Differentiation of pluripotent stem cells

Abstract

Methods for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) by contacting hiPSCs with a viral vector including polynucleotide sequences encoding ETS2 and / or ETV2, seeding the hiPSCs on a cell culture plate in the presence of a first cell culture media to provide plated hiPSCs, exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase inhibitor and culturing the seeded hiPSCs, replacing the second cell culture media with a third cell culture media including VEGF-A and a TGFβ inhibitor, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs, reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising VEGF-C; and expanding the induced hiPSCs to provide differentiated LEC cells.

Description

[0001] DIFFERENTIATION OF PLURIPOTENT STEM CELLS RELATED APPLICATIONS The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 687,722 filed August 27, 2024, the entire disclosure of which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERAL FUNDING This invention was made with government support under grant 2047903 awarded by the National Science Foundation (NSF) and grant R35 GM143055 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND OF THE INVENTION The lymphatic system, a crucial component of the vascular network, plays an indispensable role in maintaining tissue fluid homeostasis, immune cell trafficking, and lipid absorption. Dysfunction or impairment of lymphatic vessels underlies various pathological conditions spanning neurodegenerative diseases, metabolic syndrome, cardiovascular ailments, and lymphedema. Despite its critical role, therapeutic and disease modeling strategies focused on generating new lymphatic vessels remain relatively underexplored. One promising avenue in addressing this gap lies in harnessing human induced pluripotent stem cells (hiPSCs) for the differentiation and generation of lymphatic endothelial cells (LECs). The development of efficient protocols for generating lymphatic endothelial cells from hiPSCs is crucial for advancing our understanding of lymphatic biology and for exploring new therapeutic approaches for lymphatic-related diseases. While considerable progress has been made in directing hiPSCs toward blood endothelial cells, the protocols to effectively differentiate hiPSCs into LECs have been somewhat elusive, hindering advancements in lymphatic-related research and therapeutic interventions. There exists a handful of differentiation protocol to generate LEC from hiPSCs mostly relying on embryonic body (EB) intermediate or the use of murine feeder layers. Existing protocols, however, are far from optimal. Limitations stem from the inherent complexity associated with improper cell aggregation and interaction with extraneous cell population. These results in very low yield and poor isolation of final differentiated cells. Also, these protocols involve prolonged culture periods and lacks definite functional behavior. 1

[0002] 501.113WO1 ND 25-008 Accordingly, there is a need for new methods for providing sources of LECs for tissue transplantation and the treatment of lymphatic diseases and disorders. The present disclosure satisfies these needs. SUMMARY OF THE INVENTION Transcription factors, particularly ETS2 and ETV2, have emerged as pivotal regulators governing embryonic vascular development, including the specification of lymphatic endothelial cells. ETV2 has shown promise in generating blood endothelial cells efficiently. One recent study even shows that ETV2 is required for lymphangiogenesis and directly regulates VEGFR3 / FLT4 expression. Using in vitro differentiated mouse embryonic stem cells, ETV2 ChIP-Seq analysis revealed specific ETV2 binding peaks present within VEGFR3 and LYVE1 promoter / enhancer regions. The VEGFR3 promoter is a likely direct target of ETV2, containing an evolutionary conserved FOX: ETS domain that is bound by ETV2 and FOXC2 transcription factors. On the other hand, the expression and colocalization of ETS2 was identified in the nuclei of LECs. In addition, the work highlights the synergistic enhancement of ETS2 and PROX1 in expression of VEGFR3. Consistent with the effects on expression profile of VEGFR3, ETS2 induces LEC migration towards VEGF-C. In summary, ETS2 is reported as a pivotal pro-lymphangiogenic factor in collaboration with PROX1 during lymphangiogenesis. Though a fair number of studies exist to show that ETV2 and ETS2 are important regulatory components of LECs, the potential of these factors in the differentiation process toward lymphatic endothelial cells remains unexplored. This study aims to address this gap by presenting a novel methodology that reliably and efficiently generates LECs from diverse hiPSC lines using transcription factors - ETS2 and ETV2. The critical temporal activation of ETV2 and ETS2 is emphasized in this protocol, as it enables the essential interaction with Prox1, a master regulator pivotal in lymphatic specification. The resulting differentiated LECs express key lymphatic markers such as VEGFR-3, LYVE-1, and Podoplanin, exhibiting expression levels comparable to mature LECs. Moreover, the differentiated LECs, following 86 three days of culture in 3D hydrogels, demonstrate their capacity to assemble into stable lymphatic vascular networks in vitro. Additionally, these cells exhibit the ability to secrete reelin, a crucial lymphangiocrine, further highlighting their functional attributes. Overall, this protocol not only offers significant implications for advancing our fundamental understanding of lymphatic biology but also holds substantial promise for various applications, including approaches in lymphatic regeneration and personalized medicine. The methodology detailed herein lays a robust foundation for 2

[0003] 501.113WO1 ND 25-008 further exploration and development of targeted therapies for a spectrum of lymphatic-related disorders and diseases. Accordingly, a method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) and a transforming growth factor β (TGFβ) inhibitor, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising VEGF-C or VEGF-C156S; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. In another embodiment, a method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor in an amount of about 0.1 µM to 20 µM and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) in an amount of about 0.1 ng / mL to about 20 ng / mL and a transforming growth factor β (TGFβ) inhibitor in an amount of about 0.1 µM to 20 µM, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising about 25 ng / mL to about 250 ng / mL VEGF-C or VEGF-C156S; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying 3

[0004] 501.113WO1 ND 25-008 claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention. Fig.1. Schematic of deriving LECs from hiPSCs. The cultured hiPSCs were transduced with viral vector containing specific transcription factor and seeded onto matrigel-coated plate. Next, the transcription factors were activated, and cells were cultured in differentiation media. Then differentiated ECs were seeded onto fibronectin-coated plates and treated with VEGF-C for 3 more days to make them committed to lymphatic endothelial lineage. The second row represents bright-field images of the corresponding cell states. The size bar corresponds to 500 µm. Fig.2A-F. Characterization of differentiated LEC by flow cytometry. A–C Prox1 shift compared to Isotype control; D–F LYVE-1 and Podoplanin expression of three transduced group. h-iLECs were characterized by quantitative protein expression of Podoplanin, LYVE- 1, and Prox1. Among three transduced group, ETS2 showed more Podoplanin expression leading up to over 88%, followed by ETV2+ETS2 group (44.2%) and ETV2 group. Similarly, Prox1 expression was higher in ETS2 group as well, rising up to 95% whereas the ETV2 and combination group showed 69.5% and 81% expression, respectively. Fig.3. Immunofluorescent data show presence of key endothelial and lymphatic marker in h-iLECs. A–I represents ETV2, ETS2, and ETV2+ETS2 transduced group respectively. Panels are, left to right, of DAPI, ETS-related gene and Prox1 in that order. All three-cohort shows nuclear presence of ERG and PROX1. The size bar corresponds to 100 µm. Fig. 4A-D. Genotypic characterization of iLECs. A–D qRT-PCR data of LYVE-1, PDPN, Prox1, and VEGRR3 of three transduced group, respectively, in each case growth factor derived LECs (endogenous activation of ETV2 / ETS2) were used as control. The data show that all three transduced group showed higher expression of the genes of interest compared to control. Especially cohort having ETS2 showed very significant expression of all the LEC 4

[0005] 501.113WO1 ND 25-008 markers. Data represent mean ± standard deviation, n = 4 per condition. Significance levels were set at: *p < 0.5 and **p < 0.01. Three biological replicates (n = 3) were collected per condition and analyzed with real-time qRT-PCR. Fig. 5A-G. Functional properties of iLECs. A–C Capillary-like network formation of ETV2, ETS2, and ETV2+ETS2 group where Live cells are stained by cell tracker green. D, E Quantitative analysis of tube formation assay by measuring the tube length and no. of branches respectively. F, G Quantification of Reelin and VEGFC secreted by the iLECs obtained through ELISA assay. Significance level was set at: **p < 0.01. Fig.6A-B. Exemplary A ETS2 containing plasmid and B ETV2 containing plasmid for use with embodiments of the invention. Fig.7. Cells seeded for differentiation (on the left), Zoomed in images of the transduced cells expressing GFP (on the right). Fig. 8. A Standard curve for VEGFC (top), y=0.0184x; R2=0.9966, and B ELISA assay (bottom) y=0.0019x; R2=0.9977. Fig. 9. A, B, C represent tube formation analysis of ETV2, ETS2 and ETV2+ETS2 group, respectively. DETAILED DESCRIPTION OF THE INVENTION Definitions. The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001 or Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, PCR amplification, cell culturing. etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012. References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, 5

[0006] 501.113WO1 ND 25-008 moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring. As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect. The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., 6

[0007] 501.113WO1 ND 25-008 weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, … 9, 10. It also means 1.0, 1.1, 1.2. 1.3, …, 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above. 7

[0008] 501.113WO1 ND 25-008 One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation. The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo. An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect. Alternatively, the terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the 8

[0009] 501.113WO1 ND 25-008 disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment). The terms "treating", "treat" and "treatment" include (i) inhibiting the disease, pathologic or medical condition or arresting its development; (ii) relieving the disease, pathologic or medical condition; and / or (iii) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can include lowering, stopping, or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical and therapeutic administration, as appropriate. The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting. As used herein, terms "subject" or "patient" are used interchangeably to refer to an animal (e.g., birds, reptiles, and mammals). In a specific embodiment, a subject is a bird. In another embodiment, a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In certain embodiments, a subject is a non-human animal. In another embodiment, a subject is a human. As used herein, the term “a portion of” or “a portion thereof” means consecutive nucleotides of the sequence of said particular region. A portion according to the invention can comprise or consist of at least 15 or 20 consecutive nucleotides, preferably at least 100, 200, 300, 500 or 700 consecutive nucleotides, and more preferably at least 1, 2, 3, 4 or 5 consecutive kb of said particular region. For example, a portion can comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 consecutive kb of said particular region. The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%. Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of” or “consisting essentially of” are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is 9

[0010] 501.113WO1 ND 25-008 inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein. The term "promoter" refers to a polynucleotide which directs the transcription of a structural gene to produce mRNA. Typically, a promoter is located in the 5' region of a gene, proximal to the start codon of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter. The term "enhancer" refers to a polynucleotide. An enhancer can increase the efficiency with which a particular gene is transcribed into mRNA irrespective of the distance or orientation of the enhancer relative to the start site of transcription. Usually, an enhancer is located close to a promoter, a 5'-untranslated sequence, or in an intron. "Transgene", "transgenic" or "recombinant" refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man. For instance, a transgenic expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above. In case the term "recombinant" is used to specify an organism or cell, e.g., a microorganism, it is used to express that the organism or cell comprises at least one "transgene", "transgenic" or "recombinant" polynucleotide, which is usually specified later on. 10

[0011] 501.113WO1 ND 25-008 The terms "operable linkage" or "operably linked" are generally understood as meaning an arrangement in which a genetic control sequence, e.g., a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucleotide being operably linked to it, for example a polynucleotide encoding a polypeptide. Function, in this context, may mean for example control of the expression, i.e., transcription and / or translation, of the nucleic acid sequence. Control, in this context, encompasses for example initiating, increasing, governing or suppressing the expression, i.e., transcription and, if appropriate, translation. Controlling, in turn, may be, for example, tissue- and / or time-specific. It may also be inducible, for example by certain chemicals, stress, pathogens and the like. Preferably, operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed. An operably linkage does not necessarily require a direct linkage in the chemical sense. For example, genetic control sequences like enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked. Preferred arrangements are those in which the nucleic acid sequence to be expressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another. The distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette, is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. The skilled worker is familiar with a variety of ways in order to obtain such an expression cassette. However, an expression cassette may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example an endogenous promoter, for example by means of homologous recombination or else by random insertion. Such constructs are likewise understood as being expression cassettes for the purposes of the invention. The terms "express," "expressing," "expressed" and "expression" refer to expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) at a level that the resulting enzyme activity of this protein encoded for or the pathway or reaction that it refers to allows metabolic flux through this pathway or reaction in the organism in which this gene / pathway is expressed in. The expression can be done by genetic alteration of the microorganism that is used as a starting organism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to 11

[0012] 501.113WO1 ND 25-008 express a gene product at an increased level relative to that produced by the starting microorganism or in a comparable microorganism which has not been altered. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and / or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). In some embodiments, a microorganism can be physically or environmentally altered to express a gene product at an increased or lower level relative to level of expression of the gene product unaltered microorganism. For example, a microorganism can be treated with, or cultured in the presence of an agent known, or suspected to increase transcription of a particular gene and / or translation of a particular gene product such that transcription and / or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a temperature selected to increase transcription of a particular gene and / or translation of a particular gene product such that transcription and / or translation are enhanced or increased. As used herein, the term “vector” refers to any nucleic acid construct used to transfer a nucleic acid encoding a therapeutic protein into a host cell. In some embodiments, a vector includes a replicon, which functions to replicate the nucleic acid construct. Non-limiting examples of vectors useful for gene therapy include plasmids, phages, cosmids, artificial chromosomes, and viruses, which function as autonomous units of replication in vivo. In some embodiments, a vector is a viral vector for introducing a nucleic acid encoding a therapeutic protein into the host cell. Many modified eukaryotic viruses useful for gene therapy are known in the art. For example, adeno-associated viruses (AAVs) and lentiviruses are particularly well suited for use in human gene delivery because humans are a natural host for the viruses, the native viruses are not known to contribute to any diseases, and the viruses illicit a mild immune response. The term “polypeptide” or “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may 12

[0013] 501.113WO1 ND 25-008 comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or toxin. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The terms “polypeptide” and “protein” as used herein specifically encompass antibodies. The terms “expansion” or “expanding” as used herein in the context of cells or cell culture refer to an increase in the number of cells of a certain type (for example endothelial cells) from an initial population of cells, which may or may not be identical. The initial cells used for expansion need not be the same as the cells generated as a result of the expansion. For instance, the expanded cells may be produced by growth and differentiation of the initial population of cells. The term "lymphatic endothelial cell" or "LEC" as used herein refers to a cell which makes up the lymphatic endothelium (LE) that lines the lymphatic vascular system, and expresses typical LEC markers including, but not limited to, the intracellular expression of Proxl, lymphatic vessel endothelial hyaluronan receptor-1 (lyvel), VEGFR- 3, flt4, soxl8, nr2f2, neuropilin 2 (Nrp2), forkhead box C2 (Foxc2), podoplanin (Pdpn), angiopoietin 2 (Ang2), ephrin B2 (Efnb2), cxcr4, activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), protein phosphatase 1 regulatory (inhibitor) subunit 13B (Asppl) and / or core 1 synthase glycoprotein-N-acetylgalactosamine 3-beta- galactosyltransferase 1 (T-synthase); or the cell membrane expression of CD31, CD34 and / or GP38. LECs typically also produce chemokines, including but not limited to, CCL21. According to a specific embodiment, LECs express lyvel, proxl, Pdpn and / or VEGFR-3. Determination of LECs may be carried out using any method known in the art, e.g. by FACS analysis, by PCR or by ELISA, selecting cells expressing the LEC markers as described in detail above. As used herein, the phrase "stem cells" refers to cells which are capable of remaining in an undifferentiated state (e.g., totipotent, pluripotent or multipotent stem cells) for extended periods of time in culture until induced to differentiate into other cell types having a particular, specialized function (e.g., fully differentiated cells). Totipotent cells, such as embryonic cells within the first couple of cell divisions after fertilization are the only cells that can differentiate into embryonic and extra-embryonic cells and are able to develop into a viable human being. Preferably, the phrase "pluripotent stem cells" refers to cells which can differentiate into all 13

[0014] 501.113WO1 ND 25-008 three embryonic germ layers, i.e. , ectoderm, endoderm and mesoderm or remaining in an undifferentiated state. The pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS). The multipotent stem cells include adult stem cells and hematopoietic stem cells. See U.S. Patent Nos 10,865,381 and 9,404,122 to Yu et al., 10,836,997 to Ko et al., U.S. Patent Publication No. 2022 / 0204924 to Nolan et al., and PCT Patent Publication Nos. 2016 / 185457 to Yaniv et al., and 2024 / 173696 to Clarke et al. The phrase "embryonic stem cells" refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i.e. , endoderm, ectoderm and mesoderm) or remaining in an undifferentiated state. The phrase "embryonic stem cells" may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation / pre-gastrulation stage blastocyst (see PCT Patent Publication No.2006 / 040763 to Amit et al.), embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation, and cells originating from an unfertilized ova which are stimulated by parthenogenesis (parthenotes). Induced pluripotent stem cells (iPS; embryonic-like stem cells), are cells obtained by de-differentiation of adult somatic cells which are endowed with pluripotency (i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the invention, such cells are obtained from a differentiated tissue (e.g., a somatic tissue such as skin) and undergo de-differentiation by genetic manipulation, which re-program the cell to acquire embryonic stem cells characteristics. According to some embodiments of the invention, the induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in a somatic stem cell. The iPS can be generated from somatic cells by genetic manipulation of somatic cells, e.g., by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, gastric epithelial cells with transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 (Yamanaka et al.. Cell Stem Cell. 2007, l(l):39-49; Park et al. Nature 2008;451: 141-146; Takahashi et al., Cell 2007;131:861-872). Other embryonic-like stem cells can be generated by nuclear transfer to oocytes, fusion with embryonic stem cells or nuclear transfer into zygotes if the recipient cells are arrested in mitosis. The term "lymphangiogenesis" as used herein refers to the development, growth or formation of new lymphatic vessels e.g. capillaries, collecting vessels, and ducts. 14

[0015] 501.113WO1 ND 25-008 In order to induce lymphangiogenesis (e.g. formation of lymphatic capillaries, collecting vessels, and ducts), the LEC committed cells are subjected to a LEC morphogenesis signal, a LEC proliferation signal or a LEC survival signal. As used herein, the phrase "LEC morphogenesis signal" refers to any agent which induces lymphangiogenesis in the LEC committed cells. As used herein, the phrase "LEC proliferation signal" refers to any agent which induces cell division in the LEC committed cells. As used herein, the phrase "LEC survival signal" refers to any agent which reduced cell death in the LEC committed cells. Generally, the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). 15

[0016] 501.113WO1 ND 25-008 Glycogen synthase kinase (GSK) is a serine / threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. In mammals, GSK is encoded by two genes, GSK3α and GSK3β. GSK is involved in a great number of signaling pathways and has been implicated in a number of diseases including Type II diabetes, Alzheimer's disease, inflammation, cancer, and bipolar disorder. “Transforming growth factor beta (TGFβ) receptor inhibitor as used herein refers to an agent that inhibits the TGFβ receptor. TGFβ receptors are single pass serine / threonine kinase receptors. Three TGF-β receptor types include receptor types I, II and III i.e., TGF-β receptor 1, TGF-β receptor 2 and TGF-β receptor 3. Aspects of the invention are described in Saha et al., Cells Tissues Organs. 2024;213(6):464-474. doi: 10.1159 / 000539699. Epub 2024 Aug 28, including supplemental information, incorporated herein by reference in its entirety. The present disclosure provides for a two-dimensional, feeder-free, and chemically defined protocol that relies on a timely transition of hiPSCs through three distinct stages: conversion of hiPSCs into intermediate mesodermal progenitor cells (MPCs), converting the MPCs into iECs (transduced endothelial cells), and treating the cells with vascular endothelial growth factor C (VEGF-C) and Transforming growth factor-β (TGF-β) inhibitor. This stepwise protocol rapidly and uniformly converted hiPSCs into iLECs (transduced lymphatic endothelial cells). In some embodiments, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) and a transforming growth factor β (TGFβ) inhibitor, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. 16

[0017] 501.113WO1 ND 25-008 Exemplary GSK inhibitors include, but are not limited to GSK-3β inhibitors are well known in the art and include, but are not limited to, 1, 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5- methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile “CHIR99021” (Ring et al., 2003), LiCl (Klein et al., 1996), BIO-acetoxime ((2′Z,3′E)-6- Bromoindirubin-3′-oxime) (Meijer et al., 2003), N6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H- imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine “CHIR98014” (Ring et al., 2003), 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione “SB 216763” also known as GSK-3 Inhibitor IV (Coghlan et al., 2000), 3-[(3-Chloro-4- hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione “SB 415286” (Coghlan et al., 2000), 5-ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]-isoquinoline-1,3-(2H)-dione “3F8” (Zhong et al., 2009), 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one “Kenpaullone” (Schultz et al., 1999; Zaharevitz et al., 1999), 9-Bromo-7,12-dihydro- pyrido[3′,2′:2,3]azepino[4,5-b]indol-6(5H)-one “1-Azakenpaullone” (Schultz et al., 1999; Zaharevitz et al., 1999), N-(3-Chloro-4-methylphenyl)-5-(4-ni¬trophenyl)-1,3,4-oxadiazol-2- amine “TC-G 24” (Khanfar et al., 2010), 2-Methyl-5-[3-[4-(methylsulfinyl)phenyl]-5- benzofuranyl]-1,3,4-oxadiazole “TCS 2002” (Saitoh et al., 2009), N-[(4- Methoxyphenyl)methyl]-N′-(5-nitro-2-thiazolyl)urea “AR-A 014418” (Bhat et al., 2003), 3-[5- [4-(2-Hydroxy-2-methyl-1-oxopropyl)-1-piperazinyl]-2-(trifluoromethyl)phenyl]-4-(1H indol-3-yl)-1H-pyrrole-2,5-dione “TCS 21311” (Thoma et al., 2011), 3-[[6-(3-aminophenyl)- 7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol “TWS 119” (Ding et al., 2003), ((2′Z,3′E)-6- Bromoindirubin-3′-acetoxime) “BIO-acetoxime” also known as GSK-3 Inhibitor IX (Meijer et al., 2003), 4-(2-Amino-4-oxo-2-imidazolin-5-ylidene)-2-bromo-4,5,6,7- tetrahydropyrrolo[2,3-c]azepin-8-one “10Z-Hymenialdisine” (Breton et al., 1997), 2-[(3- iodophenyl)methylsulfanyl]-5-pyridin-4-yl-1,3,4-oxadiazole, also known as GSK- 3β Inhibitor II (Wada, 2009), 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, also known as GSK-3β Inhibitor I (Wada, 2009), 3-Amino-6-(4-((4-methylpiperazin-1- yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide, HCl, also known as GSK- 3β Inhibitor XXVII (US Patent Pub. No. 2006 / 0173014), 4,5-bis(1-Methyl-1H-indol-3-yl)- 1,2-dihydropyrazol-3-one, also known as GSK-3β Inhibitor XXVI (Chen et al, 2011), 3- Amino-1H-pyrazolo[3,4-b]quinoxaline “Cdk1 / 5 Inhibitor” (Andreani et al., 1996, 2000; Katoh et al., 2011) and 4-((5-Bromo-2-pyridinyl)amino)-4-oxobutanoic acid “Bikinin” (De Rybel et al., 2009). Preferably, the inhibitor of GSK is CHIR99021. (See U.S. Patent No.10,954,490 to Karp et al.). 17

[0018] 501.113WO1 ND 25-008 In some embodiments, the GSK inhibitor is selected from the group consisting of CHIR 99021, CHIR 98014, BIO-acetoxime, BIO, LiCl, SB 216763, SB 415286, AR A014418, 1- Azakenpaullone, and Bis-7-indolylmaleimide. TGFβ receptor inhibitors appropriate for use in a method of the present invention include, without limitation, SB-431542, SB-525334, A83-01, LY2157299, LY210976, GW788388, RepSox, SB-505124, D4476, GW788388, SD208, and EW-7197. Preferably, the inhibitor of TGF-beta signaling is SB431542, a small molecule inhibitor of endogenous activin and the type I receptor (TGFβ Receptor I) (Inman et al., Mot Pharmacol. 62(1):65-74 (2002). A suitable working concentration range for chemical inhibitors such as those described herein is from about 0.1 μM to about 100 μM, e.g., about 2 μM, about 5 μM, about 7 μM, about 10 μM, about 12 μM, about 15 μM, about 18 μM, or another working concentration of one or more the foregoing chemical inhibitors between about 0.1 μM to about 100 μM. In some embodiments, the working concentration range is about 0.1 μM to about 15 μM, about 0.1 μM to about 12 μM, about 0.1 μM to about 10 μM, about 0.1 μM to about 8 μM, about 0.1 μM to about 5 μM, or about 1 μM to about 15 μM, about 1 μM to about 5 μM, or about 5 μM to about 10 μM . In some embodiments, the GSK inhibitor is CHIR99021 at a concentration ranging from about 0.1 μM to about 15 μM, e.g., about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM or another concentration of CHIR99021 from about 1 μM to about 15 μM, about 1 μM to about 10 μM or about 1 μM to about 5 μM. In some embodiments, the GSK inhibitor is CHIR99021 at a concentration of about 3 μM to 5 μM, or about 4 μM. In some embodiments, the TGFβ inhibitor is SB431542 present in the cell culture media at a concentration ranging from about 1 μM to about 15 μM, e.g., about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM, or another concentration of SB431542 from about 1 μM to about 15 μM, or about 1 μM to about 10 μM. In some embodiments, the TGFβ inhibitor is SB431542 present in the cell culture media at a concentration of about 8 μM to about 12 μM, or about 10 μM. In some embodiments, the GSK inhibitor is CHIR99021 and the TGFβ inhibitor is SB431542 at a concentration ranging from about 1 μM to about 15 μM, e.g., about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 18

[0019] 501.113WO1 ND 25-008 15 μM, or another concentration of CHIR99021 and SB431542 from about 1 μM to about 15 μM. In some embodiments, the cell culture media can be supplemented with certain growth factors such as Vascular Endothelial growth factor, a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. The VEGF signaling pathway mainly includes 6 kinds of ligands (VEGF-A, -B, -C, -D, -E and PGF) and 3 kinds of receptors (VEGFR1, VEGFR2 and VEGFR3). In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL to about 250 ng / mL VEGF, about 0.1 ng / mL to about 200 ng / mL VEGF, about 0.1 ng / mL to about 150 ng / mL VEGF, about 0.1 ng / mL to about 100 ng / mL VEGF, about 0.1 ng / mL to about 75 ng / mL VEGF, about 0.1 ng / mL to about 50 ng / mL VEGF, or about 0.1 ng / mL to about 25 ng / mL VEGF. In some embodiments, the cell culture media is supplemented with about 50 ng / mL to about 200 ng / mL VEGF, about 75 ng / mL to about 150 ng / mL VEGF, or about 100 ng / mL to about 150 ng / mL VEGF. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL to about 15 ng / mL VEGF, about 5 ng / mL to about 15 ng / mL VEGF, or about 10 ng / mL to about 15 ng / mL VEGF. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 25 ng / mL, about 50 ng / mL, about 75 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 175 ng / mL, about 200 ng / mL, about 225 ng / mL, or about 250 ng / mL. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL to about 250 ng / mL VEGF-A, about 0.1 ng / mL to about 200 ng / mL VEGF-A, about 0.1 ng / mL to about 150 ng / mL VEGF-A, about 0.1 ng / mL to about 100 ng / mL VEGF-A, about 0.1 ng / mL to about 75 ng / mL VEGF-A, about 0.1 ng / mL to about 50 ng / mL VEGF-A, or about 0.1 ng / mL to about 25 ng / mL VEGF-A. In some embodiments, the cell culture media is supplemented with about 50 ng / mL to about 200 ng / mL VEGF-A, about 75 ng / mL to about 150 ng / mL VEGF-A, or about 100 ng / mL to about 150 ng / mL VEGF-A. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL to about 15 ng / mL VEGF-A, about 5 ng / mL to about 15 ng / mL VEGF-A, or about 10 ng / mL to about 15 ng / mL VEGF-A. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 25 ng / mL, about 50 ng / mL, about 75 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 175 ng / mL, about 200 ng / mL, about 225 ng / mL, or about 250 ng / mL of VEGF-A. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL to about 250 ng / mL VEGF-C or VEGF-C156S, about 0.1 ng / mL to about 200 ng / mL VEGF-C 19

[0020] 501.113WO1 ND 25-008 or VEGF-C156S, about 0.1 ng / mL to about 150 ng / mL VEGF-C or VEGF-C156S, about 0.1 ng / mL to about 100 ng / mL VEGF-C or VEGF-C156S, about 0.1 ng / mL to about 75 ng / mL VEGF-C or VEGF-C156S, about 0.1 ng / mL to about 50 ng / mL VEGF-C or VEGF-C156S, or about 0.1 ng / mL to about 25 ng / mL VEGF-C or VEGF-C156S. In some embodiments, the cell culture media is supplemented with about 50 ng / mL to about 200 ng / mL VEGF-C or VEGF- C156S, about 75 ng / mL to about 150 ng / mL VEGF-C or VEGF-C156S, or about 100 ng / mL to about 150 ng / mL VEGF-C or VEGF-C156S. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL to about 15 ng / mL VEGF-C or VEGF-C156S, about 5 ng / mL to about 15 ng / mL VEGF-C or VEGF-C156S, or about 10 ng / mL to about 15 ng / mL VEGF-C or VEGF-C156S. In some embodiments, the cell culture media is supplemented with about 0.1 ng / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 25 ng / mL, about 50 ng / mL, about 75 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 175 ng / mL, about 200 ng / mL, about 225 ng / mL, or about 250 ng / mL of VEGF-C or VEGF-C156S. VEGF-C156S is described, for example, in Forte et al., Lymphat Res Biol.2022 Dec;20(6):580- 584. In some embodiments, the cell culture media is supplemented with more than one VEGF. In some embodiments, a cell culture media can comprise about 0.1 ng / ml to about 15 ng / ml VEGF-A, about 5 ng / ml to about 12 ng / ml VEGF-A, or about 8 ng / ml to about 10 ng / ml VEGF-A. In some embodiments, a culture media comprises about 50 ng / ml to about 150 ng / ml VEGF-C or VEGF-C156S. In some embodiments, the cell culture media comprises about 100 ng / ml VEGF-C or VEGF-C156S. In some embodiments, a cell culture media can comprise about 0.1 ng / ml to about 15 ng / ml VEGF-A and about 5 µM to about 15 µM of the TGFβ inhibitor. In some embodiments, a cell culture media comprises about 1 ng / ml to about 10 ng / ml VEGF-A and about 8 µM to about 12 µM of the TGFβ inhibitor. The LECs described herein can be cultured according to approaches known in the art, and the cultured cells can be used in several of the embodied methods. For example, LECs can be cultured on collagen-coated dishes in endothelial cell basal medium in the presence of low or high fetal bovine serum or similar product, as described in Ng et al., November 2004, Microvasc Res. 68(3):258-64, incorporated herein by reference. Alternatively, LECs can be cultured on other extracellular matrix protein-coated dishes. Examples of extracellular matrix proteins that may be used include, but are not limited to, fibronectin, laminin, vitronectin, and 20

[0021] 501.113WO1 ND 25-008 collagen IV. Gelatin or any other compound or support, which similarly promotes adhesion of endothelial cells into culture vessels may be used to culture LECs, as well. Examples of culture medium that can be used to culture LECs in vitro include, but are not limited to, EGM, RPMI, M199, MCDB131, DMEM, EMEM, McCoy's 5A, Iscove's medium, modified Iscove's medium or any other medium known in the art to support the growth of endothelial cells. Examples of supplemental factors or compounds that can be added to the basal culture medium that could be used to culture LECs include, but are not limited to, ascorbic acid, heparin, endothelial cell growth factor, endothelial growth supplement, glutamine, HEPES, Nu serum, fetal bovine serum, human serum, equine serum, plasma- derived horse serum, iron-supplemented calf serum, penicillin, streptomycin, amphotericin B, basic and acidic fibroblast growth factors, insulin-growth factor, astrocyte conditioned medium, fibroblast or fibroblast-like cell conditioned medium, sodium hydrogencarbonate, epidermal growth factor, bovine pituitary extract, magnesium sulphate, isobutylmethylxanthine, hydrocortisone, dexamethasone, dibutyril cyclic AMP, insulin, transferrin, sodium selenite, oestradiol, progesterone, growth hormone, angiogenin, angiopoietin-1, Del-1, follistatin, granulocyte colony-stimulating factor (G-CSF), erythropoietin, hepatocyte growth factor (HGF) / scatter factor (SF), leptin, midkine, placental growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF) / vascular permeability factor (VPF), interleukin-3 (IL-3), interleukin 7 (IL-7), interleukin-8 (IL-8), ephrins, matrix metalloproteinases (such as MMP2 and MMP9), or any other compound known in the art to promote survival, proliferation or differentiation of endothelial cells. Further processing of the cells may also include: cell expansion (of one or more regenerative cell types) and cell maintenance (including cell sheet rinsing and media changing); sub-culturing; cell seeding; transient transfection (including seeding of transfected cells from bulk supply); harvesting (including enzymatic, non-enzymatic harvesting and harvesting by mechanical scraping); measuring cell viability; cell plating (e.g., on microtiter plates, including picking cells from individual wells for expansion, expansion of cells into fresh wells); high throughput screening; cell therapy applications; gene therapy applications; tissue engineering applications; therapeutic protein applications; viral vaccine applications; harvest of regenerative cells or supernatant for banking or screening, measurement of cell growth, lysis, inoculation, infection or induction; generation of cell lines (including hybridoma cells); 21

[0022] 501.113WO1 ND 25-008 culture of cells for permeability studies; cells for RNAi and viral resistance studies; cells for knock-out and transgenic animal studies; affinity purification studies; structural biology applications; assay development and protein engineering applications. In some embodiments, the cell culture media is STEMdiff™ APEL™2 Medium brand culture media is a fully defined, serum-free, and animal origin-free medium for the differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. (Ng et al., Nature Protocols volume 3, pages768–776 (2008)). In some embodiments, the cell culture media is Endothelial Cell Growth Medium MV2 is a low-serum (5% V / V) medium developed for the in vitro cultivation of endothelial cells from microvascular vessels, the coronary artery, and the aorta. (See Grau et al., J Neurooncol 104, 103–112 (2011)). In some embodiments, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor in an amount of about 1 µM to 10 µM and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) in an amount of about 5 ng / mL to about 15 ng / mL and a transforming growth factor β (TGFβ) inhibitor in an amount of about 5 µM to 15 µM, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising about 50 ng / mL to about 150 ng / mL VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. In some embodiments, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor in an amount of about 1 µM to 10 µM and culturing 22

[0023] 501.113WO1 ND 25-008 the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) in an amount of about 5 ng / mL to about 15 ng / mL and a transforming growth factor β (TGFβ) inhibitor in an amount of about 5 µM to 15 µM, and inducing expression of the one or more polynucleotide sequences encoding ETS2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising about 50 ng / mL to about 150 ng / mL VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. In some embodiments, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor in an amount of about 1 µM to 10 µM and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) in an amount of about 5 ng / mL to about 15 ng / mL and a transforming growth factor β (TGFβ) inhibitor in an amount of about 5 µM to 15 µM, and inducing expression of the one or more polynucleotide sequences encoding ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising about 50 ng / mL to about 150 ng / mL VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. In some embodiments, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor in an amount of about 1 µM to 10 µM and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) in an amount of about 5 ng / mL to about 15 ng / mL and a transforming growth factor β (TGFβ) inhibitor in an amount of about 5 µM to 15 µM, and inducing expression of the one or more 23

[0024] 501.113WO1 ND 25-008 polynucleotide sequences encoding ETS2 and ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising about 50 ng / mL to about 150 ng / mL VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. In some embodiments, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprises contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor CHIR 99021 in an amount of about 4 µM and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) in an amount of about 10 ng / mL and a transforming growth factor β (TGFβ) inhibitor SB-431542 in an amount of about 10 µM, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 24-48 hours or about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising about 100 ng / mL VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. In some embodiments, a method generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprising, in order, the steps of: contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor and culturing the seeded hiPSCs for about 24 hours; replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) and a transforming growth factor β (TGFβ) inhibitor, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; growing the induced hiPSCs for about 48 hours; reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising VEGF-C; and expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. 24

[0025] 501.113WO1 ND 25-008 In some embodiments, the second cell culture media comprises a GSK inhibitor present in an amount of about 0.1 µM to about 20 µM, about 0.1 µM to about 15 µM, about 0.1 µM to about 10 µM, about 0.1 µM to about 8 µM, about 2 µM to about 6 µM, or about 4 µM. In some embodiments, the GSK inhibitor is CHIR 99021. In some embodiments, the third cell culture media comprises about 0.1 ng / ml to about 50 ng / ml VEGF-A and about 0.1 µM to about 25 µM of a TGFβ inhibitor, about 0.1 ng / ml to about 25 ng / ml VEGF-A and about 0.1 µM to about 20 µM of a TGFβ inhibitor, about 0.1 ng / ml to about 15 ng / ml VEGF-A and about 5 µM to about 15 µM of a TGFβ inhibitor, about 1 ng / ml to about 10 ng / ml VEGF-A and about 8 µM to about 12 µM of a TGFβ inhibitor, In some embodiments, the TGFβ inhibitor is SB-431542. In some embodiments, the fourth cell culture media comprises about 25 ng / ml to about 200 ng / ml VEGF-C or VEGF-C156S, about 50 ng / ml to about 150 ng / ml VEGF-C or VEGF- C156S, about 75 ng / ml to about 125 ng / ml VEGF-C or VEGF-C156S, about 85 ng / ml to about 115 ng / ml VEGF-C or VEGF-C156S, about 90 ng / ml to about 110 ng / ml VEGF-C or VEGF- C156S, or about 100 ng / ml VEGF-C or VEGF-C156S. In some embodiments, the hiPSCs are seeded on a cell culture plate for about 8 to about 24 hours in the presence of a first cell culture media to provide plated hiPSCs, or about 10 to about 24 hours, about 12 to about 24 hours, about 14 to about 24 hours, about 16 to about 24 hours, or about 18 to about 24 hours. In some embodiments, the seeded hiPSCs are culturing in the second cell culture media for about 8 to about 24 hours in the presence of a first cell culture media to provide plated hiPSCs, or about 10 to about 24 hours, about 12 to about 24 hours, about 14 to about 24 hours, about 16 to about 24 hours, or about 18 to about 24 hours. In some embodiments, induced hiPSCs are grown in the third cell culture media for about 24 to about 48 hours, about 28 to about 48 hours, about 30 to about 48 hours, about 34 to about 48 hours, about 38 to about 48 hours, about 42 to about 48 hours, or about 48 hours. In some embodiments, the induced hiPSCs are expanded on cell culture plates and a fourth cell culture media to provide differentiated LEC cells for about 48 to about 72 hours, about 54 to about 72 hours, about 60 to about 72 hours, about 66 to about 72 hours, or about 72 hours. As noted, a number of viral and nonviral vectors have been developed for delivery of genetic material in various tissues and organs. In most cases, these vectors are replication incompetent and pose little threat of viral-induced disease. Rather, the viral genome has been partly or fully deleted, expanding the capacity to allow inclusion of therapeutic DNA cargo 25

[0026] 501.113WO1 ND 25-008 within the viral capsid. Some vectors include single-stranded DNA, while others include double-stranded DNA. Particularly preferred vectors in the context of the invention are lentiviral vectors, adenovirus vectors, or Adeno-associated viruses (AAV) as disclosed in Ahmed et al, JARO 18:649-670 (2017). In some embodiments, the recombinant virus comprises a lentivirus, an adenovirus, an adeno-associated virus (AAV), or a retrovirus. Methods for preparing and using recombinant viruses and recombinant virus vectors are disclosed, for example U.S. Pat. Publication No. 2022 / 0001028 to Hatfield et al.; 2022 / 0125875 to Safieddine et al.; and 2020 / 0405883 to Bilic et al. The recombinant expression vector may be a viral vector, e.g., a retroviral vector or a lentiviral vector. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009); Buchscher, et al. (1992) J. Virol. 66:2731-2739; Johann, et al. (1992) J. Virol.66:1635-1640; Sommerfelt, et al. (1990) Virol.176:58-59; Wilson, et al. (1989) J. Virol. 63:2374-2378; Miller, et al. (1991) J. Virol. 65:2220-2224 (1991); U.S. Pat. Nos. 6,013,516; 5,994,136; PCT. Patent Publication No. 2025 / 090942 to Haldar et al.; and 1994026877 to Wong-Stall et al.). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif vpr, vpu and nef are deleted making the vector biologically safe. Other examples of lentivirus vectors that may be used in the clinic, include, for example, and not by way of limitation, the LENTIVECTOR® gene delivery technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Patent No. 5,994,136 to Naldini et al. Exemplary lentivirus viral vectors are shown in Fig. 6. In some embodiments, the viral vector contains the ETS2 and / or ETV2 gene flanked by two inverted terminal repeats (ITRs). In some embodiments, the viral vector includes one or more of an enhancer, promoter, an intron, a Woodchuck Hepatitis Virus (WHP) 26

[0027] 501.113WO1 ND 25-008 Posttranscriptional Regulatory Element (WPRE) (see, e.g., Choi et al. (2014) Molecular Brain 7: 17-26; Zufferey et al. (1999) J. Virol. 73(4):2886-2992)), ETS2 and / or ETV2 coding sequence, and polyadenylation (polyA) signal. See, e.g., PCT Patent Publication No. 2014 / 151341 to Wilson et al . In addition to the major elements identified above for the recombinant viral vector, in certain embodiments, the vector can also typically include conventional control elements that are operably linked to the transgene in a manner that permits its transcription, translation and expression in a cell transfected with the plasmid vector or infected with the virus. Expression control sequences include, for example, appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized. In some embodiments, the polynucleotide is operably linked to a promoter. In certain embodiments, the promoter sequence may be included as part of the expression control sequences (regulatory sequences), e.g., located between the selected 5’ ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters (see, e.g., U.S. Patent Publication No. 2013 / 0023033 to Wilson et al.), tissue specific promoters, or a promoter responsive to physiological cues may be utilized in the vectors described herein. Examples of suitable promoters include, but are not limited to the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al (1985) Cell, 41:521- 530), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter (e.g., chicken β-actin promoter), the phosphoglycerol kinase (PGK) promoter, the EF1α promoter, the CBA promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 (methyl-CPG binding protein 2) promoter, GFAP promoter, CBh promoter and the like. In some embodiments, transgene expression is controlled by the Tet on / off system as described Das et al., Curr Gene Ther. 2016 Jun;16(3):156–167; and Qin et al. (2010). PLoS ONE 5(5): e10611. doi.org / 10.1371 / journal.pone.0010611. Also see PCT Patent Publication No. 2025 / 090942 to Haldar et al. In some embodiments, the ETS2 and ETV2 genes are human ETS2 and ETV2 genes. In some embodiments, the ETS2 gene and protein are from Mus musculus. 27

[0028] 501.113WO1 ND 25-008 In some embodiments, the ETS2 gene comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 11. In some embodiments, the ETS2 protein comprises a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 12. In some embodiments, the ETV2 gene comprises a nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments, the ETV2 protein comprises an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In some embodiments, the viral vector comprises SEQ ID NO: 13 and / or SEQ ID NO: 14. In some embodiments, the effective amount of a viral vector is a number of viral genomes sufficient to attain a multiplicity of infection (MOI) of less than 1, about 10 to about 50, or about 10,000 to about 500,000. See, for example, Iwami et al., Front Microbiol. 2012 Sep 4;3:319. Statements of certain embodiments of the invention. 1. In a first embodiment, method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprising the steps of: i) contacting the hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences encoding ETS2 and / or ETV2 operably linked to an inducible promoter sequence; ii) seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; iii) exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor and culturing the seeded hiPSCs for about 24 hours; iv) replacing the second cell culture media for a third cell culture media comprising VEGF-A and a TGFβ inhibitor and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; v) growing the induced hiPSCs for about 48 hours; vi) reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising VEGF-C; and vii) expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells. 2. The method of embodiment 1, wherein the cell culture plates comprise matrigel, and the hiPSCs are seeded on the cell culture plates in an amount of about 20,000 to 30,000 cells / cm2. 3. The method of embodiments 1 or 2, wherein the cell culture plates comprise matrigel, and the hiPSCs are seeded on the cell culture plates in an amount of about 15,000 to 25,000 cells / cm2. 28

[0029] 501.113WO1 ND 25-008 4. The method of any one of embodiments 1-3, wherein the cell culture plates comprise matrigel, and the hiPSCs are seeded on the cell culture plates in an amount of about 10,000 to 15,000 cells / cm2. 5. The method of any one of embodiments 1-4, wherein the GSK inhibitor is selected from the group consisting of CHIR 99021, CHIR 98014, BIO-acetoxime, BIO, LiCl, SB 216763, SB 415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide. 6. The method of any one of embodiments 1-5, wherein the GSK inhibitor present in an amount of about 0.1 µM to about 8 µM. 7. The method of any one of embodiments 1-6, wherein the GSK inhibitor present in an amount of about 2 µM to about 6 µM. 8. The method of any one of embodiments 1-7, wherein the GSK inhibitor present in an amount of about 4 µM. 9. The method of any one of embodiments 1-8, wherein the GSK inhibitor is CHIR 99021. 10. The method of any one of embodiments 1-9, wherein the GSK inhibitor is CHIR 99021 present in an amount of about 4 µM. 11. The method of any one of embodiments 1-10, wherein the TGFβ inhibitor is selected from the group consisting of SB-431542, SB-525334, A83-01, LY2157299, LY210976, GW788388, RepSox, SB-505124, D4476, GW788388, SD208, and EW-7197. 12. The method of any one of embodiments 1-11, wherein TGFβ inhibitor is present in an amount of about 5 µM to about 15 µM. 13. The method of any one of embodiments 1-12, wherein TGFβ inhibitor is present in an amount of about 8 µM to about 12 µM. 14. The method of any one of embodiments 1-13, wherein TGFβ inhibitor is present in an amount of about 10 µM. 15. The method of any one of embodiments 1-14, wherein the TGFβ inhibitor is SB- 431542. 16. The method of any one of embodiments 1-15, wherein the third cell culture comprises about 0.1 ng / ml to about 15 ng / ml VEGF-A. 17. The method of any one of embodiments 1-16, wherein the third cell culture comprises about 1 ng / ml to about 12 ng / ml VEGF-A. 18. The method of any one of embodiments 1-17, wherein the third cell culture comprises about 5 ng / ml to about 12 ng / ml VEGF-A. 19. The method of any one of embodiments 1-18, wherein the third cell culture comprises about 8 ng / ml to about 12 ng / ml VEGF-A. 29

[0030] 501.113WO1 ND 25-008 20. The method of any one of embodiments 1-19, wherein the third cell culture comprises about 10 ng / ml VEGF-A. 21. The method of any one of embodiments 1-20, wherein the fourth cell culture media comprises about 25 ng / ml to about 200 ng / ml VEGF-C. 22. The method of any one of embodiments 1-21, wherein the fourth cell culture media comprises about 50 ng / ml to about 150 ng / ml VEGF-C. 23. The method of any one of embodiments 1-22, wherein the fourth cell culture media comprises about 75 ng / ml to about 125 ng / ml VEGF-C. 24. The method of any one of embodiments 1-23, wherein the fourth cell culture media comprises about 85 ng / ml to about 115 ng / ml VEGF-C. 25. The method of any one of embodiments 1-24, wherein the fourth cell culture media comprises about 90 ng / ml to about 110 ng / ml VEGF-C. 26. The method of any one of embodiments 1-25, wherein the fourth cell culture media comprises about 100 ng / ml VEGF-C. 27. The method of any one of embodiments 1-26, wherein the third culture media comprises about 0.1 ng / ml to about 15 ng / ml VEGF-A and about 5 µM to about 15 µM of a TGFβ inhibitor. 28. The method of any one of embodiments 1-27, wherein the third culture media comprises about 1 ng / ml to about 10 ng / ml VEGF-A and about 8 µM to about 12 µM of a TGFβ inhibitor. 29. The method of any one of embodiments 1-28, wherein the TGFβ inhibitor is SB- 431542. 30. The method of any one of embodiments 1-29, wherein the fourth cell culture media comprises about 50 ng / ml to about 150 ng / ml VEGF-C. 31. The method of any one of embodiments 1-30, wherein the fourth cell culture media comprises about 100 ng / ml VEGF-C. 32. The method of any one of embodiments 1-31, wherein the second cell culture media is STEMdiff APEL2 brand cell culture media. 33. The method of any one of embodiments 1-32, wherein the third cell culture media is APEL2 brand cell culture media. 34. The method of any one of embodiments 1-33, wherein the fourth cell culture media is MV2 brand cell culture media. 30

[0031] 501.113WO1 ND 25-008 35. The method of any one of embodiments 1-34, wherein the one or more polynucleotide sequences encoding ETS2 and / or ETV2 operably linked to an inducible promoter sequence encodes ETS2. 36. The method of any one of embodiments 1-35, wherein the one or more polynucleotide sequences encoding ETS2 and / or ETV2 operably linked to an inducible promoter sequence encodes ETV2. 37. The method of any one of embodiments 1-36, wherein the viral vector comprises at least two viral vectors, wherein a first viral vector comprises a polynucleotide sequence encoding ETS2 and a second viral vector comprises a polynucleotide sequence encoding ETV2. 38. The method of any one of embodiments 1-37, wherein the viral vector is a lentivirus. 39. The method of any one of embodiments 1-38, wherein the ETS2 gene comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 11. 40. The method of any one of embodiments 1-39, wherein the ETV2 gene comprises a nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. 41. The method of any one of embodiments 1-40, wherein the ETS2 protein comprises an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 12. 42. The method of any one of embodiments 1-41, wherein the ETV2 protein comprises an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. 43. The method of any one of embodiments 1-42, wherein the ETS2 gene and the ETV2 gene are human ETS2 gene and the ETV2 genes. 44. The method of any one of embodiments 1-43, wherein the ETS2 gene is from Mus musculus. 45. The method of any one of embodiments 1-44, wherein the viral vector comprises SEQ ID NO: 13 and / or SEQ ID NO: 14. 46. The method of any one of embodiments 1-45, wherein the VEGF-C is selected from the group consisting of VEGF-C and VEGF-C156S. 47. The method of any one of embodiments 1-46, wherein the VEGF-C is VEGF-C156S. 48. A lymphatic endothelial cell obtained by the method of any one of embodiments 1-47. The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be 31

[0032] 501.113WO1 ND 25-008 understood that numerous variations and modifications may be made while remaining within the scope of the invention. Example 1. Robust Differentiation of Human Pluripotent Stem Cells into Lymphatic Endothelial Cells Using Transcription Factors. Efficient Transduction of Human Pluripotent Stem Cells into Lymphatic Endothelial Cells. We developed a two-dimensional, feeder-free, and chemically defined protocol that relies on a timely transition of hiPSCs through three distinct stages (Fig. 1). First is the conversion of hiPSCs into intermediate mesodermal progenitor cells (MPCs), which is mediated by the activation of Wnt and Nodal signaling pathways using the glycogen synthase kinase 3 inhibitor CHIR99021. Second, we converted the mesodermal progenitor cells into iECs (transduced endothelial cells). This is done by the transduction and activation of exogenous ETV2 / ETS2. Third, we treat the cells with vascular endothelial growth factor C (VEGF-C) and transforming growth factor-β (TGF-β) inhibitor. Our stepwise protocol rapidly and uniformly converted hiPSCs into iLECs (transduced LECs). We achieved maximum 88.5% efficiency (CD144+ / Podoplanin+) in driving the cells into LEC lineage. In contrast when endogenous ETV2 was activated via VEGF signaling, the differentiation efficiency was much lower (less than 30%, data not shown). Conversion efficiency of iECs was dependent on amount of ETV2 / ETS2 utilized and thus affecting the EC-LEC conversion as well. We tested 3 different conditions to see how the differentiation efficiency was impacted by ETV2, ETS2 or combination of both. Transduction using ETV2 produced 69.5% Prox1+cells (Fig. 2a), while transduction using ETS2 produced 95.0% Prox1+cells (Fig. 2b). Transduction using both ETV2 and ETS2 produced 81.0% Prox1+cells (Fig. 2c). Collectively, we found that ETS2 transduced group had the most efficiency followed by the combination of ETS2 and ETV2 group, and lastly ETV2 alone resulted in the least number of differentiated cells. Expression of Key Lymphatic Markers in Differentiated LECs. Next, we further examined the lymphatic endothelial differentiation of the hiPSCs with FACS analysis for key lymphatic markers. Upon differentiation, the resulting iLECs showcased a robust and mature profile of multiple transmembrane key lymphatic markers, including Podoplanin, LYVE-1 and VEGFR3. Importantly, the levels of these markers in the differentiated LECs closely paralleled those observed in mature LECs, indicating a high fidelity of phenotype and maturity in the generated cell population (Fig. 2d–f). Interestingly, we did see differences in the degree of Podoplanin and LYVE-1 expression in the three transduced groups. From the FACS data, it is 32

[0033] 501.113WO1 ND 25-008 clearly observed that ETV2 transduction produced 37.93% Podoplanin+cells (Fig. 2d) whereas ETS2 transduction produced 95.65% Podoplanin+cells (Fig. 2e). The combination of ETV2 and ETS2 group produced 48.23% Podoplanin+cells (Fig. 2f). In terms of LYVE-1 expression, we see the opposite trend. Combination of ETV2 and ETS2 produced 23.3% LYVE-1+cells, ETV2 produced 22.63% LYVE-1+cells, and ETS2 produced 9.0% LYVE- 1+cells (Fig. 2d–f). To characterize the identity of the cells derived from hiPSCs we investigated whether the differentiated cells expressed lymphatic markers at the protein level with proper localization. For endothelial cells, ERG (ETS-related gene) is expressed in the nuclei of endothelial cells, whereas in LECs, PROX-1, a transcription factor, is expressed in the nuclei. As expected, immunostaining results showed that ERG and PROX-1 both were exclusively localized in the nuclei of the differentiated cells confirming correct localization of the key EC and LEC markers in the differentiated cells (Fig. 3). Apart from protein level expression, we also looked at the gene expression of the common LEC markers, such as LYVE-1, PDPN, Prox1, and VEGFR3 (Fig. 4). Compared to growth factor derived LECs, ETV2 transduced LECs express higher level of lymphatic markers LYVE-1 (1.92 ± 0.78-fold), PDPN (2.84 ± 0.44), Prox1 (2.06 ± 0.64-fold), and VEGFR3 (2.20 ± 0.65-fold), ETV2 and ETS2 transduced LECs express higher level of lymphatic markers LYVE-1 (2.14 ± 0.60-fold), PDPN (7.75 ± 0.56-fold), Prox1 (3.28 ± 0.54- fold), and VEGFR3 (2.57 ± 0.28-fold), and ETS2 transduced LECs express the highest level of lymphatic markers LYVE-1 (3.03 ± 0.78-fold), PDPN (9.12 ± 0.61-fold), Prox1 (4 ± 0.64- fold), and VEGFR3 (3.90 ± 0.37-fold). Consistent with the differentiation efficiency data, ETS2 transduced group express the highest lymphatic markers followed by the combination of ETS2 and ETV2 group, and lastly the ETV2 group express relatively higher lymphatic markers compared to the growth factor derived LECs. Functional Capacity and Phenotypic Maturation. To validate the functionality of the LECs differentiated from hiPSCs under our culture conditions, we performed a series of in vitro studies. First, we tested in vitro activities of iLECs using a tube formation assay (Figure 5a–c, Fig. 9). Cells positive for LYVE-1 and Podoplanin were isolated by MACS from differentiating cell population at day 14, labeled with a fluorescent dye, CellTracker™ Green and subjected to tube formation assay. After 12 h of culture, all hiPSC-derived LECs readily formed tube-like structures (Fig. 5a–c). Although all the cells showed some degree of tube formation capability, ETS2 group formed 33.072 ± 1.268 mm tube length and 109 ± 12.73 branches, followed by ETS2+ETV2 group that formed 28.271 ± 2.381 mm tube length and 33

[0034] 501.113WO1 ND 25-008 106.5 ± 13.44 branches, and ETV2 group that formed 24.005 ± 0.235 mm tube length and 66.5 ± 7.78 branches (Fig. 5d, e). A number of branches are not significantly different among the different conditions, but tube length of ETS2 group was significantly greater than ETV2 group. Further, we looked at the secretory properties of our differentiated cells. LECs are known for secreting reelin, one of the major lymphangiocrine factors. In our study, we found that our differentiated LECs secrete reelin comparable to primary LECs in the ranges between 10 and 20 ng / mL (Fig. 5f). We also investigated the level of VEGF-C secreted by iLECs. We confirmed that all the iLECs and primary LECs secreted a very negligible amount of VEGF- C, in the range of pg / mL (Fig. 5g). Overall, the hiPSC-derived LECs showed phenotypic characteristics of mature LECs and faithfully recapitulated functional lymphatic behavior. Promoting the development of new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes. Yet, LECs are difficult to isolate, and they can lose their lymphatic phenotypes during in vitro culture. Therefore, generating LECs from hiPSCs represents an appealing strategy not only for lymphatic regeneration, but also for modeling human diseases in vitro. Thus far, methods to generate LECs from hiPSCs have mainly relied on either EB intermediate or the use of murine feeder layers (i.e., OP9). While generation of EB recapitulate early embryonic development, they require sorting of differentiated cells and therefore produce lower overall yield. On the other hand, co-culture with murine feeder layers introduces xenogeneic components to the final product. Alternatively, direct differentiation can be achieved using lentiviral transduction of ETV2 and ETS2, which are known to be important for blood endothelial cells. In this study, we compared the differentiation efficiency, lymphatic markers, and functionality of differentiated LECs using transduction of ETV2, ETS2, and combination of ETV2 and ETS2. We discovered that transduction with ETS2 produced the highest efficiency of Prox1+cells, which also express high lymphatic markers, such as LYVE-1 and Podoplanin. These results are consistent with previous studies that show ETS2 interaction with Prox1, the master regulator of lymphatic genes. Transduction with ETV2 produced lower efficiency of Prox1+, which express lower lymphatic markers. Interestingly, the combination of ETS2 and ETV2 produced lower efficiency of Prox1+cells, which may suggest the opposite effect of ETS2 and ETV2. LECs are also known to express different lymphatic markers depending on their locations. Lymphatic capillaries highly express LYVE-1, a receptor for hyaluronic acid, which is important for leukocytes trafficking. Lymphatic collecting vessels express Podoplanin, 34

[0035] 501.113WO1 ND 25-008 which can bind to platelet receptor CLEC-2 and is important for blood and lymphatic separation. ETS2 transduced cells highly express Podoplanin, which reflects LECs that reside at the lymphatic collecting vessels. On the other hand, ETV2 transduced cells highly express LYVE-1, which corresponds to LECs that reside at the lymphatic capillaries. Depending on the final applications, different transcription factors may be more suitable to generate different LECs with varying degree of Podoplanin and LYVE-1 expression. Regardless of the different lentivirus vectors used, all the transduced cells express key lymphatic markers comparable to primary LECs and are able to form lymphatic networks in vitro. The transduced cells also express reelin in the ranges between 10 and 20 ng / mL, which are comparable to primary LECs. Reelin is a key lymphangiocrine, which has been attributed to cardiac regeneration following heart attack. We also confirmed that the transduced LECs secrete low level of VEGF-C, comparable to primary LECs. While VEGF-C is a key soluble factor to promote lymphangiogenesis, it is well known that VEGF-C is not secreted by LECs, rather by macrophages and fibroblasts. All these results suggest that the differentiated LECs exhibit the characteristics and functionalities of LECs. Overall, the current study demonstrated an efficient method of deriving LECs, which may be useful for modeling human diseases in vitro and basic understanding of human development. For instance, differentiated LECs can be cultured in synthetic hydrogels and microfluidic devices to investigate the molecular mechanism underlying lymphatic disorders in patients with Down syndrome and lymphatic malformations. Future studies can also generate differentiated LECs that can secrete VEGF-C to provide pseudo-autocrine signaling and further enhance lymphatic regeneration in patients with lymphedema. It is worth noting that while direct differentiation using lentiviral vectors may introduce lentiviral components, future studies can take advantage of modulating ETV2 and ETS2 expression with modified mRNA. Furthermore, promising results from the current work warrants future studies to investigate a stepwise and well-defined method to differentiate LECs from hiPSCs, useful for broad applications in basic study of lymphatic biology, as well as toward various approaches in lymphatic regeneration and personalized medicine. Example 2. Material and Methods. Viral Vector Synthesis and Formulation. Lentiviruses were produced as previously described Patzke et al., Cell. 2019;179(2):498–513.e22 in HEK293T cells (ATCC) by cotransfection with three helper plasmids (pRSV-REV, pMDLg / pRRE and vesicular stomatitis virus G protein expression vector) with 12 μg of lentiviral vector DNA and 6 μg of each of the 35

[0036] 501.113WO1 ND 25-008 helper plasmid DNA per 75 cm2culture area) using calcium phosphate transfection. Lentiviral vector DNA plasmids used are pSIN4-EF1a-ETV2-IRES-Puro (61061, Addgene) (Elcheva et al., Nat Commun. 2014;5:4372) pLV-tetO-Ets2 (70272, Addgene) (Kubaczka et al., Cell Stem Cell.2015; 17(5):557–68) and FUW-M2rtTA (20342, Addgene) (Hockemeyer et al., Cell Stem Cell.2008; 3(3):346–53.). Lentiviruses were harvested with the medium 46 h after transfection, aliquoted, and frozen at −80°C. Details of lentiviral constructs are available in Table 1 and Fig. 6. Only virus preparations with >90% infection efficiency as assessed by GFP expression or puromycin resistance were used for experiments (Fig. 7). Table 1. Viral Vectors Culture of hiPSCs. Four different hiPSC lines derived from various tissue origins (2) and were obtained from WiCell Research Resources (WiCell, WI). (Table 2) The cells were maintained on growth factor reduced Matrigel (Corning) in mTeSR™ Plus medium (Stemcell Technologies). Table 2. iPSC source. The hiPSC colonies were checked regularly and passaged when reached around 70% confluency. Accutase (Innovative Cell Technologies) was used for dislodging the cells and depending on the cell line; ROCK inhibitor (10 µM) was used on the first day. Human iPSCs were routinely examined for pluripotent markers using immunofluorescence staining and flow cytometry analysis for TRA-1-60, TRA-1-81, SSEA4, and OCT4 (Table 3). All cell lines were 36

[0037] 501.113WO1 ND 25-008 routinely tested for karyotyping and mycoplasma contamination; they expressed normal karyotype and were negative for mycoplasma throughout this study. Table 3. Antibodies used for immunostaining and FACS. Transduction of hiPSCs. The hiPSCs were passaged as usual and resuspended with mTeSR™ Plus (Stemcell Technologies) media and mixed with viral vector solutions. The cells were then seeded on Matrigel coated plate (20,000–30,000 cell / cm2). The next day the media was changed into STEMdiff™ APEL™2 (Stemcell Technologies) containing 4 μM CHIR99021 (Stemcell Technologies). The ETS2 (co-transduced with rtTA) was activated the next day by addition of doxycycline to the cell culture media and ETV2 transduced cells were selected through puromycin treatment. This time the media was switched to APEL2 media containing 5 ng / mL VEGF-A and 10 μM SB431542 (Stemcell Technologies). After 48 h the cells were seeded on fibronectin-coated plates and cultured in MV2 media (PromoCell) containing 100 ng / mL VEGF-C. This treatment continued for 3 more days, and the cells were then used for various assays. Growth factor-based endothelial cell differentiation protocol was adopted from Harding et al., Stem Cell. 2017;35(4):909–19. After EC differentiation, the cells were treated with 100 ng / mL VEGF-C similar to the treatment of transduction-based differentiation. Immunofluorescence Staining. To visualize the lymphatic protein expression, LECs and differentiated LECs were seeded on tissue culture plastic around 60% confluency. Samples were fixed with 4% paraformaldehyde, blocked with 1% BSA, permeabilized with 0.1% Triton- X, and stained for Prox1 and ERG (Table 3). Samples were rinsed twice in PBS and counterstained with 300 nMDAPI (Thermo Fisher). All samples were imaged using Nikon AX- R confocal at ×20 magnification. Flow Cytometry. Differentiated LECs were analyzed for lymphatic markers using flow cytometry (FACS) following standard procedures. Briefly, cells were trypsinized and centrifuged following resuspension in FACS buffer. Suspended cells (1 × 106cells) were 37

[0038] 501.113WO1 ND 25-008 stained with the antibodies (1 μg / mL) for 30 min at room temperature: Anti-LYVE-1 antibody (R&D systems, FAB20892A), Anti-PDPN antibody APC (Biolegend, 337004), as well as their corresponding IgG isotype controls (Table 3). For intracellular staining, the cells were fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (Thermo, 00-5523-00) and then incubated with Anti-Prox1 antibody FITC (Novus Biologicals, NBP1-30045AF488) for 30 min. The cells were washed twice and resuspended in FACS buffer for analysis. Then, the cells were analyzed using flow cytometry (BD LSR FortessaX-20), and the metadata were analyzed using FlowJo. Quantitative Reverse Transcription PCR. To analyze the expression of key lymphatic genes in the differentiated LECs, quantitative reverse transcription PCR was carried out in RNA lysates prepared from cells in culture. Three biological replicates (n = 3) were collected per condition and analyzed with real-time qRT-PCR with triplicate readings. RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher) according to the manufacturer’s protocol. cDNA was then used with the TaqMan Universal PCR Master Mix and Gene Expression Assays for LYVE-1, Prox1, PDPN, VEGFR3, and GAPDH (Table 4). Each sample was prepared in triplicate, and the relative expression was normalized to GAPDH and analyzed using theΔΔCt method. Table 4. Primers Enzyme-Linked Immunosorbent Assay. Supernatant samples were collected from cell culture and standardized using Coomassie Protein Assay Kit (ThermoFisher Scientific). Then standardized protocol for ELISA kits (ab100664 – VEGFC Human ELISA Kit, ab284620 – Human Reelin SimpleStep ELISA®Kit) were followed with the samples diluted to correct values. Briefly, standard solutions of target protein were prepared in a two-fold dilution series in assay diluent (Fig. 8). Supernatant samples and standards were added to respective wells in triplicate and incubated. Then subsequently detection antibodies, enzyme conjugate, and substrate solution were added and finally the absorbance was measured at 495 nM using a microplate reader. Tube Formation Assay. Tube formation assay was performed as previously described (Hanjaya-Putra et al., Blood. 2011;118(3):804–15; Bui et al., Commun Biol. 2022;5(1):635.). 38

[0039] 501.113WO1 ND 25-008 Briefly, human differentiated LECs were stained CellTracker™ Green (ThermoFisher) plated at a density of 100,000 cells / cm2in one well of a µ-Slide 15 Well 3D plate (ibidi) on top of solidified Matrigel (10 μL) with differentiation media. After 6 h, cells were imaged using a fluorescence microscope. Numbers of branches were counted by AutoTube script in MATLAB (Montoya-Zegarra et al., Angiogenesis. 2019;22(2):223–36). Statistical Analyses. Unless otherwise stated, data were expressed as means ± SD of the mean. Statistical analyses were performed with GraphPad Prism 10.1.2(324). For each condition, at least three independent experiments were performed with three biological replicates. Statistical comparisons were made using Student’s t test for paired data, analysis of variance (ANOVA) for multiple comparisons, and with Tukey post hoc analysis for parametric data. Specifically, the Student’s t test was used to analyze differences between protein expression and gene expression among different transduced groups. Significance levels were set at the following: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Example 3. Sequences. Human ETS2 NCBI Reference Sequence: NM_001256295.2 gccactcccgcggagcctgcgggatcggggcttcccgggagcagcgcgatcagcaccacgactcggggacacagccagggccc ggtttctacaggaagcgcctcatttggagcctttttgtgatagaatgatcattagtcctaagcccattcagaggttcaagaatggggtcgg ctcaatttcagggccttattacccaagcccggctgcccttcggtgccaccagcaccactgctccgtcgctgcggaattccaaaggcag gtttggcgttagggccttggccccagagaggacgccgagcgctccacggaaagtctccgcccggctcccagggcgcacactcgcg cgcacgtggggccgaggccctgctcccggggcctcagggccagccggcgagggacccagccgagtgacagcaggaggcggag ggaagctcagagctcccggagccgcccggccagcgtccggcctccctgatcgtctctggccggcgccctcgccctcgcccggcgc gcaccgagcagccgcgggcgccgagcagccaccgtcccgaccaagcgccggccctgcccgcagcggcaggatgaatgatttcg gaatcaagaatatggaccaggtagcccctgtggctaacagttacagagggacactcaagcgccagccagcctttgacacctttgatgg gtccctgtttgctgtttttccttctctaaatgaagagcaaacactgcaagaagtgccaacaggcttggattccatttctcatgactccgccaa ctgtgaattgcctttgttaaccccgtgcagcaaggctgtgatgagtcaagccttaaaagctaccttcagtggcttcaaaaaggaacagcg gcgcctgggcattccaaagaacccctggctgtggagtgagcaacaggtatgccagtggcttctctgggccaccaatgagttcagtctg gtgaacgtgaatctgcagaggttcggcatgaatggccagatgctgtgtaaccttggcaaggaacgctttctggagctggcacctgactt tgtgggtgacattctctgggaacatctggagcaaatgatcaaagaaaaccaagaaaagacagaagatcaatatgaagaaaattcacac ctcacctccgttcctcattggattaacagcaatacattaggttttggcacagagcaggcgccctatggaatgcagacacagaattacccc aaaggcggcctcctggacagcatgtgtccggcctccacacccagcgtactcagctctgagcaggagtttcagatgttccccaagtctc ggctcagctccgtcagcgtcacctactgctctgtcagtcaggacttcccaggcagcaacttgaatttgctcaccaacaattctgggactc ccaaagaccacgactcccctgagaacggtgcggacagcttcgagagctcagactccctcctccagtcctggaacagccagtcgtcct tgctggatgtgcaacgggttccttccttcgagagcttcgaagatgactgcagccagtctctctgcctcaataagccaaccatgtctttcaa ggattacatccaagagaggagtgacccagtggagcaaggcaaaccagttatacctgcagctgtgctggccggcttcacaggaagtg gacctattcagctgtggcagtttctcctggagctgctatcagacaaatcctgccagtcattcatcagctggactggagacggatgggagt ttaagctcgccgaccccgatgaggtggcccgccggtggggaaagaggaaaaataagcccaagatgaactacgagaagctgagcc ggggcttacgctactattacgacaagaacatcatccacaagacgtcggggaagcgctacgtgtaccgcttcgtgtgcgacctccagaa cttgctggggttcacgcccgaggaactgcacgccatcctgggcgtccagcccgacacggaggactgaggtcgccgggaccaccct 39

[0040] 501.113WO1 ND 25-008 gagccggccccaggctcgtggactgagtgggaagcccatcctgaccagctgctccgaggacccaggaaaggcaggattgaaaatg tccaggaaagtggccaagaagcagtggccttattgcatcccaaaccacgcctcttgaccaggctgcctcccttgtggcagcaacggc acagctaattctactcacagtgcttttaagtgaaaatggtcgagaaagaggcaccaggaagccgtcctggcgcctggcagtccgtggg acgggatggttctggctgtttgagattctcaaaggagcgagcatgtcgtggacacacacagactatttttagattttcttttgccttttgcaa ccaggaacagcaaatgcaaaaactctttgagagggtaggagggtgggaaggaaacaaccatgtcatttcagaagttagtttgtatatat tattataatcttataattgttctcagaatcccttaacagttgtatttaacagaaattgtatattgtaatttaaaataattatataactgtatttgaaat aagaattcagacatctgaggttttatttcatttttcaatagcacatatggaattttgcaaagatttaatctgccaagggccgactaagagaag ttgtaaagtatgtattatttacatttaatagacttacagggataaggcctgtggggggtaatccctgctttttgtgtttttttgtttgtttgtttgttt gtttttggggggttttcttgccttggttgtctggcaaggactttgtacatttgggagtttttatgagaaacttaaatgttattatctgggcttatat ctggcctctgctttctcctttaattgtaaagtaaaagctataaagcagtatttttcttgacaaatggcatatgttttccacttctttgcatgcgttt aagtcagtttatacacaaaatggattttattttttagtttaactgtgtttctccgacagctcacctctctctgaccacccagccatttccttcctgt gctccacgttcttctgtgtgattaaaataagaatattatttttggaaatatgcaactccttttcagagatcaggagggatttatgtagcagctat ttttactgcaaaagtaattcactggaaaaaaaatgtaatttgtaagaaagctttatttttatctcagctctatgtaaagttaaagttactgtacag agctgaaggacggggggcggtaggggtcttgatgaaacctcttgaacgaagcacagtttgtcccatctttgttcactcgtgtgtctcaac catcttaatagcatgctgctcctttttgctcagtgtccacagcaagatgacgtgattcttattttcttggacacagactattctgaggcacaga gcggggacttaagatgggaaagagaaagcatcggagccattcattcggagaaaacgttttgatcaaaatggagacttttgtagtcgtttc aaaagagcacctgagtcatgtgtattcccggcctttataaatgacccggtcaagttggtttcaaagtccgacaggcttgtctgtttactagc tgcgtggccttggacgggtggctgacatctgtaaagaatcctcctgtgatgaaactgaggaatcgggtggccgggcaagctgggaag agcaaagccagagctgcgctgcctcaatacccacaaaagaccattcccagtatacataagcacaggatgtttttctcaagagggatgta tttatcacttggacatctgtttataatataaacagacatgtgactgggaacatcttgctgccaaaagaatcctaggcagtggctcattgtatg tgaggttgaaccacgtgaaattgccaatattaggctggcttttatctacaaagaaggagtttcatggggttcagcctaacagttatggaaa ctacagtccttataaaccattggcatggtaataaacagatcttaagtataaaaattttgtaattgggcctttactctctcaataataaagtatttt gtttatataaa (SEQ ID NO: 1) NCBI Reference Sequence: NP_001243224.1 MGSAQFQGLITQARLPFGATSTTAPSLRNSKGRFGVRALAPERTPSAPRKVSARLPGR TLARTWGRGPAPGASGPAGEGPSRVTAGGGGKLRAPGAARPASGLPDRLWPAPSPS PGAHRAAAGAEQPPSRPSAGPARSGRMNDFGIKNMDQVAPVANSYRGTLKRQPAFD TFDGSLFAVFPSLNEEQTLQEVPTGLDSISHDSANCELPLLTPCSKAVMSQALKATFS GFKKEQRRLGIPKNPWLWSEQQVCQWLLWATNEFSLVNVNLQRFGMNGQMLCNL GKERFLELAPDFVGDILWEHLEQMIKENQEKTEDQYEENSHLTSVPHWINSNTLGFG TEQAPYGMQTQNYPKGGLLDSMCPASTPSVLSSEQEFQMFPKSRLSSVSVTYCSVSQ DFPGSNLNLLTNNSGTPKDHDSPENGADSFESSDSLLQSWNSQSSLLDVQRVPSFESF EDDCSQSLCLNKPTMSFKDYIQERSDPVEQGKPVIPAAVLAGFTGSGPIQLWQFLLEL LSDKSCQSFISWTGDGWEFKLADPDEVARRWGKRKNKPKMNYEKLSRGLRYYYDK NIIHKTSGKRYVYRFVCDLQNLLGFTPEELHAILGVQPDTED (SEQ ID NO: 2) NCBI Reference Sequence: NM_005239.6 ggttacttcctccagagactgacgagtgcggtgtcgctccagctcagagctcccggagccgcccggccagcgtccggcctccctgat cgtctctggccggcgccctcgccctcgcccggcgcgcaccgagcagccgcgggcgccgagcagccaccgtcccgaccaagcgc cggccctgcccgcagcggcaggatgaatgatttcggaatcaagaatatggaccaggtagcccctgtggctaacagttacagaggga cactcaagcgccagccagcctttgacacctttgatgggtccctgtttgctgtttttccttctctaaatgaagagcaaacactgcaagaagtg ccaacaggcttggattccatttctcatgactccgccaactgtgaattgcctttgttaaccccgtgcagcaaggctgtgatgagtcaagcct taaaagctaccttcagtggcttcaaaaaggaacagcggcgcctgggcattccaaagaacccctggctgtggagtgagcaacaggtat gccagtggcttctctgggccaccaatgagttcagtctggtgaacgtgaatctgcagaggttcggcatgaatggccagatgctgtgtaac cttggcaaggaacgctttctggagctggcacctgactttgtgggtgacattctctgggaacatctggagcaaatgatcaaagaaaacca agaaaagacagaagatcaatatgaagaaaattcacacctcacctccgttcctcattggattaacagcaatacattaggttttggcacaga gcaggcgccctatggaatgcagacacagaattaccccaaaggcggcctcctggacagcatgtgtccggcctccacacccagcgtac 40

[0041] 501.113WO1 ND 25-008 tcagctctgagcaggagtttcagatgttccccaagtctcggctcagctccgtcagcgtcacctactgctctgtcagtcaggacttcccag gcagcaacttgaatttgctcaccaacaattctgggactcccaaagaccacgactcccctgagaacggtgcggacagcttcgagagctc agactccctcctccagtcctggaacagccagtcgtccttgctggatgtgcaacgggttccttccttcgagagcttcgaagatgactgcag ccagtctctctgcctcaataagccaaccatgtctttcaaggattacatccaagagaggagtgacccagtggagcaaggcaaaccagtta tacctgcagctgtgctggccggcttcacaggaagtggacctattcagctgtggcagtttctcctggagctgctatcagacaaatcctgcc agtcattcatcagctggactggagacggatgggagtttaagctcgccgaccccgatgaggtggcccgccggtggggaaagaggaa aaataagcccaagatgaactacgagaagctgagccggggcttacgctactattacgacaagaacatcatccacaagacgtcgggga agcgctacgtgtaccgcttcgtgtgcgacctccagaacttgctggggttcacgcccgaggaactgcacgccatcctgggcgtccagc ccgacacggaggactgaggtcgccgggaccaccctgagccggccccaggctcgtggactgagtgggaagcccatcctgaccagc tgctccgaggacccaggaaaggcaggattgaaaatgtccaggaaagtggccaagaagcagtggccttattgcatcccaaaccacgc ctcttgaccaggctgcctcccttgtggcagcaacggcacagctaattctactcacagtgcttttaagtgaaaatggtcgagaaagaggc accaggaagccgtcctggcgcctggcagtccgtgggacgggatggttctggctgtttgagattctcaaaggagcgagcatgtcgtgg acacacacagactatttttagattttcttttgccttttgcaaccaggaacagcaaatgcaaaaactctttgagagggtaggagggtgggaa ggaaacaaccatgtcatttcagaagttagtttgtatatattattataatcttataattgttctcagaatcccttaacagttgtatttaacagaaatt gtatattgtaatttaaaataattatataactgtatttgaaataagaattcagacatctgaggttttatttcatttttcaatagcacatatggaatttt gcaaagatttaatctgccaagggccgactaagagaagttgtaaagtatgtattatttacatttaatagacttacagggataaggcctgtgg ggggtaatccctgctttttgtgtttttttgtttgtttgtttgtttgtttttggggggttttcttgccttggttgtctggcaaggactttgtacatttggg agtttttatgagaaacttaaatgttattatctgggcttatatctggcctctgctttctcctttaattgtaaagtaaaagctataaagcagtatttttc ttgacaaatggcatatgttttccacttctttgcatgcgtttaagtcagtttatacacaaaatggattttattttttagtttaactgtgtttctccgac agctcacctctctctgaccacccagccatttccttcctgtgctccacgttcttctgtgtgattaaaataagaatattatttttggaaatatgcaa ctccttttcagagatcaggagggatttatgtagcagctatttttactgcaaaagtaattcactggaaaaaaaatgtaatttgtaagaaagcttt atttttatctcagctctatgtaaagttaaagttactgtacagagctgaaggacggggggcggtaggggtcttgatgaaacctcttgaacga agcacagtttgtcccatctttgttcactcgtgtgtctcaaccatcttaatagcatgctgctcctttttgctcagtgtccacagcaagatgacgt gattcttattttcttggacacagactattctgaggcacagagcggggacttaagatgggaaagagaaagcatcggagccattcattcgg agaaaacgttttgatcaaaatggagacttttgtagtcgtttcaaaagagcacctgagtcatgtgtattcccggcctttataaatgacccggt caagttggtttcaaagtccgacaggcttgtctgtttactagctgcgtggccttggacgggtggctgacatctgtaaagaatcctcctgtga tgaaactgaggaatcgggtggccgggcaagctgggaagagcaaagccagagctgcgctgcctcaatacccacaaaagaccattcc cagtatacataagcacaggatgtttttctcaagagggatgtatttatcacttggacatctgtttataatataaacagacatgtgactgggaac atcttgctgccaaaagaatcctaggcagtggctcattgtatgtgaggttgaaccacgtgaaattgccaatattaggctggcttttatctaca aagaaggagtttcatggggttcagcctaacagttatggaaactacagtccttataaaccattggcatggtaataaacagatcttaagtata aaaattttgtaattgggcctttactctctcaataataaagtattttgtttatataaa (SEQ ID NO: 3) NCBI Reference Sequence: NP_005230.1 MNDFGIKNMDQVAPVANSYRGTLKRQPAFDTFDGSLFAVFPSLNEEQTLQEVPTGL DSISHDSANCELPLLTPCSKAVMSQALKATFSGFKKEQRRLGIPKNPWLWSEQQVCQ WLLWATNEFSLVNVNLQRFGMNGQMLCNLGKERFLELAPDFVGDILWEHLEQMIK ENQEKTEDQYEENSHLTSVPHWINSNTLGFGTEQAPYGMQTQNYPKGGLLDSMCPA STPSVLSSEQEFQMFPKSRLSSVSVTYCSVSQDFPGSNLNLLTNNSGTPKDHDSPENG ADSFESSDSLLQSWNSQSSLLDVQRVPSFESFEDDCSQSLCLNKPTMSFKDYIQERSD PVEQGKPVIPAAVLAGFTGSGPIQLWQFLLELLSDKSCQSFISWTGDGWEFKLADPDE VARRWGKRKNKPKMNYEKLSRGLRYYYDKNIIHKTSGKRYVYRFVCDLQNLLGFT PEELHAILGVQPDTED (SEQ ID NO: 4) Human ETV2 NCBI Reference Sequence: NM_001300974.2 gcagataagcccagcttagcccagctgaccccagaccctctcccctcactccccccatgtcgcaggatcgagaccctgaggcagac agcccgttcaccaagccccccgccccgcccccatcaccccgtaaacttctcccagcctccgccctgccctcacccagcccgctgttcc ccaagcctcgctccaagcccacgccacccctgcagcagggcagccccagaggccagcacctatccccgaggctggggtcgaggc tcggccccgcccctgcctctgcaacttgagcctggctgcgacccctgctctgacgtctcggaaaattcccccttgcccaggcccttggg 41

[0042] 501.113WO1 ND 25-008 ggagggggtgcatggtatgaaatggggctgagacccccggctgggggcagaggaacccgccagagaaggagccaaattaggctt ctgtttccctgatctggcactccaaggggacacgccgacagcgacagcagagacatgctggaaaggtacaagctcatccctggcaa gcttcccacagctggactggggctccgcgttactgcacccagaagttccatggggggcggagcccgactctcaggctcttccgtggt ccggggactggacagacatggcgtgcacagcctgggactcttggagcggcgcctcgcagaccctgggccccgcccctctcggccc gggccccatccccgccgccggctccgaaggcgccgcgggccagaactgcgtccccgtggcgggagaggccacctcgtggtcgc gcgcccaggccgccgggagcaacaccagctgggactgttctgtggggcccgacggcgatacctactggggcagtggcctgggcg gggagccgcgcacggactgtaccatttcgtggggcgggcccgcgggcccggactgtaccacctcctggaacccggggctgcatgc gggtggcaccacctctttgaagcggtaccagagctcagctctcaccgtttgctccgaaccgagcccgcagtcggaccgtgccagtttg gctcgatgccccaaaactaaccaccgaggtcccattcagctgtggcagttcctcctggagctgctccacgacggggcgcgtagcagc tgcatccgttggactggcaacagccgcgagttccagctgtgcgaccccaaagaggtggctcggctgtggggcgagcgcaagagaa agccgggcatgaattacgagaagctgagccggggccttcgctactactatcgccgcgacatcgtgcgcaagagcggggggcgaaa gtacacgtaccgcttcgggggccgcgtgcccagcctagcctatccggactgtgcgggaggcggacggggagcagagacacaata aaaattcccggtcaaacctc (SEQ ID NO: 5) NCBI Reference Sequence: NP_001287903.1 MACTAWDSWSGASQTLGPAPLGPGPIPAAGSEGAAGQNCVPVAGEATSWSRAQAA GSNTSWDCSVGPDGDTYWGSGLGGEPRTDCTISWGGPAGPDCTTSWNPGLHAGGT TSLKRYQSSALTVCSEPSPQSDRASLARCPKTNHRGPIQLWQFLLELLHDGARSSCIR WTGNSREFQLCDPKEVARLWGERKRKPGMNYEKLSRGLRYYYRRDIVRKSGGRKY TYRFGGRVPSLAYPDCAGGGRGAETQ (SEQ ID NO: 6) NCBI Reference Sequence: NM_001304549.2 gcagataagcccagcttagcccagctgaccccagaccctctcccctcactccccccatgtcgcaggatcgagaccctgaggcagac agcccgttcaccaagccccccgccccgcccccatcaccccgtaaacttctcccagcctccgccctgccctcacccagcccgctgttcc ccaagcctcgctccaagcccacgccacccctgcagcagggcagccccagaggccagcacctatccccgaggctggggtcgaggc tcggccccgcccctgcctctgcaacttgagcctggctgcgacccctgctctgacgtctcggaaaattcccccttgcccaggcccttggg ggagggggtgcatggtatgaaatggggctgagacccccggctgggggcagaggaacccgccagagaacattcagaaggccttcat cgcatccatggacctgtggaactgggatgaggcatccccacaggaagtgcctccagggaacaagctggcagggcttgaaggagcc aaattaggcttctgtttccctgatctggcactccaaggggacacgccgacagcgacagcagagacatgctggaaaggtcccattcagc tgtggcagttcctcctggagctgctccacgacggggcgcgtagcagctgcatccgttggactggcaacagccgcgagttccagctgt gcgaccccaaagaggtggctcggctgtggggcgagcgcaagagaaagccgggcatgaattacgagaagctgagccggggccttc gctactactatcgccgcgacatcgtgcgcaagagcggggggcgaaagtacacgtaccgcttcgggggccgcgtgcccagcctagc ctatccggactgtgcgggaggcggacggggagcagagacacaataaaaattcccggtcaaacctc (SEQ ID NO: 7) NCBI Reference Sequence: NP_001291478.1 MDLWNWDEASPQEVPPGNKLAGLEGAKLGFCFPDLALQGDTPTATAETCWKGPIQ LWQFLLELLHDGARSSCIRWTGNSREFQLCDPKEVARLWGERKRKPGMNYEKLSRG LRYYYRRDIVRKSGGRKYTYRFGGRVPSLAYPDCAGGGRGAETQ (SEQ ID NO: 8) NCBI Reference Sequence: NM_014209.4 gcagataagcccagcttagcccagctgaccccagaccctctcccctcactccccccatgtcgcaggatcgagaccctgaggcagac agcccgttcaccaagccccccgccccgcccccatcaccccgtaaacttctcccagcctccgccctgccctcacccagcccgctgttcc ccaagcctcgctccaagcccacgccacccctgcagcagggcagccccagaggccagcacctatccccgaggctggggtcgaggc tcggccccgcccctgcctctgcaacttgagcctggctgcgacccctgctctgacgtctcggaaaattcccccttgcccaggcccttggg ggagggggtgcatggtatgaaatggggctgagacccccggctgggggcagaggaacccgccagagaacattcagaaggccttcat cgcatccatggacctgtggaactgggatgaggcatccccacaggaagtgcctccagggaacaagctggcagggcttgaaggagcc aaattaggcttctgtttccctgatctggcactccaaggggacacgccgacagcgacagcagagacatgctggaaaggtacaagctcat 42

[0043] 501.113WO1 ND 25-008 ccctggcaagcttcccacagctggactggggctccgcgttactgcacccagaagttccatggggggcggagcccgactctcaggct cttccgtggtccggggactggacagacatggcgtgcacagcctgggactcttggagcggcgcctcgcagaccctgggccccgccc ctctcggcccgggccccatccccgccgccggctccgaaggcgccgcgggccagaactgcgtccccgtggcgggagaggccacct cgtggtcgcgcgcccaggccgccgggagcaacaccagctgggactgttctgtggggcccgacggcgatacctactggggcagtg gcctgggcggggagccgcgcacggactgtaccatttcgtggggcgggcccgcgggcccggactgtaccacctcctggaacccgg ggctgcatgcgggtggcaccacctctttgaagcggtaccagagctcagctctcaccgtttgctccgaaccgagcccgcagtcggacc gtgccagtttggctcgatgccccaaaactaaccaccgaggtcccattcagctgtggcagttcctcctggagctgctccacgacggggc gcgtagcagctgcatccgttggactggcaacagccgcgagttccagctgtgcgaccccaaagaggtggctcggctgtggggcgag cgcaagagaaagccgggcatgaattacgagaagctgagccggggccttcgctactactatcgccgcgacatcgtgcgcaagagcg gggggcgaaagtacacgtaccgcttcgggggccgcgtgcccagcctagcctatccggactgtgcgggaggcggacggggagca gagacacaataaaaattcccggtcaaacctc (SEQ ID NO: 9) NCBI Reference Sequence: NP_055024.2 MDLWNWDEASPQEVPPGNKLAGLEGAKLGFCFPDLALQGDTPTATAETCWKGTSS SLASFPQLDWGSALLHPEVPWGAEPDSQALPWSGDWTDMACTAWDSWSGASQTL GPAPLGPGPIPAAGSEGAAGQNCVPVAGEATSWSRAQAAGSNTSWDCSVGPDGDTY WGSGLGEPRTDCTISWGGPAGPDCTTSWNPGLHAGGTTSLKRYQSSALTVCSEPSPQ SDRASLARCPKTNHRGPIQLWQFLLELLHDGARSSCIRWTGNSREFQLCDPKEVARL WGERKRKPGMNYEKLSRGLRYYYRRDIVRKSGGRKYTYRFGGRVPSLAYPDCAGG GRGAETQ (SEQ ID NO: 10) Mus musculus ETS2 NCBI Reference Sequence: NM_011809.3 tccggccctcccccagctccagaagagcgcgcggctaggagatcgggctggttacttccttcaccgcggcgttgagcacagagtgg acaccactgaggcccgtcagtccccgccaccttccccggccccgcgcgcccggatcggccctacggcctcgtctcgcccggccttg cgcgccgggaccgccgcgatctcctctccccgccgccctccggctggccctgcctgctgcggcgcgatgaatgactttggaatcaag aacatggaccaagtggcccctgtcgccaacagttttcgtgggacactcaagcgccagccagcctttgacaccttcgatggctctctgttt gctgtgctcccttctctcagtgaagatcagacactccaagaagtgcccacgggcctggattctgtctcccatgactcggccagctgcga gctgcctttgctcactccctgcagcaaggcagtgatgagccaagccttaaaagccaccttcagtggcttccaaaaggagcaacgacgt cttggcatccccaaaaacccctggctgtggagcgagcagcaggtgtgccagtggcttctctgggccaccaacgagttcagcctggtg aatgtgaacctgcaccagtttggcatgaacggccagatgctgtgtaacctcggcaaggagcgcttcctggagctggcgcctgactttgt gggtgacatcctctgggaacatctagagcagatgatcaaagagaaccaagaaaagacagaagaccaatatgaggaaaactctcacct caacgcggttcctcattggatcaacagcaatacattaggcttcagcatggaacaggctccatatggaatgcaggcaccaaactacccc aaagacaatctcctggacagcatgtgcccgccatcggccacgcctgcagctctgggctctgagctccagatgttgcccaagtctcggc tcaacaccgtcaatgtcaattactgttccatcagccaggacttccccagcagcaacgtgaatttgctcaacaacaattctggaaaaccca aggaccacgactctccagagaacggtggggacagcttcgagagctccgactcgctgctgaggtcctggaacagccagtcgtcccta ctggatgtacagcgggtaccttccttcgagagctttgaggaggactgtagccagtctctgtgcctcagtaagctgaccatgtccttcaag gactacatccaagagaggagcgacccagtcgagcaaggcaaaccagttattcctgcagcagtactggctggcttcactggaagcgg accaatccagttgtggcagtttcttctggagctactctctgacaagtcctgtcaatctttcatcagctggacgggggatggatgggagttc aagcttgctgaccccgatgaggttgcccgccggtgggggaagaggaaaaataaaccaaagatgaactacgagaagctgagccggg gcttacgttactactacgacaagaacatcatccacaagacttcgggcaagcgctacgtgtaccgtttcgtatgtgacctgcagaacttgc tgggcttcactccggaggaactgcatgccatcctgggcgtccagcctgatacagaagactgagggcctcaggaccaccctgagcca gccccgggcccagggactgagtgggaagcccgtcctgacccacttgctccaaagacccatgggagggcaggatggtacccctcgg gagagagccaccaagcagcagtggcctttcatccacgcctcttccacaagccaatgccccatggtggaaaccggcatagcctctctgt cctgtatgagacccccctgaagggaagagtctctgggaagccaccgtggcaccgagcaggccctgtgcattgtggctatttgagatg ctccagggagccagcatgtggtggatacacagacactgagaaacattgtttttcctttggccttttgcctccaggagtagcaaatgcaaa aagccacctctgcttgagagggcccggcaggtggagagtgtgattgtccatttcagaagtcagtttgtttgtacatactattgtatcttata attgtcctaatcctccaacagttctatttaacagaaattgtatattgtatttaaagtaattataacttgtctgaatcaagaatgcagatacctaat aggttatccttttatttttttcactcctacataggaacgtcattttacaccgatttaatctcttagaatccaactgagagaaaccgtgatattctg 43

[0044] 501.113WO1 ND 25-008 tttttgttttatttaccagcaggaatgggctctgaagggtgacctggttttttcttttgttttgctctcttgttattgttgctcattatctgaccagga ctttgtatattggtttttttttataagaaacttaatgttattatttgagggtccatatggcctctccactctcctggactgtaaaagctgtaaagcg gtatttttcttgacacatggcacatgttttccacttccctgcatgccttaagccaatttatacacaaatgtattttattttttggcgtaactgggttt tccaggcaactcccctcccccagccagtccttcccttccttcgccccaagatattctgtgtgactacaacaagaagactatttttggaaata tatgggatttatgtagcagctatttttactgcaaaagtaactcactggaaaaaaatattgtaatttgtacgaaagctttatttttatcttagtcac gtaaagggagatgtgtcgggtgggcagggctcagggcaatcaggagggatcttccatcagccttgaccttaaacgaagtgtggtgctt cctgtcttgctgacctgctatgtctcttaaccatctgggctgcatgctcctctttctgctccgtgtgctcagagatttgctcctcttatttttttgg ccatggatacccagaagcacagacagagcaagcatgctgggaaatggtgtcttggaccagttctgtacgaggaggttctcatctagtg aagagctaactccaccatgagacccccacgctttcatctagttgtgaactggcttcgccgtttaccagcatggtgacctcagatcagcaa gtgacattgataaagagccgttataccatgagaccacacgggacgtaggaccagctgggacctgctgtgccagtcctgggaggtctt ggcctgcatgccccaagagaaatgctttgtggttaagcacagggacctgtttgtcgctgggacatctgtgtgtaatataaacagacatgg gacttggagcatcctcctgtcagaggcctaatcctcagtcagtttatgtacagtggagccacatgacattgccaagtttaacctgagtctt gtcagccaacatgacggttttacctggttgaatctaacagtcatggaagtcctcagaagtctttggctgggtcttaagtatgaaggttttgt aatttggcctcctctttctcaaataataaagtatttggtttatgacaaaaaa (SEQ ID NO: 11) NCBI Reference Sequence: NP_035939.3 MNDFGIKNMDQVAPVANSFRGTLKRQPAFDTFDGSLFAVLPSLSEDQTLQEVPTGLD SVSHDSASCELPLLTPCSKAVMSQALKATFSGFQKEQRRLGIPKNPWLWSEQQVCQ WLLWATNEFSLVNVNLHQFGMNGQMLCNLGKERFLELAPDFVGDILWEHLEQMIK ENQEKTEDQYEENSHLNAVPHWINSNTLGFSMEQAPYGMQAPNYPKDNLLDSMCPP SATPAALGSELQMLPKSRLNTVNVNYCSISQDFPSSNVNLLNNNSGKPKDHDSPENG GDSFESSDSLLRSWNSQSSLLDVQRVPSFESFEEDCSQSLCLSKLTMSFKDYIQERSDP VEQGKPVIPAAVLAGFTGSGPIQLWQFLLELLSDKSCQSFISWTGDGWEFKLADPDE VARRWGKRKNKPKMNYEKLSRGLRYYYDKNIIHKTSGKRYVYRFVCDLQNLLGFT PEELHAILGVQPDTED (SEQ ID NO: 12) pSIN4-EF1a-ETV2-IRES-Puro cggaccgccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatat gtactacaaacttagtagttggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctac ttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagcc agataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagag aagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatc ctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaaccc actgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagac ccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacg caggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggc tagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagg gggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacat cagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagca accctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaag accaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataa agtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaata ggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattat tgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaag cagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttg caccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagag aaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattag ataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttt 44

[0045] 501.113WO1 ND 25-008 aagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggg gacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgac ggtatcgccacaaatggcagtattcatccacaattttaaaagaaagggggggattggggggtacagtgcaggggaaagaatagtaga cataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagaga tccactttggatcgataagctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaagg agtgggaattggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaat tgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtggggga gaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcc cgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcg ggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctg gggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctg cgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacg gggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaag ctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagtt gcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcggg tgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggca cctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtg gagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcaga cagtggttcaaagtttttttcttccatttcaggtgtcgtgaggaattcgccaccatggacctgtggaactgggatgaggcatccccacagg aagtgcctccagggaacaagctggcagggcttgaaggagccaaattaggcttctgtttccctgatctggcactccaaggggacacgc cgacagcgacagcagagacatgctggaaaggtacaagctcatccctggcaagcttcccacagctggactggggctccgcgttactg cacccagaagttccatggggggcggagcccgactctcaggctcttccgtggtccggggactggacagacatggcgtgcacagcctg ggactcttggagcggcgcctcgcagaccctgggccccgcccctctcggcccgggccccatccccgccgccggctccgaaggcgc cgcgggccagaactgcgtccccgtggcgggagaggccacctcgtggtcgcgcgcccaggccgccgggagcaacaccagctggg actgttctgtggggcccgacggcgatacctactggggcagtggcctgggcggggagccgcgcacggactgtaccatttcgtggggc gggcccgcgggcccggactgtaccacctcctggaacccggggctgcatgcgggtggcaccacctctttgaagcggtaccagagct cagctctcaccgtttgctccgaaccgagcccgcagtcggaccgtgccagtttggctcgatgccccaaaactaaccaccgaggtcccat tcagctgtggcagttcctcctggagctgctccacgacggggcgcgtagcagctgcatccgttggactggcaacagccgcgagttcca gctgtgcgaccccaaagaggtggctcggctgtggggcgagcgcaagagaaagccgggcatgaattacgagaagctgagccgggg ccttcgctactactatcgccgcgacatcgtgcgcaagagcggggggcgaaagtacacgtaccgcttcgggggccgcgtgcccagcc tagcctatccggactgtgcgggaggcggacggggagcagagacacaataaggatccgcatgcatctagggcggccaattccgccc ctctcccccccccccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgtta ttttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctc gccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccct ttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggca caaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatg cccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctag gccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataagcttgccacaacccacaaggagacgaccttccatgac cgagtacaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactac cccgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggc tcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcg gtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgc cgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccg tcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctac gagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcct gataataggcggccgctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggc tagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatctt agccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctct ggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaag tagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcat gtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggccttgacattataatagatttagcaggaattgaa 45

[0046] 501.113WO1 ND 25-008 ctaggagtggagcacacaggcaaagttctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccct cccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtg tcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtg ggctctatggcttctgaggcggaaagaaccagctgggggcgcgcccctcgaggccgccatggtcatagctgtttgacgtcaggtggc acttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcacgagacaataaccctgataaa tgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgc tcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggt aagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgac gccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacgg atggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggagga ccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccatacc aaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttccc ggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgat aaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacac gacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagac caagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaa tcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatct gctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggct tcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctaca gcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttctggccttttgca (SEQ ID NO: 13) pLV-tetO-Ets2 gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctcc ctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaat ctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatca attacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgac ccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggt aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatt atgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagt acatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag cagcgcgttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagc ctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcag tgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcgg cttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggaga gagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaa aaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggct gtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctatt gtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgc acagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaa aattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgt tccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtata gtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccag gcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgc tgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaaca 46

[0047] 501.113WO1 ND 25-008 attacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatggg caagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagt ttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgac aggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgtgcgc caattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtag acataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagag atccagtttggttatcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaa gtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaa gtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcga gtttaccactccctatcagtgatagagaaaagtgaaagtcgagctcggtacccgggtcgaggtaggcgtgtacggtgggaggcctata taagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatc cagcctccgcggccccgaattcggcttcgcgatgaatgactttggaatcaagaacatggaccaagtggcccctgtcgccaacagttttc gtgggacactcaagcgccagccagcctttgacaccttcgatggctctctgtttgctgtgctcccttctctcagtgaagatcagacactcca agaagtgcccacgggcctggattctgtctcccatgactcggccagctgcgagctgcctttgctcactccctgcagcaaggcagtgatg agccaagccttaaaagccaccttcagtggcttccaaaaggagcaacgacgtcttggcatccccaaaaacccctggctgtggagcgag cagcaggtgtgccagtggcttctctgggccaccaacgagttcagcctggtgaatgtgaacctgcaccagtttggcatgaacggccaga tgctgtgtaacctcggcaaggagcgcttcctggagctggcgcctgactttgtgggtgacatcctctgggaacatctagagcagatgatc aaagagaaccaagaaaagacagaagaccaatatgaggaaaactctcacctcaacgcggttcctcattggatcaacagcaatacatta ggcttcagcatggaacaggctccatatggaatgcaggcaccaaactaccccaaagacaatctcctggacagcatgtgtccgccatcg gccacgcctgcagctctgggctctgagctccagatgttgcccaagtctcggctcaacaccgtcaatgtcaattactgttccatcagccag gacttccccagcagcaacgtgaatttgctcaacaacaattctggaaaacccaaggaccacgactctccagagaacggtggggacagc ttcgagagctccgactcgctgctgaggtcctggaacagccagtcgtccctactggatgtacagcgggtaccttccttcgagagctttga ggaggactgtagccagtctctgtgcctcagtaagctgaccatgtccttcaaggactacatccaagagaggagcgacccagtcgagca aggcaaaccagttattcctgcagcagtactggctggcttcactggaagcggaccaatccagttgtggcagtttcttctggagctactctct gacaagtcctgtcaatctttcatcagctggacgggggatggatgggagttcaagcttgctgaccccgatgaggttgcccgccggtggg ggaagaggaaaaataaaccaaagatgaactacgagaagctgagccggggcttacgttactactacgacaagaacatcatccacaag acttcgggcaagcgctacgtgtaccgtttcgtatgtgacctgcagaacttgctgggcttcactccggaggaactgcatgccatcctggg cgtccagcctgatacagaagactgagggcctcaggaccaccctgagccagaagccgaattcgatatcaagcttatcgataatcaacct ctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcat gctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgt ggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttcc ccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgt ggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccctt cggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagt cggatctccctttgggccgcctccccgcatcgataccgtcgacctcgagacctagaaaaacatggagcaatcacaagtagcaataca gcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagac caatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagaca agatatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggccagggatcagatatccac tgacctttggatggtgctacaagctagtaccagttgagcaagagaaggtagaagaagccaatgaaggagagaacacccgcttgttac accctgtgagcctgcatgggatggatgacccggagagagaagtattagagtggaggtttgacagccgcctagcatttcatcacatggc ccgagagctgcatccggactgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactg cttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttt tagtcagtgtggaaaatctctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcc cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtag gtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcg gtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgc ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgcc acgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaa cttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtgg actcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaa atgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcagg 47

[0048] 501.113WO1 ND 25-008 cagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaag catgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgcc ccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggag gcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtatat cggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgc cggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacg tgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctg tacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtggg ggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctacgagatttc gattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcat gctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatt tttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatc atggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggt gcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaa tcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctg cggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaa atcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctc agttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg tcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcgg tgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttc ggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcag aaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgaca gttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaa ccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctaga gtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattc agctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtc agaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgc gccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccag ttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaat gccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgt ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgac (SEQ ID NO: 14) While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood 48

[0049] 501.113WO1 ND 25-008 that many variations and modifications may be made while remaining within the spirit and scope of the invention. 49

[0050] 501.113WO1 ND 25-008

Claims

What is claimed is:

1. A method for generating lymphatic endothelial cells (LECs) from human induced pluripotent stem cells (hiPSCs) comprising the steps of: i) contacting hiPSCs with a viral vector, wherein the viral vector comprises one or more polynucleotide sequences operably linked to an inducible promoter sequence and encoding ETS2 and / or ETV2; ii) seeding the hiPSCs on a cell culture plate for about 24 hours in the presence of a first cell culture media to provide plated hiPSCs; iii) exchanging the first cell culture media for a second cell culture media comprising a glycogen synthase kinase (GSK) inhibitor and culturing the seeded hiPSCs for about 24 hours; iv) replacing the second cell culture media with a third cell culture media comprising vascular endothelial growth factor -A (VEGF-A) and a transforming growth factor β (TGFβ) inhibitor, and inducing expression of the one or more polynucleotide sequences encoding ETS2 and / or ETV2 to provided induced hiPSCs; v) growing the induced hiPSCs for about 48 hours; vi) reseeding the induced hiPSCs on cell culture plates and a fourth cell culture media comprising VEGF-C; and vii) expanding the induced hiPSCs for about 72 hours to provide differentiated LEC cells.

2. The method of claim 1, wherein the cell culture plates comprises matrigel, and the hiPSCs are seeded on the cell culture plates in an amount of about 20,000 to 30,000 cells / cm2.

3. The method of claim 1, wherein the GSK inhibitor is selected from the group consisting of CHIR 99021, CHIR 98014, BIO-acetoxime, BIO, LiCl, SB-216763, SB- 415286, AR-A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide, present in an amount of about 0.1 µM to about 8 µM.

4. The method of claim 3, wherein the GSK inhibitor is CHIR 99021 present in an amount of about 4 µM. 50501.113WO1 ND 25-0085. The method of claim 1, wherein the TGFβ inhibitor is selected from the group consisting of SB-431542, SB-525334, A83-01, LY2157299, LY210976, GW788388, RepSox, SB-505124, D4476, GW788388, SD208, and EW-7197, present in an amount of about 5 µM to about 15 µM.

6. The method of claim 5, wherein the TGFβ inhibitor is SB-431542 present in an amount of about 10 µM.

7. The method of claim 1, wherein the third culture media comprises about 0.1 ng / ml to about 15 ng / ml VEGF-A and about 5 µM to about 15 µM of the TGFβ inhibitor.

8. The method of claim 7, wherein the third culture media comprises about 1 ng / ml to about 10 ng / ml VEGF-A and about 8 µM to about 12 µM of the TGFβ inhibitor.

9. The method of claim 1, wherein the fourth cell culture media comprises about 50 ng / ml to about 150 ng / ml VEGF-C.

10. The method of claim 1, wherein the fourth cell culture media comprises about 100 ng / ml VEGF-C.

11. The method of claim 1, wherein the VEGF-C is VEGF-C156S.

12. The method of claim 1, wherein the one or more polynucleotide sequences encoding ETS2 and / or ETV2 operably linked to an inducible promoter sequence encodes ETS2.

13. The method of claim 1, wherein the one or more polynucleotide sequences encoding ETS2 and / or ETV2 operably linked to an inducible promoter sequence encodes ETV2.

14. The method of claim 1, wherein the one or more polynucleotide sequences encoding ETS2 and / or ETV2 operably linked to an inducible promoter sequence encodes both ETS2 and ETV2. 51501.113WO1 ND 25-00815. The method of claim 1, wherein the viral vector comprises at least two viral vectors, wherein a first viral vector comprises a polynucleotide sequence encoding ETS2 and a second viral vector comprises a polynucleotide sequence encoding ETV2.

16. The method of claim 1, wherein the viral vector is a lentivirus.

17. Lymphatic endothelial cells (LECs) obtained using the method of claim 1. 52501.113WO1 ND 25-008

Images