

wherein the derived human mesenchymal stromal cells do not express beta 2-microglobulin and express attenuated or do not express HLA class I molecules on the cell surface.

The term "reduced immunogenicity" as used herein refers to cells that have a lower or reduced measurable response to activated T cells expressing CD8+ as compared to the response of non-genetically modified natural or wild-type (WT) MSCs. In various embodiments, the attenuated response is measured as a percentage of cytolysis, and genetically modified MSC cells that do not express β 2-microglobulin (B2M) have a lower percentage of cytolysis in the face of CD8+ T cell challenge than native or wild-type (WT) MSCs that have not been genetically modified. In various embodiments, the attenuated response is measured as a relative percentage of proliferation, whereby genetically modified MSC cells that do not express β 2-microglobulin (B2M) have a lower percentage of proliferation than native or wild-type (WT) MSCs that are not genetically modified when challenged with CD8+ T cells. In various embodiments, the attenuated response is measured in terms of secretion of cytokines, such as Interleukins (IL), Tumor Necrosis Factor (TNF), Interferon (IFN), and other known cytokines, comparing secretion of the same cytokines in genetically modified MSC cells that do not express β 2-microglobulin (B2M) to native or wild-type (WT) MSCs that have not been genetically modified.

As used herein, the term "mutation" or "mutation" refers to any change in the genome of a cell. In the context of the present method, mutations may include, but are not limited to, insertions, or deletions or substitutions. Mutations may cause loss of function or removal of the β 2-microglobulin (B2M) gene. In various embodiments, the B2M gene comprises the nucleic acid sequence set forth in SEQ ID No. 1. In various embodiments, the mutation in the B2M gene is present in the first exon. In various embodiments, the target sequence in the first exon of the B2M gene comprises the 12 th to 57 th nucleobases of SEQ ID No. 1. In various embodiments, the target sequence comprises the nucleic acid sequence set forth in SEQ ID NO.2 (TGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTG). In various embodiments, the target sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 3. In various embodiments, the target sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 4. A mutation anywhere in the B2M gene may stop expression and reduce immunogenicity, however, advantageously, mutation of the first exon results in a large number of clones that do not express B2M. The term "does not express β 2-microglobulin" as used herein refers to knocking out or eliminating the expression of the gene of β 2-microglobulin (B2M) or B2M gene. For example, a B2M gene can be knocked out by deleting or adding a nucleotide sequence to interfere with the reading frame. As another example, a gene may be knocked out by replacing or replacing a portion of the gene with an unrelated sequence. The term "expression-reduced or non-expression of HLA class I molecules" as used herein refers to the knock-down or reduction of the expression of a gene or gene product thereof. The β 2-microglobulin (B2M) gene encodes all common subunits required for surface expression of HLA-class I heterodimers (HLA-a, B, C, E, F and G). As a result of the B2M gene knockout, the activity or function of the protein may be reduced or the protein level of the HLA-class I complex may be reduced or eliminated.

The term "insertion" as used herein refers to the addition of one or more nucleotides to a DNA sequence. Insertions may range from the insertion of small fragments of a nucleotide to the insertion of large fragments of a gene (e.g., a cDNA or a gene). In various embodiments, the insertion includes one EGFP gene. The term "deletion" refers to the loss or removal of one or more nucleotides in a DNA sequence or the loss or removal of the function of a gene. In some cases, a deletion can include, for example, a loss of several nucleotides, an exon, an intron, a gene fragment, or the entire sequence of a gene. In some cases, deleting a gene refers to the removal or reduction of function or expression of a gene or its gene product. This is not only due to the deletion of sequences within or near the gene, but may also be due to other events (e.g., insertions, nonsense mutations) interfering with the expression of the gene. In various embodiments, the first allele of the β 2-microglobulin gene is mutated to delete base pairs 47 and 48 of SEQ ID No.1, and the second allele of the β 2-microglobulin gene is mutated to include an insertion of a cytosine between base pairs 46 and 47 of SEQ ID No. 1.

In various embodiments, the human pluripotent stem cells are human embryonic stem cells (hescs). hESC is a good choice from a safety point of view, but derivatives of hESC have been ethically controversial and have been limited in application.

In various embodiments, the human pluripotent stem cell is an Induced Pluripotent Stem Cell (iPSC). Methods for preparing ipscs from various sources are known. Currently, most ipscs are prepared from reprogrammed human cells. The choice of starting somatic cells will not only affect the efficiency and kinetics of reprogramming, but will also affect the utility of making GMP-grade iPSCs. Although fibroblasts are the most commonly used somatic cells, they are not very GMP compliant. Collecting skin samples by drilling biopsies is invasive and growing fibroblasts from skin biopsies is time consuming (up to 3 weeks). Derivation of fibroblasts under GMP specifications itself has become a difficult task.

In various embodiments, the human pluripotent stem cells are induced from peripheral blood cells (pbc) (ipsc). The advantage of using iPSCs is that they can be prepared from a reliable, unlimited and standardized source of starting cells, such as Peripheral Blood Mononuclear Cells (PBMCs) isolated from a donor or patient, thereby making iPSCs derivatives useful for autologous and allogeneic applications, and further, without any ethical concerns when using hescs. The use of PBC for the preparation of iPSCs (hP-iPS) is a practical option due to the ease of peripheral blood collection and the only 15 minutes required for isolation of mononuclear cells from peripheral blood samples. The simplicity of implementing GMP in sample collection, transportation and handling makes PBC an attractive starting material for deriving ipscs. Advantageously, human mesenchymal stromal cells can be induced on a large scale directly from hP-iPS cells knocked out from the B2M gene, which show negative B2M and HLA class I expression. These B2M gene knockout hP-iPS cells can serve as a permanent cell source for the preparation of reduced immunogenicity or low functional iPS derived imscs.

In various embodiments, the methods and compositions described herein further include the introduction of one or more of Oct-4, Sox2, Nanog, c-MYC, and KIf4 or other reprogramming compositions known in the art for reprogramming. As noted above, the exact method used for reprogramming is not critical to the methods and compositions described herein. However, cells differentiated from reprogrammed cells are used in, for example, human therapy, and reprogramming is not affected by the method of changing the genome, on the one hand. Thus, in such embodiments, reprogramming can be achieved without the use of viral or plasmid vectors, for example.

In various embodiments, the method further comprises determining that the mutation is present in both alleles of the B2M gene. In various embodiments, determining that the mutation is present in both alleles of the B2M gene comprises sequencing both alleles of the B2M gene to detect any insertions or deletions in the B2M gene. In various embodiments, sequencing comprises the use of primers of SEQ ID nos.5 and 6. In various embodiments, sequencing comprises the use of primers of SEQ ID nos.7 and 8. In various embodiments, sequencing comprises the use of primers selected from SEQ ID NOS.5-14. In various embodiments, sequencing comprises the use of any one of the primers selected from SEQ ID NOS.5-14. In various embodiments, determining that the mutation is present in both alleles of the B2M gene comprises determining that the mutation is present in the first exon of the B2M gene. In various embodiments, determining that the mutation is present in both alleles of the B2M gene comprises determining that a first allele of the β 2-microglobulin gene is mutated to delete base pairs 47 and 48 of SEQ ID No.1 and determining that a second allele of the β 2-microglobulin gene is mutated to comprise inserting a cytosine between base pairs 46 and 47 of SEQ ID No. 1. In other various embodiments, determining that the mutation is present in both alleles of the B2M gene comprises determining that a first allele of the β 2-microglobulin gene is mutated to delete base pairs 47 and 48 of SEQ ID No.1 and determining that a second allele of the β 2-microglobulin gene is mutated to include inserting a cytosine between base pairs 46 and 47 of SEQ ID No. 1. In various embodiments, determining that the mutation is present in both alleles comprises a detection method such as Southern blot hybridization. In various embodiments, determining that the mutation is present in both alleles comprises detecting methods such as the presence of cell sorting HLA class I molecules, wherein a loss of expression of HLA class I molecules in the mutant cell as compared to the wild-type cell indicates the presence of the mutation in both alleles. In various embodiments, determining that the mutation is present in both alleles comprises detecting the presence or absence of any of a combination of methods such as cell sorting B2M, HLA-A, HLA-B, or HLA-C, wherein a loss of expression of any of B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof, in the mutant cell as compared to the wild-type cell, indicates the presence of a mutation in both alleles.

In various embodiments, the derived human mesenchymal stromal cells having biallelic mutations in the β 2-microglobulin gene express at least one of CD29, CD44, CD73, CD90, CD105, CD166, or a combination thereof, and include low or no expression of any of CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof. In various embodiments, the derived human mesenchymal stromal cells are stained with at least one antibody of CD29, CD44, CD73, CD90, CD105, CD166, CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof, and sorted in a flow cytometer to determine which cells express at least one of CD29, CD44, CD73, CD90, CD105, CD166, CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof. In various embodiments, the derived human mesenchymal stromal cells having biallelic mutations in the β 2-microglobulin gene express any two, three, four, five, or all of CD29, CD44, CD73, CD90, CD105, CD 166. In various embodiments, the derived human mesenchymal stromal cells having biallelic mutations in the β 2-microglobulin gene comprise any two, three, four, five, six, seven, eight, or all of low or no expression of CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, and HLA-C.

In various embodiments, the allele is on one or more plasmids comprising a plasmid targeting the guide nucleic acid sequence of the β 2-microglobulin gene part and an endonuclease.

In various embodiments, the endonuclease may include CRISPR-associated endonucleases such as Cas9, Cpfl, and analogs thereof, permanently edited within or near the genomic site of the B2M gene or other DNA sequence encoding the B2M gene regulatory element. As such, the examples set forth in this disclosure may help reduce or eliminate expression of the B2M gene. In various embodiments, the endonuclease is guided by a guide nucleic acid sequence. In various embodiments, the Cas9 endonuclease or Cpf1 endonuclease is selected from streptococcus pyogenes Cas9, staphylococcus aureus Cas9, neisseria meningitidis Cas9, streptococcus thermophilus CRISPR1 Cas9, streptococcus thermophilus CRISPR3 Cas9, treponema denticola Cas9, lachnospira ND2006 Cpfl, and amino acid coccus BV3L6 Cpfl, or any endonuclease known in the art.

In various embodiments, the guide nucleic acid sequence is a single guide RNA (sgrna) comprising a target sequence (crRNA sequence) and an RNA-guided nuclease recruitment sequence (tracrRNA). In various embodiments, the plasmid comprises a pX260(addge, cambridge, massachusetts, usa) comprising a CRISPR/cas9 system targeting a portion of the β 2-microglobulin gene comprising SEQ ID No. 3. In various embodiments, the guide nucleic acid sequence comprises a crRNA-tracrRNA-Cas9 complex, which can guide the complex to a target nucleic acid sequence to which the crRNA can hybridize. Hybridization of crRNA to the target nucleic acid sequence can activate Cas9 for target nucleic acid cleavage. The target nucleic acid sequence is referred to as a pro-spacer adjacent motif (PAM) in the CRISPR system. In nature, the PAM is essential to facilitate binding of a site-directed polypeptide (such as Cas9) to the target nucleic acid sequence. In various embodiments, the plasmid comprises a pX459(addge, cambridge, massachusetts, usa) comprising a CRISPR/Cas9 system targeting a portion of the β 2-microglobulin gene comprising SEQ ID No. 4.

In various other embodiments, the guide nucleic acid sequence is a single RNA-guided endonuclease lacking tracrRNA as compared to the system described above. Indeed, Cpfl-associated CRISPR arrays can be processed into mature crRNAs without the need for additional transactivation of tracrrnas. CRISPR arrays of this type can be processed into short mature crRNAs of 42-44 nucleotides in length, each mature crRNA starting with a forward repeat of 19 nucleotides in length followed by a spacer sequence of 23-25 nucleotides in length.

In various embodiments, the methods of mutating a gene described herein include methods of cleaving deoxyribonucleic acid (DNA) at a precise target location on the B2M gene using a site-directed nuclease, thereby generating a break in single-stranded or double-stranded DNA at a specific location within the gene. Such breaks can be repaired by natural endogenous cellular processes and periodically, such as Homology Directed Repair (HDR) and non-homologous end joining (NHEJ). These two major DNA repair processes consist of a series of alternative pathways. NHEJ directly engages DNA ends caused by double strand breaks, sometimes with the loss or addition of nucleotide sequences, which may disrupt or enhance gene expression. HDR uses a homologous or donor sequence as a template to insert a defined DNA sequence at the breakpoint. The homologous sequences may be in an endogenous genome, such as a sister chromatid. Alternatively, the donor may be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a virus, which has a region of high homology to the nuclease cleavage site, but may also contain other sequences or sequence changes, including deletions, which may be incorporated into the target cleavage site. The third repair mechanism is microhomology-mediated end joining (MMEJ), also known as "surrogate NHEJ (alternative NHEJ)", whose genetic result is similar to NHEJ in that small deletions and insertions can occur in the cleavage site. MMEJ can use several base pairs of homologous sequences flanking a DNA break to drive more favorable DNA end-joining repair results.

In various embodiments, nickase variants of RNA-guided endonucleases, such as Cas9, can be used to increase the specificity of CRISPR-mediated genome editing. Wild-type Cas9 is typically guided by a single guide RNA designed to hybridize to a sequence of 20 nucleotides in a particular length in a target sequence (e.g., an endogenous genomic locus). However, few mismatches between the guide RNA and the target locus can be tolerated, effectively reducing the length of the homology sequence required for the target site to, for example, as low as 13nt homology (about 65%), thus leading to increased probability of CRISPR/Cas9 complex binding and double-stranded nucleic acid cleavage, also known as off-target cleavage, elsewhere in the target genome. Since each nicking enzyme variant of Cas9 nicks only one strand, in order to generate a double strand break, it is necessary for a pair of nicking enzymes to bind tightly on opposite strands of the target nucleic acid, thus generating a pair of nicks corresponding to the double strand break. This requires that two separate guide RNAs (one for each nickase) must be tightly bound on opposite strands of the target nucleic acid. This requirement essentially doubles the minimum homology length required for a double-strand break to occur, thus reducing the likelihood that a double-strand break will occur elsewhere in the genome, and the two guide RNA sites, if present, are unlikely to be close enough to each other to be able to form a double-strand break. As is known in the art, nickases may also be used to promote HDR to combat NHEJ. HDR can be used to efficiently mediate prospective changes by introducing selected changes to a target site in a genome using a particular donor sequence.

In various embodiments, any of these genome editing mechanisms can be used to make the desired mutation in both alleles of B2M. One of the steps of the mutation process may be to make a break in one or both of the DNAs, either double-stranded or double-stranded, by setting the vicinity of the desired mutation as a target site.

One CRISPR (clustered regularly interspaced short palindromic repeats) locus can be found in the genomes of various prokaryotes such as bacteria and archaea. In prokaryotes, the product encoded by the CRISPR locus functions as a type of immune system to help prokaryotes defend against foreign invaders such as viruses and bacteriophages. There are three phases of function of CRISPR loci: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA) and silencing of foreign invader nucleic acids. Five types of CRISPR systems (e.g., Type I, Type II, Type III, Type U, and Type V) have been identified.

One CRISPR locus contains a large number of short repeats called "repeats". When expressed, the repeat sequences may form secondary structures (e.g., hairpin structures) and/or include unstructured single-stranded sequences. The repetitive sequences usually occur in clusters, which often differ among different species. The repeated sequences are regularly spaced by unique intervening sequences called "spacers" which result in the appearance of a "repeat-spacer-repeat" locus structure. The spacer is identical or highly homologous to the target sequence. A "spacer-repeat" unit encodes a criprpr rna (crrna), which is the mature form processed into the "spacer-repeat" unit. A crRNA includes a "seed" or spacer sequence that is involved in targeting a target nucleic acid. A spacer sequence is located at the 5 'or 3' end of the crRNA.

One CRISPR locus also includes a polynucleotide sequence encoding a CRISPR-associated (Cas) gene. The Cas gene encodes an endonuclease involved in the biogenesis and interference phases of crRNA function in prokaryotes. Some Cas genes include homologous secondary and/or tertiary structures

In various embodiments, the endonuclease includes a nucleic acid sequence Cas9 associated with a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

In various embodiments, the guide nucleic acid sequence targeting the β 2-microglobulin gene portion comprises a sequence selected from SEQ ID No.3 or SEQ ID No. 4. In various embodiments, the β 2-microglobulin gene portion comprises the first exon of the B2M gene.

In various embodiments, the human pluripotent stem cell is mutated by:

(a) transfecting the human pluripotent stem cell with a first plasmid comprising an endonuclease, a guide nucleic acid sequence targeting a portion of the β 2-microglobulin gene, and at least one selectable marker to mutate both alleles of the β 2-microglobulin gene; (b) selecting cells having a mutation in the first allele of the β 2-microglobulin gene by the at least one selectable marker; (c) transfecting the cell having a mutation in the first allele of the β 2-microglobulin gene with a second plasmid comprising an endonuclease, a guide nucleic acid sequence targeting a portion of the β 2-microglobulin gene, and at least one other selectable marker to mutate the second allele of the β 2-microglobulin gene; and (d) selecting for cells having a mutation in the first allele of the β 2-microglobulin gene and a mutation in the second allele of the β 2-microglobulin gene by at least one other selectable marker.

In various embodiments, any selection marker known in the art is selected or screened to distinguish between cells that have undergone mutation and cells that have not undergone mutation. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses antibiotic resistance, wherein the mutant cells are selected as the only viable cells upon administration of the antibiotic. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses neomycin resistance, wherein the mutant cell is selected as the only cell that survives administration of neomycin. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses Green Fluorescent Protein (GFP), wherein the mutant cells are selected by fluorescence-activated cell sorting. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses neomycin resistance and a nucleic acid sequence that expresses a Green Fluorescent Protein (GFP), wherein the mutant cells are first selected as the only viable cells upon administration of neomycin and subsequent fluorescence-activated cell sorting. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses hygromycin resistance, wherein the mutant cells are selected as the only cells that survive hygromycin administration. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses mcherry, wherein the mutant cell is selected by fluorescence activated cell sorting. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses hygromycin resistance and a nucleic acid sequence that expresses mcherry, wherein the mutant cell is the only cell that survives administration of hygromycin and is subsequently first selected by fluorescence-activated cell sorting. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses puromycin resistance, wherein the mutant cell is selected as the only viable cell upon administration of puromycin. In various embodiments, the selectable marker in the first or second plasmid is a nucleic acid sequence that expresses neomycin resistance; a nucleic acid sequence expressing hygromycin resistance; a nucleic acid sequence expressing puromycin resistance; a nucleic acid sequence expressing Green Fluorescent Protein (GFP); at least one of a nucleic acid sequence expressing mcherry, wherein the selectable marker in a first plasmid is different from the selectable marker in a second plasmid to allow for differential selection of mutations in both alleles.

In various embodiments, the human pluripotent stem cell is transfected with a plasmid capable of mutating both alleles of the β 2-microglobulin gene, the plasmid comprising an endonuclease, a guide nucleic acid sequence targeting a portion of the β 2-microglobulin gene, and a selectable marker; and selecting a cell having a mutation in both alleles of the β 2-microglobulin gene by the selection marker.

In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses antibiotic resistance, wherein the mutant cells are selected as the only viable cells upon administration of the antibiotic. In various embodiments, the selectable marker comprises a nucleic acid sequence that expresses puromycin resistance, wherein the mutant cell is selected as the only viable cell upon administration of puromycin. In various embodiments, the selectable marker is a transient marker. In various embodiments, the selectable marker selected for cell sorting lacks expression of HLA class I molecules. In various embodiments, the selectable marker comprises both a nucleic acid sequence that expresses antibiotic resistance and the lack of expression of an HLA-class I molecule, wherein the mutant cell is the only cell that survives administration of the antibiotic and is subsequently first selected by cell sorting. This has the advantage of screening for cells with mutations in both alleles, without having to prepare two separate mutations and select for each mutation. In various embodiments, the antibiotic may be puromycin. In various embodiments, the selectable marker does not comprise a nucleic acid sequence that expresses a fluorescent molecule. This has the advantage of not interfering with subsequent fluorescent staining or cell imaging.

Another aspect of the invention includes genetically modified human mesenchymal stromal cells, including cells that do not express β 2-microglobulin and express attenuated or do not express HLA class I molecules located on the cell surface.

The term "genetically modified human mesenchymal stromal cells" as used herein refers to human mesenchymal stromal cells that have been differentiated from human pluripotent stem cells, wherein the B2M gene has been artificially modified to not express beta 2-microglobulin and HLA-class I molecules. The human mesenchymal stromal cells thus produced also do not express beta 2-microglobulin and HLA class I molecules. The human pluripotent stem cells of the invention may be Induced Pluripotent Stem Cells (iPSCs). An advantage of using iPSCs is that the cells may be derived from the same subject in which the mesenchymal stromal cells are to be used or administered. That is, somatic cells can be obtained from a subject, reprogrammed to induce pluripotent stem cells, and then differentiated into mesenchymal stromal cells for use in therapy or administration to the subject (e.g., autologous cells). Since the beta 2-microglobulin and HLA-class I molecule are not expressed, the risk of graft rejection or anaphylaxis can be reduced compared to using other human mesenchymal stromal cells. In addition, the use of iPSCs eliminates the need to obtain cells from embryonic sources. In various embodiments, the human pluripotent stem cells used in the disclosed methods are not embryonic stem cells. Although differentiation is generally irreversible in a physiological environment, several methods have been developed to reprogram somatic cells to iPSCs. Exemplary methods are known to those skilled in the art.

In various embodiments, the HLA class I molecule comprises any one of B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof. In various embodiments, the genetically modified human mesenchymal stromal cells lack the expression of any one of B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof. In various embodiments, the genetically modified human mesenchymal stromal cells lack expression of all of B2M, HLA-A, HLA-B, or HLA-C. Since B2M is a key component of the HLA class I complex, knockout of B2M can disrupt HLA class I expression on cells. The HLA class I complex serves to present cytoplasmic peptides to autoantigens during histocompatibility recognition. The HLA class I negative phenotype generated by the B2M gene knockout can significantly reduce the immunogenicity of such human mesenchymal stromal cells by virtue of minimal antigen presentation. Thus, the risk of allograft rejection can also be reduced, since the host's alloreactive lymphocytes cannot be activated by recognizing any alloantigens located on the cell surface of the graft carrying such cells.

In various embodiments, the genetically modified human mesenchymal stromal cells express any one of CD29, CD44, CD73, CD90, CD105, CD166, or a combination thereof, and low or no expression of any one of CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof.

In various embodiments, the derived human mesenchymal stromal cells having biallelic mutations in the β 2-microglobulin gene express any one of CD29, CD44, CD73, CD90, CD105, CD166, or a combination thereof, and include low or no expression of any one of CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, or HLA-C, or a combination thereof. In various embodiments, any two, three, four, five, or all of CD29, CD44, CD73, CD90, CD105, CD166 are expressed by derived human mesenchymal stromal cells having biallelic mutations in the β 2-microglobulin gene. In various embodiments, the human mesenchymal stromal cells derived from the beta 2-microglobulin gene having a biallelic mutation comprise any two, three, four, five, six, seven, eight, or all of under-expressed or under-expressed CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, or HLA-C. Such cells have the advantage of having the characteristics of human mesenchymal stromal cells, such as expressing any of CD29, CD44, CD73, CD90, CD105, CD 166; low or no expression of any of CD14, CD34, HLA-DR, CD25, CD 45; and the ability to promote adipogenesis, bone formation and cartilage formation, but they have the advantage of not expressing HLA class I molecules. The expression of HLA class I remains low even after differentiation into adipocytes, osteocytes and chondrocytes.

In various embodiments, the first allelic mutation of the β 2-microglobulin gene is a deletion of the 47 th and 48 th base pairs of SEQ ID No.1, and the second allelic mutation of the β 2-microglobulin gene is a mutation comprising an insertion of a cytosine between the 46 th and 47 th base pairs of SEQ ID No. 1.

In various embodiments, the first allelic mutation of the β 2-microglobulin gene is a deletion of the 47 th and 48 th base pairs of SEQ ID No.1, and the second allelic mutation of the β 2-microglobulin gene is a deletion of the 46 th base pair of SEQ ID No. 1.

In various embodiments, the first allele includes a mutation in the first exon, including the nucleic acid sequence set forth in SEQ ID No.57 (CCGAGATGTCTCGCTCCGTGGTTAGCTGTGCTCGCGCTACTCTCT). In various embodiments, the second allele includes a mutation in the first exon, including the nucleic acid sequence set forth in SEQ ID No.58 (CCGAGATGTCTCGCTCCGTGGCCCTTAGCTGTGCTCGCGCTACTCTCT). In various embodiments, the second allele includes a mutation in the first exon, including the nucleic acid sequence set forth in SEQ ID No.59 (CCGAGATGTCTCGCTCCGTGCCTTAGCTGTGCTCGCGCTACTCTCT).

In various embodiments, the cells of the invention are suitable for use in therapy. Since HLA class I expression of B2M gene knockout human mesenchymal stromal cells was blocked, there was little antigen presentation on B2M gene knockout imscs. In this case, the B2M knockout iMSC act as an allograft and do not cause host rejection of the graft. In addition, they can escape the lytic response of alloreactive T cells and prolong survival time after infusion. These B2M knock-out hP-iPSC-derived MSCs can be used as allogeneic biomaterials in the "off-the-shelf" scenario for therapeutic purposes, e.g. for regenerative medicine. These B2M gene knockout iPS cell-derived MSCs are as effective as primary human mesenchymal stromal cells in treating a number of diseases ranging from tissue damage to immune disorders.

Another aspect of the present invention includes a kit for preparing mesenchymal stromal cells with reduced immunogenicity, the kit comprising:

(a) one or more plasmids capable of mutating both alleles of the β 2-microglobulin gene of a human pluripotent stem cell;

(b) a culture medium of mesenchymal stromal cells for differentiating human pluripotent stem cells into human mesenchymal stromal cells.

In various embodiments, the kit further comprises two or more primer sequences selected from SEQ ID nos. 5-28. In various embodiments, the primer pair comprises a pair of forward and reverse primers selected from the group consisting of SEQ ID nos.5 and 6; SEQ ID nos.7 and 8; SEQ ID nos.9 and 10; SEQ ID NOS.11 and 12; SEQ ID nos.13 and 14; any pair of SEQ ID NOS.15 and 16. Wherein one or more additional primer pairs may be selected from the group consisting of SEQ ID NOS.17 and 18; SEQ ID NOS.19 and 20; SEQ ID nos.21 and 22; SEQ ID nos.23 and 24; SEQ ID nos.25 and 26; SEQ ID nos.27 and 28; SEQ ID nos.29 and 30; SEQ ID nos.31 and 32; SEQ ID NOS.33 and 34; SEQ ID nos.35 and 36; SEQ ID nos.37 and 38; SEQ ID NOS.39 and 40; SEQ ID nos.41 and 42; SEQ ID nos.43 and 44; SEQ ID NOS.45 and 46; SEQ ID NOS.47 and 48; SEQ ID NOS.49 and 50; SEQ ID nos.51 and 52; SEQ ID nos.53 and 54; SEQ ID NOS.55 and 56.

In various embodiments, the one or more plasmids include a guide nucleic acid sequence targeting the β 2-microglobulin gene portion and an endonuclease.

In various embodiments, the guide nucleic acid sequence targeting the β 2-microglobulin gene portion is as described above. In various embodiments, the endonuclease is as described above.

In various embodiments, the targeting moiety of the β 2-microglobulin gene is the first exon of B2M. In various embodiments, the one or more plasmids include a CRISPR/Cas9 system targeting the β 2-microglobulin gene portion. In various embodiments, the guide nucleic acid sequence targeting the β 2-microglobulin gene portion comprises a sequence selected from SEQ ID No.3 or SEQ ID No. 4.

In various embodiments, the plasmid comprises a pX260(addge, cambridge, massachusetts, usa) comprising a CRISPR/cas9 system targeting the β 2-microglobulin gene portion comprising SEQ ID No. 3. In various embodiments, the plasmid comprises a pX459(addge, cambridge, ma, usa) containing a CRISPR/Cas9 system targeting the β 2-microglobulin gene portion comprising SEQ ID No. 4.

In various embodiments, the endonuclease includes a nucleic acid sequence (Cas9) associated with a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention belongs. As used herein, the following definitions are provided for the understanding of the present invention.

In this document, unless indicated to the contrary, the terms "comprise" and variations thereof are to be construed as "non-exhaustive" or, in other words, to mean "including, but not limited to".

Furthermore, throughout the specification, unless the context requires otherwise, the term "comprise" or variations such as "comprises" or "comprising", is understood to imply the inclusion of any stated integer or group of integers but not the exclusion of any other integer or group of integers.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Illustrate by way of example

In various embodiments, the preparation of HLA class I negative MSCs is described. Human PBMC-derived iPS cells (hP-iPSCs) were first prepared and genetically modified by knocking out the B2M gene of hP-iPSCs with a specially designed CRISPR/Cas9 system. The B2M gene knockout hP-iPSC single cell clone is screened out by selecting a clone which is negative for expression of HLA-I complex and keeps pluripotency and inheritance normality. The selected cell clones then differentiated into iPS-derived MSCs (imscs) with B2M gene knockout, which express the corresponding MSC marker without expressing HLA-class I, and showed pluripotency to differentiate into osteoblasts, chondrocytes, and adipocytes. Importantly, for allogeneic immune cells, the iMSCs exhibit less immunogenicity as compared to wild-type iMSCs. Therefore, the invention discloses a technology for preparing low-immunogenicity MSCs by using hP-iPSCs with the B2M gene knockout as a cell resource of 'finished product (off-the-shelf'). The prepared iMSCs have great potential in the field of regenerative medicine.

Cell culture of peripheral blood cell-derived iPSCs

Human PBMC-derived induced pluripotent stem cells cultured in mTeSR ^TM1 Medium (STEMCELL Technologies, Vancouver, Canada), Matrigel^TMhESC matrigel (BD Biosciences, franklin lake, new jersey, usa) coated plates. The culture medium is replaced by new one every day, and hP-iPSCs are subcultured every week. To form embryoid bodies from hP-iPSCs, confluent iPSC colonies were separated into large cell aggregates using 1mg/mL dispase (Thermo Fisher Scientific, Waltham, Mass., USA). Those cell aggregates were cultured on low-attachment plates, Dulbecco's Modified Eagle medium as Embryoid Body (EB) medium: nutrient mixture F-12(DMEM/F12, Gibco)^TMThermo Fisher Scientific) supplemented with 20% KnockOut serum replacement (Gibco)^TM) 2mM L-glutamine (Lonza, Basel, Switzerland), 0.1mM non-essential amino acid (NEAA, Gibco)^TM) And 0.1mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, Mo., USA). The EB medium was refreshed every 2 to 3 days.

Western blot hybridization and flow cytometry analysis

For Western blot hybridization, sample proteins were analyzed by Radioimmunoprecipitation (RIPA) buffer (Nacalai Te)sque, kyoto, japan) lysed cells, analyzed in SDS-PAGE gels under reducing conditions, and electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, ca, usa). The rabbit anti-B2M antibody clone EP2978Y (dilution 1:5000, Aocam, Cambridge, UK) and the murine anti- β -actin antibody clone GT5512 (dilution 1:1000, Abcam) were used as primary antibodies. Goat anti-rabbit lgG-HRP (dilution 1:5000, Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and goat anti-mouse lgG-HRP (dilution 1:2000, Santa Cruz Biotechnology) were used as secondary antibodies. The film passes MYECL^TMImager (Thermo Fisher Scientific), developed and visualized using chemiluminescence. For flow cytometry analysis, iPS and MSC cells were stained with antibody, placed in

Running buffer (Milteny iBiotec) with BDAccuri^TMC6 flow cytometry (BD Biosciences) analysis. For data

Sampler software (BD Biosciences) analysis.

Southern blot hybridization

Southern blot hybridization was performed on each sample and 15. mu.g of genomic DNA was digested with 50U HindIII-HF (New England Biolabs, Ipswich, Mass., USA) overnight. The digested DNA was loaded on a 1% agarose gel and subjected to gel electrophoresis at 40V for 5 hours. Then use

Dry transfer System (Invitrogen)^TMThermo Fisher Scientific) transfers DNA onto a positively charged nylon membrane. The membrane was washed with 1.5M NaCl/0.5M NaOH denaturing solution and then air dried. At 130mJ/cm²And carrying out ultraviolet crosslinking. The membrane was first prehybridized in DIG Easy Hyb (Roche Diagnostics) buffer for 1 hour before hybridizing overnight with DIG-labeled probe. The membrane was then washed first with 2 × sodium citrate (SSC)/0.1% Sodium Dodecyl Sulfate (SDS) at 40 deg.CWashed twice and then twice with 0.1 XSSC/0.1% SDS at 50 ℃. Then, the cell membranes were blocked, washed with DIG detergent and blocking solution combination (Roche Diagnostics) and incubated with an anti-digoxigenin-AP (Roche Diagnostics). Finally, using MYECL^TMThe membrane was detected using a CDP-Star (Roche diagnostics) as a chemiluminescent substrate in an imager (Thermo Fisher Scientific). The probe was synthesized using a PCR DIG probe synthesis kit (Roche Diagnostics) using a donor plasmid of the B2M gene as a template. Primers used for probe synthesis are listed in table 1.

Table 1: lists of oligonucleotides and primers

RT-PCR

Total RNA was isolated from each cell sample using TRIzol reagent (Thermo Fisher Scientific). The reverse transcription was performed by the Super Script III first strand synthesis system (Invitrogen). PCR amplification was performed by KAPA Taq ReadyMix PCR kit (KaPa Biosystems, Roche Diagnostics) for 35 cycles at the corresponding annealing temperature. The PCR products were separated on a 1.5% agarose gel for analysis. The primers and their associated annealing temperatures are listed in Table 1.

Preparation of B2M Gene-knocked-out hP-iPSC clone

The B2M gene knockout hP-iPSCs are prepared by two methods. The first method is a two-color selection method, and the second method is a puromycin selection (one-shot puromycin selection) only once.

Example 1 two-color selection Using CRISPR/Cas9

Construction of plasmids

The pX260 plasmid (addge, cambridge, ma, usa) previously described (l.condet., science339,819(feb15,2013)) comprising the CRISPR/Cas9 system was used in this study. The CRISPR/Cas9 target sequence at the first exon was designed using CHOPCHOP (http:// chop. cbu. uib. no /). The B2M Target1(SEQ ID NO.3: GGCCGAGATGTCTCGCTCCG) was subcloned into pX 260. Two donor plasmids were constructed for homologous direct integration of the first exon of B2M. A plasmid designed with a promoter EF1 α (eukaryotic translation elongation factor 1 α) to drive the expression of an EGFP gene and a promoter PGK (murine phosphoglycerate kinase 1) to drive the expression of the Neo gene (neomycin resistance gene); meanwhile, another plasmid was designed to carry a promoter EF1 α to drive the expression of a mCheerry gene and a promoter SV40 (Simian Virus 40) to drive the expression of a Hygro gene (hygromycin resistance gene). Both donor sequences were flanked by homologous DNA sequences from the first exon locus of B2M (chromosome 15: nucleotides 44,710, 501-44, 711,401 and 44,711, 615-44, 712,485, GRCh38.p2 Primary Assembly).

For the two-color selection, hP-iPS cells were treated with Accutase^TM(Merck Millipore) separation, washing with phosphate buffered saline (PBS, Lonza), and resuspending in

Serum-reduced medium (Gibco)^TM) A single cell suspension was formed. 1X 10 by electric perforator (Nepa Gene, Qianye, Japan)⁶Cells were transfected with 2.5. mu.g of pX260 plasmid and 2.5. mu.g of donor plasmid with GFP. Transfected individual cells in

The medium (Biological Industries, Beit-Haemek, Israel) was recovered and inoculated into Matrigel^TMCoating on the plate. Four days after electroporation, the medium was changed back to mTeSR ^TM1 medium, 25. mu.g/mL

(G418 sulfate, Gibco)^TM) Cells were selected for 2 weeks. Using BD FACSAria^TMFlow cytometry (BD Biosciences, franklin lake, nj, usa) performed single cell seeded flow cytofluorimetric sorting (FACS) of selected cells. Accutase for selected hP-iPS cells^TM(Merck Millipore) into single cells and the GFP-positive population was seeded as one cell per well

Medium, Matrigel^TM96-well coated plates. Single cell clonal amplification, genotype detection by PCR and sequencing. Single-allele knockout single-cell clones were confirmed prior to a second round of gene knockout on the other allele. The single-allele knock-out single-cell clones were transfected with 2.5. mu.g of pX260 plasmid and 2.5. mu.g of the donor plasmid with mCherry by electroporation. Transfected cells were treated with 10. mu.g/mL hygromycin B (Gibco)^TM) Cells were selected for 2 weeks and single cell inoculation was performed. The bicolored single cell clones were collected, amplified and confirmed by genotyping.

To disrupt the B2M gene in human PBMC-derived iPS cells, B2M was first targeted by CRISPR/Cas9 with the aid of color selection. The hP-iPSCs were co-transfected with CRISPR/Cas9 plasmid pX260 targeting the B2M gene and a donor plasmid containing the EGFP and neomycin resistance gene flanked by B2M homologous sequences (fig. 1A). Transfected cells were selected for two weeks with Geneticin and sorted by GFP positive expression as single cells. A total of 18 single cell clones were amplified for analysis. All of the 18 monoclonals were confirmed by integration of the selection marker EGFP at site B2M (fig. 2A, 2B), but they still had normal B2M expression. Sequencing analysis showed that they each had one full wild type B2M allele (fig. 2C), indicating that these 18 clones were only single allele knockouts.

To disrupt the remaining wild-type B2M allele, #18 of one of the single allele knockout clones was again transfected with a plasmid targeting CRISPR/Cas9 of B2M, but accompanied by one donor containing mCherry and hygromycin selectable markers (fig. 1A). Similarly, transfected cells were subjected to two weeks of hygromycin B selection and cell sorting based on the EGFP and mCherry dual colors. A total of 6 single cell clones were collected and amplified, showing that both selectable markers were positive (fig. 1B). Furthermore, all 6 clones were confirmed by site-specific integration of the two markers at site B2M, and no wild-type B2M allele was detected by PCR (fig. 1C). This indicates that both alleles of B2M were knocked out for 6 bi-colored hP-iPSC clones. As expected, expression of B2M was not detected by Western blotting in bi-allelic knockout clones, even though the cells were treated with IFN-. gamma.for 48 hours (FIG. 1D). However, the single allele knockout clones maintained efficient B2M expression compared to the wild type clones. This finding was also supported by flow cytometry experiments, showing surface expression of B2M, and negative B2M expression only in double allele knock-out cells (fig. 1E, 3A). In addition, the double allele knockout clone was negative for HLA class I (FIG. 1E). Since B2M is a key component of the HLA class I complex, disruption of B2M blocks HLA class I expression and significantly reduces immunogenicity. Thus, selection of the B2M gene knockout hP-iPS cells using a two-color selection method was achieved.

Maintenance of pluripotency of hP-iPSCs with B2M Gene knockout

Pluripotency of the single and double allele B2M knock-out iPS cells was tested. The RT-PCR results showed that B2M single and double allele knock-out iPS cells, Oct4, Sox2, and Nanog had equivalent expression to wild-type cells (fig. 3B). hP-iPS cells are induced to form Embryoid Bodies (EBs). Differentiation markers for all three germ layers were detected in the B2M knock-out iPS derived EBs (fig. 3C, 3D), indicating that the B2M gene knock-out iPS cells have full differentiation capacity.

Example 2 Generation of B2M Gene knockout hP-iPSC clone by CRISPR/Cas9 in one step without color selection

Construction of plasmids

The pX459 plasmid (addge, cambridge, ma, usa) previously described (f.a. raneet, Nature protocols 8,2281(Nov,2013)) comprising a CRISPR/Cas9 system was used in this study. The CRISPR/Cas9 was again designed with CHOPCHOP (http:// chophop. cbu. uib. no /) at the target sequence of the first exon. The B2M Target2 SEQ ID NO.4(CGCGAGCACAGCTAAGGCCA) was subcloned into pX 459.

For the puromycin selection only once, 1 × 10 was electroporated⁶hP-iPS cells were transfected with 5. mu.g of pX459 plasmid harboring the B2M Target2 (FIG. 4A) and cultured

Medium, Matrigel^TMRecovery on coated plates was overnight. Then, the transfected cells were treated with 1. mu.g/mL puromycin (Thermo Fisher Scientific)

Selection was performed in medium for 24 hours. Survival of single cells in mTeSR ^TM1 before the culture medium, in the fresh state

Culturing in the culture medium for 3 to 4 days. Isolated into single cells and subjected to genotypic and phenotypic analysis. From those single cell clones, B2M negative clones were identified.

Although the B2M negative hP-iPS cells were well constructed by color selection, the entire process was time consuming and inefficient. Furthermore, cells with fluorescence may interfere with subsequent assays when using relevant fluorescent reagents. The possibility of the selection marker integrating into a genomic region other than B2M is also a concern, as evidenced by Southern blot hybridization (FIG. 3A). To overcome these problems, methods for knocking out B2M of hP-iPS cells without color selection were improved.

It was observed that some cells were already B2M negative after transfection with the CRISPR/Cas9 plasmid, but they could not be easily selected. Thus, the pX459 plasmid (fig. 4A) carrying the B2M Target2 was introduced as transient expression of Cas9, which can be selected by puromycin. The hP-iPS cells were transfected with plasmid pX459 targeting B2M using electroporation. When the transfected cells were stable, they were subsequently selected with puromycin for only 24 hours. Among the surviving cells, a large number of B2M negative cells were observed by flow cytometry (fig. 4B). These surviving cells were seeded as single cells and 12 single cell clones were successfully expanded. Of the 12 clones, 5 of them were detected as negative B2M by flow cytometry. B2M knock-out (B2MKO) clones #3 and #8 were randomly selected for subsequent analysis. Two of the B2M knockout clones, both B2M and HLA class I molecules were negative (FIG. 5A). By genotyping, either a deletion or an insertion in the B2M targeting site was found in those clones that were knockout of the B2M gene (fig. 5B). This specific mutation is caused by CRISPR/Cas 9-mediated cleavage, resulting in a frameshift of the B2M translational reading frame and bringing about an early stop codon that terminates B2M expression.

Off-target and karyotyping

The off-target site of the B2M CRISPR/Cas9 target was predicted by benchling (https:// benchling. com /). Corresponding primers were designed for the total predicted and coding sequence of the top three high-ranking off-target sites. Those primer sequences are contained in table 1. Genomic DNA from the B2M knockout hP-iPSCs clone was isolated using the DNeasy Blood & tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The off-target site sequences were subjected to PCR amplification, purification and Sanger sequencing analysis (AITbiotech, Singapore), respectively. For karyotyping, the hP-iPSCs colonies that were cell-covered were sent to a NUH Recommendation Laboratory (NRL) for chromosomal analysis.

The accuracy of the CRISPR/Cas 9-mediated B2M gene knockout was also checked by high-risk off-target site sequencing. Only one off-target that did not affect the reading frame was found in clone # 3. No observable off-targets were found in clone #8, which ensured the accuracy of this gene editing (table 2). In addition, karyotyping analysis also showed the normal state of chromosome of B2M knock-out clone #8 (fig. 5C). Thus, the B2M gene on the hP-iPS cells was precisely knocked out without introducing any selection marker or random genomic damage.

Table 2: sequencing results of potential off-target sites.

Maintenance of pluripotency of hP-iPSCs with B2M Gene knockout

The pluripotency of B2M knockout iPS cells was tested. The RT-PCR results showed that B2M single and double allele knock-out iPS cells, Oct4, Sox2, and Nanog, had equivalent expression to wild-type cells (fig. 6A). hP-iPS cells were also induced to form Embryoid Bodies (EBs). Differentiation markers for all 3 germ layers were detected in B2M knock-out iPS derived EBs (fig. 6B, 6C), which shows that B2M knock-out iPS cells have full differentiation capacity.

Preparation of mesenchymal stromal cells from B2M Gene knockout

Numerous studies have revealed the feasibility of obtaining Mesenchymal Stromal Cells (MSCs) from human embryonic stem cells (hESCs) or iPS cells (y. dual., Cellular physiology and biochemistry: international journel of experimental Cellular physiology, biochemistry, and pharmacology 43,611 (2017)). Mesenchymal Stromal Cells (MSCs) are differentiated directly from hP-iPSCs (Y. Dual., Cellular physiology and biochemistry: international journal of experimental Cellular physiology, biochemistry, and pharmacology 43,611(2017)) as described previously. When hP-iPSCs reached confluency, the medium was reduced in sugar Dulbecco's Modified Eagle Medium (DMEM) (Gibco)^TM) 10% fetal bovine serum (FBS, Hyclone)^TMGE Healthcare, Little Chalfount, UK), 2mM L-glutamine (Lonza). The medium was renewed initially every day for 4 days and then every two days for more than 6-12 days. Then, trypsin (Hyclone) was used^TMGE Healthcare) cells were separated at 1X 10⁶Cell/well density was seeded on Matrigel 6 well coated plates. When the cells reached confluence, the cells were detached with trypsin at 2X 10⁵Cell/well density was seeded in 6-well coated plates of 0.1% gelatin (Merck Millipore, Billerica, ma, usa). The morphology of adherent cells gradually transforms into fibroblasts. When they reached 80% cell confluence, these differentiatedCells were subcultured every 3 to 4 days as mesenchymal stromal cells for subsequent analysis.

The mesenchymal stromal cells were successfully derived from B2M knock-out iPS cells, displaying a fibroblast-like phenotype as with wild-type cells (fig. 7A). Flow cytometry confirmed that MSCs from B2M knock-out iPS cells were negative for B2M and HLA-class I (fig. 7B). Further phenotypic analysis supported that those B2M knockout cells retained the features of MSCs, e.g. they were negative for CD14, CD24, CD34, CD45 and HLA-DR, but positive for CD29, CD44, CD73, CD90, CD105 and CD166 (fig. 7C).

In vitro differentiation of hP-iPSC-derived MSCs

The pluripotency of the B2MKO iPS-derived MSCs was analyzed by inducing further differentiation of those MSCs into adipocytes, osteocytes, and chondrocytes. Adipogenesis was confirmed by positive staining of the lipid fraction of differentiated cells with oil red O (fig. 8A). Robust osteogenesis was confirmed by positive red staining for calcium deposition with alizarin red S (fig. 8B). In addition, chondrogenesis was achieved by chondrocyte aggregates and blue staining on acidic polysaccharides (fig. 8C). RT-PCR also showed upregulation of relevant markers in differentiated cells, supporting the differentiation capacity of B2MKO iPS derived MSCs. Therefore, functional mesenchymal stromal cells can be prepared from hP-iPS cells knocked out by B2M gene.

In this case, the method for preparing MSCs directly from the B2M gene knock-out hP-iPS cells can be modified. For adipogenesis, iPSC-derived MSCs were dosed at 10,000 cells/cm²Is inoculated for 2 to 4 days to reach cell confluence. The medium was then removed from the StemPro^TMAdipocyte differentiation kit (Gibco)^TM) Change to differentiation medium and change every 3 to 4 days for more than two weeks. Cells were then fixed with 4% formaldehyde (Sigma-Aldrich) and histological staining of lipid components was performed with oil red O (Sigma-Aldrich).

For osteogenesis, iPSC-derived MSC cells were plated at 5,000 cells/cm²Is inoculated for 2 to 4 days. The medium was then removed from the StemPro^TMBone cell differentiation kit (Gibco)^TM) Change to differentiation medium and change every 3 to 4 days for more than three weeks. Cells were then fixed with 4% formaldehyde (Sigma-Aldrich) and calcified parts were histologically stained with alizarin red s (merck millipore).

For chondrogenesis, iPSC-derived MSC cells were concentrated to 1.6 x 10⁷cells/mL. Cells were loaded onto the plate in the form of 5 μ L droplets and cultured as pellets for 2 hours to form cell clusters. The medium is then removed from the StemPro^TMChondrocyte differentiation kit (Gibco)^TM) The medium was changed to differentiation medium and every 3 to 4 days for more than three weeks. Cells were then fixed with 4% formaldehyde (Sigma-Aldrich) and the acidic polysaccharide was histologically stained with alcian blue 8GX (Sigma-Aldrich).

Low immunogenicity of B2MKO hP-iPSC-derived MSCs

To test the immunogenicity of hP-iPSC-derived MSCs, allogeneic human Peripheral Blood Mononuclear Cells (PBMCs) were first activated with wild-type hP-iPSC-derived MSCs (iMSCs). Fresh Peripheral Blood Mononuclear Cells (PBMCs) were used by density gradient centrifugation

PREMIUM 1.084(GE Healthcare) was isolated from the buffy coat (Healthcare donor's buffy coat) of healthy donors. PBMCs are activated by interaction with MSCs in 5% human AB serum (Valley biological, Wechester, Va., USA) and 300IU/mL IL-2(PeproTech, RockyHill, N.J., USA)

Culture medium (Gibco)^TM) Co-cultured to be activated. Activated CD3+ T cells were restimulated with MSCs at a ratio of 5:1 once every 7 days to obtain the MSC-specific Cytotoxic T Lymphocyte (CTLs) population.

Human PBMCs were co-cultured directly with wild-type imscs and re-stimulated weekly with fresh imscs. Activated PBMCs were studied deeply into their specific populations by flow cytometric analysis. Most activated donors will eventually possess a predominant population of CD3+ CD56-T cells, with CD8+ T predominating (fig. 9A). These activated PBMCs populations indicate that although mesenchymal stromal cells or iMSCs have immunosuppressive properties (m.giganiainetal, Blood 118,3254(sep22,2011)), alloreactive cytotoxic T lymphocytes have been stimulated by iMSCs.

Cytotoxicity assays

For cytotoxicity

EuTDA cytotoxic agents (PerkinElmer, Walthermer, Mass., USA) were measured in a standard 2-hour europium release assay. The test was performed according to the manufacturer's instructions. Target cells were first labeled with BATDA agent at 37 ℃ for 5 to 15 minutes and then washed three times with PBS. Placing effector cells and labeled target cells in a serum containing 5% human AB

In the medium, triplicate mixtures of different effect targets (E: T) were performed. The mixed cells were incubated in a humidified incubator (humid incubator) at 37 ℃ for 2 hours. Spontaneous and maximal release was determined by incubation of target cells without effector cells and with lysis solution, respectively. After incubation, the supernatant was transferred to mix with europium solution and passed through VICTOR^TMTime resolved fluorometer (PerkinElmer) analysis. The specific cracking percentages are calculated as follows:

in cytotoxicity assays, when both wild-type and B2MKO iMSCs were challenged with activated PBMCs, those activated PBMCs would preferentially kill wild-type iMSCs while ignoring the B2MKO iMSCs (fig. 9A). Due to the lack of HLA-class I antigen presentation process, the B2MKO iMSCs can escape from alloreactive cytotoxicity, whereas wild-type iMSCs are eliminated by alloreactivity.

Similarly, wild species compared to PBMCs in the presence of OKT3The B2M knockout cells showed the lowest proliferation rate when both biotypes and B2MKO iMSCs were challenged with activated PBMCs in the presence of OKT3 (FIG. 10). This is a statistically significant reduction. Human peripheral blood-derived mononuclear cells (hPBMCs) were stained with CFES/far red. Then this 1X 10⁵Cells were centrifuged through a gradient, added to each well, and stimulated with OKT-3 to stimulate proliferation. hPBMCs activated by OKT-3 were then cultured in wild-type and B2MKO iMSCs for 3-4 days. Proliferation levels were assessed after 18 hours. (ii) the proliferation of said hPBMCs is inhibited when they are co-cultured with naive (nave) MSCs; in iMSCs derived from the B2M knock-out, the proliferation of hPBMCs was more inhibited, as shown in FIG. 10.

For some donors, a large number of CD3-CD56+ Natural Killer (NK) cells may be accompanied by alloreactive T cells upon activation (fig. 9B). When this type of cells were used as effector cells for cytotoxicity assays, B2MKO iMSCs were also dose-dependently killed, but their lysis rate was significantly lower than the wild-type counterpart (fig. 9B). To assess the sensitivity of iMSCs to NK cells, wild-type and B2MKO iMSCs were challenged with primary NK cells.

Primary Natural Killer (NK) cells were expanded from fresh PBMC with inactivated modified K562 cells as feeder cells. The expanded NK cells were collected 7-10 days after co-culture with K562 cells and subjected to cytotoxicity assay with a purity of CD3-CD56+ cells higher than 90%.

It was observed that B2MKO ismcs cleaved slightly more than wild-type ismcs (fig. 9C), consistent with the "missing-self" theory (m.g. morvan, l.l. lanier, Nature reviews. cancer16,7(Jan, 2016)). Thus, B2MKO hP-iPSC derived MSCs showed lower immunogenicity but higher sensitivity to NK lysis.

Statistical analysis

Data were collected as described above and summarized by prism version7 software (GraphPad). Data are presented as mean (+ -SD) and analyzed using two-way analysis of variance, Tukey multiple comparison test, and independent sample t-test. Representative histograms and graphs are selected from the independent repetitive samples based on the mean.

Those skilled in the art will appreciate that variations and combinations of the features described above, not being alternatives or alternatives, may be combined to form further embodiments falling within the scope of the invention.

Sequence listing

<110> Singapore National University (National University of Singapore)

King tree (Wang, Shu)

Zhan Shijun (Zha, Shijun)

<120> low-immunogenicity engineered human mesenchymal stromal cells, preparation method and kit

<130> 2019.P01734

<150> SG 10201808423W

<151> 2018-09-26

<160> 59

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7361

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

attcctgaag ctgacagcat tcgggccgag atgtctcgct ccgtggcctt agctgtgctc 60

gcgctactct ctctttctgg cctggaggct atccagcgtg agtctctcct accctcccgc 120

tctggtcctt cctctcccgc tctgcaccct ctgtggccct cgctgtgctc tctcgctccg 180

tgacttccct tctccaagtt ctccttggtg gcccgccgtg gggctagtcc agggctggat 240

ctcggggaag cggcggggtg gcctgggagt ggggaagggg gtgcgcaccc gggacgcgcg 300

ctacttgccc ctttcggcgg ggagcagggg agacctttgg cctacggcga cgggagggtc 360

gggacaaagt ttagggcgtc gataagcgtc agagcgccga ggttggggga gggtttctct 420

tccgctcttt cgcggggcct ctggctcccc cagcgcagct ggagtggggg acgggtaggc 480

tcgtcccaaa ggcgcggcgc tgaggtttgt gaacgcgtgg aggggcgctt ggggtctggg 540

ggaggcgtcg cccgggtaag cctgtctgct gcggctctgc ttcccttaga ctggagagct 600

gtggacttcg tctaggcgcc cgctaagttc gcatgtccta gcacctctgg gtctatgtgg 660

ggccacaccg tggggaggaa acagcacgcg acgtttgtag aatgcttggc tgtgatacaa 720

agcggtttcg aataattaac ttatttgttc ccatcacatg tcacttttaa aaaattataa 780

gaactacccg ttattgacat ctttctgtgt gccaaggact ttatgtgctt tgcgtcattt 840

aattttgaaa acagttatct tccgccatag ataactacta tggttatctt ctgcctctca 900

cagatgaaga aactaaggca ccgagatttt aagaaactta attacacagg ggataaatgg 960

cagcaatcga gattgaagtc aagcctaacc agggcttttg cgggagcgca tgccttttgg 1020

ctgtaattcg tgcatttttt tttaagaaaa acgcctgcct tctgcgtgag attctccaga 1080

gcaaactggg cggcatgggc cctgtggtct tttcgtacag agggcttcct ctttggctct 1140

ttgcctggtt gtttccaaga tgtactgtgc ctcttacttt cggttttgaa aacatgaggg 1200

ggttgggcgt ggtagcttac gcctgtaatc ccagcactta gggaggccga ggcgggagga 1260

tggcttgagg tccgtagttg agaccagcct ggccaacatg gtgaagcctg gtctctacaa 1320

aaaataataa caaaaattag ccgggtgtgg tggctcgtgc ctgtggtccc agctgctccg 1380

gtggctgagg cgggaggatc tcttgagctt aggcttttga gctatcatgg cgccagtgca 1440

ctccagcgtg ggcaacagag cgagaccctg tctctcaaaa aagaaaaaaa aaaaaaaaga 1500

aagagaaaag aaaagaaaga aagaagtgaa ggtttgtcag tcaggggagc tgtaaaacca 1560

ttaataaaga taatccaaga tggttaccaa gactgttgag gacgccagag atcttgagca 1620

ctttctaagt acctggcaat acactaagcg cgctcacctt ttcctctggc aaaacatgat 1680

cgaaagcaga atgttttgat catgagaaaa ttgcatttaa tttgaataca atttatttac 1740

aacataaagg ataatgtata tatcaccacc attactggta tttgctggtt atgttagatg 1800

tcattttaaa aaataacaat ctgatattta aaaaaaaatc ttattttgaa aatttccaaa 1860

gtaatacatg ccatgcatag accatttctg gaagatacca caagaaacat gtaatgatga 1920

ttgcctctga aggtctattt tcctcctctg acctgtgtgt gggttttgtt tttgttttac 1980

tgtgggcata aattaatttt tcagttaagt tttggaagct taaataactc tccaaaagtc 2040

ataaagccag taactggttg agcccaaatt caaacccagc ctgtctgata cttgtcctct 2100

tcttagaaaa gattacagtg atgctctcac aaaatcttgc cgccttccct caaacagaga 2160

gttccaggca ggatgaatct gtgctctgat ccctgaggca tttaatatgt tcttattatt 2220

agaagctcag atgcaaagag ctctcttagc ttttaatgtt atgaaaaaaa tcaggtcttc 2280

attagattcc ccaatccacc tcttgatggg gctagtagcc tttccttaat gatagggtgt 2340

ttctagagag atatatctgg tcaaggtggc ctggtactcc tccttctccc cacagcctcc 2400

cagacaagga ggagtagctg ccttttagtg atcatgtacc ctgaatataa gtgtatttaa 2460

aagaatttta tacacatata tttagtgtca atctgtatat ttagtagcac taacacttct 2520

cttcattttc aatgaaaaat atagagttta taatattttc ttcccacttc cccatggatg 2580

gtctagtcat gcctctcatt ttggaaagta ctgtttctga aacattaggc aatatattcc 2640

caacctggct agtttacagc aatcacctgt ggatgctaat taaaacgcaa atcccactgt 2700

cacatgcatt actccatttg atcataatgg aaagtatgtt ctgtcccatt tgccatagtc 2760

ctcacctatc cctgttgtat tttatcgggt ccaactcaac catttaaggt atttgccagc 2820

tcttgtatgc atttaggttt tgtttctttg ttttttagct catgaaatta ggtacaaagt 2880

cagagagggg tctggcatat aaaacctcag cagaaataaa gaggttttgt tgtttggtaa 2940

gaacatacct tgggttggtt gggcacggtg gctcgtgcct gtaatcccaa cactttggga 3000

ggccaaggca ggctgatcac ttgaagttgg gagttcaaga ccagcctggc caacatggtg 3060

aaatcccgtc tctactgaaa atacaaaaat taaccaggca tggtggtgtg tgcctgtagt 3120

cccaggaatc acttgaaccc aggaggcgga ggttgcagtg agctgagatc tcaccactgc 3180

acactgcact ccagcctggg caatggaatg agattccatc ccaaaaaata aaaaaataaa 3240

aaaataaaga acataccttg ggttgatcca cttaggaacc tcagataata acatctgcca 3300

cgtatagagc aattgctatg tcccaggcac tctactagac acttcataca gtttagaaaa 3360

tcagatgggt gtagatcaag gcaggagcag gaaccaaaaa gaaaggcata aacataagaa 3420

aaaaaatgga aggggtggaa acagagtaca ataacatgag taatttgatg ggggctatta 3480

tgaactgaga aatgaacttt gaaaagtatc ttggggccaa atcatgtaga ctcttgagtg 3540

atgtgttaag gaatgctatg agtgctgaga gggcatcaga agtccttgag agcctccaga 3600

gaaaggctct taaaaatgca gcgcaatctc cagtgacaga agatactgct agaaatctgc 3660

tagaaaaaaa acaaaaaagg catgtataga ggaattatga gggaaagata ccaagtcacg 3720

gtttattctt caaaatggag gtggcttgtt gggaaggtgg aagctcattt ggccagagtg 3780

gaaatggaat tgggagaaat cgatgaccaa atgtaaacac ttggtgcctg atatagcttg 3840

acaccaagtt agccccaagt gaaataccct ggcaatatta atgtgtcttt tcccgatatt 3900

cctcaggtac tccaaagatt caggtttact cacgtcatcc agcagagaat ggaaagtcaa 3960

atttcctgaa ttgctatgtg tctgggtttc atccatccga cattgaagtt gacttactga 4020

agaatggaga gagaattgaa aaagtggagc attcagactt gtctttcagc aaggactggt 4080

ctttctatct cttgtactac actgaattca cccccactga aaaagatgag tatgcctgcc 4140

gtgtgaacca tgtgactttg tcacagccca agatagttaa gtggggtaag tcttacattc 4200

ttttgtaagc tgctgaaagt tgtgtatgag tagtcatatc ataaagctgc tttgatataa 4260

aaaaggtcta tggccatact accctgaatg agtcccatcc catctgatat aaacaatctg 4320

catattggga ttgtcaggga atgttcttaa agatcagatt agtggcacct gctgagatac 4380

tgatgcacag catggtttct gaaccagtag tttccctgca gttgagcagg gagcagcagc 4440

agcacttgca caaatacata tacactctta acacttctta cctactggct tcctctagct 4500

tttgtggcag cttcaggtat atttagcact gaacgaacat ctcaagaagg tataggcctt 4560

tgtttgtaag tcctgctgtc ctagcatcct ataatcctgg acttctccag tactttctgg 4620

ctggattggt atctgaggct agtaggaagg gcttgttcct gctgggtagc tctaaacaat 4680

gtattcatgg gtaggaacag cagcctattc tgccagcctt atttctaacc attttagaca 4740

tttgttagta catggtattt taaaagtaaa acttaatgtc ttcctttttt ttctccactg 4800

tctttttcat agatcgagac atgtaagcag catcatggag gtaagttttt gaccttgaga 4860

aaatgttttt gtttcactgt cctgaggact atttatagac agctctaaca tgataaccct 4920

cactatgtgg agaacattga cagagtaaca ttttagcagg gaaagaagaa tcctacaggg 4980

tcatgttccc ttctcctgtg gagtggcatg aagaaggtgt atggccccag gtatggccat 5040

attactgacc ctctacagag agggcaaagg aactgccagt atggtattgc aggataaagg 5100

caggtggtta cccacattac ctgcaaggct ttgatctttc ttctgccatt tccacattgg 5160

acatctctgc tgaggagaga aaatgaacca ctcttttcct ttgtataatg ttgttttatt 5220

cttcagacag aagagaggag ttatacagct ctgcagacat cccattcctg tatggggact 5280

gtgtttgcct cttagaggtt cccaggccac tagaggagat aaagggaaac agattgttat 5340

aacttgatat aatgatacta taatagatgt aactacaagg agctccagaa gcaagagaga 5400

gggaggaact tggacttctc tgcatcttta gttggagtcc aaaggctttt caatgaaatt 5460

ctactgccca gggtacattg atgctgaaac cccattcaaa tctcctgtta tattctagaa 5520

cagggaattg atttgggaga gcatcaggaa ggtggatgat ctgcccagtc acactgttag 5580

taaattgtag agccaggacc tgaactctaa tatagtcatg tgttacttaa tgacggggac 5640

atgttctgag aaatgcttac acaaacctag gtgttgtagc ctactacacg cataggctac 5700

atggtatagc ctattgctcc tagactacaa acctgtacag cctgttactg tactgaatac 5760

tgtgggcagt tgtaacacaa tggtaagtat ttgtgtatct aaacatagaa gttgcagtaa 5820

aaatatgcta ttttaatctt atgagaccac tgtcatatat acagtccatc attgaccaaa 5880

acatcatatc agcatttttt cttctaagat tttgggagca ccaaagggat acactaacag 5940

gatatactct ttataatggg tttggagaac tgtctgcagc tacttctttt aaaaaggtga 6000

tctacacagt agaaattaga caagtttggt aatgagatct gcaatccaaa taaaataaat 6060

tcattgctaa cctttttctt ttcttttcag gtttgaagat gccgcatttg gattggatga 6120

attccaaatt ctgcttgctt gctttttaat attgatatgc ttatacactt acactttatg 6180

cacaaaatgt agggttataa taatgttaac atggacatga tcttctttat aattctactt 6240

tgagtgctgt ctccatgttt gatgtatctg agcaggttgc tccacaggta gctctaggag 6300

ggctggcaac ttagaggtgg ggagcagaga attctcttat ccaacatcaa catcttggtc 6360

agatttgaac tcttcaatct cttgcactca aagcttgtta agatagttaa gcgtgcataa 6420

gttaacttcc aatttacata ctctgcttag aatttggggg aaaatttaga aatataattg 6480

acaggattat tggaaatttg ttataatgaa tgaaacattt tgtcatataa gattcatatt 6540

tacttcttat acatttgata aagtaaggca tggttgtggt taatctggtt tatttttgtt 6600

ccacaagtta aataaatcat aaaacttgat gtgttatctc ttatatctca ctcccactat 6660

taccccttta ttttcaaaca gggaaacagt cttcaagttc cacttggtaa aaaatgtgaa 6720

ccccttgtat atagagtttg gctcacagtg taaagggcct cagtgattca cattttccag 6780

attaggaatc tgatgctcaa agaagttaaa tggcatagtt ggggtgacac agctgtctag 6840

tgggaggcca gccttctata ttttagccag cgttctttcc tgcgggccag gtcatgagga 6900

gtatgcagac tctaagaggg agcaaaagta tctgaaggat ttaatatttt agcaaggaat 6960

agatatacaa tcatcccttg gtctccctgg gggattggtt tcaggacccc ttcttggaca 7020

ccaaatctat ggatatttaa gtcccttcta taaaatggta tagtatttgc atataaccta 7080

tccacatcct cctgtatact ttaaatcatt tctagattac ttgtaatacc taatacaatg 7140

taaatgctat gcaaatagtt gttattgttt aaggaataat gacaagaaaa aaaagtctgt 7200

acatgctcag taaagacaca accatccctt tttttcccca gtgtttttga tccatggttt 7260

gctgaatcca cagatgtgga gcccctggat acggaaggcc cgctgtactt tgaatgacaa 7320

ataacagatt taaaattttc aaggcatagt tttatacctg a 7361

<210> 2

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M exon 1 target (B2M exon 1 target)

<400> 2

tgacagcatt cgggccgaga tgtctcgctc cgtggcctta gctgtg 46

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Target1 (B2M Target 1)

<400> 3

ggccgagatg tctcgctccg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Target2 (B2M Target 2)

<400> 4

cgcgagcaca gctaaggcca 20

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Target1 forward primer (B2M Target1 forward primer)

<400> 5

aaacggccga gatgtctcgc tccggt 26

<210> 6

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Target1reverse primer (B2M Target1reverse primer)

<400> 6

taaaaccgga gcgagacatc tcggcc 26

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Target2 forward primer (B2M Target2 forward primer)

<400> 7

cacccgcgag cacagctaag gcca 24

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Target2 reverse primer (B2M Target2 reverse primer)

<400> 8

aaactggcct tagctgtgct cgcg 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M EX1 WT forward primer (B2M EX1 WT forward primer)

<400> 9

tctcgaatga aaaatgcagg tccg 24

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M EX1 WT reverse primer (B2M EX1 WT reverse primer)

<400> 10

tgacgcttat cgacgcccta aactt 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M EGFP forward primer (B2M EGFP HDR forward primer)

<400> 11

ccacaacgtc tatatcatgg ccgac 25

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M EGFP reverse primer (B2M EGFP HDR reverse primer)

<400> 12

gtttgctctg gagaatctca cgcag 25

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M mCherry HDR forward primer (B2M mCherry HDR forward primer)

<400> 13

tctagagcct gcagtctcga caagc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M mCherry HDR reverse primer (B2M mCherry HDR reverse primer)

<400> 14

gtttgctctg gagaatctca cgcag 25

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Probe EGFP forward primer (Probe EGFP Forward primer)

<400> 15

cacctacggc aagctgaccc tgaagttcat 30

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Probe EGFP reverse primer (Probe EGFP reverse primer)

<400> 16

atgtgatcgc gcttctcgtt ggggtctttg 30

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target1 forward primer (B2M Off-target1 forward primer)

<400> 17

ccccaaccag attgccccat 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target 1reverse primer (B2M Off-target 1reverse primer)

<400> 18

tccctgcagc ggatcctcaa 20

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target2 forward primer (B2M Off-target2 forward primer)

<400> 19

tccagctgca aaagggagaa gaga 24

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target2 reverse primer (B2M Off-target2 reverse primer)

<400> 20

agggaagaag ccaaagcgaa 20

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target3 forward primer (B2M Off-target3 forward primer)

<400> 21

tgcctgaggt ccaaggaaga gct 23

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target3 reverse primer (B2M Off-target3 reverse primer)

<400> 22

tgagcccaaa tccagccaca 20

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target5 forward primer (B2M Off-target5 forward primer)

<400> 23

agctgtatga cagcccctct gtgc 24

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target5 reverse primer (B2M Off-target5 reverse primer)

<400> 24

tagactcacg aggccggcga 20

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target8 forward primer (B2M Off-target8 forward primer)

<400> 25

cggtgactgt ggctgagact gagac 25

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target8 reverse primer (B2M Off-target8 reverse primer)

<400> 26

cgccgctacc ccgaagaaaa 20

<210> 27

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target16 forward primer (B2M Off-target16 forward primer)

<400> 27

aggaactggg gcttgaatgg gg 22

<210> 28

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M Off-target16 reverse primer (B2M Off-target16 reverse primer)

<400> 28

ggctgttgcc cagtcctgga aa 22

<210> 29

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Oct4 forward primer (Oct 4 forward primer)

<400> 29

gagcaaaacc cggaggagt 19

<210> 30

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Oct4 reverse primer (Oct 4 reverse primer)

<400> 30

ttctctttcg ggcctgcac 19

<210> 31

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Sox2 forward primer (Sox 2 forward primer)

<400> 31

gggaaatggg aggggtgcaa aa 22

<210> 32

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Sox2 reverse primer (Sox 2 reverse primer)

<400> 32

ttgcgtgagt gtggatggga tt 22

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NANOG forward primer (NANOG forward primer)

<400> 33

gcttgccttg ctttgaagca 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NANOG reverse primer (NANOG reverse primer)

<400> 34

ttcttgaccg ggaccttgtc 20

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Beta-actin forward primer

<400> 35

tggcacccag cacaatgaag 20

<210> 36

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Beta-actin reverse primer

<400> 36

gatggagggg ccggactc 18

<210> 37

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Pax6 forward primer (Pax 6 forward primer)

<400> 37

caataatgtt gacggtgact atc 23

<210> 38

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Pax6 reverse primer (Pax 6 reverse primer)

<400> 38

agaaggaagc gacactctg 19

<210> 39

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MHC-alpha forward primer (MHC-alpha forward primer)

<400> 39

gtcattgctg aaaccgagaa tg 22

<210> 40

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MHC-alpha reverse primer (MHC-alpha reverse primer)

<400> 40

gcaaagtact ggatgacacg ct 22

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AFP forward primer (AFP forward primer)

<400> 41

cccgaacttt ccaagccata 20

<210> 42

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AFP reverse primer (AFP reverse primer)

<400> 42

tacatgggcc acatccagg 19

<210> 43

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LPL forward primer (LPL forward primer)

<400> 43

tcaactggat ggaggag 17

<210> 44

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LPL reverse primer (LPL reverse primer)

<400> 44

ggggcttctg catactcaaa 20

<210> 45

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PPAR-gamma forward primer

<400> 45

ctcctattga cccagaaagc 20

<210> 46

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PPAR-gamma reverse primer (PPAR-gamma reverse primer)

<400> 46

gtagagctga gtcttctcag 20

<210> 47

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ALP forward primer (ALP forward primer)

<400> 47

tggagcttca gaagctcaac acca 24

<210> 48

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ALP reverse primer (ALP reverse primer)

<400> 48

atctcgttgt ctgagtacca gtcc 24

<210> 49

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OCN forward primer (OCN forward primer)

<400> 49

ggcagcgagg tagtgaagag 20

<210> 50

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OCN reverse primer (OCN reverse primer)

<400> 50

cagcagagcg acaccctaga c 21

<210> 51

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> COL2a forward primer (COL2a forward primer)

<400> 51

atgattcgcc tcggggctcc 20

<210> 52

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> COL2a reverse primer (COL2a reverse primer)

<400> 52

cattactggg aactgggcgc 20

<210> 53

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ACAN forward primer

<400> 53

cactgttacc gccacttccc 20

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ACAN reverse primer (ACAN reverse primer)

<400> 54

accagcggaa gtccccttcg 20

<210> 55

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> GAPDH forward primer (GAPDH forward primer)

<400> 55

ccccttcatt gacctcaact aca 23

<210> 56

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> GAPDH reverse primer (GAPDH reverse primer)

<400> 56

ttgctgatga tcttgaggct gt 22

<210> 57

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutation in exon 1 of allel 1 (exon 1 mutation of allele 1)

<400> 57

ccgagatgtc tcgctccgtg gttagctgtg ctcgcgctac tctct 45

<210> 58

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutation in exon 1 of allel 2 (allele 2 exon 1 mutation)

<400> 58

ccgagatgtc tcgctccgtg gcccttagct gtgctcgcgc tactctct 48

<210> 59

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 2nd mutation in exon 1 of allel 2 (2 nd mutation in exon 1 of allele 2)

<400> 59

ccgagatgtc tcgctccgtg ccttagctgt gctcgcgcta ctctct 46

Claims (21)

1. A method for preparing human mesenchymal stromal cells with reduced immunogenicity, comprising:

(a) mutating two alleles of a human pluripotent stem cell beta 2-microglobulin gene;

(b) differentiating the mutated human pluripotent stem cells into derived human mesenchymal stromal cells having a biallelic mutation in the beta 2-microglobulin gene;

wherein the derived human mesenchymal stromal cells do not express beta 2-microglobulin and express attenuated or do not express HLA class I molecules located on the cell surface.

2. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

3. The method of claim 1, wherein the human pluripotent stem cell is an Induced Pluripotent Stem Cell (iPSC).

4. The method of claim 3, wherein the induced pluripotent stem cells are derived from peripheral blood cells.

5. The method of claim 4, further comprising determining that a mutation is present in both alleles of the β 2-microglobulin gene.

6. The method of any one of claims 1-5, wherein the derived human mesenchymal stromal cells having biallelic mutations on the β 2-microglobulin gene express at least one of CD29, CD44, CD73, CD90, CD105, CD166, or a combination thereof, and comprise low or no expression of any one of CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, HLA-C, or a combination thereof.

7. A method according to any one of claims 1 to 6 wherein the allele is mutated by one or more plasmids comprising a guide nucleic acid sequence targeting the β 2-microglobulin gene part and an endonuclease.

8. The method of claim 7, wherein the endonuclease comprises a nucleic acid sequence Cas9 associated with clustered regularly spaced short palindromic repeats.

9. The method of claim 7 or 8, wherein said guide nucleic acid sequence targeting the β 2-microglobulin gene part comprises a sequence selected from the group consisting of SEQ ID No.3 or SEQ ID No. 4.

10. The method of any one of claims 1-9, wherein the human pluripotent stem cell is mutated by:

i. transfecting the human pluripotent stem cell with a first plasmid to mutate a first allele of a β 2-microglobulin gene, the plasmid comprising an endonuclease, a guide nucleic acid sequence targeting a portion of the β 2-microglobulin gene, and at least one selectable marker;

selecting cells having a mutation in the first allele of the β 2-microglobulin gene by the at least one selectable marker;

transfecting a cell having a mutation in a first allele of a β 2-microglobulin gene with a second plasmid to mutate a second allele of the β 2-microglobulin gene, the plasmid comprising an endonuclease, a guide nucleic acid sequence targeting a portion of the β 2-microglobulin gene, and at least one other selectable marker;

selecting, by the at least one other selectable marker, a cell having a mutation in a first allele of a β 2-microglobulin gene and a mutation in a second allele of a β 2-microglobulin gene.

11. The method of any one of claims 1-9, wherein the human pluripotent stem cell is transfected with a plasmid comprising an endonuclease, a guide nucleic acid sequence targeting a portion of the β 2-microglobulin gene, and a selectable marker to mutate both alleles of the β 2-microglobulin gene; selecting for cells having a mutation in both alleles of the β 2-microglobulin gene by means of the selection marker.

12. Genetically modified human mesenchymal stromal cells, including cells that do not express β 2-microglobulin and express attenuated or do not express HLA-class I molecules located on the cell surface.

13. The cell of claim 12, expressing at least one of CD29, CD44, CD73, CD90, CD105, CD166, or a combination thereof, and comprising low or no expression of any one of CD14, CD34, HLA-DR, CD25, CD45, B2M, HLA-A, HLA-B, HLA-C, or a combination thereof.

14. The cell of claim 12 or 13, wherein the first allelic mutation of the β 2-microglobulin gene is a deletion of the 47 th and 48 th base pairs of SEQ ID No.1 and the second allelic mutation of the β 2-microglobulin gene comprises an insertion of a cytosine between the 46 th and 47 th base pairs of SEQ ID No. 1.

15. The cell of claim 12 or 13, wherein the first allelic mutation of the β 2-microglobulin gene is a deletion of the 47 th and 48 th base pairs of SEQ ID No.1 and the second allelic mutation of the β 2-microglobulin gene is a deletion of the 46 th base pair of SEQ ID No. 1.

16. Use of the cell of claims 12-15 in therapy.

17. A kit for preparing mesenchymal stromal cells of reduced immunogenicity, the kit comprising:

(a) one or more plasmids capable of mutating both alleles of the human pluripotent stem cell β 2-microglobulin gene;

(b) a culture medium of mesenchymal stromal cells for differentiating human pluripotent stem cells into human mesenchymal stromal cells.

18. The kit of claim 17, further comprising two or more primer sequences selected from SEQ ID nos. 5-28.

19. The kit of claim 17 or 18, wherein the one or more plasmids comprise a guide nucleic acid sequence targeting the β 2-microglobulin gene portion and an endonuclease.

20. The kit of claim 19, wherein the endonuclease comprises a nucleic acid sequence Cas9 associated with a clustered, regularly spaced, short palindromic repeat.

21. The kit of claim 19 or 20, wherein said guide nucleic acid sequence targeting the β 2-microglobulin gene portion comprises a sequence selected from the group consisting of SEQ ID No.3 and SEQ ID No. 4.

Images