Compositions and methods for induced stem cell differentiation to astrocytes

Abstract

A method of differentiating nonhuman primate induced pluripotent stem cells (iPSCs) into astrocytes may include providing a modified vector comprising a piggyBac vector backbone configured to express SOX9 and NFIB. The piggyBac vector backbone may include terminal inverted repeats and transposase recognition sites that support stable genomic integration of the inserted differentiation factor genes. Introduction of the vector into nonhuman primate iPSCs, such as Macaca fascicularis iPSCs, may initiate transcriptional programs associated with astrocyte-lineage commitment and maturation. Expression of SOX9 and NFIB may facilitate the formation of astrocytic morphology and the development of molecular features associated with astrocytes, including process elaboration and lineage-specific marker expression. The method may further include culturing the transfected cells under conditions that support astrocytic differentiation and expansion, resulting in populations of astrocytes or induced astrocytes suitable for research, disease modeling, and / or therapeutic applications.

Description

Docket No. 0138-705.600INTERNATIONAL APPLICATIONCOMPOSITIONS AND METHODS FOR INDUCED STEM CELL DIFFERENTIATION TO ASTROCYTESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 722,168, filed November 19, 2024, which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 0138- 705.600_Sequence_Listing.xml, created November 17, 2025, which is 30,000 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0003] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.TECHNICAL FIELD

[0004] This disclosure relates generally to the field of cellular differentiation, and more specifically to the field of lentiviral transduction for cellular differentiation. Described herein are compositions and methods for induced stem cell differentiation.BACKGROUND

[0005] Induced pluripotent stem cells (iPSCs) have the potential to be differentiated into different cell types including neuronal cells. This process has been successfully conducted using human iPSCs. Induced pluripotent stem cells, or iPSCs, are used throughout contemporary biomedical research as a source of cells capable of developing into many different cell types. In conventional laboratory practice, the process of differentiation from iPSCs to a target lineage is carried out by culturing the cells in defined environments that mimic developmental conditions. This approach relies on sequential exposure to combinations ofDocket No. 0138-705.600INTERNATIONAL APPLICATION growth media, nutrients, and environmental factors that guide undifferentiated cells toward particular functional identities.

[0006] For neuronal differentiation in particular, iPSCs are maintained under conditions that encourage transformation into cells resembling early neural tissue. Overtime, these cells adopt characteristic shapes and molecular profiles associated with the developing nervous system. Culture media and physical conditions are adjusted at each stage to support cell survival, organization, and gradual maturation. Once differentiation is achieved, the resulting cells exhibit features of neural identity and are maintained in vitro for further study. They are used as model systems to observe cellular behavior, communication, and responses to external stimuli. Through established laboratory protocols, researchers can generate populations of cells that resemble neurons or related support cells, enabling investigation of neural development and function in a controlled setting.SUMMARY

[0007] In some aspects, the techniques described herein relate to a vector for differentiating nonhuman primate iPSCs from Macaca fascicularis into astrocytes, wherein the vector includes a nucleic acid sequence including a sequence encoding: a PiggyBac vector backbone including: one or more terminal inverted repeats; and one or more transposase recognition sites for genomic integration of an inserted sequence; and a differentiation factor gene integrated within the piggyBac vector backbone, the differentiation factor gene configured to induce astrocyte differentiation through an activation of one or more astrocyte-lineage regulatory pathways and an expression of one or more molecular features associated with astrocytic maturation.

[0008] In some aspects, the techniques described herein relate to Macaca fascicularis NFIB.

[0009] In some aspects, the techniques described herein relate to a vector for differentiating nonhuman primate iPSCs to one or more neuronal lineage cells, wherein the vector includes a nucleic acid sequence including a sequence encoding: a PiggyBac vector backbone; and a differentiation factor gene configured to promote differentiation of the one or more neuronal lineage cells.

[0010] In some aspects, the techniques described herein relate to Macaca fascicularis NFIB.Docket No. 0138-705.600 INTERNATIONAL APPLICATION

[0011] In some aspects, the techniques described herein relate to a vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis to one or more neuronal lineage cells, the vector including a nucleic acid sequence including: a piggyBac vector backbone including: one or more terminal inverted repeats; and one or more transposase recognition sites for genomic integration of an inserted sequence; and a bidirectional Tet-responsive promoter positioned between a first differentiation factor gene and a second differentiation factor gene, the first differentiation factor gene and the second differentiation factor gene are configured to be inserted into the piggyBac vector backbone for the nonhuman primate iPSCs to the one or more neuronal lineage cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology are described below in connection with various embodiments, with reference made to the accompanying drawings.

[0013] FIG. 1 shows a schematic of a PiggyBac vector backbone, according to some embodiments.

[0014] FIG. 2 is a flow chart of an embodiment of a method for transfecting nonhuman primate (NHP) iPSCs, according to some embodiments.

[0015] FIG. 3 shows a schematic of a PiggyBac vector backbone modified to express differentiation factor genes Macaca fascicularis SOX9 (SEQ ID NO. 2) and Macaca fascicularis NFIB (SEQ ID NO. 3), according to some embodiments.

[0016] FIG. 4 is a flow chart of an embodiment of a method for differentiating NHP iPSCs to a neuronal lineage, according to some embodiments.

[0017] FIG. 5 is a flow chart showing an embodiment of a method for reprogramming nonhuman primate peripheral blood mononuclear cells (PBMCs) into iPSCs, according to some embodiments.

[0018] FIG. 6 is a schematic of an embodiment of a method for reprogramming nonhuman primate PBMCs into iPSCs, according to some embodiments.

[0019] FIG. 7 is a block diagram illustrating a vector, according to some embodiments.

[0020] FIG. 8 is a block diagram further illustrating the vector from FIG. 7, according to some embodiments.

[0021] FIG. 9 is a block diagram illustrating a vector, according to some embodiments.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0022] FIGs. 10A-10C show immunofluorescence images showing differentiated astrocyte cells at day in vitro 7 (DIV7) in FIG. 10A, differentiated astrocyte cells at day in vitro 14 (DIV14) in FIG. 10B, and differentiated astrocyte cells at day in vitro 21 (DIV21) in FIG. 10C, each stained for DAPI to visualize nuclei and TUJ1 to indicate early neuronal lineage- associated morphology during astrocytic differentiation, according to some embodiments.

[0023] FIG. 11 illustrates a phase-contrast micrograph showing differentiated astrocyte cells at day in vitro 14-21 (DIV14-DIV21) showing the cellular morphology and network organization of the cultured cells under live-cell imaging conditions, according to some embodiments.

[0024] FIG. 12 illustrates a bar graph showing the percentage of TUJl-positive cells at day in vitro 7 (DIV7), day in vitro 14 (DIV14), and day in vitro 21 (DIV21) during astrocyte differentiation, according to some embodiments.

[0025] FIG. 13 illustrates a bar graph showing total neurite length per well at day in vitro 7 (DIV7), day in vitro 14 (DIV14), and day in vitro 21 (DIV21) during differentiation of Macaca fascicularis induced pluripotent stem cells (iPSCs) into astrocyte cells, according to some embodiments.

[0026] FIG. 14 illustrates a bar graph showing total cell count per well at day in vitro 7 (DIV7), day in vitro 14 (DIV14), and day in vitro 21 (DIV21) during differentiation of Macaca fascicularis induced pluripotent stem cells (iPSCs) into astrocyte cells, according to some embodiments.

[0027] FIG. 15A illustrates a phase-contrast micrograph showing cocultured NGN2-induced neurons (NGN2 cells) at day in vitro 7 (NGN2 DIV7) together with astrocyte cells at day in vitro 14 (Astro DIV14), according to some embodiments.

[0028] FIG. 15B illustrates a phase-contrast micrograph showing cocultured NGN2-induced neurons at day in vitro 14 (NGN2 DIV14) together with astrocyte cells at day in vitro 21 (Astro DIV21), according to some embodiments.

[0029] FIG. 16A illustrates a phase-contrast micrograph showing a coculture of astrocyte cells and NGN2-induced neurons, in which the NGN2 cells are at day in vitro 7 (NGN2 DIV7) and the astrocyte cells are at day in vitro 14 (Astro DIV14), according to some embodiments.

[0030] FIG. 16B illustrates a phase-contrast micrograph showing a coculture of astrocyte cells and NGN2-induced neurons, in which the NGN2 cells are at day in vitro 14 (NGN2 DIV14) and the astrocyte cells are at day in vitro 21 (Astro DIV21), according to some embodiments.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0031] FIG. 17A illustrates a phase-contrast micrograph of a coculture containing NGN2- induced neurons at day in vitro 7 (NGN2 DIV7) and astrocyte cells at day in vitro 14 (Astro DIV14).

[0032] FIG. 17B illustrates a phase-contrast micrograph of a coculture containing NGN2- induced neurons at day in vitro 14 (NGN2 DIV14) and astrocyte cells at day in vitro 21 (Astro DIV21).

[0033] The illustrated embodiments are merely examples and are not intended to limit the disclosure. The schematics are drawn to illustrate features and concepts and are not necessarily drawn to scale.DETAILED DESCRIPTION

[0034] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology will now be described in connection with various embodiments. The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the claimed subject matter. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0035] Conventionally, stems cells from humans have been differentiated using lentivirus- based systems. However, lentivirus-based systems are not efficient when differentiating NHP stem cells. Species-specific differences may prevent the use of human iPSC methods for nonhuman primate iPSCs. Using lentivirus-based systems, NHP have a low transduction efficiency, for example about 1% to about 5%. Further, using lentiviruses are undesirable since the genetic material is incorporated into the genome, which could result in mutations that may be imperceptible to the user / researcher but nonetheless impactful on the results from the experiments. Others have attempted to use Sendai virus, but these have similar drawbacks as lentiviruses. For example, Sendai virus in nonhuman primate cells is not as efficient as its usage in human cells.

[0036] The above technical problems presented by using lentivirus-based systems and / or Sendai viruses may be solved by technical solutions. For examples, the systems and methods described herein may solve the above technical problems with technical solutions that includeDocket No. 0138-705.600INTERNATIONAL APPLICATION using piggyBac vectors for transfecting NHP iPSCs. Using piggyBac vectors provide several technical advantages when transfecting NHP iPSCs. For example, Piggybac vectors can be removed from the genome without leaving a footprint, and piggyBac vectors have high transposition activity in at least particular cell types. As shown and described elsewhere herein, a piggyBac vector has been modified herein to express particular differentiation factor genes to enable differentiation of NHP iPSCs into pre-defined neuronal lineages. For example, one or more differentiation factors may be used to differentiate NHP iPSCs into NGN2 cells (iN cells), astroglia, astrocytes, induced astrocytes (iA cells), and / or oligodendrocytes.

[0037] As used herein, the term “astrocyte” refers to a differentiated glial cell of the central nervous system that contributes to neuronal support, homeostatic regulation, and maintenance of the blood-brain barrier. Astrocytes exhibit characteristic stellate morphology and express lineage-specific markers including, but not limited to, glial fibrillary acidic protein (GFAP), SI 00 calcium-binding protein P (S100P), and aquaporin-4 (AQP4). Astrocytes regulate neurotransmitter uptake, modulate ion balance, participate in synaptic signaling, and / or provide metabolic and trophic support to neurons.

[0038] The term “induced astrocyte” (iA cell) refers to an astrocyte that has been derived in vitro from pluripotent stem cells, including induced pluripotent stem cells (iPSCs), or from reprogrammed somatic cells through the introduction or expression of one or more transcription factors associated with astrocytic differentiation. Induced astrocytes exhibit the molecular, structural, and functional characteristics of mature astrocytes, including, but not limited to, expression of GFAP, S100P, and AQP4, while demonstrating functional responses such as calcium signaling, glutamate uptake, and / or cytokine release.

[0039] The term “astroglia” refers broadly to the astrocytic lineage, encompassing astrocyte progenitors, immature astrocytes, and mature astrocytes. Astroglia may include both proliferative precursors and differentiated forms present during various stages of development. In contrast, iA cells as described herein are terminally differentiated and correspond to mature astrocytes rather than progenitor forms.

[0040] As used herein, the term “neuronal lineage cell” refers to any cell type that arises from the ectodermal lineage during neural differentiation. Neuronal lineage cells include, but are not limited to, neurons, astrocytes, induced astrocytes (iA cells), oligodendrocytes, and / or induced neurons (iN cells, such as NGN2 cells).

[0041] The term “induced neuron” (iN cell) refers to a neuron generated in vitro from pluripotent or somatic cells through directed differentiation or reprogramming. InducedDocket No. 0138-705.600INTERNATIONAL APPLICATION neurons include, but are not limited to, NGN2-induced neurons (NGN2 cells) that express neuronal markers such as TUJ1, MAP2, and / or synapsin-1 and exhibit functional properties including, but not limited to, spontaneous electrical activity and synaptic formation.

[0042] The term “oligodendrocyte” refers to a myelinating glial cell type that arises from the neuronal lineage and functions to insulate axons with myelin sheaths. Oligodendrocytes express characteristic markers including, but not limited to, OLIG2, MBP, and / or PLP1 and are responsible for maintaining efficient action potential conduction in the central nervous system.

[0043] As used herein, the term “differentiation factor gene” refers to a gene whose expression contributes to the transition of pluripotent or multipotent cells into a defined lineage. Differentiation factor genes may include, but are not limited to, transcription factors, signaling molecules, or regulatory elements that direct cell fate determination. In certain embodiments, the differentiation factor genes comprise Macaca fascicularis SOX9 and Macaca fascicularis NFIB, which together activate transcriptional networks that initiate and stabilize astrocytic differentiation.

[0044] The term “PiggyBac vector backbone” refers to a transposable DNA element comprising terminal inverted repeats and internal transposase recognition sites that allow transposase-mediated genomic insertion and excision of an inserted genetic cargo. The PiggyBac system integrates transgenes at TTAA target sites within the host genome and is capable of footprint-free excision upon reintroduction of transposase. The PiggyBac backbone may further include, but is not limited to, additional sequences such as a selectable marker, a constitutive or inducible promoter, and / or one or more origins of replication.

[0045] The term “bidirectional Tet-responsive promoter” refers to a regulatory DNA sequence that drives transcription of two genes in opposite orientations under the control of a tetracycline-responsive transactivator. In the presence of an inducer such as doxycycline, the promoter activates coordinated expression of both genes positioned on either side of the promoter sequence.

[0046] The term “nonhuman primate induced pluripotent stem cell” (NHP iPSC) refers to a stem cell derived from a nonhuman primate, including, but not limited to, Macaca fascicularis, that possesses the ability to self-renew and to differentiate into derivatives of all three germ layers: ectoderm, mesoderm, and / or endoderm. These cells are reprogrammed from somatic cells, including, but not limited to, peripheral blood mononuclear cells, through the introduction of reprogramming factors.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0047] As used herein, the term “TTAA site” refers to a four-nucleotide genomic target sequence recognized by the PiggyBac transposase for integration and excision events. Integration occurs precisely at the TTAA motif, and excision restores the original TTAA sequence without residual insertions or deletions.

[0048] The term “transposase recognition site” refers to a specific nucleotide sequence within the PiggyBac vector backbone that interacts with the PiggyBac transposase enzyme to mediate excision and integration of the transposon. These regions may range in length from about 200 base pairs to about 500 base pairs, for example about 250 base pairs to about 450 base pairs, or about 275 base pairs to about 350 base pairs.

[0049] The term “terminal inverted repeat” refers to a short DNA sequence located at both ends of the PiggyBac transposon that is identical in sequence but oriented in reverse complement fashion. These inverted repeats serve as binding sites for the PiggyBac transposase, allowing recognition, cleavage, and reinsertion of the transposon. Each terminal inverted repeat may range in length from about 10 base pairs to about 25 base pairs, for example about 12 base pairs to about 22 base pairs, or about 15 base pairs to about 18 base pairs.

[0050] As used herein, the terms “comprising,” “including,” and “having” are used in an open-ended sense and do not exclude additional elements or steps not recited. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, “nonhuman primates” may be used to refer to primate species that are used in genetic research or more generally primates that are not humans. Exemplary species, to which the methods and systems described herein may apply, include, but are not limited to, rhesus macaques (Macaca mulatto), cynomolgus macaques (Macaco fascicular is), baboons (Papio sppi), common marmosets (Callithrix jacchus), squirrel monkeys (Saimiri spp.), African green monkeys (Chlor ocebus sabaeus), pigtailed macaques (Macaca nemestrina), and owl monkeys (Aotus lemurinus). In some embodiments, the systems and methods described herein may be applied to cells from cynomolgus macaques or Macaca fascicularis.

[0052] As used herein, the term “DAPI” refers to 4',6-diamidino-2-phenylindole, a fluorescent DNA-binding dye commonly used in cell and tissue imaging. DAPI intercalates into the minor groove of double-stranded DNA with a preference for adenine-thymine-rich regions and emits a blue fluorescence when excited by ultraviolet light. DAPI staining is used to visualize and quantify cell nuclei, assess nuclear morphology, and identify total cell number in fixed or live samples.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0053] As used herein, the term “TUJ1” refers to an antibody that recognizes class III P- tubulin, a cytoskeletal protein selectively enriched in neuronal cells. TUJ1 immunostaining is widely used to identify neuronal lineage cells, quantify neuronal purity, and visualize neurite structures including axons and dendrites. TUJ1 labeling highlights filamentous microtubule networks within the cytoplasm and neurites, enabling assessment of neuronal morphology, process extension, and network formation during differentiation.

[0054] NHP iPSCs from NHP PBMCs

[0055] NHP iPSCs may be derived from NHP PBMCs. For example, a method of deriving iPSCs from NHP PBMCs may include obtaining tissue or blood from a first nonhuman primate; isolating PBMCs from the first nonhuman primate; and deriving iPSCs from the PBMCs from the first nonhuman primate.

[0056] FIG. 5 shows an embodiment of deriving iPSCs from nonhuman primate PBMCs. The method 500 includes: obtaining peripheral blood mononuclear cells (PBMCs) from at least one individual of a population comprising a single species of nonhuman primate S510; culturing the PBMCs to expand blood progenitor cells (e.g., including CD34+ cells) in a hematopoietic stem cell (HSC) expansion medium for a pre-determined time period S520; transfecting the cultured PBMCs to reprogram the cultured cells into iPSCs S530; transferring the transfected cells into a container comprising a plurality of feeder cells S540; transferring the cells to a second container S550; on selected days after transfer to the second container, performing at least one of: adding medium to the cells; changing the medium in the cells; or adding one or more cell growth factors to the cells S560; and when a first cell colony appears; passaging the cells by placing each colony in a third container coated with an extracellular matrix S570. As used herein, medium may include, but is not limited to, DMEM medium, RPMI ™ medium, F12™ medium, and the like. In some embodiments, specialty media are detailed below or elsewhere herein.

[0057] The method 500 may further include expanding the first colony for use in one or more of: an in vitro test, an in vivo test, or creation of a genome library for the individual primate.

[0058] In some embodiments, transfecting includes transfecting with a combination of transcription factors so that the cells are induced to overexpress the transcription factors. For example, the transcription factors may include: c-myc, KLf4, Sox2, or Oct3 / 4. Additional or alternative transcription factors may be used and are within the scope of the present disclosure. For example, transcription factors such as Klf2, Nanog, Tfcp2Ll and Stat3 may additionally or alternatively be used.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0059] In some embodiments, the expanding may include washing the iPSCs with a buffer (e.g., phosphate-buffered saline) and incubating the iPSCs with a cell detachment solution, for example trypsin, a chelating agent, collagenase, or the like. In some embodiments, the incubating occurs at about 37 degrees Celsius for a predetermined time period. The predetermined time period may be about 30 seconds to about 10 minutes; about 1 minute to about 60 minutes; about 1 minute to about 5 minutes; etc.

[0060] In some embodiment of method 500, transferring the transfected cells to a container with feeder cells occurs no more than about one day post-transfection. The feeder cells may comprise mouse embryonic fibroblast (MEF) cells, SNL feeder cells, or the like.

[0061] In some embodiments, transferring the cells to the second container occurs no more than about one day after transferring to the container with the feeder cells.

[0062] In some embodiments, the expanding comprises aspirating and dissociating the iPSCs into a single cell suspension.

[0063] As described elsewhere herein, the iPSCs may be differentiated into main lineages. The main lineages may include at least one of: ectoderm, mesoderm, and endoderm. Additionally, or optionally, the main lineages, for example the ectoderm lineage, may be further differentiated into final tissues, for example NGN2 neurons, astroglia, astrocytes, oligodendrocytes, and the like. The final tissues may be used for in vitro testing, autologously translated into the first nonhuman primate for in vivo testing, and / or used in genome library generation.

[0064] FIG. 6 shows an embodiment of a method 600 for deriving iPSCs from nonhuman primate PBMCs. For example, peripheral blood mononuclear cells (PBMCs) are obtained from an individual from a species of nonhuman primate, and the PBMCs are isolated from the animals’ whole blood. The PBMCs may be isolated from the animals’ whole blood using density gradient centrifugation (DGC), fluorescence active cells sorting, magnetic bead-based separation, buoyancy activated cell sorting (BACS), and the like. In an embodiment, PBMCs are isolated using DGC. In an embodiment, PBMCs are isolated using BACS.

[0065] In some embodiments, a method 500 for reprogramming NHP PBMCs into iPSCs includes culturing 610 the PBMCs to expand blood progenitor cells (e.g., CD34+ cells) in a hematopoietic stem cell (HSC) expansion medium for a period of about three days to about 10 days, about 5 days to about 10 days, about 6 days to about 10 days, about 7 days to about 10 days, about eight days to about 10 days, or about nine days. For example, a composition for the expansion of hemopoietic stems (HSPC) and / or progenitor cells of nonhuman primates mayDocket No. 0138-705.600INTERNATIONAL APPLICATION include IL3, IL6, FLT-3, TPO, and SCF (e.g., prior to transfection with reprogramming transcription factors). In some embodiments, animal serum (e.g., Fetal Bovine Serum) in the expansion medium may be replaced or combined with poly vinyl alcohol (PVA) to improve the efficiency of expansion of HSPC from nonhuman primates. CD34 is expressed not only by HSCs but by a multitude of other non-hematopoietic cell types, including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. Thus, several different cell types may potentially exhibit progenitor activity and be expanded

[0066] Further, a method 600 for reprogramming PBMCs into iPSCs may include transfecting blood progenitor cells with transcription factors 620. The NHP PBMCs are reprogrammed into NHP iPSCs. The method for reprogramming the PBMCs into iPSCs may include transfecting the PBMCs with one or more transcription factors so that the cells are induced to overexpress the transcription factors. Transcription factors are one of the groups of proteins that read and interpret DNA. They bind to the DNA and help initiate a program of increased or decreased gene transcription. The stem cell derivation protocol is optimized for nonhuman primates specifically as there are different approaches taken to generate optimized stem cells for nonhuman primates. In some embodiments, transcription binding sites of these transcription factors may be mapped on individualized primate genomes to improve efficiency of the reprogramming process in NHPs, especially since transcription binding sites in human genomes are not completely conserved in NHP species. Thus, the use of human iPSCs reprogramming kits is not efficient for iPSC generation in NHP species. This is also important considering the fact that NHP genomes are very heterogenous and existing mutations in NHP genomes result in inefficient binding of transcription factors (from human iPSC generation kits) in nonhuman primate cells.

[0067] Various transfection methods may be used, including but not limited to: electroporation, calcium-phosphate exposure, liposome-based transfection, viral-mediated transfection (also known as transduction), and the like. In an embodiment, a method for transfecting PBMCs includes transduction. Transduction may be accomplished using, for example, a Sendai virus, an adenoviral, an oncoretroviral, or a lentiviral vector. In some variations, transfection includes using a Sendai virus vector. In some instances, transfecting PBMCs includes liposome mediated transfection. For example, the transcription factors may include: c-myc, KLf4, Sox2, or Oct3 / 4. Additional or alternative transcription factors may be used and are within the scope of the present disclosure.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0068] Further, a method 600 for reprogramming PBMCs into iPSCs may include coculturing the cells (e.g., transfected and untransfected) with feeder cells 630. The feeder cells may comprise mouse embryonic fibroblast (MEF) cells, SNL feeder cells, or the like. The ratio of transfected cells to feeder cells may be about 1 :2 to about 1 :8, about 1 :3 to about 1 :7, about 1 :4 to about 1 :6, for example about 1 :5. Co-culturing may occur on a subsequent day after transfection, for example about 20 hours to about 36 hours, about 18 hours to about 30 hours, about 18 hours to about 36 hours, about 22 hours to about 26 hours, etc. after transfection.

[0069] Co-culturing may occur in Essential-8™ medium (Thermofisher Scientific). In an embodiment, the Essential-8™ medium may include a tankyrase 1 / 2 inhibitor. The tankyrase 1 / 2 inhibitor may be present in the medium at a concentration of about 1 uM to about 3 uM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc. In some embodiments, the tankyrase 1 / 2 inhibitor may maintain the NHP iPSCs in a pluripotent state and significantly reduce spontaneous differentiation of the NHP iPSCs.

[0070] Further, a method 600 for reprogramming PBMCs into iPSCs may include transferring unadhered cells to new medium. For example, the transfer may occur about 24 hours to about 72 hours, about 18 hours to about 78 hours, about 18 hours to about 30 hours, about 30 hours to about 42 hours, about 42 hours to about 52 hours, about 52 hours to about 66 hours, about 66 hours to about 78 hours, etc. after co-culturing. The unadhered cells may be cultured in a basal medium, for example MEM medium, DMEM medium, RPMI medium, F12 ™ medium, and the like. Further, a method 600 for reprogramming PBMCs into iPSCs may optionally include adding medium to the adherent cells 640. The added media may comprise a medium that is feeder-free and xeno-free that supports the reprogramming of somatic cells and the spontaneous or directed differentiation of pluripotent stem cells (PSCs). For example, the added medium may be Essential-6® medium (Thermofisher Scientific). In an embodiment, adding medium occurs about 48 hours to 96 hours, about 42 hours to about 102 hours, etc. after transfection.

[0071] Further, a method 600 for reprogramming PBMCs into iPSCs may include optionally replacing the medium on the adherent cells. The medium may be replaced after about 90 hours to about 144 hours, about 96 hours to about 144 hours, about 90 hours to about 126 hours, about 114 hours to about 126 hours, etc. after transfection. The replacing medium may be a medium that is feeder-free and xeno-free that supports the reprogramming of somatic cells andDocket No. 0138-705.600INTERNATIONAL APPLICATION the spontaneous or directed differentiation of pluripotent stem cells (PSCs). For example, the added medium may be Essential-6® medium (Thermofisher Scientific).

[0072] Further, a method 600 for reprogramming PBMCs into iPSCs may include optionally replacing the medium with medium comprising one or more growth factors 650. For example, the medium may comprise a medium that is feeder-free and xeno-free that supports the reprogramming of somatic cells and the spontaneous or directed differentiation of pluripotent stem cells (PSCs). For example, the added medium may be Essential-6® medium (Thermofisher Scientific). The growth factor may comprise basic fibroblast growth factor, DJ- 1, epidermal growth factor, and the like. In an embodiment, the growth factor comprises basic fibroblast growth factor. The concentration of the growth factor (i.e., bFGF) may be about 100 ng / ul to about 200 ng / ul. In some embodiments, this particular growth factor and / or concentration is advantageous for the NHP iPSC derivation process since commercially available human bFGF is not as efficient in cross-reactivity with NHP cells. Medium replacement may occur about 144 hours to about 192 hours, about 160 hours to about 250 hours, about 162 hours to about 174 hours, about 188 hours to about 198 hours, about 210 hours to about 222 hours, about 232 hours to about 246 hours, etc. after transfection. Medium replacement may be repeated daily for one or more days or a plurality of days, for example repeated once, twice, thrice, or more.

[0073] Further, a method 600 for reprogramming PBMCs into iPSCs may include optionally replacing the medium daily for one or more days or for a plurality of days 680. For example, the medium may be replaced between about 240 hours post-transfection to about 440 hours post-transfection. The medium may be replaced with basal medium, Essential-6®, Essential- 8®, a medium comprising tumor growth factor-b (TGF-b) and / or b-FGF, or similar medium. In some embodiments, the medium may be replaced daily until visual identification of a first colony of iPSC.

[0074] Further, a method 600 for reprogramming PBMCs into iPSCs may include culturing the first colony of iPSC on a matrix 670, for example an extracellular matrix, Matrigel®, Cultrex®, Geltrex®, and the like. The first colony may be cultured in medium comprising a serine / threonine kinase inhibitor. For example, the medium may include Essential-8®. The serine / threonine kinase may include a Rho inhibitor or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, for example, Thiazovivin (TZV) or similar products. A concentration of the inhibitor may be about 1 uM to about 3 uM.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0075] Further, a method 600 for reprogramming PBMCs into iPSCs may include passaging the cells after about 12 hours to about 48 hours. Passaging 660 may include refreshing the medium and adding a Wnt pathway inhibitor, tankyrasel / 2 inhibitor, or the like. The inhibitor may be at a concentration of about 1 uM to about 3uM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc.

[0076] Optionally, iPSCs may be passaged and / or expanded by washing with a buffer (e.g., phosphate buffered saline) and incubating the cells with a cell detachment solution. The cell detachment solution may comprise collagenase (or recombinant enzymes thereof), trypsin (or recombinant enzymes thereof), a chelating agent (e.g., ethylenediamine tetra-acetic acid), and the like.

[0077] Optionally, iPSCs may be expanded or maintained by culturing in medium on feeder cells or an extracellular matrix, as described elsewhere herein. The medium may comprise a serine / threonine kinase may comprise a Rho inhibitor or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor. The inhibitor may be at a concentration of about 1 nM to about 3 nM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc.

[0078] In embodiments with subsequent passaging, the iPSCs may be incubated in a container, without feeder calls, the container having a substrate coated thereon. The substrate may be configured to retain pluripotency of the cells, a stable karyotype of the cells, and / or an expression of pluripotency markers by the cells. For example, the substrate may comprise laminin, a recombinant laminin fragment (e.g., 511 e8 fragment), vitronectin, recombinant vitronectin, or a combination thereof. The medium may comprise Essential-8®, E8-specific supplement (e.g., TGF-b, bFGF, etc.), and a Wnt pathway inhibitor, tankyrasel / 2 inhibitor, or the like. The inhibitor may be at a concentration of about 1 nM to about 3 nM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc. The subsequent passaging may result in colonies with little-to-no differentiation.

[0079] DIFFERENTIATION OF NHP iPSCs INTO NEURONAL LINEAGES

[0080] In some embodiments described herein, the methods may be used for differentiating NHP iPSCs to neuronal lineage cells, for example astrocytes. In some embodiments, the differentiation may be from NHP iPSCs to astrocytes or induced astrocytes (iA cells). In some embodiments, the NHP species include cynomolgus macaques (Macaco fascicularis), rhesus macaques (Macaco mulatto), and pig-tailed macaque (Macaco nemestrina).Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0081] In some embodiments, the differentiated neuronal lineage cells can be used for in vitro safety and efficacy assessments as well as discovery of human drugs in iPSC-derived neuronal lineage cells in NHPs.

[0082] To differentiate NHP iPSCs into neuronal lineage cells, for example astrocytes or other neuronal lineage, a method may include optionally transitioning NHP iPSCs from a feeder-based culture condition (e.g., culturing iPSCs on MEF or other feeder cells) into a feeder-free condition. This transition into a feeder-free condition of NHP iPSCs may reduce or remove various small molecules that are secreted by the feeder cells into the culture media. For example, small molecules secreted by feeder cells can interrupt the process of differentiation of iPSCs to neural cells including astrocytes neurons.

[0083] As shown in FIGs. 1-3, a vector 100, and associated method, may be used for differentiating NHP iPSCs into various cell types. The vector 100 (e.g., SEQ ID NO. 1) may be used for differentiating NHP iPSCs, for example Macaca fascicularis iPSCs. The vector 100 may be a piggyBac vector. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NO. 7) encoding a selectable marker 110 (e.g., antibiotic resistance gene) for the selection of successfully transfected cells. For example, the nucleic acid sequence may encode the selectable marker 110 Puromycin (PuroR) between about 1113 base pairs and about 1712 base pairs as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NO. 15) encoding a modified rtTA protein 120 that binds to promoters containing the let operator in the presence of doxycycline. For example, SEQ ID NO. 15 may be between about 1788 base pairs and about 2531 base as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NOs. 5, 6) encoding a Tet-responsive promoter 140. For example, the Tet-responsive promoter 140 may be a unidirectional promoter (SEQ ID NO. 5), for example as shown in FIGs. 1 and 3, or a bi-directional promoter (SEQ ID NO. 6) as shown in FIG. 3. The Tet-responsive promoter 140 may be between about 3815 base pairs and about 4193 base pairs as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NOs. 9, 11) encoding one or more origins of replication (Ori) that enable the vector to be replicated. As shown in SEQ ID NO. 1, an fl Ori 180 (e.g., SEQ ID NO. 11) may be between about 3 base pairs to about 458 base pairs for phage based single stranded DNA replication and packaging. Further, for example, as shown in SEQ ID NO. 1, an Ori 160 (e.g., SEQ ID NO. 9) may be between about 5852 base pairs to about 6440 base pairs for plasmid amplification in bacterial cells in preparation for transfection. The vector 100 may include nucleic acid sequence (e.g., SEQ ID NO. 10) encoding a second selectable marker 170Docket No. 0138-705.600INTERNATIONAL APPLICATION(e.g., antibiotic resistance gene) for selection of those bacterial cells that are amplifying the vector. SEQ ID NO. 10 may be between about 6611 base pairs and about 7471 base pairs, as shown in SEQ ID NO. 1. For example, the nucleic acid sequence may encode Ampicillin (AmpR), which confers resistance to ampicillin, carbenicillin, and related antibiotics. The promoter 190 (e.g., SEQ ID NO. 12) for the selectable marker 170 may be between about 7472 base pairs and about 7576 base pairs, as shown in SEQ ID NO. 1. The vector 100 may optionally include a nucleic acid sequence (e.g., SEQ ID NOs. 13, 14) encoding a T7 promoter 184 for bacteriophage T7 RNA polymerase and / or a T3 promoter 194 for bacteriophage T3 RNA polymerase. For example, SEQ ID NO. 13 may be between about 623 base pairs and about 641 base pairs; and / or SEQ ID NO. 14 may be between about 5411 base pairs to about 5429 base pairs, as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NO. 7) for encoding a constitutive promoter 130 (e.g., Ubiquitin C promoter or UbC) to drive the expression of one or more differentiation factor genes in the target cells. For example, SEQ ID NO. 7 may be between about 2545 base pairs and about 3754 base pairs, as shown in SEQ ID NO. 1.

[0084] Producing a modified vector 100 may include replacing a reporter gene 150, for example TurboGFP or SEQ ID NO. 9 (as shown in FIG. 1), with a differentiation factor gene 154 or inserting a differentiation factor gene 154 upstream or downstream of a report gene 150 (as shown in FIG. 3). For example, the reporter gene 150 may be used as a marker of transduction efficiency (i.e., cells that are not transduced may not survive in the presence of the compound used to transcribe the selectable marker). Although TurboGFP is shown and described herein, one of skill in the art will appreciate that other reporter genes may be used without departing from the intent and scope of the present disclosure. Alternative reporter genes include, but are not limited to, enhanced Green Fluorescent Protein (eGFP, excitation at 488 nm, emission at 507 nm), mCherry (excitation at 587 nm, emission at 610 nm), mVenus (excitation at 515 nm, emission at 528 nm), mKate2 (excitation at 588 nm, emission at 633 nm), TagRFP (excitation at 555 nm, emission at 584 nm), AmCyanl (excitation at 458 nm, emission at 489 nm), mCerulean3 (excitation at 433 nm, emission at 475 nm), tdTomato (excitation at 554 nm, emission at 581 nm), mRuby2 (excitation at 559 nm, emission at 600 nm), sfGFP (Superfolder GFP, excitation at 485 nm, emission at 510 nm), and the like. The reporter gene 150 may be, for example, between about 4214 base pairs and about 4894 base pairs in length, as shown in SEQ ID NO. 1.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0085] The differentiation factor gene 154 may be Macaca fascicularis NFIB 164 or SEQ ID NO. 3 (as shown in FIG. 3), and / or Macaca fascicularis SOX9 162 or SEQ ID NO. 2 (as shown in FIG. 3), inserted into vector 100, for example a piggyBac vector backbone (e.g., SEQ ID NO. 1, OTPL007-pbvector). Although sequences for Macaca fascicularis are shown and described herein, one of skill in the art will appreciate that sequences derived from Homo sapiens may also apply and / or be used in the transduction of NHP iPSCs given the sequence similarity between NHP genomes and the Homo sapiens ’ genome.

[0086] As shown in FIG. 3, to modify the vector 100 with differentiation factor gene 164 or SEQ ID NO. 3, the differentiation factor gene 164 or SEQ ID NO. 3 may be inserted upstream (or optionally downstream) of the reporter gene 150. To modify the vector 100 with differentiation factor gene 162 or SEQ ID NO. 2, the reporter gene 150 may be replaced with the differentiation factor gene 162 or SEQ ID NO. 2. In some embodiments, a bidirectional Tet-responsive promoter 140 (e.g., SEQ ID NO. 6) may be positioned between a first differentiation factor gene 154 and a second differentiation factor gene 154, as shown in FIG. 3. The vector 100 as modified as shown in FIG. 3 may be used to differentiate nonhuman primate iPSCs to iA cells. In some embodiments, SEQ ID NO. 2 may be positioned in vector 100 between about 3865 base pairs and about 5190 base pairs. In some embodiments, SEQ ID NO. 3 may be positioned in vector 100 between about 5696 base pairs and about 7225 base pairs.

[0087] The vector 100 may be modified using restriction enzymes, Gibson Assembly, or the like to replace the reporter gene 150 with the differentiation factor gene 154. For example, using Gibson Assembly, the differentiation factor gene and the vector 100 may each include overlapping sequences, complementary to each another. The vector 100 may be linearized (e.g., using restriction digestions or PCR amplification) to create opens ends in the vector 100 for insertion of the gene. The gene and linearized vector may be mixed with an enzymatic reaction mixture, including for example, exonuclease, DNA polymerase, and DNA ligase. The reaction may be an isothermal reaction. For example, the reaction may be incubated at about 45 degrees to about 55 degrees to enable the gene to be inserted into the vector. The recombinant vector may be transformed into bacterial cells that can replicate the vector using the Ori in the vector. Bacterial cells with successful replication of the vector may be selected using a selectable marker, for example Ampicillin resistance (i.e., using the AmpR gene of the vector).Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0088] The vectors may respond to doxycycline induction while having continuous puromycin selection. The Tet-On 3G is a modified rTtA protein that binds tight to the promoters containing the Tet operator in the presence of doxycycline. The vector 100 exhibits increased sensitivity to doxycycline, allowing for tighter control over gene expression levels compared to earlier Tet-On systems. By incorporating the puromycin resistance gene (PuroR), as shown in FIG. 3, cells expressing the incorporated differentiation factor 162, 164 (i.e., have been transduced) can be selectively cultured.

[0089] In some embodiments, a method 200 of transfecting NHP iPSCs may include transfecting the NHP iPSCs (e.g., electroporation the NHP iPSCs) with a vector expressing a differentiation factor gene 154 (e.g., any of SEQ ID NOs. 2-3, etc.) atblock S210, and selecting for the transfected NHP iPSCs using a selectable marker (e.g., puromycin selection) at block S230, for example a selectable marker associated with vector 100. Th method 200 may optionally further include expanding the transfected NHP iPSCs on a matrix at block S220, for example an extracellular matrix, Matrigel®, Cultrex®, Geltrex®, and the like. The transfected NHP iPSCs may be expanded in medium (e.g.,B8 medium, E8 basal medium + E8 Supplement, etc.). The medium may include or be supplemented with thiazovivin.

[0090] In some embodiments, a method 400 of differentiating NHP iPSCs into a neuronal lineage, for example astrocytes (iA cells), may include providing a vector expressing one or more differentiation factor genes (e.g., SEQ ID NOs. 2-3, etc.) at block S410; and transfecting the nonhuman primate iPSCs with the vector in medium at block S420, as described above with respect to FIG. 2. Method 400 may further include culturing the transfected cells with expansion medium at block S430; optionally replacing the expansion medium with a second medium at block S440; replacing the second medium with a maturation medium at block S450; and producing neuronal lineage cells at block S460. The method 400 may be used with a modified vector 100 having a piggyBac vector backbone modified to express one or more differentiation factor genes. In some embodiments, method 400 may also be used with a lentiviral vector, Sendai virus vector, or Adenovirus vector, although with reduced efficiency.

[0091] Culturing the transfected cells with expansion medium at block S430 of method 400 may include culturing the transfected cells with the expansion medium between and / or during about day zero to about day three or on about day one to about day two. The expansion medium may include one or more of: a medium supplemented with about 5% to about 15% fetal bovine serum, about 0.1% to about 5% N2 supplement, and about 0.1% to about 5% L-glutamine (e.g.,Docket No. 0138-705.600INTERNATIONAL APPLICATIONGlutamax or the like). The medium may include Neurobasal medium, DMEM / F-12, RPMI 1640, IMDM, KOSR, and the like.

[0092] In some embodiments, the expansion medium may be optionally replaced with a second medium, as shown in block S440 in FIG. 4. The second medium may be applied to the cells all at once or gradually over time. When replaced gradually, the mixed medium (expansion medium and second medium) may be fully replaced between about day five to about day seven; or on about day six. The second medium may be applied to the cells between about day two to about day six, or between about day 3 to about day five. The second medium may include a growth factor medium, for example basic fibroblast growth factor (bFGF) medium, a neurobasal medium, or a combination thereof. The second medium may include one or more of: a B27 supplement, NEAA, L-glutamine, fetal bovine serum (FBS), FGF, Ciliary neurotrophic factor (CNTF), and / or bone morphogenetic protein 4 (BMP4). The second medium may include about 0.2% to about 8%, about 0.5% to about 5%; about 1% to about 3%; etc. B27 supplement. The second medium may include about 0.1% to about 5% NEAA; about 0.5% to about 2% NEAA; about 0.5% to about 1.5% NEAA; etc. The second medium may include about 0.1% to about 10% L-glutamine (e.g., Glutamax); about 0.5% to about 5% L- glutamine; about 0.5% to about 2% L-glutamine; about 0.5% to about 1.5% L-glutamine; etc. The second medium may include about 0.1% to about 10% FBS; about 0.5% to about 5% FBS; about 0.5% to about 2% FBS; about 0.5% to about 1.5% FBS; etc. The second medium may include about 0.8 ng / mL FGF to about 16 ng / mL FGF; about 6 ng / ml FGF to about 10 ng / ml FGF; about 5 ng / ml FGF to about 10 ng / ml FGF; about 4 ng / ml FGF to about 12 ng / ml FGF; etc. The second medium may include about 0.5 ng / mL CNTF to about 15 ng / ml CNTF; about 3 ng / ml CNTF to about 7 ng / ml CNTF; about 1 ng / ml CNTF to about 10 ng / ml CNTF; about 2 ng / ml CNTF to about 8 ng / ml CNTF; etc. The second medium may include about 0.1 ng / ml BMP4 to about 20 ng / ml BMP4; about 5 ng / ml BMP4 to about 15 ng / ml BMP4; about 8 ng / ml BMP4 to about 12 ng / ml BMP4; etc.

[0093] In some embodiments, as shown in block S450 in FIG. 4, the expansion medium or optionally the second medium may be replaced with a maturation medium. For example, the maturation medium may be added to the cells on about day six to about day eight or on about day seven or on about day eight. The expansion medium and / or the second medium may be fully replaced by the maturation medium or replaced fractionally with the maturation medium. For example, initially, about 10% to about 60%; about 25% to about 50%; about 40% to about 75%; or about 40% to about 60% of the medium may be replaced with the maturation medium.Docket No. 0138-705.600INTERNATIONAL APPLICATIONOptionally, the cells may be dissociated with an enzyme treatment (e.g., Accutase, trypsin, etc.) and replated on an extracellular matrix, as described elsewhere herein. The maturation medium may include one or more of: N2 supplement, sodium pyruvate, L-glutamine, A-acetyl-cysteine, dbcAMP, epidermal growth factor-like growth factor (EGF-like growth factor), CNTF, and / or BMP4.

[0094] The maturation medium may a medium, for example DMEM / F-12, IMDM, RPMI 1640, Neurobasal. For example, two or more medias may be mixed or combined to form the maturation medium. In some embodiments, DMEM / F-12 may be mixed with Neurobasal medium. For example, the mix may be 1 : 1 to 2: 1 or 1 :2. The maturation medium may include about 0.1% to about 5% N2 supplement; 0.5% to about 2% N2 supplement; 0.5% to about 1.5% N2 supplement; about 1% to about 3% N2 supplement; etc. The maturation medium may include about 0.1% to about 5% sodium pyruvate; 0.5% to about 2% sodium pyruvate; 0.5% to about 1.5% sodium pyruvate; about 1% to about 3% sodium pyruvate; etc. The maturation medium may include about 0.1% to about 5% L-glutamine; 0.5% to about 2% L-glutamine; 0.5% to about 1.5% L-glutamine; about 1% to about 3% L-glutamine; etc. The maturation medium may include about 1 mg / ml A-acetyl-cysteine to about 10 mg / ml A-acetyl-cysteine; about 2 mg / ml / ' -acetyl -cysteine to about 8 mg / ml / ' / -acetyl -cysteine; about 2.5 mg / ml N- acetyl-cysteine to about 7.5 mg / ml A-acetyl -cysteine; about 4 mg / ml / ' / -acetyl -cysteine to about 6 mg / ml A -acetyl -cysteine; etc. The maturation medium may include about 100 mg / ml dbcAMP to about 1,000 mg / ml dbcAMP; about 250 mg / ml dbcAMP to about 750 mg / ml dbcAMP; about 400 mg / ml dbcAMP to about 600 mg / ml dbcAMP; about 450 mg / ml dbcAMP to about 550 mg / ml dbcAMP; etc. The maturation medium may include about 0.5 ng / mL EGF- like growth factor to about 15 ng / ml EGF-like growth factor; about 3 ng / ml EGF-like growth factor to about 7 ng / ml EGF-like growth factor; about 1 ng / ml EGF-like growth factor to about 10 ng / ml EGF-like growth factor; about 2 ng / ml EGF-like growth factor to about 8 ng / ml EGF- like growth factor; etc. The maturation medium may include about 0.1 ng / ml CNTF to about 20 ng / ml CNTF; about 5 ng / ml CNTF to about 15 ng / ml CNTF; about 8 ng / ml CNTF to about 12 ng / ml CNTF; etc. The second medium may include about 0.1 ng / ml BMP4 to about 20 ng / ml BMP4; about 5 ng / ml BMP4 to about 15 ng / ml BMP4; about 8 ng / ml BMP4 to about 12 ng / ml BMP4; etc. In some embodiments, the maturation medium is optionally replaced every or about one day to about five days or about two days to about three days.

[0095] Producing neuronal cells, as shown in block S460 in FIG. 4, may occur after about ten days to about thirty days; about ten days to about 15 days; about 15 days to about 25 days;Docket No. 0138-705.600INTERNATIONAL APPLICATION about 20 days to about 30 days; about 14 days to about 21 days; about 14 days to about 28 days; about 21 days to about 28 days; etc. The neuronal lineage cells (e.g., astrocytes) at block S460 of method 400 may be identified using immunostaining of anti-GFAP and / or anti-CD44.

[0096] FIG. 7 illustrates an embodiment of a vector 700 designed for differentiating nonhuman primate iPSCs from Macaca fascicularis into astrocytes. The vector 700 includes a nucleic acid sequence 710 that defines a PiggyBac vector backbone 712 and one or more inserted sequences that facilitate directed astrocytic differentiation. The configuration shown may vary depending on the vector configuration, the differentiation conditions, and the host cell type.

[0097] The PiggyBac vector backbone 712 comprises structural elements that facilitate transposition and genomic integration of inserted sequences into the genome of the target cell. The PiggyBac vector backbone 712 includes terminal inverted repeats 714 and transposase recognition sites 716. The terminal inverted repeats 714 may each have a length between about 200 base pairs and about 800 base pairs, for example between about 250 base pairs and about 600 base pairs, between about 300 base pairs and about 550 base pairs, or between about 325 base pairs and about 500 base pairs. In some embodiments, the pair of terminal inverted repeats 714 may collectively span between about 400 base pairs and about 1600 base pairs, for example between about 500 base pairs and about 1400 base pairs, or between about 600 base pairs and about 1200 base pairs. The transposase recognition sites 716 may be located adjacent to or partially overlapping the terminal inverted repeats 714 and may comprise recognition motifs ranging from about 8 base pairs to about 40 base pairs, for example about 10 base pairs to about 30 base pairs, about 12 base pairs to about 28 base pairs, or about 15 base pairs to about 25 base pairs. In certain variations, the PiggyBac transposase binding region defined by the transposase recognition sites 716 may extend over a total length of about 50 base pairs to about 200 base pairs, for example about 75 base pairs to about 150 base pairs, or about 90 base pairs to about 125 base pairs.

[0098] The PiggyBac vector backbone 712 may have a total sequence length between about 6 kilobases and about 10 kilobases, for example about 7 kilobases to about 9 kilobases, or about 7.5 kilobases to about 8.5 kilobases. In some embodiments, the vector backbone may be circularized and propagated in bacterial cells prior to transfection, using a replication origin (Ori) sequence having a length between about 400 base pairs and about 900 base pairs, for example about 500 base pairs to about 800 base pairs, or about 550 base pairs to about 700 base pairs. The vector may further include a selectable marker, such as an ampicillin resistanceDocket No. 0138-705.600INTERNATIONAL APPLICATION gene, having a coding length between about 700 base pairs and about 1200 base pairs, for example about 800 base pairs to about 1100 base pairs, or about 850 base pairs to about 1000 base pairs.

[0099] Integrated within the PiggyBac vector backbone 712 is at least one differentiation factor gene 718. The differentiation factor gene 718 encodes a transcriptional regulator or other factor that drives differentiation of nonhuman primate iPSCs into astrocytes by activating astrocyte-lineage regulatory pathways. In some embodiments, the differentiation factor gene 718 comprises Macaca fascicularis SOX9, Macaca fascicularis NFIB, or homologous sequences derived from other primate species. The differentiation factor gene 718 may be inserted into the PiggyBac vector backbone using Gibson Assembly or restriction enzyme ligation. The inserted differentiation factor gene may have a sequence length between about 0.8 kilobases and about 4.0 kilobases, for example about 1.0 kilobases to about 3.5 kilobases, about 1.2 kilobases to about 3.0 kilobases, or about 1.4 kilobases to about 2.8 kilobases. In some embodiments, the differentiation factor gene 718 may be flanked by regulatory sequences such as promoters, enhancers, and polyadenylation signals having a combined length between about 300 base pairs and about 1500 base pairs, for example about 400 base pairs to about 1200 base pairs, or about 500 base pairs to about 1000 base pairs.

[0100] The differentiation factor gene 718 activates one or more astrocyte-lineage regulatory pathways and induces expression of astrocytic markers such as glial fibrillary acidic protein (GFAP), SlOOp, and aquaporin-4. Expression of these markers may be detected within about 5 days to about 35 days after transfection, for example about 7 days to about 30 days, about 10 days to about 25 days, or about 14 days to about 21 days, depending on the expression level, promoter strength, and stability of the integrated transgene. In some embodiments, astrocytic differentiation may result in the appearance of morphological features such as stellate processes or branching extensions after about 10 days to about 40 days, for example about 12 days to about 35 days, or about 15 days to about 30 days of culture.

[0101] In operation, the vector 700 may be introduced into nonhuman primate iPSCs using electroporation, lipofection, or nanoparticle-assisted delivery. The amount of DNA used for transfection may range between about 0.2 pg / mL and about 20 pg / mL, for example about 0.5 pg / mL to about 10 pg / mL, about 1 pg / mL to about 8 pg / mL, or about 2 pg / mL to about 6 pg / mL. In some embodiments, the electroporation parameters may include a voltage between about 700 volts and about 1500 volts, for example about 800 volts to about 1400 volts, or about 900 volts to about 1300 volts, with a pulse duration between about 5 milliseconds and aboutDocket No. 0138-705.600INTERNATIONAL APPLICATION50 milliseconds, for example about 10 milliseconds to about 40 milliseconds, or about 15 milliseconds to about 30 milliseconds. The post-transfection recovery period may range between about 8 hours and about 48 hours, for example about 10 hours to about 36 hours, or about 12 hours to about 24 hours before the cells are transferred to a growth or selection medium.

[0102] In certain embodiments, the PiggyBac vector backbone 712 may further include an inducible or constitutive promoter to regulate expression of the differentiation factor gene 718. For example, a constitutive promoter such as the human ubiquitin C (UbC) promoter or cytomegalovirus (CMV) promoter may be used. The promoter may have a sequence length of about 400 base pairs to about 2000 base pairs, for example about 500 base pairs to about 1500 base pairs, or about 700 base pairs to about 1200 base pairs. The vector may also include an antibiotic resistance gene, such as puromycin resistance, for selection of transfected cells, with an open reading frame between about 600 base pairs and about 1000 base pairs, for example about 700 base pairs to about 900 base pairs, or about 750 base pairs to about 850 base pairs.

[0103] After successful genomic integration mediated by the PiggyBac transposase, expression of the differentiation factor gene 718 directs lineage-specific differentiation, resulting in formation of induced astrocytes (iA cells). The differentiated cells may exhibit positive staining intensity (e.g., GFAP or another reporter molecular) of about 50% to about 95% of total viable cells, for example about 60% to about 90%, or about 70% to about 85%, depending on the duration and conditions of culture.

[0104] The PiggyBac vector system enables stable genomic integration without leaving residual sequences after excision, thereby maintaining the genomic integrity of the host cells. The excision efficiency may range between about 85% and about 100%, for example about 90% to about 99%, and / or about 92% to about 98% using optimized transposase systems. The arrangement shown in FIG. 7 may be implemented using any PiggyBac-based system comprising terminal inverted repeats 714, transposase recognition sites 716, and / or one or more differentiation factor genes 718 that together direct nonhuman primate iPSCs toward astrocytic lineage commitment. Variations in promoter architecture, transgene placement, and / or sequence composition may be used without departing from the intended function of the vector in differentiating nonhuman primate iPSCs into astrocytes and / or other glial derivatives.

[0105] The vector 700 provides technical advantages in differentiating nonhuman primate iPSCs into astrocytes. These advantages result from the structure of the PiggyBac vector backbone 712, the configuration of the differentiation factor gene 718, and the mechanism ofDocket No. 0138-705.600INTERNATIONAL APPLICATION transposition that enables stable and efficient transgene expression in Macaca fascicularis iPSCs.

[0106] The PiggyBac vector backbone offers high genomic integration efficiency compared to conventional lentiviral or Sendai virus-based delivery systems. The terminal inverted repeats 714 and transposase recognition sites 716 function together to allow precise insertion of the differentiation factor gene 718 into host genomic TTAA sites. Integration efficiency may range from about 10% to about 50%, for example about 15% to about 40%, or about 20% to about 35%, which represents a substantial improvement over lentiviral -based systems in nonhuman primate cells, where efficiencies are typically about 1% to about 5%.

[0107] The PiggyBac transposon system also provides a footprint-free excision mechanism, allowing removal of the integrated transgene without residual sequences. Following expression of a transposase, excision efficiency may range from about 85% to about 100%, for example about 90% to about 99%, or about 92% to about 98%. This property enables transient differentiation induction without permanent genomic alteration, reducing the risk of insertional mutagenesis or unpredictable gene disruption.

[0108] Another technical benefit is the large cargo capacity of the PiggyBac vector, which allows multiple differentiation factor genes, regulatory elements, and / or selectable markers to be included within a single construct. The total insert size can range from about 5 kilobases to about 15 kilobases, for example about 6 kilobases to about 12 kilobases, or about 7 kilobases to about 10 kilobases, without measurable loss of integration efficiency. This capacity supports the use of two differentiation factor genes, such as SOX9 and NFIB, which together direct astrocytic lineage specification in Macaca fascicularis iPSCs.

[0109] The vector further demonstrates species-specific efficiency in Macaca fascicularis cells, overcoming the reduced transduction rates observed when using human-optimized lentiviral systems. The transposase recognition sequences 716 and promoter elements used in the PiggyBac construct interact effectively with the transcriptional machinery of nonhuman primate cells, ensuring stable and sustained expression of the differentiation factor gene 718. This allows reliable differentiation into astrocytes within about 10 days to about 30 days, for example about 14 days to about 25 days, or about 15 days to about 21 days after transfection.

[0110] In addition, the PiggyBac system minimizes gene silencing and positional effects by integrating preferentially into transcriptionally active genomic regions, resulting in stable and uniform gene expression. Expression stability may be maintained in more than about 80% ofDocket No. 0138-705.600INTERNATIONAL APPLICATION the cell population through at least ten passages, for example about 85% to about 95%, or about 88% to about 93%, depending on the growth conditions and transgene configuration.

[0111] A further advantage of the vector configuration is the inclusion of a bidirectional Tet- responsive promoter positioned between the first and second differentiation factor genes, which allows controlled and tunable gene expression. Doxycycline-inducible expression levels can be adjusted between about 0.01 pg / mL and about 5 pg / mL, for example about 0.1 pg / mL to about 3 pg / mL, or about 0.5 pg / mL to about 2 pg / mL, permitting precise timing of differentiation initiation and regulation of transcription factor activity. The PiggyBac system also provides scalability and reproducibility in experimental applications. Because the integration process is non-viral, efficiency remains consistent across multiple nonhuman primate iPSC lines without reliance on host immune mechanisms. Differentiation yields of astrocytes may range between about 60% and about 95% of total cells, for example about 70% to about 90%, or about 75% to about 85%, depending on the composition of the culture medium and the electroporation parameters used.

[0112] In some embodiments, the vector 700 improves biosafety by eliminating the use of viral components, thereby reducing the risk of replication-competent vector formation. The reversible integration system provides additional experimental flexibility, allowing conditional expression and subsequent removal of transgenes as needed for downstream applications. In some embodiments, the vector 700 offers technical benefits including efficient genomic integration, reversible excision without residual sequences, large transgene capacity, speciesspecific compatibility, stable expression, inducible gene control, and / or high reproducibility. These advantages address the limitations of traditional viral transduction systems and provide a reliable and safe platform for generating functional astrocytes from Macaca fascicularis iPSCs for use in research, disease modeling, and translational studies.

[0113] FIG. 8 shows an embodiment of a vector 700 designed for differentiating nonhuman primate iPSCs from Macaca fascicularis into astrocytes. The vector 700 includes a nucleic acid sequence 710 comprising a PiggyBac vector backbone 712 that facilitates genomic integration of a differentiation factor gene 718 into the genome of a host cell. The PiggyBac vector backbone 712 facilitates genomic integration of the differentiation factor gene 718 through a cut, copy, and / or paste transposition process that depends on recognition between the terminal inverted repeats and the transposase recognition sites located within the vector. The PiggyBac vector 700 is introduced into the host cell along with a transposase enzyme, which may be provided on a helper plasmid or as recombinant protein. The transposase bindsDocket No. 0138-705.600INTERNATIONAL APPLICATION to the terminal inverted repeats of the PiggyBac vector backbone 712 and forms a complex that mediates excision and reinsertion of the transposon into genomic DNA.

[0114] Once bound, the transposase cleaves the DNA at the junctions of the inverted repeats, releasing the transposon containing the differentiation factor gene 718, the selectable marker 804, and the promoter 808. The excised transposon is inserted into the host genome at a TTAA site. The transposase makes staggered cuts in the genomic DNA and integrates the transposon through ligation, allowing stable incorporation into the chromosome. The terminal inverted repeats of the PiggyBac vector backbone 712 may have a length between about 13 base pairs and about 20 base pairs, for example about 14 base pairs to about 18 base pairs, about 15 base pairs to about 19 base pairs, or about 16 base pairs to about 17 base pairs. The transposase recognition sequences may range from about 200 base pairs to about 400 base pairs, for example about 225 base pairs to about 375 base pairs, about 250 base pairs to about 350 base pairs, or about 275 base pairs to about 325 base pairs. The distance between the inverted repeats may range from about 5 kilobases to about 15 kilobases, for example about 6 kilobases to about 14 kilobases, about 7 kilobases to about 12 kilobases, or about 8 kilobases to about 11 kilobases, depending on the inserted cargo and vector configuration.

[0115] Integration efficiency may range from about 10 percent to about 60 percent, for example about 15 percent to about 50 percent, about 20 percent to about 45 percent, or about 25 percent to about 40 percent. The process results in stable integration without sequence truncation or rearrangement, maintaining the structural integrity of the inserted differentiation factor gene 718. The integration may occur within transcriptionally active genomic regions, leading to consistent expression levels across more than about 80 percent to about 95 percent of the transfected cells, for example about 85 percent to about 92 percent, about 88 percent to about 94 percent, or about 90 percent to about 93 percent. The PiggyBac transposase-mediated process is reversible. Upon re-expression of the transposase, the integrated transposon can be excised precisely, restoring the original TTAA sequence in the genome without leaving residual nucleotides. Excision efficiency may range between about 85 percent and about 100 percent, for example about 88 percent to about 99 percent, about 90 percent to about 98 percent, or about 92 percent to about 96 percent. In some embodiments, the PiggyBac transposon mechanism is non-viral and avoids insertional mutagenesis and / or one or more recombination events commonly associated with lentiviral or retroviral systems. The non-viral integration pathway provides higher fidelity and allows repeated and / or conditional use within the same cells. The integrated differentiation factor gene 718 may remain transcriptionally active forDocket No. 0138-705.600INTERNATIONAL APPLICATION about ten to about thirty cell passages, for example about twelve to about twenty -five passages, about fourteen to about twenty passages, or about sixteen to about eighteen passages, depending on the culture conditions and promoter used. The PiggyBac system therefore provides a versatile and efficient platform for stable, reversible, and high-fidelity genomic integration of transgenes in nonhuman primate iPSCs. This enables controlled expression of differentiation factor genes involved in astrocyte-lineage specification and supports reliable generation of astrocytes from Macaca fascicularis iPSCs under laboratory and preclinical conditions.

[0116] The PiggyBac vector backbone 712 provides the structural framework required for efficient transposition and stable expression of inserted genes in nonhuman primate iPSCs. The PiggyBac vector backbone 712 includes a second sequence 802 encoding a selectable marker 804 and a third sequence 806 encoding a promoter 808. The second sequence 802 encoding the selectable marker 804 enables the identification and propagation of successfully transfected or integrated cells. The selectable marker 804 may encode an antibiotic resistance gene such as puromycin resistance (PuroR), neomycin resistance (NeoR), hygromycin resistance (HygR), or ampicillin resistance (AmpR). The coding region of the selectable marker 804 may have a sequence length between about 600 base pairs and about 1300 base pairs, for example between about 700 base pairs and about 1200 base pairs, about 800 base pairs and about 1100 base pairs, and / or about 850 base pairs and about 1000 base pairs. In some embodiments, the selectable marker 804 may further include a polyadenylation signal sequence downstream of the coding region having a length between about 50 base pairs and about 250 base pairs, for example about 75 base pairs to about 200 base pairs, and / or about 100 base pairs to about 150 base pairs.

[0117] In an embodiment, the selectable marker 804 may also include a promoter-specific regulatory region that enhances antibiotic resistance gene expression in mammalian cells. For example, the selectable marker 804 may be operably linked to a phosphoglycerate kinase (PGK) promoter or a simian virus 40 (SV40) early promoter. The combined promoter and coding region of the selectable marker 804 may have a total length between about 900 base pairs and about 2500 base pairs, for example about 1000 base pairs to about 2200 base pairs, or about 1200 base pairs to about 2000 base pairs. In some variations, the selectable marker 804 may include a Kozak consensus sequence positioned upstream of the start codon to enhance translation efficiency, the sequence length being about 6 base pairs to about 20 base pairs, for example about 8 base pairs to about 15 base pairs, and / or about 10 base pairs to about 12 base pairs.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0118] The third sequence 806 encodes a promoter 808 that drives transcription of the differentiation factor gene 718 or other elements within the vector. The promoter 808 may be constitutive, inducible, or tissue-specific depending on the experimental configuration. Constitutive promoters may include the cytomegalovirus (CMV) immediate early promoter, the elongation factor 1 -alpha (EFla) promoter, or the ubiquitin C (UbC) promoter, each capable of driving continuous expression across diverse cell types. Inducible promoters may include tetracycline-responsive (Tet-On or Tet-Off) systems, metallothionein promoters, or heat shock protein promoters. Tissue-specific promoters may include glial fibrillary acidic protein (GFAP) promoters for astrocyte lineage targeting or neuron-specific enolase (NSE) promoters for neuronal differentiation applications.

[0119] The promoter 808 may have a sequence length between about 400 base pairs and about 2000 base pairs, for example about 500 base pairs to about 1800 base pairs, about 600 base pairs to about 1500 base pairs, or about 700 base pairs to about 1200 base pairs. In embodiments using a bidirectional promoter, such as a Tet-responsive bidirectional promoter, the promoter 808 may have a total length between about 700 base pairs and about 1800 base pairs, for example about 800 base pairs to about 1600 base pairs, and / or about 900 base pairs to about 1400 base pairs, depending on the inclusion of operator binding sites and enhancer domains.

[0120] In some embodiments, the promoter 808 may be positioned upstream of the differentiation factor gene 718 or operably linked to multiple transcription units to allow coexpression of more than one differentiation factor gene. The spacing between the promoter 808 and the transcription start site may be between about 20 base pairs and about 100 base pairs, for example about 30 base pairs to about 80 base pairs, or about 40 base pairs to about 60 base pairs. The promoter 808 may also include an enhancer sequence positioned between about 100 base pairs and about 1000 base pairs upstream of the transcription start site, for example about 200 base pairs to about 800 base pairs, or about 300 base pairs to about 600 base pairs, to facilitate high-level transcription.

[0121] The PiggyBac vector backbone 712 may have a total sequence length between about 6 kilobases and about 12 kilobases, for example about 7 kilobases to about 11 kilobases, or about 8 kilobases to about 10 kilobases. Within the backbone, the second sequence 802 and third sequence 806 may be separated by a spacer region that allows independent transcription of the selectable marker 804 and differentiation factor gene 718. This spacer may have a lengthDocket No. 0138-705.600INTERNATIONAL APPLICATION between about 100 base pairs and about 800 base pairs, for example about 200 base pairs to about 700 base pairs, or about 300 base pairs to about 500 base pairs.

[0122] In another embodiment, the PiggyBac vector backbone 712 may also include additional elements such as a multiple cloning site (MCS), an origin of replication (Ori) for bacterial propagation, or a reporter gene such as green fluorescent protein (GFP) or mCherry for visualization of successful transfection. The origin of replication may have a length between about 400 base pairs and about 1000 base pairs, for example about 500 base pairs to about 900 base pairs, or about 600 base pairs to about 800 base pairs. The reporter gene may range in length between about 600 base pairs and about 1200 base pairs, for example about 700 base pairs to about 1100 base pairs, or about 800 base pairs to about 1000 base pairs.

[0123] Together, the second sequence 802 and third sequence 806 contribute to the functionality of the PiggyBac vector backbone 712 by providing selectable and transcriptional control elements within a single construct. These features support efficient screening, stable gene expression, and reproducible differentiation outcomes when introducing the vector 700 into nonhuman primate iPSCs. The selectable marker 804 allows for positive selection of successfully transfected or integrated cells. In some embodiments, the selectable marker 804 confers resistance to an antibiotic such as puromycin, ampicillin, or neomycin, enabling selective survival of cells that contain the integrated vector. The selectable marker 804 may have a coding region between about 600 base pairs and about 1200 base pairs, for example about 700 base pairs to about 1000 base pairs, and / or about 800 base pairs to about 950 base pairs.

[0124] The promoter 808 drives expression of the inserted differentiation factor gene 718 within the host cells. The promoter 808 may be a constitutive promoter such as a ubiquitin C (UbC) promoter, cytomegalovirus (CMV) promoter, and / or elongation factor- la (EFla) promoter, and / or alternatively, an inducible promoter such as a Tet-responsive promoter. The promoter sequence may have a length of about 400 base pairs to about 1500 base pairs, for example about 500 base pairs to about 1200 base pairs, and / or about 600 base pairs to about 1000 base pairs. The differentiation factor gene 718 is inserted into the PiggyBac vector backbone 712 using molecular cloning methods such as Gibson Assembly 810 and / or restriction enzyme 815 digestion and ligation. In Gibson Assembly, overlapping sequences between the differentiation factor gene 718 and the vector backbone 712 facilitate ligation. The reaction is performed at an isothermal temperature between about 45°C and about 60°C, for example about 48°C to about 55°C, and / or about 50°C to about 52°C. Restriction enzymeDocket No. 0138-705.600INTERNATIONAL APPLICATION cloning may alternatively be used, employing one or more restriction sites flanking the multiple cloning region of the PiggyBac vector backbone 712.

[0125] The differentiation factor gene 718 includes sequences derived from Macaca fascicularis SOX9 825 and Macaca fascicularis NFIB 820, which are essential for the induction of astrocyte differentiation. Each differentiation factor gene may have a sequence length between about 1.0 kilobases and about 3.0 kilobases, for example about 1.2 kilobases to about 2.5 kilobases, and / or about 1.4 kilobases to about 2.0 kilobases. When co-expressed, SOX9 and NFIB initiate transcriptional programs that activate astrocyte-lineage regulatory pathways, including those associated with glial fibrillary acidic protein (GFAP), S100P, and aquaporin-4.

[0126] The PiggyBac vector backbone 712 facilitates stable genomic integration through the action of a transposase that recognizes inverted terminal repeats and transposase recognition sites, allowing for precise excision and insertion of a transposon into the host genome. The terminal inverted repeats may range from about 10 base pairs to about 25 base pairs, for example about 12 base pairs to about 22 base pairs, about 14 base pairs to about 20 base pairs, or about 15 base pairs to about 18 base pairs, providing structural motifs required for transposase binding and catalytic activity. The transposase recognition sites may range from about 150 base pairs to about 500 base pairs, for example about 200 base pairs to about 450 base pairs, about 225 base pairs to about 400 base pairs, or about 250 base pairs to about 350 base pairs, stabilizing the interaction between the transposase and the DNA during the transposition process.

[0127] When the PiggyBac transposase is expressed in the host cell, the enzyme binds to the inverted terminal repeats and cleaves the vector DNA at the boundaries of the transposable element. The excised transposon, which includes the inserted differentiation factor gene and associated regulatory elements, is then inserted at a TTAA target site within the genome. The transposase performs staggered cuts in both donor and acceptor DNA, aligning the overhangs before ligating the transposon into the chromosomal DNA. The result is a stable insertion that maintains the integrity and orientation of the inserted genetic sequence.

[0128] The spacing between the inverted terminal repeats and the transposase recognition sites may range from about 100 base pairs to about 600 base pairs, for example about 150 base pairs to about 500 base pairs, about 200 base pairs to about 400 base pairs, or about 250 base pairs to about 350 base pairs. This spacing supports proper assembly of the transposase-DNA complex and ensures efficient excision and integration kinetics.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0129] The PiggyBac vector backbone is capable of carrying large genetic inserts, typically ranging from about 5 kilobases to about 15 kilobases, for example about 6 kilobases to about14 kilobases, about 7 kilobases to about 12 kilobases, or about 8 kilobases to about 11 kilobases. Larger constructs containing multiple transcriptional units or bidirectional promoters can also be integrated without compromising structural stability. Integration efficiency may fall within a range of about 10 percent to about 60 percent, for example about15 percent to about 50 percent, about 20 percent to about 45 percent, or about 25 percent to about 40 percent, depending on the cell type, DNA amount, and transposase concentration used during transfection. Once integrated, the inserted gene typically remains stably expressed for about 15 cell passages to about 40 cell passages, for example about 18 passages to about 35 passages, about 20 passages to about 30 passages, or about 22 passages to about 28 passages, with minimal risk of epigenetic silencing or structural rearrangement. The integration occurs preferentially at TTAA motifs, which are dispersed throughout transcriptionally active genomic regions, supporting long-term gene expression without disrupting essential endogenous loci.

[0130] The PiggyBac transposon mechanism also permits excision of the integrated sequence upon reintroduction of the transposase, restoring the original genomic sequence. Excision efficiency may range from about 85 percent to about 100 percent, for example about 88 percent to about 98 percent, about 90 percent to about 97 percent, or about 92 percent to about 95 percent, with no detectable residual bases or mutations at the excision site. This transposase-dependent process provides a stable, reversible, and precise means of integrating differentiation factor genes into the genome of nonhuman primate iPSCs. Because the mechanism operates independently of viral infection or replication, it avoids the safety, recombination, and silencing issues typically associated with lentiviral or retroviral delivery systems. The PiggyBac vector backbone therefore supports reproducible and long-term expression of genes such as Macaca fascicularis SOX9 and Macaca fascicularis NFIB, which are involved in the transition of pluripotent cells into neuronal lineages, including astrocytes and induced astrocytes.

[0131] Following transposition, the integrated construct stably expresses the differentiation factor genes in nonhuman primate iPSCs, initiating the differentiation process. The assembled PiggyBac vector 830 may be delivered to nonhuman primate iPSCs via electroporation or chemical transfection. Electroporation may be performed using a voltage between about 800 volts and about 1400 volts, for example about 900 volts to about 1200 volts, or about 1000Docket No. 0138-705.600INTERNATIONAL APPLICATION volts to about 1100 volts. Following electroporation, the cells are recovered in a growth medium and subjected to antibiotic selection to isolate successfully transfected colonies. Differentiation proceeds through activation of astrocyte-lineage pathways, with visible morphological and molecular changes observed within about 10 days to about 30 days, for example about 12 days to about 25 days, or about 14 days to about 21 days post-transfection. The resulting induced astrocytes (iA cells) exhibit hallmark astrocytic features, including stellate morphology and expression of GFAP and S100P markers.

[0132] In some embodiments, the PiggyBac vector backbone 712 corresponds to SEQ ID NO. 1. The vector may further include a bidirectional Tet-responsive promoter positioned between the SOX9 and NFIB genes to allow inducible expression. Alternatively, a constitutive promoter 808 may drive continuous expression of the differentiation factors for stable lineage conversion. The described configuration provides a modular system in which the selectable marker 804 and promoter 808 are integrated components of the PiggyBac vector backbone 712, while the differentiation factor gene 718 is inserted using either restriction enzyme digestion or Gibson Assembly. The combination of these structural and functional features allows efficient transfection, stable integration, and reproducible differentiation of Macaca fascicularis iPSCs into astrocytes and / or induced astrocytes (iA cells).

[0133] FIG. 9 illustrates an embodiment of a vector 900 for differentiating nonhuman primate iPSCs 920 derived from Macaca fascicularis 922 into one or more neuronal lineage cells 924, including astrocytes 926 and induced astrocytes (iA cells) 928. The vector 900 includes a nucleic acid sequence 910 comprising a PiggyBac vector backbone 712 that provides structural and functional elements for stable genomic integration of inserted differentiation factor genes.

[0134] The PiggyBac vector backbone 712 includes one or more terminal inverted repeats 914 and one or more transposase recognition sites 916, which together enable site-specific recognition, excision, and insertion of the vector into a host genome. The inverted repeats 914 are positioned at both ends of the transposable element and provide binding sequences for the PiggyBac transposase enzyme. Each inverted repeat may range from about 10 base pairs to about 25 base pairs, for example about 12 base pairs to about 22 base pairs, about 14 base pairs to about 20 base pairs, or about 15 base pairs to about 18 base pairs in length. The transposase recognition sites 916 provide additional sequence elements that facilitate the integration of the inserted transposon at a TTAA target sequence within the host genome. These regions may have a sequence length between about 200 base pairs and about 500 base pairs, for exampleDocket No. 0138-705.600INTERNATIONAL APPLICATION about 250 base pairs to about 450 base pairs, about 275 base pairs to about 400 base pairs, or about 300 base pairs to about 350 base pairs.

[0135] The nucleic acid sequence 910 further includes a bidirectional Tet-responsive promoter 918 positioned between a first differentiation factor gene and a second differentiation factor gene. The bidirectional Tet-responsive promoter 918 is designed to regulate inducible expression of both genes in opposite transcriptional orientations. In the presence of doxycycline, the Tet-responsive system activates transcription, allowing simultaneous or balanced expression of both differentiation factor genes that drive neuronal lineage specification. The bidirectional promoter may range from about 700 base pairs to about 1800 base pairs, for example about 800 base pairs to about 1600 base pairs, about 900 base pairs to about 1400 base pairs, or about 1000 base pairs to about 1200 base pairs, depending on the number and spacing of Tet operator sequences.

[0136] The first differentiation factor gene and the second differentiation factor gene may include coding sequences corresponding to astrocyte-lineage transcriptional regulators such as Macaca fascicularis SOX9 and Macaca fascicularis NFIB. Each differentiation factor gene may have a sequence length between about 1.0 kilobases and about 3.0 kilobases, for example about 1.2 kilobases to about 2.6 kilobases, about 1.4 kilobases to about 2.2 kilobases, or about 1.5 kilobases to about 2.0 kilobases. When expressed under control of the bidirectional Tet- responsive promoter 918, these genes cooperate to activate astrocyte-lineage regulatory pathways, leading to the expression of markers such as glial fibrillary acidic protein (GFAP), S100P, and aquaporin-4.

[0137] The PiggyBac vector backbone 712 allows for stable genomic integration of the differentiation factor genes through a transposase-mediated mechanism. The transposase recognizes the terminal inverted repeats 914 and transposase recognition sites 916, excises the vector from its plasmid form, and inserts it into a TTAA site within the host cell genome. The integration may occur with an efficiency of about 10 percent to about 60 percent, for example about 15 percent to about 50 percent, about 20 percent to about 45 percent, or about 25 percent to about 40 percent, depending on the concentration of transposase enzyme and the amount of vector DNA introduced into the cells.

[0138] The vector 900 may be introduced into the nonhuman primate iPSCs 920 through electroporation or chemical transfection. Electroporation may be carried out at voltages ranging from about 800 volts to about 1400 volts, for example about 900 volts to about 1200 volts, about 950 volts to about 1100 volts, or about 1000 volts to about 1050 volts, depending on theDocket No. 0138-705.600INTERNATIONAL APPLICATION size of the plasmid and the cell type. Following transfection, the cells may be cultured in a selective medium containing an antibiotic corresponding to the selectable marker encoded within the vector. Selection typically occurs over about 3 days to about 10 days, for example about 5 days to about 9 days, about 6 days to about 8 days, or about 7 days.

[0139] After successful genomic integration and induction with doxycycline, the transfected nonhuman primate iPSCs 920 begin to differentiate into one or more neuronal lineage cells 924, specifically astrocytes 926 and induced astrocytes (iA cells) 928. Differentiation may occur over about 10 days to about 30 days, for example about 12 days to about 25 days, about 14 days to about 22 days, or about 16 days to about 20 days. The resulting cells display astrocytic morphology, including stellate shapes and branching processes, and express astrocyte-specific proteins detectable through immunostaining. The described system allows for temporal and / or spatial control of gene expression through the bidirectional Tet-responsive promoter 918, enabling tunable induction of astrocyte differentiation pathways. The PiggyBac transposon system provides stable and reversible integration, supporting long-term and reproducible generation of astrocytes from Macaca fascicularis iPSCs for use in modeling neurological function, disease studies, or preclinical applications involving nonhuman primate neuronal cells.

[0140] The vector 900 provides technical advantages for differentiating Macaca fascicularis iPSCs into neuronal lineage cells, particularly astrocytes and induced astrocytes (iA cells). The combination of the PiggyBac vector backbone, the bidirectional Tet-responsive promoter, and the integrated differentiation factor genes allows for high-efficiency, stable, and tunable expression of lineage-determining factors in nonhuman primate cells. The PiggyBac vector backbone facilitates stable genomic integration without the limitations of viral-based delivery systems. Because PiggyBac transposition operates through a transposase-mediated, footprint- free mechanism, the vector inserts the transgene precisely at TTAA target sites and can later be removed without leaving residual sequences. This allows researchers to achieve long-term, heritable expression of differentiation factors while maintaining genomic integrity. Integration efficiency may range from about 10 percent to about 60 percent, for example about 15 percent to about 50 percent, or about 20 percent to about 45 percent, resulting in reliable establishment of stably transfected iPSC lines.

[0141] As noted above, the PiggyBac vector backbone facilitates stable genomic integration without the limitations of viral-based delivery systems by relying on a transposase-mediated mechanism that precisely inserts the desired genetic cargo into the host genome throughDocket No. 0138-705.600INTERNATIONAL APPLICATION recognition of specific nucleotide sequences. Unlike viral systems such as lentivirus or retrovirus, which integrate genetic material through reverse transcription and semi-random insertion events that can disrupt endogenous genes or regulatory regions, the PiggyBac system performs targeted and reversible integration using a defined TTAA sequence as the insertion site. In operation, the PiggyBac transposase recognizes the terminal inverted repeats located at both ends of the vector backbone and binds to these motifs to form a DNA-transposase complex that mediates the excision and integration of the transposable element. The transposase identifies these inverted repeat sequences, which are typically between about 10 base pairs and about 25 base pairs in length, for example about 12 base pairs to about 22 base pairs, about 14 base pairs to about 20 base pairs, or about 15 base pairs to about 18 base pairs, by their highly conserved nucleotide arrangement that defines the transposon boundaries. Upon binding, the transposase undergoes a conformational change that aligns its catalytic domains with the DNA ends, creating a synaptic complex that positions both termini of the transposon for coordinated cleavage.

[0142] The enzyme then performs double-stranded breaks at the junctions between the inverted repeats and the flanking donor DNA, thereby excising the transposon as a linear DNA fragment. During this process, the transposase introduces staggered nicks at the TTAA target sequences in the host genome, typically separated by four nucleotides, to create compatible single-stranded overhangs for integration. The excised transposon is subsequently ligated into these TTAA sites through a cut-and-paste mechanism that restores genomic continuity and inserts the transgene precisely at the integration locus.

[0143] Formation of the DNA-transposase complex may occur in a concentration-dependent manner, with optimal complex assembly achieved at a transposase-to-DNA ratio ranging from about 1 :1 to about 3: 1, for example about 1.5: 1 to about 2.5: 1, or about 2: 1. The stability of the complex is further influenced by ionic conditions, such as magnesium ion concentrations ranging from about 0.5 mM to about 5 mM, for example about 1 mM to about 4 mM, or about 2 mM to about 3 mM, which promote active-site coordination during the transposition reaction.

[0144] The PiggyBac transposase exhibits high catalytic activity across a broad range of temperatures, generally from about 30 degrees Celsius to about 39 degrees Celsius, for example about 32 degrees Celsius to about 37 degrees Celsius, or about 34 degrees Celsius to about 36 degrees Celsius, making it suitable for mammalian cell applications. Once integration occurs, the inserted sequence becomes stably incorporated into the host genome without disrupting neighboring loci. The process is reversible; subsequent reintroduction of the transposaseDocket No. 0138-705.600INTERNATIONAL APPLICATION excises the transposon by cleaving at the same inverted repeats, restoring the original TTAA sequence without introducing mutations or insertions at the excision site.

[0145] This coordinated recognition, cleavage, and integration mechanism allows for efficient genomic incorporation of large DNA fragments, typically ranging from about 5 kilobases to about 15 kilobases, for example about 6 kilobases to about 14 kilobases, or about 7 kilobases to about 12 kilobases, while maintaining sequence fidelity and integration stability. The ability of the PiggyBac transposase to bind both termini, catalyze excision, and precisely insert transgenes through a single enzymatic cycle provides a reliable non-viral platform for stable genetic modification in nonhuman primate induced pluripotent stem cells.

[0146] This complex excises the transposon region, which contains the differentiation factor genes and associated promoter elements, from the plasmid donor and inserts it directly into a TTAA site within the host genome. Because TTAA sites are evenly distributed throughout the genome, integration occurs in regions compatible with stable expression without causing chromosomal rearrangements or random integration artifacts. The transposase then ligates the transposon precisely into the chromosomal DNA, preserving the orientation and sequence integrity of the inserted material.

[0147] This process enables the PiggyBac system to achieve high-efficiency genomic integration while avoiding the need for viral packaging, infection, and reverse transcription steps. The result is a clean, non-viral method that eliminates the risks of vector recombination, replication-competent viral reactivation, and unpredictable insertional mutagenesis. Furthermore, because PiggyBac transposition occurs entirely in the nucleus and does not rely on cellular proliferation for integration, it is suitable for both dividing and non-dividing cells, a property that is often a limiting factor for viral vectors. The PiggyBac system also supports footprint-free excision, meaning that the integrated transposon can be removed from the genome by reintroducing the transposase without leaving behind residual sequences or mutations at the excision site. This reversibility allows for transient or conditional expression studies, providing flexibility for both short-term experiments and long-term stable cell line generation. Integration stability can persist for more than about 20 cell passages to about 40 cell passages, for example about 25 to about 35 passages, without measurable transgene silencing or loss of copy number.

[0148] In addition to its high stability and reversibility, the PiggyBac backbone supports large genetic cargo sizes, enabling integration of fragments ranging from about 5 kilobases to about 15 kilobases, for example about 7 kilobases to about 13 kilobases, or about 8 kilobasesDocket No. 0138-705.600INTERNATIONAL APPLICATION to about 12 kilobases, depending on the complexity of the inserted sequences. This exceeds the typical cargo capacity of lentiviral vectors, which is limited to approximately 8 kilobases. The PiggyBac transposition mechanism thus accommodates larger promoter regions, multiple gene cassettes, and selectable markers within a single construct while maintaining integration efficiency. By combining precise transposase recognition, high cargo tolerance, and reversible site-specific integration, the PiggyBac vector backbone provides a stable, controllable, and non-viral alternative to conventional delivery methods. This stability ensures consistent and long-term expression of differentiation factor genes such as Macaca fascicularis SOX9 and Macaca fascicularis NFIB, enabling reproducible induction of neuronal lineage differentiation in Macaca fascicularis iPSCs without the genetic instability and safety challenges associated with viral vectors.

[0149] The bidirectional Tet-responsive promoter provides precise temporal and quantitative control of gene expression. By regulating transcription in opposite orientations, this promoter enables simultaneous induction of two differentiation factor genes, such as Macaca fascicularis SOX9 and Macaca fascicularis NFIB. These genes act in coordination to activate astrocytelineage regulatory pathways that lead to the formation of astrocytic phenotypes. Because the system responds to doxycycline, researchers can control when differentiation begins and to what extent the differentiation factors are expressed, improving experimental reproducibility and reducing off-target lineage activation.

[0150] The system’s modular configuration allows for flexible cloning and rapid modification using either restriction enzyme-based ligation or Gibson Assembly. This flexibility permits replacement of differentiation factor genes or regulatory elements without redesigning the entire construct. Each component, the promoter, transposase recognition sites, and inverted repeats, can be interchanged with variants optimized for different nonhuman primate species or for specific research purposes. This modular compatibility enhances adaptability for various applications, including disease modeling and drug screening using nonhuman primate neuronal cells. Compared to lentiviral and / or retroviral systems, the PiggyBac-based approach minimizes the risk of insertional mutagenesis and transgene silencing. Because the system does not rely on viral packaging or reverse transcription, there is no viral genome integration, random concatemer formation, or reactivation of replication- competent particles. The non-viral nature of the PiggyBac vector also simplifies regulatory compliance for translational research, as it reduces biosafety concerns associated with viral delivery.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0151] When applied to Macaca fascicularis iPSCs, the vector achieves consistent and efficient differentiation into astrocytes and iA cells with characteristic morphological and molecular features, including stellate structures and expression of GFAP, SlOOp, and ALDH1L1. The induced astrocytes generated through this system provide a physiologically relevant platform for studying neurodevelopment, neurodegeneration, and cell-cell interactions in primate models. In some embodiments, the technical advantages of the vector 900 include high transfection efficiency, stable genomic integration with minimal off-target effects, reversible insertion capability, controlled inducible expression of lineage-defining genes, and / or broad flexibility for nonhuman primate neuronal differentiation applications. These features collectively provide a robust and precise genetic engineering platform for producing astrocyte-lineage cells from Macaca fascicularis iPSCs in both research and / or preclinical contexts.

[0152] EXAMPLES

[0153] FIG. 10A shows an immunofluorescence micrograph 1002 of Macaca fascicularis induced pluripotent stem cells (iPSCs) differentiated into astrocyte cells at day in vitro 7 (DIV7). The staining includes DAPI, which marks the cell nuclei, and TUJ1, used here to visualize neurite-like extensions characteristic of early-stage astrocytic differentiation. DAPI is a fluorescent DNA-binding dye that intercalates into adenine-thymine-rich regions of the minor groove, producing bright nuclear fluorescence and enabling visualization of total cell number, nuclear morphology, and spatial distribution of cells within the culture. TUJ1 refers to an antibody recognizing class III P-tubulin, a cytoskeletal protein expressed in cells of neuronal lineage and, in certain early differentiation contexts, in cells exhibiting neurite-like cytoskeletal organization. TUJ1 staining highlights filamentous microtubule structures, including, but not limited to, cell bodies, primary processes, and extended branching networks. The combined use of DAPI and TUJ1 permits simultaneous assessment of nuclear localization and cytoskeletal architecture, allowing evaluation of lineage commitment, process development, and overall structural organization in differentiated cell populations. As used herein, the term “DAPI” refers to 4',6-diamidino-2-phenylindole, a fluorescent DNA-binding dye commonly used in cell and tissue imaging. DAPI intercalates into the minor groove of double-stranded DNA with a preference for adenine-thymine-rich regions and emits a blue fluorescence when excited by ultraviolet light. DAPI staining is used to visualize and quantify cell nuclei, assess nuclear morphology, and identify total cell number in fixed or live samples. The developing astrocytes display elongated and branched processes extending from the cellDocket No. 0138-705.600INTERNATIONAL APPLICATION bodies, forming interconnected networks. The image demonstrates efficient induction of astrocytic lineage cells from iPSCs within seven days of differentiation, with extensive process formation and cell clustering indicative of early astrocytic maturation. The scale bar represents 100 micrometers.

[0154] FIG. 10B shows an immunofluorescence micrograph 1004 of Macaca fascicularis induced pluripotent stem cells (iPSCs) differentiated into astrocyte cells at day in vitro 14 (DIV14). The staining includes DAPI for nuclear visualization and TUJ1 to identify filamentous cytoskeletal networks that persist during astrocytic maturation. At this stage, the astrocytes exhibit extensive interwoven processes forming dense cellular networks, reflecting ongoing morphological and structural maturation. The increased density and complexity of these processes compared to earlier time points indicate progressive differentiation and stabilization of astrocytic identity. The image demonstrates continued expansion and organization of the astrocytic culture by DIV14. The scale bar represents 100 micrometers.

[0155] FIG. 10C shows an immunofluorescence micrograph 1006 of Macaca fascicularis induced pluripotent stem cells (iPSCs) differentiated into astrocyte cells at day in vitro 21 (DIV21). The staining includes DAPI for nuclear visualization and TUJ1 to mark cytoskeletal filaments and process extensions. At this later stage of differentiation, the astrocyte culture displays a dense and intricate meshwork of interconnected cellular processes spanning the imaging field. The increased structural density and the overlapping, reticulated morphology reflect advanced maturation and stabilization of astrocytic phenotypes. Compared to earlier time points, the astrocytes exhibit more elaborate branching and tighter intercellular associations, characteristic of mature astrocyte networks in vitro. The scale bar represents 100 micrometers.

[0156] High purity was observed among the differentiated cell populations, with greater than 95 percent of cells exhibiting TUJ1 -positive immunoreactivity at day in vitro 7 (DIV7). This level of purity indicates that a significant proportion of the cells successfully adopted neuronal or astrocytic lineage characteristics within the first week of differentiation, reflecting a high degree of transcriptional activation and cytoskeletal remodeling associated with early lineage commitment. TUJ1 staining revealed extensive filamentous networks and defined cell bodies, confirming expression of class III P-tubulin as an indicator of mature cytoskeletal assembly.

[0157] Electrophysiological activity (E-Phys) was detected using microelectrode array (MEA) recordings from DIV7 through DIV21, demonstrating the development and persistence of functional excitability in the differentiated cultures. Spontaneous spike trains were recordedDocket No. 0138-705.600INTERNATIONAL APPLICATION across multiple electrodes as early as DIV7, indicating rapid establishment of electrically active cell populations. Over the subsequent two weeks, the cultures exhibited progressive increases in firing frequency, spike amplitude, and interconnectivity, consistent with network maturation and synaptic coupling. MEA recordings revealed both spontaneous and synchronized burst activity, suggesting that the cells had formed functional networks capable of coordinated electrical signaling.

[0158] The sustained electrophysiological activity across DIV7 to DIV21 confirms that the differentiated astrocytes and associated neuronal lineage cells developed stable membrane potentials and ion channel functionality. This functional profile supports the conclusion that the differentiation protocol not only achieves morphological and molecular fidelity but also yields physiologically competent cells suitable for downstream neurobiological and pharmacological applications.

[0159] FIG. 11 shows a phase-contrast image 1102 of Macaca fascicularis induced pluripotent stem cells (iPSCs) differentiated into astrocyte cells between day in vitro 14 (DIV14) and day in vitro 21 (DIV21). The astrocytes display elongated and stellate morphologies with extended cytoplasmic processes forming an extensive interconnected network characteristic of mature astrocytic cultures. The cells adhere to the substrate and exhibit branching and overlapping processes, creating a dense, reticulated pattern across the imaging field. The increased process complexity and organization relative to earlier time points indicate progressive cytoskeletal maturation and stable astrocytic phenotype development. The homogeneity of the culture and the absence of undifferentiated or proliferative morphologies confirm successful differentiation and maintenance of astrocytic identity under the applied culture conditions.

[0160] FIG. 12 shows a quantitative analysis bar graph 1202 of TUJl-positive cell populations derived from Macaca fascicularis induced pluripotent stem cells (iPSCs) during differentiation into astrocyte cells. The y-axis represents the percentage of TUJl-positive cells, and the x-axis denotes the time points measured at day in vitro 7 (DIV7), day in vitro 14 (DIV14), and day in vitro 21 (DIV21). The results indicate a high proportion of TUJl-positive cells (>95 percent) at DIV7, demonstrating early lineage specification and strong expression of the class III P-tubulin cytoskeletal marker during the initial stages of differentiation. The percentage remains above approximately 85 percent at DIV14, reflecting continued expression of TUJ1 during intermediate network formation.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0161] At DIV21, the percentage decreases to above approximately 70 percent. This reduction is consistent with the expected maturation-dependent shift in cytoskeletal marker profiles as astrocyte-lineage cells progress toward later developmental states. TUJ1 is highly expressed during early lineage transitions and robust neurite extension, but its expression can decline as astrocyte-lineage cells remodel their cytoskeleton, reduce neurite-like projections, and transition toward more stabilized glial morphologies. Additionally, increases in total cell count by DIV21 result in a larger denominator for the TUJ1 -positive fraction, and the emergence of GF AP -negative, non-neuronal astrocyte-lineage populations can further reduce the proportion of TUJ1 -positive cells. Together, these factors explain the observed decrease in TUJ1 -positive percentage at DIV21 relative to earlier timepoints.

[0162] The data confirm efficient and stable induction of astrocytic lineage cells expressing TUJ1 during early and mid-stage differentiation, with some expected reduction in TUJ1 expression as astrocytes mature and reorganize their cytoskeletal profile. Each bar represents the mean value from multiple biological replicates, and error bars indicate standard deviation.

[0163] FIG. 13 shows quantitative measurements of a bar graph 1302 of total neurite length per well obtained from Macaca fascicularis iPSCs undergoing differentiation into astrocyte cells. The y-axis represents total neurite length per well, and the x-axis represents the days in vitro (DIV7, DIV14, DIV21). The data demonstrate a progressive increase in total neurite length from approximately 1.5>< 106micrometers per well at DIV7 to approximately 3.0x l06micrometers per well at DIV14, with sustained network complexity around 2.8x 10® micrometers per well at DIV21. This trend indicates active process extension and stabilization of cytoskeletal architecture over the 21 -day differentiation period. The increase in neurite length corresponds to astrocytic process elaboration and network maturation, consistent with the morphological development observed in FIGs. 10A-10C. Each bar represents the mean value of multiple biological replicates, and error bars denote standard deviation.

[0164] FIG. 14 shows a quantitative analysis bar graph 1402 of total cell number, represented as nuclei per well, in Macaca fascicularis iPSC-derived astrocyte cultures across day in vitro 7 (DIV7), day in vitro 14 (DIV14), and day in vitro 21 (DIV21). As used herein, the terms day in vitro 7 (DIV7), day in vitro 14 (DIV14), and day in vitro 21 (DIV21) refer to the number of days that a cell culture has been maintained under in vitro conditions following the initiation of a differentiation protocol. DIV7 denotes seven days of continuous culture from the start of differentiation, DIV14 denotes fourteen days of culture, and DIV21 denotes twenty-one days of culture. These time points correspond to defined stages of lineage progression,Docket No. 0138-705.600INTERNATIONAL APPLICATION morphological development, and maturation within the differentiation timeline. DIV7 generally represents an early differentiation stage, DIV14 represents an intermediate stage, and DIV21 represents a later or more advanced stage of cellular maturation, process elaboration, and network development.

[0165] The y-axis indicates the total number of nuclei per well, corresponding to cell count, while the x-axis indicates the differentiation time point. The results demonstrate an increase in total cell number over time, with approximately 3,000 to 4,000 cells per well at DIV7, 4,000 to 5,000 cells per well atDIV14, and 8,000 to 12,000 cells per well atDIV21. The data indicate progressive proliferation and survival of differentiated astrocytes throughout the 21 -day culture period. The observed increase in cell count correlates with process expansion and structural maturation of the astrocytic network shown in FIGs. 10A-10C. Each bar represents the mean of multiple biological replicates, and error bars denote standard deviation.

[0166] The data illustrated in FIGs. 15A through 17B demonstrate the morphological progression, coculture behavior, and maturation characteristics of NGN2-induced neurons and astrocyte-lineage cells generated under the disclosed differentiation and maturation conditions. From day 0 to day 7, cells were cultured in a differentiation medium composed of Neurobasal- B27 base supplemented with 1 percent FBS, bFGF, CNTF, BMP4, doxycycline, and ROCK inhibitor (day 0 only). This early-stage environment facilitated the induction of neuronal and astrocytic differentiation programs while supporting initial survival and lineage commitment.

[0167] Beginning on day 7, cells were transitioned to a maturation medium containing Neurobasal-N2 base supplemented with EGF, CNTF, BMP4, cAMP, and continued doxycycline administration. This medium supported progressive maturation, neurite extension, and functional integration of neuronal and astrocyte-lineage populations over later timepoints.

[0168] FIGs. 15A and 15B depict NGN2-derived neuronal cells cultured in the presence of differentiating astrocyte-lineage cells at two stages. FIG. 15A shows NGN2 neurons at DIV7 paired with astrocyte-lineage cells at DIV14, while FIG. 15B shows NGN2 neurons at DIV14 paired with astrocyte-lineage cells at DIV21. Across these images, the astrocyte-lineage cells exhibit flat, elongated morphologies consistent with early and intermediate differentiation states, whereas NGN2 neurons show progressive neurite development over the two timepoints.

[0169] FIGs. 16A and 16B depict astrocyte-lineage populations alone at corresponding maturation stages. The morphological shift from DIV14 (FIG. 16A) to DIV21 (FIG. 16B) demonstrates increased density and spreading, although immunocytochemical analysis confirmed that the astrocyte-lineage cells did not express GFAP at any timepoint. This lack ofDocket No. 0138-705.600INTERNATIONAL APPLICATIONGFAP expression indicates that the cells occupy an early or alternative astrocytic lineage state rather than fully mature GFAP-positive astrocytes.

[0170] FIGs. 17A and 17B illustrate cocultures ofNGN2 neurons and astrocyte-lineage cells, highlighting the influence of astrocyte maturation on neuronal network organization. AtNGN2 DIV7 and Astro DIV14 (FIG. 17A), the coculture displays early neuronal-glial interactions with moderate neurite outgrowth. By NGN2 DIV14 and Astro DIV21 (FIG. 17B), the coculture shows markedly increased cell density, more extensive neuritic networks, and more developed astrocyte-lineage morphologies supporting enhanced structural integration. Thus, the illustrations in FIGs. 15A-17B demonstrate that the staged differentiation and maturation media promote time-dependent morphological changes in both neuronal and astrocyte-lineage populations while confirming that astrocyte-lineage cells remain GFAP-negative throughout the measured period. The images further show that later-stage astrocyte-lineage cells support denser and more complex neuronal networks in coculture, consistent with functional maturation under the described culture conditions.

[0171] As noted above, FIG. 15A shows a phase-contrast image 1502 of a coculture of NGN2-induced neurons (iN cells) at day in vitro 7 (NGN2 DIV7) and astrocyte cells differentiated at day in vitro 14 (Astro DIV14). At this stage, the NGN2 cells exhibit early neuronal morphology with compact cell bodies and emerging neurite extensions. The astrocyte cells in the coculture display larger, more flattened somas and early process formation characteristic of mid-stage astrocytic differentiation. The combined culture environment supports early neuron-astrocyte interactions, with initial process contact and network establishment visible throughout the image field.

[0172] FIG. 15B shows a phase-contrast image 1504 of a later coculture time point of NGN2- induced neurons at day in vitro 14 (NGN2 DIV14) and astrocyte cells differentiated at day in vitro 21 (Astro DIV21). The NGN2 cells display elongated processes and more defined neuronal morphology compared to earlier stages, while the astrocyte cells exhibit expanded somas and increased process density consistent with later-stage astrocytic maturation. The coculture demonstrates more extensive neurite-astrocyte interactions and a denser cellular network, reflecting coordinated maturation of both cell populations.

[0173] FIG. 16A shows a phase-contrast image 1602 of a coculture containing NGN2- induced neurons at day in vitro 7 and astrocyte cells at day in vitro 14. At this early coculture stage, the NGN2 cells exhibit small, rounded somas with emerging neurite extensions, while the astrocyte cells display flattened, polygonal morphologies typical of mid-stage astrocyticDocket No. 0138-705.600INTERNATIONAL APPLICATION differentiation. The early neurites make initial contacts with nearby astrocytic processes, indicating the beginning of neuron-astrocyte interactions within the maturing culture.

[0174] FIG. 16B shows a phase-contrast image 1604 of a later coculture containing NGN2- induced neurons at day in vitro 14 and astrocyte cells at day in vitro 21. At this stage, the NGN2 cells exhibit more elongated processes and maturing neuronal morphology, and the astrocyte cells display greater process complexity and more pronounced branching consistent with advanced astrocytic maturation. The coculture presents a denser, more interconnected network, reflecting increased structural integration and coordinated development between the neuronal and astrocytic cell populations.

[0175] FIG. 17A shows an image 1702 of a coculture of NGN2-induced neurons at DIV7 with astrocyte cells at DIV14. At this stage, the coculture contains early-stage NGN2 neurons displaying small somas, limited neurite extension, and sparse network formation. The astrocyte population exhibits intermediate differentiation characteristics, including enlarged somas and broad, flattened morphologies. The neuronal and astrocytic populations begin forming initial points of contact, and the field shows moderate cell density typical of developing mixed cultures.

[0176] FIG. 17B shows an image 1704 of a coculture of NGN2-induced neurons at DIV 14 with astrocyte cells at DIV21. In contrast to FIG. 17A, the coculture exhibits substantially higher overall cell density. The NGN2 DIV14 neurons demonstrate more extensive neurite outgrowth and increased network complexity, while the DIV21 astrocytes show advanced maturation with more elaborate branching and a denser mesh of glial processes. The image reflects a more structurally integrated coculture environment with widespread neuron-glia interactions and a confluent appearance across the field of view.

[0177] Embodiment 1. A vector for differentiating nonhuman primate iPSCs from Macaca fascicularis into astrocytes, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a PiggyBac vector backbone comprising: one or more terminal inverted repeats; and one or more transposase recognition sites for genomic integration of an inserted sequence; and a differentiation factor gene integrated within the piggyBac vector backbone, the differentiation factor gene configured to induce astrocyte differentiation through an activation of one or more astrocyte-lineage regulatory pathways and an expression of one or more molecular features associated with astrocytic maturation.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0178] Embodiment 2. The vector of embodiment 1, wherein the PiggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter.

[0179] Embodiment 3. The vector of embodiment 1, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using Gibson Assembly.

[0180] Embodiment 4. The vector of embodiment 1, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using restriction enzymes.

[0181] Embodiment 5. The vector of embodiment 1, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

[0182] Embodiment 6. The vector of embodiment 1, wherein differentiating comprises transfecting.

[0183] Embodiment 7. The vector of embodiment 1, wherein the differentiation factor gene is SEQ ID NO. 2 and SEQ ID NO. 3.

[0184] Embodiment 8. The vector of embodiment 1, wherein the differentiation factor gene comprises Macaca fascicularis SOX9 and Macaca fascicularis NFIB

[0185] Embodiment 10. The vector of embodiment 1, wherein the nonhuman primate iPSCs are differentiated into iA cells.

[0186] Embodiment 11. The vector of embodiment 1, wherein the PiggyBac vector backbone comprises SEQ ID NO. 1.

[0187] Embodiment 12. The vector of embodiment 1, wherein the PiggyBac vector backbone comprises a PiggyBac vector.

[0188] Embodiment 13. A vector for differentiating nonhuman primate iPSCs to one or more neuronal lineage cells, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a PiggyBac vector backbone; and a differentiation factor gene configured to promote differentiation of the one or more neuronal lineage cells.

[0189] Embodiment 14. The vector of embodiment 12, wherein the PiggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter.

[0190] Embodiment 15. The vector of embodiment 12, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using Gibson Assembly.

[0191] Embodiment 16. The vector of embodiment 12, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using restriction enzymes.Docket No. 0138-705.600INTERNATIONAL APPLICATION

[0192] Embodiment 17. The vector of embodiment 12, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

[0193] Embodiment 18. The vector of embodiment 12, wherein differentiating comprises transfecting.

[0194] Embodiment 19. The vector of embodiment 12, wherein the differentiation factor gene is SEQ ID NO. 2 and SEQ ID NO. 3.

[0195] Embodiment 20. The vector of embodiment 12, wherein the differentiation factor gene comprises Macaca fascicularis SOX9 and Macaca fascicularis NFIB.

[0196] Embodiment 22. The vector of embodiment 12, wherein the one or more neuronal lineage cells comprising at least one of astrocytes or induced astrocytes (iA cells). 23.

[0197] Embodiment 24. A vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis to one or more neuronal lineage cells, the vector comprising a nucleic acid sequence comprising: a piggyBac vector backbone comprising: one or more terminal inverted repeats; and one or more transposase recognition sites for genomic integration of an inserted sequence; and a bidirectional Tet-responsive promoter positioned between a first differentiation factor gene and a second differentiation factor gene, the first differentiation factor gene and the second differentiation factor gene are configured to be inserted into the piggyBac vector backbone for the nonhuman primate iPSCs to the one or more neuronal lineage cells.

[0198] Embodiment 25. The vector of embodiment 21, wherein the one or more neuronal lineage cells comprise at least one of astrocytes or induced astrocytes (iA cells).

[0199] References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” “some embodiments,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0200] As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. For example, the term “cell” may include, and is contemplated to include, a plurality of cells. At times, theDocket No. 0138-705.600INTERNATIONAL APPLICATION claims and disclosure may include terms such as “a plurality,” “one or more,” or “at least one;” however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived.

[0201] The term “about” or “approximately,” when used before a numerical designation or range (e.g., to define a length or pressure), indicates approximations which may vary by ( + ) or ( - ) 5%, 1% or 0.1%. All numerical ranges provided herein are inclusive of the stated start and end numbers. The term “substantially” indicates mostly (i.e., greater than 50%) or essentially all of a device, substance, or composition.

[0202] As used herein, the term “comprising” or “comprises” is intended to mean that the devices, systems, and methods include the recited elements, and may additionally include any other elements. “Consisting essentially of’ shall mean that the devices, systems, and methods include the recited elements and exclude other elements of essential significance to the combination for the stated purpose. Thus, a system or method consisting essentially of the elements as defined herein would not exclude other materials, features, or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of’ shall mean that the devices, systems, and methods include the recited elements and exclude anything more than a trivial or inconsequential element or step. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0203] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. Other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

Docket No. 0138-705.600INTERNATIONAL APPLICATIONCLAIMSWHAT IS CLAIMED IS:

1. A vector for differentiating nonhuman primate iPSCs from Macaca fascicularis into astrocytes, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a PiggyBac vector backbone comprising: one or more terminal inverted repeats; and one or more transposase recognition sites for genomic integration of an inserted sequence; and a differentiation factor gene integrated within the piggyBac vector backbone, the differentiation factor gene configured to induce astrocyte differentiation through an activation of one or more astrocyte-lineage regulatory pathways and an expression of one or more molecular features associated with astrocytic maturation.

2. The vector of claim 1, wherein the PiggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter.

3. The vector of claim 1, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using Gibson Assembly.

4. The vector of claim 1, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using restriction enzymes.

5. The vector of claim 1, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

6. The vector of claim 1, wherein differentiating comprises transfecting.

7. The vector of claim 1, wherein the differentiation factor gene is SEQ ID NO. 2 and SEQ ID NO. 3.

8. The vector of claim 1, wherein the differentiation factor gene comprises Macaca fascicularis SOX9 and Macaca fascicularis FIB.

9. The vector of claim 1, wherein the nonhuman primate iPSCs are differentiated into iA cells.Docket No. 0138-705.600INTERNATIONAL APPLICATION10. The vector of claim 1, wherein the PiggyBac vector backbone comprises SEQ ID NO.1.

11. The vector of claim 1, wherein the PiggyBac vector backbone comprises a PiggyBac vector.

12. A vector for differentiating nonhuman primate iPSCs to one or more neuronal lineage cells, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a PiggyBac vector backbone; and a differentiation factor gene configured to promote differentiation of the one or more neuronal lineage cells.

13. The vector of claim 12, wherein the PiggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter.

14. The vector of claim 12, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using Gibson Assembly.

15. The vector of claim 12, wherein the differentiation factor gene is configured to be inserted into the PiggyBac vector backbone using restriction enzymes.

16. The vector of claim 12, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

17. The vector of claim 12, wherein differentiating comprises transfecting.

18. The vector of claim 12, wherein the differentiation factor gene is SEQ ID NO. 2 and SEQ ID NO. 3.

19. The vector of claim 12, wherein the differentiation factor gene comprises Macaca fascicularis SOX9 and Macaca fascicularis FIB.

20. The vector of claim 12, wherein the one or more neuronal lineage cells comprise at least one of astrocytes or induced astrocytes (iA cells).

21. A vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis to one or more neuronal lineage cells, the vector comprising a nucleic acid sequence comprising: a piggyBac vector backbone comprising: one or more terminal inverted repeats;Docket No. 0138-705.600 INTERNATIONAL APPLICATION one or more transposase recognition sites for genomic integration of an inserted sequence; and a bidirectional Tet-responsive promoter positioned between a first differentiation factor gene and a second differentiation factor gene, the first differentiation factor gene and the second differentiation factor gene are configured to be inserted into the piggyBac vector backbone for the nonhuman primate iPSCs to the one or more neuronal lineage cells.

22. The vector of claim 21, wherein the one or more neuronal lineage cells comprise at least one of astrocytes or induced astrocytes (iA cells).

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