Modified laminin and use thereof

Abstract

The present invention relates to the technical field of biomedicine, and specifically relates to a modified laminin. By attaching an amino acid linker to the N-terminus of laminin, the interactions between the laminin and culture plates and between the laminin and cells are enhanced, the adherent activity of cells is improved, thereby achieving suspension culture without the need for pre-coating in the cell culture process.

Description

Reformed laminin and application TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a reformed laminin with changed activity, a corresponding composition and application thereof in cell culture. BACKGROUND

[0002] Induced pluripotent stem cells (iPSCs) are becoming increasingly important in the development of cellular models and advancing regenerative medicine, but the time and expense required for iPSC generation remains a major challenge for researchers. To address this issue, the rise of iPSC banks offers a promising solution that can help standardize the quality and reliability of these cells.

[0003] However, establishing an efficient iPSC bank still needs to overcome problems including rapid expansion of cells, maintaining their stemness and differentiation potential, and ensuring the stability of the genome and karyotype, and laminin can provide a clear surface for the in vitro feeder-free culture of pluripotent stem cells (PSCs), and can flexibly adapt to various culture requirements to provide a solution for the iPSC bank building requirements. However, traditional laminins such as Laminin 511 or Laminin 521 need to be pre-coated before cell seeding during pluripotent stem cell culture. For example, Laminin 511, as a laminin, contains a core functional region that binds to cell surface integrin proteins. After coating Laminin 511 protein on the surface of the culture plate and seeding iPSC cells, Laminin 511 can make the seeded iPSC cells adhere and proliferate by binding to the integrin protein on the cell surface, but mixing Laminin 511 protein with iPSC cell liquid and directly seeding cannot make the cells adhere. This coating method is cumbersome and not suitable for large-scale culture by automated equipment, so it is crucial to develop Laminin protein that does not need pre-coating for large-scale iPSC culture.

[0004] SUMMARY

[0005] The present inventors have obtained a reformed laminin with changed cell adhesion activity by studying the sequence of laminin. When using this protein for cell (especially stem cell) culture, there is no need for pre-coating treatment of laminin in advance, and only mixing with cells can achieve direct cell adhesion, which is convenient and fast for stem cell culture. Accordingly, the present application specifically provides the following technical solutions:

[0006] The present application first provides an amino acid linker, which has the following general formula:

[0007] X a (G n S) m Y b ,

[0008] Where X = K or R, Y = K or R, a = 0-20, b = 0-20, m = 0-5, n = 1-5;

[0009] Preferably, a = 2-10, b = 0-10, m = 0-3, n = 1-4; and a, b, m, and n are all integers;

[0010] The amino acid linker can promote cell adhesion.

[0011] The present invention also provides a modified laminin protein, which is assembled from laminin protein and the aforementioned amino acid linkers.

[0012] The present invention also provides a fusion protein comprising the aforementioned modified Laminin protein.

[0013] Furthermore, the Laminin protein is a Laminin protein with integrin protein-binding activity; even further, the Laminin protein with integrin protein-binding activity includes the full-length Laminin protein, any truncated form of the Laminin protein, especially the Laminin E8 fragment, and the E8 sequence further truncated from the N-terminus of any one or more of the α, β, or γ chains in the E8.

[0014] Furthermore, the assembly includes a connection; preferably, the connection is a joint connection or a direct connection; more preferably, a direct connection.

[0015] Furthermore, the amino acid linker is attached to the N-terminus of the Laminin protein.

[0016] Furthermore, the amino acid linker is attached to the N-terminus of one or more chains of the α-chain, β-chain, or γ-chain of the Laminin protein; preferably, the amino acid linker is attached to the N-terminus of the γ1 chain of the Laminin protein.

[0017] Furthermore, the Laminin proteins include, but are not limited to, Laminin 511 and Laminin 521 proteins. In this disclosure, Laminin 511 and Laminin 521 proteins include full-length Laminin 511 and Laminin 521 proteins, truncated Laminin 511 proteins or truncated Laminin 521 proteins, and mutants thereof with conserved amino acid substitutions.

[0018] Further, the Laminin 511 protein is assembled by an α5 chain, a β1 chain and a γ1 chain.

[0019] Further, the Laminin 521 protein is assembled by an α5 chain, a β2 chain and a γ1 chain.

[0020] In some embodiments, the amino acid linker is connected to the N-terminus of any one of the α5 chain, the β1 chain or the γ1 chain of the Laminin 511 protein; in other embodiments, the amino acid linker is connected to the N-terminus of any one of the α5 chain, the β2 chain or the γ1 chain of the Laminin 511 protein.

[0021] Further, the Laminin protein comprises a Laminin 511 or a Laminin 521 protein.

[0022] The present application also provides a truncated Laminin 511 protein, which is assembled by a truncated α5 chain, a truncated β1 chain and / or a truncated γ1 chain of the Laminin 511 protein.

[0023] Preferably, the sequence of the truncated α5 chain, the truncated β1 chain and the truncated γ1 chain has at least 90% homology, more preferably at least 95% homology, and even more preferably at least 98% homology with the sequence shown in SEQ ID NO. 1-3, respectively; preferably, the homology is from conservative amino acid substitution.

[0024] More preferably, the sequence of the truncated α5 chain, the truncated β1 chain and the truncated γ1 chain comprises the sequence shown in SEQ ID NO. 1-3, respectively.

[0025] Further preferably, the sequence of the truncated α5 chain, the truncated β1 chain and the truncated γ1 chain is the sequence shown in SEQ ID NO. 1-3, respectively.

[0026] The present application also provides a composition or complex comprising or connected to the engineered Laminin protein or the truncated Laminin 511 protein, or the fusion protein described above.

[0027] Further, the composition can be in the form of an extracellular matrix material, a culture substrate or a scaffold material, etc.

[0028] In some embodiments, the composition can further comprise collagen and / or gelatin, etc.

[0029] Further, the composition is a pharmaceutical composition.

[0030] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0031] The present application also provides a nucleic acid encoding any of the aforementioned amino acid linker, engineered Laminin protein, fusion protein or truncated Laminin 511 protein.

[0032] The present application also provides a recombinant vector comprising the aforementioned nucleic acid.

[0033] Further, the recombinant vector is a recombinant expression vector.

[0034] In some embodiments, the nucleic acid sequence further comprises an amino acid linker, a polyadenylation signal sequence, a termination sequence, etc.

[0035] The present application also provides a host cell comprising the aforementioned recombinant vector.

[0036] The present application also provides a kit comprising the aforementioned engineered Laminin protein or truncated Laminin 511 protein, or fusion protein; preferably further comprising a container.

[0037] The present application also provides an in vitro cell (preferably stem cell) expansion culture method, comprising culturing cells (preferably stem cells) in the presence of the aforementioned engineered Laminin protein, fusion protein, recombinant vector, host cell or composition or complex.

[0038] The present application also provides an in vitro method for maintaining stem cell pluripotency, comprising culturing stem cells in the presence of the aforementioned engineered Laminin protein, fusion protein, recombinant vector, host cell or composition or complex.

[0039] The present application also provides an in vitro stem cell directed differentiation method, comprising culturing stem cells in the presence of the aforementioned engineered Laminin protein, fusion protein, recombinant vector, host cell or composition or complex.

[0040] The present application also provides an organoid culture method, comprising culturing stem cells in the presence of the aforementioned engineered Laminin protein, fusion protein, recombinant vector, host cell or composition or complex.

[0041] Further, the aforementioned method can not include the step of pre-coating the culture vessel with the aforementioned engineered Laminin protein or fusion protein before seeding the in vitro cells (preferably stem cells) into the culture vessel.

[0042] In view of the special activity of the modified protein, the application also provides an in vitro cell (preferably stem cell) suspension culture method, which is directly carried out by adhering the cells after mixing the modified Laminin protein or the fusion protein with the cells (preferably stem cells), without pre-coating the corresponding modified Laminin protein or fusion protein in advance.

[0043] Further, the aforementioned cell is a stem cell; in some preferred aspects, the stem cell is a mammalian stem cell, including but not limited to: an ES cell, an iPS cell, or an adult stem cell, etc.

[0044] The application also provides the use of the aforementioned amino acid linker in promoting the adhesion of cells (preferably stem cells).

[0045] Further, the cell adhesion is the adhesion activity of the cells promoted by the binding of the surface integrin of the cells to the Laminin protein (preferably the Laminin protein having integrin binding activity) connected by the amino acid linker.

[0046] Further, the Laminin protein having integrin binding activity includes a full-length Laminin protein, any truncated form thereof, in particular, a Laminin E8 fragment, and an E8 sequence further truncated from the N-terminus of any one or more of the alpha, beta or gamma chains in E8.

[0047] Further, the connection mode of the amino acid linker to the Laminin protein is the same as described above.

[0048] Advantages of the application:

[0049] By introducing an amino acid linker at the N-terminus of the Laminin protein having integrin binding activity, the application can realize the direct seeding of the modified Laminin protein and cells in a suitable ratio without pre-coating the plate before cell seeding, so as to make the cells adhere and support the convenient suspension culture method.

[0050] The modified Laminin protein of the application only needs to use half of the amount of traditional pre-coating material, saves the operation and coating time required for pre-coating, and realizes the advantages of cost and performance. At the same time, it can be compatible with an automated system, simplifying the workflow, reducing the coating steps and improving the overall efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings required to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0052] Figure 1, schematic diagram of the structure of full-length and truncated fragments of Laminin protein;

[0053] Figure 2, immunofluorescence staining results of the truncated Laminin511 protein of the present application pre-coated to maintain the expression of stemness genes after a certain number of passages of iPSCs;

[0054] Figure 3, flow detection results of the truncated Laminin511 protein of the present application pre-coated to maintain the expression of stemness genes after a certain number of passages of iPSCs;

[0055] Figure 4, the truncated Laminin511 protein of the present application supports iPSC adhesion in pre-coating mode;

[0056] Figure 5, effect of Laminin511 of different linkers in the modified form supporting iPSC suspension culture mode;

[0057] Figure 6, concentration exploration of the modified Laminin511 of the present application in suspension culture;

[0058] Figure 7, effect of the modified Laminin511 of the present application supporting long-term culture of iPSCs;

[0059] Figure 8, effect of full-length and different chain modified Laminin511 supporting iPSC suspension culture mode;

[0060] Figure 9, effect diagram of the modified Laminin511 protein of the present application pre-coated to maintain the expression of stemness genes after a certain number of passages of iPSCs;

[0061] Figure 10, effect of the modified Laminin521 protein of the present application supporting iPSC suspension culture mode.

[0062] Detailed description of the application

[0063] While the application can be susceptible to embodiment in many different forms, there are described in this disclosure specific illustrative implementations of the application as it is presently perceived. It is to be understood that the application is not limited to the particular implementation described as modifications can be made without departing from the spirit or scope of the application. Additionally, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The following terms or definitions are provided solely to aid in the understanding of the application. These definitions should not be construed to limit the scope or applicability of the application.

[0064] Unless otherwise defined, all technical and scientific terms used in the Brief Description of the Invention have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. Although the terms used in the following description are believed to be well understood by one of ordinary skill in the art, to the extent there is any uncertainty, the following definitions are set forth to facilitate a clearer understanding of the application.

[0065] The terms "comprising," "including," "having," "containing," or "involving," and the like, are to be construed as being inclusive (and therefore open ended) and not exclusive or exhaustive. The term "consisting of" is considered to be a preferred embodiment of the term "comprising." If a group is defined to comprise at least one of a plurality of elements, and a subset of said plurality is explicitly disclosed, for example, as preferred embodiments, it is understood that the subgroup is a subset of the group and thus the group is defined to include the subgroup.

[0066] The indefinite articles "a" or "an," as used herein in a context to refer to a

[0067] Furthermore, the terms first, second, third, (a), (b), (c), and the like as used in the specification and claims are used as names for clarity not as a means to limit the order or the orientation of the elements described. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of use in other combinations and orientations.

[0068] The term "and / or" shall be taken as a specific disclosure of each of the elements listed after such phrases in a definition of a group of elements conjunctively, and of each of the elements conjunctively and disjunctively with respect to the other elements of the group. Thus, for example, the phrase "A and / or B" as used herein shall be interpreted to mean either or both of A and B. Likewise, the phrase "A, B, and / or C" shall be interpreted to mean A, B, C, or any combination of these. The term "consisting only of" is used as an open-ended transition term.

[0069] The terms "for example" and "e.g." are used to provide examples, and are not to be construed as a limitation of the scope of the application. The term "or" is used in the inclusive sense (and not the exclusive sense), so that when used, for example, in a list of two or more items, the term "or" means that any of the items can be present alone, or in combination with any one or more of the other items. In addition, it is specifically intended that the various implementations, embodiments, examples and features described herein can be combined or

[0070] The terms "or more," "at least," "more than," and the like, e.g., "at least one," are understood to include but not be 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, 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 than the stated value. Any larger number or fraction in between is also included.

[0071] Conversely, the term "no more than" includes every value 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, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

[0072] The terms "a plurality", "at least two", "two or more", "at least a second", and the like, shall be understood to include but not be limited to 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 in between is also included.

[0073] The terms "about", "substantially" mean an interval of accuracy that a person skilled in the art would understand as guaranteeing the technical effect in question of the characteristic. This term generally means ±10% of the indicated numerical value, preferably ±5%.

[0074] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include any integer within the recited range and, to the extent practicable, fractions thereof (e.g., one-tenth and one-hundredth of an integer), in addition to the exact number.

[0075] Laminin of the present invention

[0076] Laminin is a heterotrimeric molecule composed of three subunit chains of α chain, β chain and γ chain. There are known five subtypes of α chain, α1 to α5, three subtypes of β chain, β1 to β3, and three subtypes of γ chain, γ1 to γ3, and there are at least 12 or more isoforms of their combinations (see Table 1 below). The Laminin constituting the present application can be any of the isoforms. That is, the Laminin constituting the present application or the Laminin fragment forming a heterotrimer can be composed of one α chain selected from α1 to α5 or a fragment thereof, one β chain selected from β1 to β3 or a fragment thereof, and one γ chain selected from γ1 to γ3 or a fragment thereof. Specifically, it is preferable to use the 12 isoforms described in Table 1 and all isoforms other than these and fragments thereof. For example, Laminin 511 represents a heterotrimeric molecule composed of α5, β1 and γ1 three chains, Laminin 521 represents a heterotrimeric molecule composed of α5, β2 and γ1 three chains, their truncated forms, and their mutants having conservative amino acid substitutions.

[0077] Table 1

[0078] The Laminin constituting the present application can be full-length or a fragment thereof. That is, it can be a full-length Laminin composed of full-length α chain, full-length β chain and full-length γ chain, or a Laminin fragment composed of a fragment shorter than full-length of at least one of α chain, β chain and γ chain, which is a truncated Laminin.

[0079] The Laminin fragment is capable of forming a heterotrimer, and importantly, has Integrin binding activity, which ensures cell adhesion activity.

[0080] Since the domain of Laminin Integrin binding activity has been thoroughly studied, the trimeric fragment having Integrin binding activity is clear and easily obtainable in the art. For example, the Laminin fragment forming a heterotrimer can be confirmed by supplying the Laminin fragment to SDS-PAGE and counting the number of bands, and the Laminin fragment having Integrin binding activity can be confirmed by solid-phase binding assay.

[0081] In some embodiments, the laminin fragment having Integrin binding activity constituting the present application can be any heterotrimer composed of an alpha chain, a beta chain and a gamma chain, and the molecular weight thereof is not particularly limited. In some preferred embodiments of the present application, the protein fragment is an E8 fragment of laminin. For example, the E8 fragment of Laminin-511 laminin is a fragment having strong cell adhesion activity among fragments obtained by digesting laminin alibl gail with elastase. The laminin E8 is generally a fragment in which globular domains 4 and 5 are removed from the C-terminal fragment of the alpha chain, and a trimer of the C-terminal fragment of the beta chain and the C-terminal fragment of the gamma chain, and the molecular weight of the trimer is about 150 kDa to about 170 kDa. The E8 alpha chain is generally composed of about 770 amino acids, and about 230 amino acids on the N-terminal side thereof are involved in trimer formation; the E8 beta chain is generally composed of about 220 to about 230 amino acids; and the E8 gamma chain is generally composed of about 240 to about 250 amino acids. In other preferred embodiments of the present application, the protein fragment is a sequence in which each chain is further appropriately truncated based on the E8 fragment. It is known in the art that the C-terminus of the alpha chain, the beta chain or the gamma chain determines the Integrin binding activity, and thus the active truncated sequence can be a sequence in which, for example, the N-terminus of the alpha chain, the beta chain or the gamma chain is further truncated, and the sequence further forms a trimer, without affecting the Integrin binding activity. In some more specific embodiments of the present application, the truncated sequence specifically includes the sequences of the alpha chain, the beta chain and the gamma chain described in SEQ ID NOs. 1 to 4 (corresponding to Laminin 511 and 521), in which the alpha chain and the gamma chain of Laminin 521 and Laminin 511 are the same; and the beta chain is different, as shown in SEQ ID NO. 2 and SEQ ID NO. 4, respectively.

[0082] As can be seen from the foregoing, the "laminin having Integrin binding activity" in the present application includes not only the E8 sequence of laminin, but also an E8 sequence in which the N-terminus of any one or more of the alpha, beta or gamma chains of E8 is further truncated. Thus, the "laminin having Integrin binding activity" of the present application includes a full-length laminin protein, a laminin E8 fragment, and a fragment in which the N-terminus of any one or more of the alpha, beta or gamma chains of E8 is further truncated.

[0083] For example, the truncated Laminin 511 fragment specifically provided in embodiments 1-3 of the present application is also part of the present application, which is a kind of protein: a heterotrimer assembled by a truncated a5 chain, a truncated b1 chain and a truncated g1 chain in Laminin protein, and the truncation is a sequence truncation based on retaining the core function of Integrin binding. In some specific embodiments, the truncated a5 chain, truncated b1 / b2 chain and truncated g1 chain sequences are as follows, respectively:

[0084] The truncated a5 chain sequence is shown in SEQ ID NO. 1; the truncated b1 chain sequence is shown in SEQ ID NO. 2; and the truncated g1 chain sequence is shown in SEQ ID NO. 3, wherein:

[0085] SEQ ID NO. 1 (a5 truncated chain):

[0086] The SEQ ID NO. 2 (b1 truncated chain):

[0087] The SEQ ID NO. 3 (g1 truncated chain):

[0088] It can be understood that the amino acid changes in the above amino acid homology include substitution, insertion or deletion of amino acids, which are conservative and do not change the Integrin binding activity. Taking conservative substitution as an example, conservative substitution refers to substitution of one amino acid with another amino acid within the same category, such as substitution of one acidic amino acid with another acidic amino acid, substitution of one basic amino acid with another basic amino acid, or substitution of one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).

[0089] Therefore, in some more specific embodiments, the truncated a5 chain sequence can also be:

[0090] a polypeptide having at least 90% homology, preferably 95% homology, and more preferably 98% homology with the polypeptide sequence SEQ ID NO. 1, the homology being achieved by conservative amino acid substitution;

[0091] In some more specific embodiments, the truncated b1 chain can also be:

[0092] a polypeptide having at least 90% homology, preferably 95% homology, and more preferably 98% homology with the polypeptide sequence SEQ ID NO. 2, the homology being achieved by conservative amino acid substitution;

[0093] In some more specific embodiments, the truncated γ1 chain can also be:

[0094] a polypeptide having at least 90% homology, preferably 95% homology, more preferably 98% homology with the polypeptide sequence of SEQ ID NO. 3, the homology being achieved by conservative amino acid substitution.

[0095] The origin of the laminin is not particularly limited, and various biologically derived laminins can be used. A laminin of mammalian origin is preferred. As the mammal, for example, a human, a mouse, a rat, a cow, a pig, etc. can be mentioned, without limitation. Among them, a laminin of human origin is particularly preferred. In the case where human stem cells are cultured in order to obtain a regenerative medical material for humans, a culture environment satisfying the xeno-free condition of eliminating xeno-derived components from the culture system is required, and therefore, a laminin of human origin is preferably used.

[0096] The laminin can be a natural type or a modified type in which one or more amino acid residues are conservatively modified while maintaining the biological activity thereof (conservative amino acid substitution as described above), and these modifications do not change the integrin binding activity.

[0097] The method for producing the laminin is not particularly limited, and for example, a method of purifying from a laminin high-expression cell, a method of producing as a recombinant protein, etc. can be mentioned. The method for producing the laminin fragment is also not particularly limited, and for example, a method of purifying by digesting a full-length laminin with a protein-decomposing enzyme such as elastase and fractionating the desired fragment, a method of producing as a recombinant protein, etc. can be mentioned. From the viewpoints of the production amount, the uniformity of quality, the production cost, etc., both the laminin and the laminin fragment are preferably produced as a recombinant protein.

[0098] The recombinant laminin and the recombinant laminin fragment can be produced by appropriately using a publicly known gene recombination technique. As the method for producing the recombinant laminin and the recombinant laminin fragment, for example, a method of obtaining DNAs encoding each full-length protein or partial protein of the α chain, the β chain, and the γ chain, respectively, inserting them into expression vectors, respectively, co-introducing the obtained three kinds of expression vectors into a suitable host cell, and allowing them to express, and purifying the protein forming a trimer by a publicly known method can be mentioned.

[0099] Modified laminin and amino acid linker of the present application

[0100] The "engineered laminin" or "engineered laminin" of the present application means the same, which is further assembled (such as connected) with the amino acid linker of the present application on the basis of the aforementioned laminin.

[0101] Therefore, the engineered laminin of the present application can include any one of the laminins of the types described in Table 1 or fragments thereof further connected with the amino acid linker of the present application at the end, especially the N-terminus.

[0102] Regarding the amino acid linker, it has been proved that the different types of amino acid linker connection cases given in the examples of the present application can enhance the interaction between Laminin 511 / 521 protein and the culture plate and between the cells, and realize the suspension culture method without pre-coating in the stem cell culture process. Therefore, the general formula of the amino acid linker can be summarized as follows:

[0103] X a (G n S) m Y b ;

[0104] X = K or R, Y = K or R, a = 0-20, b = 0-20, m = 0-5, n = 1-5;

[0105] Preferably, a = 2-10, b = 0-10, m = 0-3, n = 1-4; and a, b, m, n are all integers.

[0106] For example, in some embodiments of the application, the engineered laminin is an engineered Laminin 511. In some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RR; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-KK; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RRRRRRRRRR; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-KKKKKKKKKK; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RRRGGGSRR; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-KKKGGGSKK; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RRRRRGGGS; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-KKKKKGGGS; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RRRRRGGGSGGGSRRRRR; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-KKKKKGGGSGGGSKKKKK; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RRGGGGSRRRRRR; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-KKGGGGSKKKKKK; in some specific embodiments of the application, the engineered Laminin 511 protein is Laminin 511-RRRRRRRRRRGGGSGGGSGGGSKKKKKKKKKK.

[0107] As for the assembly connection of the amino acid linker, considering the basic Integrin binding activity of laminin, the amino acid linker cannot be connected at the C-terminal of the protein, i.e. only the N-terminal of the protein. However, through the tests in the examples, the desired technical effect can be achieved when the amino acid linker is connected to different chains (such as the a chain, the b chain or the g chain) of the laminin, therefore, the selection of the amino acid linker on the trimer chain is not limited, and can be at the N-terminal of any one or more of the a chain, the b chain or the g chain.

[0108] For example, in some embodiments of the present application, the modified Laminin 511 protein can be a5-X a (G n S) m Z b / β1 / γ1; the modified Laminin 511 protein can be a5 / β1-X a (G n S) m Z b / γ1; the modified Laminin 511 protein can be a5 / β1 / γ1-X a (G n S) m Z b ; in some other embodiments of the present application, the modified Laminin 521 protein can be a5-X a (G n S) m Z b / β2 / γ1; the modified Laminin 521 protein can be a5 / β2-X a (G n S) m Z b / γ1; the modified Laminin 521 protein can be a5 / β2 / γ1-X a (G n S) m Z b .

[0109] The present application demonstrates the use of the above-mentioned amino acid linker in promoting cell (especially stem cell) adhesion, and this use is also within the protection scope of the present application. In this use, the adhesion of the cell is the direct adhesion activity promoted by the combination of the modified Laminin protein and the surface integrin protein of the cell, without the need for the previous pre-coating treatment of the Laminin protein.

[0110] The fusion protein of the present application, in addition to comprising the aforementioned modified Laminin, can also comprise or be connected to other protein components or other components, which are not limited.

[0111] The composition or complex of the present application, especially such a composition, is based on the inclusion of the aforementioned modified Laminin protein.

[0112] In some aspects, the composition can be in the form of an extracellular matrix material, a culture substrate or a scaffold material, etc.

[0113] The extracellular matrix material of the present application, particularly the extracellular matrix material containing the above-mentioned engineered laminin (such as engineered Laminin 511 / Laminin 521) and containing collagen and / or gelatin and the like. The extracellular matrix material of the present application can be composed of the engineered laminin and collagen, or the engineered laminin and gelatin, or the engineered laminin, collagen and gelatin, or can contain other components in addition thereto. As the other components in addition thereto, there is no particular limitation as long as it is a component that can be used in cell culture, and for example, an extracellular matrix component other than collagen and gelatin is preferred, and examples include fibronectin, Matrigel, proteoglycan, hyaluronic acid, tenascin, elastin, laminin, fibrinogen (fibrin) and the like.

[0114] The collagen or gelatin used in the extracellular matrix material of the present application is not particularly limited, and a publicly known collagen or gelatin for cell culture use can be preferably used. The extracellular matrix material of the present application can be provided in a liquid, gel, sponge, sheet and the like, and can be coated on a plate or used as a three-dimensional matrix.

[0115] 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 differentiation induction of stem cells using laminin that is suitable as a scaffold for stem cells. In addition, the extracellular matrix material of the present application can be used not only for three-dimensional culture of cells, but also as a tissue regeneration induction device for embedding in a living body. In addition, by binding an arbitrary laminin isoform to a three-dimensional matrix composed of collagen or gelatin having a hardness suitable for stem cells for which differentiation induction is intended, a three-dimensional culture environment most suitable for differentiation induction of the stem cells can be provided.

[0116] The culture substrate of the present application is a culture substrate coated with the modified laminin of the present application (such as modified Laminin 511 / Laminin 521), and collagen and / or gelatin. The cells cultured using the culture substrate of the present application are not particularly limited, and can be any cells as long as they are culturable. Preferred are mammalian cells, and more preferred are mammalian stem cells. The stem cells include somatic stem cells, pluripotent stem cells, and the like. As the somatic stem cells, there can be mentioned neural stem cells, mesenchymal stem cells, hematopoietic stem cells, cardiac stem cells, liver stem cells, small intestinal stem cells, and the like. As the pluripotent stem cells, there can be mentioned ES cells (embryonic stem cells), iPS cells (induced pluripotent stem cells), mGS cells (multipotency germ stem cells), fusion cells of ES cells and somatic cells, and the like. As the mammals, there can be mentioned humans, mice, rats, cows, pigs, and the like; and humans are preferred. The culture substrate of the present application is also useful in culturing cells that are cultured using existing feeder cells without using feeder cells.

[0117] The method for producing the culture substrate of the present application is not particularly limited, and for example, a mixed solution of collagen and / or gelatin and the modified laminin can be coated on a culture vessel, or the modified laminin of the present application can be further coated after coating collagen or gelatin or a mixture of both on a culture vessel. The culture vessel is not particularly limited as long as it is a culture vessel that can be used in cell culture, and there can be mentioned, for example, glass or plastic culture dishes, flasks, multi-well plates, culture slides, microcarriers, polyvinylidene fluoride membranes, and the like.

[0118] The collagen or gelatin used for coating is not particularly limited, and a publicly known collagen or gelatin for cell culture use can be preferably used. In the case of cell culture for regenerative medicine, a collagen or gelatin whose safety for medical use is confirmed is preferably used, and a collagen or gelatin derived from humans is preferably used.

[0119] The scaffold material of the present application is a scaffold for constructing a three-dimensional tissue structure for differentiation induction of stem cells. The scaffold of the present application is only required to contain the aforementioned modified laminin of the present application, and collagen and / or gelatin. The material constituting the scaffold of the present application is not particularly limited as long as it functions as a scaffold for cells, and can be any material. Examples include natural polymers such as collagen, gelatin, fibrin, hyaluronic acid, alginic acid, starch, chitin, pectic acid, and the like; inorganic materials such as polylactic acid, polyglycolic acid, a copolymer of lactic acid and glycolic acid, hydroxyapatite, calcium phosphate, and the like. The form of the scaffold can be any form such as a gel-like form, a sponge-like form, a film-like form, a net-like form, a non-woven fabric-like form, a woven fabric-like form, and the like three-dimensional structure. In the case where the scaffold is constituted of gelatin or gelatin itself, the scaffold of the present application can be produced by binding the modified laminin (such as Laminin511 / Laminin521) of the present application to the scaffold. In the case where the scaffold is constituted of a material other than collagen and gelatin, the scaffold of the present application can be produced by coating the surface of the scaffold with collagen and / or gelatin and binding the modified laminin thereon. In addition, the collagen and gelatin used for the scaffold of the present application are the same as the aforementioned culture substrate of the present application.

[0120] The composition of the present application is a pharmaceutical composition, and is a pharmaceutical composition comprising the aforementioned modified laminin 511 and a pharmaceutically acceptable carrier. The pharmaceutical composition can contain other agents depending on the condition to be treated. The pharmaceutical composition can be prepared in any suitable form, and generally contains the modified laminin 511 and a pharmaceutically acceptable carrier. The carrier can be an injectable carrier, a topical carrier, a transdermal carrier, and the like. The preparation can advantageously be in a form for topical administration, such as an ointment, a gel, an emulsion, a spray, a dispersion, a suspension, or a paste, and the like.

[0121] The nucleic acid of the present application can encode any of the aforementioned amino acid linker, modified Laminin protein, fusion protein, or truncated Laminin 511 protein, and the like. The form of the nucleic acid is not limited, and in some embodiments, the nucleic acid is an isolated nucleic acid, in some embodiments, the nucleic acid is a chemically synthesized nucleic acid, and the like.

[0122] The recombinant vector of the present application comprises a nucleic acid sequence expressing any of the aforementioned modified laminin (such as modified Laminin511 / Laminin521) protein;

[0123] The term "vector", as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors (such as mammalian non-episomal vectors) when introduced into a host cell are integrated into the genome of the host and are replicated along with the host genome. Additionally certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply, "expression vectors". In general, the expression vectors of the present disclosure will be circular double stranded DNA plasmids. However, the disclosure is intended to include other forms of expression vectors which serve equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), although certain preferred embodiments will be described in terms of plasmid vectors. Thus, the description herein, which follows, is intended to apply equally to all such forms of recombinant expression vectors, unless specifically stated otherwise.

[0124] In some embodiments, the nucleic acid sequence can further comprise an amino acid linker, a polyadenylation signal sequence, or a termination sequence, etc.

[0125] The host cell of the present disclosure comprises the recombinant vector described above.

[0126] The term "host cell", as used herein, 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, for rapid production of large amounts of the expression vectors of the present disclosure, or a eukaryotic cell for functional studies.

[0127] The terms "host cell" and "recombinant host cell", as used herein, can be used interchangeably. It should be understood that such terms encompass not only a particular subject cell, but also the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either naturally-occurring or induced mutations, the progeny of the cell may

[0128] The in vitro cell (especially stem cell) expansion culture method of the present disclosure comprises culturing the in vitro cell (especially stem cell) in the presence of the engineered laminin (e.g., engineered Laminin 511 / Laminin 521) of any one of the preceding embodiments, or in the presence of the composition of any one of the preceding embodiments.

[0129] The method for maintaining pluripotency of stem cells in vitro of the present application comprises culturing cells, particularly stem cells, in the presence of the engineered laminin (such as Laminin511 / Laminin521) of any one of the preceding items or in the presence of the composition of any one of the preceding items.

[0130] The method for directed differentiation of stem cells in vitro of the present application comprises culturing cells, particularly stem cells, in the presence of the engineered laminin (such as Laminin511 / Laminin521) of any one of the preceding items or in the presence of the composition of any one of the preceding items.

[0131] The method for culturing organoids of the present application comprises culturing cells, particularly stem cells, in the presence of the engineered laminin (such as Laminin511 / Laminin521) of any one of the preceding items or in the presence of the composition of any one of the preceding items.

[0132] It can be understood that, based on the activity of the engineered laminin of the present application, in each of the aforementioned methods, the engineered Laminin protein can not be pre-coated on the culture vessel before the stem cells are inoculated into the culture vessel, i.e., the step of pre-coating the engineered Laminin protein on the culture vessel can be excluded. Of course, this does not exclude the pre-coating step, but only expresses that the step can be excluded. It can be understood that the method of the present application can still have the pre-coating step for further improving the adhesion effect.

[0133] In addition, in view of the special activity of the engineered protein, the present application also relates to a method for culturing cells in vitro in suspension, which is to mix the engineered Laminin protein with cells (preferably stem cells) and then directly perform cell adherent culture without pre-coating the engineered Laminin protein in advance.

[0134] In fact, the method of the present application is a method for culturing cells, particularly stem cells, in the presence of the engineered laminin (such as Laminin511 / Laminin521) of the present application. It can be a method for adding the engineered laminin of the present application and collagen and / or gelatin to the culture medium respectively and culturing, a method for adding a complex of the engineered laminin of the present application and collagen or a complex of the engineered laminin of the present application and gelatin to the culture medium and culturing, or a method for adding the engineered laminin of the present application to the culture medium using a culture device coated with collagen and / or gelatin and culturing. Preferably, it is a method for culturing using the culture medium material of the present application or the scaffold of the present application.

[0135] The cell herein is preferably a stem cell. Stem cells refer to cells having self-replication ability and pluripotency, including adult stem cells, pluripotent stem cells, etc. As adult stem cells, one can cite neural stem cells, mesenchymal stem cells, hematopoietic stem cells, cardiac stem cells, liver stem cells, small intestine stem cells, etc. As pluripotent stem cells, one can cite ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), mGS cells (multipotent germ stem cells), fusion cells of ES cells and somatic cells, etc. Mammals are not particularly limited, and one can cite humans, mice, rats, cows, pigs, etc. Among them, humans are preferred. That is, the culture method of the present application is preferably used for the culture of human stem cells.

[0136] Finally, it should be noted that, based on 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 is involved in the regulation of important physiological processes such as cell adhesion, migration, differentiation, phenotype maintenance, and matrix-mediated signal transduction", since the present application has been fully confirmed that the modified laminin-521 of the present application has the same or even better activity as the intact or full-length laminin-521. Therefore, based on the practical application or method of the basic activity of the full-length protein, theoretically, it is suitable for the modified laminin-521 of the present application, and it belongs to the protection scope of the present patent, which is not listed here.

[0137] The present application is further described by the following examples and the accompanying drawings, which are only intended to illustrate specific embodiments of the present application and should not be construed as limiting the scope of the present application in any way.

[0138] Example 1, design of recombinant truncated Laminin 511 protein

[0139] The full-length Laminin protein has a large molecular weight (about 750 kDa), and is a well-known protein that is difficult to prepare. The industry usually chooses fragments with high recombinant expression levels, such as E8 fragments, to support the culture and proliferation of stem cells. The applicant assisted in the design of recombinant Laminin 511 protein by predicting the structure of Laminin 511; based on structural simulation and design, the present application selected a Laminin 511 truncation that retains integrin binding activity and ensures stable trimer formation as the basis for designing and modifying Laminin 511 protein in the present application (see Figure 1). In specific operations, a truncated α5 chain, a truncated β1 chain, and a truncated γ1 chain were used as the basis, a polyhistidine tag was connected to the N-terminus of the β1 chain for protein purification, and a trimer was prepared by recombinant expression, and the sequences of the truncated α5 chain, the truncated β1 chain, and the truncated γ1 chain are shown in SEQ ID NO. 1-3, respectively.

[0140] Example 2, Preparation of recombinant truncated Laminin 511 protein

[0141] 1. Construction of recombinant expression vector

[0142] Construction of recombinant expression vector pHEK-a5 of a5 chain of Laminin 511 protein: Using basic molecular biology means (for example, see: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York 1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. 1989), the a5 truncated chain shown in SEQ ID NO. 1 was cloned into the recombinant expression vector pcDNA3.1(+) (Invitrogen Corporation, Catalog No: V790-20) to obtain the truncated a5 chain recombinant expression vector pHEK-a5, which was identified and stored at -20°C for standby.

[0143] Construction of recombinant expression vector pHEK-b1 of b1 chain of Laminin 511 protein: Using basic molecular biology means, the b1 truncated chain shown in SEQ ID NO. 2 was cloned into the recombinant expression vector pcDNA3.1(+) (Invitrogen Corporation, Catalog No: V790-20) to obtain the truncated b1 chain recombinant expression vector pHEK-b1, which was identified and stored at -20°C for standby.

[0144] Construction of recombinant expression vector pHEK-g1 of g1 chain of Laminin 511 protein: Using basic molecular biology means, the g1 truncated chain shown in SEQ ID NO. 3 was cloned into the recombinant expression vector pcDNA3.1(+) (Invitrogen Corporation, Catalog No: V790-20) to obtain the truncated g1 chain recombinant expression vector pHEK-g1, which was identified and stored at 4°C.

[0145] 2. Preparation of recombinant protein

[0146] The constructed recombinant plasmids pHEK-a5, pHEK-b1 and pHEK-g1 were transfected into HEK293 cells in equal proportions using PEI transfection reagent to complete the recombinant expression of the heterotrimer. The Laminin 511 heterotrimer recombinant protein was separated and purified using Ni-NTA affinity chromatography method, and the obtained Laminin 511 heterotrimer recombinant protein was sterile filtered, lyophilized and stored at 4°C for standby.

[0147] Example 3, Precoating activity detection of recombinant truncated Laminin 511 protein

[0148] Reagents: Laminin 511 prepared in Example 2, ROCK inhibitor (Y-27632), DMEM-F12; mTeSR TM Plus medium (STEM CELL); Gentle Cell Dissociation Reagent (STEM CELL), PBS (Ca- / Mg-) (Cytiva Cat. SH30256.01), D-PBS (Ca 2+ / Mg 2+ ) (Cytiva Cat. SH30264.01); Antibodies: OCT4 (CST), Nanog (CST), PE-OCT4 (Biolegend), PE-SOX2 (Biolegend).

[0149] 1. Experimental procedure:

[0150] 1) Plating: Laminin 511 prepared in Example 2 was reconstituted to 500 pg / ml using sterile deionized water or water for injection; 0.5 pg / cm 2 of initial Laminin protein coating concentration was used on the surface of the culture, and the detailed calculation of Laminin 511 addition amount is shown in Table 1 below.

[0151] Table 1

[0152] The above volume is calculated according to the working concentration of 0.5 pg / cm 2 .

[0153] The corresponding volume of laminin-DPBS mixture was added to each well, and gently shaken to ensure that the matrix was evenly distributed in the well; the culture plate was transferred to the incubator at 37°C for 2h or overnight at 4°C. The recombinant laminin 511 solution was aspirated and discarded when the cells were ready to be plated.

[0154] 2) iPSC culture: iPSC cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and thawed for 10 seconds; the frozen test tube was disinfected with 75% alcohol and transferred to the bench top; the cell solution was transferred to a new 15 mL centrifuge tube containing 9 mL DMEM-F12; the 15 mL centrifuge tube was centrifuged at 300 g for 5 minutes at room temperature. Discard the cell supernatant, gently resuspend the iPSC with 1 mL of mTeSR™-Plus containing 10 μM Y-27632, and transfer it to the coated 12-well plate obtained in 1). Shake the plate to evenly distribute the cells, with a final cell density of 1 x 10 5

[0155] 3) iPSC passage: discard the culture supernatant and rinse with 1 mL of PBS (Ca- / Mg-); discard the PBS and add 0.5 mL of mild cell dissociation reagent. Incubate at room temperature for 6-8 minutes, or keep under microscopic observation until the cells are not bound to the plate; add an equal volume of DMEM-F12, gently mix the cells, and transfer them to a 15 mL centrifuge tube for centrifugation at 300 g for 5 minutes; before centrifuging the cells, prepare a laminin 511-coated 12-well plate; discard the supernatant and resuspend the cells with mTeSR-Plus containing 10 μM Y-27632; transfer the cells to the laminin 511-coated 12-well plate. Gently shake the plate to evenly distribute the cells, and incubate at room temperature for 10 minutes; place the 12-well plate in a 37°C incubator for 4 days, with daily medium changes.

[0156] 3) Detect the expression of stemness genes such as Sox2 and OCT4 in cells.

[0157] Results observation:

[0158] The recombinant truncated laminin 511 constructed can at least maintain the stemness of the 5th generation of iPSCs. Immunofluorescence staining results (Figure 2) show that iPSCs express high levels of pluripotency-related markers OCT4 and Nanog. Flow cytometry results (Figure 3) show that iPSCs can express high levels of pluripotency-related markers OCT4 and SOX2.

[0159] In addition, bright field photography was performed on the fourth day of the first generation, i.e. P1, to observe the cell adhesion effect and cell proliferation capacity, respectively. The results are shown in Figure 4. As can be seen from Figure 4, the recombinant truncated laminin 511 can maintain the adhesion effect and proliferation capacity of iPSCs at 0.5 μg / cm 2 ​The coating concentration of the pre-coating can make the stem cells adhere normally. However, if the Laminin 511 protein is directly mixed with the iPSC cell liquid for inoculation, that is, in a suspension mode, the cells cannot adhere and grow.

[0160] Example 4, modification of recombinant protein

[0161] Since the above-mentioned recombinant truncated Laminin 511 protein cannot adhere to the iPSC cell liquid for inoculation, in order to save the coating step and improve efficiency, the applicant attempts to further modify the recombinant truncated Laminin 511 protein, hoping to obtain a suspension culture method without protein pre-coating.

[0162] To solve this problem, the applicant further introduces an amino acid linker containing arginine R or lysine K to the N-terminal sequence of the recombinant truncated Laminin 511 protein (including any of the aforementioned truncated chains), in an attempt to enhance the interaction between Laminin 511 protein and the culture plate and the cells, and hopes to realize an iPSC suspension culture method without pre-coating 511 protein.

[0163] The preparation method of the recombinant protein with the added linker refers to the similar method of Example 2, except that the corresponding amino acid linker is added to the N-terminal of the α5 chain, β1 chain or γ1 chain sequence.

[0164] Taking the N-terminal linker of the γ1 chain as an example, the applicant designs and prepares a series of amino acid linkers with different sequences (Table 2), which all satisfy the general formula: X a (G n S) m Z b The obtained modified recombinant Laminin 511 protein with the linker is as follows:

[0165] Table 2

[0166] Example 5, effect evaluation of various modified recombinant Laminin 511 proteins

[0167] 1. Experimental instruments and materials

[0168] Instruments: biosafety cabinet, cell incubator, low-temperature horizontal centrifuge, inverted microscope, EnSight (PerkinElmer, HH3400), microscope, flow cytometer.

[0169] Materials: sterile pipette tips, sterile EP tubes and other consumables.

[0170] The reagents are as follows:

[0171] Laminin 511: engineered Laminin 511 in Table 2 above. Negative control is unengineered recombinant truncated Laminin 511 protein obtained from Example 2

[0172] 2. iPSC cell suspension culture

[0173] Take iPSC out of liquid nitrogen or dry ice, thaw in water at 37°C; sterilize the frozen test tube with 75% alcohol, then transfer it to the bench top; transfer the cell solution to a new 15 mL centrifuge tube containing 9 mL of DMEM-F12; centrifuge the 15 mL centrifuge tube at 300g for 5 minutes at room temperature; add the specified volume of laminin to the iPSC suspension containing 1 mL of mTeSR plus with 10 mM Y-27632 (laminin addition amount for different culture dishes refer to Example 3) according to the addition amount of 0.5 pg / cm2, mix well by blowing and sucking, and then transfer it to a 12-well plate. Shake the plate to evenly distribute the cells, and the final cell density is 1 x 10 2 5 6 cells per well, and stand at room temperature for 10 minutes (note that the number of cells is determined according to the growth rate of the cultured cells, and it is ensured that the cells are passaged within 4-5 days); return the 12-well plate to the incubator at 37°C; the medium containing the ROCK inhibitor should be removed after 12-16 hours, and continue to culture in the medium without the inhibitor.

[0174] 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 mild cell dissociation reagent. Stand at room temperature for 6-8 minutes, or keep under microscope observation until the cells are not bound to the plate; add an equal volume of DMEM-F12, mix the cells gently, and transfer them to a 15 mL centrifuge tube to centrifuge at 300g for 5 minutes; resuspend the cells with mTeSR™-Plus containing 10 mM Y-27632; transfer the cells to a 12-well plate after mixing with the specified volume of laminin. Shake the plate gently to evenly distribute the cells, and stand at room temperature for 10 minutes; place the 12-well plate in the incubator at 37°C for culture for 4 days, and change the medium every day.

[0175] 3. Observation of results:

[0176] Take photographs in bright field and perform CTG detection on the fourth day of the first generation (P1) and the fourth day of the second generation (P2) and the fourth day of the third generation (P3), respectively, to observe the cell adhesion effect and cell proliferation capacity. CTG detection is performed according to the instructions of the Promega CTG kit (Cell Counting-Lite 2.0 Luminescent Cell Viability Assay).​

[0177] 4. Experimental results

[0178] Various modified human laminin 511 can make stem cells adhere and significantly proliferate without pre-coating, while human laminin 511 protein without amino acid linker cannot make stem cells adhere at the same protein concentration (see Figure 5); and as can be seen from Figure 5, laminin with different linkers can promote cell adhesion, but laminin with different linkers differs in promoting cell adhesion, and in different application scenarios, the appropriate amino acid linker can be selected according to the required degree of cell adhesion.

[0179] Take Laminin 511-K10 in Table 2 as an example to explore different use concentrations. According to the addition amount of 0.125 μg / cm 2 , 0.25 μg / cm 2 , 0.5 μg / cm 2 and 1.0 μg / cm 2 , add sufficient amount of Laminin 511-K10 to the stem cell suspension for culture, and as shown in the results of Figure 6, when the use concentration of Laminin 511-K10 is 0.25 μg / cm 2 , the effect in Example 3 (Figure 4) can be achieved, i.e. the use concentration of Laminin 511 protein can be halved compared to the pre-coating method (see Figure 6). At the same time, Laminin 511-K10 can maintain long-term culture of iPSCs, and the cell morphology is not abnormal (see Figure 7).

[0180] Further, take K10 as an example to verify the N-terminal addition of amino acid linker to the α5 chain or β1 chain of truncated Laminin 511 protein and the α5 chain or β1 or γ1 chain of full-length Laminin 511 protein. Refer to Example 2 and Example 3 to prepare modified truncated Laminin 511 α5-K10 and Laminin 511 β1-K10 proteins, and prepare modified full-length Laminin 511 α5FL-K10 (full-length α5, β1 and γ1 sequences are shown in SEQ ID NO. 5, 6, 7), Laminin 511 β1FL-K10 and Laminin 511 γ1FL-K10 proteins; and verify the effect of supporting stem cell suspension culture method. The results show that when the use concentration of modified truncated Laminin 511 α5-K10 and Laminin 511 β1-K10 proteins is 0.25 μg / cm 2When the concentration is 0.5 μg / cm 2 , the effect comparable to Laminin 511-K10 can be achieved, while the full-length Laminin 511 protein without the addition of the amino acid linker (BioLamina, item number LN511-02) cannot make the cells adhere and grow at the concentration of 0.5 μg / cm 2 in the iPSC cell liquid mixed with seeding, i.e. in the suspension mode (Figure 8). The results show that the amino acid linker of the present application is also applicable to the full-length Laminin protein, and has the same effect when connected to the N-terminus of the a chain, the b chain or the g chain of the Laminin protein.

[0181] Example 6, Long-term maintenance of iPSC stemness gene expression by the modified Laminin 511 protein

[0182] This example further demonstrates whether the modified Laminin 511 protein has long-term maintenance activity of iPSC stemness. The reagents used in this example include: the modified Laminin 511-K10 obtained in Example 4, ROCK inhibitor (Y-27632), DMEM F12 (Gibco); mTeSR TM Plus medium (STEM CELL); Gentle Cell Dissociation Reagent (STEM CELL), PBS (Ca 2+ / Mg 2+ ), antibodies: OCT4 (CST), Nanog (CST), SSEA4 (CST), PE-OCT4 (Biolegend), PE-SOX2 (Biolegend), SSEA4 (CST).

[0183] The specific steps of iPSC cell culture are as follows:

[0184] 1) The specific steps of plating and iPSC culture are consistent with Example 5.

[0185] 2) After seeding the cells, the cells were photographed every day to observe the growth state of the cells. The cells can cover the entire cell culture plate at 4 days after seeding.

[0186] 3) Continue to culture the cells to the tenth generation (P10), and then perform immunofluorescence staining and flow cytometry detection on the cells in multiple wells, respectively.

[0187] As shown in the immunofluorescence results in FIG. 9, the iPSCs cultured on the modified Laminin 511 coated plates for a long time (P10) still maintain high expression of the stem cell genes OCT4, Nanog and SOX2, indicating that the cells are still in the stem cell state and no differentiation phenomenon occurs. As shown in the flow cytometry results, the iPSCs cultured on the modified Laminin 511 coated plates for a long time (P10) still maintain high expression of the stem cell genes OCT4, SOX2 and SSEA4, indicating that the cells are still in the stem cell state and no differentiation phenomenon occurs.

[0188] Example 7, modification of Laminin 521 protein and evaluation of effects

[0189] To verify that the modification method of the present application is also applicable to other Laminin proteins, in this example, Laminin 521 protein, which supports the adhesion and proliferation of stem cells, was selected as the basic Laminin, and the effects of the suspension culture method were evaluated on truncated Laminin 521 γ1-K10 (Laminin 521 truncated sequence: truncated α5 chain and truncated γ1 sequence same as truncated Laminin 511, i.e., SEQ ID NO. 1 and 3, truncated β2 chain sequence as shown in SEQ ID NO. 4: AAQARAEQLRDEARDLLQAAQDKLQRLQELEGTYEENERALESKAAQLDGLEARMRSVLQAINLQVQIYNTCQ) and full-length Laminin 521 γ1FL-K10 (wherein the full-length α5, β2 and γ1 sequences are shown in SEQ ID NO. 5, 8 and 7, respectively). The specific operation is similar to the procedures in Examples 4 and 5.

[0190] The evaluation results are shown in FIG. 10. The Laminin 521 with the amino acid linker of the present application connected at the N-terminus can also achieve the suspension culture method without pre-coating the plates in stem cell culture, while the Laminin 521 truncated body (Acrobiosystems company, item number LA5-H5215) or Laminin 521 full-length (BioLamina company, item number CT521) without the amino acid linker of the present application cannot be used in the suspension manner to make the stem cells adhere and grow (FIG. 10).

[0191] In summary, the present application can realize the cell adhesion without plate coating before cell inoculation by introducing amino acid linker at the N-terminal of Laminin protein, directly mixing Laminin 511 or Laminin 521 protein with cells according to appropriate ratio and directly inoculating cells, which can support the convenient suspension culture method. The modified Laminin protein only needs to use half of the amount of traditional pre-coating material compared with the unmodified Laminin protein, while saving the operation and coating time required for pre-coating, and has more cost and performance advantages in stem cell culture. In addition, the modified Laminin can maintain the high expression of iPSC stemness genes OCT4, Nanog, SOX2 and SSEA4 for a long time, so that the cells are in the stemness state and do not differentiate.

[0192] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application, and to thereby enable others skilled in the art to best utilize the application and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. An amino acid linker, characterized in that, The amino acid linker has the general formula: X a (G n S) m Y b ; wherein X=K or R, Y=K or R, a=0-20, b=0-20, m=0-5, n=1-5; Preferably, a=2-10, b=0-10, m=0-3, n=1-4; The amino acid linker can promote cell adhesion.

2. A modified Laminin protein, characterized in that, The modified Laminin protein is assembled from a Laminin protein and the amino acid linker of claim 1.

3. A fusion protein, characterized in that, The fusion protein comprises the modified Laminin protein of claim 2.

4. The engineered Laminin protein or fusion protein of claims 2-3, wherein, The amino acid linker is assembled at the N-terminus of the Laminin protein.

5. The engineered Laminin protein or fusion protein of claim 4, wherein, The amino acid linker is assembled at the N-terminus of any one or more of the α chain, β chain or γ chain of the Laminin protein.

6. The modified Laminin protein or fusion protein according to any one of claims 2-5, wherein, The Laminin protein comprises Laminin 511 or Laminin 521 protein, any truncation thereof, and mutants thereof with conservative amino acid substitutions.

7. A truncated Laminin 511 protein, characterized in that, The truncated Laminin 511 protein is assembled from a truncated α5 chain, a truncated β1 chain and / or a truncated γ1 chain. Preferably, the truncated α5 chain, the truncated β1 chain and the truncated γ1 chain have at least 90%, preferably at least 95%, more preferably at least 98% homology to the sequences shown in SEQ ID NO. 1-3, respectively, the homology being from conservative amino acid substitutions.

8. A composition or composite, characterized in that, The composition or complex comprises the modified Laminin protein or fusion protein of any one of claims 2-6, or the truncated Laminin 511 protein of claim 7.

9. A nucleic acid, characterized in that, The nucleic acid encodes the amino acid linker, the modified Laminin protein, the fusion protein or the truncated Laminin 511 protein of any one of claims 1-7.

10. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid of claim 9.

11. A kit characterized in that, The composition or complex comprises the modified Laminin protein or fusion protein of any one of claims 2-6, or the truncated Laminin 511 protein of claim 7.

12. A method for in vitro cell suspension culture, or a method for in vitro cell expansion culture, or a method for maintaining stem cell pluripotency in vitro, or a method for directed differentiation of stem cells in vitro, or a method for organoid culture, characterized in that, The method comprises culturing cells in the presence of the modified Laminin protein or fusion protein of any one of claims 2-6, or in the presence of the composition or complex of claim 8; preferably, the method does not comprise a step of pre-coating the culture vessel with the modified Laminin protein or the fusion protein or the composition comprising the same before seeding the stem cells into the culture vessel.

13. Use of the amino acid linker of claim 1 in promoting cell adhesion; preferably, the cell adhesion is the adhesive activity of the cell after the Laminin protein assembled with the amino acid linker binds to the surface Integrin protein of the cell.

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