Modified laminin 521 protein with enhanced activity and use thereof,

Abstract

Provided is a modified recombinant Laminin 521 protein. By means of designs such as sequence fragment truncation and label linking, the recombinant protein has a higher activity compared with a full-length protein, and can maintain stem cell stemness, karyotype stability, etc. at a lower use concentration. In addition, the recombinant protein has a more accurate assembly proportion and a more accurate aggregation form, and is easier to prepare and lower in terms of production cost.

Description

Activity-enhanced modified Laminin 521 protein and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410610671.3 filed with the Patent Office of China on May 16, 2024, entitled “Activity-enhanced modified Laminin 521 protein and its application,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biomedicine, and specifically to a modified Laminin 521 protein with enhanced activity, a composition, and applications thereof. Background Art

[0004] Laminin 521 (LN521), also known as laminin 521, is a heterotrimeric glycoprotein composed of three chains: α5, β2, and γ1. It is expressed in stem cells and most basement membranes, particularly in the nerve sheath, muscle-nerve junction, and glomerular basement membrane (GBM). As a key cell adhesion protein in the natural stem cell niche, laminin 521 plays an important role in supporting stem cell growth and maintaining the stability of basement membrane structure and properties.

[0005] Laminin 521 maintains stem cell pluripotency, promotes cell spreading, supports the clonal expansion of pluripotent human stem cells, and supports the growth of skin keratinocytes. It also regulates important physiological processes such as cell adhesion, migration, differentiation, phenotypic maintenance, and matrix-mediated signaling. Compared to laminin 511, laminin 521 can support the maintenance and expansion of stem cell monolayers in the absence of apoptosis inhibitors, making it a superior alternative to laminin 511 and more suitable for the culture of human pluripotent stem cells.

[0006] Laminin 521 is currently being used in regenerative medicine for stem cell expansion and culture, directed stem cell differentiation, organoid culture, and the maintenance of differentiated primary cell growth. However, as a more widely used stem cell matrix, laminin 521 is difficult to express and requires high concentrations (15-20 μg / ml), resulting in a very high price tag. This significantly limits the further development and accessibility of stem cell therapies.

[0007] Therefore, seeking a recombinant Laminin 521 protein with higher activity and lower preparation cost for use in regenerative medicine is still a breakthrough that is urgently needed in current technology and the market.

[0008] In view of this, this application is filed.

[0009] SUMMARY OF THE INVENTION

[0010] To address the above-mentioned technical problems, the present application has obtained a modified recombinant Laminin 521 protein with higher activity through design research and activity verification of the natural Laminin 521 sequence. Based on this, the present application provides the following technical solutions:

[0011] The present application first provides an (isolated) remodeled Laminin 521 protein, wherein the remodeled Laminin 521 protein is assembled from a truncated α5 chain, a truncated β2 chain and / or a truncated γ1 chain in the Laminin 521 protein.

[0012] In some aspects, the sequences of the truncated α5 chain, the truncated β2 chain, and the truncated γ1 chain are respectively as follows:

[0013] Truncated α5 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO.1;

[0014] Truncated β2 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO. 2;

[0015] Truncated γ1 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO. 3;

[0016] In some embodiments, the α5 chain is a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to the polypeptide sequence of SEQ ID NO. 1; and preferably, the homology is derived from conservative amino acid substitutions;

[0017] In some embodiments, the β2 chain is a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to the polypeptide sequence SEQ ID NO. 2; and preferably, the homology is derived from conservative amino acid substitutions;

[0018] In some embodiments, the γ1 chain is a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to the polypeptide sequence SEQ ID NO. 3; and preferably, the homology is derived from conservative amino acid substitutions.

[0019] In some aspects, the remodeled Laminin 521 protein further comprises a tag sequence;

[0020] In some specific embodiments, the N-terminus of the truncated β2 chain is connected to, for example, an Fc tag;

[0021] In some embodiments, the N-terminus of the truncated γ1 chain is linked to, for example, an Fc tag.

[0022] The present application also provides a composition comprising any of the above-mentioned modified Laminin 521 proteins.

[0023] In some aspects, the composition can be in the form of an extracellular matrix material, a culture substrate, or a scaffold material;

[0024] In some specific embodiments, such compositions may further comprise components such as collagen and / or gelatin.

[0025] In some aspects, the composition is a pharmaceutical composition;

[0026] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0027] The present application also provides a recombinant vector comprising a nucleic acid sequence for expressing any of the aforementioned modified Laminin 521 proteins;

[0028] In some aspects, the recombinant vector is a recombinant expression vector;

[0029] In some embodiments, the nucleic acid sequence further comprises a linker sequence, a polyadenylation signal sequence, a termination sequence, and the like.

[0030] The present application also provides a host cell comprising the above-mentioned recombinant vector.

[0031] The present application also provides a kit, which comprises any of the aforementioned modified Laminin 521 proteins; preferably, it also comprises a container.

[0032] The present application also provides a method for maintaining stem cell pluripotency in vitro, comprising: administering any of the aforementioned modified Laminin 521 proteins, or administering any of the aforementioned compositions.

[0033] The present application also provides an in vitro stem cell expansion and culture method, characterized in that it comprises: a step of administering any of the aforementioned modified Laminin 521 proteins, or a step of administering any of the aforementioned compositions.

[0034] The present application also provides an in vitro stem cell directed differentiation method, comprising: a step of administering any of the aforementioned modified Laminin 521 proteins, or a step of administering the aforementioned composition.

[0035] The present application also provides an organoid culture method, comprising: a step of administering the modified Laminin521 protein described in any one of the aforementioned items, or a step of administering the composition described in any one of the aforementioned items.

[0036] The modified Laminin 521 protein of the present application has at least the following advantages:

[0037] 1) Compared with the full-length Laminin 521 protein, the modified Laminin 521 protein has higher activity and can maintain similar functions at lower concentrations. It can effectively maintain the stemness gene expression and karyotype stability of stem cells, making it a more economical stem cell extracellular matrix material.

[0038] 2) Compared with the full-length Laminin 521 protein, the truncated Laminin 521 protein of the present application has a higher expression level, is relatively easier to prepare and obtain, and has a lower production cost.

[0039] 3) Through the selection of truncation intervals and tag design, the truncated α5 / β2 / γ1 subunits prepared in this application have a more correct assembly ratio and a better aggregation form. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0041] Figure 1, 521 protein electrophoresis diagram; wherein, a is the electrophoresis diagram of the modified 521 protein of the present application, and b is the electrophoresis diagram of the full-length 521 protein.

[0042] Figure 2, HPLC-MALS graph of 521 protein; wherein, a is the HPLC-MALS graph of the modified 521 protein of the present application, and b is the HPLC-MALS graph of the full-length 521 protein.

[0043] Figure 3. Comparison of the wall adhesion performance between the modified 521 protein and the full-length 521 protein.

[0044] Figure 4. Modified 521 protein can support the rapid expansion of single-cell hPSCs.

[0045] Figure 5. The modified 521 protein can maintain the stemness of iPSCs after several generations of passages.

[0046] Figure 6. The modified 521 protein maintains the karyotype stability of iPSCs after several passages.

[0047] Detailed Description of the Invention

[0048] Although the present application can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that verifies the principle of the present application.It should be emphasized that the present application is not limited to the specific embodiment illustrated.In addition, any section heading used herein is only for organizational purposes and is not to be interpreted as limiting the subject matter described.The following terms or definitions are provided only to help understand the present application.These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0049] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as that generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0050] The terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0051] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" include a plural of that noun.

[0052] Furthermore, the terms first, second, third, (a), (b), (c), and the like, in the specification and claims, are used to distinguish similar elements and are not necessarily intended to describe a sequential or chronological order. It is understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described herein can be practiced in other sequences than described or illustrated herein.

[0053] The term "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] The terms "such as" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly enumerated in the specification.

[0055] The terms "or more", "at least", "more than", etc., for example, "at least one" should be understood to include but are not limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 800000, 800000, 800000, 800000, 800000 Also includes any larger numbers or fractions in between.

[0056] Conversely, the term "no more than" includes every value that is less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0057] The terms "plurality", "at least two", "two or more", "at least a second", etc., should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Any larger number or fraction therebetween is also included.

[0058] The terms "approximately" and "substantially" represent an accuracy range that can be understood by those skilled in the art and can still ensure the technical effect of the characteristics discussed. This term usually means ±10%, preferably ±5%, of the deviation from the indicated value.

[0059] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the recited range, and where appropriate, fractions thereof (e.g., tenths and hundredths of integers).

[0060] The modified laminin 521 or Laminin 521 disclosed herein.

[0061] As used herein, the term "Laminin 521" or "laminin 521" refers to a protein formed by the binding of α5, β2, and γ1 chains. This term should be understood to include both recombinant laminin 521 and naturally derived heterotrimeric laminin 521. In preferred embodiments, laminin 521 comprises recombinant laminin 521.

[0062] The terms "modified laminin 521" or "modified laminin 521" or "modified Laminin 521" used in this article have the same meaning and are used to distinguish between natural laminin 521 or full-length laminin 521. This type of modification is mainly achieved by shortening the length of the α5, β2 and γ1 chain sequences in the laminin 521 protein.

[0063] In some aspects, the remodeled Laminin 521 protein is a type of protein assembled from a truncated α5 chain, a truncated β2 chain and / or a truncated γ1 chain in the Laminin 521 protein, wherein the truncation is performed while retaining its core function.

[0064] Therefore, this type of modified protein can be assembled from a truncated α5 chain and a non-truncated β2 chain and a γ1 chain; or it can be assembled from a truncated β2 chain and a non-truncated α5 chain and a γ1 chain; or it can be assembled from a truncated γ1 chain and a non-truncated β2 chain and an α5 chain; or it can be assembled from a truncated α5 chain, a β2 chain and a non-truncated γ1 chain; or it can be assembled from a truncated α5 chain, a γ1 chain and a non-truncated β2 chain; or it can be assembled from a truncated α5 chain, a truncated β2 chain and a truncated γ1 chain. As long as the protein can be assembled and has basic activity, all types of truncation combinations are within the scope of this application.

[0065] In some embodiments, such modified proteins are most preferably assembled from truncated α5 chain, β2 chain, and γ1 chain.

[0066] In some embodiments, the sequences of the truncated α5 chain, truncated β2 chain, and truncated γ1 chain are as follows:

[0067] Truncated α5 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO.1;

[0068] Truncated β2 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO.2;

[0069] Truncated γ1 chain: a polypeptide having at least 80% homology to the polypeptide sequence of SEQ ID NO. 3.

[0070] It is understood that, on the basis of satisfying the chain activity, the corresponding homology limitation is within the scope of protection of this application.

[0071] In some embodiments, the basic sequences of SEQ ID NO. 1, 2 and 3 described herein are as follows:

[0072] Said SEQ ID NO.1 (α5 truncated chain):

[0073] Said SEQ ID NO.2 (β2 truncated chain):

[0074] Said SEQ ID NO.3 (γ1 truncated chain):

[0075] In some embodiments, the amino acid changes in the above amino acid homology include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another amino acid within the same class, for example, the substitution of an acidic amino acid with another acidic amino acid, the substitution of a basic amino acid with another basic amino acid, or the substitution of a neutral amino acid with another neutral amino acid.

[0076] Exemplary substitutions are shown in the table below (amino acid substitutions).

[0077] Therefore, in some preferred embodiments, the truncated α5 chain may be:

[0078] A polypeptide having at least 90% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0079] A polypeptide having at least 91% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0080] A polypeptide having at least 92% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0081] A polypeptide having at least 93% homology to the polypeptide sequence of SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0082] A polypeptide having at least 94% homology to the polypeptide sequence of SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0083] A polypeptide having at least 95% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0084] A polypeptide having at least 96% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0085] A polypeptide having at least 97% homology to the polypeptide sequence of SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0086] A polypeptide having at least 98% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0087] A polypeptide having at least 99% homology to the polypeptide sequence SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0088] A polypeptide having at least 100% homology to the polypeptide sequence of SEQ ID NO. 1, wherein the homology is a conservative amino acid substitution;

[0089] In some preferred embodiments, the truncated β2 chain may be:

[0090] A polypeptide having at least 90% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0091] A polypeptide having at least 91% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0092] A polypeptide having at least 92% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0093] A polypeptide having at least 93% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0094] A polypeptide having at least 94% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0095] A polypeptide having at least 95% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0096] A polypeptide having at least 96% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0097] A polypeptide having at least 97% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0098] A polypeptide having at least 98% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0099] A polypeptide having at least 99% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0100] A polypeptide having at least 100% homology to the polypeptide sequence of SEQ ID NO. 2, wherein the homology is a conservative amino acid substitution;

[0101] In some preferred embodiments, the truncated γ1 chain may be:

[0102] A polypeptide having at least 90% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0103] A polypeptide having at least 91% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0104] A polypeptide having at least 92% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0105] A polypeptide having at least 93% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0106] A polypeptide having at least 94% homology to the polypeptide sequence of SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0107] A polypeptide having at least 95% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0108] A polypeptide having at least 96% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0109] A polypeptide having at least 97% homology to the polypeptide sequence of SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0110] A polypeptide having at least 98% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0111] A polypeptide having at least 98% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0112] A polypeptide having at least 9100% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is conservative amino acid substitution.

[0113] In some ways, in order to further stabilize the activity of the modified protein, the present application attempts to connect an Fc tag to the end of a portion of the truncated chain of the modified Laminin 521 protein, which has been proven to play a structural stabilizing role.

[0114] It is understood that, given a known sequence, the modified Laminin 521 protein of the present application can be prepared by any method known in the art, without limitation. For example, recombinant expression, the most common method, can be used to separately express the truncated α5 chain, the truncated β2 chain, and the truncated γ1 chain and then assemble the proteins, or directly co-express the three chains and then assemble them.

[0115] The composition disclosed in the present invention is a type of composition, which is based on the modified Laminin 521 protein described above.

[0116] In some aspects, the composition can be in the form of an extracellular matrix material, a culture substrate, or a scaffold material.

[0117] The extracellular matrix material of the present application is to contain the above-mentioned remodeled laminin 521, and the extracellular matrix material comprising collagen and / or gelatin etc. The extracellular matrix material of the present application can be made up of remodeled laminin and collagen, can also be made up of remodeled laminin and gelatin, can also be made up of remodeled laminin, collagen and gelatin, and can also contain other components in addition to this. As other components in addition to this, as long as it is a composition that can be used in cell culture, it is not particularly limited, for example, it is preferably an extracellular matrix component other than collagen and gelatin, and for example fibronectin, Matrigel, proteoglycan, hyaluronic acid, tenascin, elastin, laminin, fibrinogen (fibrin) etc. are enumerated.

[0118] The collagen or gelatin used in the extracellular matrix material of the present application are not particularly limited, and can preferably use the known collagen or gelatin for cell culture purposes. The extracellular matrix material of the present application can be provided in the form of liquid, gel, sponge, sheet, etc., and can also be coated on a plate or utilized in the form of a three-dimensional matrix.

[0119] The extracellular matrix material of the present application can be used as a three-dimensional culture matrix for constructing a three-dimensional tissue structure for regenerative medicine by inducing differentiation of stem cells through the presence of laminin suitable as a scaffold for stem cells. In addition, the extracellular matrix material of the present application can not only be used for the three-dimensional culture of cells, but can also be used as a tissue regeneration induction device for embedding a living body. In addition, by making any laminin isoform be attached to a three-dimensional matrix consisting of collagen or gelatin with a hardness suitable for the stem cells wishing to undergo differentiation induction, a three-dimensional culture environment that is most suitable for the differentiation induction of the stem cells can be provided.

[0120] The culture substrate of the present application is a culture substrate coated with the modified laminin 521 of the present application and collagen and / or gelatin. The cells cultured using the culture substrate of the present application are not particularly limited, and any cells can be used as long as they are culturable cells. Preferably, they are mammalian cells, more preferably mammalian stem cells. Stem cells include adult stem cells, pluripotent stem cells, etc. As adult stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, cardiac stem cells, liver stem cells, small intestinal stem cells, etc. can be listed. As pluripotent stem cells, ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), mGS cells (pluripotent germline stem cells), fusion cells of ES cells and somatic cells, etc. can be listed. As mammals, humans, mice, rats, cattle, pigs, etc. can be listed; preferably humans. The culture substrate of the present application is also useful in culturing cells that are cultured using existing feeder cells without using feeder cells.

[0121] The method for manufacturing the culture substrate of the present application is not particularly limited. For example, a mixed solution of collagen and / or gelatin and modified laminin can be coated on a culture vessel, or the modified laminin of the present application can be further coated after collagen or gelatin or a mixture of the two is coated on the culture vessel. The culture vessel is not limited as long as it can be used for cell culture. Examples include glass or plastic culture dishes, flasks, multi-well plates, culture slides, microcarriers, and polymer membranes such as polyvinylidene fluoride membranes.

[0122] The collagen or gelatin used for coating is not particularly limited, and known collagen or gelatin for cell culture purposes can be preferably used. When used for cell culture for regenerative medicine, collagen or gelatin with confirmed safety for medical use is preferably used, and collagen or gelatin derived from humans is preferably used.

[0123] The scaffold material of the present application is used to induce stem cell differentiation and construct a three-dimensional tissue structure. The scaffold of the present application can be any material containing the modified laminin of the present application, as well as collagen and / or gelatin. The material constituting the scaffold of the present application can be any material, without particular limitation, as long as it functions as a scaffold for cells. Examples include natural polymers such as collagen, gelatin, fibrin, hyaluronic acid, alginic acid, starch, chitin, and pectic acid, inorganic materials such as polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, hydroxyapatite, and calcium phosphate. The scaffold can have any form, such as a gel, sponge, film, mesh, non-woven fabric, or woven fabric. In cases where the scaffold is composed of collagen or gelatin alone, the scaffold of the present application can be manufactured by binding the modified laminin 521 of the present application to the scaffold. In the case of using materials other than collagen and gelatin to form the scaffold, the scaffold of the present application can be manufactured by coating the surface of the scaffold with collagen and / or gelatin and combining it with modified laminin. In addition, the collagen and gelatin that can be used for the scaffold of the present application are the same as those of the culture substrate of the present application mentioned above.

[0124] The compositions disclosed herein are pharmaceutical compositions comprising the modified laminin 521 described above and a pharmaceutically acceptable carrier. Depending on the condition to be treated, the pharmaceutical composition may contain other agents. The pharmaceutical composition may be prepared in any suitable form and generally comprises the modified laminin 521 and a pharmaceutically acceptable carrier. The carrier may be an injectable carrier, a topical carrier, a transdermal carrier, or the like. The formulation may advantageously be in a form for topical administration, such as an ointment, gel, emulsion, spray, dispersion, suspension, or paste.

[0125] The recombinant vector disclosed herein comprises a nucleic acid sequence for expressing any of the aforementioned modified Laminin 521 proteins;

[0126] The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is attached. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be cloned. Another type of vector is a viral vector, in which additional DNA segments can be cloned into the viral genome. Certain vectors are capable of automatically replicating in the host cell into which they are introduced. Other vectors (such as mammalian non-episomal vectors) integrate into the host cell's genome after being introduced into the host cell, thereby replicating along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively attached. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally speaking, expression vectors used in recombinant DNA technology are usually in the form of plasmids. Since plasmids are the most commonly used vector form, "plasmid" and "vector" can be used interchangeably in this specification. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functions. In some embodiments, the recombinant vector herein is a recombinant expression vector, which can be used to express the truncated α5 chain, truncated β2 chain, and truncated γ1 chain of the modified Laminin 521 protein.

[0127] In some specific embodiments, the nucleic acid sequence may further comprise a linker sequence, a polyadenylation signal sequence or a termination sequence.

[0128] The host cell of the present disclosure comprises the above-mentioned recombinant vector.

[0129] As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present disclosure (e.g., a recombinant expression vector) has been introduced. The cell can be a prokaryotic cell, which can be used, for example, to rapidly produce large quantities of the expression vector of the present disclosure, or a eukaryotic cell for functional studies.

[0130] As used herein, the terms "host cell" and "recombinant host cell" are used interchangeably. It should be understood that these terms refer not only to the specific subject cell, but also to the progeny or potential progeny of that cell. Due to variation or environmental influences, certain changes may occur during passage, and thus, such progeny may not actually be identical to the parent cell, but they are still included within the scope of the terms as used herein.

[0131] The in vitro stem cell expansion and culture method disclosed herein is characterized by comprising the step of administering any of the aforementioned modified Laminin 521 proteins, or the step of administering any of the aforementioned compositions.

[0132] The method for maintaining stem cell pluripotency in vitro disclosed herein comprises the step of administering any of the aforementioned modified Laminin 521 proteins, or the step of administering any of the aforementioned compositions.

[0133] The in vitro stem cell directed differentiation method disclosed herein comprises the step of administering any one of the aforementioned modified Laminin521 proteins, or the step of administering the aforementioned composition.

[0134] The organoid culture method disclosed herein comprises the step of administering the modified Laminin 521 protein described in any one of the preceding items, or administering the composition described in any one of the preceding items.

[0135] As long as the method disclosed herein is a method for culturing cells (especially stem cells) in the presence of the modified laminin 521 of the present application, it belongs to the method category of the present application. It can be a method in which the modified laminin 521 of the present application and, for example, collagen and / or gelatin are added to the culture medium and cultured, or a method in which the composite of the modified laminin 521 of the present application and collagen or the composite of the modified laminin 521 of the present application and gelatin are added to the culture medium and cultured, or a method in which the modified laminin of the present application is added to the culture medium and cultured using a culture vessel coated with collagen and / or gelatin. It is preferably a method in which the culture substrate or the scaffold of the present application above is used to culture.

[0136] The cells herein are preferably stem cells. Stem cells refer to cells with self-replication ability and pluripotency, including adult stem cells, pluripotent stem cells, etc. As adult stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, cardiac stem cells, liver stem cells, small intestinal stem cells, etc. can be listed. As pluripotent stem cells, ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), mGS cells (pluripotent germline stem cells), fusion cells of ES cells and somatic cells, etc. can be listed. Mammals are not particularly limited, and humans, mice, rats, cattle, pigs, etc. can be listed. Among them, humans are preferred. That is, the culture method of the present application is preferably used for the culture of human stem cells.

[0137] Finally, it should be noted that, given the known basic activity of "Laminin 521 can maintain the pluripotency of stem cells, promote cell proliferation, support the clonal expansion of pluripotent human stem cells and the growth of skin keratinocytes, and participate in regulating important physiological processes such as cell adhesion, migration, differentiation, phenotypic maintenance, and matrix-mediated signal transduction," since this application has fully demonstrated that the modified Laminin 521 of this application has the same or even higher activity as the intact or full-length Laminin 521. Therefore, practical applications or methods based on the basic activity of the full-length protein are theoretically applicable to the modified Laminin 521 of this application and fall within the scope of protection of this patent, and are not exhaustively listed here.

[0138] The present application is further described by the accompanying drawings and the following examples. The drawings and examples are only intended to illustrate specific embodiments of the present application and should not be understood as limiting the scope of the present application in any way.

[0139] Example 1: Design and modification of protein sequences

[0140] This example uses AlphaFold2 to predict the structure of Laminin 521 to assist in the design and selection of sequence intervals. In addition, during the formation of the trimeric structure of Laminin 521, considering that the disulfide bond between β2 and γ1 plays a role in assisting the formation of β2 and γ1 dimers and thus stabilizing the trimeric structure of Laminin 521, this application attempts to introduce a human IgG Fc tag that can form a covalent dimer to further promote and stabilize the trimeric structure of Laminin 521. In specific operations, the human IgG Fc tag is designed to be fused to the N-terminus of β2 and γ1. After the above design, the optimal sequences are screened and are shown in SEQ ID NO.1-3, respectively, where the N-termini of SEQ ID NO.2 and 3 are connected to the Fc tag sequence.

[0141] Example 2: Preparation of modified recombinant protein

[0142] 1. Construction of recombinant expression vector

[0143] Construction of the recombinant expression vector pHEK-A5 for the α5 chain of laminin 521 protein: A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 was synthesized by Shanghai Bioengineering Co., Ltd. A restriction endonuclease BamHI site and a Kozak sequence were added to the 5' end, and a stop codon TAA and a restriction endonuclease XhoI site were added to the 3' end. The synthesized product was double-digested with restriction endonucleases BamHI and XhoI, followed by agarose gel electrophoresis and recovery of the desired fragment. The target fragments were ligated with pcDNA3.1(+) (Invitrogen, catalog number: V790-20) that had also been double-digested with restriction endonucleases BamHⅠ and XhoⅠ to construct the recombinant eukaryotic expression vector pHEK-A5, which was then transformed into Escherichia coli DH5α. After culturing at 37°C for 16 h, single colonies were picked for plasmid extraction. The extracted plasmids were double-digested with restriction endonucleases BamHⅠ and XhoⅠ and identified by agarose gel electrophoresis. Positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the clones with correct sequencing were selected for plasmid amplification and extraction. The extracted plasmids were sterile filtered and stored at -20°C for later use.

[0144] Construction of the recombinant expression vector pHEK-B2 for the β2 chain of laminin 521 protein: A polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 (linked to an Fc tag nucleic acid sequence) was synthesized by Shanghai Bioengineering Co., Ltd. A restriction endonuclease BamHI site and a Kozak sequence were added to the 5' end, and a TAA stop codon and an XhoI restriction endonuclease site were added to the 3' end. The synthesized product was double-digested with restriction endonucleases BamHI and XhoI, followed by agarose gel electrophoresis and recovery of the desired fragment. The target fragments were ligated with pcDNA3.1(+) (Invitrogen, catalog number: V790-20) that had also been double-digested with restriction endonucleases BamHⅠ and XhoⅠ to construct the recombinant eukaryotic expression vector pHEK-B2, which was then transformed into Escherichia coli DH5α. After culturing at 37°C for 16 h, single colonies were picked for plasmid extraction. The extracted plasmids were double-digested with restriction endonucleases BamHⅠ and XhoⅠ and identified by agarose gel electrophoresis. Positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and clones with correct sequencing were selected for plasmid amplification and extraction. The extracted plasmids were sterile filtered and stored at -20°C for later use.

[0145] Construction of the recombinant expression vector pHEK-G1 for the γ1 chain of laminin 521 protein: A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 (conjunct to an Fc tag nucleic acid sequence) was synthesized by Shanghai Bioengineering Co., Ltd. A restriction endonuclease BamHI site and a Kozak sequence were added to the 5' end, and a TAA stop codon and an XhoI restriction endonuclease site were added to the 3' end. The synthesized product was double-digested with restriction endonucleases BamHI and XhoI, followed by agarose gel electrophoresis and recovery of the desired fragment. The target fragments were ligated with pcDNA3.1(+) (Invitrogen, catalog number: V790-20) that had also been double-digested with restriction endonucleases BamHⅠ and XhoⅠ to construct the recombinant eukaryotic expression vector pHEK-G1, which was transformed into Escherichia coli DH5α. After culturing at 37°C for 16 h, single colonies were picked for plasmid extraction. The extracted plasmids were double-digested with restriction endonucleases BamHⅠ and XhoⅠ and identified by agarose gel electrophoresis. Positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the clones with correct sequencing were selected for plasmid amplification and extraction. The extracted plasmids were sterile filtered and stored at -20°C for later use.

[0146] 2. Preparation of recombinant protein

[0147] The constructed recombinant plasmids pHEK-A5, pHEK-B2 and pHEK-G1 were transfected into HEK293 cells at a mass ratio of 1:1:1 using PEI transfection reagent for recombinant expression of heterotrimers. The day before transfection, HEK293 cells were plated at 1×10 6 / mL passage, culture at 37℃. Count the cells on the day of transfection and adjust the cell density to 1.8×10 6 -2.2×10 6 / ml, with an activity rate of more than 95%.

[0148] The amount of each component in the transfection complex was calculated based on the cell density: the corresponding relationship between the plasmid dose and the number of cells was 1×10 6 1 μg of plasmid corresponds to 1 μg of cells. The PEI dosage corresponds to 3 times the DNA mass. The prepared transfection complex was slowly added to 3 L of HEK293 cell suspension. Incubate at 37°C, 5% CO2, and 135 rpm for 96 hours, then collect the supernatant. The cell culture medium used was CD 293 TGE medium (ACRObiosystems, Cat. No. CM-1156-11).

[0149] Laminin 521 heterotrimeric recombinant protein was isolated and purified using Protein A affinity chromatography: the cell culture supernatant was collected by centrifugation at 1000g for 30 min at 4°C, filtered and bound to a Protein A chromatography column (Situofan Bioengineering Technology (Guangzhou) Co., Ltd., Cat. No.: 17543803) pre-equilibrated with binding buffer (20mM Tris-HCl, 150mM NaCl, pH 8.0), and then the target protein was eluted with elution buffer (100mM glycine, 150mM NaCl, pH3.5). After the protein eluate was collected, 1 / 40 volume of neutralization buffer (2M Tris-HCl, pH8.0) was added for neutralization, and SDS-PAGE (Figure 1.a) and HPLC analysis (Figure 2.a) were performed. SDS-PAGE analysis revealed three purified bands located around 80 kDa (α5), 60 kDa (β2), and 40 kDa (γ1), respectively, consistent with the theoretical molecular weights of 76.7 kDa (α5), 51.3 kDa (β2), and 35.6 kDa (γ1), with a purity of ≥95%. HPLC-MALS analysis also confirmed a purity of ≥90% for the heterotrimeric complex.

[0150] In order to compare the advantages of the modified protein of the present application in terms of expression, assembly and production, this example also performed SDS-PAGE and HPLC-MALS analysis on multiple commercial full-length Laminin 521 proteins (including self-recombinantly expressed full-length proteins). TM Taking laminin 521 (Cat. No. CT521) as an example, the results showed that the ratio of the three subunits of this protein after electrophoresis was not 1:1:1 (theoretical molecular weight is 400kDa (α5) | 196kDa (β2) | 178kDa (γ1)) (Figure 1b); and HPLC-MALS results showed that its main peak molecular weight was between 1288-2654kDa, which was inconsistent with the molecular weight of full-length laminin 521 (774kDa), and the fluctuation range was large, making it impossible to determine the aggregation form (Figure 2b). In fact, this result is also very common, because the molecular weight of the full-length 521 protein is too large, which puts great pressure on the host cell itself during recombinant expression, easily leading to imbalanced problems such as low expression of individual subunits; in addition, it is very difficult to fold the full-length protein completely correctly, which can easily lead to problems such as incorrect folding and even aggregation.

[0151] In comparison, the truncated modified Laminin 521 protein of the present application exhibited three distinct bands in SDS-PAGE, with the three subunits maintaining a ratio very close to 1:1:1 (Figure 1a). Furthermore, HPLC-MALS results showed a major peak molecular weight of 175 kDa (Figure 2a), consistent with the theoretical molecular weight, indicating that it possesses the correct assembly and folding form. Therefore, the modified Laminin 521 of the present application, as a reagent with the correct assembly and folding form, not only better ensures its basal activity during use, but also ensures batch-to-batch stability.

[0152] Example 3. Comparison of performance of modified protein and full-length protein in stem cell culture

[0153] The reagents used in this example include: modified laminin 521 obtained in Example 2, full-length laminin 521 (Biolamina), ROCK inhibitor (Y-27632), DMEM F12 (Gibco); mTeSR TM -Plus medium(STEM CELL); Gentle Cell Dissociation Reagent(STEM CELL), PBS(Ca- / Mg-), D-PBS(Ca 2+ / Mg 2+ ), Cell Counting-Lite 2.0 Luminescent Cell Viability Assay (Vazyme), Antibodies: OCT4 (CST), Nanog (CST), SSEA4 (CST), PE-OCT4 (Biolegend), PE-SOX2 (Biolegend).

[0154] The specific experimental steps are as follows:

[0155] 1) Experimental setup: The modified laminin 521 was set at three working concentrations: 10 μg / ml, 5 μg / ml, and 2.5 μg / ml, labeled as ACRO-10, ACRO-5, and ACRO-2.5; the full-length laminin 521 was set at two concentrations: 10 μg / ml and 5 μg / ml, labeled as full-length-10 and full-length-5.

[0156] 2) Plating: Dilute the modified laminin 521 of the present application to 400 μg / ml using sterile deionized water or water for injection; 2+ and Mg 2+ DPBS is used as a solution and diluted to 100 μg / ml for storage. It is recommended to use 0.5 μg / cm 2A detailed guide to calculating the amount of Laminin 521 to be added based on the initial coating concentration is given in the table below.

[0157] The above volumes are calculated based on a working concentration of 5 μg / ml.

[0158] 1) Add the designated volume of laminin-DPBS mixture to each well and gently shake to ensure the matrix is ​​spread throughout the well. Transfer the plate to a 37°C incubator for 2 hours or 4°C overnight.

[0159] 2) iPSC culture: iPSC cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd. iPSCs were removed from liquid nitrogen or -80°C and thawed in 37°C water for 10 seconds; the frozen test tubes were sterilized with 75% alcohol and transferred to the table; the cell solution was transferred to a new 15mL centrifuge tube containing 9mL DMEM-F12; the 15mL centrifuge tube was centrifuged at 300g for 5 minutes at room temperature; the laminin solution coated in the 12-well plate was discarded. The cell supernatant was discarded and 1mL mTeSR containing 10μM Y-27632 was used. TM Gently resuspend the iPSCs and transfer them to a coated 12-well plate. Shake the plate to evenly distribute the cells. The final cell density is 1 × 10 cells per well. 5 The cells were plated and allowed to stand at room temperature for 10 minutes. The 12-well plate was returned to the 37°C incubator. The Y-27632-containing medium was removed after 12-16 hours and the cells were continued to be cultured in mTeSR without inhibitors. TM -Plus for 4 days, changing the medium every day.

[0160] 3) iPSC passaging: Discard the culture supernatant and rinse with 1 mL of PBS (Ca- / Mg-); discard the PBS and add 0.5 mL of a mild cell dissociation reagent. Let it stand at room temperature for 6-8 minutes, or keep observing under a microscope, until the cells are no longer attached to the plate; add an equal volume of DMEM-F12, gently mix the cells, and transfer them to a 15 mL centrifuge tube and centrifuge at 300 g for 5 minutes; before centrifuging the cells, prepare a laminin-coated 12-well plate; discard the supernatant and wash with mTeSR containing 10 μM Y-27632 TM Resuspend the cells in 5% Glutamic A (-Plus) and transfer the cells to a laminin-coated 12-well plate. Gently shake the plate to evenly distribute the cells and let it rest at room temperature for 10 minutes. Place the 12-well plate in a 37°C incubator and incubate for 4 days, changing the medium daily. See Figure 3 for the results.

[0161] 4) Observation: Brightfield photography and CTG assay were performed on the fourth day of culture for the first generation (P1), the second generation (P2), and the third generation (P3) to observe cell adhesion and cell proliferation, respectively. CTG assays were performed according to the instructions for the Promega CTG kit (Cell Counting-Lite 2.0 Luminescent Cell Viability Assay). The results are shown in Figure 3.

[0162] As shown in Figure 3, when the modified laminin 521 was coated at a concentration of 10 μg / ml, its iPSC adhesion (bright field) and proliferation (CTG assay) performance was consistent with that of full-length laminin 521. Further dilutions of the modified laminin 521 to 5 μg / ml and 2.5 μg / ml showed no significant difference in iPSC adhesion and proliferation compared to the 10 μg / ml concentration. However, further dilution of the full-length laminin 521 to a low concentration of 5 μg / ml significantly decreased iPSC adhesion, resulting in virtually no adherent cells by the second passage, making further use impossible. These data demonstrate that the modified laminin 521 exhibits superior adhesion to full-length laminin 521 and remains effective even at low concentrations.

[0163] Example 4: Modified protein maintains iPSC stemness gene expression and karyotype for a long time

[0164] The reagents used included: modified laminin 521 obtained in Example 2, ROCK inhibitor (Y-27632), DMEM F12 (Gibco); mTeSR TM -Plus medium(STEM CELL); Gentle Cell Dissociation Reagent(STEM CELL), PBS(Ca- / Mg-), D-PBS(Ca 2+ / Mg 2+ ), antibodies: OCT4(CST), Nanog(CST), SSEA4(CST), PE-OCT4(Biolegend), PE-SOX2(Biolegend).

[0165] The specific steps for iPSC cell culture are as follows:

[0166] 1) The specific steps of plating and iPSC culture are the same as those in Example 3.

[0167] 2) After inoculation, take brightfield photos of the cells daily to monitor their growth. By day 4, the cells should have completely covered the entire cell culture plate. See Figure 4 for the results.

[0168] 3) Continue culturing the cells until the third passage (P3), plate cells in several more wells, and then perform immunofluorescence staining and flow cytometry analysis.

[0169] a. The experimental steps of immunofluorescence staining are as follows:

[0170] 1) Discard the supernatant from the cells in the 24-well plate, add 500 μl PBS to wash, discard the supernatant, and add 350 μl 4% paraformaldehyde to fix at room temperature for 15 minutes;

[0171] 2) Discard the supernatant, add 500 μl PBS to wash, discard the supernatant, add 350 μl 0.5% Triton X-100, and incubate at room temperature for 15 min;

[0172] 3) Discard the supernatant, add 500 μl PBS for washing, discard the supernatant, add 2% BSA and incubate at room temperature for 1 hour;

[0173] 4) Discard the supernatant, add 500 μl PBS for washing, discard the supernatant, and add 2% BSA containing primary antibodies (OCT4, Nanog, SOX2) and incubate overnight at 4°C. The primary antibody dilution ratio is 1:100.

[0174] 5) Discard the supernatant and wash three times with 500 μl of PBS, each wash on a shaker for 5 minutes. Discard the supernatant and add 2% BSA containing secondary antibody and incubate at room temperature for 1 hour. The secondary antibody dilution ratio is 1:200.

[0175] 6) Discard the supernatant and add 500 μl PBS for three washes, each for 5 minutes on a shaker. Discard the supernatant and add DAPI solution for 5 minutes.

[0176] 7) Discard the supernatant and add 500 μl PBS for washing twice, each time on a shaker for 5 minutes;

[0177] 8) Discard the supernatant, add 500 μl PBS, and observe and photograph under a microscope.

[0178] b. The experimental steps of flow cytometry are as follows:

[0179] 1) Discard the supernatant from the cells in a 12-well plate, wash with 500 μl of PBS, and digest for 6-8 minutes in 350 μl of digestion buffer. Resuspend the cells, centrifuge, discard the supernatant, and fix with 350 μl of 4% paraformaldehyde at room temperature for 15 minutes.

[0180] 2) After centrifugation, discard the supernatant, add 500 μl PBS for washing, discard the supernatant, add 350 μl 0.5% Triton X-100, and incubate at room temperature for 15 minutes.

[0181] 3) After centrifugation, discard the supernatant, wash with 500 μl of PBS, and incubate with 2% BSA containing the primary antibody (OCT4, SOX2, SSEA4) or a control antibody at 4°C in the dark for 1 hour. The antibody dilution ratio is 1:100.

[0182] 4) After centrifugation, discard the supernatant, add 100 μl of PBS, resuspend the cells, transfer to a 96-well V-shaped plate and prepare for flow cytometry.

[0183] c. The experimental steps of karyotyping are as follows:

[0184] 1) Cell culture: Cells were cultured normally, and the cell growth status was observed. Cells were seeded into 6-well culture plates and cultured for 44-48 hours. Experiments were performed when cells reached the logarithmic growth phase (confluence 50%-60%).

[0185] 2) Colchicine Treatment: Aspirate the culture medium and add fresh culture medium containing colchicine (final concentration 0.3 μg / ml) to each cell. Continue incubating at 37°C for 3 hours. After incubation, digest the cells as usual, remove the supernatant by centrifugation, resuspend the cells, and gently disperse them evenly.

[0186] 3) Hypotonic treatment: Add 3 mL of preheated hypotonic solution (pre-heated in a 37°C water bath) dropwise, and evenly distribute the cells in the suspension. Then add 5 mL of hypotonic solution dropwise. Gently invert the centrifuge tube twice to make it horizontal, and place it in a 37°C water bath for 15 minutes.

[0187] 4) Pre-fixation: Add about 200 μL of freshly prepared fixative solution and pre-fix for 2 minutes; centrifuge at 1000 rpm for 8 minutes.

[0188] 5) Fixation: A. Remove the supernatant and tap the bottom of the tube to thoroughly resuspend the cells with the remaining solution, ensuring that the cells are individually dispersed. B. Add 3 mL of fixative solution dropwise along the tube wall, gently disperse the cells with a pipette, then add 5-8 mL of fixative solution dropwise. Fix in a refrigerator at 4°C for 30 min. C. Centrifuge at 1000 rpm for 8 minutes, tap the bottom of the tube to thoroughly resuspend the cells, ensuring that the cells are individually dispersed. Repeat the fixative solution twice. D. With 0.3 mL of fixative solution remaining, gently disperse the cells with a pipette.

[0189] 6) Preparation: Pipette the cell suspension onto a pre-chilled glass slide. Pass the slide over an alcohol burner several times and dry at room temperature. Observe under a microscope, ensuring that the added suspension does not overlap.

[0190] 7) Staining: Remove the slides and add diluted Giemsa solution until the slides are covered. Stain for 25 minutes. Rinse with tap water to terminate staining and dry at 37°C.

[0191] 8) Sealing: Seal the slides with sealing medium for long-term storage.

[0192] 9) Microscopic observation: First use a low-power microscope to perform a comprehensive inspection, then switch to a high-power microscope, observe and take pictures with a 100X oil immersion lens.

[0193] Experimental results:

[0194] As shown in the brightfield images in Figure 4, iPSCs digested into single cells for cell culture can reach confluence after 4 days of culture on modified laminin-521, allowing for the next passage. This data also demonstrates that modified laminin-521 can effectively promote the rapid proliferation of stem cells.

[0195] Figure 5 shows immunofluorescence results showing that long-term (P3) iPSC culture on modified laminin 521-coated plates maintains high expression of the stem cell genes OCT4, Nanog, and SOX2, indicating that the cells remain in a stem cell state and have not differentiated. Flow cytometry results show that long-term (P3) iPSC culture on modified laminin 521-coated plates maintains high expression of the stem cell genes OCT4, SOX2, and SSEA4, indicating that the cells remain in a stem cell state and have not differentiated.

[0196] As shown in the karyotype analysis results in Figure 6, the karyotype of iPSCs did not change after long-term culture (P10) on modified laminin 521-coated plates, indicating that modified laminin 521 can maintain the karyotype of iPSCs without changes in the long term.

[0197] In summary, the modified laminin 521 of the present application performs better in stem cell culture than the full-length laminin 521. When the revived cells are initially cultured, it is not necessary to adapt to high-concentration culture (10 μg / ml). Its cell adhesion effect and proliferation ability are not significantly different from those of the full-length laminin 521. At low concentrations (5 μg / ml and 2.5 μg / ml), it can still maintain the adherence culture of stem cells very well, and its performance is far superior to that of the full-length Laminin 521. In addition, the modified Laminin 521 can maintain the high expression of iPSC stem genes OCT4, Nanog, SOX2 and SSEA4 for a long time, so that the cells are in a stem state, do not differentiate, and can maintain the karyotype of iPSCs for a long time without changing; finally, due to its smaller molecular weight, the purified yield of the modified Laminin 521 protein is significantly higher than that of the full-length laminin 521, and its cost is lower, which is conducive to better customer use and promotion.

[0198] The foregoing descriptions of specific exemplary embodiments of the present application are for purposes of illustration and description. These descriptions are not intended to limit the present application to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments are selected and described for the purpose of explaining the specific principles of the present application and their practical application, thereby enabling those skilled in the art to realize and utilize the various exemplary embodiments of the present application and various options and modifications. The scope of the present application is intended to be defined by the claims and their equivalents.

Claims

1. A modified Laminin 521 protein, characterized in that: The remodeled Laminin 521 protein is assembled from a truncated α5 chain, a truncated β2 chain and / or a truncated γ1 chain in the Laminin 521 protein.

2. The modified Laminin 521 protein according to claim 1, characterized in that: The sequences of the truncated α5 chain, truncated β2 chain and truncated γ1 chain are as follows: Truncated α5 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO.1; Truncated β2 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO.2; Truncated γ1 chain: a polypeptide having at least 80% homology to the polypeptide sequence SEQ ID NO.

3.

3. The modified Laminin 521 protein according to claim 2, characterized in that: The truncated α5 chain is a polypeptide having at least 90% homology to the polypeptide sequence SEQ ID NO.1, wherein the homology is derived from conservative amino acid substitutions; The truncated β2 chain is a polypeptide having at least 90% homology to the polypeptide sequence SEQ ID NO.2, wherein the homology is derived from conservative amino acid substitutions; The truncated γ1 chain is a polypeptide having at least 90% homology to the polypeptide sequence SEQ ID NO. 3, wherein the homology is derived from conservative amino acid substitutions.

4. The remodeled Laminin 521 protein according to claim 1, further comprising a tag sequence; Preferably, the N-termini of the truncated β2 and truncated γ1 chains are respectively connected to Fc tags.

5. A composition, characterized in that A method comprising the modified Laminin 521 protein according to any one of claims 1 to 4.

6. The composition according to claim 5, characterized in that The composition is an extracellular matrix material, a culture substrate or a scaffold material.

7. The composition according to claim 5, characterized in that The composition is a pharmaceutical composition.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a nucleic acid sequence for expressing the modified Laminin 521 protein according to any one of claims 1 to 4.

9. A kit, characterized in that: The invention comprises the modified Laminin 521 protein according to any one of claims 1 to 4.

10. A method for maintaining stem cell pluripotency in vitro, or a method for expanding and culturing stem cells in vitro, or a method for directed differentiation of stem cells in vitro, or a method for culturing organoids in vitro, characterized in that: include: A step of administering the remodeled Laminin 521 protein according to any one of claims 1 to 4, or a method of administering the composition according to any one of claims 5 to 6.

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