Methods and systems for differentiating cells using optogenetics

Abstract

Provided herein are methods and systems for differentiating cells in a population of cells into a desired cell lineage. In some embodiments, the methods and systems use light (e.g., optogenetics) to control differentiation of cells to a desired cell lineage. In some cases, the methods and systems involve differentiating cells in a suspension culture to produce cells of a desired cell lineage on a large scale.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 347,780, filed on June 1, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Transcription factors can regulate the differentiation of cells (e.g., stem cells) into different cell types. Expression of transcription factors such as MyoD can cause cells to differentiate into myocytes, while expression of other transcription factors such as PPARγ can cause cells to differentiate into adipocytes or adipocyte-like cells. It may be difficult to differentiate a cell (e.g., stem cell) group into a single cell lineage, for example, with high efficiency and time precision. Summary of the invention

[0004] The methods and systems described herein address an unmet need for efficiently differentiating a population of cells (eg, stem cells) into a specified or desired cell lineage (or cell type), for example, using optogenetics.

[0005] In one aspect, a method for differentiating at least one cell of a cell population into a desired cell lineage is provided, the method comprising: (a) providing or obtaining a cell population in a suspension culture; and (b) controlling the differentiation of at least one cell of the cell population with light, thereby differentiating at least one cell into a desired cell lineage. In some cases, the controlled differentiation of (b) comprises irradiating at least one cell with light of a first wavelength. In some cases, the controlled differentiation of (b) comprises removing light of a first wavelength from at least one cell. In some cases, the at least one cell is genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one transcription factor, at least one differentiation factor, or both that achieves differentiation into a desired cell lineage. In some cases, the expression of the at least one transcription factor, the at least one differentiation factor, or both is induced by irradiating or removing light. In some cases, the exogenous nucleic acid comprises at least one promoter operably connected to the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the at least one cell is genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one light-controllable transcriptional regulatory factor. In some cases, the promoter is an inducible promoter. In some cases, the at least one light-controllable transcriptional regulator is a light-controllable transcriptional activator. In some cases, the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain. In some cases, the irradiation induces the light-controllable transcriptional activator to bind to the inducible promoter and activate the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the removal induces the light-controllable transcriptional activator to bind to the inducible promoter and activate the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the promoter is a constitutive promoter. In some cases, the at least one light-controllable transcriptional regulator is a light-controllable transcriptional repressor. In some cases, the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain. In some cases, the irradiation induces the light-controllable transcriptional repressor to dissociate from the constitutive promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the removal of light induces the light-controllable transcriptional repressor to dissociate from the constitutive promoter, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the exogenous nucleic acid further comprises a blocking sequence downstream of the at least one promoter, which, when present, blocks expression of the at least one first transcription factor, the at least one differentiation factor, or both. In some cases, the at least one cell further comprises a nucleic acid sequence encoding at least one light-controllable recombinase.In some cases, the at least one light-controllable recombinase is a light-activatable recombinase. In some cases, the light-activatable recombinase comprises a recombinase or a portion thereof fused to a light-activatable domain. In some cases, the blocking sequence is flanked by a recombinase recognition site recognized by the at least one light-controllable recombinase. In some cases, the irradiation activates the light-controllable recombinase, thereby causing excision of the blocking sequence and inducing expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the at least one 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 at least one cell is a fibroblast. In some cases, the at least one cell is a human cell, a bovine cell, or a mouse cell. In some cases, the desired cell lineage is selected from: adipocytes, myocytes, and chondrocytes. In some cases, the at least one transcription factor is selected from: PPARγ, CEBPα, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, and any combination thereof. In some cases, the irradiation further includes multiple cells of the cell mass irradiated with light to differentiate each of the multiple cells into a desired cell lineage; or the removal further includes removing light from the cell mass to differentiate each of the multiple cells into a desired cell lineage. In some cases, the suspension culture has a volume of at least 100 milliliters (mL). In some cases, the suspension culture is contained in a bioreactor container. In some cases, the bioreactor container has a cumulative volume of at least 100 milliliters (mL). In some cases, the cell mass grows on the surface of a microcarrier. In some cases, the microcarrier is coated with one or more extracellular matrix components.

[0006] On the other hand, a system for differentiating a cell population is provided, the system comprising: (a) a cell population in suspension culture, wherein at least one cell of the cell population is engineered to contain an exogenous nucleic acid, the exogenous nucleic acid comprising a nucleic acid sequence, the nucleic acid sequence encoding at least one transcription factor, at least one differentiation factor, or both that achieves differentiation into a desired cell lineage, wherein the expression of the at least one transcription factor, the at least one differentiation factor, or both is controlled by light; and (b) one or more light sources configured to irradiate at least one cell of the cell population with light of a first wavelength. In some cases, the exogenous nucleic acid comprises at least one promoter operably connected to the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the at least one cell is genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one light-controllable transcriptional regulator. In some cases, the promoter is an inducible promoter. In some cases, the at least one light-controllable transcriptional regulator is a light-controllable transcriptional activator. In some cases, the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain. In some cases, the light-controllable transcriptional activator binds to the inducible promoter and activates the inducible promoter after being irradiated by light of the first wavelength, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the light-controllable transcriptional activator binds to the inducible promoter and activates the inducible promoter after removing the light of the first wavelength, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the promoter is a constitutive promoter. In some cases, the at least one light-controllable transcriptional regulatory factor is a light-controllable transcriptional repressor. In some cases, the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain. In some cases, the light-controllable transcriptional repressor dissociates from the constitutive promoter after being irradiated by light of the first wavelength, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the light-controllable transcriptional repressor dissociates from the constitutive promoter after removing the light of the first wavelength, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the exogenous nucleic acid further comprises a blocking sequence downstream of the at least one promoter, which, when present, blocks the expression of the at least one transcription factor, the at least one differentiation factor, or both. In some cases, the at least one cell of the cell population further comprises a nucleic acid sequence encoding at least one light-controllable recombinase. In some cases, the at least one light-controllable recombinase is a light-activatable recombinase. In some cases, the light-activatable recombinase comprises a recombinase fused to a light-activatable domain.In some cases, the blocking sequence is flanked by a recombinase recognition site recognized by the at least one light-controllable recombinase. In some cases, the blocking sequence is configured to be excised by the at least one light-controllable recombinase, thereby inducing the expression of the at least one transcription factor. In some cases, the at least one 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 at least one cell is a fibroblast. In some cases, the at least one cell is a human cell, a bovine cell, or a mouse cell. In some cases, the desired cell lineage is selected from: adipocytes, myocytes, and chondrocytes. In some cases, the at least one transcription factor is selected from: PPARγ, CEBPα, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, and any combination thereof. In some cases, the system further comprises a plurality of microcarriers, wherein the cell population grows on the surface of the plurality of microcarriers. In some cases, the plurality of microcarriers are coated with one or more extracellular matrix components. In some cases, the one or more light sources include one or more light emitting diodes (LEDs). 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 disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and accompanying drawings which illustrate illustrative embodiments utilizing the principles of the present disclosure, in which:

[0010] Figure 1A and Figure 1B Depicted are non-limiting examples of systems for differentiating cells into desired cell lineages using photodimerization domains coupled to transcriptional activators according to embodiments herein.

[0011] Figure 2A and Figure 2B Depicted are non-limiting examples of systems for differentiating cells into desired cell lineages using photodissociable domains coupled to transcriptional activators according to embodiments herein.

[0012] Figure 3A and Figure 3BDepicted are non-limiting examples of systems for differentiating cells into desired cell lineages using photodimerization domains coupled to transcriptional repressors according to embodiments herein.

[0013] Figure 4A and Figure 4B Depicted are non-limiting examples of systems for differentiating cells into desired cell lineages using photodissociable domains coupled to transcriptional repressors according to embodiments herein.

[0014] Figure 5 Non-limiting examples of schematic diagrams depicting nucleic acid sequences for expressing transcription factors suitable for use in the methods and systems described herein.

[0015] Figure 6 Non-limiting examples of inducible recombinase systems suitable for use with the methods and systems described herein are depicted.

[0016] Fig. 7A and Figure 7B Depicted are non-limiting examples of constructs suitable for use with the methods and systems described herein.

[0017] Fig. 8A and Figure 8B Depicted are non-limiting examples of constructs suitable for use with the methods and systems described herein.

[0018] Fig.9A and Fig. 9B Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in two-dimensional culture that constitutively express adipogenic transcription factors according to the methods and systems described herein.

[0019] Fig.10 Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in suspension culture that exhibit constitutive expression of adipogenic transcription factors according to the methods and systems described herein.

[0020] Fig.11A and Fig. 11B Depicted are data demonstrating the use of light to activate a light-activatable recombinase according to the methods and systems described herein.

[0021] Fig.12 Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in two-dimensional culture expressing adipogenic transcription factors under the control of constitutively expressed recombinant enzymes according to the methods and systems described herein.

[0022] Fig.13A and Fig. 13B Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in two-dimensional culture expressing adipogenic transcription factors under the control of constitutively expressed recombinant enzymes according to the methods and systems described herein.

[0023] Fig.14 Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in two-dimensional culture expressing adipogenic transcription factors under the control of light-activatable recombinases according to the methods and systems described herein.

[0024] Fig.15A and Fig. 15B Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in two-dimensional culture expressing adipogenic transcription factors under the control of light-activatable recombinases according to the methods and systems described herein.

[0025] Fig.16 Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in suspension culture expressing adipogenic transcription factors under the control of light-activatable recombinases according to the methods and systems described herein.

[0026] Fig.17 Depicted are data demonstrating lipid droplet accumulation in fibroblasts grown in suspension culture expressing adipogenic transcription factors under the control of light-activatable recombinases according to the methods and systems described herein.

[0027] Fig.18 Depicted are data from control experiments according to the methods and systems described herein.

[0028] Fig.19A and Fig.19B Depicted are data demonstrating the expression of genes associated with adipogenesis in fibroblasts grown in suspension culture that have been differentiated into adipocyte-like cells using the methods and systems described herein. DETAILED DESCRIPTION

[0029] The developmental fate of a cell (e.g., a stem cell) can be determined by expressing a specific transcription factor in the cell, which initiates a developmental program, causing it to differentiate into a specific cell type. However, it may be difficult to control (e.g., time control) a cell (e.g., a stem cell) group to differentiate into one or more specified or desired cell lineages or cell types in a controlled manner using existing technology. For example, stem cells differentiate into specified or desired cell types (e.g., wherein the specified or desired cell type includes all or part of a three-dimensional tissue) and may require high time or spatiotemporal precision. As described herein, controlling the (e.g., time) expression of transcription factors in stem cells can allow a single stem cell group (e.g., simultaneously and / or patterned) to differentiate into a cell group comprising one or more cell types. For example, the methods and systems disclosed herein can be used to differentiate a single cell (e.g., a stem cell) group into a single specified or desired cell type, for example, while minimizing or preventing all or part of a single cell group from differentiating into a cell type other than a specified or desired cell type. The methods and systems provided herein generally use optogenetics to control cells (e.g., stem cells) to differentiate into desired cell lineages. In some cases, the methods and systems provided herein use light-controllable transcriptional regulators (e.g., light-controllable transcriptional activators, light-controllable transcriptional repressors, etc.) that are controlled by light (e.g., by irradiating cells (e.g., stem cells) with light of a specific wavelength or wavelength range, or by removing light of a specific wavelength or wavelength range from cells (e.g., stem cells)) to control the expression of transcription factors and / or differentiation factors, resulting in cells (e.g., stem cells) differentiating into desired cell lineages. In other cases, the methods and systems disclosed herein use light-controllable recombinases (e.g., light-activatable recombinases) that are controlled by light to differentiate cells (e.g., stem cells) into desired cell lineages.

[0030] Generally, the method and system provided herein use optogenetics to activate the desired differentiation pathway in cells (e.g., stem cells). Compared with traditional methods (e.g., using expensive chemical factors), it is relatively cheap to use optogenetics to differentiate cells. In addition, since there is no need to add exogenous chemical factors, the method and system provided herein are safe alternatives to traditional methods, particularly when producing differentiated cells for individual downstream use (e.g., for individual consumption or for therapeutic purposes). In addition, using light to differentiate cells allows a subset of individual cells or cell groups to be illuminated with light, rather than a typical entire cell group as in the case of adding chemical factors. This provides precise time and space control of differentiation in a cell group. In addition, in some embodiments, the method and system provided herein are adjustable, because light can be easily provided or removed to induce or block the expression of differentiation pathways (e.g., such as when using a light-inducible promoter). In addition, the amount of gene expression can be controlled, for example, by adjusting the light intensity.

[0031] Advantageously, the methods and systems provided herein can differentiate cells (e.g., stem cells) in suspension cultures into desired cell lineages. The use of suspension cultures allows large-scale production of desired cell types, which cannot be achieved with cells in adherent cultures (e.g., in the wells of tissue culture plates). Therefore, when it is necessary to mass-produce cells of a particular type or lineage, the methods and systems provided herein are particularly advantageous.

[0032] In some embodiments, the methods and systems provided herein use optogenetics to control cell differentiation into desired cell lineages or cell types (e.g., temporally and / or spatially). For example, in some embodiments, the promoters regulating (e.g., expressing) one or more transcription factors and / or differentiation factors (e.g., participating in differentiation pathways) can be controlled by light. In some cases, the methods and systems are directed to using a light-controlled transcriptional regulatory factor that can be combined with a promoter (or a region adjacent to a promoter or near a promoter) or dissociated from a promoter (or a region adjacent to a promoter or near a promoter) to activate the expression of one or more transcription factors and / or differentiation factors. In some cases, the light-controlled transcriptional regulatory factor is a transcriptional regulatory factor fused with a light-controlled domain (e.g., an optical switch). In some cases, the light-controlled transcriptional regulatory factor is a light-controlled transcriptional activator. The light-controlled transcriptional activator is a transcriptional activator fused with a light-controlled domain (e.g., an optical switch). In some cases, the light-controlled transcriptional regulatory factor is a light-controlled transcriptional repressor. The light-controlled transcriptional repressor is a transcriptional repressor fused with a light-controlled domain. Advantageously, such methods and systems do not result in permanent activation of differentiation pathways, and differentiation pathways can be turned on or off simply by providing or removing light, as described herein.

[0033] In some embodiments, the methods and systems provided herein use one or more light-controllable recombinases. The recombinase recognizes a specific DNA sequence, and if there are two recognition sequences that are correctly arranged, it can excise the DNA between the two sites or flip the direction of the DNA between the two sites. By briefly activating the recombinase, the DNA can be permanently changed, which provides the prospect of permanently turning on differentiation genes with only a short activation period. Such methods and systems can lead to permanent activation of, for example, a differentiation program.

[0034] Disclosed herein are methods and systems for differentiating cell (e.g., stem cell) groups (e.g., separate cell (e.g., stem cell) groups), such as using suspension cell culture techniques. In some cases, the systems described herein may be configured to (and / or used to) differentiate a first cell (e.g., stem cell) group into cells of a desired cell lineage (e.g., wherein the cells of the desired cell lineage include one or more phenotypes, gene expression profiles, and / or epigenetic states that are different from the phenotypes, gene expression profiles, and / or epigenetic states of undifferentiated cells). In some cases, the systems described herein may be used to perform the methods described herein. In some cases, the systems described herein may be used to promote differentiation of a cell group into a specified or desired cell type, for example, by establishing and / or maintaining conditions necessary or beneficial for differentiation of all or a portion of a cell group into a specified or desired cell type. In some cases, the systems described herein can be used to promote cell groups to differentiate into specified or desired cell types, by establishing and / or maintaining conditions necessary to prevent or be beneficial to prevent all or part of a cell group from differentiating into a cell type different from a specified or desired cell type (e.g., a cell type containing another phenotype, gene expression profile, and / or epigenetic state different from a specified or desired cell type). In some cases, methods and systems include the use of light-controllable transcriptional regulatory factors and / or light-controllable recombinases. Provided herein is a method for differentiating a cell (e.g., stem cell) group. The method can include providing or obtaining a cell (e.g., stem cell) (e.g., engineered cell (e.g., stem cell)) group, and controlling at least one cell (e.g., stem cell) of a cell (e.g., stem cell) group with light to differentiate into a desired cell lineage. In some cases, differentiation can involve irradiating at least one cell (e.g., stem cell) of a cell (e.g., stem cell) group with light of a first wavelength to differentiate at least one cell (e.g., stem cell) into a desired cell lineage. In some cases, differentiation can involve removing light of a specific wavelength (or removing light completely) to differentiate at least one cell (e.g., a stem cell) into a desired cell lineage. In some cases, a population of cells (e.g., stem cells) is in suspension culture (e.g., in a bioreactor).

[0035] The expression of at least one transcription factor and / or differentiation factor in the cell can cause the cell to differentiate into a specific cell type. It is contemplated herein that any transcription factor or combination of transcription factors that causes the cell to differentiate into a desired cell lineage when expressed, and / or any differentiation factor or combination of differentiation factors. In some cases, cell differentiation into a specified or desired cell lineage or cell type (e.g., adipocytes, myocytes, or chondrocytes) can be induced by transcription factors, and the non-limiting examples of the transcription factors include PPARγ, CEBPα, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX, SOX9, SOX5, SOX6, or any combination thereof. In a non-limiting example, when cells are required to differentiate into adipocytes (e.g., adipocytes), cells can be induced to express transcription factors such as, but not limited to, PPARγ and / or CEBPα. In another non-limiting example, when cells are required to differentiate into muscle cells (e.g., myocytes), cells can be induced to express transcription factors such as, but not limited to, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, and / or PAX3. In another non-limiting example, when cells are required to differentiate into chondrocytes (e.g., chondrocytes), cells can be induced to express transcription factors such as, but not limited to, SOX9, SOX5, and / or SOX6. In some embodiments, at least one transcription factor is selected from: PPARγ, CEBPα, MYOD, MYOG, and combinations thereof. In some cases, at least one differentiation factor may include at least one chromatin remodeling factor. In some cases, the chromatin remodeling factor may be SMARCD3 and / or JMJD3. In some cases, the expression of transcription factors and / or differentiation factors can be regulated, for example, by light.

[0036] 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 differentiation factors (e.g., chromatin remodeling factors) in a cell (e.g., a stem cell) (e.g., to differentiate the cell (e.g., a stem cell) into a desired cell lineage).

[0037] In some aspects, the cell population described herein (e.g., engineered cell population) may include at least one exogenous nucleic acid. Exogenous nucleic acid may include 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. The promoter sequence may be constitutively active. In alternative embodiments, the promoter sequence may be conditionally active. For example, a conditionally active promoter sequence may be adjustable or inducible, such as by light. In some aspects, the cell described herein comprises at least two exogenous nucleic acids. In some aspects, the cell described herein comprises at least three, at least four, at least five or more exogenous nucleic acids. Each exogenous nucleic acid may include at least one nucleic acid sequence encoding one or more transcription factors and / or differentiation factors. The nucleic acid sequence encoding the transcription factor and / or differentiation factor may be operably connected to one or more promoters (e.g., constitutive, inducible) as described herein. In some cases, when more than one transcription factor and / or differentiation factor is used to differentiate cells into a desired cell lineage, the expression of each of the more than one transcription factor and / or differentiation factor can be under the control of the same single promoter. In such cases, each gene encoding a transcription factor and / or differentiation factor can be combined into a single bicistronic or multicistronic transcript. A single transcript can encode a self-cleaving 2A peptide between each individual protein, or can contain an internal ribosome entry site (IRES), or a combination thereof. In other cases, the expression of transcription factors and / or differentiation factors can be under the control of different promoters.

[0038] In some cases, at least one cell (e.g., stem cell) in a cell (e.g., stem cell) population described herein may be genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one light-switchable transcriptional regulator. In some cases, at least one light-switchable transcriptional regulator is a light-switchable transcriptional activator. The light-switchable transcriptional activator may be a transcriptional activator fused or otherwise associated with a light-switchable domain, such as an optical switch (e.g., as described herein). In some cases, at least one light-switchable transcriptional regulator may be a light-switchable transcriptional repressor. The light-switchable transcriptional repressor may be a transcriptional repressor fused or otherwise associated with a light-switchable domain, such as an optical switch (e.g., as described herein).

[0039] In various aspects, the light-switchable transcriptional regulator is a light-switchable transcriptional activator. In one embodiment, the light-switchable transcriptional activator binds or otherwise associates with a promoter sequence (e.g., an inducible promoter) upon irradiation with light (e.g., at a specific wavelength or wavelength range), thereby inducing expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors, as described herein).

[0040] Figure 1A , 1B 2A and 2B describe various embodiments of using light-controllable activators to control the expression of one or more transcription factors and / or differentiation factors. Figure 1A As shown, in one embodiment, the transcription activator is fused to the first photoheterodimerization domain (e.g., photoheterodimerization domain 1). A second photoheterodimerization domain (e.g., photoheterodimerization domain 2) capable of dimerizing with the first photoheterodimerization domain is fused to a DNA binding domain. The DNA binding domain binds to a DNA sequence within or near a promoter sequence. In the absence of light, the first and second photoheterodimerization domains do not associate, and the expression of the transcription factor and / or differentiation factor is turned off. After irradiation with light (e.g., at an appropriate wavelength or wavelength range), the first and second photoheterodimerization domains heterodimerize, bringing the transcription activator into close contact with the inducible promoter. Then, the transcription activator induces the activity of the inducible promoter, and the transcription factor and / or differentiation factor is expressed, resulting in differentiation into the desired cell lineage. In this case, those cells irradiated with light differentiate into the desired cell lineage, while those cells not irradiated with light or not irradiated with light of an appropriate wavelength remain undifferentiated.

[0041] In another embodiment, Figure 1B As shown, the transcription activator is fused with a light homodimerization domain and a DNA binding domain. In this case, the DNA binding domain is combined with a DNA sequence in or near the promoter sequence after homodimerization with another DNA binding domain. When irradiated with light (for example, at an appropriate wavelength or wavelength range), the light homodimerization domain homodimerizes, which makes two DNA binding domains in close contact so that they homodimerize. After homodimerization, the DNA binding domain is combined with the promoter sequence, so that the transcription activator is in close contact with the inducible promoter. Then, the transcription activator induces the activity of the inducible promoter, and the transcription factor and / or differentiation factor are expressed, resulting in differentiation into the desired cell lineage. In this case, those cells irradiated with light differentiate into the desired cell lineage, while those cells not irradiated with light or not irradiated with light of appropriate wavelength remain undifferentiated.

[0042] In various aspects, in the absence of light or in the absence of light of a particular wavelength, the light-controllable transcriptional activator binds or associates with a promoter sequence (e.g., an inducible promoter), thereby inducing expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In the presence of light, the light-controllable transcriptional activator does not bind to or dissociates from a promoter sequence (e.g., an inducible promoter), thereby preventing or reducing expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein).

[0043] like Figure 2A As shown, in one embodiment, the transcription activator is fused to the first photodissociation domain (e.g., photodissociation domain 1). The second photodissociation domain (e.g., photodissociation domain 2) is fused to the DNA binding domain. The DNA binding domain binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second photodissociation domains associate, bringing the transcription activator into close contact with the inducible promoter, and the expression of the transcription factor and / or differentiation factor is initiated. When irradiated with light (e.g., at an appropriate wavelength or wavelength range), the first and second photodissociation domains dissociate, releasing the transcription activator from the inducible promoter, thereby shutting down the expression of the transcription factor and / or differentiation factor. In this case, those cells irradiated with light remain undifferentiated, while those cells not irradiated with light or not irradiated with light of an appropriate wavelength differentiate into desired cell lineages.

[0044] In another embodiment, Figure 2B As shown, the transcription activator is fused with the photodissociation domain and the DNA binding domain. In this case, the DNA binding domain is combined with the DNA sequence in or near the promoter sequence after homodimerization with another DNA binding domain. In the absence of light, the photodissociation domain homodimerizes with another photodissociation domain, allowing the DNA binding domain to homodimerize and bind to the inducible promoter, and the transcription activator is in close contact with the inducible promoter. Then, the transcription activator induces the activity of the inducible promoter, and the transcription factor and / or the differentiation factor are expressed, resulting in differentiation into the desired cell lineage. After irradiation with light (for example, at an appropriate wavelength or wavelength range), the photodissociation domain dissociates, which releases the transcription activator from the inducible promoter and turns off the expression of the transcription factor and / or the differentiation factor. In this case, those cells irradiated with light remain undifferentiated, and those cells not irradiated with light or not irradiated with light of appropriate wavelengths are differentiated into the desired cell lineage.

[0045] In various aspects, the light-controllable transcriptional regulator is a light-controllable transcriptional repressor. In one embodiment, the light-controllable transcriptional repressor is combined with a promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcription start site) or otherwise associated after being irradiated with light (e.g., at a specific wavelength or wavelength range), thereby repressing the constitutive promoter and preventing or reducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In the absence of light, the light-controllable transcriptional repressor is not combined with a promoter sequence (e.g., a sequence between a constitutive promoter or a constitutive promoter and a transcription start site) or dissociated therefrom, thereby allowing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In this case, those cells irradiated with light (e.g., stem cells) remain undifferentiated, while those cells not irradiated with light (e.g., stem cells) differentiate into desired cell lineages.

[0046] Figure 3A , 3B 4A and 4B describe various embodiments of using light-switchable transcriptional repressors to control the expression of one or more transcription factors and / or differentiation factors. Figure 3A As shown, in one embodiment, the transcriptional repressor is fused to the first photoheterodimerization domain (e.g., photoheterodimerization domain 1). The second photoheterodimerization domain (e.g., photoheterodimerization domain 2) capable of dimerizing with the first photoheterodimerization domain is fused to the DNA binding domain. The DNA binding domain binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second photoheterodimerization domains do not associate, and the expression of the transcription factor and / or differentiation factor is initiated (e.g., because the gene encoding the transcription factor and / or differentiation factor is under the control of a constitutive promoter). After irradiation with light (e.g., at an appropriate wavelength or wavelength range), the first and second photoheterodimerization domains dimerize, bringing the transcriptional repressor into close contact with the promoter sequence or the sequence between the promoter and the transcription start site. The transcriptional repressor blocks the activity of the constitutive promoter, and the expression of the transcription factor and / or differentiation factor is turned off. In this case, those cells irradiated with light remain undifferentiated, while those cells not irradiated with light or not irradiated with light of an appropriate wavelength differentiate into the desired cell lineage.

[0047] In another embodiment, Figure 3BAs shown, the transcription repressor is fused with a light homodimerization domain and a DNA binding domain. In this case, the DNA binding domain is combined with a DNA sequence in or near a promoter sequence after homodimerization with another DNA binding domain. In the absence of light, the first and second light homodimerization domains do not associate, and the expression of transcription factors and / or differentiation factors is initiated (for example, because the gene encoding the transcription factor and / or differentiation factor is under the control of a constitutive promoter). After irradiation with light (for example, at an appropriate wavelength or wavelength range), the first and second light homodimerization domains dimerize, so that the transcription repressor is in close contact with the sequence between the promoter sequence or the promoter and the transcription start site. The transcription repressor blocks the activity of the constitutive promoter, and the expression of the transcription factor and / or differentiation factor is turned off. In this case, those cells irradiated with light remain undifferentiated, and those cells not irradiated with light or not irradiated with light of appropriate wavelengths differentiate into desired cell lineages.

[0048] In various aspects, the light-controllable transcriptional repressor binds or associates with a promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcription start site) in the absence of light, thereby preventing or reducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In the presence of light, the light-controllable transcriptional repressor does not bind to or dissociate from a promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcription start site), thereby activating the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In this case, those cells (e.g., stem cells) that are irradiated with light differentiate into the desired cell lineage, while those cells (e.g., stem cells) that are not irradiated with light remain undifferentiated.

[0049] like Figure 4A As shown, in one embodiment, the transcriptional repressor is fused to the first photodissociation domain (e.g., photodissociation domain 1). The second photodissociation domain (e.g., photodissociation domain 2) is fused to the DNA binding domain. The DNA binding domain binds to a DNA sequence in or near the promoter sequence. In the absence of light, the first and second photodissociation domains associate, thereby bringing the transcriptional repressor into close contact with the promoter sequence or the sequence between the promoter and the transcription start site, and the expression of the transcription factor and / or differentiation factor is turned off. When irradiated with light (e.g., at an appropriate wavelength or wavelength range), the first and second photodissociation domains dissociate, releasing the transcriptional repressor from the promoter sequence, thereby initiating the expression of the transcription factor and / or differentiation factor. In this case, those cells irradiated with light differentiate into the desired cell lineage, while those cells not irradiated with light or not irradiated with light of an appropriate wavelength remain undifferentiated.

[0050] In another embodiment, Figure 4BAs shown, the transcription repressor is fused with a photodissociation domain and a DNA binding domain. In this case, the DNA binding domain is combined with a DNA sequence in or near the promoter sequence after homodimerization with another DNA binding domain. In the absence of light, the photodissociation domain homodimerizes with another photodissociation domain, thereby allowing the DNA binding domain to homodimerize and bind to the sequence between the promoter sequence or the promoter and the transcription start site, and the transcription repressor is in close contact with the sequence between the promoter sequence or the promoter and the transcription start site. The transcription repressor blocks the activity of the constitutive promoter, and the expression of the transcription factor and / or the differentiation factor is turned off. When irradiated with light (for example, at an appropriate wavelength or wavelength range), the photodissociation domain dissociates, which releases the transcription repressor from the sequence between the promoter or the promoter and the transcription start site, and the expression of the transcription factor and / or the differentiation factor is activated. In this case, those cells irradiated with light differentiate into the desired cell lineage, while those cells not irradiated with light or not irradiated with light of appropriate wavelength remain undifferentiated.

[0051] Non-limiting examples of DNA binding domains that can be used in any of these embodiments include Gal4 (which binds to the Gal4 UAS DNA sequence), TetR (which binds to the TetO DNA sequence), and CymR (which binds to the CuO DNA sequence).

[0052] In some cases, a transcriptional regulator (e.g., a transcriptional activator, a transcriptional repressor) is fused to a light-controllable domain. In some cases, the light-controllable domain is an optogenetic switch. In some cases, the optogenetic switch is an optogenetic switch based on dimerization. In some cases, the optogenetic switch based on dimerization is an optogenetic switch based on heterodimerization, as described herein. In other cases, the optogenetic switch based on dimerization is an optogenetic switch based on homodimerization, as described herein. Any light-controllable domain can be used, including any light-controllable domain described herein. Non-limiting examples of optogenetic dimerization systems suitable for the methods and systems provided herein are described in Table 2.

[0053] Non-limiting examples of transcriptional activators include VP16 of herpes simplex virus (HSV), and the transcriptional activation domain of p65. Non-limiting examples of transcriptional repressors include the KRAB (Krüppel-associated box) domain of Kox1.

[0054] In other aspects of the present disclosure, controlling differentiation may involve the use of light-controllable recombinases. In this case, the expression of one or more transcription factors and / or differentiation factors may be under the control of a (e.g., constitutive) promoter. A blocking sequence may be inserted between a (e.g., constitutive) promoter and a sequence encoding one or more transcription factors and / or differentiation factors so that when there is a blocking sequence, the expression of one or more transcription factors and / or differentiation factors is blocked. After removing the blocking sequence, the expression of one or more transcription factors and / or differentiation factors is activated. The removal of the blocking sequence may be achieved by using a recombinase (e.g., light-controllable recombinase). In this case, the blocking sequence is flanked by a recombinase recognition site so that when irradiated with light, a light-controllable (or light-activatable) recombinase excises the blocking sequence, thereby allowing the expression of one or more transcription factors and / or differentiation factors. By fusing a recombinase with a light-controllable domain (e.g., an optogenetic switch (e.g., as described herein)), the recombinase may be activated by light (e.g., at a specific wavelength or wavelength range). In this case, the irradiation of a cell (e.g., a stem cell) causes the cell (e.g., a stem cell) to differentiate into a desired cell lineage.

[0055] Figure 5 Describe the non-limiting examples of nucleic acid boxes applicable to the disclosure of this paper.Promoter 503 can be operably connected with the nucleic acid sequence encoding transcription factor 504.In some cases, blocking sequence 502 can be between promoter sequence and the nucleic acid sequence encoding transcription factor (for example, wherein the blocking sequence is at promoter downstream).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 an expression cassette or multiple expression cassettes, and each expression cassette contains transcription terminator sequence.In some cases, blocking sequence can be flanked by recombinase recognition site 501 (for example, wherein the recombinase recognition site is recognized by light-controllable recombinase).In this case, in the presence of activated recombinase, excision blocking sequence, thus allow the expression of transcription factor.In some cases, recombinase can be activatable, for example, can be activated by light, as described herein, thus allow control recombinase activity. In such cases, the recombinase can be expressed in the cell (e.g., stem cell), but can be in an inactive state until the cell (e.g., stem cell) is exposed to light of a specific wavelength or wavelength range. Upon exposure of the cell (e.g., stem cell) to light of a specific wavelength or wavelength range, the recombinase can be activated, resulting in excision of the blocking sequence and expression of the transcription factor (thereby causing the cell (e.g., stem cell) to differentiate into a desired cell lineage).

[0056] In an alternative embodiment, rather than excising the blocking sequence, an activatable recombinase is used to flip the nucleic acid sequence so that it is under the control of the promoter, thereby resulting in expression of the transcription factor and / or differentiation factor.

[0057] In some cases, more than one recombinase can be used so that different transcriptional programs are activated depending on which recombinase is activated. For example, a cell can express more than one light-controllable recombinase (e.g., each fused to a different light-controllable domain). When differentiation into cell lineage A is desired, the cell can be exposed to light of a first wavelength or wavelength range, thereby activating a first recombinase fused to a first light-controllable domain, resulting in the expression of a first transcription factor and / or differentiation factor, and the cell differentiates into cell lineage A. When differentiation into cell lineage B is desired, the cell can be exposed to light of a second different wavelength or wavelength range, thereby activating a second recombinase fused to a second different light-controllable domain, resulting in the expression of a second transcription factor and / or differentiation factor, and the cell differentiates into cell lineage B. In a cell population, the method can be used to (e.g., temporarily) control the differentiation of cells into a specific or desired cell lineage or cell type.

[0058] In some cases, a light-activatable system can be used to activate the recombinase. The light-activatable system can be as described in Table 1.

[0059] Table 1. Light-activatable systems

[0060]

[0061] In various aspects, a combination of light-controllable domains (e.g., a first light-controllable domain and a second light-controllable domain) can be used (e.g., each of the light-controllable domains can be fused to a portion of the recombinase). In some cases, the first light-controllable domain and the second light-controllable domain can be binding partners, such that upon irradiation with light of a specific wavelength or within a specific spectral range, the first and second light-controllable domains heterodimerize or hetero-oligomerize. Upon irradiation with light of a specific wavelength or within a specific spectral range, the first and second light-controllable domains heterodimerize or hetero-oligomerize, thereby bringing the protein domains (or their functional domains or functional portions) into close contact with each other, so that the recombinase is activated.

[0062] In various aspects, the light-controllable domain includes 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 part or domain thereof; such as 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.

[0063] In some cases, a combination of light-controllable domains is used, wherein the first light-controllable domain is cryptochrome 2 (or a variant or functional portion thereof), and the second light-controllable domain is CIB1 (or a variant or functional portion thereof; e.g., CIBN). In some cases, a combination of light-controllable domains is used, wherein the first light-controllable domain is BphP1 (or a variant or functional portion thereof), and the second light-controllable domain is QPAS1 (or a variant or functional portion thereof). In some cases, the light-controllable domains (or a combination of light-activatable domains) are selected from Table 2. In some cases, the light-controllable domain may 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 more) with any of the light-controllable domains described in Table 2.

[0064] Table 2. Non-limiting examples of light-controllable domain systems

[0065]

[0066]

[0067] In another aspect of the methods described herein, the recombinase can be activated 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 light-induced system. The recombinase can mediate irreversible excision. 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.

[0068] In another embodiment, the dimerization and activation of the recombinase can be induced by light (e.g., optogenetic dimerization). The first half of the recombinase and the second half of the recombinase can be fused with a light-controllable domain. In various aspects, the light-controllable domain can include a light-oxygen-voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a cryptochrome (CRY) domain, a FAD (BLUF) photoreceptor domain using blue light, a phytochrome (PHY) domain, a CIB1 (cryptochrome interacting basic-helix-loop-helix protein 1) (or its functional part or 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. The dimerization and activation of the recombinase can be regulated by any light-controllable system described in Table 2. Alternatively, dimerization and activation can be induced by temperature.

[0069] In various aspects, the method may involve exposing a cell (e.g., genetically engineered to express a fusion protein comprising a recombinase and a light-controllable domain) to light of a specific wavelength or light within a specific spectral range. The wavelength of light can be selected so that the light can activate the light-controllable domain. For example, Table 2 provides non-limiting examples of light parameters for different light-controllable domain systems. The wavelength of light can be one or more of infrared light, near infrared light, visible light (e.g., red light, green light, blue light), ultraviolet light, or a combination thereof. Infrared light can include light of about 780nm to 1mm wavelength. Near infrared light can include light of about 740nm to about 780nm wavelength. Red light can include light of about 620nm to 750nm, 600nm to 690nm, or about 650nm wavelength. Green light can include light of about 577nm to about 492nm wavelength. Blue light can include light of 492nm to about 455nm, or about 440nm to about 473nm wavelength. Ultraviolet light can include light of about 10nm to 400nm, or about 280 to 315nm. In various aspects, the wavelength of light is from 100nm to 1mm.

[0070] In some embodiments, the methods and systems involve irradiating cells with light of a particular intensity or range of intensities. For example, the methods provided herein can involve irradiating cells with light of an intensity of about 2 μW / mm 2 In other examples, the methods provided herein can involve irradiating cells with light having an intensity of about 8 μW / mm 2 In some cases, the methods provided herein may involve irradiating cells with light having an intensity of about 2 μW / mm 2 About 8μW / mm 2 It will be appreciated that the level of light intensity may vary and may depend on the type of optogenetic switch used.

[0071] In some embodiments, the methods provided herein involve irradiating cells with light in an illumination mode (such as a pulsed mode). For example, the method can involve irradiating cells for a period of time, turning off the illumination of the cells for a period of time, and then repeating the on / off cycle multiple times.

[0072] In another embodiment, the recombinase comprises a single-chain polypeptide. The single-chain polypeptide can be fused to a light-controllable domain to produce a light-controllable recombinase. Exposure to light of a specific wavelength can produce the activity of the recombinase. For example, irradiation of the Cre system LiCre based on AsLOV2 with blue light leads to activation of the recombinase.

[0073] In another embodiment, the recombinase can be fused to the PhoCl protein or a derivative thereof. Irradiation with violet light (about 400 nm) results in the cleavage of PhOCl. In some cases, the PhoCl domain may be present between the blocker domain and the recombinase domain in the fusion protein, as shown in 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 nucleus. For example, the blocker domain can be a steroid receptor domain that interacts with Hsp90 to prevent the recombinase from entering the nucleus. Exposing a fusion protein comprising a blocker domain, a PhoCl domain, and a recombinase to violet light can result in the cleavage of the PhoCl domain, allowing the recombinase to enter the nucleus and reach the genomic DNA, resulting in recombinase activity.

[0074] Recombinases, transcriptional regulatory factors, transcription factors, differentiation factors and other elements described herein can be encoded by nucleic acids. In some embodiments, nucleic acids comprising recombinases, transcriptional regulatory factors, transcription factors, differentiation factors and other elements described herein can be expression cassettes or can be contained in expression cassettes. As used herein, "expression cassette" means a recombinant nucleic acid construct comprising one or more nucleic acids described herein, wherein the recombinant nucleic acid construct is operably associated with at least one control sequence (e.g., a promoter).

[0075] In some cases, engineered cells (e.g., engineered cells described herein, e.g., before differentiation or after differentiation) may include a positive selection cassette or a negative selection cassette. For example, by including a negative selection cassette (e.g., in the nucleic acid sequence of cells that were not excised due to recombinase activity) or a positive selection cassette (e.g., in the nucleic acid sequence of cells that were excised due to recombinase activity) and applying one or more factors sufficient to induce the respective positive or negative selection cassettes, cells that do not belong to a specified or desired cell lineage or cell type (e.g., undifferentiated cells or cells that differentiate into a cell lineage or type other than a specified or desired cell lineage) can be selectively killed or eliminated from a cell population. In some cases, the inclusion of a positive selection cassette in the nucleic acid sequence of cells excised during recombinase activity can be used to remove or destroy cells in which recombinase activity has not been successful (e.g., after providing a stimulus sufficient to activate the positive selection cassette).

[0076] 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 be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, such as a nucleic acid that can replicate extrachromosomally. A vector that can direct the expression of a gene operably connected thereto is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc.

[0077] In the vector, 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 and the selection gene that promotes transformant recognition, which are usually given by the origin of replication, can be incorporated in addition. Virus-derived vectors, such as slow viruses, retroviruses, adenoviruses, adeno-associated viruses, etc., can be used. Plasmid vectors can be linearized to be integrated into the chromosome position. The vector can include a sequence (for example, AttP-AttB recombination) that instructs site-specific integration into a restricted position in the genome or a set of restriction sites. In addition, the vector can include a sequence derived from a transposable element.

[0078] In some aspects, the nucleic acid introduced into the eukaryotic cell is operably linked to a promoter and / or a poly A (poly A) signal 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 operably linked to a poly A signal at the 3' end. In some aspects, the nucleic acid comprises a 2A peptide sequence and / or an internal ribosome entry site.

[0079] In some embodiments, the expression cassette includes a nucleotide sequence encoding a selective marker that can be used to select transformed host cells. As used herein, "selective marker" means a nucleotide sequence that, when expressed, gives a different phenotype to the host cell expressing the marker, thereby distinguishing the transformed cell from those cells that do not have the marker. Such a nucleotide sequence can encode a selectable or screenable marker, depending on whether the marker confers 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).

[0080] Advantageously, the methods herein provide for differentiation of cells (e.g., stem cells) suspended (rather than in the form of, for example, two-dimensional adherent cell cultures). In some cases, the methods provided herein do not require the use of a feeder layer of cells. In some cases, the methods provided herein do not require the use of an extracellular matrix component. In some cases, the methods provided herein relate to growing or maintaining cells (e.g., stem cells) in a bioreactor. In some cases, the methods provided herein may relate to the use of microcarriers (e.g., beads). In some cases, microcarriers may be coated with an extracellular matrix component (e.g., to promote cell (e.g., stem cell) attachment thereto).

[0081] In various fields, suspension culture has the volume of at least about 100 milliliters (mL).For example, suspension culture can have at least about 150mL, at least about 200mL, at least about 250mL, at least about 300mL, at least about 350mL, at least about 400mL, at least about 450mL, at least about 500mL, at least about 550mL, at least about 600mL, at least about 650mL, at least about 700mL, at least about 750mL, at least about 800mL, at least about 850mL, at least about 900mL, at least about 950mL, at least about 1000mL, at least about 2000mL, at least about 3000mL, at least about 4000mL or at least about 5000mL volume.In some cases, suspension culture has the volume less than about 1000mL.In some cases, suspension culture has the volume greater than about 1000mL.

[0082] Any suitable cell culture medium for differentiating cells (e.g., stem cells) can be used. In some embodiments, the cell culture medium comprises any of the following in appropriate combinations: isotonic saline, buffer, amino acids, serum or serum replacement, sugars (e.g., glucose), and other exogenously added factors. In some embodiments, the cell culture medium comprises DMEM, F12, αMEM, Hepatostim in the presence or absence of serum. TM , RPMI or a combination thereof. Suitable serum includes calf serum, fetal bovine serum, horse serum, etc. In some embodiments, a serum supplement is used.

[0083] In some cases, the methods and systems provided herein can be used to differentiate cells (e.g., stem cells) into desired cell lineages. In some cases, the cells used in the methods and systems provided herein are stem cells. The stem cells used in the methods and systems provided herein can be any desired stem cells. In some cases, stem cells are pluripotent stem cells. In some cases, stem cells are multipotent stem cells. In some cases, stem cells are embryonic stem cells. In some cases, stem cells are mesenchymal stem cells. In some cases, stem cells are satellite cells or muscle stem cells. In some cases, stem cells are adipose stem cells. In certain embodiments, stem cells described herein are mammalian stem cells. In some cases, mammalian stem cells are selected from: human stem cells, (cattle) stem cells of cattle, (sheep) stem cells of sheep and (pig) stem cells of pigs. In some cases, stem cells are avian stem cells, such as but not limited to chicken stem cells. In some cases, stem cells are fish stem cells, such as but not limited to tuna stem cells or salmon stem cells. In some cases, cells can be non-stem cells (such as fibroblasts) that can be transdifferentiated into desired cell lineages. Any cell that can differentiate (e.g., any differentiable cell) can be used in the methods and systems provided herein. Any type of cell can be used in the methods and systems provided herein, including but not limited to human cells, bovine cells, or mouse cells.

[0084] In another aspect of the method disclosed herein, a cell (e.g., stem cell) group is deposited on a solid support (such as a microcarrier). In some cases, a cell (e.g., stem cell) group can grow on the surface of a microcarrier. In some cases, a microcarrier (or a portion thereof, a cell group as described herein grows thereon) can be coated with one or more extracellular matrix components. The solid support can be biodegradable. The solid support can include natural materials. Natural materials include, but are not limited to, extracellular matrix components, silk, gelatin and alginate. The solid support can include synthetic materials. The solid support can include any surface or support (e.g., hydrogel) that a cell (e.g., stem cell) can adhere to.

[0085] The solid support can be coated with one or more extracellular matrix components. For example, the solid support can be coated with or incorporated with collagen, hyaluronic acid, fibrin, fibronectin, integrin, laminin, proteoglycans, glycosaminoglycans, gelatin, vitronectin or any other extracellular matrix protein.

[0086] Further provided herein is a system (e.g., configured to implement the method provided herein). In some cases, the system is included in a cell (e.g., stem cell) group in a suspension culture. In some cases, at least one cell (e.g., stem cell) of a cell (e.g., stem cell) group is genetically engineered as described herein (e.g., to contain exogenous nucleic acid, the exogenous nucleic acid comprises a nucleic acid sequence encoding at least one transcription factor and / or differentiation factor), which is differentiated into a desired cell lineage. In some cases, the expression of at least one transcription factor and / or differentiation factor can be controlled by light (e.g., using a light-controllable transcriptional regulatory factor and / or a light-controllable recombinase, for example, as described herein). In some aspects, the system may further include one or more light sources, which are configured to irradiate at least one cell (e.g., stem cell) of a cell (e.g., stem cell) group with a specific wavelength or wavelength range. In some cases, a cell (e.g., stem cell) group can grow in a suspension culture in a bioreactor. In some cases, one or more light sources are present on a bioreactor (e.g., on the surface of the wall of a bioreactor).

[0087] In some embodiments, the one or more light sources 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. 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.

[0088] The bioreactor can be any type of culture vessel suitable for growing cells in suspension culture. In various aspects, the bioreactor vessel comprises a total volume of at least about 50 mL, at least about 100 mL, at least about 150 mL, at least about 200 mL, at least about 250 mL, at least about 300 mL, at least about 400 mL, at least about 500 mL, at least about 600 mL, at least about 700 mL, at least about 750 mL, at least about 800 mL, at least about 900 mL, at least about 1000 mL, at least about 2000 mL, at least about 3000 mL, at least about 5000 mL or more.

[0089] The cells used in the systems provided herein can be any desired cells, including stem cells and non-stem cells (e.g., fibroblasts). In some cases, stem cells are pluripotent stem cells. In some cases, stem cells are multipotent stem cells. In some cases, stem cells are embryonic stem cells. In some cases, stem cells are mesenchymal stem cells. In some cases, stem cells are satellite cells or muscle stem cells. In some cases, stem cells are adipose stem cells. In certain embodiments, stem cells described herein are mammalian stem cells. In some cases, mammalian stem cells are selected from: human stem cells, cattle (cattle) stem cells, sheep (sheep) stem cells and pig (pig) stem cells. In some cases, stem cells are avian stem cells, such as but not limited to chicken stem cells. In some cases, stem cells are fish stem cells, such as but not limited to tuna stem cells or salmon stem cells.

[0090] Unless otherwise defined, all technical terms, symbols and other technical and scientific terms or nomenclature 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 these definitions herein should not be construed as representing a substantial difference from what is generally understood in the art.

[0091] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the present disclosure. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and each numerical value within the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and a single number within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this applies.

[0092] As used in the 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.

[0093] As used herein, the term "about" a number refers to the number plus or minus 10% of the number. The term "about" a range refers to the range minus 10% of its lower limit and plus 10% of its upper limit.

[0094] Generally, "sequence identity" refers to the precise nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, the technology used to determine sequence identity includes determining the nucleotide sequence of the polynucleotide and / or determining the amino acid sequence encoded therein, 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 aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity can also be determined, for example, by using the advanced BLAST computer program available from the National Institutes of Health, including version 2.2.9, 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 the percent identity over the entire length of the compared proteins. Default parameters are provided to optimize searches with short query sequences 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 of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993).

[0095] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0096] Example

[0097] Example 1: Recombinase-mediated excision to activate fate box expression

[0098] A stem cell group containing an exogenous nucleic acid sequence is suspended and cultured for differentiation. A pair of recognition sites (recognition sites are recognized by a light-controllable recombinase) is placed in the construct so that when the corresponding recombinase is active in the cell, the muscle expression box (e.g., fate determination box) is permanently turned on. In this scheme, a recognition sequence of the recombinase is placed between the leftmost (e.g., most upstream) related constitutive promoter and the transcription start site of the gene controlled by the promoter. The fate box is placed at the opposite end of the construct; the fate box is preceded by the recognition sequence and transcription start site of the recombinase, but there is no promoter. In this state, the fate box should not be expressed because there is no promoter directly upstream to recruit the transcription machinery due to the presence of the blocking sequence. When the cell is irradiated with light of a wavelength sufficient to activate the light-controllable recombinase and the recombinase becomes active in the cell, all sequences between its two recognition sites are excised, so that the fate box immediately downstream of the rightmost (most downstream) recognition site is under the control of the leftmost (most upstream) constitutive promoter. In this case, if the recombinase is activated, the entire sequence between the promoter and the muscle expression box is excised and the muscle expression box is expressed.

[0099] Example 2: Photoinduced differentiation of fibroblasts into adipocyte-like cells in suspension culture

[0100] This example demonstrates light-induced differentiation of fibroblasts into adipocyte-like cells in suspension culture. In this example, light-activatable Cre recombinase was used to excise blocking sequences and drive expression of adipocyte transcription factors, peroxisome proliferator-activated receptor gamma (PPARg) and CCAAT / enhancer binding protein alpha (CEBPa).

[0101] Plasmid / construct

[0102] The plasmids created for this study were designed in Geneious (Biomatters). The different components of the plasmids were obtained by restriction digestion, PCR or via DNA synthesis and assembled via Gibson cloning. Table 3 describes the plasmids used in these studies. pCMV-PPARg, Cre reporter plasmid and SV40 lentivirus were obtained from commercial sources.

[0103] Table 3. Plasmid / viral vectors

[0104]

[0105] Plasmid 703 (in Figure 8B ) is contained in the constitutive human Ef1α promoter (P Ef1α) under the control of bovine transcription factor PPARg (UniProt: O18971-1) and fluorescent protein mScarletI (GenBank: APD76536.1). The coding sequences of PPARg and mScarletI are connected by an IRES (internal ribosome entry site) sequence to allow co-expression of the two genes. PPARg-IRES-mScarletI is connected to the promoter via a sequence containing three SV40 polyadenylation sequences (SV40pA) flanked by loxP sites. The sequence containing three SV40 polyadenylation sequences is excised by recombination of the two loxP sites via Cre recombinase, and the expression of PPARg and mScarletI is initiated. Transcription from the Ef1α promoter is terminated via a rabbit β-globin polyadenylation signal (rBGpA). The plasmid further contains an expression cassette for the neomycin resistance gene (Neo, GenBank: WP_004614937.1) containing the constitutive human cytomegalovirus immediate early promoter (P CMV ) and the bovine growth hormone polyadenylation signal (bGHpA), which allows selection of cells with genomic integration of the plasmid element. The entire nucleotide sequence comprising the element is flanked by the 5' and 3' ITR sequences of the PiggyBac transposon.

[0106] Plasmid 704 (in Figure 7B ) is contained in the constitutive human Ef1α promoter (P Ef1α ) and the nucleic acid sequence encoding the bovine transcription factors PPARg (UniProt: O18971-1) and CEBPa (GenBank: NP_789741.2) and the fluorescent protein mScarletI (GenBank: APD76536.1) under the control of . The coding sequences of PPARg and CEBPa are connected via a T2A self-cleaving peptide, and PPARg-T2A-CEBPa is connected to mScarletI via an IRES (internal ribosome entry site) sequence to allow co-expression of the three genes. PPARg-T2A-CEBPa-IRES-mScarletI is connected to the promoter via a sequence containing three SV40 polyadenylation sequences (SV40pA) flanked by loxP sites. The sequence containing the three SV40 polyadenylation sequences is excised by recombination of the two loxP sites by Cre recombinase, and the expression of PPARg, CEBPa and mScarletI is initiated. Transcription from the Ef1α promoter is terminated via the rabbit β-globin polyadenylation signal (rBGpA). The plasmid further contains an expression cassette for a neomycin resistance gene (Neo, GenBank: WP_004614937.1) containing a constitutive human cytomegalovirus immediate early promoter (P CMV) and the bovine growth hormone polyadenylation signal (bGHpA), which allows selection of cells with genomic integration of the plasmid element. The entire nucleotide sequence comprising the element is flanked by the 5' and 3' ITR sequences of the PiggyBac transposon.

[0107] Plasmid 361 contains an expression cassette comprising the constitutive human cytomegalovirus immediate early promoter (P CMV ), the coding sequence for the Cre recombinase (UniProt: P06956, residues 2-343) linked to a nuclear localization signal (UniProt: Q2HJ27, residues 320-328), and the bovine growth hormone polyadenylation signal (bGHpA).

[0108] Plasmid 709 (in Fig. 7A and Fig. 8A ) comprises an expression cassette comprising a constitutive bovine Ef1α promoter (P Ef1α ), coding sequences for light-inducible Cre recombinase and fluorescent protein mTagBFP2 (GenBank: HCE4034942.1), and bovine growth hormone polyadenylation signal (bGHpA). The coding sequences of light-inducible Cre recombinase and mTagBFP2 are connected by an IRES (internal ribosome entry site) sequence to allow co-expression of the two genes. The light-inducible Cre recombinase contains three nuclear localization signals (UniProt: Q2HJ27, residues 320-328) fused to the N-terminus of an N-terminal truncated form of LiCre containing AsLOV2 (Genbank: 4WF0_A, residues 11-148) and Cre (UniProt: P06956, residues 20-339) (as described in Duplus-Bottin et al., "A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch", eLife 10: e61268 (2021)). The entire nucleotide sequence containing the element is flanked by the 5' and 3' IR / DR sequences of the Sleeping Beauty transposon.

[0109] P CMV -PPARg lentiviral vector contains a nucleic acid sequence encoding the bovine transcription factor PPARg (UniProt:O18971-1) under the control of the constitutive human cytomegalovirus immediate early promoter (PCMV). The lentiviral vector further contains a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a constitutive mouse phosphoglycerate kinase 1 promoter (PmPGK ) is a nucleic acid sequence encoding a puromycin resistance gene under the control of.

[0110] Cell lines

[0111] Isolation of Fibroblasts from Wagyu

[0112] Adipose stem cells were extracted from fresh adipose tissue of adult Wagyu bulls. Briefly, adipose tissue was minced with a sterile scalpel, added to 25 mL of digestion solution (2 mg / ml collagenase II (Sigma, C2-BIOC), 1% penicillin / streptomycin / amphotericin B (Lonza, 17-745E), 10 μM ROCK inhibitor Y-27632 (Tocris, 1 254)), and a total volume of 40 ml was obtained in a 50 ml centrifuge tube, and incubated at 37 ° C for 60 minutes, inverted every 2 minutes. The tube was centrifuged at 300 x g for 5 minutes, a precipitate was produced at the bottom, and a fat plug was produced at the top, which was discarded together with the supernatant. The pellet was washed with 25 mL of growth medium (DMEM (Sigma, SLM-021) containing 10% fetal bovine serum (Avantor, 89510-186) and 1% penicillin / streptomycin / amphotericin B), then resuspended in 10 mL of growth medium and transferred to a T75 tissue culture flask. The cells were incubated undisturbed at 37° C. and 5% CO2 for 24 hours and then expanded for multiple passages, with the growth medium replaced every 2 days.

[0113] Generation of SV40 background cell lines (SV40 cells)

[0114] Produce cells that stably express SV40-LgT (large T antigen) because they confer a doubling time advantage and immortalize the cells. P3 Wagyu fibroblast cell line was cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin / amphotericin B and maintained under standard conditions. Cells were transduced with lentivirus (carrying LgT under the CMV promoter, GentargetInc LVP016-Hygro San Diego, CA) in 24-well plates, 20 μL of virus was added to each well, and then incubated for 24 hours. After incubation, the culture medium was replaced with fresh culture medium. After approximately 48 hours, cells were subjected to antibiotic selection (hygromycin, InvivoGen, San Diego, CA) for one week to promote the survival of stably transduced cells with only antibiotic resistance genes. These cells were then amplified, and after reaching sufficient numbers, some were cryopreserved for future use, involving resuspension in freezing medium, controlled rate freezing, and storage in liquid nitrogen.

[0115] Integration of plasmids using PiggyBac / Sleeping Beauty and selection using Geneticin / cell sorting

[0116] One day before transfection, 500,000 SV40 cells were seeded in growth medium in a T75 flask. On the day of transfection, Lipofectamine TM Each transposon plasmid was co-transfected with a transposase plasmid (HypB for PiggyBac and hySB100X for Sleeping Beauty) using 3000 transfection reagent (ThermoFisher). Each flask contained 9 μg of transposon plasmid, 0.9 μg of the corresponding transposase plasmid, 22 μL of P3000, and 27 μL of Lipofectamine TM 3000. Also carry out the transfection without transposase to monitor the reduction of transient transfection signal before cell sorting.Change culture medium 48 hours after transfection.For the cells transfected by plasmid 703 and plasmid 704, 4mg / mL geneticin is included in the growth medium for selection.Simultaneously, the control flask containing the same number of but unengineered cells is selected with geneticin.All cells in the control flask were killed at the 6th day, which shows that the cells surviving in the experimental flask are engineered.The engineered cells are amplified in the growth medium without geneticin for experiment.

[0117] Isolation of cell populations stably expressing plasmids

[0118] Cells were separated and dissociated into single cell suspensions using TrypLE by incubation at 37 °C for 5 minutes. After centrifugation, cells were resuspended in PBS and filtered through a 100 μm cell strainer. Cell sorting of cell populations with PiggyBac and Sleeping Beauty transposon integration was performed using a SONY cell sorter SH800S according to the manufacturer's instructions. To sort plasmid 709 in 703 and 704 transfected cells, the mScarlet-negative BFP low population and the mScarlet-negative BFP high population were gated to purify Cre recombinase-expressing cells with intact loxP-3xSTOP-loxP sequences upstream of the transcription factor. All sorted cell populations were expanded in growth medium.

[0119] Bioreactor preparation

[0120] A 100 mL spinner flask (Bellco) with an impeller was coated with Sigmacote (Sigma Aldrich), excess Sigmacote was removed, and the flasks were dried by removing the side caps and lids, inverting the flasks and placing them in a fume hood overnight. The next day, the flasks were rinsed with 100 ml of fresh MilliQ water and dried. The dry reactor was assembled and autoclaved for 30 minutes in a fast / dry cycle.

[0121] Microcarrier preparation

[0122] Coat a 50 mL falcon tube with Sigmacote, dry, wash with fresh MilliQ water and invert to dry before use. Use 0.2 g of Cytodex 3 microcarriers per 100 mL flask. Place the microcarriers from each spinner flask into a coated 50 mL falcon tube and add 45 mL of Mg / Ca-free 2+ Rehydrate with PBS without Mg / Ca. Invert the falcon tube several times until the microcarriers are completely suspended and then leave at room temperature for at least 3 hours. Remove the supernatant and rehydrate with PBS without Mg / Ca. 2+ After removing the supernatant, add PBS without Mg / Ca 2+ PBS to the 40mL mark and sterilize the microcarriers by autoclaving in a wet cycle for 15 minutes. The rehydrated Cytodex 3 can be stored at 4°C or used directly. To use the microcarriers, remove 25mL of PBS supernatant and 25mL of growth medium containing DMEM / F12 high glucose, 10% FBS, 1% penicillin / streptomycin / amphotericin B. Allow the microcarriers to rest and repeat the medium wash 2 times. For a 100mL container, bring the volume to 20mL.

[0123] Suspension culture

[0124] Microcarrier solution (20mL) is inverted and poured into each spinner flask, and the falcon tube is rinsed with 20mL of growth medium containing DMEM / F12 high glucose+10%FBS+1% penicillin / streptomycin / amphotericin B, and it is added to the flask.Then the spinner flask is placed on a rotating plate, and rotated at least 2 hours at 30rpm before adding cells. Wash cells, digest with trypsin and count. Add 2-3 million cells to each reactor, and use growth medium to make the volume reach 100mL. Close the spinner flask and rotate to mix cells and microcarriers gently.Then the flask is placed in an incubator and rotated at 30rpm per hour for 1 minute, for 3 hours, and then left to attach to the microcarriers overnight without rotating.

[0125] The next day, the rotating flasks were continuously placed on a rotator at 30 rpm. For each experiment, one flask was exposed to light, while the other flask was not exposed to any light as a control. The blue light (peak intensity at about 465 nm) used to illuminate the flasks was applied in 20 second pulses every 80 seconds, with a power density of about 8 μW / mm at the reactor wall. 2. All flasks were covered with light-proof jackets and substrates as well as aluminum foil to prevent light leakage into the dark control flasks. The irradiated flasks were exposed to the irradiation pattern for 48 hours. After 2 days of suspension culture, the growth medium was replaced with StemPro Adipogenic Differentiation Kit Medium (Gibco) supplemented with 1% Penicillin / Streptomycin / Amphotericin B. Thereafter, 70% StemPro medium was replaced on day 3. To this end, the spinner flask was removed from the spin plate and the microcarriers were allowed to settle for 5 minutes before replacing 70 mL of warm StemPro medium. 1 mL of microcarrier / medium was sampled per well for imaging, and imaging was performed in triplicate for each sample. As indicated, 2 mL of microcarrier / medium was sampled in duplicate for RNA. Cultures were sampled in StemPro medium after 6 days.

[0126] dyeing

[0127] For staining with BODIPY 493 / 503, the wells were filled with PBS, most of the supernatant was removed without exposing the cells to air, and 1 mL / well of PBS (24-well plate) containing 260 ng / ml of BODIPY 493 / 503 was gently added to the wells. The plates were incubated in a cell culture incubator at 37°C, 5% CO2, and about 95% humidity for 30 minutes. 1 mL of supernatant was gently removed from each well, and 1 mL of PBS was gently added back to each well for imaging.

[0128] For staining with Hoechst, 1 μg / ml Hoechst in PBS was added to the samples together with BODIPY 493 / 503 stain for 30 minutes.

[0129] Imaging

[0130] All 2D and 3D images were acquired using a Leica dmi8 THUNDER microscope. More specifically, a 20x zoom lens with a 0.8 aperture objective was used. To improve image quality and reduce noise, THUNDER's Instant Computational Clearing (ICC) method was applied to each image at the time of acquisition. For all fluorescent stains, the intensities, exposure times, and excitation / emission wavelengths of the Leica filter cubes were as follows:

[0131] Hoechst:

[0132] Strength: 31%

[0133] Exposure time: 100ms

[0134] Excitation wavelength: 390nm

[0135] Emission wavelength: 460nm

[0136] BODIPY 493 / 503:

[0137] Strength: 35%

[0138] Exposure time: 300ms

[0139] Excitation wavelength: 510nm

[0140] Emission wavelength: 535nm

[0141] 2D Imaging

[0142] For all 2D assays, cells were imaged in 96-well glass bottom plates. On 96-well plates, 6 × 6 tile scans were performed. For all tile scans, a Leica AFC laser autofocus was used to ensure that the image was in focus across the wells.

[0143] 3D Imaging

[0144] For the determination of MBX, SV40 and SV40-PPARG cells in comparison growth medium and Stempro medium (see below), images were obtained on 6-well glass bottom plates. All other 3D images were taken in 24-well glass bottom plates. For all suspension experiments on both types of plates, 2 Z-stack images were taken manually at different positions in each well to ensure that the microcarriers were imaged. For each Z-stack, a location with a high density of microcarriers was selected to ensure that similar samples were obtained in different wells and experiments.

[0145] Image analysis

[0146] For all image analyses, images were exported from Leica.lif files into separate .tif files using Python after post-processing on Leica as described below. In addition, all plotting was performed using Python. In all suspension experiments, the normalized BODIPY spot area occupied by each image was selected as the key metric because BODIPY spots are often clustered together. Therefore, the occupied area is considered a reliable measure of BODIPY stain and, therefore, a reliable measure of lipid droplet accumulation. In all analyses, any segmented BODIPY objects with a diameter less than or equal to 2 pixels in size were filtered out.

[0147] 2D image analysis

[0148] First, the tile scans were stitched together into a single image using the Mosaic Merge function of the Leica software. The exported image was loaded into the CellProfiler pipeline. To segment the BODIPY spots in 2D, the RobustBackground segmentation algorithm of CellProfile was used.

[0149] 3D Image Analysis

[0150] Z-stack images were processed using Leica's maximum projection function. This means that for each pixel in each image, the maximum intensity value for each channel across all z planes is represented, resulting in a 2D image. These exported images were also loaded into CellProfiler. To segment the BODIPY stain on microcarriers, the Otsu segmentation algorithm of CellProfiler was found to be optimal. Therefore, all maximum projection images were segmented using the Otsu algorithm. It was found that the CellProfiler implementation of Otsu performed better than the Leica implementation for segmenting Bodipy stain on microcarriers. For data normalization, microcarrier counts were performed manually for each image.

[0151] qPCR

[0152] RNA was isolated from the collected cell samples using the Direct-zol RNA Miniprep Kit and TRI Reagent from Zymo Research according to strict RNase-free practices. TM High-Capacity RNA-to-cDNA TM The kit quantifies the extracted RNA and transcribes it into cDNA. For qPCR, use Applied Biosystems TM PowerUp TM SYBR TM Green Master Mix amplified cDNA. The prepared reaction mixture was loaded into the Quantstudio 7Pro instrument. Amplification conditions were set as follows: UDG activation at 50°C for 2 minutes, double-lock DNA polymerase activation at 95°C for 2 minutes, and 40 cycles of denaturation at 95°C for 15 seconds and annealing / extension at 60°C for 1 minute. The program was run in standard mode according to the manufacturer's instructions. The fold change of light relative to darkness was calculated via 2^-ΔΔCt.

[0153] Fibroblasts constitutively expressing PPARg grown in adipogenic medium differentiate into adipocytes. Cytosolic cells

[0154] This example demonstrates that fibroblasts constitutively expressing the adipogenic master regulator PPARg differentiate into adipocytes in both two-dimensional and suspension cultures when grown in adipogenic medium, but not when grown in adipogenic medium without constitutive PPARg expression.

[0155] In this example, wild-type bovine fibroblasts and fibroblasts that have been transduced with SV40 large T antigen (SV40-LgT) (as described above) are used. SV40-LgT gene expression confers a proliferation advantage and reduces the doubling time of fibroblasts. One day before transduction, 300,000 wild-type (WT) or SV40-expressing fibroblasts were inoculated into a 100 mm culture dish containing DMEM / F12+10% FBS+1% penicillin / streptomycin / amphotericin B growth medium. The next day, the growth medium was replaced, and then a CMV-PPARg lentiviral vector (Vector Builder) at an MOI of 10 and 8 μg / mL of polybrene were added for transduction. The cells were incubated with the lentiviral vector for 48 hours and then selected with 1.5 μg / mL of puromycin in the growth medium. A control plate containing the same number of cells that were not transduced with the viral vector was selected with puromycin at the same time. All cells in the control plate were killed on day 6, indicating that the surviving cells in the transduction plate were transduced with the CMV-PPARg viral vector. The transduced cells were expanded in growth medium containing DMEM / F12+10% FBS+1% penicillin / streptomycin / amphotericin B but without puromycin for experiments.

[0156] To demonstrate the differentiation of fibroblasts into adipocytes in 2D culture, WT cells, WT cells transduced with CMV-PPARg, SV40 cells, and SV40 cells transduced with PPARg were first plated at 3000 cells / cm 2 Seed in growth medium containing DMEM / F12+10%FBS+1% penicillin / streptomycin / amphotericin B for 24 hours to allow cells to attach. The next day, the medium was replaced with StemPro adipogenic differentiation medium. The medium was replaced after 3 days by gently removing the supernatant without disturbing the oil droplets in the cells. The cells were cultured in adipogenic medium for 6 days. Lipid staining was performed by staining with BODIPY 493 / 503 as described above. Imaging and image analysis were performed as described above.

[0157] like Fig.9A and Fig. 9B As shown, PPARg was expressed in WT cells ( Fig.9A , panel F) and cells expressing SV40 ( Fig.9A, Panel H) resulted in enhanced lipid droplet formation (stained with BODIPY 493 / 503) in the presence of StemPro medium, a medium that induces adipogenesis, but not in the presence of growth medium ( Fig.9A , Panels B and D). Adipogenic medium alone did not lead to enhanced lipid droplet formation ( Fig.9A , Panels E and G), demonstrating that PPARg expression in fibroblasts grown in adipogenic medium is essential for lipid droplet accumulation and, thus, differentiation of fibroblasts into adipocyte-like cells in 2-dimensional cell culture. Fig. 9B Depicted Fig.9A Quantification of Fig. 9B As shown, when cells constitutively expressing PPARg were grown in growth medium, lipid droplet accumulation was minimal. Similarly, when cells were grown in adipogenic medium without PPARg expression, there was also minimal lipid droplet accumulation. In contrast, when cells constitutively expressing PPARg were grown in adipogenic medium, there was robust lipid droplet accumulation.

[0158] These results were further confirmed in three-dimensional suspension cultures. Spinner flasks were prepared as described above. Fibroblasts containing SV40 cells with and without constitutive expression of PPARg (as described above) and 3T3-L1 MBX cells were cultured in a growth medium containing DMEM / F12+10%FBS+1% penicillin / streptomycin / amphotericin B. Cells were seeded onto Cytodex 3 microcarriers as described above, and cells were suspended and cultured as described above, except that in some spinner flasks, the growth medium was replaced with StemPro one day after inoculation. Growth medium and StemPro were replaced on the 3rd day, and samples were harvested on the 6th day. Cells / microcarriers were stained according to the above-mentioned BODIPY 493 / 503 stain. 3T3-L1 MBX cells were used as positive controls. 3T3-L1 MBX cells are fibroblast lines that are derived to ensure that close to 100% differentiation into adipocyte-like cells is achieved when grown in adipogenic medium.

[0159] like Fig.10 As shown, 3T3-L1 MBX cells as well as SV40-CMV PPARg cells showed lipid droplet accumulation when grown in adipogenic medium ( Fig.10 , panels D and F, respectively), but not when grown in growth medium ( Fig.10 , panels A and C, respectively). When grown in growth medium or adipogenic medium, non-transduced cells did not express adipocyte-like lipid droplets ( Fig.10, panels B and E, respectively), demonstrating that the expression of PPARg plus adipogenic medium is necessary for the adipogenic differentiation of fibroblasts. This example demonstrates that fibroblasts can be differentiated into adipogenicity in suspension culture.

[0160] Validation of light-activatable recombinase constructs

[0161] In this example, a plasmid encoding a light-activatable recombinase was tested and validated for subsequent experiments. One day before transfection, SV40 cells were seeded at a density of 6000 cells / well in growth medium in 96-well glass-like polymer bottom plates. On the day of transfection, Lipofectamine TM 3000 transfection reagent (ThermoFisher), plasmid 709 (light-activatable Cre recombinase) or plasmid 361 (wild-type Cre recombinase) and Cre recombinase reporter plasmid (pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, lifescienceMarket, catalog number PVT11052) were co-transfected. Each well contained 30 ng Cre plasmid, 30 ng Cre reporter plasmid, 0.12 μL p3000 and 0.2 μL Lipofectamine TM 3000. The Cre recombinase reporter plasmid expresses the fluorescent protein dsRed until Cre recombinase activity excises the coding sequence of dsRed located between the two loxP sites and initiates the expression of the fluorescent protein eGFP. Treat cells under red or green light, because room light activates the recombinase and permanently changes the DNA. Replace the medium in each well 24 hours after transfection, and then start irradiation in the selected wells (2μW / mm 2 or 8μW / mm 2 The cells were harvested 48 hours after the start of irradiation for flow cytometry analysis (Attune CytPix, Thermo Fisher Scientific). The recombination efficiency was calculated as follows: % recombination = % of cells expressing EGFP / % of EGFP and % of cells expressing mCherry, and Fig.11A or calculate the multiple of recombination change in light relative to darkness and make a graph, such as Fig. 11B shown.

[0162] like Fig.11A and Fig. 11BAs shown, when Cre recombinase is absent, there is minimal leaky recombination. Constitutive expression of Cre (CMV-Cre) results in approximately 100% recombination efficiency, validating the reporter plasmid. The light-activatable recombinase (plasmid 709) was validated and showed a light power-dependent increase in recombination for both light intensities tested, with peak recombination at 8 μW / mm 2 .

[0163] Using constitutively expressed recombinases to excise the blocking sequence and drive the expression of adipogenic transcription factors Fibroblasts differentiate into adipocytes.

[0164] In this example, fibroblasts were differentiated into adipocytes in two-dimensional cell culture using constitutive Cre recombinase.This example validates a plasmid for differentiating fibroblasts into adipocytes.

[0165] One day before transfection, 6000 cells / well of cells stably expressing plasmid 703 or plasmid 704 (see Table 3) were seeded into 96-well glass polymer bottom plates in growth medium (containing DMEM / F12 high glucose, 10% FBS, 1% penicillin / streptomycin / amphotericin B). On the day of transfection, lipofectamine was used according to the manufacturer's instructions. TM Transfection of CMV-Cre plasmid (plasmid 361) was performed using P3000 transfection reagent (ThermoFisher). Untransfected controls were exposed to transfection reagent alone. Each transfection well contained 60 ng CMV-Cre, 0.12 μL P3000, and 0.2 μL Lipofectamine TM 3000. At 48 hours post-transfection, the medium in some wells was replaced with StemPro Adipogenic Differentiation Medium for 6 days. Growth medium and adipogenic medium were replaced after 3 days. BODIPY 493 / 503 staining of lipid droplets was performed on day 6 according to the procedure outlined above.

[0166] like Fig.12 As shown, in the corresponding StemPro or growth medium experiments, when plasmids 703 and 361 ( Fig.12 , panels B and F) or with plasmids 704 and 361 ( Fig.12 , panels D and H) were significantly higher in cells transfected with plasmid 703 ( Fig.12 , panels A and E) or 704( Fig.12 , panels C and G) in the transfected cells. This indicates that the constitutively expressed Cre recombinase can remove the blocking sequences of plasmids 703 and 704, so that the expression of transcription factors (PPARg of plasmid 703; or PPARg and CEBPa of plasmid 704) is activated and differentiation into adipocyte-like cells occurs. Fig.12 , panels F and H, respectively) compared to growth medium ( Fig.12 , panels B and D), expression of PPARg alone and PPARg+CEBPa resulted in substantially more lipid droplet accumulation, once again emphasizing that transcription factors and adipogenic media are required for fibroblast differentiation into adipocytes. Fig.13A and Fig. 13B Describes Fig.12 Quantification of the results shown.

[0167] The fibroblasts were transformed into adipogenic cells using a light-activatable recombinase to excise the blocking sequence and drive the expression of adipogenic transcription factors. Differentiate cells into adipocytes.

[0168] In this example, light-activatable Cre recombinase was used to differentiate fibroblasts into adipocytes in both two-dimensional cell culture and three-dimensional suspension culture.

[0169] In this experiment, fibroblasts (SV40 cells) expressing SV40-LgT were stably transfected with plasmid 704 (a construct that achieves PPARg+CEBPa expression upon recombination) using the PiggyBac system. Cells were selected with antibiotics so that the entire population expressed plasmid 704. These cells were then stably transfected with plasmid 709 (a construct that drives constitutive expression of a light-activatable recombinase) using the Sleeping Beauty system. Cells expressing plasmids 704 and 709 were sorted into populations with high expression of plasmid 709 and low expression of plasmid 709. Cells were treated under red or green light because room light activates the recombinase and permanently changes the DNA. The cells were then cultured in 2D and exposed to dark conditions; 2 μW / mm 2 Blue light (465nm) with 20 seconds on, 60 seconds off pulse mode; or 8μW / mm 2 Blue light (465 nm) with a pulse pattern of 20 seconds on, 60 seconds off. The light was turned off after 48 hours of irradiation and the medium was changed to adipogenic medium for 6 days. The cells were stained with BODIPY 493 / 503. Fig.14 As shown, increasing light power induced cells with high expression of light-activatable recombinase ( Fig.14 , panels A, B, and C) and cells with low expression of the light-activatable recombinase ( Fig.14 , Panels D, E, and F) showed increased lipid droplet accumulation. This data suggests that light induces fibroblast differentiation into adipocytes in a power-dependent manner. Fig.15A and Fig. 15B Describes Fig.14 Quantification of the results shown.

[0170] These results were further confirmed in three-dimensional suspension cultures. In this experiment, fibroblasts (SV40 cells) expressing SV40 LgT were stably transfected with plasmid 704 (a construct that achieves PPARg+CEBPa expression upon recombination) using the PiggyBac system. Cells were selected with antibiotics so that the entire population expressed plasmid 704. These cells were then stably transfected with plasmid 709 (a construct that drives constitutive expression of a light-activatable recombinase) using the Sleeping Beauty system. Cells expressing plasmids 704 and 709 were sorted into populations with high expression of plasmid 709 and low expression of plasmid 709. Cells were treated under red or green light because indoor light activates the recombinase and permanently changes the DNA. Cells were attached to Cytodex 3 microcarriers in 100 mL of growth medium and cultured in spinner flasks. During this period, the cells were kept in the dark or irradiated with 8 μW / mm 2 Blue light (465 nm) was irradiated for 48 hours in a pulsed mode of 20 seconds on and 60 seconds off. After 48 hours, the irradiation was turned off and the medium was replaced with 100 ml of StemPro adipogenic medium. The medium was replaced after 3 days. The cells + microcarriers were then stained with Hoechst (to stain the nuclei) and BODIPY493 / 503 (to stain lipid droplets). Fig.16 As shown, irradiation with blue light induced cells with high expression of the light-activatable recombinase ( Fig.16 , panel H) and cells with low expression of the light-activatable recombinase ( Fig.16 , panel F) increased lipid droplet accumulation. This data suggests that light can be used to induce fibroblast differentiation into adipocytes in suspension culture. Images are z-projections, and representative images are shown in Fig.16 The area covered by BODIPY 493 / 503 stain (normalized to the number of microcarriers in the image) was quantified and displayed in Fig.17 middle.

[0171] As a control, SV40 cells (not expressing light-activatable recombinase or adipogenic transcription factors) were attached to Cytodex 3 microcarriers in 100 mL of growth medium and cultured in spinner flasks. During this period, the cells were stimulated with 8 μW / mm 2 Blue light (465 nm) was irradiated for 48 hours in a pulsed mode of 20 seconds on and 60 seconds off. After 48 hours, the irradiation was turned off and the medium was replaced with 100 ml of StemPro adipogenic medium. The medium was replaced after 3 days. The cells + microcarriers were then stained with Hoechst (to stain nuclei) and BODIPY 493 / 503 (to stain lipid droplets). Images are z-projections. The area covered by the BODIPY 493 / 503 stain (normalized to the number of microcarriers in the image) was quantified and plotted on Fig.18 This data demonstrates that irradiation of SV40 cells with blue light alone does not increase lipid droplet accumulation in the absence of expression of light-activatable recombinase and adipogenic transcription factors, and that expression of light-activatable recombinase (at appropriate expression levels) and constructs encoding adipogenic transcription factors are required.

[0172] To demonstrate that genes related to adipogenesis are expressed in cells differentiated into adipocyte-like cells using light induction, Fig.16 Samples were obtained from the cells depicted in panels B and F. RNA was extracted and cDNA was generated. qPCR was run to interrogate the expression of two key adipogenic genes, FABP4 and LPL. FABP4 encodes a fatty acid binding protein found in adipocytes, and LPL encodes lipoprotein lipase expressed in adipose tissue. Fig.19A and Fig.19B showed that adipocyte-related genes were upregulated in light compared with darkness, reflecting the Fig.16 Panels B and F show increased lipid accumulation in response to light. Fig.19A and Fig.19B Shown are fold changes in expression in light relative to darkness.

[0173] In summary, these experiments demonstrate that 8μW / mm 2 Blue light activated the photoactivatable recombinase that excised the blocking sequence, resulting in PPARg / CEBPa expression and inducing lipid accumulation, demonstrating that light causes fibroblast differentiation toward an adipocyte phenotype. They also demonstrated that the level of photoactivatable recombinase expression is important for controlling differentiation. Compared to cells with low photoactivatable recombinase expression in the dark, cells that highly expressed the photoactivatable recombinase showed greater leaky photoactivatable recombinase activation in the dark, resulting in greater PPARg / CEBPa expression and lipid droplet accumulation in the dark. This may be because when the expression of the photoactivatable recombinase is too high, the probability that the two halves of the photoactivatable recombinase will come together increases to the point where the mechanism is less dependent on light. Therefore, having an appropriate level of expression of the photoactivatable recombinase is important for achieving control of the differentiation of fibroblasts into adipocyte-like cells in suspension using light.

[0174] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present disclosure, those skilled in the art will now conceive of many variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used to implement the present disclosure. The following claims are intended to define the scope of the present disclosure, and thus encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for differentiating at least one cell of a cell population into a desired cell lineage, the method comprising: (a) providing or obtaining said cell population in suspension culture; and (b) controlling differentiation of the at least one cell of the cell population using light, thereby causing the at least one cell to differentiate into a desired cell lineage.

2. The method of claim 1, wherein the controlling differentiation of (b) comprises irradiating the at least one cell with light of a first wavelength.

3. The method of claim 1, wherein the controlling differentiation of (b) comprises removing light of the first wavelength from the at least one cell.

4. The method of any one of claims 1-3, wherein the at least one cell is genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one transcription factor, at least one differentiation factor, or both that effects differentiation into the desired cell lineage.

5. The method of claim 4, wherein expression of the at least one transcription factor, the at least one differentiation factor, or both is induced by the irradiating or the removing of light.

6. The method of claim 5, wherein the exogenous nucleic acid comprises at least one promoter operably linked to the at least one transcription factor, the at least one differentiation factor, or both.

7. The method of claim 6, wherein the at least one cell is genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one light-switchable transcriptional regulator.

8. The method of claim 7, wherein the promoter is an inducible promoter.

9. The method of claim 8, wherein the at least one light-switchable transcriptional regulator is a light-switchable transcriptional activator.

10. The method of claim 9, wherein the light-switchable transcriptional activator comprises a transcriptional activator fused to a light-switchable domain.

11. The method of claim 10, wherein the irradiation induces the light-switchable transcriptional activator to bind to and activate the inducible promoter, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

12. The method of claim 10, wherein the removal induces the light-switchable transcriptional activator to bind to and activate the inducible promoter, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

13. The method of claim 7, wherein the promoter is a constitutive promoter.

14. The method of claim 13, wherein the at least one light-switchable transcriptional regulator is a light-switchable transcriptional repressor.

15. The method of claim 14, wherein the light-switchable transcriptional repressor comprises a transcriptional repressor fused to a light-switchable domain.

16. The method of claim 15, wherein the irradiation induces dissociation of the light-controllable transcriptional repressor from the constitutive promoter, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

17. The method of claim 15, wherein the removal of light induces dissociation of the light-controllable transcriptional repressor from the constitutive promoter, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

18. The method of claim 13, wherein the exogenous nucleic acid further comprises a blocking sequence downstream of the at least one promoter, which, when present, blocks expression of the at least one first transcription factor, the at least one differentiation factor, or both.

19. The method of claim 18, wherein the at least one cell further comprises a nucleic acid sequence encoding at least one light-switchable recombinase.

20. The method of claim 19, wherein the at least one light-switchable recombinase is a light-activatable recombinase.

21. The method of claim 20, wherein the light-activatable recombinase comprises a recombinase or a portion thereof fused to a light-activatable domain.

22. The method of any one of claims 18-21, wherein the blocking sequence is flanked by recombinase recognition sites recognized by the at least one light-switchable recombinase.

23. The method of claim 22, wherein the irradiation activates the light-controllable recombinase, thereby resulting in excision of the blocking sequence and inducing expression of the at least one transcription factor, the at least one differentiation factor, or both.

24. The method of any one of the preceding claims, wherein the at least one cell is a stem cell.

25. The method of claim 24, wherein the stem cell is a pluripotent stem cell or a multipotent stem cell.

26. The method of any one of claims 1-23, wherein the at least one cell is a fibroblast.

27. The method of any one of the preceding claims, wherein the at least one cell is a human cell, a bovine cell, or a mouse cell.

28. The method of any one of the preceding claims, wherein the desired cell lineage is selected from the group consisting of: adipocytes, myocytes, and chondrocytes.

29. The method according to any one of the preceding claims, wherein the at least one transcription factor is selected from the group consisting of: PPARγ, CEBPα, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, and any combination thereof.

30. The method of any of the preceding claims, wherein the irradiating further comprises irradiating a plurality of cells of the cell population with light to differentiate each of the plurality of cells into the desired cell lineage; or the removing further comprises removing light from the cell population to differentiate each of the plurality of cells into the desired cell lineage.

31. The method of any one of the preceding claims, wherein the suspension culture has a volume of at least 100 milliliters (mL).

32. The method of any one of the preceding claims, wherein the suspension culture is contained in a bioreactor vessel.

33. The method of claim 32, wherein the bioreactor vessel has a total volume of at least 100 milliliters (mL).

34. A method according to any one of the preceding claims, wherein the cell population is grown on the surface of microcarriers.

35. The method of claim 34, wherein the microcarriers are coated with one or more extracellular matrix components.

36. A system for differentiating a cell population, the system comprising: (a) a population of cells in suspension culture, wherein at least one cell of the population of cells is engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one transcription factor, at least one differentiation factor, or both that effects differentiation into a desired cell lineage, wherein expression of the at least one transcription factor, the at least one differentiation factor, or both is controlled by light; and (b) one or more light sources configured to illuminate at least one cell of the population of cells with light of a first wavelength.

37. The system of claim 36, wherein the exogenous nucleic acid comprises at least one promoter operably linked to the at least one transcription factor, the at least one differentiation factor, or both.

38. The system of claim 37, wherein the at least one cell is genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one light-switchable transcriptional regulator.

39. The system of claim 38, wherein the promoter is an inducible promoter.

40. The system of claim 39, wherein the at least one light-switchable transcriptional regulator is a light-switchable transcriptional activator.

41. The system of claim 40, wherein the light-switchable transcriptional activator comprises a transcriptional activator fused to a light-switchable domain.

42. The system of claim 41, wherein the light-controllable transcriptional activator binds to and activates the inducible promoter upon irradiation with light of the first wavelength, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

43. The system of claim 41, wherein the light-controllable transcriptional activator binds to and activates the inducible promoter upon removal of the first wavelength of light, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

44. The system of claim 38, wherein the promoter is a constitutive promoter.

45. The system of claim 44, wherein the at least one light-switchable transcriptional regulator is a light-switchable transcriptional repressor.

46. ​​The system of claim 45, wherein the light-switchable transcriptional repressor comprises a transcriptional repressor fused to a light-switchable domain.

47. The system of claim 46, wherein the light-controllable transcriptional repressor dissociates from the constitutive promoter upon illumination by light of the first wavelength, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

48. The system of claim 46, wherein the light-controllable transcriptional repressor dissociates from the constitutive promoter upon removal of the first wavelength of light, thereby causing expression of the at least one transcription factor, the at least one differentiation factor, or both.

49. The system of claim 37, wherein the exogenous nucleic acid further comprises a blocking sequence downstream of the at least one promoter, which, when present, blocks expression of the at least one transcription factor, the at least one differentiation factor, or both.

50. The system of claim 49, wherein the at least one cell of the cell population further comprises a nucleic acid sequence encoding at least one light-switchable recombinase.

51. The system of claim 50, wherein the at least one light-controllable recombinase is a light-activatable recombinase.

52. The system of claim 51, wherein the light-activatable recombinase comprises a recombinase fused to a light-activatable domain.

53. The system of any one of claims 49-52, wherein the blocking sequence is flanked by recombinase recognition sites recognized by the at least one light-switchable recombinase.

54. The system of claim 53, wherein the blocking sequence is configured to be excised by the at least one light-controllable recombinase, thereby inducing expression of the at least one transcription factor.

55. The system of any one of claims 36-54, wherein the at least one cell is a stem cell.

56. The system of any one of claims 36-55, wherein the stem cell is a pluripotent stem cell or a multipotent stem cell.

57. The system of any one of claims 36-54, wherein the at least one cell is a fibroblast.

58. The system of any one of claims 36-57, wherein the at least one cell is a human cell, a bovine cell, or a mouse cell.

59. The system of any one of claims 36-58, wherein the desired cell lineage is selected from the group consisting of: adipocytes, myocytes, and chondrocytes.

60. The system of any one of claims 36-59, wherein the at least one transcription factor is selected from the group consisting of: PPARγ, CEBPα, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, and any combination thereof.

61. The system of any one of claims 36-60, further comprising a plurality of microcarriers, wherein the cell population is grown on the surface of the plurality of microcarriers.

62. The system of claim 61, wherein the plurality of microcarriers are coated with one or more extracellular matrix components.

63. The system of any one of claims 36-62, wherein the one or more light sources comprise one or more light emitting diodes (LEDs).

64. The system of any one of claims 36-63, wherein the one or more light sources comprise one or more lasers.

65. The system of any one of claims 36-64, wherein the one or more light sources comprise an incandescent light source.

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