Methods and systems for co-differentiating cells using optogenetics

Abstract

Provided herein are methods and systems for co-differentiating differentiable cells in a differentiable cell population into two or more different cell lineages. In some embodiments, the methods and systems use photoactivatable or chemically activatable recombinase to drive the expression of one or more transcription factors and / or differentiation factors.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 385,716, filed December 1, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Transcription factors can regulate the differentiation of cells into different cell types. For example, expression of transcription factors such as MyoD can lead to the differentiation of cells into muscle cells, while expression of other transcription factors such as PPARγ can lead to the differentiation of cells into adipocytes. However, using existing technologies, it is currently impossible to co-differentiate cells from the same cell population into multiple different cell lineages with high temporal and spatial precision. Summary of the Invention

[0004] There is an unmet need for methods and systems for co-differentiating cells within a cell population into multiple cell lineages with high temporal and spatial precision. The methods and systems described herein address this unmet need.

[0005] In one aspect, a method for co-differentiating a population of differentiable cells is provided, the method comprising: (a) providing or obtaining a population of differentiable cells; (b) controlling the differentiation of a first differentiable cell in the population of differentiable cells with light, thereby causing the first differentiable cell to differentiate into a first cell lineage; and (c) causing a second differentiable cell in the population of differentiable cells to differentiate into a second cell lineage, wherein the first cell lineage and the second cell lineage are different, thereby causing the population of differentiable cells to co-differentiate. In some cases, the control of differentiation in (b) comprises irradiating the first differentiable cell with light of a first wavelength or wavelength range. In some cases, the control of differentiation in (b) comprises removing light of the first wavelength or wavelength range from the first differentiable cell. In some cases, the differentiation in (c) comprises irradiating the second differentiable cell in the population of differentiable cells with light of a second wavelength or wavelength range, thereby causing the second differentiable cell to differentiate into a second cell lineage; or the differentiation in (c) comprises removing light of the second wavelength or wavelength range from the second differentiable cell, thereby causing the second differentiable cell to differentiate into a second cell lineage. In some cases, the first wavelength or wavelength range and the second wavelength or wavelength range are different. In some cases, the differentiation in (c) comprises contacting a second differentiable cell in the differentiable cell population with a chemical inducer so that the second differentiable cell differentiates into a second cell lineage; or the differentiation in (c) comprises removing the chemical inducer from the second differentiable cell in the differentiable cell population so that the second differentiable cell differentiates into a second cell lineage. In some cases, each differentiable cell in the differentiable cell population is engineered to contain an exogenous nucleic acid comprising: (i) a nucleic acid sequence encoding at least one first transcription factor or a first differentiation factor that causes differentiation to a first cell lineage; and (ii) a nucleic acid sequence encoding at least one second transcription factor or a second differentiation factor that causes differentiation to a second cell lineage. In some cases, the control in (b) comprises regulating the expression of at least one first transcription factor or a first differentiation factor, and the differentiation in (c) comprises regulating the expression of at least one second transcription factor or a second differentiation factor. In some cases, the exogenous nucleic acid comprises at least one promoter operably linked to a nucleic acid sequence encoding at least one first transcription factor or a first differentiation factor and a nucleic acid sequence encoding at least one second transcription factor or a second differentiation factor. In some cases, at least one promoter is a constitutive promoter. In some cases, the exogenous nucleic acid further comprises: (iii) a blocking sequence downstream of at least one promoter, which, when present, blocks expression of at least one first transcription factor or first differentiation factor and / or at least one second transcription factor or second differentiation factor. In some cases, the nucleic acid sequence encoding at least one first transcription factor or first differentiation factor, or a portion thereof, is present in the exogenous nucleic acid in reverse such that at least one first transcription factor or first differentiation factor is not expressed.In some cases, the nucleic acid sequence encoding at least one second transcription factor or second differentiation factor, or a portion thereof, is present in the exogenous nucleic acid in reverse, so that at least one second transcription factor or second differentiation factor is not expressed. In some cases, each differentiable cell in the differentiable cell population further comprises: (iv) an exogenous nucleic acid sequence encoding a first activatable recombinase; and (v) an exogenous nucleic acid sequence encoding a second activatable recombinase. In some cases, the first activatable recombinase is a photoactivatable recombinase. In some cases, the second activatable recombinase is a photoactivatable recombinase. In some cases, the first activatable recombinase is different from the second activatable recombinase. In some cases, the second activatable recombinase is a chemically activatable recombinase. In some cases, the expression of the first activatable recombinase, the expression of the second activatable recombinase, or both are induced by a chemical inducer, or by light. In some cases, the flanks of the blocking sequence are a first recombinase recognition site recognized by the first activatable recombinase, a second recombinase recognition site recognized by the second activatable recombinase, or both. In some cases, the control in (b), the differentiation in (c), or both result in the excision of the blocking sequence, thereby inducing the expression of at least one first transcription factor or first differentiation factor, at least one second transcription factor or second differentiation factor, or both. In some cases, (i) the nucleic acid sequence encoding at least one first transcription factor or first differentiation factor, or a portion thereof, is present in an inverted orientation in the exogenous nucleic acid; (ii) the nucleic acid sequence encoding at least one second transcription factor or second differentiation factor, or a portion thereof, is present in an inverted orientation in the exogenous nucleic acid sequence; or (iii) both are flanked by a first recombinase recognition site recognized by a first activatable recombinase, a second recombinase recognition site recognized by a second activatable recombinase, or both. In some cases, the control in (b), the differentiation in (c), or both result in (i) inversion of the nucleic acid sequence encoding at least one first transcription factor or first differentiation factor, or a portion thereof, such that the at least one first transcription factor or first differentiation factor is expressed; (ii) inversion of the nucleic acid sequence encoding at least one second transcription factor or second differentiation factor, or a portion thereof, such that the at least one second transcription factor or second differentiation factor is expressed; or (iii) both. In some cases, the method further includes (d) differentiating a third differentiable cell in the differentiable cell population into a third cell lineage, wherein the third cell lineage is different from the first cell lineage and the second cell lineage. In some cases, the differentiation in (d) includes (i) irradiating the third differentiable cell with light of a third wavelength or wavelength range; (ii) removing light of the third wavelength or wavelength range from the third differentiable cell; (ii) contacting the third differentiable cell with a chemical inducer; or (iv) removing the chemical inducer from the third differentiable cell. In some cases, the third wavelength or wavelength range is different from the first wavelength or wavelength range, the second wavelength or wavelength range, or both. In some cases, the differentiable cell population comprises stem cells.In some cases, stem cells are multipotent stem cells or pluripotent stem cells. In some cases, the differentiable cell group comprises mature somatic cells that can be transdifferentiated under certain conditions. In some cases, the differentiable cells are human differentiable cells or cattle differentiable cells. In some cases, the first cell lineage, the second cell lineage and / or the third cell lineage are selected from: adipocytes, myocytes and chondrocytes. In some cases, at least one first transcription factor, at least one second transcription factor or both are selected from: PPARγ, CEBPα, MYOD, MYOG, MYF5, MRF4 (MYF6), HEYL, KLF4, PAX3, PRDM16, SREBP1, SOX9, SOX5, SOX6 and any combination thereof. In some cases, the control in (b) and the differentiation in (c) occur substantially simultaneously. In some cases, the control in (b) and the differentiation in (c) occur sequentially. In some cases, the control in (b) precedes the differentiation in (c), or the differentiation in (c) precedes the control in (b). In some cases, the first differentiable cell and the second differentiable cell are adjacent to each other. In some cases, the control in (b) further includes irradiating multiple first differentiable cells with the light of the first wavelength or wavelength range so that each of the multiple first differentiable cells is differentiated into the first cell lineage; or the control in (b) further includes removing the light of the first wavelength or wavelength range from the multiple first differentiable cells so that each of the multiple first differentiable cells is differentiated into the first cell lineage. In some cases, the differentiation in (c) further includes differentiating multiple second differentiable cells so that each of the multiple second differentiable cells is differentiated into the second cell lineage. In some cases, the differentiable cell group is deposited on a solid support. In some cases, the differentiable cell group is deposited on a solid support in multiple layers. In some cases, the solid support is coated with one or more extracellular matrix components or parts thereof.

[0006] On the other hand, a system for making a differentiable cell group co-differentiate is provided, the system comprising: (a) a differentiable cell group, wherein each differentiable cell in the differentiable cell group is engineered to contain an exogenous nucleic acid, the exogenous nucleic acid comprising: (i) encoding a nucleic acid sequence of at least one first transcription factor or a first differentiation factor that causes differentiation to a first cell lineage; and (ii) encoding a nucleic acid sequence of at least one second transcription factor or a second differentiation factor that causes differentiation to a second cell lineage; and (b) one or more light sources configured to control the differentiation of the first differentiable cell in the differentiable cell group with light of a first wavelength or wavelength range, so that the first differentiable cell differentiates into a first cell lineage, wherein the first cell lineage and the second cell lineage are different. In some cases, the system is configured to use one or more light sources to irradiate the first differentiable cell with light of a first wavelength or wavelength range, so that the first differentiable cell differentiates into a first cell lineage. In some cases, the system is configured to remove the light of the first wavelength or wavelength range from the first differentiable cell, so that the first differentiable cell differentiates into a first cell lineage. In some cases, the system is further configured to use one or more light sources to illuminate the second differentiable cells in the differentiable cell population with light of a second wavelength or wavelength range, so that the second differentiable cells differentiate into a second cell lineage; or the system is further configured to remove the light of the second wavelength or wavelength range from the second differentiable cells, so that the second differentiable cells differentiate into the second cell lineage. In some cases, the first wavelength or wavelength range and the second wavelength or wavelength range are different. In some cases, the system further includes a chemical inducer that causes the second differentiable cells in the differentiable cell population to differentiate into the second cell lineage; or the system is further configured to remove the chemical inducer from the second differentiable cells, so that the second differentiable cells differentiate into the second cell lineage. In some cases, the exogenous nucleic acid comprises at least one promoter operably linked to at least one first transcription factor or first differentiation factor and at least one second transcription factor or second differentiation factor. In some cases, at least one promoter is a constitutive promoter. In some cases, the exogenous nucleic acid further comprises: (iii) a blocking sequence downstream of at least one promoter, which, when present, blocks expression of at least one first transcription factor or first differentiation factor and at least one second transcription factor or second differentiation factor. In some cases, the nucleic acid sequence encoding at least one first transcription factor or first differentiation factor, or a portion thereof, is present in the exogenous nucleic acid in an inverted manner such that at least one first transcription factor or first differentiation factor is not expressed. In some cases, the nucleic acid sequence encoding at least one second transcription factor or second differentiation factor, or a portion thereof, is present in the exogenous nucleic acid in an inverted manner such that at least one second transcription factor or second differentiation factor is not expressed. In some cases, each differentiable cell in the population of differentiable cells further comprises: (iv) a nucleic acid sequence encoding a first activatable recombinase; and (v) a nucleic acid sequence encoding a second activatable recombinase.In some cases, the first activatable recombinase is a photoactivatable recombinase. In some cases, the second activatable recombinase is a photoactivatable recombinase. In some cases, the first activatable recombinase is different from the second activatable recombinase. In some cases, the second activatable recombinase is a chemically activatable recombinase. In some cases, the expression of the first activatable recombinase, the expression of the second activatable recombinase, or both can be induced by a chemical inducer, or can be induced by light. In some cases, the flanks of the blocking sequence are a first recombinase recognition site recognized by the first activatable recombinase, a second recombinase recognition site recognized by the second activatable recombinase, or both. In some cases, the blocking sequence is configured to be excised by the first activatable recombinase, the second activatable recombinase, or both, thereby regulating the expression of at least one first transcription factor or the first differentiation factor, at least one second transcription factor or the first differentiation factor, or both. In some cases, (i) the nucleic acid sequence encoding at least one first transcription factor or first differentiation factor, or a portion thereof, is present in the exogenous nucleic acid in an inverted orientation; (ii) the nucleic acid sequence encoding at least one second transcription factor or second differentiation factor, or a portion thereof, is present in the exogenous nucleic acid sequence in an inverted orientation; or (iii) both are flanked by a first recombinase recognition site recognized by a first activatable recombinase, a second recombinase recognition site recognized by a second activatable recombinase, or both. In some cases, (i) the nucleic acid sequence encoding at least one first transcription factor or first differentiation factor, or a portion thereof, is configured to be inverted by the first activatable recombinase, the second activatable recombinase, or both, such that the at least one first transcription factor or first differentiation factor is expressed; (ii) the nucleic acid sequence encoding at least one second transcription factor or second differentiation factor, or a portion thereof, is configured to be inverted by the first activatable recombinase, the second activatable recombinase, or both, such that the at least one second transcription factor or second differentiation factor is expressed; or (iii) both. In some cases, the exogenous nucleic acid further comprises a nucleic acid sequence encoding at least one third transcription factor or third differentiation factor that causes differentiation to a third cell lineage. In some cases, the system is further configured to use one or more light sources to illuminate the third differentiable cell in the differentiable cell population with a third wavelength or wavelength range of light, so that the third differentiable cell is differentiated into a third cell lineage; or wherein the system is further configured to remove the light of the third wavelength or wavelength range, so that the third differentiable cell is differentiated into a third cell lineage, wherein the third wavelength or wavelength range is different from the first wavelength or wavelength range and / or the second wavelength or wavelength range, and wherein the third cell lineage is different from the first cell lineage and / or the second cell lineage. In some cases, the differentiable cell is a stem cell. In some cases, the stem cell is a pluripotent stem cell or a multipotent stem cell. In some cases, the differentiable cell is a mature somatic cell that can be transdifferentiated under certain conditions. In some cases, the differentiable cell is a human differentiable cell or a cattle differentiable cell.In some cases, the first cell lineage, the second cell lineage and / or the third cell lineage are selected from: adipocytes, myocytes and chondrocytes. In some cases, at least one first transcription factor, at least one second transcription factor, at least one third transcription factor or any combination thereof are selected from: PPARγ, CEBPα, MYOD, MYOG, MYF5, MRF4 (MYF6), HEYL, KLF4, PAX3, PRDM16, SREBP1, SOX9, SOX5, SOX6 and any combination thereof. In some cases, the first differentiable cell and the second differentiable cell are adjacent to each other. In some cases, the system further includes a solid support, wherein the differentiable cell group is deposited on the solid support. In some cases, the differentiable cell group is deposited on the solid support in multiple layers. In some cases, the one or more lights include one or more light emitting diodes (LEDs). In some cases, the one or more LEDs include at least two different LEDs. In some cases, at least two different LEDs emit light of different wavelengths or wavelength ranges. In some cases, the one or more light sources include one or more lasers. In some cases, the one or more light sources include incandescent light sources.

[0007] Incorporation by reference

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings, in which:

[0010] Figure 1 Depicted are non-limiting examples of schematic diagrams of nucleic acid sequences for transcription factor expression suitable for use with the methods and systems described herein.

[0011] Figure 2 Depicted are non-limiting examples of inducible recombinase systems suitable for use with the methods and systems described herein.

[0012] Figure 3 Non-limiting examples of using recombinases to invert nucleic acid sequences encoding transcription factors or differentiation factors such that the desired transcription factors or differentiation factors are expressed are described.

[0013] Figure 4Schematic illustration of a non-limiting example of co-differentiation of a population of differentiable cells into multiple cell lineages using the methods and systems described herein.

[0014] Figures 5A-5C Non-limiting examples of constructs and co-differentiation mechanisms described herein are schematically illustrated.

[0015] Figure 6A and Figure 6B Depicted are spatial and temporal optogenetic control of green fluorescent protein (GFP) expression.

[0016] Figures 7A-7C Demonstrated changes in gene expression in response to different light parameters: blue light ( Figure 7A )、UV light( Figure 7B ) and red light ( Figure 7C ).

[0017] Figure 8 demonstrated co-differentiation of differentiable cell populations using blue light and doxycycline.

[0018] Figure 9 Co-differentiation of differentiable cell populations using UV light and doxycycline is demonstrated.

[0019] Figure 10A and Figure 10B Shown via the Blu-ray system ( Figure 10A ) and UV light systems ( Figure 10B ) quantitative data of adipose differentiation.

[0020] Figure 11 Transcription factors or differentiation factors capable of inducing muscle differentiation are shown. DETAILED DESCRIPTION

[0021] The developmental fate of differentiable cells can be determined by expressing in cells a specific transcription factor or differentiation factor that causes it to differentiate into a developmental program of a specific cell type. However, it is currently impossible to use existing technology to make a differentiable cell group differentiate into a variety of cell types (e.g., co-differentiation) in a controlled manner (e.g., temporally, spatially). For example, making differentiable cells shaped into three-dimensional tissues requires differentiable cells to be differentiated into a variety of cell types with high spatiotemporal precision. Controlling the temporal and spatial expression of transcription factors or differentiation factors in differentiable cells allows differentiable cells to be co-differentiated into a variety of cell types (e.g., simultaneously) within a single group. The methods and systems disclosed herein generally use at least one light-activated recombinase to make the first cell differentiate into a first cell lineage. The second cell can be differentiated into a second cell lineage by any mechanism, as described herein, including by using a second light-activated recombinase, by using an inducible promoter (e.g., light-induced, chemical-induced) to express one or more transcription factors and / or differentiation factors, by contacting cells with appropriate culture medium factors, by differentiation mediated by cell-cell contact, etc.

[0022] In some embodiments, provided herein are methods and systems that use optogenetics to (e.g., spatially, temporally) control the co-differentiation of cells to a variety of cell lineages. For example, in some embodiments, the promoter (e.g., light-inducible promoter) of a transcription factor can be induced by irradiating light with a specific wavelength or wavelength range. In some embodiments, provided herein are methods and systems that use recombinases. Recombinases recognize specific DNA sequences, and if there are two recognition sequences that are appropriately arranged, they can excise the DNA between the two sites or invert the direction of the DNA. By simply activating the recombinase, permanent changes can be made to DNA, which provides the prospect of permanently opening differentiation genes with only a short activation phase.

[0023] Disclosed herein are methods and systems for co-differentiating differentiable cells within a population of differentiable cells into multiple cell lineages. In some cases, the methods and systems include the use of a recombinase, such as a recombinase that can be activated by light and / or by chemical means.

[0024] method

[0025] Provided herein is a method for making a differentiable cell group co-differentiate.Methods can include providing or obtaining a differentiable cell (e.g., stem cell) group, controlling the differentiation of the first differentiable cell in the differentiable cell group with light so that the first differentiable cell is differentiated into a first cell lineage, and the second differentiable cell is differentiated into a second cell lineage. In some cases, the first differentiable cell lineage and the second differentiable cell lineage are different, so that the method allows the first differentiable cell and the second differentiable cell to be co-differentiated into different cell lineages within the same differentiable cell group. In some cases, controlling the differentiation of the first differentiable cell with light involves irradiating the first differentiable cell with a first wavelength or wavelength range of light, so that the first differentiable cell is differentiated into the first cell lineage. In other cases, controlling the differentiation of the first differentiable cell with light involves removing the light of the first wavelength or wavelength range from the first differentiable cell. For example, the light source can be turned off so that the first differentiable cell is no longer irradiated by the light of any wavelength. In other cases, light can be changed from the first wavelength or wavelength range to a different wavelength or wavelength range so that the cell is no longer irradiated by the light of the first wavelength or wavelength range.

[0026] In some cases, the methods described herein relate to codifferentiating cells within a differentiable cell population into multiple cell lineages. In some cases, the methods relate to codifferentiating differentiable cells within a differentiable cell population into at least two, at least three, at least four, or at least five different cell lineages.

[0027] The expression of a transcription factor and / or differentiation factor or a combination of transcription factors and / or differentiation factors in a differentiable cell can cause the differentiable cell to differentiate into a specific cell type. Contemplated herein are any transcription factors and / or differentiation factors or combinations of transcription factors and / or differentiation factors that, when expressed, cause the differentiable cell to differentiate into a desired cell lineage. In a non-limiting example, when differentiable cells need to differentiate into adipocytes (e.g., adipocytes), the differentiable cells can be induced to express transcription factors or differentiation factors such as, but not limited to, SREBP1, PPARγ, and / or CEBPα. In another non-limiting example, when differentiable cells need to differentiate into muscle cells (e.g., myocytes), the differentiable cells can be induced to express transcription factors or differentiation factors such as, but not limited to, MYOD, MYOG, MYF5, MRF4 (MYF6), HEYL, KLF4, and / or PAX3. In another non-limiting example, when the differentiable cells are to differentiate into cartilage-forming cells (e.g., chondrocytes), the differentiable cells can be induced to express transcription factors or differentiation factors such as, but not limited to, SOX9, SOX5, and / or SOX6. In some embodiments, at least one transcription factor is selected from the group consisting of: PPARγ, CEBPα, MYOD, MYOG, and combinations thereof. In some cases, the expression of the transcription factor and / or differentiation factor can be regulated, for example, by a chemical inducer or by light.

[0028] It should be understood that the present disclosure is not limited to the expression of transcription factors. Any differentiation factor that is necessary or sufficient to co-differentiate a population of differentiable cells can be used in the methods and systems provided herein, alone or in combination with any other factor. For example, non-transcription factors such as chromatin remodeling factors can be used. In some cases, the chromatin remodeling factor can be SMARCD3 and / or JMJD3.

[0029] In certain aspects, the methods described herein can involve expressing at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten transcription factors and / or other differentiation factors (e.g., chromatin remodeling factors) in differentiable cells (e.g., to cause the differentiable cells to differentiate into a desired cell lineage).

[0030] In some aspects, the differentiable cells described herein comprise at least one exogenous nucleic acid. Exogenous nucleic acid may comprise a sequence encoding any transcription factor and / or other differentiation factor (e.g., chromatin remodeling factor) as described herein. The sequence encoding the transcription factor and / or other differentiation factor may be operably connected to a promoter. Nucleic acid as described herein may comprise a promoter sequence. The promoter sequence may have constitutive activity. In alternative embodiments, the promoter sequence may have conditional activity. For example, the promoter sequence may be induced by a chemical inducing agent or by light.

[0031] In some aspects, the differentiable cells described herein comprise at least two exogenous nucleic acids. In some aspects, the differentiable cells described herein comprise at least three, at least four or at least five exogenous nucleic acids. Each exogenous nucleic acid can comprise at least one nucleic acid sequence encoding a transcription factor and / or a differentiation factor. As described herein, the nucleic acid sequence encoding a transcription factor and / or a differentiation factor can be operably linked to a promoter. In some cases, when using more than one transcription factor and / or differentiation factor to differentiate differentiable cells into desired cell lineages, the expression of each of more than one transcription factor and / or differentiation factor can be controlled by the same promoter. In such cases, each gene encoding a transcription factor and / or differentiation factor can be combined into a single transcript. This single transcript can encode a self-cleaving 2A peptide between each separated protein, or can contain an internal ribosome entry site.

[0032] In a non-limiting example, the exogenous nucleic acid may be Figure 1In some cases, the blocking sequence 102 can be arranged as described above.Promoter 103 can be operably connected with the nucleic acid sequence 104 encoding transcription factor or differentiation factor. In some cases, blocking sequence 102 can be between promoter sequence and the nucleic acid sequence encoding transcription factor or differentiation factor. Blocking sequence can be any nucleic acid sequence that stops the transcription of the nucleic acid sequence encoding transcription factor, such as nucleic acid sequence and / or transcription terminator sequence of one or more termination codons of encoding. In some cases, blocking sequence can comprise expression cassette or multiple expression cassettes, each containing transcription terminator sequence. In some cases, the flank of blocking sequence can be recombinase recognition site 101. In such scenarios, blocking sequence is excised under the presence of recombinase, allows the expression of transcription factor or differentiation factor. When " flank " of target sequence (for example, blocking sequence) is recombinase recognition site, it means that there is at least the first recombinase recognition site in target sequence upstream (for example, 5 '), and there is at least the second recombinase recognition site in target sequence downstream (for example, 3 '). When used in relation to a recombinase recognition site, the term "flanking" includes scenarios where the recombinase recognition site is directly adjacent to or directly linked to the target sequence, as well as scenarios where there are intervening sequences of any length between the recombinase recognition site and the target sequence.

[0033] In some cases, as described herein, recombinase can for example be by chemical activator or can be by light activation, therefore allow the control of recombinase activity.In such cases, recombinase can be expressed in differentiable cells, but can be in inactive state, until differentiable cells are exposed to activator (for example, chemical activator, specific wavelength or wavelength range of light). After differentiable cells are exposed to activator (for example, chemical activator, specific wavelength or wavelength range of light), recombinase can be activated, resulting in the excision of blocking sequence and the expression of transcription factor or differentiation factor (thereby causing differentiable cells to be differentiated into required cell lineage). In some cases, recombinase is a light-activated recombinase (for example, comprising a light-activated domain) by the light activation of specific wavelength or wavelength range. In some cases, light-activated recombinase can be by the light inactivation of a wavelength or wavelength range different from the wavelength or wavelength range for activating recombinase. In some cases, recombinase is a chemically activated recombinase.

[0034] In an alternative embodiment, rather than excising the blocking sequence, the activatable recombinase can be used to invert the nucleic acid sequence such that the nucleic acid sequence is controlled by the promoter, thereby resulting in expression of the transcription factor and / or differentiation factor. Figure 3The non-limiting example of this scenario has been described.In this scenario, the coding causes one or more transcription factors and / or differentiation factors of the differentiation to muscle cell lineage when expressed in cell to be present in exogenous nucleic acid in reverse (making one or more transcription factors and / or differentiation factors not expressed).The muscle cell lineage box also serves as blocking sequence so that the adipocyte lineage transcription factor and / or differentiation factor in downstream are not expressed.The flank of the nucleotide sequence encoding muscle cell lineage transcription factor and / or differentiation factor is two groups of recombinase recognition sites (for example, recA, recB).After the activatable recombinase activation (for example, by light, by chemical activation) of recognition recA site, recA recognition recombinase will invert the nucleotide sequence encoding muscle cell lineage transcription factor and / or differentiation factor, so that nucleotide sequence is in correct direction, and the expression of muscle cell lineage transcription factor and / or differentiation factor occurs.The expression of muscle cell lineage transcription factor and / or differentiation factor causes this cell differentiation into muscle cell. Alternatively, when what is desired is fat, activate (for example, by light activation) the second different activatable recombinase, this recombinase recognition recB site. After recB recognition recombinase activation, muscle cell lineage box is excised, and adipocyte lineage transcription factor and / or differentiation factor is expressed. The expression of adipocyte lineage transcription factor and / or differentiation factor causes this cell to differentiate into adipocyte.

[0035] In some cases, more than one recombinase can be used in a population of differentiable cells such that different transcriptional programs are activated depending on which recombinase is activated, such as Figure 3 . For example, differentiable cells can express a light-activated recombinase (recombinase A) and a chemically activated recombinase (recombinase B). When it is necessary to differentiate into cell type A (e.g., muscle cells), the differentiable cells can be exposed to a chemical activator, thereby activating recombinase A, resulting in the expression of transcription factor A and the differentiation of differentiable cells into cell type A. When it is necessary to differentiate into cell type B (e.g., adipocytes), the differentiable cells can be exposed to light of a specific wavelength or wavelength range, thereby activating recombinase B, resulting in the expression of transcription factor B and the differentiation of differentiable cells into cell type B. In a differentiable cell population, this method can be used to temporally and / or spatially control the co-differentiation of differentiable cells into different cell lineages. In some cases, this method can be used to cause differentiable cells to co-differentiate into different cell lineages at the same time.

[0036] In some cases, differentiable cells can express two recombinases, each of which can be activated by the light of different wavelengths or wavelength ranges. In alternative embodiments, differentiable cells can express two recombinases, one of which can be activated by the light of a specific wavelength or wavelength range, and another of which can be activated by a chemical activator. In another embodiment, differentiable cells can express three recombinases, two of which can be activated by the light of different wavelengths or wavelength ranges, and the third of which can be activated by a chemical activator. In some cases, the recombinase can be activated substantially simultaneously or simultaneously. In other cases, the recombinase can be activated successively. In some scenarios, only a kind of recombinase is used to cause differentiation to the first cell lineage. The second different cell lineage can be achieved by any other mechanism, such as by using an inducible promoter (for example, can be induced by chemicals, can be induced by light) to control the expression of transcription factors and / or differentiation factors, by exposing the cells to specific culture medium or culture medium factors, or by differentiation mediated by cell-cell contact.

[0037] In some cases, the recombinase can be activated using an activatable system. The activatable system can be as described in Table 1.

[0038] Table 1: Activatable Systems

[0039]

[0040]

[0041] In various aspects, a combination of photoactivatable domains (e.g., a first photoactivatable domain and a second photoactivatable domain) can be used (e.g., each of the photoactivatable domains can be fused to a portion of the recombinase). In this scenario, the first photoactivatable domain and the second photoactivatable domain are binding partners such that upon irradiation with light of a specific wavelength or within a specific spectral range, the first and second photoactivatable domains heterodimerize or heterooligomerize. The first and second photoactivatable domains heterodimerize or heterooligomerize upon irradiation with light of a specific wavelength or within a specific spectral range, thereby bringing the protein domains (or their functional domains or functional portions) into close contact with each other, such that the recombinase is activated. In some cases, upon irradiation with light of a wavelength or wavelength range different from the wavelength or wavelength range used to heterodimerize or heterooligomerize the first and second photoactivatable domains, the first and second photoactivatable domains can dissociate from each other (e.g., thereby inactivating the recombinase).

[0042] In various aspects, the photoactivatable domain comprises a light-oxygen-voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a cryptochrome (CRY) domain, a blue light using FAD (BLUF) photoreceptor domain, a phytochrome (PHY) domain, CIB1 (cryptochrome interacting basic helix-loop-helix protein 1) (or a functional portion or domain thereof; e.g., CIBN (N-terminal domain of CIB1)), a PIF (phytochrome interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin binding domain (CBD), or a combination thereof.

[0043] In some cases, a combination of photoactivatable domains is used, wherein the first photoactivatable domain is cryptochrome 2 (or a variant or functional portion thereof) and the second photoactivatable domain is CIB1 (or a variant or functional portion thereof; e.g., CIBN). In some cases, a combination of photoactivatable domains is used, wherein the first photoactivatable domain is BphP1 (or a variant or functional portion thereof) and the second photoactivatable domain is QPAS1 (or a variant or functional portion thereof). In some cases, the photoactivatable domain (or combination of photoactivatable domains) is selected from Table 2. In some cases, the photoactivatable domain can have an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any of the photoactivatable domains described in Table 2.

[0044] Table 2. Non-limiting examples of photoactivatable domain systems

[0045]

[0046]

[0047] In another aspect of the methods described herein, the recombinase can be activated by a chemical inducer or by light of a specific wavelength or wavelength range. The recombinase can be constitutively expressed in an inactive form. The recombinase can be conditionally expressed in an inactive form by a chemically induced or light-induced system (e.g., by using an inducible (e.g., chemicals, light-induced) promoter). The recombinase can mediate irreversible excision or can invert the nucleic acid sequence. The recombinase can be a serine integrase, such as ΦC31, TP901, and Bxb1. The recombinase can be a tyrosine recombinase, such as Cre, VCre, and Flp.

[0048] In one embodiment, the recombinase is split into a first part and a second part. Addition of a chemical inducer results in dimerization and activation of the recombinase. The chemical inducer can be any of the chemical inducers described herein, including but not limited to rapamycin or a derivative thereof, ABA, GA, tetracycline or a derivative thereof, cumate or a derivative thereof, or vanillic acid or a derivative thereof. The recombinase can be any recombinase, including but not limited to Cre, VCr, Flp, ΦC31, TP901, and Bxb1.

[0049] In some embodiments, a chemical and / or light inducer controls the expression of the recombinase, and a chemical and / or light activator controls the activation of the recombinase. In some cases, both the chemical inducer and the activator are plant hormones. For example, the expression of the split recombinase can be regulated by GA, while the dimerization and activation of the split recombinase are mediated by ABA, or vice versa.

[0050] In another embodiment, the dimerization of recombinase and activation are induced by light (for example, optogenetics dimerization). The first half of recombinase and the second half of recombinase can be fused with a light-activated domain. In various aspects, the light-activated domain includes light-oxygen-voltage (LOV) photoreceptor domain, LOV2 photoreceptor domain, cryptochrome (CRY) domain, blue light (BLUF) photoreceptor domain using FAD, phytochrome (PHY) domain, CIB1 (cryptochrome interacting basic helix-loop-helix protein 1) (or its functional part or domain; For example CIBN), PIF (phytochrome interacting factor) domain, Dronpa domain, UVR8 photoreceptor domain, COP1 domain, BphP1 domain, QPAS-1 domain, cobalamin binding domain (CBD) or its combination. The dimerization of recombinase and the regulation of activation can utilize any one of the light-activated systems described in Table 2. Dimerization and activation can alternatively be induced by temperature. In some cases, inactivation of the recombinase can be achieved by irradiating the cells with light of a wavelength or range of wavelengths that is different from the wavelength or range of wavelengths used to activate the recombinase.

[0051] In various aspects, the method involves exposing differentiable cells (e.g., genetically engineered to express a fusion protein comprising a recombinase and a photoactivatable domain) to light of a specific wavelength or light within a specific spectral range. The wavelength or wavelength range of the light is selected so that the light can activate the photoactivatable domain. For example, Table 2 provides non-limiting examples of light parameters for different photoactivatable domain systems. The wavelength or wavelength range of the light can be one or more of infrared, near-infrared, visible light (e.g., red, green, blue), ultraviolet light, or a combination thereof. Infrared light can include light with a wavelength or wavelength range of about 780nm to 1mm. Near-infrared light can include light with a wavelength or wavelength range of about 740nm to about 780nm. Red light can include light with a wavelength or wavelength range of about 620nm to 750nm, 600nm to 690nm, or about 650nm. Green light can include light with a wavelength or wavelength range of about 577nm to about 492nm. Blue light can include light with a wavelength or wavelength range of 492nm to about 455nm or about 440nm to about 473nm. Ultraviolet light can include light having a wavelength or wavelength range of about 10 nm to 400 nm or about 280 to 315 nm. In various aspects, the light has a wavelength or wavelength range of 100 nm to 1 mm.

[0052] In various aspects, the method involves irradiating cells with light having one or more illumination parameters. In some cases, the one or more illumination parameters include light intensity and / or illumination time pattern. In some cases, the illumination intensity can be about 0 μW / mm 2 to about 100 μW / mm 2 In some cases, the irradiation intensity can be at least or at most about 0 μW / mm 2 , 0.1μW / mm 2 , 0.2μW / mm 2 , 0.3μW / mm 2 , 0.4μW / mm 2 , 0.5μW / mm 2 , 0.6μW / mm 2 , 0.7μW / mm 2 , 0.8μW / mm 2 , 0.9μW / mm 2 , 1μW / mm 2 , 1.2μW / mm 2 , 1.4μW / mm 2 , 1.6μW / mm 2 , 1.8μW / mm 2 , about 2μW / mm 2 , about 3μW / mm 2 , about 4μW / mm 2 , about 5μW / mm 2, about 6μW / mm 2 , about 8μW / mm 2 , about 10μW / mm 2 , about 20μW / mm 2 , about 30μW / mm 2 , about 40μW / mm 2 , about 50μW / mm 2 , about 60μW / mm 2 , about 70μW / mm 2 , about 80μW / mm 2 , about 90μW / mm 2 or about 100 μW / mm 2 .

[0053] In some cases, the time pattern may include a stimulation duration and an interstimulation duration. In some cases, the time pattern includes a light stimulation duration of at least about one tenth of a second, at least about 1 second, at least about 1 minute, at least about 5 minutes, at least about 30 minutes, or at least about 1 hour. In some cases, the stimulation duration may be at least about 5 minutes. In some cases, the time pattern includes an interstimulation duration of at least about 1 second, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, or more. In some cases, the interstimulation duration may be from about 20 minutes to about 250 minutes.

[0054] In another embodiment, the recombinase comprises a single-chain polypeptide. This single-chain polypeptide can be fused to a photoactivatable domain to produce a photoactivatable recombinase. Exposure to light of a specific wavelength or wavelength range can activate the recombinase. For example, irradiation of the AsLOV2-based Cre system, LiCre, with blue light results in activation of the recombinase.

[0055] In another embodiment, the recombinase can be fused to the PhoCl protein or its derivatives. Irradiation with violet light (about 400 nm) results in the cleavage of PhoCl. In some cases, the PhoCl domain can be present between the blocker domain and the recombinase domain in the fusion protein, such as Figure 2 The blocker domain can be any domain that prevents the recombinase from functioning, such as a domain that prevents the recombinase from entering the cell nucleus. For example, the blocker domain can be a steroid receptor domain that interacts with Hsp90 to prevent the recombinase from entering the cell nucleus. Exposing a fusion protein comprising a blocker domain, a PhoCl domain, and a recombinase to ultraviolet light can cause the PhoCl domain to break, allowing the recombinase to enter the cell nucleus and reach the genomic DNA, resulting in recombinase activity.

[0056] In some cases, the expression of the recombinases and / or transcription factors and / or differentiation factors described herein can also be regulated, as described herein. For example, the differentiable cells can contain an exogenous nucleic acid comprising a nucleic acid sequence encoding the recombinase and / or transcription factor and / or differentiation factor. The recombinase and / or transcription factor and / or differentiation factor can be expressed in the differentiable cells after exposure to a chemical inducer or light of a specific wavelength or wavelength range.

[0057] Any suitable system for inducing gene expression can be used to induce the expression of gene of interest (for example, transcription factor, differentiation factor) herein.The example of chemically inducible system includes but is not limited to Tet inducible system, cumate inducible system, acetaldehyde inducible system, vanillic acid inducible system and derivative thereof, rapamycin inducible system and derivative thereof, and plant hormone signal transduction system and derivative thereof.The transcription of gene of interest (for example, the gene encoding transcription factor or differentiation factor) can be induced by the presence or absence of chemical inducer.Chemical inducer can include but is not limited to tetracycline or derivatives thereof, cumate or derivatives thereof, acetaldehyde, vanillic acid or derivatives thereof, rapamycin or derivatives thereof, abscisic acid, gibberellin or auxin.Chemical inducer can be food safety additive.

[0058] In some cases, chemical inducers regulate the transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) by affecting the binding of a protein to a DNA motif that regulates transcription. In some cases, chemical inducers regulate the transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) by affecting the dimerization of two proteins so that the DNA binding and transcriptional regulatory domains come together.

[0059] Tet inducible systems are derived from the E. coli tetracycline resistance operon and use the antibiotic tetracycline or a derivative such as doxycycline as an inducing agent. The expression of a gene of interest (e.g., a transcription factor, a differentiation factor) can be inducible by tetracycline or its derivatives. Tetracycline derivatives include, but are not limited to, doxycycline, minocycline, methacycline, and tigecycline.

[0060] Addition of tetracycline or its derivatives can lead to transcriptional activation via the Tet repressor protein (TetR). TetR binds as a homodimer to the Tet operator (TetO) DNA motif in the promoter of an operon, repressing the operon's transcription. When bound to tetracycline, the conformation of the TetR dimer changes, preventing it from binding to the TetO element and releasing the operon's transcriptional repression.

[0061] In some embodiments, the Tet system can be modified to work in mammalian cells. The Tet-Off system is used to regulate the transcription of genes of interest (e.g., genes encoding transcription factors and / or differentiation factors) in another form. In this variant, the coding sequence of a protein of interest (e.g., transcription factor, differentiation factor) is placed downstream of a synthetic promoter, which is composed of a plurality of TetO elements upstream of a minimal promoter (e.g., derived from a CMV promoter) with a TATA box that initiates transcription but does not have an enhancer element. Cells constitutively express a fusion protein of TetR and a transcription activator (such as the herpes simplex virus VP16 activation domain). In the absence of tetracycline, the TetR-activator fusion protein is tightly bound to the TetO elements in the inducible promoter, activating the transcription of the gene of interest. When tetracycline is present, the TetR fusion can no longer be combined with the TetO elements, and transcription from the promoter stops.

[0062] Tet-On system is used to regulate the transcription of gene of interest (for example, the gene encoding transcription factor or differentiation factor) in another form. In this variant, VP16 activation domain is fused with TetR mutant (retro-TetR, rTetR) with reverse tetracycline-dependent behavior. In the presence of tetracycline, rTetR is combined with TetO, and in the absence of tetracycline, it is not combined with TetO. Therefore, when tetracycline is added, the expression from the promoter as above is turned on. rTetR or its variants can include rTetR, high performance V16 rTetR sequence, wild-type TetR fused with transcription repressors such as KRAB domains.

[0063] In some cases, transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) is induced by the presence of cumate or its derivatives. In one configuration, addition of cumate or its derivatives induces expression of the gene of interest. In the absence of cumate, the CymR repressor protein binds to one or more CuO operator sequences positioned between a strong constitutive promoter and the gene of interest, repressing transcription of the gene of interest. Addition of cumate or its derivatives can activate expression of the gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) by interacting with at least one CymR repressor protein and preventing it from binding to CuO, thereby alleviating transcriptional repression. In another configuration, transcription of the gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) is induced by the absence of cumate or its derivatives. In this example, a chimeric CymR protein fused to a transcriptional activator domain binds to a CuO operator sequence upstream of a minimal promoter preceding the gene of interest in the absence of cumate, thereby inducing expression. When cumate or a derivative thereof is supplied to the cell, it interacts with the chimeric CymR fusion protein and prevents its binding to CuO, resulting in the gene of interest no longer being expressed. Further embodiments of the cumate transcription induction system are possible, such as those using mutant "retro-inverso" CymR proteins that bind to CuO in the presence (but not in the absence) of cumate.

[0064] In some cases, transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) is induced by the presence of acetaldehyde. In this example of acetaldehyde-inducible regulation (AIR), a repressor from the fungus Aspergillus nidulans that binds to an operator element is placed between a constitutive promoter and the transcription start site, thereby repressing transcription. In the presence of acetaldehyde, the repressor binds to the acetaldehyde and results in transcription of the gene of interest (e.g., a gene encoding a transcription factor or differentiation factor).

[0065] In some cases, transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) is induced by the presence or absence of vanillic acid. The VanR protein binds to the VanO sequence and dissociates in the presence of vanillic acid. In some cases, a VAC-ON version is created by fusing VanR to a transcriptional repressor such as KRAB, or a VAC-OFF version is created by fusing it to an activator such as VP16.

[0066] In some cases, transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) is induced by the presence of rapamycin or its derivatives. Rapamycin causes dimerization of FKBP and FRB, bringing together the transcriptional activation and DNA binding domains fused to those proteins and leading to transcriptional activation.

[0067] In some cases, the transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor) is induced by a plant hormone signal transduction system. Chemical inducers can include abscisic acid (ABA), gibberellins (GA), or derivatives thereof. ABA triggers the dimerization of ABI and PYL1 to induce the transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor). In some cases, the DNA binding domain is fused to AB1 or its derivatives, and the transcription activator domain or its derivatives are fused to PYL1. In some cases, GA dimerizes GID1 and GAI to induce the transcription of a gene of interest (e.g., a gene encoding a transcription factor or differentiation factor). In some cases, the DNA binding domain is fused to GID1 or its derivatives, and the transcription activator domain or its derivatives are fused to GAI. The DNA binding domain and the transcription activation domain can include GAL4 and VP16, respectively; dCas9 and VPR chimeric activators; or any other combination of DNA binding domains and transcription activation domains.

[0068] In some cases, the chemical inducer is an auxin. In such scenarios, a DNA binding domain fused to a transcriptional repressor, such as a KRAB domain, and tagged with a degron is constitutively expressed and blocks expression from a promoter containing sequences homologous to the DBD. TIR1 is also constitutively expressed. Upon addition of the auxin, the repressor fusion protein is degraded, thereby releasing transcriptional repression of the gene of interest.

[0069] In other aspects, transcription is controlled using a light-activated system regulated by CcasS / R or UirS / R. These systems may require the addition of phycocyanin as a cofactor. In another case, transcription is controlled by a CarH / CarO system that is regulated by green light. This system may require the addition of B12 as a cofactor.

[0070] In some embodiments, a promoter with constitutive activity is used to drive the expression of the recombinase or to open the transcription factor expression after the recombinase is activated. In some cases, the constitutive promoter is a viral promoter. In some cases, the promoter is a eukaryotic promoter. In a non-limiting example, the promoter can be a cytomegalovirus immediate early promoter (CMV promoter), a SV40 early promoter (SV-40), an elongation factor 1 alpha (EF1a), ubiquitin c (UBC), phosphoglycerate kinase (PGK) or beta actin (ACTB). In some cases, the recombinase can be expressed under the control of an inducible promoter (e.g., a chemically inducible promoter, a light-inducible promoter).

[0071] In some cases, the gene of interest is one or more transcription factors or differentiation factors as described herein.

[0072] On the other hand of the method described herein, at least one of the cells in the cell mass can be differentiated into a desired cell lineage by being exposed to certain conditions that allow differentiation into a desired cell lineage. In one embodiment, at least one cell can be differentiated into a desired cell lineage by contacting the culture medium containing one or more medium factors (e.g., at an appropriate concentration) that affect the differentiation to the desired cell lineage. In some embodiments, the cell can be differentiated into a desired cell lineage by deducting or removing at least one medium factor from the culture medium, thereby making the cell differentiation into the desired cell lineage. In some embodiments, at least one cell can be differentiated into a desired cell lineage by differentiation mediated by cell-cell contact.

[0073] In some embodiments, the different medium preparations comprising at least one medium factor can promote the differentiation of differentiable cells to specific cell types or cell lineages. In some embodiments, medium components can include dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), insulin and indomethacin, triiodothyronine (t3), Asc-2-P and basic FGF (bFGF-2), transferrin, T3, cortisol, pioglitazone, ascorbic acid, calcium pantothenate, biotin, rosiglitazone or its combination. In some embodiments, medium components can include fetal calf serum (FCS), VEGF, FGF, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or its combination. In some embodiments, the medium components may include FCS, transforming growth factor-β1 (TGF-β1), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), ascorbic acid, bFGF, bone morphogenetic protein-4 (BMP-4), copper sulfate, proline, glycine, ascorbic acid, alanine, heparin, angiotensin II, sphingosine phosphorylcholine, or a combination thereof.

[0074] In some embodiments, the culture medium is supplemented with a concentration of at least about 1 μM, at least about 5 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 110 μM, at least about 120 μM, at least about 130 μM, at least about 140 μM, at least about 150 μM, at least about 160 μM, at least about 170 μM, at least about 180 μM, at least about 190 μM, at least about 200 μM, at least about 210 μM, at least about 220 μM, at least about 230 μM, at least about 240 μM, at least about 250 μM, at least about 260 μM, at least about 270 μM, at least about 280 μM, at least about 290 μM, at least about 300 μM, at least about 310 μM, at least about 320 μM, at least about 330 μM, at least about 340 μM, at least about 350 μM, at least about 360 μM, at least about 370 μM, at least about 380 μM, at least about 390 μM, at least about At least about 100 mM, at least about 200 mM, at least about 300 mM, at least about 400 mM, at least about 500 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, or at least about 100 mM of various medium factors.

[0075] In some embodiments, medium preparation includes non-serum medium preparation.In some embodiments, various medium preparations from conventional medium can be improved, to realize proliferation, differentiation or other required qualities.In some embodiments, medium preparation includes synthetic medium or its improvement.The example of synthetic medium includes minimum essential medium (MEM), Essential8 culture medium, Eagle basal medium (BME), Ham's F12, Ham's F-10, Fischer's culture medium, CMRL-1066 culture medium, Click's culture medium, culture medium 199, Dulbecco's modified Eagle's culture medium (DMEM), RPMI-1640, L-15 culture medium, McCoy's5A modified culture medium, William's culture medium E, and Iscove's modified Dulbecco's culture medium (IMDM).

[0076] The differentiable cells used in the methods provided herein can be any desired differentiable cells. In some cases, the differentiable cells are stem cells. In some cases, the stem cells are totipotent stem cells. In some cases, the stem cells are pluripotent stem cells. In some cases, the stem cells are multipotent stem cells. In some cases, the stem cells are oligopotent stem cells. In some cases, the stem cells are unipotent stem cells. In some cases, the stem cells are embryonic stem cells. In some cases, the stem cells are mesenchymal stem cells. In some cases, the stem cells are satellite cells or muscle stem cells. In some cases, the stem cells are adipose stem cells. In some cases, the stem cells are endothelial stem cells. In some cases, the stem cells are induced pluripotent stem cells (iPSCs). In some cases, the differentiable cells are progenitor cells. In some cases, the differentiable cells are transition amplifying cells (transit-amplifying cells, TACs). In some cases, the differentiable cells are cells that can proliferate and divide (for example, proliferate and divide for example for a period of time under certain conditions). In some cases, the differentiable cells are not stem cells. For example, the differentiable cells can be mature somatic cells that can transdifferentiate into another cell type (for example, under appropriate conditions) that is different from the original cell type. In some cases, the differentiable cells are fibroblasts. In some cases, the differentiable cells are chondrocytes. In some cases, the differentiable cells are epithelial cells. In some cases, the differentiable cells are erythroid-megakaryocytes. In some cases, the differentiable cells are beta cell progenitor cells. In some cases, the differentiable cells are liver cells (e.g., hepatocytes). In some cases, the differentiable cells are exocrine cells. In some cases, the differentiable cells are non-sensory cells. In some cases, the differentiable cells are non-cardioblastic mesoderm. In some cases, the differentiable cells are cardiac fibroblasts. In some cases, the differentiable cells are skin fibroblasts. In some cases, the differentiable cells are cardiomyocytes. In some cases, the differentiable cells are fibroblast-adipocyte progenitor cells.

[0077] In certain embodiments, the differentiable cells described herein are mammalian cells. In some cases, the mammalian cells are selected from the group consisting of: human cells, bovine (cow) cells, ovine (sheep) cells, porcine (pig) cells, and mouse cells. In some cases, the differentiable cells are avian cells, such as, but not limited to, chicken cells. In some cases, the cells are fish cells, such as, but not limited to, tuna cells or salmon cells.

[0078] In another aspect of the methods disclosed herein, the differentiable cell population is deposited on a solid support. The solid support can allow two-dimensional growth or shaping of differentiable cells. The solid support can allow three-dimensional growth or shaping of differentiable cells. The solid support can be biodegradable. The solid support can comprise natural materials. Natural materials include but are not limited to extracellular matrix components, silk, gelatin and alginate. The solid support can comprise synthetic materials. The solid support can include any surface or scaffold (e.g., hydrogel) to which differentiable cells can adhere.

[0079] The solid support can be coated with one or more extracellular matrix components or parts or fragments thereof. For example, the solid support can be coated with or incorporate collagen, hyaluronic acid, fibrin, fibronectin, integrin, laminin, proteoglycans, glycosaminoglycans, gelatin, vitronectin, or any other extracellular matrix protein or part or fragment thereof.

[0080] The differentiable cell population can be deposited on the solid support in at least one layer. The differentiable cell population can be deposited on the solid support in at least one, two, three, four, five, six, seven, eight, nine, ten or more layers.

[0081] Recombinases and transcription factors and / or differentiation factors as described herein can be encoded by nucleic acids. In some embodiments, the nucleic acids comprising recombinases and transcription factors and / or differentiation factors can be expression cassettes or can be contained within expression cassettes. As used herein, "expression cassette" means a recombinant nucleic acid construct comprising one or more nucleic acids as described herein, wherein the recombinant nucleic acid construct is operably associated with at least one control sequence (e.g., a promoter).

[0082] In certain embodiments, nucleic acid is a component of a vector that can be used to transfer nucleic acid into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid connected thereto. One type of vector is a genomic integration vector, or "integration vector," which can become integrated into the chromosomal DNA of a host cell. Another type of vector is an "add-on" vector, for example, a nucleic acid that can be replicated outside the chromosome. The vector that can guide the expression of a gene operably connected thereto is referred to as an "expression vector" in this article. Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc.

[0083] In the vector, the regulatory elements (such as promoters, enhancers and polyadenylation signals) for controlling transcription can be derived from mammals, microorganisms, viruses or insect genes. The ability to replicate in the host (which is usually given by a replication origin) and the selection gene that helps the identification of the transformant can be additionally included. The vector derived from a virus (such as a slow virus, a retrovirus, adenovirus, adeno-associated virus, etc.) can be adopted. The plasmid vector can be linearized for integration into the chromosome position. The vector can include a sequence that guides the site-specific integration (for example, AttP-AttB recombination) of a defined position or a restricted site set in the genome. In addition, the vector can include a sequence derived from a transposable element.

[0084] In some aspects, the nucleic acid introduced into the eukaryotic cell is operably linked to a promoter and / or to a polyA signal as known in the art. In some embodiments, the nucleic acid having a 5' end and a 3' end is operably linked to a promoter at the 5' end and to a polyA signal at the 3' end. In some aspects, the nucleic acid comprises a 2A peptide sequence and / or an internal ribosome entry site.

[0085] In some embodiments, the expression cassette includes a nucleotide sequence encoding a selectable marker, which can be used to select transformed host cells. As used herein, "selectable marker" means giving different phenotypes to the host cell expressing the marker when expressed, and therefore allows the nucleotide sequence for distinguishing such transformed cells from cells without the marker. This nucleotide sequence can encode a selectable or screenable marker, depending on whether the marker is given a trait that can be selected by chemical means (such as by using a selection agent (e.g., antibiotics, etc.)), or whether the marker is simply a trait that can be identified by observation or testing (such as by screening (e.g., fluorescence)).

[0086] system

[0087] Provided herein are systems for co-differentiating a population of differentiable cells. The system can include a population of differentiable cells, wherein each differentiable cell in the population is engineered to contain an exogenous nucleic acid comprising: a nucleic acid sequence encoding at least one first transcription factor or differentiation factor that causes differentiation toward a first cell lineage; and a nucleic acid sequence encoding at least one second transcription factor or differentiation factor that causes differentiation toward a second cell lineage. The system can further include light configured to illuminate a first differentiable cell in the population with light of a first wavelength or range of wavelengths to cause the first differentiable cell to differentiate into a first cell lineage.

[0088] In some cases, the systems described herein involve co-differentiation of a population of differentiable cells into multiple cell lineages. In some cases, the systems involve co-differentiation of a population of differentiable cells into at least two, three, four, or five cell lineages. In some embodiments, the first cell lineage and the second cell lineage are different. In some systems, at least the first cell lineage, the second cell lineage, the third cell lineage, the fourth cell lineage, and / or the fifth cell lineage are different.

[0089] In some cases, the differentiable cells described herein comprise at least one exogenous nucleic acid. The exogenous nucleic acid may comprise a sequence encoding a transcription factor and / or a differentiation factor. The sequence encoding the transcription factor and / or differentiation factor may be operably linked to a promoter. The nucleic acids described herein may comprise a promoter sequence. The promoter sequence may be constitutively active. In alternative embodiments, the promoter sequence may be conditionally active. For example, the promoter sequence may be regulated by a chemical inducer or by light.

[0090] In the systems described herein, the differentiable cells comprise at least one nucleic acid sequence comprising a sequence encoding at least one transcription factor and / or differentiation factor. In some embodiments, the at least one transcription factor is selected from the group consisting of: PPARγ, CEBPα, MYOD, MYOG, MYF5, MRF4 (MYF6), HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, PRDM16, SREBP1, and any combination thereof. In certain aspects, the systems described herein may involve at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 transcription factors and / or differentiation factors. The expression of the transcription factors can be regulated by chemical inducers or by light.

[0091] In some embodiments, the differentiable cells described herein comprise at least two exogenous nucleic acids. In some embodiments, the differentiable cells described herein comprise at least three, four, or five exogenous nucleic acids. Each exogenous nucleic acid can comprise at least one sequence encoding a transcription factor and / or a differentiation factor. The sequence encoding the transcription factor can be operably linked to a promoter.

[0092] The systems described herein include activatable recombinases. The activatable recombinases can include an activator system as described in Table 1.

[0093] In some cases, the population of differentiable cells can comprise at least two, at least three, at least four, or at least five exogenous nucleic acid sequences encoding at least two, at least three, at least four, or at least five transcription factors and / or differentiation factors.

[0094] The differentiable cells may further comprise at least one recombinase. The differentiable cells may further comprise one, two, three, four, five, or more recombinases. At least one recombinase may be chemically activatable as described herein. At least one recombinase may be photoactivatable as described herein. In another embodiment, at least two recombinases are photoactivatable.

[0095] In various aspects, a combination of photoactivatable domains (e.g., a first photoactivatable domain and a second photoactivatable domain) can be used. In this scenario, the first photoactivatable domain and the second photoactivatable domain are binding partners such that upon irradiation with light of a specific wavelength or within a specific spectral range, the first and second photoactivatable domains heterodimerize or heterooligomerize. The first and second photoactivatable domains heterodimerize or heterooligomerize upon irradiation with light of a specific wavelength or within a specific spectral range, thereby bringing the protein domains (or their functional domains or functional portions) into close contact with each other, such that the recombinase is activated. In some cases, the first and second photoactivatable domains can be dissociated (e.g., thereby inactivating the recombinase) by irradiating the cell with light of a wavelength or wavelength range different from the wavelength or wavelength range used to heterodimerize or heterooligomerize the first and second photoactivatable domains.

[0096] In various aspects, the photoactivatable domain comprises a light-oxygen-voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a cryptochrome (CRY) domain, a FAD-using blue light (BLUF) photoreceptor domain, a phytochrome (PHY) domain, CIB1 (or a functional portion of its domain; e.g., CIBN), a PIF (phytochrome interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin binding domain (CBD), or a combination thereof.

[0097] In some cases, a combination of photoactivatable domains is used, wherein the first photoactivatable domain is cryptochrome 2 (or a variant or functional portion thereof) and the second photoactivatable domain is CIB1 (or a variant or functional portion thereof or a functional domain thereof; e.g., CIBN). In some cases, a combination of photoactivatable domains is used, wherein the first photoactivatable domain is BphP1 (or a variant or functional portion thereof) and the second photoactivatable domain is QPAS1 (or a variant or functional portion thereof). In some cases, the photoactivatable domain (or combination of photoactivatable domains) is selected from Table 2. In some cases, the photoactivatable domain can have an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any of the photoactivatable domains described in Table 2.

[0098] In various aspects, the system involves exposing differentiable cells (e.g., genetically engineered to express a fusion protein comprising a recombinase and a photoactivatable domain) to light of a specific wavelength or within a specific spectral range. The wavelength or wavelength range of the light is selected so that the light can activate the photoactivatable domain. For example, Table 2 provides non-limiting examples of light parameters for different photoactivatable domain systems. The wavelength or wavelength range of the light can be one or more of infrared, near infrared, visible light (e.g., red, green, blue), ultraviolet light, or a combination thereof. Infrared light can include light with a wavelength or wavelength range of about 780nm to 1mm. Near infrared light can include light with a wavelength or wavelength range of about 740nm to about 780nm. Red light can include light with a wavelength or wavelength range of about 620nm to 750nm, 600nm to 690nm, or about 650nm. Green light can include light with a wavelength or wavelength range of about 577nm to about 492nm. Blue light can include light with a wavelength or wavelength range of 492nm to about 455nm or about 440nm to about 473nm. Ultraviolet light can include light having a wavelength or wavelength range of about 10 nm to 400 nm or about 280 to 315 nm. In various aspects, the light has a wavelength or wavelength range of 100 nm to 1 mm.

[0099] In some cases of the systems described herein, the recombinase can be configured to be expressed in an inactive form. The recombinase can include a chemically induced or light-induced system for an inactive form. The recombinase can mediate irreversible excision or inversion of a nucleic acid sequence. The recombinase can be a serine integrase, such as φC31, TP901, and Bxb1. The recombinase can be a tyrosine recombinase, such as Cre, VCre, and Flp.

[0100] In one embodiment, the recombinase is split into a first portion and a second portion. Addition of a chemical activator results in dimerization and activation of the recombinase. The chemical activator can be any of the chemical activators described herein, including but not limited to rapamycin or a derivative thereof, ABA, GA, tetracycline or a derivative thereof, cumate or a derivative thereof, or vanillic acid or a derivative thereof. The recombinase can be any recombinase, including but not limited to Cre, VCr, Flp, φC31, TP901, and Bxb1.

[0101] In another embodiment, the first part of the recombinase and the second part of the recombinase can be fused to a light-activated domain. In various aspects, the light-activated domain includes a light-oxygen-voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a cryptochrome (CRY) domain, a blue light (BLUF) photoreceptor domain using FAD, a phytochrome (PHY) domain, CIB1 (or its functional part or domain; e.g., CIB1), a PIF (phytochrome interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin binding domain (CBD), or a combination thereof. The dimerization and activation of the recombinase can be regulated by any of the light-activated systems described in Table 2. Dimerization and activation can alternatively be induced by temperature.

[0102] In another embodiment, the recombinase comprises a single-chain polypeptide. The single-chain polypeptide can be fused to a photoactivatable domain to produce a photoactivatable recombinase. Exposure to light of a specific wavelength or wavelength range can activate the recombinase. For example, irradiation of the AsLOV2-based Cre system with blue light results in activation of the recombinase.

[0103] In another embodiment, the recombinase can be fused to the PhoCl protein or its derivatives. Irradiation with violet light (about 400 nm) results in the cleavage of PhoCl. In some cases, the PhoCl domain can be present between the blocker domain and the recombinase domain in the fusion protein, such as Figure 2 The blocker domain can be any domain that prevents the recombinase from functioning, such as a domain that prevents the recombinase from entering the cell nucleus. For example, the blocker domain can be a steroid receptor domain that interacts with Hsp90 to prevent the recombinase from entering the cell nucleus. Exposing a fusion protein comprising a blocker domain, a PhoCl domain, and a recombinase to ultraviolet light can cause the PhoCl domain to break, allowing the recombinase to enter the cell nucleus and generate recombinase activity.

[0104] In various aspects, the system includes a light source configured to expose differentiable cells (e.g., genetically engineered to express a fusion protein comprising a recombinase and a photoactivatable domain) to light of a specific wavelength or light within a specific spectral range. The wavelength or wavelength range of the light is selected so that the light can activate the photoactivatable domain. For example, Table 2 provides non-limiting examples of light parameters for different photoactivatable domain systems. The wavelength or wavelength range of the light can be one or more of infrared, near infrared, visible light (e.g., red, green, blue), ultraviolet light, or a combination thereof. Infrared light can include light with a wavelength or wavelength range of about 780nm to 1mm. Near infrared light can include light with a wavelength or wavelength range of about 740nm to about 780nm. Red light can include light with a wavelength or wavelength range of about 620nm to 750nm, 600nm to 690nm, or about 650nm. Green light can include light with a wavelength or wavelength range of about 577nm to about 492nm. Blue light can include light with a wavelength or wavelength range of 492 to about 455 nm, or about 440 to about 473 nm. Ultraviolet light can include light with a wavelength or wavelength range of about 10 nm to 400 nm, or about 280 to 315 nm. In various aspects, the wavelength or wavelength range of light is 100 nm to 1 mm.

[0105] In various aspects, the system involves irradiating cells with light having one or more illumination parameters. In some cases, the one or more illumination parameters include light intensity and / or illumination time pattern. In some cases, the illumination intensity can be about 0 μW / mm 2 to about 100 μW / mm 2 In some cases, the irradiation intensity can be at least or at most about 0 μW / mm 2 , 0.1μW / mm 2 , 0.2μW / mm 2 , 0.3μW / mm 2 , 0.4μW / mm 2 , 0.5μW / mm 2 , 0.6μW / mm 2 , 0.7μW / mm 2 , 0.8μW / mm 2 , 0.9μW / mm 2 , 1μW / mm 2 , 1.2μW / mm 2 , 1.4μW / mm 2 , 1.6μW / mm 2 , 1.8μW / mm 2 , about 2μW / mm 2 , about 3μW / mm 2 , about 4μW / mm 2 , about 5μW / mm2 , about 6μW / mm 2 , about 8μW / mm 2 , about 10μW / mm 2 , about 20μW / mm 2 , about 30μW / mm 2 , about 40μW / mm 2 , about 50μW / mm 2 , about 60μW / mm 2 , about 70μW / mm 2 , about 80μW / mm 2 , about 90μW / mm 2 or about 100 μW / mm 2 . In some cases, the temporal pattern can include a stimulation duration and an interstimulus duration. In some cases, the temporal pattern includes a light stimulation duration of at least about one tenth of a second, at least about 1 second, at least about 1 minute, at least about 5 minutes, at least about 30 minutes, or at least about 1 hour. In some cases, the stimulation duration can be at least about 5 minutes. In some cases, the temporal pattern includes an interstimulus duration of at least about 1 second, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, or more. In some cases, the interstimulus duration can be from about 20 minutes to about 250 minutes.

[0106] In various aspects, the system and method provided herein can be used for various applications. In some embodiments, the system and method provided herein can be used for producing cultured meat or cultivating meat. For example, the system and method provided herein can be used for producing cultured meat or cultivating meat compositions comprising two or more different cell types, and the two or more different cell types are integrated into a single continuous tissue and integrated in a spatially shaped manner so that the cell types in different regions of the composition can be controlled or biased towards one cell type or another cell type. In other aspects, the system and method provided herein can be used for producing tissue (e.g., tissue engineering). For example, the system and method provided herein can be used for producing tissue comprising a plurality of different cell types with spatial specificity (e.g., for biomedical purposes (e.g., skin grafts, pacemakers), for research and development (R&D), for in vitro tissue experiments (including the growth of organoids for drug testing or disease modeling)).

[0107] In some embodiments, the one or more lights include one or more light emitting diodes (LEDs). In some cases, the one or more LEDs include at least two different LEDs. In some cases, the at least two different LEDs emit light of different wavelengths or wavelength ranges. Additionally or alternatively, the one or more light sources include one or more lasers. Additionally or alternatively, the one or more light sources include incandescent light sources.

[0108] The differentiable cells used in the systems provided herein can be any desired differentiable cells. In some cases, the differentiable cells are stem cells. In some cases, the stem cells are totipotent stem cells. In some cases, the stem cells are pluripotent stem cells. In some cases, the stem cells are multipotent stem cells. In some cases, the stem cells are oligopotent stem cells. In some cases, the stem cells are unipotent stem cells. In some cases, the stem cells are embryonic stem cells. In some cases, the stem cells are mesenchymal stem cells. In some cases, the stem cells are satellite cells or muscle stem cells. In some cases, the stem cells are adipose stem cells. In some cases, the stem cells are endothelial stem cells. In some cases, the stem cells are induced pluripotent stem cells (iPSCs). In some cases, the differentiable cells are progenitor cells. In some cases, the differentiable cells are transition amplifying cells (TACs). In some cases, the differentiable cells are cells that can proliferate and divide (for example, proliferate and divide for example for a period of time under certain conditions). In some cases, the differentiable cells are not stem cells. For example, the differentiable cells can be mature somatic cells that can transdifferentiate into another cell type (for example, under appropriate conditions) that is different from the original cell type. In some cases, the differentiable cells are fibroblasts. In some cases, the differentiable cells are chondroblasts. In some cases, the differentiable cells are epithelial cells. In some cases, the differentiable cells are erythroid-megakaryocytes. In some cases, the differentiable cells are beta cell progenitor cells. In some cases, the differentiable cells are liver cells (e.g., hepatocytes). In some cases, the differentiable cells are exocrine cells. In some cases, the differentiable cells are non-sensory cells. In some cases, the differentiable cells are non-cardioblastic mesoderm. In some cases, the differentiable cells are cardiac fibroblasts. In some cases, the differentiable cells are skin fibroblasts. In some cases, the differentiable cells are cardiomyocytes. In some cases, the differentiable cells are fibroblast-adipocyte progenitor cells.

[0109] In certain embodiments, the differentiable cells described herein are mammalian cells. In some cases, the mammalian cells are selected from the group consisting of: human cells, bovine (cow) cells, ovine (sheep) cells, porcine (pig) cells, and mouse cells. In some cases, the differentiable cells are avian cells, such as, but not limited to, chicken cells. In some cases, the cells are fish cells, such as, but not limited to, tuna cells or salmon cells.

[0110] In another aspect of the system disclosed herein, the system further includes a solid support configured to deposit a differentiable cell population. The solid support can be configured to allow the two-dimensional growth or shaping of differentiable cells. The solid support can be configured to allow the three-dimensional growth or shaping of differentiable cells. The solid support can be biodegradable. The solid support can comprise natural materials. Natural materials include but are not limited to extracellular matrix components, silk, gelatin and alginate. The solid support can comprise synthetic materials. The solid support can include any surface or scaffold (e.g., hydrogel) to which differentiable cells can adhere.

[0111] The solid support can be coated with one or more extracellular matrix components or fragments thereof. For example, the solid support can be coated with collagen, hyaluronic acid, fibrin, fibronectin, integrin, laminin, proteoglycans, glycosaminoglycans, gelatin, vitronectin, or any other extracellular matrix protein or part or fragment thereof.

[0112] The differentiable cell population can be deposited on the solid support in at least one layer. The differentiable cell population can be deposited on the solid support in at least one, two, three, four, five, six, seven, eight, nine, ten or more layers.

[0113] Unless otherwise defined, all terms of art, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial departure from the general understanding of the art.

[0114] Throughout this application, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a hard limit to the scope of the present disclosure. Therefore, the description of a range should be deemed to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, a description such as a range from 1 to 6 should be deemed to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, this principle applies.

[0115] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "sample" includes a plurality of samples, including mixtures thereof.

[0116] As used herein, the term "about" a number refers to the number plus or minus 10%. The term "about" a range refers to the range minus 10% of its lower limit and plus 10% of its upper limit.

[0117] The term "differentiable cell" refers to any cell that can be divided into a second different cell type from a first cell type under certain conditions. In some cases, differentiable cell is a cell that can (for example, under certain conditions) divide and self-renew over a period of time. In some cases, differentiable cell includes stem cells, such as but not limited to totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells or induced pluripotent stem cells (iPSC). In some cases, differentiable cell includes progenitor cells. In some cases, differentiable cell includes transition amplification cells (TAC). In some cases, the term differentiable cell includes cells that can temporarily or permanently proliferate and divide. In some cases, the term differentiable cell includes a mature somatic cell that can be transdifferentiated into a cell type different from the original cell type under certain conditions.

[0118] "Sequence identity" generally refers to the precise nucleotide and nucleotide or amino acid and amino acid consistency of two polynucleotides or polypeptide sequences respectively. Generally, the technology for determining sequence identity includes determining the nucleotide sequence of the polynucleotide and / or determining the amino acid sequence encoded by it, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". Whether it is a nucleic acid or an amino acid sequence, the percent identity of two sequences is the number of exact matches between the two sequences compared divided by the length of the longer sequence and multiplied by 100. Percent identity can also be determined, for example, by using advanced BLAST computer programs (including the 2.2.9 version available from the National Institutes of Health) to compare sequence information. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990), and as discussed in Altschul, et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine sequence identity over the entire length of the compared proteins. Default parameters are provided to optimize short query sequence searches in, for example, the blastp program. The program also allows the use of SEG filters to mask segments of the query sequence as determined by the SEG program in Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993).

[0119] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0120] Example

[0121] Example 1: Recombinase-mediated excision activates fate box expression

[0122] Cultivate a differentiable cell group containing an exogenous nucleic acid sequence for co-differentiation. Two pairs of recognition sites (each pair is recognized by a different recombinase) are placed in the construct so that when the corresponding recombinase is active in the cell, one fate determination box or another fate determination box is permanently opened. In this scheme, a recognition sequence of each recombinase is placed between the leftmost constitutive promoter and the transcription start site of the gene controlled by the promoter. The two fate boxes are placed at opposite ends of the construct; each is preceded by a recognition sequence and a transcription start site of a type of recombinase, but without a promoter. In this state, the fate box should not be expressed because there is no adjacent promoter upstream to recruit the transcription machinery due to the presence of the blocking sequence. When the recombinase is active in the cell, all sequences between its two recognition sites are excised, so that the fate box immediately downstream of its rightmost recognition site is under the control of the leftmost constitutive promoter. In this case, if recombinase 2 is activated, the entire sequence between the promoter and the muscle box is excised, the muscle box is expressed, and the fat box remains in the genome, but is silent because the muscle box is interposed between it and the promoter. If recombinase 1 is activated (regardless of whether recombinase 2 is active), all sequences between its recognition sites are excised, including the muscle box, and the fat box is expressed.

[0123] Adding a transcriptional inducer induces expression of both recombinase 1 and recombinase 2 in all cells. Both recombinases are expressed but inactive. Adding a chemical inducer activates recombinase 2 expression in all cells. The entire sequence between the recombinase 2 recognition sites is activated. Muscle cell fate is permanently activated in all cells, while expression of the fat fate is silenced.

[0124] A subset of cells was illuminated with light to activate recombinase 2. In the irradiated cells, the muscle cell transcription factor was ablated, and the fat fate was permanently activated.

[0125] Example 2: Co-differentiation of differentiable cell populations into fat or muscle using light and chemical inducers

[0126] Figure 4 is a schematic diagram providing an overview of the process for co-differentiating a population of differentiable cells (fibroblasts in this example) into fat and muscle as described in this example. Figures 5A-5CA non-limiting example of the co-differentiation mechanism and constructs described in this example is depicted. Briefly, a light mask is used so that only a portion of the cells in the well receive light. All cells are engineered to express a light-responsive promoter that drives Cre recombinase expression, a doxycycline-responsive promoter that drives Bxb1 recombinase expression, and a DNA construct containing myogenic and adipogenic transcription factors as well as Cre and Bxb1 recognition sites. First, a portion of the cells on the left side of the well are irradiated. This results in Cre recombinase and GFP expression. Cre recombinase recognizes LoxP sites and recombines the DNA, causing MYOG to be excised and the adipogenic transcription factors PPARG and CEBPA to be expressed, leading to fibroblast-to-adipocyte transdifferentiation. After 2 days, doxycycline is added to the medium, resulting in Bxb1 expression in the cells. Bxb1 recognizes the attP / attB target sites in cells that have not been recombined due to light-induced Cre expression, resulting in the excision of the DNA between these target sites and the generation of attL / attR sites in their place. Through this process, MYOG expression occurs in cells that have not been recombined due to light-induced Cre expression. MYOG expression enables transdifferentiation of fibroblasts into myotubes. As light-responsive Cre expression and recombination are generally not 100% efficient, MYOG expression and thus myotube formation are expected to occur in both the dark and illuminated parts of the well.

[0127] Table 3 describes the plasmids and lentiviral vectors used in this example.

[0128] Table 3: Plasmids and lentiviral vectors used in this example.

[0129]

[0130] 1. Engineering SV40 fibroblasts to express Cre recombinase and GFP in response to light (P_740)

[0131] Stable integration of the light-inducible gene expression system is achieved by the Sleeping Beauty transposon. 500,000 SV40 fibroblasts (fibroblasts stably expressing the SV40 large T antigen) were resuspended in 100 μL of P1 primary cell nucleofection agent solution (Lonza, V4XP-1024) with supplement 1 containing 4.5 μg of plasmid DNA (P_738:P_740 ratios of 10:1, 5:1, 2:1 and 1:1; P_733:P_734:P_740 or P_735:P_736:P_740 ratios of 3:1:1, 5:5:1, 2:2:1 and 1:1:1) and 0.5 μg of Sleeping Beauty transposase SB100X mRNA (Vector Builder, R009S). Lonza Procedure for Unit X CA-137 Nucleofection was performed in a Nucleocuvette. After incubation at room temperature for 30 minutes, cells were transferred to growth medium and cultured.

[0132] 48 hours after transfection, cells transfected with the same light-inducible gene expression system were pooled and expanded in T75 flasks. Light-responsive cells were incubated in the dark or treated with green light (UV and red light-inducible systems) or red light (blue light-inducible systems) to minimize the induction of GFP expression by ambient light. Cells transfected with P-738 were exposed to 2.5 μW / mm2 of light in a pulsed mode of 20 seconds on and 60 seconds off. 2 After adding 10 μM phycocyanobilin (SiChem, SC-1800), cells transfected with P_733 and P_734 were exposed to 1.5 μW / mm in a pulsed mode of 402 seconds on and 3198 seconds off. 2 Cells transfected with P_735 and P_736 were exposed to 0.15 μW / mm2 red light in a pulsed mode of 1 minute on and 29 minutes off. 2 310nm UV light. After irradiation for 2 days, TrypLE was used to detach the cells and dissociate them into single cell suspensions. After centrifugation, the cells were resuspended in PBS and filtered through a 70μm cell strainer. SONY cell sorter SH800S was used to sort cells of low GFP and high GFP populations. All sorted cell populations were amplified in growth medium in the dark. After 7 days of amplification in the dark, GFP-negative populations were sorted to remove cells with residual Cre-GFP expression. In order to evaluate light-inducible gene expression, a portion of the cells were cultured in the dark or in the light for 2 days, and GFP expression was measured by flow cytometry. Approximately 30% UV-responsive cells, 35% blue-light-responsive cells, and 8% red-light-responsive cells expressed GFP, indicating that not all sorted cells responded to light stimulation.

[0133] II. Engineering SV40 fibroblasts to express Bxb1 recombinase (P_854)

[0134] Lentiviral vectors were generated by adding plasmid P_854 together with Lenti-X packaging single dose (Takara) to 1,000,000 Lenti-X293T cells (Takara, 632180).

[0135] The day before transduction, will contain: A) P_738, P_740 (blue light responsive promoter); Or B) P_735, P_736, P_740 (UV light responsive promoter) 50,000 cells are seeded in each well of 12-well plates in growth medium. On the day of transduction, growth medium is replaced, and then the P_854 lentiviral vector with an MOI of 10 and the polybrene of 8 μg / ml are added for transduction. Cells are incubated with lentiviral vector for 24 hours and then treated with 2 μg / ml of doxycycline (Sigma, D3072-1ML). 48 hours after doxycycline treatment, cell sorting (Sony SH800S) of BFP positive groups is carried out to separate the transduced groups. The sorted cell groups are amplified in the dark in a growth medium without doxycycline.

[0136] III. Engineering cells to express transcription factors (P_745) in response to light and chemical stimuli

[0137] The day before transfection, will contain (i) P_738, P_740 and P_854, or (ii) 100,000 cells of P_735, P_736, P_740 and P_854 in growth medium and be seeded in each hole of 6-well plates.On the day of transfection, use 10 μ L Lipofectamine stem cell transfection reagent (ThermoFisher, STEM00015) to come with 0.25 μ g PiggyBac hyPB plasmid (Vector Builder, VB900088-2874gzt) cotransfection 2.5 μ g P_745 according to manufacturer's specification sheets every hole.After transfection, change medium 48 hours.After amplification in the dark, use TrypLE to make cell detachment and dissociate into single cell suspension.After centrifugation, cell is resuspended in PBS and filtered by 70 μ m cell sieve filter. The mScarlet-positive cells were sorted using a SONY SH800S cell sorter.

[0138] IV. Light-mediated spatial and temporal control of gene expression

[0139] SV40 fibroblasts stably engineered with P_740 and P_738 were plated in growth medium. The next day, 2.5 μW / mm 2 The cells were spatially irradiated with blue light (465 nm) using a mask. After 48 hours of irradiation, 5 μg / ml Hoechst 33342 was incorporated into the cells and real-time imaging was performed on a Leica Dmi8 thunder microscope using a 20x0.8NA objective lens. Figure 6A and Figure 6BAs shown in Figure 3 (Scale bar: 500 μm), only cells exposed to light expressed the fluorescent protein, demonstrating the spatial and temporal control of gene expression (e.g., cell differentiation pattern) using the methods and systems described herein.

[0140] To further demonstrate light-mediated gene expression control, various light parameters were tested. 3800 cells containing (i) P_738 and P_740, or (ii) P_733, P_734 and P_740, or (iii) P_735, P_736 and P_740 were seeded in each well of a 96-well plate in growth medium the day before irradiation. Medium supplemented with 10 μM phycocyanobilin (SiChem, SC-1800) was added to the wells with P_733, P_734 and P_740 cells. Cells transfected with P_738 and P_740 were exposed to dark conditions or 5 (L1), 2.5 (L2), 1 (L3) or 0.5 (L4) μW / mm with a pulsed mode of on for 20 seconds and off for 60 seconds. 2 The cells transfected with P_733, P_734 and P_7400 were exposed to a constant irradiation of 10 μW / mm 2 The light-responsive promoter was kept inactive by infrared (820 nm) exposure or by exposure to 4 (L1), 2 (L2), 1 (L3), or 0.5 (L4) μW / mm with a pulse pattern of 1 min on and 29 min off. 2 Cells transfected with P_735, P_736, and P_740 were exposed to dark conditions or 0.15 (UV 1), 0.09 (UV 2), or 0.04 (UV 3) μW / mm2 in a pulsed mode with 1 minute on and 29 minutes off. 2 UV light. Figures 7A-7C As shown, the ratio of GFP positive cells was assessed by flow cytometry. The results demonstrated dynamic GFP expression across multiple light systems (e.g., blue light, UV light, and red light). For blue light responsive promoters, L2 was selected for subsequent experiments. For UV responsive systems, UV1 was selected for subsequent experiments.

[0141] V. Codifferentiation using light and doxycycline

[0142] 96-well plates were coated with 100 μg / ml collagen overnight, washed with PBS and dried. Figure 8 ) or B) P_735P_736P_740P_854P_745 (UV light responsive system, Figure 9) cells were seeded in growth medium at 4000 cells per well in a 96-well plate. The next day, the medium was changed to co-differentiation medium and the cells were treated with 2.5 μW / mm2 of pulsing mode with 20 seconds on and 60 seconds off. 2 Blue light (465nm) or 0.15μW / mm in pulse mode of 1 minute on and 29 minutes off 2 The cells were irradiated with 310 nm UV light for 2 days. The next day, half of the co-differentiation medium was replaced and doxycycline was added to a final concentration of 2 μg / ml. After 3 days, half of the co-differentiation medium was replaced. After 4 days, the cells were fixed with 4% PFA, permeabilized with PBS containing 0.2% Triton-X100, and then blocked with PBS containing 2% BSA and 0.1% Tween 20. The cells were stained overnight at 4°C with mouse anti-myosin heavy chain antibody (MHC) (DSHB, A4.1025-c; 1:100). The next day, the cells were stained with donkey anti-mouse AF647 antibody (Thermo Fisher Scientific, A32787; 1:2000), BODIPY (Cayman Chemical Company, 25892) and DAPI (Abcam, ab228549; 10 μM) at room temperature for 1 hour, then washed and imaged. Images were taken on a Leica DMi8 thunder microscope using a 10x 0.3NA objective. Figure 8 They demonstrated that fibroblasts irradiated with blue light preferentially differentiated into adipocytes, whereas cells that did not undergo recombinant Cre recombinase expression induced by light remained responsive to doxycycline and differentiated toward muscle. Figure 9 They demonstrated that fibroblasts irradiated with UV light preferentially differentiated into adipocytes, whereas cells that did not undergo recombination by light-induced Cre recombinase expression remained responsive to doxycycline and differentiated toward muscle.

[0143] Figure 10A and Figure 10B Describes the Figure 8 and Figure 9 Quantitative measurement of lipid accumulation in cells. Figure 10A and Figure 10B Proof using blue light ( Figure 10A ) or UV light ( Figure 10B ) Irradiated fibroblasts preferentially differentiate into adipocytes. Scale bar: 500 μm. The lower image is a magnification of the upper BODIPY and MHC pore regions.

[0144] Figure 11 The constitutive expression of different transcription factors or differentiation factors in fibroblasts is depicted. SV40 fibroblasts were transduced with one of the following lentiviral vectors: CMV -MYF5、P CMV -MYOD1, PCMV -MYOG, P CMV -MYF6 or empty vector. Cells were incubated with lentiviral vectors for 48 hours and then selected with puromycin. After selection, the transduced cells were cultured in growth medium without puromycin. Images were taken 5 days after transduction. Cells expressing MYOD1 and MYOG showed obvious morphological changes and no longer replicated further. MYF5 and MYF6 also showed morphological changes, but not as obvious as MYOD1 and MYOG. Therefore, this suggests that various transcription / differentiation factors can be expressed in fibroblasts, which leads to the transdifferentiation of fibroblasts into myotubes. Scale bar: 100 μm.

[0145] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided as examples only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be employed. The appended claims are intended to define the scope of the invention and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for co-differentiating a differentiable cell population, the method comprising: (a) providing or obtaining a differentiable cell population; (b) controlling differentiation of a first differentiable cell in the population of differentiable cells using light, thereby causing the first differentiable cell to differentiate into a first cell lineage; (c) differentiating a second differentiable cell in the population of differentiable cells into a second cell lineage, wherein the first cell lineage and the second cell lineage are different, thereby allowing the differentiable cell population to co-differentiate. 2 . The method of claim 1 , wherein the controlling of differentiation in (b) comprises irradiating the first differentiable cell with light of a first wavelength or range of wavelengths.

3. The method of claim 1, wherein the controlling of differentiation in (b) comprises removing light of a first wavelength or range of wavelengths from the first differentiable cell.

4. The method of any one of claims 1 to 3, wherein the differentiating in (c) comprises irradiating the second differentiable cells in the differentiable cell population with light of a second wavelength or range of wavelengths so as to cause the second differentiable cells to differentiate into the second cell lineage; or the differentiating in (c) comprises removing the light of the second wavelength or range of wavelengths from the second differentiable cells so as to cause the second differentiable cells to differentiate into the second cell lineage. The method of claim 4 , wherein the first wavelength or wavelength range and the second wavelength or wavelength range are different.

6. The method of claim 1 , wherein the differentiation in (c) comprises contacting the second differentiable cell in the differentiable cell population with a chemical inducer so as to cause the second differentiable cell to differentiate into the second cell lineage; or the differentiation in (c) comprises removing the chemical inducer from the second differentiable cell in the differentiable cell population so as to cause the second differentiable cell to differentiate into the second cell lineage.

7. The method of any one of claims 1 to 6, wherein each differentiable cell in the population of differentiable cells is engineered to contain an exogenous nucleic acid comprising: (i) a nucleic acid sequence encoding at least one first transcription factor or first differentiation factor that causes differentiation to said first cell lineage; and (ii) a nucleic acid sequence encoding at least one second transcription factor or a second differentiation factor that causes differentiation to said second cell lineage.

8. The method of claim 7, wherein the controlling in (b) comprises regulating the expression of the at least one first transcription factor or first differentiation factor, and the differentiating in (c) comprises regulating the expression of the at least one second transcription factor or second differentiation factor.

9. The method of claim 8, wherein the exogenous nucleic acid comprises at least one promoter operably linked to the nucleic acid sequence encoding the at least one first transcription factor or first differentiation factor and the nucleic acid sequence encoding the at least one second transcription factor or second differentiation factor.

10. The method of claim 9, wherein the at least one promoter is a constitutive promoter.

11. The method of claim 10, wherein the exogenous nucleic acid further comprises: (iii) a blocking sequence downstream of the at least one promoter, which, when present, blocks the expression of the at least one first transcription factor or first differentiation factor and / or the at least one second transcription factor or second differentiation factor.

12. The method of claim 10, wherein the nucleic acid sequence encoding the at least one first transcription factor or first differentiation factor, or a portion thereof, is present in the exogenous nucleic acid in an inverted manner such that the at least one first transcription factor or first differentiation factor is not expressed.

13. The method according to claim 10 or 12, wherein the nucleic acid sequence encoding the at least one second transcription factor or second differentiation factor or a portion thereof is present in reverse in the exogenous nucleic acid such that the at least one second transcription factor or second differentiation factor is not expressed.

14. The method of any one of claims 8-13, wherein each differentiable cell in the population of differentiable cells further comprises: (iv) an exogenous nucleic acid sequence encoding a first activatable recombinase; and (v) an exogenous nucleic acid sequence encoding a second activatable recombinase.

15. The method of claim 14, wherein the first activatable recombinase is a photoactivatable recombinase.

16. The method of claim 14 or 15, wherein the second activatable recombinase is a photoactivatable recombinase.

17. The method of any one of claims 14-16, wherein the first activatable recombinase and the second activatable recombinase are different.

18. The method of claim 14 or 15, wherein the second activatable recombinase is a chemically activatable recombinase.

19. The method of any one of claims 14-18, wherein expression of the first activatable recombinase, expression of the second activatable recombinase, or both, is induced by a chemical inducer, or is induced by light.

20. The method of claim 19, wherein the blocking sequence is flanked by a first recombinase recognition site recognized by the first activatable recombinase, a second recombinase recognition site recognized by the second activatable recombinase, or both.

21. The method of claim 20, wherein the controlling in (b), the differentiating in (c), or both, results in excision of the blocking sequence, thereby inducing expression of the at least one first transcription factor or first differentiation factor, the at least one second transcription factor or second differentiation factor, or both.

22. The method of claim 19, wherein (i) the nucleic acid sequence encoding the at least one first transcription factor or first differentiation factor, or the portion thereof, is present in reverse orientation within the exogenous nucleic acid; (ii) the nucleic acid sequence encoding the at least one second transcription factor or second differentiation factor, or the portion thereof, is present in reverse orientation within the exogenous nucleic acid sequence; or (iii) both are flanked by a first recombinase recognition site recognized by the first activatable recombinase, a second recombinase recognition site recognized by the second activatable recombinase, or both.

23. A method according to claim 22, wherein the controlling in (b), the differentiating in (c) or both result in (i) inversion of the nucleic acid sequence or the portion thereof encoding the at least one first transcription factor or first differentiation factor, such that the at least one first transcription factor or first differentiation factor is expressed; (ii) inversion of the nucleic acid sequence or the portion thereof encoding the at least one second transcription factor or second differentiation factor, such that the at least one second transcription factor or second differentiation factor is expressed; or (iii) both.

24. The method of any one of the preceding claims, further comprising (d) differentiating a third differentiable cell in the population of differentiable cells into a third cell lineage, wherein the third cell lineage is different from the first cell lineage and the second cell lineage.

25. The method of claim 24, wherein the differentiating in (d) comprises (i) irradiating the third differentiable cells with light of a third wavelength or range of wavelengths; (ii) removing the light of the third wavelength or range of wavelengths from the third differentiable cells; (iii) contacting the third differentiable cells with a chemical inducer; or (iv) removing the chemical inducer from the third differentiable cells.

26. The method of claim 25, wherein the third wavelength or wavelength range is different from the first wavelength or wavelength range, the second wavelength or wavelength range, or both.

27. The method of any one of the preceding claims, wherein the population of differentiable cells comprises stem cells.

28. The method of claim 27, wherein the stem cells are multipotent stem cells or pluripotent stem cells.

29. The method of any one of claims 1-26, wherein the population of differentiable cells comprises mature somatic cells capable of transdifferentiation under certain conditions.

30. The method of any one of the preceding claims, wherein the differentiable cells are human differentiable cells or bovine differentiable cells.

31. The method of any one of the preceding claims, wherein the first cell lineage, the second cell lineage, and / or the third cell lineage are selected from the group consisting of: adipocytes, myocytes, and chondrocytes.

32. The method of any one of the preceding claims, wherein the at least one first transcription factor, the at least one second transcription factor, or both are selected from the group consisting of: PPARγ, CEBPα, MYOD, MYOG, MYF5, MRF4 (MYF6), HEYL, KLF4, PAX3, PRDM16, SREBP1, SOX9, SOX5, SOX6, and any combination thereof.

33. The method of any preceding claim, wherein the controlling in (b) and the differentiating in (c) occur substantially simultaneously.

34. The method of any preceding claim, wherein the controlling in (b) and the differentiating in (c) occur sequentially.

35. The method of claim 34, wherein the controlling in (b) precedes the differentiating in (c), or the differentiating in (c) precedes the controlling in (b).

36. The method of any one of the preceding claims, wherein the first differentiable cell and the second differentiable cell are adjacent to each other.

37. A method according to any of the preceding claims, wherein the controlling in (b) further comprises irradiating a plurality of first differentiable cells with light of the first wavelength or wavelength range so as to cause each of the plurality of first differentiable cells to differentiate into the first cell lineage; or the controlling in (b) further comprises removing light of the first wavelength or wavelength range from the plurality of first differentiable cells so as to cause each of the plurality of first differentiable cells to differentiate into the first cell lineage.

38. The method of any one of the preceding claims, wherein the differentiating in (c) further comprises differentiating a plurality of second differentiable cells such that each of the plurality of second differentiable cells differentiates into the second cell lineage.

39. The method of any one of the preceding claims, wherein the differentiable cell population is deposited onto a solid support.

40. The method of claim 39, wherein the differentiable cell population is deposited on the solid support in multiple layers.

41. The method of claim 39 or 40, wherein the solid support is coated with one or more extracellular matrix components or portions thereof.

42. A system for co-differentiating a population of differentiable cells, the system comprising: (a) A population of differentiable cells, wherein each differentiable cell in the population of differentiable cells is engineered to contain an exogenous nucleic acid comprising: (i) a nucleic acid sequence encoding at least one first transcription factor or first differentiation factor that causes differentiation to a first cell lineage; and (ii) a nucleic acid sequence encoding at least one second transcription factor or a second differentiation factor that causes differentiation to a second cell lineage; and (b) one or more light sources configured to control the differentiation of a first differentiable cell in the population of differentiable cells using light of a first wavelength or range of wavelengths to cause the first differentiable cell to differentiate into the first cell lineage, wherein the first cell lineage and the second cell lineage are different.

43. The system of claim 42, wherein the system is configured to use the one or more light sources to illuminate the first differentiable cell with light of the first wavelength or range of wavelengths to cause the first differentiable cell to differentiate into the first cell lineage.

44. The system of claim 42, wherein the system is configured to remove light of the first wavelength or range of wavelengths from the first differentiable cell to cause the first differentiable cell to differentiate into the first cell lineage.

45. The system of any one of claims 42-44, wherein the system is further configured to use the one or more light sources to illuminate second differentiable cells in the differentiable cell population with light of a second wavelength or range of wavelengths to cause the second differentiable cells to differentiate into the second cell lineage; or the system is further configured to remove light of the second wavelength or range of wavelengths from the second differentiable cells to cause the second differentiable cells to differentiate into the second cell lineage.

46. ​​The system of claim 45, wherein the first wavelength or wavelength range and the second wavelength or wavelength range are different.

47. The system of claim 42, further comprising a chemical inducer that causes a second differentiable cell in the differentiable cell population to differentiate into the second cell lineage; or the system is further configured to remove the chemical inducer from the second differentiable cell to cause the second differentiable cell to differentiate into the second cell lineage.

48. The system of any one of claims 42-47, wherein the exogenous nucleic acid comprises at least one promoter operably linked to the at least one first transcription factor or first differentiation factor and the at least one second transcription factor or second differentiation factor.

49. The system of claim 48, wherein the at least one promoter is a constitutive promoter.

50. The system of claim 48 or 49, wherein the exogenous nucleic acid further comprises: (iii) a blocking sequence downstream of the at least one promoter, which, when present, blocks the expression of the at least one first transcription factor or first differentiation factor and the at least one second transcription factor or second differentiation factor.

51. The system of claim 48 or 49, wherein the nucleic acid sequence encoding the at least one first transcription factor or first differentiation factor, or a portion thereof, is present in reverse in the exogenous nucleic acid such that the at least one first transcription factor or first differentiation factor is not expressed.

52. The system of claim 48 or 49, wherein the nucleic acid sequence encoding the at least one second transcription factor or second differentiation factor, or a portion thereof, is present in reverse in the exogenous nucleic acid such that the at least one second transcription factor or second differentiation factor is not expressed.

53. The system of any one of claims 50-52, wherein each differentiable cell in the population of differentiable cells further comprises: (iv) a nucleic acid sequence encoding a first activatable recombinase; and (v) a nucleic acid sequence encoding a second activatable recombinase.

54. The system of claim 53, wherein the first activatable recombinase is a photoactivatable recombinase.

55. The system of claim 53 or 54, wherein the second activatable recombinase is a photoactivatable recombinase.

56. The system of any one of claims 53-55, wherein the first activatable recombinase and the second activatable recombinase are different.

57. The system of claim 53 or 54, wherein the second activatable recombinase is a chemically activatable recombinase.

58. The system of any one of claims 53-57, wherein expression of the first activatable recombinase, expression of the second activatable recombinase, or both are inducible by a chemical inducer, or are inducible by light.

59. The system of claim 58, wherein the blocking sequence is flanked by a first recombinase recognition site recognized by the first activatable recombinase, a second recombinase recognition site recognized by the second activatable recombinase, or both.

60. The system of claim 59, wherein the blocking sequence is configured to be excised by the first activatable recombinase, the second activatable recombinase, or both, thereby regulating expression of the at least one first transcription factor or first differentiation factor, the at least one second transcription factor or first differentiation factor, or both.

61. The method of claim 59, wherein (i) the nucleic acid sequence encoding the at least one first transcription factor or first differentiation factor, or the portion thereof, is present in reverse orientation within the exogenous nucleic acid; (ii) the nucleic acid sequence encoding the at least one second transcription factor or second differentiation factor, or the portion thereof, is present in reverse orientation within the exogenous nucleic acid sequence; or (iii) both are flanked by a first recombinase recognition site recognized by the first activatable recombinase, a second recombinase recognition site recognized by the second activatable recombinase, or both.

62. A method according to claim 61, wherein (i) the nucleic acid sequence encoding the at least one first transcription factor or first differentiation factor, or the portion thereof, is configured to be inverted by the first activatable recombinase, the second activatable recombinase, or both, such that the at least one first transcription factor or first differentiation factor is expressed; (ii) the nucleic acid sequence encoding the at least one second transcription factor or second differentiation factor, or the portion thereof, is configured to be inverted by the first activatable recombinase, the second activatable recombinase, or both, such that the at least one second transcription factor or second differentiation factor is expressed; or (iii) both.

63. The system of any one of claims 42-62, wherein the exogenous nucleic acid further comprises a nucleic acid sequence encoding at least one third transcription factor or a third differentiation factor that causes differentiation to a third cell lineage.

64. The system of claim 63, wherein the system is further configured to use the one or more light sources to illuminate third differentiable cells in the differentiable cell population with light of a third wavelength or wavelength range to cause the third differentiable cells to differentiate into the third cell lineage; or wherein the system is further configured to remove light of the third wavelength or wavelength range to cause the third differentiable cells to differentiate into the third cell lineage, wherein the third wavelength or wavelength range is different from the first wavelength or wavelength range and / or the second wavelength or wavelength range, and wherein the third cell lineage is different from the first cell lineage and / or the second cell lineage.

65. The system of any one of claims 42-64, wherein the differentiable cells are stem cells.

66. The system of claim 65, wherein the stem cells are multipotent stem cells or pluripotent stem cells.

67. The system of any one of claims 42-64, wherein the differentiable cells are mature somatic cells capable of being transdifferentiated under certain conditions.

68. The system of any one of claims 42-67, wherein the differentiable cells are human differentiable cells or bovine differentiable cells.

69. The system of any one of claims 42-68, wherein the first cell lineage, the second cell lineage, and / or the third cell lineage are selected from the group consisting of: adipocytes, myocytes, and chondrocytes.

70. The system of any one of claims 42-69, wherein the at least one first transcription factor, the at least one second transcription factor, the at least one third transcription factor, or any combination thereof, is selected from the group consisting of: PPARγ, CEBPα, MYOD, MYOG, MYF5, MRF4 (MYF6), HEYL, KLF4, PAX3, PRDM16, SREBP1, SOX9, SOX5, SOX6, and any combination thereof.

71. The system of any one of claims 42-70, wherein the first differentiable cell and the second differentiable cell are adjacent to each other.

72. The system of any one of claims 42-71, further comprising a solid support, wherein the differentiable cell population is deposited onto the solid support.

73. The system of claim 72, wherein the differentiable cell population is deposited on the solid support in multiple layers.

74. The system of any one of claims 42-73, wherein the one or more lights comprise one or more light emitting diodes (LEDs).

75. The system of claim 74, wherein the one or more LEDs comprise at least two different LEDs.

76. The system of claim 75, wherein the at least two different LEDs emit light of different wavelengths or wavelength ranges.

77. The system of any one of claims 42-73, wherein the one or more light sources comprise one or more lasers.

78. The system of any one of claims 42-73, wherein the one or more light sources comprise an incandescent light source.

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