A biospherical structure and method for generating the same

Abstract

Provided are biospheres comprising: (a) a three-dimensional scaffold matrix for biological growth; (b) a first type of cell configured to be in contact with the three-dimensional scaffold matrix to grow to generate the biosphere with an internal volume; and (c) a second type of cell disposed in the internal volume. Also provided are methods of creating the same.

Description

A BIOSPHERICAL STRUCTURE AND METHOD FOR GENERATING THE SAMECROSS REFERENCE TO RELATED PATENT APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 667,236, filed July 3, 2024, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to a biological structure and methods for producing the biological structure, and, more particularly, a biological structure having one type of cell that is suitable for containing another type of cell and method for producing the biological structure having one type of cell that is suitable for containing another type of cell.BACKGROUND OF THE DISCLOSURE

[0003] State of the art technologies to date have not provided a technology for holding and providing constant motion to satisfy the motion requirements of, for example, biological structures having one type of cell that are suitable for containing another type of cell. The inventors have discovered the need for such a technology.SUMMARY OF THE DISCLOSURE

[0004] The inventors have invented a technology (including a method, system, and device) for producing a biological structure such as a biosphere or biocontainer that includes a first type of cell grown on a scaffold matrix. This biological structure is suitable for containing a second type of cell. In various embodiments, the second type of cell may be isolated from the environment of the biological structure by the first type of cell.

[0005] According to an aspect of the disclosure, a biosphere is provided having a three- dimensional scaffold matrix for biological growth, a first type of cell configured to be in contact with the three-dimensional scaffold matrix and grown to generate the biosphere with an internal volume, and a second type of cell disposed in the internal volume.

[0006] The three-dimensional scaffold matrix of the biosphere may include collagen and laminin. The three-dimensional scaffold matrix may also include collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

[0007] The first type of cell included in the biosphere may include a somatic cell. The somatic cell can, for example, be selected from a cumulus cell or a fibroblast cell. The first type of cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a protein selected from a growth factor, an extracellular matrix protein, or a cytokine. The extracellular matrix protein can, for example, be selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGF[3, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof. The cytokine can, for example, be a leukemia inhibitor factor (LIF).

[0008] The second type of cell included in the biosphere may include a somatic cell. The somatic cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein or a selectable marker. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a selectable marker. In certain embodiments, the selectable marker is selected from a fluorescent protein (e.g., a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP)), a drug resistance marker, an auxotrophic marker, or a [3- galactosidase (LacZ).

[0009] The second type of cell of the biosphere may include an induced pancreatic beta cell, a stem cell, or a dopaminergic cell. The stem cell can, for example, be selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell. If included, the stem cell may be induced to differentiate following incorporation in the biosphere.

[0010] According to another aspect of the disclosure, a method of generating a biosphere is provided. This method may include steps of mixing a first type of cell with a culture media and a three-dimensional scaffold matrix for biological growth to generate a biosphere culture and incubating the biosphere culture. The first type of cell is configured to be in contact with the three-dimensional scaffold matrix and grow to generate the biosphere with an internal volume that is configured to receive a second type of cell.

[0011] The method may also include the step of mixing the second type of cell with the first type of cell prior to generation of the biosphere.

[0012] The method may also include the step of disposing the second type of cell within the internal volume of the biosphere.

[0013] The method may also include the step of micropipetting the second type of cell within the internal volume of the biosphere.

[0014] In some examples, the three-dimensional scaffold matrix used in the method may include collagen and laminin. In addition, the three-dimensional scaffold matrix may include collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

[0015] The first type of cell included in the biosphere may include a somatic cell. The somatic cell can, for example, be selected from a cumulus cell or a fibroblast cell. The first type of cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a protein selected from a growth factor, an extracellular matrix protein, or a cytokine. The extracellular matrix protein can, for example, be selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGF[3, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof. The cytokine can, for example, be a leukemia inhibitor factor (LIF).

[0016] The second type of cell included in the biosphere may include a somatic cell. The somatic cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein or a selectable marker. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a selectable marker. In certain embodiments, the selectable marker is selected from a fluorescent protein (e.g., a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP)), a drug resistance marker, an auxotrophic marker, or a b- galactosidase (LacZ).

[0017] In other examples, the second type of cell used in the method may include an induced pancreatic beta cell or a stem cell. The stem cell can, for example, be selected from an inducedpluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell. If a stem cell is used, the stem cell may be induced to differentiate following incorporation in the biosphere.

[0018] Additional features, advantages, and embodiments of the disclosure may be set forth or apparent from consideration of the detailed description and drawings. Moreover, it is to be understood that the foregoing summary of the disclosure and the following detailed description and drawings provide non-limiting examples that are intended to provide further explanation without limiting the scope of the disclosure as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are included to provide a further understanding of the disclosure, are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the detailed description serve to explain the principles of the disclosure. No attempt is made to show structural details of the disclosure in more detail than may be necessary for a fundamental understanding of the disclosure and the various ways in which it may be practiced.

[0020] FIG. 1 shows a three-dimensional rendering of a nonlimiting embodiment of bovine cumulus cells along with a matrix for producing a biosphere according to the principles of the disclosure.

[0021] FIG. 2 shows a three-dimensional rendering of a nonlimiting embodiment of the biosphere having a first type of cell grown on the matrix of FIG. 1 and suitable for containing a second type of cell according to the principles of the disclosure.

[0022] FIG. 3 shows images of cumulus cells (cultured in the same conditions as shown in FIG. 2), fibroblast cells expressing GFP, and cumulus cells and fibroblast cells expressing GFP cultured together in a ratio of 1 : 1 for 4 days, both seeded in DMEM containing 20% FBS, 1% non-essential amino acids, 55 mM [3-mercaptoethanol, 1% penicillin / streptomycin, and mineral oil, on low attachment 8-well chambers to elicit biosphere formation.

[0023] FIG. 4 shows images of cumulus cells (cultured in the same conditions as shown in FIG. 2), fibroblast cells expressing GFP, and cumulus cells and fibroblast cells expressing GFP cultured together in a ratio of 1 : 1 for 4 days, both seeded in DMEM containing 20% FBS, 1%non-essential amino acids, 55 mM [3-mercaptoethanol, 1% penicillin / streptomycin, and mineral oil, on low attachment 8-well chambers to elicit biosphere formation.

[0024] The present disclosure is further described in the detailed description that follows.DETAILED DESCRIPTION OF THE DISCLOSURE

[0025] The disclosure and its various features and advantageous details are explained more fully with reference to the non-limiting embodiments and examples that are described or illustrated in the accompanying drawings and detailed in the following description. It should be noted that features illustrated in the drawings are not necessarily drawn to scale, and features of one embodiment can be employed with other embodiments as those skilled in the art would recognize, even if not explicitly stated. Descriptions of well-known components and processing techniques may be omitted so as to not unnecessarily obscure the embodiments of the disclosure. The examples are intended merely to facilitate an understanding of ways in which the disclosure can be practiced and to further enable those skilled in the art to practice the embodiments of the disclosure. Accordingly, the examples and embodiments should not be construed as limiting the scope of the disclosure. Moreover, it is noted that like reference numerals represent similar parts throughout the several views of the drawings.

[0026] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0028] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0029] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, aconcentration of 1 mg / mL includes 0.9 mg / mL to 1 .1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0030] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0031] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0032] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0033] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integeror group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.

[0034] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03.

[0035] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.

[0036] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art ( .g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0037] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0038] As used herein, the term “vector” is a replicon in which another nucleic acid segment can be operably inserted so as to bring about the replication or expression of the segment.

[0039] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0040] As used herein, “somatic cell” refers to any cell of a living organism other than a reproductive cell (i.e., the sperm and egg cells). In certain embodiments, the somatic cell is selected from a skin cell, a bone cell, a blood cell, a connective tissue cell, a cumulus cell, a granulosa cell, and / or a cell from the reproductive tract (e.g., a cell from the oviduct or a cell from the uterus).

[0041] As used herein, “selectable marker” is an agent, such as a nucleic acid sequence, that allows one to select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity. Examples of selectable markers include but are not limited to: (1) nucleic acid sequences that encode products which provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid sequences that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid sequences that encode products which suppress the activity of a gene product; (4) nucleic acid sequences that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and cell surface proteins); (5) nucleic acid sequences that bind products which are otherwise detrimental to cell survival and / or function; (6)nucleic acid sequences that otherwise inhibit the activity of any of the nucleic acid sequences described in Nos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acid sequences that bind products that modify a substrate (e.g. restriction endonucleases); (8) nucleic acid sequences that can be used to isolate or identify a desired molecule (e.g. specific protein binding sites); (9) nucleic acid sequences that encode a specific nucleotide sequence which can be otherwise nonfunctional (e.g., for PCR amplification of subpopulations of molecules); (10) nucleic acid sequences, which when absent, directly or indirectly confer resistance or sensitivity to particular compounds; and / or (11) nucleic acid sequences that encode products which are toxic in recipient cells. Examples of toxic gene products are well known in the art, and include, but are not limited to, restriction endonucleases (e.g., Dpnl), apoptosis-related genes (e.g. ASK1 or members of the bcl-2 / ced-9 family), retroviral genes including those of the human immunodeficiency virus (HIV), defensins such as NP-1, inverted repeats or paired palindromic nucleic acid sequences, bacteriophage lytic genes such as those from (0X174 or bacteriophage T4; antibiotic sensitivity genes such as rpsL, antimicrobial sensitivity genes such as pheS, plasmid killer genes, eukaryotic transcriptional vector genes that produce a gene product toxic to bacteria, such as GATA-1, and genes that kill hosts in the absence of a suppressing function, e.g., kicB, ccdB, X174 E (Liu, Q. et al., Curr. Biol. 8: 1300-1309 (1998), and other genes that negatively affect replicon stability and / or replication. A toxic gene can alternatively be selectable in vitro, e.g., a restriction site.

[0042] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.BIOSPHERES AND METHODS OF GENERATING THE SAME

[0043] The present disclosure provides a technological solution that includes a biological structure such as a biosphere that includes a first type of cell grown on a three-dimensional matrix and where the biological structure is configured to contain a second type of cell. In addition, the disclosure provides protocols and reagents for producing these biological structures. In various embodiments, the biological structures provide a mechanism for integrating the second type of cell for providing a conducive environment for growth of the second type of cells, and the like.

[0044] More particularly, the present disclosure provides a technological solution that includes the use of cells and / or other biological materials to create a biosphere structure that allows for other types of cells to be housed within, and to grow, proliferate, differentiate, and function biologically. This biosphere provides a physical integrity with protective structure and compartmented spaces for other types of cells to settle in, proliferate, differentiate, and to exert their biological functions. This biosphere allows cells to be housed within, protected from their environment and to function biologically. Importantly, this biosphere with compartmentalized spaces with strong and flexible walls will provide a new biological structure of housing biologically active cells to grow and to conduct their biological functions.

[0045] It is an advantage of embodiments described herein that the biological structures provide a variety of different ways of implementation both in research and in human health. In research, the biosphere can provide a tool to study cellular differentiation, cellular functions, and interactions. In human health, the biosphere has the potential of providing patients with biologically active pharmaceutical reagents for treatment. Of note, the biological structures described herein may utilize different shapes to be placed in different environments for growth and development. This biosphere can be created with the inclusion of many types of cells and extracellular matrices.

[0046] FIG. 1 shows a nonlimiting embodiment of a three-dimensional (“3D”) matrix 10 for constructing a biosphere 20 (shown in FIG. 2). As shown in FIG. 1, the 3D matrix 10 includes a plurality of interconnecting strands or filaments 12. The filaments 12 may include any suitable bio-compatible material capable of forming such filaments. Examples of suitable materials includes laminin, collagen IV, entactin, heparin sulfate proteoglycans, other proteins, sugars, lipids, and the like.

[0047] In certain embodiments, the three-dimensional matrix is a scaffold-based structure. The scaffold-based structure can, for example, comprise a gel-like material or a structural scaffold. The gel-like material can, for example, be selected from a hydrogel, an agarose, a basement membrane extract, or an extracellular matrix. The extracellular matrix can, for example, be synthetic or natural. By synthetic, it is meant that the extracellular matrix is created under laboratory conditions. By natural, it is meant that the extracellular matrix is derived and / or isolated from a particular organism. Gel-like materials are known in the art, and are available commercially, see, e.g., MyoGel (pharmasana.co.uk); OBAGEL® (Obatala; New Orleans, LA),CETUREGEL™ (Yeasen Biotechnology; Shanghai, China), JELLAGEL® (Jellagen; Wales), GROWDEX® (UPM Biomedicals; Helsinki, Finland), GELTREX™ (Thermo Fisher; Waltham, MA), and MATRIGEL® (Corning Life Sciences; Corning, NY). In a particular example, a suitable 3D matrix includes GELTREX™ manufactured by Thermo Fisher Scientific Inc.

[0048] FIG. 2 shows a nonlimiting embodiment of the biosphere 20. As shown in FIG. 2, the biosphere 20 includes a first type of cell 22 growing on the 3D matrix and that includes interior spaces that are suitable for containing a second type of cell 24. In the example shown, the first type of cell 22 has grown to confluence to form and cover the surfaces of the filaments shown in FIG. 1. This confluent growth acts to reduce the spacing between the filaments shown in FIG. 1 so that the second type of cell may be physically trapped within the biosphere 20. In other examples, the confluent growth of the first type of cell 22 may be configured to provide a permissible surface for the second type of cell to adhere.

[0049] As described herein, this structure may be generated in a variety of ways. For example, the two types of cells may be mixed and added to a growth media containing the 3D matrix 10. The first type of cell 22 may include a cell type that adheres to, grows on, and forms the filaments 12. The second type of cell may randomly enter the 3D matrix 10 and become trapped as the spacing between the filaments 12 is reduced by the growth of the first type of cell 22. In another example, the second type of cell may be introduced after the first type of cell 22 has begun to grow on the 3D matrix but prior to the spacing prohibiting entry of the second type of cell. In yet another example, the second type of cell may be mechanically introduced into a mature biosphere 20 via a micropipette or the like.

[0050] Thus, provided herein are biospheres. The biospheres can, for example, comprise (a) a three-dimensional scaffold matrix for biological growth; (b) a first type of cell configured to be in contact with the three-dimensional scaffold matrix to grow to generate the biosphere with an internal volume; and (c) a second type of cell disposed in the internal volume.

[0051] In certain embodiments, the three-dimensional scaffold matrix comprises collagen and laminin. The three-dimensional scaffold matrix can, for example, further comprise collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

[0052] In certain embodiments, the first type of cell comprises a somatic cell. The somatic cell can, for example, be selected from a cumulus cell or a fibroblast cell. The first type of cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein.The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a protein selected from a growth factor, an extracellular matrix protein, or a cytokine. The extracellular matrix protein can, for example, be selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGF[3, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof. The cytokine can, for example, be a leukemia inhibitor factor (LIF).

[0053] In certain embodiments, the second type of cell comprises a somatic cell. The somatic cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a selectable marker. In certain embodiments, the selectable marker is selected from a fluorescent protein (e.g., a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP)), a drug resistance marker, an auxotrophic marker, or a b-galactosidase (LacZ).

[0054] In certain embodiments, the second type of cell is an induced pancreatic beta cell, a stem cell, or a dopaminergic cell. The stem cell can, for example, be selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell. If included, the stem cell may be induced to differentiate following incorporation in the biosphere.

[0055] Also provided are methods of generating a biosphere. The methods comprise: (a) mixing a first type of cell with a culture media and a three-dimensional scaffold matrix for biological growth to generate a biosphere culture; and (b) incubating the biosphere culture, wherein the first type of cell is configured to be in contact with the three-dimensional scaffold matrix and grow to generate the biosphere with an internal volume that is configured to receive a second type of cell.

[0056] In certain embodiments, the methods further comprise mixing the second type of cell with the first type of cell prior to generation of the biosphere. In certain embodiments, the methods further comprise disposing the second type of cell within the internal volume of the biosphere. In certain embodiments, the methods further comprise micropipetting the second type of cell within the internal volume of the biosphere.

[0057] In certain embodiments, three-dimensional scaffold matrix comprises collagen and laminin. The three-dimensional scaffold matrix can, for example, further comprise collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

[0058] In certain embodiments, the first type of cell comprises a cumulus cell or a fibroblast cell. The first type of cell can, for example, be transfected with a vector comprising a nucleic acid sequence encoding a protein. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a growth factor, an extracellular matrix protein, or a cytokine. The extracellular matrix protein can, for example, be selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGF[3, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof. The cytokine can, for example, be a leukemia inhibitor factor (LIF).

[0059] In certain embodiments, the second type of cell comprises a somatic cell. The methods can, for example, further comprise transfecting the somatic cell with a vector comprising a nucleic acid sequence encoding a protein. The protein can, for example, be a biologically functional protein. In some instances, the nucleic acid sequence encodes a selectable marker. In certain embodiments, the selectable marker is selected from a fluorescent protein (e.g., a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP)), a drug resistance marker, an auxotrophic marker, or a [3- galactosidase (LacZ).

[0060] In certain embodiments, the second type of cell is an induced pancreatic beta cell, a stem cell, or a dopaminergic cell. The stem cell can, for example, be selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell. The methods can, for example, further comprise inducing the stem cell to differentiate following incorporation in the biosphere.EMBODIMENTS

[0061] Embodiment l is a biosphere comprising (a) a three-dimensional scaffold matrix for biological growth; (b) a first type of cell configured to be in contact with the three-dimensional scaffold matrix and grow to generate the biosphere with an internal volume; and (c) a second type of cell disposed in the internal volume.

[0062] Embodiment 2 is the biosphere of Embodiment 1, wherein the three-dimensional scaffold matrix comprises collagen and laminin.

[0063] Embodiment 3 is the biosphere of Embodiment 2, wherein the three-dimensional scaffold matrix further comprises collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

[0064] Embodiment 4 is the biosphere of any one of Embodiments 1-3, wherein the first type of cell comprises a somatic cell.

[0065] Embodiment 5 is the biosphere of Embodiment 4, wherein the somatic cell is a cumulus cell or a fibroblast cell.

[0066] Embodiment 6 is the biosphere of any one of Embodiments 1-5, wherein the first type of cell is transfected with a vector comprising a nucleic acid sequence encoding a protein.

[0067] Embodiment 7 is the biosphere of Embodiment 6, wherein the protein is selected from a growth factor, an extracellular matrix protein, or a cytokine.

[0068] Embodiment 8 is the biosphere of Embodiment 7, wherein the extracellular matrix protein is selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGFb, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof.

[0069] Embodiment 9 is the biosphere of Embodiment 7, wherein the cytokine is leukemia inhibitory factor (LIF).

[0070] Embodiment 10 is the biosphere of any one of Embodiments 1-9, wherein the second type of cell comprises a somatic cell.

[0071] Embodiment 1 1 is the biosphere of Embodiment 10, wherein the somatic cell is transfected with a vector comprising a nucleic acid sequence encoding a protein or a selectable marker.

[0072] Embodiment 12 is the biosphere of Embodiment 11, wherein the selectable marker is selected from a fluorescent protein, a drug resistance marker, an auxotrophic marker, or a [3- galactosidase.

[0073] Embodiment 13 is the biosphere of Embodiment 12, wherein the fluorescent protein is selected from a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP).

[0074] Embodiment 14 is the biosphere of any one of Embodiments 1-9, wherein the second type of cell is an induced pancreatic beta cell, a stem cell, or a dopaminergic cell.

[0075] Embodiment 15 is the biosphere of Embodiment 14, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell.

[0076] Embodiment 16 is the biosphere of Embodiment 14 or 15, wherein the stem cell is induced to differentiate following incorporation in the biosphere.

[0077] Embodiment 17 is a method of generating a biosphere, the method comprising: (a) mixing a first type of cell with a culture media and a three-dimensional scaffold matrix for biological growth to generate a biosphere culture; and (b) incubating the biosphere culture, wherein the first type of cell is configured to be in contact with the three-dimensional scaffold matrix and grow to generate the biosphere with an internal volume that is configured to receive a second type of cell.

[0078] Embodiment 18 is the method of Embodiment 17, further comprising: (c) mixing the second type of cell with the first type of cell prior to generation of the biosphere.

[0079] Embodiment 19 is the method of Embodiment 17, further comprising: (c) disposing the second type of cell within the internal volume of the biosphere.

[0080] Embodiment 20 is the method of Embodiment 19, further comprising: (c) micropipetting the second type of cell within the internal volume of the biosphere.

[0081] Embodiment 21 is the method of any one of Embodiments 17-20, wherein the three- dimensional scaffold matrix comprises collagen and laminin.

[0082] Embodiment 22 is the method of Embodiment 21, wherein the three-dimensional scaffold matrix further comprises collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

[0083] Embodiment 23 is the method of any one of Embodiments 17-22, wherein the first type of cell comprises a somatic cell.

[0084] Embodiment 24 is the method of Embodiment 23, wherein the somatic cell is a cumulus cell or a fibroblast cell.

[0085] Embodiment 25 is the method of any one of Embodiments 17-24, wherein the first type of cell is transfected with a vector comprising a nucleic acid sequence encoding a protein.

[0086] Embodiment 26 is the method of Embodiment 25, wherein the protein is selected from a growth factor, an extracellular matrix protein, or a cytokine.

[0087] Embodiment 27 is the method of Embodiment 26, wherein the extracellular matrix protein is selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGF[3, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof.

[0088] Embodiment 28 is the method of Embodiment 26, wherein the cytokine is leukemia inhibitor factor (LIF).

[0089] Embodiment 29 is the method of any one of Embodiments 17-28, wherein the second type of cell comprises a somatic cell.

[0090] Embodiment 30 is the method of Embodiment 29, further comprising transfecting the somatic cell with a vector comprising a nucleic acid sequence encoding a protein or a selectable marker.

[0091] Embodiment 31 is the method of Embodiment 30, wherein the selectable marker is selected from a fluorescent protein, a drug resistance marker, an auxotrophic marker, or a [3- galactosidase.

[0092] Embodiment 32 is the method of Embodiment 31, wherein the fluorescent protein is selected from a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP).

[0093] Embodiment 33 is the method of any one of Embodiments 17-28, wherein the second type of cell is an induced pancreatic beta cell, a stem cell, or a dopaminergic cell.

[0094] Embodiment 34 is the method of Embodiment 33, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell.

[0095] Embodiment 35 is the method of Embodiment 33 or 34, further comprising inducing the stem cell to differentiate following incorporation in the biosphere.

[0096] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.EXAMPLESExample 1: BIOSPHERE CREATION

[0097] Example l is a protocol for creating the biosphere 20 with cumulus cells. The cells were taken from the incubator and placed into a centrifuge tube. The tube was centrifuged at 300 x g for 5 minutes resulting in a cell pellet at the bottom of the centrifuge tube. The media was then aspirated from the tube. New media and a thawed matrix solution were added together. The solution was pipetted up and down to mix the solution together. The new media mixture was added to the cells, which allowed the cells to be resuspended in the growth agent for the spheroid. The cells were then dispensed into a 6-well plate with a cell repellent surface, which allowed the cells to not stick to the surface and to attach to each other. The cells were placed back into the incubator to allow for further cell growth. This process allowed for the cells to grow on each other and form a spheroid structure.

[0098] With reference to FIGS. 1 and 2, cumulus cell 3-D culture were used to generate the biospheres 20. A solution of the matrix 10 was thawed on ice in a 4°C refrigerator overnight. An aliquot of 500 pl of the matrix solution was placed in each of ten 1.5 mL tubes on ice. The cumulus cells were washed with phosphate buffered saline (PBS) twice and trypsinized by adding four drops of trypsin solution, incubated for 5-10 minutes at 38.5°C and the flask wastapped gently to detach the cells. The cells were centrifuged at 500 x g for 5 minutes and the supernatant was removed, and the cells were divided into two 1.5 mL tubes.

[0099] The cumulus cells were plated by adding 1 mL of regular DMEM medium into one of the 1.5 mL tubes. An aliquot of 500 pl of the cumulus cell solution was taken from the tube and added to two wells of a 4-well plate, covered with mineral oil, and placed in an incubator. 200 pl of the matrix solution was added into the other tube and mixed gently by pipetting up and down three times. 50 pl of the cells were plated with the matrix solution onto each well of a 4- well plate. The 4-well plate was placed into the incubator for 20-30 minutes. The 4-well plate was removed and checked to determine if the matrix solution was solidified. An aliquot of 450 pl of regular DMEM medium was added into each well and then covered in mineral oil. The 4- well plate was placed back into the incubator.Example 2: BIOSPHERE CREATION WITH GFP TAGGED SOMATIC CELLS

[0100] Example 2 is a protocol for creating the biosphere 20 with somatic cells tagged with a green fluorescent protein (GFP). Somatic cells, such as fibroblast cells, are transfected with a vector encoding a GFP reporter. The GFP reporter gene is confirmed to be successfully integrated and expressed by visualization of GFP. The GFP-tagged cells are expanded and frozen in aliquots. The matrix solution is thawed on ice in a 4°C refrigerator overnight. The following day, 500 pl of the matrix solution is added to each of ten 1.5 ml tubes. Any unused tubes are stored at -20°C.

[0101] Bovine cumulus cells are trypsinized by washing the cells in a T-12.5 flask with PBS twice. 4 drops of trypsin solution are added to the cells. The cells are incubated for 5-10 minutes at 38.5°C. The flask is tapped gently to detach the cells. The cells are divided into two 1.5 ml tubes. The cells are centrifuged at 500 x g for 5 minutes, and the supernatant is removed from each tube. The tubes with the cell pellets are placed on ice. The GFP-tagged somatic cells are produced in the same manner as the bovine cumulus cells.

[0102] A mixture of bovine cumulus cells, GFP-tagged somatic cells and matrix solution is produced by dividing the GFP-tagged somatic cells into the same 1.5 ml tubes as the bovine cumulus cells. The ratio of the two types of cells is 1 : 1. The cells are mixed by gently pipetting up and down. The cells are centrifuged at 500 x g for 5 minutes, and then the supernatant is removed. 200 pl of matrix solution is added into each tube and mixed gently by pipetting up and down. 50 pl of the mix is seeded into each well of a 4-well plate. The 4-well plate is placed intothe incubator at 37°C for 10-15 minutes. The 4-well plate is removed and checked to determine if the matrix solution is solidified. 450 pl of DMEM + 10% FBS is added into each well after the matrix solution is solidified. Each well is covered with mineral oil and the 4-well plate is incubated at 37°C in 5% CO2. The cells are monitored for growth. The formation of compartments is visualized by staining the bovine cumulus cells, and the growth and distribution of the GFP-tagged somatic cells within the compartments is visualized by GFP fluorescence microscopy.Example 3: BIOSPHERE CREATION WITH PANCREATIC BETA-CELLS

[0103] Example 3 is a protocol for creating the biosphere 20 with induced pancreatic betacells. An advantage of this embodiment is that using the biosphere technology to grow pancreatic beta-cells could potentially help diabetic patients. In this example, embryonic stem cells (ES cells) are first cultured within the biospheres and then induced to differentiate into functioning pancreatic beta-cells.

[0104] ES cells are grown and harvested according to standard ES culture protocols. The ES cells are selected to differentiate into the biosphere 20. The ES cells are differentiated into betacells according to conventional protocols. The differentiated beta-cells are expanded and harvested, and a mixture of bovine cumulus cells, beta-cells and matrix solution is prepared as detailed in the above-mentioned protocol for the GFP-tagged somatic cells. The growth of betacells is monitored within the biospheres 20. Studies are conducted, including confocal microscopy, on the growth and function of the beta-cells.Example 4: BIOSPHERE CREATION FOR EMBRYONIC STEM CELL GROWTH AND DIFFERENTIATION

[0105] Example 4 is a protocol for creating the biosphere 20 to grow and differentiate ES cells. An advantage of this embodiment is that the biospheres 20 may be configured to provide a favorable environment for the growth and differentiation of ES cells due to the 3D supporting structure and the compartmentalized micro-environment within the biosphere 20.

[0106] In this example, ES cells are grown and harvested according to standard ES culture protocols. Prepare and make mix of bovine cumulus cells, ES cells and matrix solution as detailed in the protocol for the GFP-tagged somatic cells. Monitor the growth of the ES cells inside the biospheres. Induce differentiation of the ES cells inside the biospheres using published protocols, transcription factors and reagents to targeted cell types. Conduct studiesincluding confocal microscope on the growth, differentiation and function of the differentiated cells induced from the ES cells.Example 5: BIOSPHERE CREATION FOR A MIX OF 1:1 CUMULUS CELLS AND FIBROBLASTS

[0107] GFP expressing cells: Primary bovine fibroblast cells were derived from skin ear biopsy of a male donor. At passage 2, cells were infected with Sendai virus EmGFP at an MOI of 5 (INVITROGEN™; CYTOTUNE™ EmGFP Sendai Fluorescence Reporter, Catalog Number A16519). Two days later cells were passaged. After four (4) days of transduction, cells were harvested for being used for the experiment.

[0108] Cumulus cells: Cumulus cells were isolated from cumulus-oocyte complexes (COCs) obtained from a local abattoir and supplied by ART Inc. (Madison, Wisconsin, USA). The COCs were transported in buffered maturation medium (Boviteq in vitro maturation medium; Boviteq; Madison, WI) and incubated for 20 to 24 hours. Upon arrival at the laboratory, the COCs were washed with a commercial washing medium (Boviplus, Minitube). To dissociate cumulus cells, the complexes were incubated in a hyaluronidase solution (Fujifilm Irvine Scientific Inc; Santa Ana, CA) for 3 minutes, followed by thorough pipetting. Oocytes were then separated, and cumulus cells were collected in 1.5 m tubes containing 1 mL of Boviplus. These cells were centrifuged at 2,000 rpm for 5 minutes, and this centrifugation was repeated to ensure complete removal of the maturation medium. The resulting pellet was resuspended in 300 pL of Boviplus. After confirming there was no mycoplasma contamination, the cumulus cells were counted, totaling 470xl05for subsequent experimental procedures.

[0109] Once cells were counted and washed, cumulus cells and fibroblast-GFP cells were mixed in a 1 : 1 ratio, and then pelleted by centrifugation for 10 minutes at room temperature. The tube containing the cell pellet was placed on ice for 10 minutes. After cooling down the cell pellet, the supernatant was discarded and Matrigel was added for a final density of 800x105in 200 pL.

[0110] Different seeding surfaces, different volumes (50, 10, and 5 pL droplets), and media were tested. Bio-inert or glass low attachment, with or without mineral oil, U / flat bottom were included. From the different seeding surfaces, the only one that allows a well-formed biosphere at day 4 was the sample seeded on Bio-inert (Ibidi, Cat. No. 80800) or glass (Ibidi, Cat. No. 80807) bottom chambers with mineral oil and cultured in DMEM containing 20% FBS, 1% non-essential amino acids, 55mM b-mercaptoethanol, 1% penicillin / streptomycin (FIGs. 3 and 4). This media is suitable for a wide variety of cells and also supports cumulus cell growth. From the different volumes tested, 5 mL droplets containing 2 x 104cells each resulted in the optimal cell density mixture for Fibroblasts and cumulus cells (1 x 104each cell type). lOmL and 50mL droplets generated bigger but more loose structures with higher fragility at day 4, which were easy to disrupt by pipetting.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A biosphere comprising:(a) a three-dimensional scaffold matrix for biological growth;(b) a first type of cell configured to be in contact with the three-dimensional scaffold matrix to grow to generate the biosphere with an internal volume; and(c) a second type of cell disposed in the internal volume.

2. The biosphere of claim 1, wherein the three-dimensional scaffold matrix comprises collagen and laminin.

3. The biosphere of claim 2, wherein the three-dimensional scaffold matrix further comprises collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

4. The biosphere of claim 1, wherein the first type of cell comprises a somatic cell.

5. The biosphere of claim 4, wherein the somatic cell is selected from a cumulus cell or a fibroblast cell.

6. The biosphere of claim 1, wherein the first type of cell is transfected with a vector comprising a nucleic acid sequence encoding a protein.

7. The biosphere of claim 6, wherein the protein is selected from a growth factor, an extracellular matrix protein, or a cytokine.

8. The biosphere of claim 7, wherein the extracellular matrix protein is selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGFP, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof.

9. The biosphere of claim 7, wherein the cytokine is leukemia inhibitory factor (LIF).

10. The biosphere of claim 1, wherein the second type of cell comprises a somatic cell.

11. The biosphere of claim 10, wherein the somatic cell is transfected with a vector comprising a nucleic acid sequence encoding a protein or a selectable marker.

12. The biosphere of claim 11 , wherein the selected marker is selected from a fluorescent protein, a drug resistance marker, an auxotrophic marker, or a P-galactosidase.

13. The biosphere of claim 12, wherein the fluorescent protein is selected from a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP).

14. The biosphere of claim 1, wherein the second type of cell is an induced pancreatic beta cell, a stem cell, or a dopaminergic cell.

15. The biosphere of claim 14, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell.

16. The biosphere of claim 14, wherein the stem cell is induced to differentiate following incorporation in the biosphere.

17. A method of generating a biosphere, the method comprising:(a) mixing a first type of cell with a culture media and a three-dimensional scaffold matrix for biological growth to generate a biosphere culture; and(b) incubating the biosphere culture, wherein the first type of cell is configured to be in contact with the three-dimensional scaffold matrix and grow to generate the biosphere with an internal volume that is configured to receive a second type of cell.

18. The method of claim 17, further comprising:(c) mixing the second type of cell with the first type of cell prior to generation of the biosphere.

19. The method of claim 17, further comprising:(c) disposing the second type of cell within the internal volume of the biosphere.

20. The method in claim 19, further comprising:(d) micropipetting the second type of cell within the internal volume of the biosphere.

21. The method of claim 17, wherein the three-dimensional scaffold matrix comprises collagen and laminin.

22. The method of claim 21, wherein the three-dimensional scaffold matrix further comprises collagen IV, entactin, heparin sulfate proteoglycans, and / or combinations thereof.

23. The method of claim 17, wherein the first type of cell comprises a somatic cell.

24. The method of claim 23, wherein the somatic cell is selected from a cumulus cell or a fibroblast cell.

25. The method of claim 17, wherein the first type of cell is transfected with a vector comprising a nucleic acid sequence encoding a protein.

26. The method of claim 21, wherein the protein is selected from a growth factor, an extracellular matrix protein, or a cytokine.

27. The method of claim 26, wherein the extracellular matrix protein is selected from fibronectin, proteoglycan, integrin, brevican, neurocan, perlecan, TGFBR3 protein, type VIII collagen, lumican, fibromodulin, elastin, type IV collagen, keratocan, nidogen-1, syndecan-3, tenascin, glypican 1, fibrillin, glypican 5, type XV collagen alpha 1, glypican 4, glypican 6, syndecan 1, syndecan 4, syndecan 2, glypican 3, SPOCK 1, decorin, vitronectin, CD44, tenascin X, tenascin C, tenascin R, reelin, TGFP, FREM2, beta laminin 1, type V collagen alpha 3, or combinations thereof.

28. The method of claim 26, wherein the cytokine is leukemia inhibitor factor (LIF).

29. The method of claim 17, wherein the second type of cell comprises a somatic cell.

30. The method of claim 29, further comprising transfecting the somatic cell with a vector comprising a nucleic acid sequence encoding a protein or a selectable marker.

31. The method of claim 30, wherein the selectable marker is selected from a fluorescent protein, a drug resistance marker, an auxotrophic marker, or a P-galactosidase.

32. The method of claim 31, wherein the fluorescent protein is selected from a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or a green fluorescent protein (GFP).

33. The method of claim 17, wherein the second type of cell is an induced pancreatic beta cell, a stem cell, or a dopaminergic cell.

34. The method of claim 33, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), a hematopoietic stem cell, a cord blood stem cell, or an adult stem cell.

35. The method of claim 33, further comprising inducing the stem cell to differentiate following incorporation in the biosphere.

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