Endothelial cell factors and methods thereof

Abstract

To provide a composition and a method for generating an endothelial niche cell.SOLUTION: A mode of a technique according to this application includes a composition, a kit, a vector and a method regarding generation or modification of an endothelial niche cell. One aspect is a method for generating / modifying an endothelial niche cell including a process of expressing one or a plurality of transcription factors in an endothelial cell, where one or a plurality of transcription factors derive from Ets family, Sox family, and / or nuclear hormone (NHR) family.SELECTED DRAWING: Figure 4F

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 62 / 647,433, filed March 23, 2018, the entire contents of which are incorporated herein by reference.

[0002] Government support This invention was made with government support under Grant Nos. DK111790 and HL048801 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. This ASCII copy, created on March 22, 2019, is named 701039-091810-seq_ST25.txt and is 32,536 bytes in size.

[0004] Technical Field The technology described herein relates to compositions and methods for generating endothelial niche cells. [Background technology]

[0005] background Hematopoietic stem and progenitor cells (HSPCs) are a rare cell population capable of reconstituting the entire blood system after transplantation. As a functional component of bone marrow transplantation, these cells provide curative treatment for many hematologic and immune disorders. Unfortunately, transplantation is not a viable treatment option for many individuals, especially those lacking an immunocompatible donor. A long-term goal of hematological research is to culture and expand HSPCs in vitro for use in transplantation and / or genetic modification. While umbilical cord blood-derived HSPCs can be expanded in vitro to some extent, maintaining and inducing self-renewal of adult-derived HSPCs has been shown to be challenging in the absence of niche signals.

[0006] A strategy aimed at in vitro expansion involves co-culturing HSPCs with supportive cells to recapitulate aspects of the microenvironment, or "niche," that supports HSPCs in vivo. In adult bone marrow, multiple cell types cooperate to form the HSPC niche, the major contributors of which are thought to be endothelial cells (ECs) and perivascular mesenchymal stromal cells. Different endothelial cell subtypes within the bone marrow may regulate HSPC homeostasis in different ways. Arterial ECs (AECs) are thought to have low permeability and promote HSPC quiescence, while sinusoidal ECs (SECs) are highly leaky and support HSPC differentiation and mobilization. In addition, during hematopoietic recovery after myelosuppression, ECs play a critical role in niche reconstitution and reconstitution of multilineage hematopoiesis. HSPCs can also be supported outside the bone marrow during embryonic development and under stressful conditions that induce extramedullary hematopoiesis in tissues such as the liver, spleen, and skull. As in bone marrow, ECs are thought to function as a critical central component of the HSPC niche in these tissues.

[0007] Researchers have focused on developing in vitro cultures that allow HSPCs to be grown in the laboratory along with other cell types to support their maintenance or expansion for subsequent use in transplantation, but to date, these in vitro cultures have been only partially successful. Summary of the Invention

[0008] overview In studying the vascular HSPC niche in zebrafish embryos, we investigated combinations of transcription factors (Ets, Sox, and nuclear hormone families) that are normally expressed in endogenous niche endothelial cells. Ectopic expression of human orthologs of these same transcription factors generated an ectopic vascular niche in zebrafish embryos that recruited and maintained HSPCs.

[0009] To advance these findings toward clinical application, transcription factors (first identified in zebrafish studies) can be expressed in human endothelial cells to reprogram these cells into HSPC niche-like bodies. These niche endothelial cells can then be used in co-culture with HSPCs to expand or extend the HSPC population for subsequent transplantation.

[0010] For example, transcription factors known to bind to Ets, Sox, and nuclear hormone motifs can be expressed in niche endothelial cells. In the Ets family, these factors include etv2, fli1a, and ets1, with the corresponding human factors being ETV2, FLI1, and ETS1. In the Sox family, these transcription factors include sox18 and sox7, with the corresponding human factors being SOX7 and SOX18. In the nuclear hormone family, these transcription factors include rxraa and nr2f2, with the corresponding human factors being RXRA and NR2R2.

[0011] The present invention provides a method for generating artificial niche endothelial cells to stimulate blood stem cells. Transcription factors include the Ets family (etv2, fli1a, and ets1), the SOX family (sox18, sox7), and the nuclear hormone family (rxraa, nr2f2), and their corresponding human factors (ETV2, FLI1, ETS1, SOX7, SOX18, RXRA, and NR2F2).

[0012] The method involves expressing transcription factors in endothelial cells (eg, human) to reprogram these cells into HSPC niche-like bodies.

[0013] In another embodiment, niche endothelial cells are used in co-culture with HSPCs to expand HSPC numbers or extend culture times for subsequent use in transplantation.

[0014] One aspect provides a method for generating / modifying endothelial niche cells, the method comprising expressing one or more transcription factors in endothelial cells, wherein the one or more transcription factors are derived from the Ets family, the Sox family, and / or the nuclear hormone receptor family.

[0015] Another aspect provides modified endothelial niche cells comprising one or more exogenous nucleic acid sequences encoding one or more transcription factors, wherein the one or more transcription factors are from the Ets family, the Sox family, and / or the nuclear hormone family.

[0016] Another aspect provides a composition comprising the modified endothelial niche described herein.

[0017] Another aspect provides a method for culturing HSPCs, the method comprising culturing the HSPCs in the presence of a population of modified endothelial niche cells.

[0018] Another aspect provides a method of treating a subject, the method comprising transplanting into the subject a composition comprising a population of HSPCs and modified endothelial niche cells.

[0019] Another aspect provides a method for improving HSPC engraftment, comprising administering to a subject in need thereof a composition comprising a population of HSPCs and modified endothelial niche cells.

[0020] Another aspect provides a co-culture comprising modified endothelial niche cells and HSPCs.

[0021] Another aspect provides a kit for culturing HSPCs, comprising a population of modified endothelial niche cells, reagents, and instructions for their use.

[0022] Another aspect provides a kit for generating modified endothelial niche cells, comprising a vector containing one or more exogenous nucleic acid sequences encoding one or more transcription factors of the Ets family, Sox family, or nuclear hormone family, and instructions for use thereof.

[0023] Another aspect provides a method for generating an ectopic vascular niche, the method comprising administering modified endothelial niche cells to a target site in a subject in need thereof.

[0024] Another aspect provides a method for extramedullary hematopoiesis, comprising transplanting modified niche endothelial cells into a site outside the bone marrow (e.g., the forearm) of a subject, thereby creating an artificial niche.

[0025] Another aspect provides a vector comprising one or more exogenous nucleic acid sequences encoding one or more transcription factors of the Ets family, Sox family, or nuclear hormone family, operably linked to a promoter. [Brief explanation of the drawings]

[0026] [Figure 1A]Figures 1A–1E are a collection of images and graphs demonstrating the endothelial expression signature in the fetal HSPC niche. (Figure 1A) A schematic diagram shows the sectioning strategy used to perform RNA tomography (tomo-seq) in the hematopoietic tissues (top) and CHT of zebrafish embryos (bottom, a double transgenic embryo carrying the HSPC markers cd41:GFP and runx1:mCherry is shown). (Figure 1B) The cross-sectional schematic and (continued) hierarchical clustering heatmap reveal clusters of gene expression corresponding to different tissues along the dorsal-ventral axis of the zebrafish tail. (Figure 1C) A schematic diagram shows the strategy of using kdr1:GFP transgenic embryos and FACS to isolate ECs from whole embryos for analysis by RNA-seq. (Figure 1D) Individual tomo-seq expression traces are shown for endothelial commonly expressed genes (left) and CHT EC-enriched genes (right). (Figure 1E) Images show whole-mount in situ hybridization (WISH) of the endothelial common gene kdr1 (top) and CHT EC-enriched genes identified by tomo-seq and tissue-specific RNA-seq. Arrows indicate expression in the dorsal vessels, and arrowheads indicate expression in the CHT. Scale bars represent 250 μm unless otherwise noted. [Figure 1B-1] See legend to Figure 1A. [Figure 1B-2] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2A]Figures 2A-C are a collection of images and graphs illustrating endothelial niche-specific cis-regulatory elements. (Figure 2A) Images and schematic diagrams show four cell populations isolated from mrc1a:GFP, kdr1:mCherry double-positive embryos for analysis by ATAC-seq. (Figure 2B) Gene tracks indicate regions of chromatin that were uniquely open in the mCherry+; GFP+ CHT EC fraction (boxes and arrows). (Figure 2C) Images show embryos injected with a CHT EC enhancer-GFP reporter construct corresponding to the boxed region in Figure 2B. Arrowheads indicate GFP expression in CHT ECs. Unless otherwise noted, the scale bar represents 250 µm. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A]Figures 3A-3F are a collection of images and graphs demonstrating that Ets, Sox, and NHR binding sites are required for selective expression in niche ECs. (Figure 3A) The gene track indicates the region of chromatin upstream of mrc1a (box and arrowhead below) that was uniquely open in the double-positive CHT EC fraction but not in the other three cell populations. The bars indicate the location of the 125 bp enhancer sequence and the 1.3 kb sequence used to create the mrc1l:GFP reporter transgene. (Figure 3B) The image shows transient GFP expression in an F0 embryo injected with a 125 bp enhancer sequence linked to a minimal promoter and GFP. (Figure 3C) The image shows an F0 embryo co-injected with kdr1:mCherry and mrc1a 125 bp:GFP plasmids. (Figure 3D) The image shows an embryo expressing a stably integrated mrc1a 125 bp:GFP transgene. (Figure 3E) The wild-type sequence of the 125-bp mrc1a enhancer is shown, with annotations highlighting Ets, Sox, and NHR binding motifs (see, for example, SEQ ID NO: 12). A schematic diagram illustrates enhancer-reporter constructs in which each class of motif or regulatory region was targeted by mutation. Xs indicate the location of the targeted site. mp-GFP: Mouse beta-globin minimal promoter fused to GFP. (Figure 3F) Graphs show the incidence of embryos exhibiting GFP expression in CHT ECs after injection of the wild-type sequence or mutant variants of the mrc1a 125-bp (top) or sele 158-bp (bottom) enhancer. Data are normalized to the respective wild-type control in each experiment (44% (155 / 356) for the mrc1a 125 bp enhancer and 23% (176 / 775) for the sele 158 bp enhancer). Mean + / - standard error (s.e.m.), one-way ANOVA; **P<0.01, ***P<0.001. Scale bars represent 250 μm unless otherwise noted. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 4A] Figures 4A-4F are a collection of images and graphs demonstrating that overexpression of defined factors induces ectopic vascular gene expression outside the CHT. (Figure 4A) A schematic diagram illustrates the strategy used for transcription factor overexpression experiments. (Figure 4B) Images show embryos injected with control DNA (left) or a pool of seven transcription factors from the Ets, Sox, and NHR families (FLI1, ETV2, ETS1, SOX7, Sox18, Nr2f2, and RXRA) (right) and subsequently stained by WISH for mrc1a (top) or sele (bottom). In all panels of Figures 4A-4E, arrows indicate areas of ectopic expression, and arrowheads indicate domains of normal expression. (Figure 4C) Images show mrc1a:GFP; kdr1:mCherry double transgenic embryos injected with control DNA or the seven-factor pool. (Figure 4D) Injection of a three-factor pool containing ETV2, SOX7, and Nr2f2 results in ectopic expression of mrc1a:GFP (arrows). (Figure 4E) Images show WISH for mrc1a in control embryos (top) or after injection of a three-factor pool containing ETV2, SOX7, and Nr2f2 (middle) or ETS1, SOX7, and Nr2f2 (bottom). (Figure 4F) Graph reports quantification of the proportion of injected embryos that showed ectopic mrc1a expression after overexpression of transcription factors. Chi-square test; **P<0.01, ***P<0.001. Scale bars represent 250 μm in Figures 4B and 4E and 100 μm in Figures 4C and 4D. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 5A] Figures 5A-5E are a collection of images and graphs demonstrating that HSPCs localize to areas of ectopic niche endothelial gene expression. (Figure 5A) Images show that runx1:mCherry+ HSPCs localized outside the CHT within the dorsal ectopic area of ​​mrc1a:GFP expression in embryos injected with a pool of ETV2, SOX7, and Nr2f2 (top) or a pool of ETS1, SOX7, and Nr2f2 (bottom). Enlarged insets of grayscale images for each channel are shown on the right. In all panels of Figures 5A-5E, arrows indicate ectopic expression or localization, while arrowheads indicate normal expression or localization. (Figure 5B) WISH for runx1 demonstrates HSPC localization in control (top) and three-factor-injected embryos (bottom). (Figure 5C) ECs ectopically expressing mrc1a:GFP associate with cxcl12a:DsRed2+ stromal cells, similar to ECs in the CHT. The asterisk indicates notochord expression of cxcl12a:DsRed. (Figure 5D) Time-lapse images show that runx1:mCherry+ HSPCs first arrive at the CHT and then divide. Time is shown in hh:mm. (Figure 5E) Time-lapse images from a different embryo show that runx1:mCherry+ HSPCs divide and exit the circulation. Scale bars represent 100 μm in Figures 5A-C and 30 μm in Figures 5D-E. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 6A]Figures 6A-6B are a collection of images and graphs showing the conserved endothelial expression signature in the HSPC niche. (Figure 6A) The heat map shows the expression of 29 CHT EC genes in different cell populations, including the adult zebrafish kidney marrow. The spectral scale indicates normalized expression between 0 (low) and 1 (high). (Figure 6B) The heat map shows the expression of CHT EC gene orthologs in ECs from different organs of mice at different stages of development and postnatal adult transition. Arrows indicate hematopoietic tissues at each developmental stage. Black brackets indicate genes enriched in fetal liver ECs at E14-17 and in the subsequent adult bone marrow. Spectral bars report z-scores. BM: bone marrow. [Figure 6B] See legend to Figure 6A. [Figure 7A] Figures 7A-7B are a collection of images and graphs showing RNA tomography and niche-specific endothelial gene expression. (Figure 7A) The graph shows tomo-seq expression traces for individual tissue-specific genes. Images showing whole-mount in situ hybridization (WISH) for 35 CHT-enriched genes are available on the World Wide Web at zfin.org. (Figure 7B) WISH validates the CHT-enriched expression (arrowheads) of CHT EC genes identified using a combination of tomo-seq and tissue-specific RNA-seq. Unless otherwise noted, the scale bar represents 250 µm. [Figure 7B-1] See legend to Figure 7A. [Figure 7B-2] See legend to Figure 7A. [Figure 8A]Figures 8A-8E are a collection of images and graphs showing that the GFP reporter transgene selectively labels ECs in the HSPC niche. (Figure 8A) The image shows a double transgenic embryo carrying the endothelial-common marker kdr1:mCherry and the mrc1a:GFP transgene, which are selectively expressed in CHT ECs. A magnification of the boxed area is shown on the right. (Figure 8B) The image shows a runx1:mCherry+ HSPC interacting directly with an mrc1a:GFP+ EC within the CHT niche (arrow). The center panel shows a magnification of the boxed area. A further magnification (bottom) shows an HSPC within a pocket of mrc1a:GFP+ ECs. (Figure 8C) Cxcl12a:DsRed2+ stromal cells closely contact mrc1a:GFP+ ECs in the CHT. (Figure 8D) The image shows a double transgenic embryo carrying the endothelial-common marker kdr1:mCherry and the sele:GFP transgene, which are selectively expressed in CHT ECs. A magnification of the boxed area is shown on the right. (Figure 8E) The image shows runx1:mCherry+ HSPCs directly interacting with sele:GFP+ ECs within the CHT niche (arrows). A magnification of the boxed area is shown on the right. Scale bars represent 250 μm in Figures 8A and 8D and 100 μm in Figures 8B, 8C, and 8E. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 9A]Figures 9A–9C are a collection of images and graphs showing endothelial-common regulatory elements and genome-wide motif enrichment analysis. (Figure 9A) Gene tracks indicate regions of open chromatin in both mCherry+GFP+ (CHT ECs) and mCherry+GFP- (non-CHT ECs) populations (box and straight arrow). (Figure 9B) Images show embryos injected with the endothelial-common enhancer-GFP reporter construct corresponding to the boxed region in Figure 9A. Arrows indicate GFP expression in non-CHT ECs, and arrowheads indicate expression in CHT ECs. (Figure 9C) Images show transcription factor binding motifs most enriched in the CHT EC region (top) or the endothelial-common region (bottom). Unless otherwise noted, scale bars represent 250 μm. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10A]Figures 10A–10E are a collection of images and graphs depicting the CHT endothelial cis-regulatory element. (Figure 10A) The graph reports the anatomical site of endothelial expression in F0 embryos injected with mrc1a 125 bp:GFP and kdr1:mCherry plasmids. (Figure 10B) The gene track indicates the region of chromatin upstream of sele (box and arrowhead below) that was uniquely open in the double-positive CHT EC fraction but not in the other three cell populations. The bars indicate the location of the 158 bp enhancer sequence and the 5.3 kb sequence used to generate the sele:GFP reporter transgene. (Figure 10C) The images show transient F0 (top) and stable F2 (bottom) expression of the sele 158bp:GFP construct. (Figure 10D) The wild-type sequence of the 158-bp sele enhancer is shown, with annotations (top) highlighting the Ets, Sox, and NHR binding motifs (see, for example, SEQ ID NO:13). A schematic diagram illustrates sequence variants in which each class of motif or regulatory region was targeted by mutation. Xs indicate the location of the targeted site. mp-GFP fused to GFP: mouse beta-globin minimal promoter. (Figure 10E) Images show electrophoretic mobility shift assays using recombinant Nr2f2-GST incubated with DNA sequences spanning the NHR motifs present in the 125-bp mrc1a (left two gels) or 158-bp sele (right gel) enhancer sequences. Arrows indicate DNA:protein binding, and arrowheads indicate supershifted DNA:protein complexes. The labeled DNA:protein complex was abolished by the unlabeled wild-type probe (lane 4), but not by the probe with a mutation that disrupted the NHR motif (arrow with asterisk). Scale bars represent 250 μm unless otherwise noted. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 11A] Figures 11A-C are a collection of images and graphs showing that transcription factor overexpression induces an ectopic CHT endothelial program. (Figure 11A) Images show WISH for mrc1a in the yolk sphere of control (left) and seven-factor-injected embryos (right). In all panels of Figures 11A-C, arrows indicate ectopic expression, and arrowheads indicate normal expression domains. (Figure 11B) Images show ectopic expression of the mrc1a:GFP and kdr1:mCherry transgenes in the yolk extension of seven-factor-injected embryos. A magnification of the boxed area is shown below. (Figure 11C) Images show WISH for sele, gpr182, and lgmn in control embryos (left) and embryos injected with a combination of ETV2, SOX7, and Nr2f2 (right). Scale bars in Figures 11A-C represent 100 μm. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12A] Figures 12A-12D are a collection of images and graphs showing that CHT niche endothelial gene expression is ectopically induced by overexpression of transcription factors. (Figures 12A-12B) Injection of ETV2 alone induces ectopic expression of the endogenous mrc1a gene (Figure 12A) and the mrc1a:GFP transgene (Figure 12B). In all panels of Figures 12A-12D, arrows indicate ectopic expression, and black arrowheads indicate normal expression domains. (Figure 12C) Injection of human ETV2 alone induces ectopic expression of zebrafish transcription factors, including sox7, sox18, fli1a, and etv2. (Figure 12D) Injection of a three-factor pool containing ETS1, SOX7, and Nr2f2 results in ectopic expression of mrc1a:GFP. Scale bars represent 250 μm in FIGS. 12A and 12C and 50 μm in FIGS. 12B and 12D. [Figure 12B] See legend to Figure 12A. [Figure 12C]See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13A] Figures 13A-13B are a collection of images and graphs showing niche endothelial transgene expression in adult kidney ECs. (Figure 13A) The image shows a vascular segment (white arrow) excised from the kidney of a kdr1:mCherry, mrc1a:GFP double transgenic adult zebrafish. (Figure 13B) The image shows serial sections of an adult kidney isolated from a sele:GFP transgenic fish. The sections were stained with H&E (left) and an antibody against GFP (right). The black arrows indicate GFP+ vascular endothelial cells. The scale bars represent 50 μm in Figure 13A and 100 μm in Figure 13B. [Figure 13B] See legend to Figure 13A. [Figure 14] Schematic diagram showing hematopoietic stem cell self-renewal and differentiation. LT-HSC indicates long-term hematopoietic stem cells. CMP indicates common myeloid progenitors. MEP indicates megakaryocyte-erythroid progenitors. GMP indicates granulocyte-macrophage progenitors. CLP indicates common lymphoid progenitors. [Figure 15A] Figures 15A-15B are a collection of images showing visualization of niche colonization by HSPCs in vivo. Figure 15A shows visualization of the dorsal aorta and caudal hematopoietic tissue. Figure 15B shows HSPCs surrounded by five endothelial cells and attached to stromal cells. [Figure 15B] See legend to Figure 15A. [Figure 16] 1 is a collection of images showing the use of RNA tomography (tomo-seq) to examine gene expression in the HSPC niche. [Figure 17] This is a collection of images and graphs showing how Tomo-seq + endothelial RNA-seq identified approximately 20 genes that are selectively enriched in niche endothelial cells. [Figure 18]1 is a collection of images showing that sele and mrc1a promoter-GFP fusions label endothelial cells in the HSPC niche. [Figure 19] This is a collection of images showing that specific ATAC-seq peaks near the original 20 genes can drive expression in niche endothelial cells. 13 of the 19 cloned ATAC-seq peaks can drive GFP expression in CHT endothelial cells (when linked to a minimal promoter). [Figure 20] Image showing the most enriched TF binding motifs in open chromatin of niche endothelial cells. Motif enrichment analysis was performed using HOMER on 6,710 unique ATAC-seq peaks. [Figure 21] 1 is a collection of images and graphs showing that Ets, Sox, and NHR sites are required for niche expression of the 158 bp sele enhancer (see, e.g., SEQ ID NO: 14). [Figure 22] 1 is a collection of images and graphs showing that Ets, Sox, and NHR motifs are required for niche expression of the 125 bp mrc1a enhancer (see, e.g., SEQ ID NO: 15). [Figure 23] A collection of images showing that mouse TFs can bind to zebrafish sequences in vitro. The same mutations that disrupt enhancer:GFP expression abolish TF binding. [Figure 24] 1 is an image showing that transcription factors known to bind Ets, Sox, and NH motifs are expressed in niche endothelial cells. [Figure 25] 1 is a collection of images showing the reprogramming of niche endothelial cells. [Figure 26] A collection of images and graphs showing that injection of a pool of three TFs results in ectopic niche endothelial gene expression. [Figure 27A] 27A-27C are a collection of images showing that ectopic vascular compartments can recruit runx1+ HSPCs. [Figure 27B]See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 28] A collection of images and heatmaps showing that the HSPC niche endothelial signature is also found in the adult marrow. [Figure 29] 1 is a heatmap showing that similar niche endothelial signatures were found in mammalian fetal liver and bone marrow. [Figure 30] Figure 1 shows that RNA tomography of the HSPC niche revealed the following data: approximately 20 genes were selectively enriched in HSPC niche endothelial cells; ectopic expression of only three TFs was able to induce niche endothelial gene expression and recruit HSPCs; and Ets, Sox, and nuclear hormone motifs were required for expression in niche endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description Embodiments of the technology described herein include compositions, kits, vectors, and methods related to generating or modifying endothelial niche cells. One aspect includes a method of generating / modifying endothelial niche cells, comprising expressing one or more transcription factors in endothelial cells, wherein the one or more transcription factors are from the Ets family, the Sox family, and / or the nuclear hormone receptor (NHR) family.

[0028] In some embodiments, at least one transcription factor is selected from the Ets family. In some embodiments, at least one transcription factor is selected from the Sox family. In some embodiments, at least one transcription factor is selected from the NHR family.

[0029] In some embodiments, at least one transcription factor is selected from the Ets family and at least one transcription factor is selected from the Sox family. In some embodiments, at least one transcription factor is selected from the Ets family and at least one transcription factor is selected from the NHR family. In some embodiments, at least one transcription factor is selected from the Sox family and at least one transcription factor is selected from the NHR family.

[0030] In some embodiments, at least one transcription factor is selected from the Ets family, at least one transcription factor is selected from the Sox family, and at least one transcription factor is selected from the NHR family. In some embodiments, at least one transcription factor is selected from the Ets family, at least one transcription factor is selected from the Sox family, or at least one transcription factor is selected from the NHR family.

[0031] ETS Family In some embodiments of any aspect, the endothelial niche cells express transcription factors of the ETS family. The ETS (E26 transformation-specific or E-26) family is one of the largest transcription factor families and is unique to animals. The ETS family is classified into 12 subfamilies: (1) ELF (e.g., ELF1, ELF2 / NERF, ELF4 / MEF); (2) ELG (e.g., GABPα, ELG); (3) ERG (e.g., ERG, FL1, FEV); (4) ERF (e.g., ERF / PE2, ETV3 / PE1); (5) ESE (e.g., ELF3 / ESE1 / ESX, ELF5 / ESE2, ESE3 / EHF); (6) ETS (e.g., ETS1, ETS, POINTED) ( (7) PDEF (e.g., SPDEF / PDEF / PSE); (8) PEA3 (e.g., ETV4 / PEA3 / E1AF, ETV5 / ERM, ETV1 / ER81); (9) ERF71 (e.g., ETV2 / ER71); (10) SPI (e.g., SPI1 / PU.1, SPIB, SPIC); (11) TCF (e.g., ELK1, ELK4 / SAP1, ELK3 / NET / SAP2, LIN); (12) TEL (e.g., ETV6 / TEL, ETV7 / TEL2, YAN).

[0032] All ETS family members are identified through a highly conserved DNA-binding domain, the ETS domain, a winged-helix-turn-helix structure that binds to DNA sites with a central GGA(A / T) DNA sequence. Ets family DNA motifs may also contain a central TTCCT sequence (e.g., on the DNA strand complementary to the first motif). Along with DNA-binding function, evidence suggests that ETS domains are also involved in protein-protein interactions.

[0033] The ETS family is present throughout the body and is involved in a wide variety of functions, including regulation of cell differentiation, cell cycle control, cell migration, cell proliferation, apoptosis (programmed cell death), and angiogenesis.

[0034] Non-limiting examples of human Ets family members associated with endothelial niche cells include ETV2, FLI1, and ETS1. Corresponding factors in zebrafish include etv2, fli1, and ets1.

[0035] ETV2 may also be referred to herein as ETS variant 2, ETS translocation variant 2, Ets-related protein 71, Ets variant gene 2, ETSRP71, or ER71.

[0036] Friend leukemia integration 1 transcription factor (FLI1), also known as transcription factor ERGB, is a protein encoded by the FLI1 gene in humans. FLI1 may also be referred to herein as Fli-1, proto-oncogene, ETS transcription factor, Friend leukemia integration 1 transcription factor, Friend leukemia virus integration 1, transcription factor ERGB, Ewing's sarcoma breakpoint region, proto-oncogene Fli-1, BDPLT21, EWSR2, or SIC-1.

[0037] ETS1 or protein C-ets-1 is a protein encoded by the ETS1 gene in humans. The protein encoded by this gene belongs to the ETS transcription factor family. ETS1 may also be referred to herein as ETS proto-oncogene 1 transcription factor, avian erythroblastosis virus E26 (V-Ets) proto-oncogene homolog-1, V-Ets avian erythroblastosis virus E26 proto-oncogene homolog 1, protein C-Ets-1, EWSR2, P54, V-Ets avian erythroblastosis virus E26 proto-oncogene homolog 1, Ets protein, C-Ets-1, or ETS-1.

[0038] In some embodiments of any aspect, cells are generated or engineered to express an Ets family member selected from the group consisting of ETV2, FLI1, and ETS1.

[0039] In some embodiments, the Ets gene or protein can be ETV2 or the corresponding zebrafish etv2. In some embodiments, the Ets gene or protein can be FLI1 or the corresponding zebrafish fli1. In some embodiments, the Ets gene or protein can be ETS1 or the corresponding zebrafish ets1.

[0040] In some embodiments, the Ets gene or protein can be ETV2 and FLI1 or corresponding zebrafish factors. In some embodiments, the Ets gene or protein can be ETV2 and ETS1 or corresponding zebrafish factors. In some embodiments, the Ets gene or protein can be ETS1 and FLI1 or corresponding zebrafish factors. In some embodiments, the Ets gene or protein can be ETV2, FLI1 and ETS1 or corresponding zebrafish factors. In some embodiments, the Ets gene or protein can be ETV2, FLI1 or ETS1 or corresponding zebrafish factors.

[0041] The amino acid sequences of the polypeptides described herein are assigned NCBI accession numbers for various species, such as human, mouse, rat, and zebrafish. Specifically, the NCBI accession numbers of non-limiting examples of the amino acid sequences of human EVT2 (e.g., SEQ ID NO: 1), human FLI1 (e.g., SEQ ID NO: 2), and human ETS1 (e.g., SEQ ID NO: 3) are included herein.

[0042] SEQ ID NO: 1 (Homo sapiens ETV2, NCBI accession number AAI40747, 342 amino acids (aa)): TIFF0007683068000001.tif48165

[0043] SEQ ID NO:2 (Homo sapiens FLI1, NCBI accession number AAH10115.1, 452 aa): TIFF0007683068000002.tif55165

[0044] SEQ ID NO:3 (Homo sapiens ETS1, NCBI accession number CAG47050.1, 441 aa): TIFF0007683068000003.tif55165

[0045] In some embodiments, an ETV2 amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence. In some embodiments, an FLI1 amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence. In some embodiments, an ETS amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence.

[0046] SOX Family Sox genes encode a family of transcription factors that bind to the minor groove in DNA and belong to a superfamily of genes characterized by homologous sequences called HMG boxes (high mobility group), which are DNA-binding domains that are highly conserved across eukaryotic species. Homologs have been identified in insects, nematodes, amphibians, reptiles, birds, and some mammals.

[0047] Sox genes are defined by containing the HMG box of a gene involved in sex determination called SRY, located on the Y chromosome (Sox stands for Sry-related HMG box). There are 20 SOX genes in humans and mice. This family is subdivided according to homology within the HMG domain and other structural motifs, as well as according to functional assays. In humans, members of the SOX group include: (1) SoxA (e.g., SRY); (2) SoxB1 (e.g., SOX1, SOX2, SOX3); (3) SoxB2 (e.g., SOX14, SOX21); (4) SoxC (e.g., SOX4, SOX11, SOX12); (5) SoxD (e.g., SOX5, SOX6, SOX13); (6) SoxE (e.g., SOX8, SOX9, SOX10); (7) SoxF (e.g., SOX7, SOX17, SOX18); (8) SoxG (e.g., SOX15); and (9) SoxH (e.g., SOX30).

[0048] The developmentally important Sox family does not have a single function; many members have the ability to regulate a variety of different aspects of development. Many Sox genes are involved in sex determination, but some are also important in processes such as neurogenesis. Sox proteins bind to the sequence WWCAAW and similar sequences (W = A or T). Sox family DNA motifs can also contain a central ATTGT sequence (e.g., on the DNA strand complementary to the first motif).

[0049] Non-limiting examples of human Sox family members associated with endothelial niche cells include SOX18 and SOX7. Corresponding factors in zebrafish or Xenopus include sox18 and sox7.

[0050] SOX18 may also be referred to herein as SRY-box 18, SRY (sex determining region Y)-box 18, transcription factor SOX-18, SRY box 18, HLTRS, or HLTS.

[0051] SOX7 may also be referred to herein as SRY-box 7, SRY (sex determining region Y)-box 7, transcription factor SOX-7, or SRY box 7.

[0052] In some embodiments of any aspect, cells are generated or engineered to express a Sox family member selected from the group consisting of SOX18 and SOX7. In some embodiments, the Sox gene or protein can be SOX18 or the corresponding zebrafish sox18 or Xenopus SOX18. In some embodiments, the Ets gene or protein can be SOX7 or the corresponding zebrafish sox7. In some embodiments, the Sox gene or protein can be SOX18 and SOX7 or the corresponding zebrafish or Xenopus factors. In some embodiments, the Sox gene or protein can be SOX18 or SOX7 or the corresponding zebrafish or Xenopus factors.

[0053] The amino acid sequences of the polypeptides described herein are assigned NCBI accession numbers for various species, such as human, mouse, rat, and zebrafish. Specifically, the NCBI accession numbers of non-limiting examples of the amino acid sequences of human SOX18 (e.g., SEQ ID NO: 4), human SOX7 (e.g., SEQ ID NO: 5), and Xenopus laevis SOX18 (e.g., SEQ ID NO: 8) are included herein.

[0054] SEQ ID NO:4 (Homo sapiens SOX18, NCBI accession number BAA94874.1, 384 aa) TIFF0007683068000004.tif47165

[0055] SEQ ID NO:5 (Homo sapiens SOX7, NCBI accession number CAC84226.1, 388aa) TIFF0007683068000005.tif48165

[0056] SEQ ID NO:8 (Xenopus tropicalis SOX18, NCBI accession number AAI67402.1, 362 aa) TIFF0007683068000006.tif47165

[0057] In some embodiments, the SOX7 amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence. In some embodiments, the SOX18 amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence.

[0058] NHR Family The nuclear hormone receptor (NHR) family, also known as nuclear receptors, is a class of proteins found in cells that sense steroid and thyroid hormones, as well as certain other molecules. In response, these receptors, along with other proteins, act to regulate the expression of specific genes, thereby controlling the development, homeostasis, and metabolism of organisms. The unique property of nuclear receptors that distinguishes them from other classes of receptors is their ability to directly interact with and regulate the expression of genomic DNA. As a result, nuclear receptors play a key role in both embryonic development and adult homeostasis. Non-limiting examples of DNA motifs for NHR family members include RRGGTCA, where R represents a purine (e.g., A or G).

[0059] At least 48 nuclear receptors have been identified in humans and classified into the following subfamilies: (1) thyroid hormone receptor-like (e.g., thyroid hormone receptor, retinoic acid receptor, peroxisome proliferator-activated receptor, Rev-ErbA RAR-related orphan receptor, liver X receptor-like, vitamin D receptor-like, NR with two DNA-binding domains, RORA); (2) retinoid X receptor-like (e.g., hepatocyte nuclear factor 4, retinoid X receptor, testis receptor, TLX, PNR, COUP, EAR, RXRA, NR2F2); (3) estrogen receptor-like (e.g., estrogen receptor, estrogen-related receptor, 3-ketosteroid receptor); (4) nerve growth factor IB-like (e.g., NGFIB, NURR1, NOR1); (5) steroidogenic factor-like (e.g., SF1, LRH1); (6) germline nuclear factor-like (e.g., GCNF); and (7) other nuclear receptors (e.g., DX, SHP1).

[0060] Non-limiting examples of human NHR family members associated with endothelial niche cells include RXRA and NR2F2. Corresponding factors in zebrafish include rxraa and nr2f2.

[0061] RXRA is a nuclear receptor that belongs to the RXR transcription factor group. RXRA may also be referred to herein as retinoid X receptor alpha, nuclear receptor subfamily 2 group B member 1, retinoic acid receptor RXR-alpha, NR2B1, retinoid X nuclear receptor alpha, or retinoid X receptor alpha.

[0062] Retinoid X receptors (RXRs) are a type of nuclear receptor activated by 9-cis retinoic acid and 9-cis-13,14-dihydroretinoic acid, which are considered to be the major endogenous mammalian RXR-selective agonists. There are three retinoic acid X receptors (RXRs): RXR-alpha, RXR-beta, and RXR-gamma, encoded by the RXRA, RXRB, and RXRG genes, respectively. RXRs heterodimerize with subfamily 1 nuclear receptors, including CAR, FXR, LXR, PPAR, PXR, RAR, TR, and VDR. Similar to other type II nuclear receptors, RXR heterodimers bind to hormone response elements in the absence of ligand in complex with corepressor proteins. Binding of an agonist ligand to RXR results in the dissociation of the corepressor and the recruitment of coactivator proteins, thereby promoting the transcription of downstream target genes into mRNA and ultimately into protein.

[0063] NR2F2 is a nuclear receptor belonging to the COUP transcription factor group. NR2F2 may also be referred to herein as nuclear receptor subfamily 2 group F member 2, apolipoprotein AI-regulatory protein 1, COUP transcription factor II, COUP transcription factor 2, TFCOUP2, ARP-1, ARP1, chicken ovalbumin upstream promoter transcription factor 2, chicken ovalbumin upstream promoter transcription factor I, nuclear receptor subfamily 2 group F member 2, ADP-ribosylation factor-related protein 1, apolipoprotein AI-regulatory protein 1, COUP-TF II, COUPTFII, COUP-TF2, COUPTF2, COUPTFB, CHTD4, NF-E3, or SVP40.

[0064] The chicken ovalbumin upstream promoter transcription factor (COUP-TF) protein is a member of the nuclear receptor family of intracellular transcription factors. COUP-TF exists in two variants, COUP-TFI and COUP-TFII, encoded by NR2F1 and NR2F2, respectively. COUP-TF plays an important role in the development of organisms.

[0065] In some embodiments of any aspect, cells are generated or modified to express an NHR family member selected from the group consisting of RXRA and NR2F2. In some embodiments, the NHR gene or protein can be RXRA or the corresponding zebrafish rxraa. In some embodiments, the NHR gene or protein can be NR2F2 or the corresponding zebrafish nr2f2. In some embodiments, the NHR gene or protein can be RXRA and NR2F2 or the corresponding zebrafish factor. In some embodiments, the NHR gene or protein can be RXRA or NR2F2 or the corresponding zebrafish factor.

[0066] The amino acid sequences of the polypeptides described herein are assigned NCBI accession numbers for various species, such as humans, mice, rats, and zebrafish.Specifically, the NCBI accession numbers of non-limiting examples of the amino acid sequences of human RXRA (e.g., SEQ ID NO: 6), human NR2F2 (e.g., SEQ ID NO: 7), and zebrafish Nr2f2 (e.g., SEQ ID NO: 9) are included herein.

[0067] SEQ ID NO:6 (Homo sapiens RXRA isoform A, NCBI accession number NP_002948.1, 462 aa) TIFF0007683068000007.tif55165

[0068] SEQ ID NO:7 (Homo sapiens NR2F2 isoform A, NCBI accession number NP_066285.1, 414 aa) TIFF0007683068000008.tif55165

[0069] SEQ ID NO:9 (Danio rerio NR2F2, NCBI accession number AAI62484.1, 428aa) TIFF0007683068000009.tif55165

[0070] In some embodiments, an RXRA amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence. In some embodiments, an NR2F2 amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence.

[0071] In some embodiments, the transcription factor can be selected from the group consisting of ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, and NR2F2 and corresponding zebrafish or Xenopus factors. In some embodiments, the transcription factor can be ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2 or corresponding zebrafish or Xenopus factors. In some embodiments, the transcription factor can be ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, and NR2F2 or corresponding zebrafish or Xenopus factors.

[0072] In some embodiments, the transcription factors can be ETV2, SOX7, and NR2F2 or corresponding zebrafish or Xenopus factors. In some embodiments, the transcription factors can be ETS1, SOX7, and NR2F2 or corresponding zebrafish or Xenopus factors. In some embodiments, the transcription factor can be ETV2 alone or corresponding zebrafish or Xenopus factors.

[0073] In some embodiments, the transcription factors can be at least one factor, at least two factors, at least three factors, at least four factors, at least five factors, at least six factors, or at least seven factors selected from the group consisting of ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, and NR2F2 or corresponding zebrafish or Xenopus factors.

[0074] In some embodiments, the transcription factor can be at least one factor selected from the group consisting of ETV2 and FLI1, ETS1, SOX18, SOX7, RXRA and NR2F2 or corresponding zebrafish or Xenopus factors. In some embodiments, the transcription factor can be at least one factor selected from the group consisting of ETS1 and ETV2, FLI1, SOX18, SOX7, RXRA and NR2F2 or corresponding zebrafish or Xenopus factors.

[0075] Hematopoietic development The development of the hematopoietic system, including its cell populations and molecular pathways, is highly conserved between fish and mammals. HSPCs are born in the aorta-gonad-mesonephros (AGM) region and migrate to a transient fetal niche, the fetal liver in mammals or the vascular network in the tail of fish, called the caudal hematopoietic tissue (CHT). HSPCs remain at these developmental sites for several days and expand before migrating to the adult niche—the bone marrow in mammals or the renal medulla in fish.

[0076] The CHT is primarily composed of low-flow sinusoids surrounded by mesenchymal stromal cells. HSPCs initially reside within the vascular network and express CXCL12a +HSPCs enter the CHT niche by directly interacting with stromal cells. In a characteristic vascular remodeling process, endothelial cells (ECs) reorganize to form a supportive pocket around HSPCs, which, together with stromal cells and possibly other cell types, forms a niche for stem cells (endothelial cells surrounding HSPCs may be referred to herein as endothelial niche cells). In mammals and zebrafish, specific signaling molecules, adhesion proteins, and transcription factors mediate communication and physical interactions between stem cells and ECs in this niche. Collectively, these studies suggest that ECs within the vascular niche of hematopoietic organs express niche-specific gene programs. However, to date, there has been no comprehensive investigation of the transcriptional circuitry that determines the niche properties of ECs in the HSPC niche. Understanding this regulation could lead to new strategies to improve the efficacy and utility of bone marrow transplantation therapies.

[0077] Endothelial niche cells As described herein, endothelial niche cell is the endothelial cell that provides the niche that is beneficial for the differentiation of HSPC.Endothelial niche cell is typically found in bone marrow.However, as described herein, the exogenous expression of certain transcription factors (such as ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, NR2F2) can make endothelial niche cell be found in non-bone marrow tissue, thereby providing extramedullary hematopoiesis.

[0078] In some embodiments of any aspect, the endothelial niche cells comprise cells that express one or more genes selected from the group consisting of sele, exoc312a, snx8a, cltca, aqp7, ap1b1, lgmn, prcp, cldn11a, lyve1b, adra1d, hyal2a, hyal2b, tll1, i113ra2, glu1a, hexb, slc16a9a, and sepp1a. In some embodiments, the endothelial cells are human.

[0079] In some embodiments of any aspect, the endothelial niche cells are generated or modified to express transcription factors including at least one of human transcription factors ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2. In some embodiments, the transcription factors include at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family. In some embodiments, the transcription factors include ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, and NR2F2.

[0080] In some embodiments of any aspect, the transcription factor is expressed from at least one vector.In some embodiments, the vector comprises an exogenous nucleic acid sequence or sequences encoding one or more transcription factors.In some embodiments, the exogenous nucleic acid sequence is integrated into the genome of endothelial cells.By way of non-limiting example, the exogenous nucleic acid sequence can be integrated into the genome using a viral vector (e.g., AAV, lentivirus) or CRISPR technology.

[0081] One aspect provides a composition comprising modified endothelial niche cells comprising one or more exogenous nucleic acid sequences encoding one or more transcription factors, wherein the one or more transcription factors are from the Ets family, Sox family, and / or nuclear hormone family. In some embodiments of any aspect, the composition may comprise modified endothelial niche cells. In some embodiments, the composition is a therapeutic agent, or the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a culture dish, a 3D cell system, or a suspension system. In some embodiments, the composition comprises a scaffold.

[0082] Another aspect provides a method for culturing HSPCs, comprising culturing HSPCs in the presence of a population of modified endothelial niche cells. In some embodiments of any aspect, this method is performed in vitro. In some embodiments, the modified endothelial niche cells secrete factors (e.g., growth factors) that affect the growth and / or expansion of HSPCs.

[0083] In some embodiments, HSPCs cultured in the presence of modified endothelial niche cells can be cultured for at least 3 days longer than HSPCs cultured in the absence of such modified endothelial niche cells, hi some embodiments, HSPCs cultured in the presence of modified endothelial niche cells can be cultured for at least 1 day longer, at least 2 days longer, at least 3 days longer, at least 4 days longer, at least 5 days longer, at least 6 days longer, at least 7 days longer, at least 8 days longer, at least 9 days longer, at least 10 days longer, at least 11 days longer, at least 12 days longer, at least 13 days longer, or at least 14 days longer than HSPCs cultured in the absence of such modified endothelial niche cells.

[0084] In some embodiments, cells are cultured on a biologically compatible scaffold. Non-limiting examples of biologically compatible scaffolds include hydrogels, biopolymers, or other biomaterials that exhibit the ability to grow cells in vitro in preparation for transplantation. In some embodiments, HSPCs cultured in the presence of modified endothelial niche cells have improved engraftment when administered to a subject compared to the engraftment of substantially similar HSPCs not cultured with modified endothelial niche cells. As used herein, "engraftment" refers to the process by which transplanted HSPCs begin to grow and generate healthy blood cells. Engraftment is an important milestone in recovery from HSPC transplantation.

[0085] Another aspect provides a method for treating a subject, comprising transplanting a composition comprising a population of modified endothelial niche cells into the subject.As a non-limiting example, this method can be used to treat myelofibrosis or other hematopoietic diseases that cause the breakdown of endogenous bone marrow niches, non-limiting examples of which are disclosed herein.In some embodiments, this method can comprise transplanting a composition comprising a population of HSPCs into the subject.In some embodiments, this method can comprise transplanting a composition comprising a population of HSPCs and modified endothelial niche cells into the subject.

[0086] Another aspect provides a method for enhancing HSPC engraftment, comprising administering a composition comprising a population of HSPCs and modified endothelial niche cells to a subject in need thereof. In some embodiments of any aspect, HSPC engraftment is increased by at least 10% compared to engraftment of substantially similar HSPCs in the absence of modified endothelial niche cells. In some embodiments of any aspect, HSPC engraftment is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to engraftment of substantially similar HSPCs in the absence of modified endothelial niche cells.

[0087] Another aspect provides a co-culture comprising modified endothelial niche cells and HSPCs. In some embodiments of any aspect, the endothelial cells are produced by the methods described herein.

[0088] Another aspect provides a method for generating an ectopic vascular niche, comprising administering modified endothelial niche cells to a target site in a subject in need thereof. As used herein, "ectopic vascular niche" refers to the site of atypical endothelial niche cells. For example, the vascular niche can be found outside the bone marrow. The ectopic vascular niche containing the generated or modified endothelial niche cells can be present anywhere in the body. The ectopic vascular niche can be found at the site where HSPCs, generated or modified endothelial niche cells and / or their associated transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) are injected.

[0089] Another aspect provides a method for extramedullary hematopoiesis, comprising transplanting engineered niche endothelial cells into a site (e.g., the forearm) outside the bone marrow of a subject, thereby creating an artificial niche. As used herein, "extramedullary hematopoiesis" refers to hematopoiesis that occurs in an organ outside the bone marrow. In some embodiments of any aspect, the endothelial cells are produced by the methods described herein.

[0090] Myelofibrosis In some embodiments of any aspect, the generated or modified endothelial niche cells or their associated transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) can be used to treat myelofibrosis.

[0091] Myelofibrosis is a rare type of chronic leukemia. Myelofibrosis often belongs to a group of diseases called chronic forms of myeloproliferative disorders. Chronic myeloproliferative disorders are a group of slowly progressing blood cancers in which the bone marrow produces too many abnormal red blood cells, white blood cells, or platelets, which accumulate in the blood. Non-limiting examples of chronic myeloproliferative neoplasms include chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis (also called chronic idiopathic myelofibrosis), essential thrombocythemia, chronic neutrophilic leukemia, and chronic eosinophilic leukemia.

[0092] Myelofibrosis is a serious bone marrow disorder that interferes with the body's normal blood cell production. The result is extensive scarring in the bone marrow, leading to severe anemia, weakness, fatigue, and often an enlarged spleen. Many subjects or patients with myelofibrosis gradually deteriorate, and some may eventually develop a more serious form of leukemia. Myelofibrosis can occur when blood stem cells (e.g., HSPCs) exhibit genetic mutations. Several specific genetic mutations have been identified in people with myelofibrosis. The most common is the Janus kinase 2 (JAK2) gene.

[0093] While the cause of myelofibrosis is unknown in many cases, certain factors are known to increase the risk. Increasing age may be associated with the development of myelofibrosis. While anyone can develop myelofibrosis, it is most commonly diagnosed in people over the age of 50. Patients with other blood cell disorders are at higher risk of developing myelofibrosis. A small number of people with myelofibrosis develop the condition as a complication of essential thrombocythemia or polycythemia vera. Exposure to certain chemicals may increase the risk of myelofibrosis. Myelofibrosis has been associated with exposure to industrial chemicals such as toluene and benzene. Exposure to radiation may increase the risk of myelofibrosis. People exposed to high levels of radiation, such as survivors of atomic bomb attacks, are at higher risk of myelofibrosis. Some people who received a radioactive contrast agent called Thorotrast, which was used until the 1950s, developed myelofibrosis.

[0094] Myelofibrosis can lead to multiple complications. Complications of myelofibrosis can include increased pressure on blood flow to a patient's liver. Normally, blood flows from the spleen through a large blood vessel called the portal vein. Increased blood flow from an enlarged spleen can cause high blood pressure in the portal vein (e.g., portal hypertension). This then forces excess blood into the small veins of the stomach and esophagus, which can cause these veins to rupture and bleed. Pain can be another complication of myelofibrosis. A severely enlarged spleen can cause abdominal and back pain. Myelofibrosis can promote growths in other areas of the body. Myelofibrosis can be associated with bleeding complications. As the disease progresses, platelet counts tend to fall below normal (thrombocytopenia), impairing platelet function. Insufficient platelet counts can lead to easy bleeding. Myelofibrosis can also be associated with bone and joint pain. Myelofibrosis can cause hardening of the bone marrow and inflammation of the connective tissue found around bones. This can cause bone and joint pain. Myelofibrosis can also be associated with the development of acute leukemia. Some patients with myelofibrosis develop acute myeloid leukemia, a rapidly progressing type of blood and bone marrow cancer.

[0095] Bone marrow transplantation is currently the only approved treatment for myelofibrosis. Additional treatments may only improve symptoms of myelofibrosis (e.g., anemia, enlarged spleen). Ruxolitinib, a JAK inhibitor that targets the genetic mutations found in most cases of myelofibrosis, can be used to alleviate symptoms of enlarged spleen.

[0096] Treatment method As described herein, the level of functional hematopoiesis can be decreased in myelofibrosis and / or in subjects with myelofibrosis. As used herein, "functional hematopoiesis" refers to hematopoiesis that produces normal levels and ratios of blood cells (e.g., red blood cells, white blood cells, platelets). In some embodiments of any aspect, the level of hematopoiesis can be decreased in myelofibrosis or myeloproliferative disorders and / or in subjects with myelofibrosis or myeloproliferative disorders. Thus, in one aspect of any embodiment, described herein is a method for treating myelofibrosis or myeloproliferative disorders in a subject in need thereof, comprising administering HSPCs, modified endothelial cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) to a subject determined to have a decreased level of functional hematopoiesis compared to a reference. In one aspect of any embodiment, described herein is a method of treating myelofibrosis or a myeloproliferative disorder in a subject in need thereof, comprising: (a) determining a level of functional hematopoiesis in a sample obtained from the subject; and (b) administering to the subject HSPCs, modified endothelial cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) if the level of functional hematopoiesis is decreased compared to a reference.

[0097] In some embodiments of any aspect, the method comprises administering HSPCs, modified endothelial cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) to a subject previously determined to have a decreased level of functional hematopoiesis compared to a reference. Described herein are methods of treating myelofibrosis or a myeloproliferative disorder in a subject in need thereof, comprising: (a) first determining the level of functional hematopoiesis in a sample obtained from the subject; and (b) thereafter, if the level of functional hematopoiesis is decreased compared to the reference, administering HSPCs, modified endothelial cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) to the subject.

[0098] In one aspect of any embodiment, described herein is a method for treating myelofibrosis or myeloproliferative disorder in a subject in need thereof, comprising: (a) determining whether the subject has a reduced level of hematopoiesis; and (b) administering HSPCs, modified endothelial cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) to the subject if the level of functional hematopoiesis is reduced compared to the reference. In some embodiments of any aspect, determining whether the subject has a reduced level of functional hematopoiesis can include (i) obtaining or having obtained a sample from the subject, and (ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of hematopoiesis in the subject. In some embodiments of any aspect, determining whether the subject has a reduced level of functional hematopoiesis can include performing or having performed an assay on the sample obtained from the subject to determine / measure the level of hematopoiesis in the subject. In some embodiments of any aspect, determining whether a subject has reduced levels of functional hematopoiesis can include ordering or requesting an assay on a sample obtained from the subject to determine / measure the level of hematopoiesis in the subject. In some embodiments of any aspect, determining whether a subject has reduced levels of functional hematopoiesis can include receiving results of an assay on a sample obtained from the subject that determines / measures the level of hematopoiesis in the subject. In some embodiments of any aspect, determining whether a subject has reduced levels of functional hematopoiesis can include receiving a report, result, or other measure identifying the subject as having reduced levels of functional hematopoiesis.

[0099] In one aspect of any embodiment, described herein is a method for treating myelofibrosis or myeloproliferative disorder in a subject in need thereof, comprising: (a) determining that the subject has a reduced level of functional hematopoiesis; and (b) instructing or instructing the subject to administer HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) if the level of functional hematopoiesis is reduced compared to a reference. In some embodiments of any aspect, determining whether the subject has a reduced level of functional hematopoiesis can include (i) obtaining or having obtained a sample from the subject, and (ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of functional hematopoiesis in the subject. In some embodiments of any aspect, determining whether the subject has a reduced level of functional hematopoiesis can include performing or having performed an assay on the sample obtained from the subject to determine / measure the level of functional hematopoiesis in the subject. In some embodiments of any aspect, determining whether the subject has a functional level of functional hematopoiesis can include ordering or requesting an assay on a sample obtained from the subject to determine / measure the level of functional hematopoiesis in the subject. In some embodiments of any aspect, instructing or directing the subject to administer a particular treatment can include providing a report of the assay results. In some embodiments of any aspect, instructing or directing the subject to administer a particular treatment can include providing a report of the assay results and / or a treatment recommendation that takes the assay results into account.

[0100] Administration In some embodiments, the methods described herein relate to the treatment of subjects who have or have been diagnosed with myelofibrosis or myeloproliferative disorders.Subjects with myelofibrosis or myeloproliferative disorders can be identified by physicians using current methods for diagnosing myelofibrosis or myeloproliferative disorders.Symptoms and / or complications of myelofibrosis or myeloproliferative disorders that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, anemia, splenomegaly (i.e., an enlarged, painful spleen), fatigue, weakness or shortness of breath, pain or fullness under the left ribs, easy bruising, easy bleeding, excessive sweating during sleep (night sweats), fever and / or bone pain.Tests that can aid in the diagnosis of myelofibrosis or myeloproliferative disorders include, but are not limited to, blood tests (e.g., complete blood count) or bone marrow biopsy.Myelofibrosis or myeloproliferative disorders can also be detected using physical examinations, imaging tests or genetic tests. A family history of myelofibrosis or myeloproliferative disorder, or exposure to risk factors for myelofibrosis or myeloproliferative disorder (e.g., industrial chemicals, radiation) can also aid in determining whether a subject is likely to have myelofibrosis or myeloproliferative disorder or in making a diagnosis of myelofibrosis or myeloproliferative disorder.

[0101] The compositions and methods described herein can be administered to subjects with or diagnosed as having myelofibrosis or myeloproliferative disorders. In some embodiments, the methods described herein include administering to a subject an effective amount of a composition described herein, such as HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2), to alleviate the symptoms of myelofibrosis or myeloproliferative disorders. As used herein, "alleviating the symptoms of myelofibrosis or myeloproliferative disorders" refers to improving any condition or symptom associated with myelofibrosis or myeloproliferative disorders. Compared to comparable untreated controls, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique. Various means of administering the compositions described herein to a subject are known to those skilled in the art. Such methods may include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration may be local or systemic.

[0102] The term "effective amount," as used herein, refers to the amount of HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a pharmacological composition to provide the desired effect. The term "therapeutically effective amount," therefore, refers to the amount of HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) sufficient to provide a certain anti-myofibrosis or anti-myoproliferative disorder effect when administered to a typical subject. As used herein, an effective amount also includes, in various contexts, an amount sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slow the progression of disease symptoms), or ameliorate disease symptoms. Therefore, identifying an exact "effective amount" is typically not feasible. However, in any given instance, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0103] Effective doses, toxicity, and therapeutic effects can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). Doses can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods exhibiting a high therapeutic index are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Doses can also be determined in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) that achieves half-maximal inhibition of symptoms) determined in cell culture or in an appropriate animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be observed by a suitable bioassay. The dosage can be determined by a physician and adjusted, if necessary, to suit the observed effects of treatment.

[0104] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredient of the pharmaceutical composition comprises the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of HSPCs, modified endothelial niche cells and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein.

[0105] Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents and / or dispersion media.The use of such carriers and diluents is well known in the art.Some non-limiting examples of substances that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C 12 Alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. In some embodiments, the carrier inhibits degradation of the active agent, such as the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein.

[0106] In some embodiments, pharmaceutical compositions comprising HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein can be in parenteral dosage forms. Because administration of parenteral dosage forms typically bypasses a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, dry forms that can be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, injectable suspensions, and emulsions. In addition, controlled-release parenteral dosage forms, including, but not limited to, DUROS®-type dosage forms and dose-dumping dosage forms, can be prepared for administration to a patient.

[0107] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosed HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) are well known to those skilled in the art. Examples include, but are not limited to, sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0108] In some embodiments of any aspect, the HSPCs, modified endothelial niche cells and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein are administered as monotherapy, e.g., the subject is not receiving another treatment for myelofibrosis or myeloproliferative disorder.

[0109] In some embodiments of any aspect, the methods described herein can further include administering a second agent and / or treatment to the subject, e.g., as part of a combination therapy. Non-limiting examples of second agents and / or treatments include radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryophenone; Statins; kallistatins; CC-1065 (including synthetic analogs of adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitrogenmaster amides such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunol Bicine, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, pi uromycin, queramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, Azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine;Bestravsil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremophor-free albumin-modified paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE® vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate;irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)), and VEGF-A that reduce cell proliferation, and may include pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0110] Additionally, treatment methods may further include the use of radiation or radiotherapy. Additionally, treatment methods may further include the use of surgical procedures.

[0111] The methods described herein may further comprise administering a second drug and / or treatment to the subject, for example, as part of a combination therapy.As a non-limiting example, when treating a subject for pain or inflammation according to the methods described herein, the subject may also be administered a second drug and / or treatment that is known to be beneficial to subjects suffering from pain or inflammation.In some embodiments, the second drug is an anti-inflammatory agent. Examples of such agents and / or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs, such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g., cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclomethasone); methotrexate; sulfasalazine; leflunomide; anti-TNF drugs; cyclophosphamide; pro-resolving drugs; mycophenolate; or sedatives (e.g., endorphins, enkephalins, and dynorphins), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

[0112] In certain embodiments, a composition comprising an effective dose of the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein can be administered once to a patient. In certain embodiments, a composition comprising an effective dose of the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) can be administered repeatedly to a patient. For systemic administration, a subject may be administered a therapeutic amount of a composition comprising HSPCs, modified endothelial niche cells and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2), e.g., 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0113] In some embodiments, after the initial treatment regimen, treatment can be performed less frequently. For example, after 3 months of biweekly treatment, treatment can be repeated once a month for 6 months or 1 year or more. Treatment according to the methods described herein can reduce the level of markers or symptoms of a condition, such as HSPCs, altered endothelial niche cells and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0114] The dosage of the compositions described herein can be determined by a physician and, if necessary, adjusted to suit the observed effects of treatment. Regarding the duration and frequency of treatment, a physician typically monitors the subject to determine when treatment provides therapeutic benefit and whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or develop other alternatives to the treatment regimen. The dosing schedule can vary from once a week to daily, depending on numerous clinical factors, such as the subject's sensitivity to HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2). The desired dose or activation amount can be administered all at once or in divided doses, e.g., 2 to 4 divided doses, administered over a period of time, e.g., at appropriate intervals throughout the day, or according to any other suitable schedule. In some embodiments, administration can be chronic, for example, one or more daily doses and / or treatments over several weeks or months. Exemplary dosing and / or treatment schedules include daily, twice-daily, three-times-daily, or four or more times-daily administration for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more. The composition comprising HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) can be administered over a period of time, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0115] The dose range for administering HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) according to the methods described herein depends, for example, on the form, its efficacy, and the degree to which symptoms, markers, or indicators of the conditions described herein are desired to be reduced, such as, for example, the degree to which myelofibrosis or myeloproliferative disorder is desired to be alleviated, or the degree to which functional hematopoiesis is desired to be induced. The dose should not be so high as to cause adverse side effects, such as excessive hematopoiesis or excessive extramedullary hematopoiesis. Generally, the dose will vary depending on the age, condition, and sex of the patient, and can be determined by one skilled in the art. The dose can also be adjusted by an individual physician in the event of any complications.

[0116] For example, the efficacy of HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) in treating a condition described herein or in inducing a response described herein (e.g., functional hematopoiesis) can be determined by a physician. However, a treatment is considered to be "effective treatment" within the meaning of the present specification if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically acceptable symptoms are improved or even improved, or a desired response is induced, for example, by at least 10% after treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or occurrence of a condition treated according to the methods described herein or any other suitable measurable parameter, such as a blood count. Efficacy can also be measured by the lack of an individual's deterioration or need for medical intervention (i.e., halting progression of the disease) as assessed by hospitalization. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or animals), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) alleviating the severity of the disease, e.g., reversing symptoms. An effective amount for treating a disease means an amount that, when administered to a subject in need thereof, is sufficient to provide effective treatment for the disease as defined herein. The effect of an agent can be determined by assessing physical indicators of the condition or the desired response. It is well within the capabilities of those skilled in the art to observe the effect of administration and / or treatment by measuring any one or any combination of such parameters. The effect can be evaluated in an animal model for the treatment of the conditions described herein, such as myelofibrosis or myeloproliferative disorder. When using an experimental animal model, the effect of treatment is proven when a statistically significant change in the marker is observed.

[0117] kit One aspect described herein provides a kit for culturing HSPCs, comprising a population of modified endothelial niche cells, a reagent, and instructions for use thereof. Another aspect provides a kit for generating modified endothelial niche cells, comprising a vector containing one or more exogenous nucleic acid sequences encoding one or more transcription factors of the Ets family, Sox family, or nuclear hormone family, and instructions for use thereof. Kit components that can be included in one or more of the kits described herein are described herein.

[0118] In some embodiments, the kit comprises an effective amount of reagents for culturing HSPCs and / or endothelial niche cells. As will be understood by those skilled in the art, the reagents can be provided in a lyophilized or concentrated form that can be diluted with the cultured cells before use. Preferred formulations include those that are non-toxic to the cells and / or do not affect the growth rate or viability, etc. The reagents can be provided in aliquots or unit doses.

[0119] In some embodiments, the kit further comprises a vector comprising a nucleic acid encoding a gene for one or more transcription factors of the Ets family, Sox family, or nuclear hormone family under the control of a promoter.

[0120] In some embodiments, the components described herein can be provided individually or in any combination as a kit. The kit includes the components described herein, such as a composition comprising HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described throughout the specification, such as a composition comprising a vector containing genes for one or more transcription factors of the Ets family, Sox family, or nuclear hormone family under the control of a promoter. In addition, the kit optionally includes informational materials. The kit can also include a culture dish and / or a substrate for coating the culture dish, such as laminin, fibronectin, poly-L-lysine, or methylcellulose.

[0121] In some embodiments, the compositions in the kit may be provided in a watertight or airtight container, which in some embodiments is substantially free of other components of the kit. For example, HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) components may be provided in two or more containers, e.g., in a container containing enough reagents for a predetermined number of experiments, e.g., one, two, three, or more. One or more components described herein may be provided in any form, e.g., liquid, dried, or lyophilized. The components described herein are preferably substantially pure and / or sterile. When the components described herein are provided in a liquid solution, the liquid solution is preferably an aqueous solution, and a sterile aqueous solution is preferred.

[0122] The informational material may be a description, instruction, advertisement, or other material relating to the methods described herein. The informational material of the kit is not limited in terms of its form. In one embodiment, the informational material may include information about the generation, concentration, expiration date, batch or manufacturing site information, etc. of endothelial niche cells and / or HSPCs. In one embodiment, the informational material relates to methods of use or administration of the components of the kit.

[0123] The kit may include components for detecting markers of HSPC differentiation and / or endothelial niche cell differentiation. Furthermore, the kit may include one or more antibodies that bind to cell markers, or primers for RT-PCR or PCR reactions, such as semi-quantitative or quantitative RT-PCR or PCR reactions. Such components can be used to evaluate the activation of endothelial niche cell and / or HSPC maturation markers or the disappearance of immature cell markers. When the detection reagent is an antibody, it can be provided as a dry preparation, such as a lyophilized product, or in solution. The antibody or other detection reagent can be linked to a label, such as a radioactive, fluorescent (e.g., GFP), or colorimetric label, for use in detection. When the detection reagent is a primer, it can be provided as a dry preparation, such as a lyophilized product, or in solution.

[0124] The kit will typically be provided with its various components in a single package, e.g., a fabric-based, e.g., cardboard, or polymeric, e.g., Styrofoam, box. The enclosure may be configured to maintain a temperature differential between the interior and exterior, e.g., it may provide insulating properties that maintain the reagents at a preselected temperature for a preselected period of time.

[0125] vector In some embodiments, one or more of the factors described herein are expressed in a recombinant expression vector or plasmid. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring a transgene to a host cell. The term "vector" includes plasmids, minichromosomes, phages, naked DNA, and the like. See, for example, U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and 5,919,670, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" are used interchangeably, as the most commonly used form of vector is the plasmid. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses).

[0126] A cloning vector is one that is capable of autonomous replication or integration into the genome of a host cell, and is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a determinable manner and to which a desired DNA sequence can be ligated so that the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the plasmid increases in copy number within the host cell, e.g., a host bacterium, or only once per host before the host replicates by mitosis. In the case of a phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0127] An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation so that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in identifying cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity is detectable by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that visually affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operably linked.

[0128] As used herein, a coding sequence and a regulatory sequence are said to be "operably" connected when they are covalently linked such that expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. If it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably connected when induction of a promoter within the 5' regulatory sequence results in transcription of the coding sequence, and when the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a promoter region will be operably connected to a coding sequence if it is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.

[0129] When a nucleic acid molecule encoding any of the factors / polypeptides described herein is expressed in a cell, various transcription control sequences (e.g., promoter / enhancer sequences) can be used to induce its expression. The promoter can be a native promoter that provides normal regulation of the expression of the gene, i.e., the promoter of the gene in its endogenous relationship. In some embodiments, the promoter can be constitutive, i.e., the promoter is not regulated and continuous transcription of the gene linked to it occurs. Various conditional promoters can also be used, for example, promoters that are controlled by the presence or absence of a certain molecule.

[0130] The exact nature of regulatory sequences required for gene expression may vary between species or cell types, but generally may include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences will include a promoter region, including a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences, if desired. Vectors of the present invention may optionally include a 5' leader or signal sequence. The selection and design of an appropriate vector is within the ability and discretion of one skilled in the art.

[0131] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically modified by introducing heterologous DNA (RNA) into the cells. The heterologous DNA (RNA) is placed under the functional control of transcriptional elements to achieve expression of the heterologous DNA in the host cell.

[0132] In some embodiments, the vector is a pME Gateway vector (Invitrogen™). In some embodiments, the vector is a p5E Gateway™ vector. In some embodiments, the vector is a pGEX2TK™ vector. In some other embodiments, the vector is a TOPO-TA™ vector.

[0133] Without limitation, the genes described herein can be contained in one vector or separate vectors.For example, at least one gene from Ets family (for example, ETV2, FLI1, ETS1) and at least one gene from SOX family (for example, SOX18, SOX7) and at least one gene from NHR family (for example, RXRA, NR2F2) can be contained in the same vector.

[0134] In some embodiments, at least one gene from the Ets family (e.g., ETV2, FLI1, ETS1) and at least one gene from the SOX family (e.g., SOX18, SOX7) may be included in a first vector, and at least one gene from the NHR family (e.g., RXRA, NR2F2) may be included in a second vector.

[0135] In some embodiments, at least one gene from the NHR family (e.g., RXRA, NR2F2) and at least one gene from the SOX family (e.g., SOX18, SOX7) may be included in a first vector, and at least one gene from the Ets family (e.g., ETV2, FLI1, ETS1) may be included in a second vector.

[0136] In some embodiments, at least one gene from the Ets family (e.g., ETV2, FLI1, ETS1) and at least one gene from the NHR family (e.g., RXRA, NR2F2) may be included in a first vector, and at least one gene from the SOX family (e.g., SOX18, SOX7) may be included in a second vector.

[0137] In some embodiments, at least one gene from the Ets family (e.g., ETV2, FLI1, ETS1) may be included in a first vector, at least one gene from the SOX family (e.g., SOX18, SOX7) may be included in a second vector, and at least one gene from the NHR family (e.g., RXRA, NR2F2) may be included in a third vector.

[0138] In some embodiments, the promoter operably linked to the gene can be a zebrafish ubi promoter.

[0139] In some embodiments, one or more of the recombinantly expressed genes may be integrated into the genome of the cell.

[0140] Nucleic acid molecules encoding enzymes of the invention can be introduced into a cell or group of cells using methods and techniques standard in the art. For example, the nucleic acid molecules can be introduced by standard protocols, such as transformation, including chemical transformation, and electroporation, transduction, particle bombardment, etc. Expression of nucleic acid molecules encoding enzymes of the invention can also be achieved by integrating the nucleic acid molecules into the genome.

[0141] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise indicated or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to aid in the description of specific embodiments and are not intended to limit the invention described in the claims, the scope of which is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0142] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0143] The terms "decrease," "reduced," "reduction," or "inhibition" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce," "reduction," or "decrease" or "inhibition" typically refers to a decrease of at least 10% compared to a reference level (e.g., the absence of a particular treatment or agent), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "decrease" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease may preferably be to a level that is accepted as within the normal range for individuals without the particular disorder.

[0144] The terms "increased," "increase," "enhancement," or "activation" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhancement," or "activation" can mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, including 100%, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more, compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0145] As used herein, "subject" refers to a human or animal. Usually, animals are vertebrates, such as primates, rodents, livestock animals, or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0146] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Advantageously, non-human mammals can be used as subjects corresponding to animal models of myelofibrosis or myeloproliferative disorders. The subject may be male or female.

[0147] The subject may be a person who has been previously diagnosed or identified as suffering from or having a condition requiring treatment (e.g., myelofibrosis or myeloproliferative disorder) or one or more complications related to such a condition, and optionally has already received treatment for myelofibrosis or myeloproliferative disorder or one or more complications related to myelofibrosis or myeloproliferative disorder. Alternatively, the subject may be a person who has not previously been diagnosed with myelofibrosis or myeloproliferative disorder or one or more complications related to myelofibrosis or myeloproliferative disorder. For example, the subject may be a person who shows one or more risk factors for myelofibrosis or myeloproliferative disorder or one or more complications related to myelofibrosis or myeloproliferative disorder, or a subject who does not show risk factors.

[0148] A "subject in need" of treatment for a particular condition can be a subject who has the condition, a subject who has been diagnosed with the condition, or a subject who is at risk of developing the condition.

[0149] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues interconnected by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0150] In the various embodiments described herein, it is further intended to encompass variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will understand that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," substituting a chemically similar amino acid for the altered amino acid while maintaining the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles in accordance with the present disclosure.

[0151] Certain amino acids can be replaced by residues with similar physicochemical properties, for example, by substituting one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for each other), or by substituting one polar residue for another (e.g., Lys for Arg; Glu for Asp; or Gln for Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., transcription factor activity and specificity of the native or reference polypeptide, is maintained.

[0152] Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class. Specific conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0153] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described herein below. Functional fragments may include conservative substitutions of the sequences disclosed herein.

[0154] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of the native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from that of the native or reference polypeptide by one or more deletions, insertions, or substitutions. DNA sequences encoding variant polypeptides include sequences encoding variant proteins or fragments thereof that contain one or more nucleotide additions, deletions, or substitutions compared to the native or reference DNA sequence but retain activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0155] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using computer programs commonly used for this purpose (e.g., BLASTp or BLASTn, freely available on the World Wide Web, with default settings).

[0156] Alterations to the native amino acid sequence can be achieved by any of a number of techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence adjacent to restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide site-directed mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered by the required substitution, deletion, or insertion. Techniques for making such modifications are well established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Additionally, any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to the polypeptide to improve its stability or promote oligomerization.

[0157] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, a nucleic acid can be DNA. In another aspect, a nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0158] The term "expression" refers to the cellular processes involved in the production of RNA and protein, and, where appropriate, protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as appropriate. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or the translation of mRNA into a polypeptide.

[0159] In some embodiments, expression of the biomarkers, targets, or genes / polypeptides described herein is tissue-specific. In some embodiments, expression of the biomarkers, targets, or genes / polypeptides described herein is global. In some embodiments, expression of the biomarkers, targets, or genes / polypeptides described herein is systemic.

[0160] "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed (from DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following its coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0161] In the context of the present invention, a "marker" refers to an expression product, e.g., a nucleic acid or polypeptide, that is differentially present in a sample taken from a subject with myelofibrosis or a myeloproliferative disorder compared to a comparable sample taken from a control subject (e.g., a healthy subject). The term "biomarker" is used interchangeably with the term "marker."

[0162] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term "detection" or "measurement" refers to the observation of a signal, for example, from a probe, a label, or a target molecule, indicating the presence of an analyte in a sample. Any method known in the art for detecting a specific label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some embodiments of any aspect, measurement can be a quantitative observation.

[0163] In some embodiments of any aspect, polypeptide, nucleic acid or cell described herein can be modified.As used herein, " modification " refers to the aspect of being manipulated by human hands.For example, a polypeptide is considered to be " modified " if it is manipulated by human hands so that at least one aspect of the polypeptide, for example, its sequence, is different from the aspect when it exists in nature.As is common practice and understood by those skilled in the art, the descendants of modified cells are typically referred to as " modified ", even if actual manipulation is performed on previous cells.

[0164] In some embodiments of any aspect, the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein are exogenous. In some embodiments of any aspect, the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein are ectopic. In some embodiments of any aspect, the HSPCs, modified endothelial niche cells, and / or transcription factors (e.g., ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2) described herein are not endogenous.

[0165] The term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced by a human process into a biological system, such as a cell or organism in which it is not normally found and in which it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that has been introduced by a human process into a biological system, such as a cell or organism in which it is found in relatively low amounts and in which it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to establish ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, "ectopic" refers to a substance found in an unusual location and / or amount. An ectopic substance may be one that is normally found in a particular cell, but it may be found in much lower amounts and / or at a different time. Ectopic also includes substances, such as polypeptides or nucleic acids, that are not naturally found or expressed in a particular cell in its natural environment.

[0166] In some embodiments, a nucleic acid encoding a polypeptide described herein (e.g., an ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2 polypeptide) is contained in a vector. In some aspects described herein, a nucleic acid sequence encoding a particular polypeptide described herein, or any module thereof, is operably linked to a vector. The term "vector," as used herein, refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replication when linked to appropriate control elements and can transfer a genetic sequence into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc.

[0167] In some embodiments of any aspect, the vector is recombinant, e.g., it comprises sequences derived from at least two different sources. In some embodiments of any aspect, the vector comprises sequences derived from at least two different species. In some embodiments of any aspect, the vector comprises sequences derived from at least two different genes, e.g., it comprises a nucleic acid encoding a fusion protein or expression product operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., promoter, suppressor, activator, enhancer, response element, etc.).

[0168] In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence is altered or modified to include alternative codons such that the altered or modified nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence but is transcribed and / or translated with improved efficiency in a desired expression system. In some embodiments of any aspect, the expression system is an organism (or cells obtained from such an organism) other than the source of the native / wild-type sequence. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a mammal or mammalian cell, such as a mouse, mouse cell, or human cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a human cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a yeast or yeast cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a bacterial cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in E. coli cells.

[0169] As used herein, the term "expression vector" refers to a vector that induces the expression of RNA or polypeptide from the sequence linked to the transcriptional regulatory sequence on the vector.The sequence to be expressed will often, but not necessarily, be heterologous to the cell.Expression vectors can contain additional elements, for example, expression vectors can have two replication systems, so they can be maintained in two organisms, for example, in human cells for expression and in prokaryotic hosts for cloning and amplification.

[0170] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. A viral vector can contain a nucleic acid encoding a polypeptide described herein in place of a non-essential viral gene. The vector and / or particle can be used to transfer any nucleic acid into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0171] It should be understood that in some embodiments, the vector described herein can be combined with other suitable compositions and treatments.In some embodiments, the vector is episomal.The use of suitable episomal vector provides a way to maintain the nucleotide of interest in the subject as high copy number extrachromosomal DNA, thereby eliminating the potential effect of chromosomal integration.

[0172] As used herein, the terms "treat," "treatment," "treating," or "alleviation" refer to therapeutic treatment whose purpose is to ameliorate, alleviate, reduce, inhibit, slow, or halt the progression or severity of a condition associated with a disease or disorder, e.g., myelofibrosis or a myeloproliferative disorder. The term "treatment" includes the reduction or alleviation of at least one adverse effect or symptom of a condition, disease, or disorder associated with myelofibrosis or a myeloproliferative disorder. Treatment is typically "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is reduced or halted. That is, "treatment" includes not only an improvement in symptoms or markers, but also a halt or at least slowing of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desirable clinical results, whether detectable or undetectable, include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease, delay or slowing of the progression of the disease, alleviation or palliation of the disease state, remission (whether partial or complete), and / or reduced mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0173] As used herein, the term "pharmaceutical composition" refers to an active ingredient combined with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals, under a reasonable benefit / risk ratio, and without excessive toxicity, irritation, allergic response, or other problems or complications. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or modified carrier, e.g., a carrier in which the active ingredient would not be found in nature.

[0174] As used herein, the term "administration" refers to the introduction of a compound disclosed herein into a subject by a method or route that achieves at least partial delivery of the agent to a desired site. Pharmaceutical compositions comprising a compound disclosed herein can be administered by any suitable route that results in effective treatment in the subject. In some embodiments, administration includes physical human activity, such as injection, ingestion, application, and / or operation of a delivery device or instrument. Such activity can be performed, for example, by a physician and / or the subject being treated.

[0175] As used herein, "contacting" refers to any suitable means of delivering or exposing an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery into cell culture medium, perfusion, injection, or other delivery methods known to those skilled in the art. In some embodiments, contacting includes physical human activity, such as injection, dispensing, mixing, and / or pouring, and / or manipulation of a delivery device or instrument.

[0176] The terms "statistically significant" or "significant" refer to statistical significance, and typically mean a difference of 2 standard deviations (2 SD) or greater.

[0177] Other than in the working examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0178] As used herein, the term "comprises" means that in addition to the defined elements being present, other elements may also be present. The use of "comprises" indicates inclusion rather than limitation.

[0179] The term "consisting of" refers to compositions, methods, and each element thereof described herein, exclusive of any element not mentioned in the description of that embodiment.

[0180] As used herein, the term "consisting essentially of" refers to elements required for a particular embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0181] As used herein, the term "corresponding" refers to the amino acid or nucleotide at a designated position in a first polypeptide or nucleic acid, or the amino acid or nucleotide equivalent to the designated amino acid or nucleotide in a second polypeptide or nucleic acid. The equivalent designated amino acid or nucleotide can be determined by alignment of candidate sequences using homology search programs known in the art, such as BLAST.

[0182] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells, and / or particles, in which the first entity binds to a second target entity with higher specificity and affinity than the first entity binds to a third, non-target entity. In some embodiments, specific binding can refer to the affinity of a first entity for a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or more times higher than the affinity for a third, non-target entity. A reagent specific for a target is one that exhibits specificity for the target under the conditions of the assay used.

[0183] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and," unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia," and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0184] Groupings of alternative elements or aspects of the invention disclosed herein are not intended to be limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or excluded from, a group for reasons of convenience and / or patentability. Notwithstanding any such inclusion or exclusion, the specification shall be deemed to include the modified group and therefore to constitute a written description of all Markush groups used in the appended claims.

[0185] Unless otherwise defined herein, scientific and technical terms used in connection with this application have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as such may vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WWNorton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0186] Other terms are defined herein within the description of various aspects of the invention.

[0187] All patents and other publications, including references, issued patents, published patent applications, and copending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications, which may be used in connection with the technology described herein. These publications are provided solely for the purpose of their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute an admission as to the accuracy of the dates or contents of these documents.

[0188] The descriptions of the embodiments of the present disclosure are not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Individual embodiments and examples of the present disclosure are described herein for illustrative purposes only, and various equivalent modifications are possible within the scope of the present disclosure, as will be recognized by those skilled in the relevant art. For example, while method steps or functions are shown in a certain order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The techniques of the disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as necessary, to utilize the compositions, functions, and concepts of the above-mentioned references and applications to provide still further embodiments of the present disclosure. Furthermore, in consideration of biological functional equivalence, certain modifications can be made to protein structures in terms of type and amount without affecting their biological or chemical action. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be encompassed within the scope of the appended claims.

[0189] Individual elements of any of the above embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with particular embodiments of the present disclosure have been described in connection with those embodiments, other embodiments may also exhibit such advantages, and not all embodiments are required to exhibit such advantages in order to be within the scope of the present disclosure.

[0190] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

[0191] Some aspects of the technology described herein can be defined according to any of the following numbered paragraphs: 1. A method for generating / modifying endothelial niche cells, comprising expressing one or more transcription factors in endothelial cells, wherein the one or more transcription factors are derived from the Ets family, Sox family, and / or nuclear hormone receptor family. 2. The method of any one of the preceding paragraphs, wherein the endothelial niche cells express one or more genes including sele, exoc312a, snx8a, cltca, aqp7, ap1b1, lgmn, prcp, cldn11a, lyve1b, adra1d, hya12a, hya12b, tll1, i113ra2, glu1a, hexb, slc16a9a, or sepp1a. 3. The method of any one of the preceding paragraphs, wherein the endothelial cells are human. 4. The method of any one of the preceding paragraphs, wherein the transcription factor comprises at least one of the human transcription factors ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, or NR2F2. 5. The method of any one of the preceding paragraphs, wherein the transcription factors include at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family. 6. The method of any one of the preceding paragraphs, wherein the transcription factors include ETV2, FLI1, ETS1, SOX18, SOX7, RXRA, and NR2F2. 7. The method of any one of the preceding paragraphs, wherein the transcription factor is expressed from at least one vector. 8. The method of any one of the preceding paragraphs, wherein the vector comprises an exogenous nucleic acid sequence encoding one or more transcription factors. 9. The method of any one of the preceding paragraphs, wherein the exogenous nucleic acid sequence is integrated into the genome of the endothelial cell. 10. A modified endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding one or more transcription factors, wherein the one or more transcription factors are derived from the Ets family, the Sox family, and / or the nuclear hormone family. 11. 11. A composition comprising the modified endothelial niche cells of paragraph 10. 12. The composition of any one of the preceding paragraphs, further comprising a therapeutic agent or a pharmaceutically acceptable carrier. 13. The composition of any one of the preceding paragraphs, further comprising a culture dish, a 3D cell system, or a suspension system. 14. The composition of any one of the preceding paragraphs, comprising a scaffold. 15. A method for culturing HSPCs, the method comprising culturing the HSPCs in the presence of a population of modified endothelial niche cells. 16. The method of any one of the preceding paragraphs, which is carried out in vitro. 17. The method of any one of the preceding paragraphs, wherein the modified endothelial niche cells secrete factors that affect the growth and / or expansion of HSPC cells. 18. The method of any one of the preceding paragraphs, wherein HSPCs cultured in the presence of modified endothelial niche cells can be cultured for at least 3 (e.g., at least 4, at least 5, at least 6, at least 7) ​​days longer than HSPCs cultured in the absence of such modified endothelial niche cells. 19. The method of any one of the preceding paragraphs, wherein the cells are cultured on a biologically compatible scaffold. 20. The method of any one of the preceding paragraphs, wherein HSPCs cultured in the presence of the modified endothelial niche cells, when administered to a subject, exhibit improved engraftment compared to engraftment of substantially similar HSPCs not cultured with the modified endothelial niche cells. twenty one. A method of treating a subject, the method comprising transplanting a composition comprising a population of HSPCs and modified endothelial niche cells into the subject. twenty two. A method for improving HSPC engraftment, comprising administering to a subject in need thereof a composition comprising a population of HSPCs and modified endothelial niche cells. twenty three. The method of any one of the preceding paragraphs, wherein engraftment of HSPCs is improved by at least 10% compared to engraftment of substantially similar HSPCs in the absence of the modified endothelial niche cells. twenty four. Co-cultures containing modified endothelial niche cells and HSPCs. twenty five. 25. The co-culture of paragraph 24, wherein the endothelial cells are produced by the method of any one of the preceding paragraphs. 26. A kit for culturing HSPCs, including a population of modified endothelial niche cells, reagents, and instructions for their use. 27. A kit for generating modified endothelial niche cells, comprising a vector containing one or more exogenous nucleic acid sequences encoding one or more transcription factors of the Ets family, Sox family, or nuclear hormone family, and instructions for use thereof. 28. A method for generating an ectopic vascular niche, comprising administering modified endothelial niche cells to a target site in a subject in need thereof. 29. A method for extramedullary hematopoiesis, comprising transplanting modified niche endothelial cells into a site outside the bone marrow (e.g., the forearm) in a subject, thereby creating an artificial niche. 30. The method of any one of the preceding paragraphs, wherein the endothelial cells are produced by any of the methods of the preceding paragraphs. 31. A vector comprising one or more exogenous nucleic acid sequences encoding one or more transcription factors of the Ets family, Sox family, or nuclear hormone family operably linked to a promoter. [Example]

[0192] Example 1 Induction of transcription factors in the vascular hematopoietic stem cell niche in vivo The hematopoietic niche is a supportive in vivo microenvironment composed of various cell types, including specialized vascular endothelial cells, that directly interact with hematopoietic stem and progenitor cells (HSPCs) and promote stem cell function. The molecular factors that determine niche endothelial cells and their hematopoietic-promoting activity remain largely unknown. Using multidimensional gene expression analysis and chromatin accessibility assays, we define a conserved gene expression signature and cis-regulatory profile specific to sinusoidal endothelial cells within the HSPC niche. Using enhancer mutations and transcription factor overexpression, we uncovered a transcriptional code containing members of the Ets, Sox, and nuclear hormone receptor families that is sufficient to induce ectopic niche endothelial cells that recruit HSPCs and support their homeostasis in vivo. Collectively, these studies have important implications for generating more effective artificial vascular niches for blood stem cells or for tailoring these niches in therapeutic settings.

[0193] result An endothelial gene expression signature unique to the fetal HSPC niche ECs from different organs express distinct genes, but whether this is regulated by organ-specific transcriptional programs remains poorly understood. To investigate the regulation of gene expression in the CHT niche, we performed RNA tomography (tomo-seq) on zebrafish tails at 72 hours post-fertilization (hpf; see, e.g., Figure 1A). This tomo-seq analysis revealed gene expression clusters corresponding to individual tissues along the dorsoventral axis of the tail, including the spinal cord, notochord, muscle, and epidermis, as well as individual blood and immune cell populations (see, e.g., Figures 1B, 7A, and 7B). In total, 144 genes showed enriched expression in several cryosections spanning the CHT (see, e.g., Table 3). Whole-mount in situ hybridization (WISH) was used to confirm CHT expression for 35 of these genes (see, e.g., Table 3; images available on the World Wide Web at zfin.org). To determine which of the 144 genes were expressed by ECs, we isolated ECs for bulk and single-cell RNA-seq using the endothelial-common transgene kdr1:GFP and fluorescence-activated cell sorting (FACS) (see, e.g., Figure 1C). Additionally, we cross-referenced these genes with macrophage and neutrophil RNA-seq datasets (see, e.g., Theodore et al., Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization. Stem Cell Reports 8, 1226–1241, 2017). Twenty-nine genes were identified as selectively enriched in CHT ECs (see, e.g., Table 1). In contrast to the endothelial-common genes, the tomo-seq expression traces of these CHT endothelial genes lacked strong peaks corresponding to expression in dorsal blood vessels (see, e.g., Figure 1D).CHT EC-enriched expression of 25 of the 29 genes was confirmed by WISH (see, eg, Figure 1E, Figure 7A, Figure 7B and Table 1).

[0194] To selectively isolate CHT ECs, we engineered transgenic lines to label these cells. To generate GFP reporter transgenes, we cloned 1.3 or 5.3 kb of upstream regulatory sequences for two CHT endothelial genes, mrc1a and sele, and then combined them with the endothelial consensus marker kdr1:mCherry. For both the mrc1a:GFP and sele:GFP transgenes, the highest levels of expression were observed in the venous sinusoidal vessels of the CHT, with lower levels of GFP expression observed in the posterior cardinal vein above the vitelline extension and in a few vessels in the head (see, for example, Figures 8A-8E). GFP expression was also observed in mesenchymal cells of the lateral caudal fin; however, these cells are frequent sites of ectopic transgene expression and do not represent endogenous expression of these genes. Notably, robust GFP expression was observed in sinusoidal ECs that directly interact with HSPCs, confirming that the mrc1a:GFP and sele:GFP transgenes labeled ECs within the HSPC niche (see, for example, Figures 8A-8E). + EC, cxcl12a:DsRed2 + ECs were observed to adhere closely to stromal cells and form pockets surrounding HSPCs—a cellular behavior characteristic of ECs within the CHT niche (see, for example, Figures 8A-8E).

[0195] Endothelial niche-specific cis-regulatory elements To investigate the transcriptional regulation of niche-specific gene expression within CHT ECs, double-positive mrc1a:GFP / kdr1:mCherry embryos were dissected and identified as four distinct populations using FACS for RNA-seq and ATAC-seq analysis. + / mCherry + (CHT EC), GFP - / mCherry + (EC outside CHT), GFP+ / mCherry - (mesenchymal cells in the caudal fin), and GFP - / mCherry - We isolated the negative remainder of the embryo (see, for example, Figure 2A). By comparing chromatin-accessible regions between the four populations, we identified 6,848 uniquely open regions in CHT ECs. Of the 29 CHT EC genes, 26 of the 29 had ATAC-seq elements within 100 kb of their transcription start sites, which were found exclusively in CHT ECs (see, for example, Figure 2B and Table 1). To test whether these regions might be tissue-specific enhancers, we cloned sequences for 15 of these elements, fused them to a minimal promoter and GFP, and injected them into zebrafish embryos. Twelve of the 15 constructs showed GFP expression enriched in CHT ECs at 60–72 hpf (see, for example, Figure 2C and Table 2). As a control, we cloned regions of chromatin predicted to contain endothelial consensus regulatory elements based on their proximity to previously annotated vascular-specific genes and their accessibility in both CHT and non-CHT EC fractions (e.g., Figures 9A-9C and Table 2). Many of these regions have previously been shown to contain endothelial enhancers (e.g., Quillien et al., "Robust Identification of Developmentally Active Endothelial Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq." Cell Reports 20, 709-720, 2017). For six of the six endothelial consensus regions, mosaic GFP expression in ECs was observed throughout embryos not committed to the CHT (e.g., Figures 9A-9C). Thus, this zebrafish system allowed for rapid in vivo validation of niche-specific endothelial enhancers predicted by ATAC-seq analysis.

[0196] Transcription factor binding sites for niche endothelial expression To identify transcription factors that can bind to CHT EC enhancers, we performed motif enrichment analysis on 6,848 chromatin regions that were uniquely accessible in CHT ECs. This analysis revealed that Ets, Sox (specifically, SoxF factors), and nuclear hormone receptor (specifically, NR2F2 / RORA / RXRA factors; hereafter abbreviated as NHR) binding motifs were most enriched in these 6,848 regions (see, e.g., Figures 9A-9C). In contrast, there were 4,522 endothelial consensus elements (i.e., regions of chromatin accessible to both CHT and non-CHT ECs) throughout the genome that were enriched for Ets sites but not for SoxF or NHR binding motifs (see, e.g., Figures 9A-9C). Notably, all 12 CHT-EC elements that induced GFP expression in the in vivo reporter assay contained Ets, SoxF, and NHR sites, whereas one of the three CHT-EC elements and three of the six endothelial common regions that did not induce GFP expression lacked NHR binding sites (see, e.g., Table 2 ).

[0197] To determine the minimal sequences sufficient to induce CHT EC expression, we cloned a 125-base pair (bp) sequence upstream of mrc1a and a 158-bp sequence upstream of sele and included these sequences in our original transgenes. These sequences corresponded to the strongest ATAC-seq signals in these regions (see, for example, Figure 3A and Figures 10A-E). When linked to a minimal promoter, these elements induced GFP expression in CHT ECs in 44% (125-bp mrc1a sequence; 155 of 356) and 23% (158-bp sele sequence; 176 of 775) of injected embryos (see, for example, Figure 3B and Figures 10A-E). In contrast to injection of the kdr1:mCherry construct, which showed mosaic-like expression in ECs throughout the embryo, expression of the mrc1a and sele enhancer-GFP constructs was restricted to CHT ECs (see, for example, Figure 3C and Figures 10A-10E). Furthermore, when stable transgenic lines were established using these short constructs, GFP was specifically expressed in CHT ECs (see, for example, Figure 3D and Figures 10A-10E). Mammalian Mrc1 is primarily expressed by macrophages and venous sinusoidal ECs. Zebrafish possess two homologous genes, mrc1a and mrc1b. A recent study of mrc1a reported that it was expressed in both macrophages and ECs when its promoter was linked to an intronic enhancer that showed conservation with mrc1b (see, for example, Jung et al. Development of the larval lymphatic system in zebrafish. Development 144, 2070-2081, 2017). In contrast, the 125 bp enhancer element specifically induced expression in CHT ECs, demonstrating the specificity of the enhancer.

[0198] The regulatory sequences of both the 125-bp mrc1a and 158-bp sele contained Ets, SoxF, and NHR motifs (see, e.g., Figure 3E and Figures 10A–10E). To test whether these transcription factor binding sites were required for expression, we generated mutants in which each class of motif was disrupted by mutation. In each instance, disruption of the Ets, SoxF, or NHR motif resulted in a significant reduction or complete loss of GFP expression in CHT ECs (see, e.g., Figure 3F). In control constructs in which mutations were targeted to the intervening sequences between the Ets, SoxF, and NHR motifs, GFP expression was not disrupted (see, e.g., Figure 3F). Studies of arterial-venous specification in zebrafish have shown that the NHR Nr2f2, also known as COUP-TFII, promotes venous endothelial cell fate (see, e.g., Aranguren et al. Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis. Biochemical and Biophysical Research Communications 410, 121–126, 2011). To test whether Nr2f2 can directly bind to enhancer sequences, we performed in vitro gel electrophoretic mobility shift assays. Incubation of mouse NR2F2-GST protein with labeled probes derived from either the 125-bp mrc1a or 158-bp sele zebrafish enhancer resulted in a DNA:protein complex that was supershifted by the addition of NR2F2 antibody and could be counteracted by an unlabeled competitor probe (see, e.g., Figures 10A–10E). However, unlabeled probes with mutated NHR motifs were unable to counteract the wild-type probes, indicating that NR2F2 binds to these NHR motifs in a sequence-specific manner.

[0199] Defined factors induce niche endothelial expression To determine which transcription factors are expressed in CHT ECs in vivo and can bind to Ets, Sox, and NHR motifs, we examined RNA-seq data from CHT ECs. The most highly expressed factors were fli1a, etv2, ets1, sox18, sox7, nr2f2, and rxraa (see Table 4 for an example). To test whether these seven factors can induce the expression of niche endothelial genes outside the CHT, we created constructs in which the orthologs of each transcription factor were under the control of the ubiquitous (ubi) promoter (see Figure 4A for an example). We then injected this pool of seven ubi-inducing factors into single-cell stage zebrafish embryos and examined the expression of mrc1a and sele by WISH at 60–72 hpf. Notably, 17% of these embryos (12 of 69) had ectopic vascular compartments of mrc1a expression outside the CHT, on the dorsal side of the trunk and tail, and on the yolk (see, for example, Figure 4B, Figures 11A-C). Control-injected embryos showed no ectopic expression (0 of 56). Similar results were obtained in WISH for sele or when factors were injected into mrc1a:GFP / kdr1:mCherry double transgenic embryos (see, for example, Figures 4B-C, Figures 11A-C). Ectopically expressing mrc1a vessels were often larger than their normal counterparts in these regions and had sinusoidal morphology similar to the CHT (see, for example, Figure 4C). DIC microscopy was used to visualize blood flow through these regions (data not shown). These data suggest that a small number of transcription factors are sufficient to ectopically induce the niche endothelial gene program.

[0200] Mutational analysis of the 125-bp mrc1a and 158-bp sele enhancers demonstrated that factors from the Ets, Sox, and NHR families are required for expression. Therefore, we investigated whether a combination of just three factors (one from each family) is sufficient to induce niche endothelial gene expression. ETV2 is a pioneer factor required for the specification of early mesodermal precursors to a vascular cell fate. Forced expression of ETV2 in nonvascular cells induces reprogramming to an early endothelial fate capable of generating multiple types of vasculature. Previous studies in zebrafish have demonstrated the importance of SoxF factors (sox7 and sox18) and nr2f2 during arterial and venous specification (see, for example, Swift et al., "SoxF factors and Notch regulate nr2f2 gene expression during venous differentiation in zebrafish." Developmental biology 390, 116-125, 2014). The combination of three of these factors—ETV2, SOX7, and Nr2f2—could be sufficient to induce ectopic niche endothelial gene expression. Consistent with this, when these three factors were injected, significant ectopic mrc1a expression was observed (see, e.g., Figures 4D-F). The incidence of ectopic vessels produced by the three factors was higher than that of the seven-factor pool, suggesting that the concentration of the factors has functional significance. Injected embryos also showed ectopic expression of sele, gpr182, and lgmn, indicating that the three-factor pool induced a niche endothelial program (see, e.g., Figures 11A-C).

[0201] To assess the contribution of individual transcription factors, we injected each factor alone and performed WISH for mrc1a. Embryos injected with SOX7 or Nr2f2 alone showed the least ectopic expression (see, e.g., Figure 4F). Injection of ETV2 alone resulted in ectopic expression of mrc1a, but at a lower frequency than when ETV2 was injected in combination with SOX7 and Nr2f2, indicating that additional factors enhance ectopic induction (see, e.g., Figure 4F, Figures S12-S12D). Injection of ETV2 alone induced ectopic expression of the endogenous zebrafish sox7 gene as well as sox18, fli1a, and etv2, demonstrating that human ETV2 can induce various zebrafish endothelial gene programs, including arterial, venous, and niche endothelial genes (see, e.g., Figures S12-S12D). Because endogenous ETS1 and Nr2f2 are widely expressed outside the CHT (e.g., in the spinal cord region), it was difficult to visualize whether they were similarly induced by ETV2 overexpression (see, e.g., Figures 12-12D). ETS1 has the ability to bind to the consensus ETS motif enriched in the CHT and is expressed at appropriate levels in niche ECs. In contrast to ETV2, overexpression of ETS1 alone did not result in widespread vascular induction or ectopic mrc1a expression (see, e.g., Figure 4F). However, injection of ETS1, SOX7, and Nr2f2 resulted in significant ectopic expression of mrc1a, indicating the combinatorial activity of these transcription factors (see, e.g., Figures 4E, 4F, 12-12D). Notably, the zebrafish genes encoding each of the seven early transcription factors had regions of chromatin that bound them that were uniquely accessible in the CHT EC fraction, and contained Ets, SoxF, and NHR sites (see, e.g., Table 5), indicating that these factors regulate each other as well as the autoregulatory loops established during reprogramming.Thus, overexpression of a combination of three factors, including either ETV2 or ETS1 with Sox and NHR factors, mimics the endogenous expression of these factors in niche ECs at 72 hpf and induces robust specialization of the niche endothelial gene program.

[0202] Ectopic vascular territories recruit HSPCs Next, we tested whether ectopic CHT EC gene expression regions could recruit and support HSPCs. We injected ETV2, SOX7, and Nr2f2 pools into mrc1a:GFP / runx1:mCherry double-positive transgenic embryos and compared them with runx1:mCherry. + The localization of HSPCs was examined. Of note, in 12 of 22 embryos that had ectopic vascular compartments of mrc1a:GFP, HSPCs were observed to localize to these regions (see, for example, Figure 5A). In contrast, 0 of 27 control embryos had ectopically localized HSPCs. + Similar ectopic localization of HSPCs was observed by runx1 WISH and in embryos injected with a combination of ETS1, SOX7, and Nr2f2 (see, eg, Figures 5A, 5B).

[0203] To assess whether these ectopic regions provide a supportive environment for HSPCs, we used high-resolution live-cell confocal microscopy to examine in more detail the behavior of HSPCs and their interactions with ECs at these sites. This analysis demonstrated that HSPCs express mrc1a:GFP in ectopic regions. + We found that ectopic mrc1a:GFP directly adheres to ECs and can be found in both the intraluminal and extravascular spaces. + ECs often express cxcl12a:DsRed2, similar to those observed in CHT. +They attached to stromal cells and formed pockets surrounding HSPCs (see, for example, Figures 5A and 5C). Using time-lapse microscopy, it is noteworthy that the initial recruitment, residence, and division of HSPCs was observed at these sites (see, for example, Figure 5D). As HSPCs divided, daughter cells migrated out of the ectopic site and entered the circulation, presumably to migrate into the niche (see, for example, Figure 5E). Together, these data demonstrate that reprogramming with three factors that induce a niche endothelial gene program generates a functional ectopic niche capable of recruiting and maintaining HSPCs.

[0204] A conserved endothelial signature in the HSPC niche To determine whether a similar endothelial expression signature exists in the adult zebrafish niche, we tested the mrc1a:GFP and sele:GFP transgenes and found that both strains had GFP expression in ECs in the renal medullary niche (see, e.g., Figures 13A and 13B). Additionally, we examined single-cell RNA-seq datasets from zebrafish renal medullary niches (see, e.g., Tang et al. Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing. The Journal of Experimental Medicine 214, 2875-2887, 2017). This analysis revealed that 23 of 29 CHT EC genes were highly enriched in vascular ECs in the renal medullary niche (see, e.g., Figure 6A). To determine whether this pro-hematopoietic vascular niche signature is conserved in mammals, we investigated an RNA-seq dataset containing gene expression data for ECs from multiple mouse organs, including heart, kidney, liver, lung, and bone marrow, at five developmental stages (E11-13, E14-15, E16-17, P2-P4, and adult). Orthologs of 21 of the 29 CHT EC genes were enriched in ECs from mammalian hematopoietic organs—fetal liver and / or adult bone marrow—compared to their expression in ECs from non-hematopoietic organs at the same developmental stage (see Figure 6B). Notably, some of the expression patterns reflected the temporal dynamics of HSPC ontogeny, showing robust expression in fetal liver ECs at E14-17 stages, followed by increased expression in adult bone marrow ECs and a concomitant and expected decrease in liver EC expression (P2-P4 and adult; see Figure 6B, for example). To determine whether a transcriptional program similar to that evident in zebrafish controls these genes in mouse, we examined the expression of transcription factors from the Ets, Sox, and NHR families using the same RNA-seq dataset.In both fetal liver ECs (E14-17) and adult bone marrow ECs, the most highly expressed factors were Ets1, Sox18, and Nr2f2 (see, e.g., Table 5). Together, these data indicate that conserved transcriptional programs regulate the prohematopoietic niche properties of ECs in the fetal and adult HSPC niches.

[0205] Consideration These data support a model in which a transcriptional program composed of Ets, SoxF, and NHR family factors determines the properties and intrinsic ability of vascular niche ECs to maintain and expand blood stem cells. This is a conserved feature of the hematopoietic niche at multiple developmental stages and across species. The niche endothelial expression signature identified herein includes genes with known niche functions (e.g., the adhesion receptor E-selectin and the cysteine ​​proteinase cathepsin L, which have been linked to Cxcl12 turnover in the bone marrow). In addition, there are numerous genes not previously associated with the HSPC niche, including various genes with scavenger functions or activities related to endocytosis and endocytosis, including mrc1a, stab1 / 2, dab2, ap1b1, pxk, and snx8. These molecules may regulate ligand and receptor turnover in the niche or function to remove potentially harmful substances from the niche microenvironment, such as waste products, modified proteins, or virus-, bacterial-, or fungal-related substances. Consistent with their common origin, CHT ECs share gene expression with lymphoid ECs, including genes such as lyve1b. A recent study of mouse bone marrow examined differential gene expression between SECs and AECs. The niche EC signature disclosed herein is consistent with bone marrow venous SECs. While AECs can also support hematopoiesis, the studies herein demonstrate that SECs are sufficient to induce vascular niche formation and promote stem cell expansion. Extramedullary hematopoiesis, which occurs during stress, may involve the localized induction of this SEC niche program. Other transcription factors have been shown to induce the expression of CHT genes that maintain hematopoietic cells, including tfec and klf6a, but enriched binding motifs were not observed for these factors. It is possible that their targets are distinct in different cells of the CHT, or that these factors act downstream of our program, which determines the diverse population of tissue-specific vascular niches.

[0206] Experiments using parabiotic mice have shown that niche size determines the number of HSPCs (see, e.g., Chen et al. Mobilization as a preparative regimen for hematopoietic stem cell transplantation. Blood 107, 3764–3771, 2006). Functional ectopic niches, called ossicles, are used to establish bone marrow equivalents when transplanted into mice, and SECs are believed to reside within these structures. Collectively, these studies and the examples presented herein support a method for increasing HSPC numbers in vivo by generating ectopic vascular niches in novel safe havens within the body. This approach provides a basis for novel treatments for diseases in which the endogenous bone marrow niche is impaired (e.g., myelofibrosis). Broadly, this example advances our fundamental understanding of the vascular niche responsible for blood stem cell homeostasis and regeneration and may guide novel therapeutic strategies for culturing and expanding HSPCs for transplantation.

[0207] method Animal models In this study, we used wild-type AB, casper, or casper-EKK, as well as the transgenic lines cd41:EGFP, runx1:mCherry [runx1+23:NLS-mCherry], kdr1(flk1):GFP [kdr1:GRCFP], kdr1:mCherry [kdr1:Hsa.hras-mCherry], and cxcl12a(sdf1a):DsRed2. Alternative gene names are shown in parentheses, and the full name of the transgene is shown in square brackets.

[0208] Genome analysis For RNA tomography (tomo-seq), 72 hpf embryos were euthanized with tricaine overdose, and the portion of the tail containing the CHT was manually excised using a scalpel. The tissue was oriented in a cryomold in optimal cutting temperature (OCT) tissue freezing medium, with its ventral side facing the bottom of the mold. After rapid freezing on dry ice, 40 individual 8-μm-thick cryosections were obtained along the dorsal-ventral axis using a cryostat. RNA was extracted from the individual cryosections using TRIzol™, barcoded during the reverse transcription step, and then pooled for library preparation and sequencing (see, e.g., Junker et al. Genome-wide RNA Tomography in the zebrafish embryo. Cell 159, 662-675, 2014). For single-cell and bulk RNA-seq, kdr1:GFP embryos were dissected using Liberase™ and GFP was isolated by FACS. +Cells were isolated. For bulk RNA-seq, total RNA was isolated using TRIzol™ and GenElute LPA™ carriers. Libraries were prepared using Ribogone™ and the SMARTer Universal Low Input RNA Kit™ from 50 ng of total RNA / sample as input. For single-cell sequencing, approximately 2,000 cells were encapsulated and libraries were prepared for sequencing. For ATAC-seq, embryos were dissected using Liberase™, and a minimum of 12,000 cells (maximum 50,000) were isolated by FACS. Cells were then lysed, and isolated nuclei were incubated in a transposition reaction. All sequencing was performed using an Illumina Hiseq 2500™. For RNA-seq, quality control was performed using Fast QC™ and Cutadapt™ to remove adapter sequences and low-quality regions. High-quality reads were aligned to UCSC build danRer7 of the zebrafish genome using Tophat™ 2.0.1158 without de novo splicing from the calls. Transcript abundance and differential expression were calculated using Cufflinks™ 2.2.159. FPKM values ​​were used to normalize and quantify each transcript. For ATAC-seq, reads were aligned to UCSC build danRer7 of the zebrafish genome using Bowtie2™ (version 2.2.1) with the following parameters: --end-to-end, -N0-, -L20. Regions of ATAC-seq peaks were identified using the MACS2™ (version 2.1.0) peak-finding algorithm with the following parameters: --nomodel --shift -100 --extsize 200. For peak calling, an initial q-value threshold of 0.05 for enrichment was used, and a more stringent q-value of 14 was used to identify peaks that differed between different samples. Genome-wide motif enrichment analysis was performed using HOMER™, and motif annotation was performed using Consite™.Genomic expression analysis of adult renal medulla was performed using data available on the World Wide Web at molpath.shinyapps.io / zebrafishblood / .

[0209] Whole-mount in situ hybridization (WISH) In situ hybridization was performed using standard protocols. Embryos were then transferred to glycerol for scoring and imaging. In situ probes were generated by PCR amplification using cDNA or plasmid (for transcription factors from other species) templates followed by reverse transcription using digoxigenin-linked nucleotides. Primer sequences for all WISH probes used herein are provided in Table 7. WISH images of the 35 CHT-enriched genes identified by tomo-seq can be found on the World Wide Web at zfin.org.

[0210] Gene transfer and enhancer-GFP reporter assay Transgenic lines were established. For the mrc1a:GFP and sele:GFP transgenes, 1.3 kb and 5.3 kb of sequence upstream of the transcription start site, respectively, were PCR amplified from genomic DNA and cloned into the p5E Gateway™ vector with TOPO-TA™, all combined with GFP and a polyA tail, flanked by Tol2 sites. For the 125 bp mrc1a and 158 bp sele enhancers, these elements were PCR amplified from genomic DNA and cloned into the p5E Gateway™ vector with TOPO-TA™, all combined with the mouse beta-globin minimal promoter fused to GFP with a polyA tail, flanked by Tol2 sites. Tol2 RNA was injected into single-cell stage embryos, and at least two independent lines showing similar expression were established for each construct (Tg(mrc1a(1.3 kb):GFP); Tg(sele(5.3 kb):GFP); Tg(mrc1a(125 bp):GFP); and Tg(sele(158 bp):GFP). The CHT EC and EC consensus ATAC-seq elements were similarly amplified by PCR using genomic DNA and then fused to the beta-globin minimal promoter and GFP. Mutant variants of the 125 bp mrc1a and 158 bp sele were isolated by annealing overlapping oligos followed by T4 DNA polymerase reaction to generate blunt-ended products, which were then (after A-tailing with Klenow fragment using the same workflow as for the ATAC-seq elements) transfected with p5E The gene was generated by cloning into the Gateway™ vector. The transcription factor binding motif was disrupted by exchanging the purine nucleotide in the central binding site for a pyrimidine or vice versa. Injected F0 embryos were scored between 60 and 72 hpf. Control and experimental groups were blinded before scoring, and all experiments were performed at least three times by independent personnel. GFP expression in CHT ECs or EC common expression was scored as significant if observed in at least 10% of F0-injected embryos.Embryos scored negative either had no GFP expression or only small amounts of ectopic expression in muscle cells. The sequences of the primers used to amplify the mrc1a and sele regulatory elements and the 15 CHT EC and 6 EC consensus ATAC-seq elements are provided in Table 7. The sequences of the overlapping oligos used to generate the enhancer variants are provided in Table 8. The fidelity of all constructs was confirmed by sequencing prior to injection.

[0211] Transcription factor overexpression studies For transcription factor overexpression studies, open reading frames of human (FLI1, ETV2, ETS1, SOX7, and RXRA), Xenopus (Sox18), or zebrafish (Nr2f2) genes were cloned into the pME Gateway vector (Invitrogen™) and then combined with the zebrafish ubi promoter and polyA tail, all flanked by Tol2 sites. The fidelity of all constructs was confirmed by sequencing prior to injection. Single-cell stage embryos were injected with a pool of ubi-induced transcription factors (1 nl at 25 ng / µl total DNA and Tol2 RNA) and then screened for ectopic niche endothelial gene expression or ectopic HSPC localization between 24 and 72 hpf. For control and single-factor injections, empty Tol2 Gateway™ transport vectors were used as filler DNA in the injection mixture. Expression of transcription factors was confirmed by WISH using species-specific in situ probes. Ectopic expression was scored as vascular staining or vascular GFP expression outside the normal gene expression domain. Control and experimental samples were blinded before scoring, and all experiments were performed at least three times by independent personnel.

[0212] Microscopy and image analysis Time-lapse microscopy was performed using a Yokogawa CSU-X1™ spinning disk mounted on an inverted Nikon Eclipse Ti™ microscope equipped with an Andor iXon™ EMCCD dual camera and a temperature-controlled (maintained at 28.5°C) motorized xy stage to facilitate simultaneous tiling and imaging of multiple specimens. Screening of injected enhancer-GFP constructs and imaging of WISH embryos were performed using a Nikon SMZ18™ stereomicroscope equipped with a Nikon DS-Ri2™ camera. All images were acquired using NIS-Elements™ and processed using Imaris™ or Adobe Photoshop™ software. Embryos were mounted for imaging. Briefly, specimens were mounted in 0.8% LMP agarose containing tricaine (0.16 mg / ml) in glass-bottom 6-well plates and covered with E3 medium containing tricaine (0.16 mg / ml).

[0213] Flow cytometry, renal medulla extraction, dissection, and histology Embryos were prepared for FACS. Briefly, embryos were minced using a razor blade in cold PBS and then incubated in Liberase™ for 20 minutes at 37°C. Disaggregated cells were then filtered through a 40-μm mesh filter and transferred to 2% FBS. FACS was performed using a FACS Aria™ instrument. Transgene-positive and -negative control embryos were used to set gates, and SYTOX Blue™ was used as a live / dead stain. At least 12,000 cells (maximum 50,000) were collected per sample for ATAC-seq experiments, and at least 10,000 cells (maximum 300,000) were collected per sample for RNA-seq experiments. Renal medulla were collected from adult zebrafish by manual dissection and then fixed in 4% PFA (for histology) or disaggregated by gentle pipetting (for live-cell imaging). For histology, renal marrow was paraffin-embedded before sectioning, and sections were stained alternately with H&E or antibodies against GFP. - Pdpn- Cd31 + The cells were stored.

[0214] Electrophoretic mobility shift assay The Nr2f2 fragment was cloned into the pGEX2TK™ vector to generate GST-tagged Nr2f2, and fidelity was verified by sequencing. The pGEX2TK-Nr2f2 protein plasmid was transformed into E. coli BL21 competent cells. The protein was expressed and purified, and the purified protein was quantified against BSA. EMSA was performed. Probes were generated by annealing 100 pmol of sense and antisense oligonucleotides, with 1–2 pmol of probe used in each reaction. All primer and probe sequences are provided in Table 9. Gel shift reactions were performed at 4°C in 20% glycerol, 20 mM Tris (pH 8.0), 10 mM KCl, 1 mM DTT, 12.5 ng poly(dI / C), 6.25 pmol of random single-stranded oligonucleotides, BSA, and the above amounts of probe. Samples containing Nr2f2 protein were run on a 6% gel to resolve protein-DNA complexes. For reactions with cold competitors, 20x unlabeled probe was included in the reaction. To obtain supershifts, anti-NR2F2 antibody (R&D Biosystems™; Cat. No. PP-H7147-00) was used in an amount equal to the Nr2f2 protein.

[0215] The GEO accession number for the mammalian genome data reported herein is GSE100910. The zebrafish genome data reported herein have been submitted to the NCBI™ Gene Expression Omnibus.

[0216] Real-time imaging can demonstrate blood circulation through regions of ectopic niche endothelial gene expression. At 72 hpf, blood cells can be observed circulating through regions of blood vessels in the dorsal trunk that ectopically express mrc1a:GFP in 72 hpf embryos injected with a pool of seven transcription factors at the single-cell stage.

[0217] Real-time imaging can demonstrate the early recruitment of HSPCs to areas of ectopic niche endothelial expression. runx1 + HSPCs can be observed initially residing in dorsal blood vessels (see, for example, the arrow in Figure 5D) ectopically expressing mrc1a:GFP in 72 hpf embryos injected with a pool of ETV2, SOX7, and Nr2f2 at the single-cell stage. The black arrowheads indicate the localization of HSPCs to the CHT. The elapsed time is 6.5 hours, with 2-minute time intervals. A time series is shown in Figure 5D.

[0218] Real-time imaging can demonstrate HSPC proliferation and escape from the ectopic niche endothelial gene expression region. + HSPCs can be localized to blood vessels (e.g., see arrows in Figure 5E) that ectopically express mrc1a:GFP in 72 hpf embryos injected with a pool of ETV2, SOX7, and Nr2f2 at the single-cell stage. HSPCs divide several times and migrate into the circulation. Black arrowheads indicate HSPC localization to the CHT. The elapsed time is 2.6 hours, with a time interval of 2 minutes. A time series is shown in Figure 5E.

[0219] (Table 1) CHT EC enriched genes TIFF0007683068000010.tif200163

[0220] Table 2. In vivo screening of predicted enhancer elements TIFF0007683068000011.tif74163

[0221] Table 1 shows the CHT EC genes identified by tomo-seq and tissue-specific RNA-seq. An asterisk (*) indicates that the gene is located within 100 kb of the TSS, and some genes are associated with multiple elements. TIFF0007683068000012.tif4128 shows that no expression was observed by WISH. TIFF0007683068000013.tif4128 indicates that WISH was not attempted, but CHT expression was reported on the World Wide Web available at zfin.org. A double S (§) indicates that WISH was not attempted.

[0222] Table 2 shows the CHT EC-specific and EC-shared ATAC-seq elements fused to minimal promoters and GFP and injected into single-cell stage zebrafish embryos. Asterisks (*) indicate the coordinates of MACS2 peaks. TIFF0007683068000014.tif4128 shows expression within CHT ECs for CHT EC elements and within blood vessels throughout the embryo for EC common elements. TIFF0007683068000015.tif4128 shows the absence of an NHR motif.

[0223] Table 3. CHT-enriched genes identified by tomo-seq TIFF0007683068000016.tif216140TIFF0007683068000017.tif224125TIFF0007683068000018.tif84142

[0224] An asterisk (*) in Table 3 indicates that CHT expression was not observed by WISH. TIFF0007683068000019.tif5128 indicates that WISH was not attempted.

[0225] Table 4. Expression of transcription factors in CHT ECs TIFF0007683068000020.tif34165

[0226] Table 4 shows FPHM expression values ​​in the CHT ECS for highly expressed members of the Ets, Sox, and NHR transcription factor families. Asterisks (*) in Table 4 indicate regions within 100 kb of the TSS, with some genes associated with multiple elements.

[0227] Table 5. Expression of transcription factors in the mouse hematopoietic niche TIFF0007683068000021.tif146165

[0228] Table 6: Primers used for synthesis of WISH probes (all in order of appearance, respectively: "forward" primers disclosed as SEQ ID NOs: 16-84, "reverse" primers disclosed as SEQ ID NOs: 85-153) TIFF0007683068000022.tif215130TIFF0007683068000023.tif138142

[0229] The asterisk (*) in Table 6 indicates the T3 sequence. The dagger symbol in Table 6 indicates that TIFF0007683068000024.tif4128 was added to the 5' end of each forward primer. TIFF0007683068000025.tif5128 is the T7 sequence TIFF0007683068000026.tif4128 was added to the 5' end of each reverse primer.

[0230] Table 7: Primers used to clone promoter and enhancer elements ("forward" primers disclosed as SEQ ID NOs: 154-178, "reverse" primers disclosed as SEQ ID NOs: 179-203, all in order of appearance, respectively). TIFF0007683068000027.tif153165

[0231] Table 8: Sequences and primers for mutational variants of the 125 bp mrc1a and 158 bp sele enhancer elements ("Full fragment sequence" disclosed as SEQ ID NOs: 204-208, 13, and 209-212, "Forward primer" disclosed as SEQ ID NOs: 213-222, "Reverse primer" disclosed as SEQ ID NOs: 223-232, all in order of appearance, respectively). TIFF0007683068000028.tif195165

[0232] In Table 8, lowercase letters indicate the base pair changes used to disrupt the transcription factor binding motif.

[0233] Table 9: Primers used for cloning and EMSA probe synthesis (all in order of appearance, respectively: "forward" primers disclosed as SEQ ID NOs: 233-239, "reverse" primers disclosed as SEQ ID NOs: 240-246) TIFF0007683068000029.tif62165

[0234] Table 9 shows the primers used to clone mouse Nr2f2 into the pGEX2TK vector and DNA probes derived from the zebrafish mrc1a and sele enhancers.

[0235] Sequence information SEQUENCE LISTING <110> THE CHILDREN'S MEDICAL CENTER CORPORATION <120> ENDOTHERIAL CELL FACTORS AND METHODS THEREOF <150> US 62 / 647,433 <151> 2018-03-23 <160> 246 <170> PatentIn version 3.5 <210> 1 <211> 342 <212> PRT <213> Homo sapiens <400> 1 Met Asp Leu Trp Asn Trp Asp Glu Ala Ser Pro Gln Glu Val Pro Pro 1 5 10 15 Gly Asn Lys Leu Ala Gly Leu Glu Gly Ala Lys Leu Gly Phe Cys Phe 20 25 30 Pro Asp Leu Ala Leu Gln Gly Asp Thr Pro Thr Ala Thr Ala Glu Thr 35 40 45 Cys Trp Lys Gly Thr Ser Ser Ser Leu Ala Ser Phe Pro Gln Leu Asp 50 55 60 Trp Gly Ser Ala Leu Leu His Pro Glu Val Pro Trp Gly Ala Glu Pro 65 70 75 80 Asp Ser Gln Ala Leu Pro Trp Ser Gly Asp Trp Thr Asp Met Ala Cys 85 90 95 Thr Ala Trp Asp Ser Trp Ser Gly Ala Ser Gln Thr Leu Gly Pro Ala 100 105 110 Pro Leu Gly Pro Gly Pro Ile Pro Ala Ala Gly Ser Glu Gly Ala Ala 115 120 125 Gly Gln Asn Cys Val Pro Val Ala Gly Glu Ala Thr Ser Trp Ser Arg 130 135 140 Ala Gln Ala Ala Gly Ser Asn Thr Ser Trp Asp Cys Ser Val Gly Pro 145 150 155 160 Asp Gly Asp Thr Tyr Trp Gly Ser Gly Leu Gly Gly Glu Pro Arg Thr 165 170 175 Asp Cys Thr Ile Ser Trp Gly Gly Pro Ala Gly Pro Asp Cys Thr Thr 180 185 190 Ser Trp Asn Pro Gly Leu His Ala Gly Gly Thr Thr Ser Leu Lys Arg 195 200 205 Tyr Gln Ser Ser Ala Leu Thr Val Cys Ser Glu Pro Ser Pro Gln Ser 210 215 220 Asp Arg Ala Ser Leu Ala Arg Cys Pro Lys Thr Asn His Arg Gly Pro 225 230 235 240 Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu Leu His Asp Gly Ala Arg 245 250 255 Ser Ser Cys Ile Arg Trp Thr Gly Asn Ser Arg Glu Phe Gln Leu Cys 260 265 270 Asp Pro Lys Glu Val Ala Arg Leu Trp Gly Glu Arg Lys Arg Lys Pro 275 280 285 Gly Met Asn Tyr Glu Lys Leu Ser Arg Gly Leu Arg Tyr Tyr Tyr Arg 290 295 300 Arg Asp Ile Val Arg Lys Ser Gly Gly Arg Lys Tyr Thr Tyr Arg Phe 305 310 315 320 Gly Gly Arg Val Pro Ser Leu Ala Tyr Pro Asp Cys Ala Gly Gly Gly 325 330 335 Arg Gly Ala Glu Thr Gln 340 <210> 2 <211> 452 <212> PRT <213> Homo sapiens <400> 2 Met Asp Gly Thr Ile Lys Glu Ala Leu Ser Val Val Ser Asp Asp Gln 1 5 10 15 Ser Leu Phe Asp Ser Ala Tyr Gly Ala Ala Ala His Leu Pro Lys Ala 20 25 30 Asp Met Thr Ala Ser Gly Ser Pro Asp Tyr Gly Gln Pro His Lys Ile 35 40 45 Asn Pro Leu Pro Pro Gln Gln Glu Trp Ile Asn Gln Pro Val Arg Val 50 55 60 Asn Val Lys Arg Glu Tyr Asp His Met Asn Gly Ser Arg Glu Ser Pro 65 70 75 80 Val Asp Cys Ser Val Ser Lys Cys Ser Lys Leu Val Gly Gly Gly Glu 85 90 95 Ser Asn Pro Met Asn Tyr Asn Ser Tyr Met Asp Glu Lys Asn Gly Pro 100 105 110 Pro Pro Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro Ala 115 120 125 Asp Pro Thr Leu Trp Thr Gln Glu His Val Arg Gln Trp Leu Glu Trp 130 135 140 Ala Ile Lys Glu Tyr Ser Leu Met Glu Ile Asp Thr Ser Phe Phe Gln 145 150 155 160 Asn Met Asp Gly Lys Glu Leu Cys Lys Met Asn Lys Glu Asp Phe Leu 165 170 175 Arg Ala Thr Thr Leu Tyr Asn Thr Glu Val Leu Leu Ser His Leu Ser 180 185 190 Tyr Leu Arg Glu Ser Ser Leu Leu Ala Tyr Asn Thr Thr Ser His Thr 195 200 205 Asp Gln Ser Ser Arg Leu Ser Val Lys Glu Asp Pro Ser Tyr Asp Ser 210 215 220 Val Arg Arg Gly Ala Trp Gly Asn Asn Met Asn Ser Gly Leu Asn Lys 225 230 235 240 Ser Pro Pro Leu Gly Gly Ala Gln Thr Ile Ser Lys Asn Thr Glu Gln 245 250 255 Arg Pro Gln Pro Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg 260 265 270 Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu 275 280 285 Glu Leu Leu Ser Asp Ser Ala Asn Ala Ser Cys Ile Thr Trp Glu Gly 290 295 300 Thr Asn Gly Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg Arg 305 310 315 320 Trp Gly Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys Leu Ser 325 330 335 Arg Ala Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His 340 345 350 Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala Gln Ala 355 360 365 Leu Gln Pro His Pro Thr Glu Ser Ser Met Tyr Lys Tyr Pro Ser Asp 370 375 380 Ile Ser Tyr Met Pro Ser Tyr His Ala His Gln Gln Lys Val Asn Phe 385 390 395 400 Val Pro Pro His Pro Ser Ser Met Pro Val Thr Ser Ser Ser Phe Phe 405 410 415 Gly Ala Ala Ser Gln Tyr Trp Thr Ser Pro Thr Gly Gly Ile Tyr Pro 420 425 430 Asn Pro Asn Val Pro Arg His Pro Asn Thr His Val Pro Ser His Leu 435 440 445 Gly Ser Tyr Tyr 450 <210> 3 <211> 441 <212> PRT <213> Homo sapiens <400> 3 Met Lys Ala Ala Val Asp Leu Lys Pro Thr Leu Thr Ile Ile Lys Thr 1 5 10 15 Glu Lys Val Asp Leu Glu Leu Phe Pro Ser Pro Asp Met Glu Cys Ala 20 25 30 Asp Val Pro Leu Leu Thr Pro Ser Ser Lys Glu Met Met Ser Gln Ala 35 40 45 Leu Lys Ala Thr Phe Ser Gly Phe Thr Lys Glu Gln Gln Arg Leu Gly 50 55 60 Ile Pro Lys Asp Pro Arg Gln Trp Thr Glu Thr His Val Arg Asp Trp 65 70 75 80 Val Met Trp Ala Val Asn Glu Phe Ser Leu Lys Gly Val Asp Phe Gln 85 90 95 Lys Phe Cys Met Asn Gly Ala Ala Leu Cys Ala Leu Gly Lys Asp Cys 100 105 110 Phe Leu Glu Leu Ala Pro Asp Phe Val Gly Asp Ile Leu Trp Glu His 115 120 125 Leu Glu Ile Leu Gln Lys Glu Asp Val Lys Pro Tyr Gln Val Asn Gly 130 135 140 Val Asn Pro Ala Tyr Pro Glu Ser Arg Tyr Thr Ser Asp Tyr Phe Ile 145 150 155 160 Ser Tyr Gly Ile Glu His Ala Gln Cys Val Pro Pro Ser Glu Phe Ser 165 170 175 Glu Pro Ser Phe Ile Thr Glu Ser Tyr Gln Thr Leu His Pro Ile Ser 180 185 190 Ser Glu Glu Leu Leu Ser Leu Lys Tyr Glu Asn Asp Tyr Pro Ser Val 195 200 205 Ile Leu Arg Asp Pro Leu Gln Thr Asp Thr Leu Gln Asn Asp Tyr Phe 210 215 220 Ala Ile Lys Gln Glu Val Val Thr Pro Asp Asn Met Cys Met Gly Arg 225 230 235 240 Thr Ser Arg Gly Lys Leu Gly Gly Gln Asp Ser Phe Glu Ser Ile Glu 245 250 255 Ser Tyr Asp Ser Cys Asp Arg Leu Thr Gln Ser Trp Ser Ser Gln Ser 260 265 270 Ser Phe Asn Ser Leu Gln Arg Val Pro Ser Tyr Asp Ser Phe Asp Ser 275 280 285 Glu Asp Tyr Pro Ala Ala Leu Pro Asn His Lys Pro Lys Gly Thr Phe 290 295 300 Lys Asp Tyr Val Arg Asp Arg Ala Asp Leu Asn Lys Asp Lys Pro Val 305 310 315 320 Ile Pro Ala Ala Ala Leu Ala Gly Tyr Thr Gly Ser Gly Pro Ile Gln 325 330 335 Leu Arg Gln Phe Leu Leu Glu Leu Leu Thr Asp Lys Ser Cys Gln Ser 340 345 350 Phe Ile Ser Trp Thr Gly Asp Gly Trp Glu Phe Lys Leu Ser Asp Pro 355 360 365 Asp Glu Val Ala Arg Arg Trp Gly Lys Arg Lys Asn Lys Pro Lys Met 370 375 380 Asn Tyr Glu Lys Leu Ser Arg Gly Leu Arg Tyr Tyr Tyr Asp Lys Asn 385 390 395 400 Ile Ile His Lys Thr Ala Gly Lys Arg Tyr Val Tyr Arg Phe Val Cys 405 410 415 Asp Leu Gln Ser Leu Leu Gly Tyr Thr Pro Glu Glu Leu His Ala Met 420 425 430 Leu Asp Val Lys Pro Asp Ala Asp Glu 435 440 <210> 4 <211> 384 <212> PRT <213> Homo sapiens <400> 4 Met Gln Arg Ser Pro Pro Gly Tyr Gly Ala Gln Asp Asp Pro Pro Ala 1 5 10 15 Arg Arg Asp Cys Ala Trp Ala Pro Gly His Gly Ala Ala Ala Asp Thr 20 25 30 Arg Gly Leu Ala Ala Gly Pro Ala Ala Leu Ala Ala Pro Ala Ala Pro 35 40 45 Ala Ser Pro Pro Ser Pro Gln Arg Ser Pro Pro Arg Ser Pro Glu Pro 50 55 60 Gly Arg Tyr Gly Leu Ser Pro Ala Gly Arg Gly Glu Arg Gln Ala Ala 65 70 75 80 Asp Glu Ser Arg Ile Arg Arg Pro Met Asn Ala Phe Met Val Trp Ala 85 90 95 Lys Asp Glu Arg Lys Arg Leu Ala Gln Gln Asn Pro Asp Leu His Asn 100 105 110 Ala Val Leu Ser Lys Met Leu Gly Lys Ala Trp Lys Glu Leu Asn Ala 115 120 125 Ala Glu Lys Arg Pro Phe Val Glu Glu Ala Glu Arg Leu Arg Val Gln 130 135 140 His Leu Arg Asp His Pro Asn Tyr Lys Tyr Arg Pro Arg Arg Lys Lys 145 150 155 160 Gln Ala Arg Lys Ala Arg Arg Leu Glu Pro Gly Leu Leu Leu Pro Gly 165 170 175 Leu Ala Pro Pro Gln Pro Pro Pro Glu Pro Phe Pro Ala Ala Ser Gly 180 185 190 Ser Ala Arg Ala Phe Arg Glu Leu Pro Pro Leu Gly Ala Glu Phe Asp 195 200 205 Gly Leu Gly Leu Pro Thr Pro Glu Arg Ser Pro Leu Asp Gly Leu Glu 210 215 220 Pro Gly Glu Ala Ala Phe Phe Pro Pro Pro Ala Ala Pro Glu Asp Cys 225 230 235 240 Ala Leu Arg Pro Phe Arg Ala Pro Tyr Ala Pro Thr Glu Leu Ser Arg 245 250 255 Asp Pro Gly Gly Cys Tyr Gly Ala Pro Leu Ala Glu Ala Leu Arg Thr 260 265 270 Ala Pro Pro Ala Ala Pro Leu Ala Gly Leu Tyr Tyr Gly Thr Leu Gly 275 280 285 Thr Pro Gly Pro Tyr Pro Gly Pro Leu Ser Pro Pro Pro Glu Ala Pro 290 295 300 Pro Leu Glu Ser Ala Glu Pro Leu Gly Pro Ala Ala Asp Leu Trp Ala 305 310 315 320 Asp Val Asp Leu Thr Glu Phe Asp Gln Tyr Leu Asn Cys Ser Arg Thr 325 330 335 Arg Pro Asp Ala Pro Gly Leu Pro Tyr His Val Ala Leu Ala Lys Leu 340 345 350 Gly Pro Arg Ala Met Ser Cys Pro Glu Glu Ser Ser Leu Ile Ser Ala 355 360 365 Leu Ser Asp Ala Ser Ser Ala Val Tyr Tyr Ser Ala Cys Ile Ser Gly 370 375 380 <210> 5 <211> 388 <212> PRT <213> Homo sapiens <400> 5 Met Ala Ser Leu Leu Gly Ala Tyr Pro Trp Pro Glu Gly Leu Glu Cys 1 5 10 15 Pro Ala Leu Asp Ala Glu Leu Ser Asp Gly Gln Ser Pro Pro Ala Val 20 25 30 Pro Arg Pro Pro Gly Asp Lys Gly Ser Glu Ser Arg Ile Arg Arg Pro 35 40 45 Met Asn Ala Phe Met Val Trp Ala Lys Asp Glu Arg Lys Arg Leu Ala 50 55 60 Val Gln Asn Pro Asp Leu His Asn Ala Glu Leu Ser Lys Met Leu Gly 65 70 75 80 Lys Ser Trp Lys Ala Leu Thr Leu Ser Gln Lys Arg Pro Tyr Val Asp 85 90 95 Glu Ala Glu Arg Leu Arg Leu Gln His Met Gln Asp Tyr Pro Asn Tyr 100 105 110 Lys Tyr Arg Pro Arg Arg Lys Lys Gln Ala Lys Arg Leu Cys Lys Arg 115 120 125 Val Asp Pro Gly Phe Leu Leu Ser Ser Leu Ser Arg Asp Gln Asn Ala 130 135 140 Leu Pro Glu Lys Arg Ser Gly Ser Arg Gly Ala Leu Gly Glu Lys Glu 145 150 155 160 Asp Arg Gly Glu Tyr Ser Pro Gly Thr Ala Leu Pro Ser Leu Arg Gly 165 170 175 Cys Tyr His Glu Gly Pro Ala Gly Gly Gly Gly Gly Gly Thr Pro Ser 180 185 190 Ser Val Asp Thr Tyr Pro Tyr Gly Leu Pro Thr Pro Pro Glu Met Ser 195 200 205 Pro Leu Asp Val Leu Glu Pro Glu Gln Thr Phe Phe Ser Ser Pro Cys 210 215 220 Gln Glu Glu His Gly His Pro Arg Arg Ile Pro His Leu Pro Gly His 225 230 235 240 Pro Tyr Ser Pro Glu Tyr Ala Pro Ser Pro Leu His Cys Ser His Pro 245 250 255 Leu Gly Ser Leu Ala Leu Gly Gln Ser Pro Gly Val Ser Met Met Ser 260 265 270 Pro Val Pro Gly Cys Pro Pro Ser Pro Ala Tyr Tyr Ser Pro Ala Thr 275 280 285 Tyr His Pro Leu His Ser Asn Leu Gln Ala His Leu Gly Gln Leu Ser 290 295 300 Pro Pro Pro Glu His Pro Gly Phe Asp Ala Leu Asp Gln Leu Ser Gln 305 310 315 320 Val Glu Leu Leu Gly Asp Met Asp Arg Asn Glu Phe Asp Gln Tyr Leu 325 330 335 Asn Thr Pro Gly His Pro Asp Ser Ala Thr Gly Ala Met Ala Leu Ser 340 345 350 Gly His Val Pro Val Ser Gln Val Thr Pro Thr Gly Pro Thr Glu Thr 355 360 365 Ser Leu Ile Ser Val Leu Ala Asp Ala Thr Ala Thr Tyr Tyr Asn Ser 370 375 380 Tyr Ser Val Ser 385 <210> 6 <211> 462 <212> PRT <213> Homo sapiens <400> 6 Met Asp Thr Lys His Phe Leu Pro Leu Asp Phe Ser Thr Gln Val Asn 1 5 10 15 Ser Ser Leu Thr Ser Pro Thr Gly Arg Gly Ser Met Ala Ala Pro Ser 20 25 30 Leu His Pro Ser Leu Gly Pro Gly Ile Gly Ser Pro Gly Gln Leu His 35 40 45 Ser Pro Ile Ser Thr Leu Ser Ser Pro Ile Asn Gly Met Gly Pro Pro 50 55 60 Phe Ser Val Ile Ser Ser Pro Met Gly Pro His Ser Met Ser Val Pro 65 70 75 80 Thr Thr Pro Thr Leu Gly Phe Ser Thr Gly Ser Pro Gln Leu Ser Ser 85 90 95 Pro Met Asn Pro Val Ser Ser Ser Glu Asp Ile Lys Pro Pro Leu Gly 100 105 110 Leu Asn Gly Val Leu Lys Val Pro Ala His Pro Ser Gly Asn Met Ala 115 120 125 Ser Phe Thr Lys His Ile Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly 130 135 140 Lys His Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys 145 150 155 160 Arg Thr Val Arg Lys Asp Leu Thr Tyr Thr Cys Arg Asp Asn Lys Asp 165 170 175 Cys Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr 180 185 190 Gln Lys Cys Leu Ala Met Gly Met Lys Arg Glu Ala Val Gln Glu Glu 195 200 205 Arg Gln Arg Gly Lys Asp Arg Asn Glu Asn Glu Val Glu Ser Thr Ser 210 215 220 Ser Ala Asn Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu 225 230 235 240 Ala Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu 245 250 255 Asn Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala 260 265 270 Asp Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His 275 280 285 Phe Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly 290 295 300 Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val 305 310 315 320 Lys Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser 325 330 335 Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu 340 345 350 Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly 355 360 365 Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser 370 375 380 Asn Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu 385 390 395 400 Glu Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala 405 410 415 Lys Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys 420 425 430 Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp 435 440 445 Thr Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Met Thr 450 455 460 <210> 7 <211> 414 <212> PRT <213> Homo sapiens <400> 7 Met Ala Met Val Val Ser Thr Trp Arg Asp Pro Gln Asp Glu Val Pro 1 5 10 15 Gly Ser Gln Gly Ser Gln Ala Ser Gln Ala Pro Pro Val Pro Gly Pro 20 25 30 Pro Pro Gly Ala Pro His Thr Pro Gln Thr Pro Gly Gln Gly Gly Pro 35 40 45 Ala Ser Thr Pro Ala Gln Thr Ala Ala Gly Gly Gln Gly Gly Pro Gly 50 55 60 Gly Pro Gly Ser Asp Lys Gln Gln Gln Gln Gln His Ile Glu Cys Val 65 70 75 80 Val Cys Gly Asp Lys Ser Ser Gly Lys His Tyr Gly Gln Phe Thr Cys 85 90 95 Glu Gly Cys Lys Ser Phe Phe Lys Arg Ser Val Arg Arg Asn Leu Ser 100 105 110 Tyr Thr Cys Arg Ala Asn Arg Asn Cys Pro Ile Asp Gln His His Arg 115 120 125 Asn Gln Cys Gln Tyr Cys Arg Leu Lys Lys Cys Leu Lys Val Gly Met 130 135 140 Arg Arg Glu Ala Val Gln Arg Gly Arg Met Pro Pro Thr Gln Pro Thr 145 150 155 160 His Gly Gln Phe Ala Leu Thr Asn Gly Asp Pro Leu Asn Cys His Ser 165 170 175 Tyr Leu Ser Gly Tyr Ile Ser Leu Leu Leu Arg Ala Glu Pro Tyr Pro 180 185 190 Thr Ser Arg Phe Gly Ser Gln Cys Met Gln Pro Asn Asn Ile Met Gly 195 200 205 Ile Glu Asn Ile Cys Glu Leu Ala Ala Arg Met Leu Phe Ser Ala Val 210 215 220 Glu Trp Ala Arg Asn Ile Pro Phe Phe Pro Asp Leu Gln Ile Thr Asp 225 230 235 240 Gln Val Ala Leu Leu Arg Leu Thr Trp Ser Glu Leu Phe Val Leu Asn 245 250 255 Ala Ala Gln Cys Ser Met Pro Leu His Val Ala Pro Leu Leu Ala Ala Ala 260 265 270 Ala Gly Leu His Ala Ser Pro Met Ser Ala Asp Arg Val Val Ala Phe 275 280 285 Met Asp His Ile Arg Ile Phe Gln Glu Gln Val Glu Lys Leu Lys Ala 290 295 300 Leu His Val Asp Ser Ala Glu Tyr Ser Cys Leu Lys Ala Ile Val Leu 305 310 315 320 Phe Thr Ser Asp Ala Cys Gly Leu Ser Asp Val Ala His Val Glu Ser 325 330 335 Leu Gln Glu Lys Ser Gln Cys Ala Leu Glu Glu Tyr Val Arg Ser Gln 340 345 350 Tyr Pro Asn Gln Pro Thr Arg Phe Gly Lys Leu Leu Leu Arg Leu Pro 355 360 365 Ser Leu Arg Thr Val Ser Ser Ser Val Ile Glu Gln Leu Phe Phe Val 370 375 380 Arg Leu Val Gly Lys Thr Pro Ile Glu Thr Leu Ile Arg Asp Met Leu 385 390 395 400 Leu Ser Gly Ser Ser Phe Asn Trp Pro Tyr Met Ala Ile Gln 405 410 <210> 8 <211> 362 <212> PRT <213> Xenopus tropicalis <400> 8 Met His Arg Pro Glu Pro Ser Tyr Cys Arg Glu Glu Pro Thr Pro Cys 1 5 10 15 Gln Gly Val Asn Ser Thr Trp Val Pro Pro Ala Asp Thr Val Pro Glu 20 25 30 Thr Ser Pro Thr Pro Ser Ser Pro Pro Ala Pro Asp Ser Pro Thr Pro 35 40 45 Ser Pro Gln Pro Gly Tyr Gly Tyr Ser Pro Cys Glu Glu Lys Pro Gly 50 55 60 Asp Pro Arg Ile Arg Arg Pro Met Asn Ala Phe Met Val Trp Ala Lys 65 70 75 80 Asp Glu Arg Lys Arg Leu Ala Gln Gln Asn Pro Asp Leu His Asn Ala 85 90 95 Val Leu Ser Lys Met Leu Gly Gln Ser Trp Lys Asn Leu Ser Ser Ala 100 105 110 Glu Lys Arg Pro Phe Val Glu Glu Ala Glu Arg Leu Arg Val Gln His 115 120 125 Leu Gln Asp His Pro Asn Tyr Lys Tyr Arg Pro Arg Arg Lys Lys Gln 130 135 140 Ala Lys Lys Leu Lys Arg Val Asp Pro Ser Pro Leu Leu Arg Asn Glu 145 150 155 160 Gly Tyr Arg Gly Gln Ala Met Ala Asn Leu Ser His Phe Arg Asp Leu 165 170 175 His Pro Leu Gly Gly Ser Gly Asp Leu Glu Ser Tyr Gly Leu Pro Thr 180 185 190 Pro Glu Met Ser Pro Leu Asp Val Val Glu Pro Ser Glu Pro Ala Phe 195 200 205 Phe Pro Pro His Met Arg Glu Glu Ala Asp Pro Gly Pro Phe Arg Thr 210 215 220 Tyr Gln His Gly Val Asp Phe Gly Gln Glu Lys Thr Leu Arg Glu Ile 225 230 235 240 Ser Leu Pro Tyr Ser Ser Ser Pro Ser His Met Gly Gly Phe Leu Arg 245 250 255 Thr Pro Thr Ala Ser Ala Phe Tyr Tyr Asn Pro His Gly Gly Ser Pro 260 265 270 Ala Cys Thr Pro Leu Gly Gln Leu Ser Pro Pro Pro Glu Ala Pro Ala 275 280 285 Leu Glu Ala Met Asp His Leu Gly Pro Ala Glu Leu Trp Gly Asp Phe 290 295 300 Asp Arg Asn Glu Phe Asp Gln Tyr Leu Asn Met Ser Arg Thr Gln Gly 305 310 315 320 Pro Gly Tyr Pro Phe Pro Met Ser Lys Leu Gly Ala Pro Arg Thr Ile 325 330 335 Pro Cys Glu Glu Ser Ser Leu Ile Ser Ala Leu Ser Asp Ala Ser Thr 340 345 350 Ala Met Tyr Tyr Thr Pro Cys Ile Thr Gly 355 360 <210> 9 <211> 428 <212> PRT <213> Danio rerio <400> 9 Met Ala Met Val Val Trp Arg Gly Ser Gln Asp Asp Val Ala Glu Thr 1 5 10 15 His Gly Thr Leu Ser Ser Gln Thr Gln Gly Gly Leu Ser Leu Pro Thr 20 25 30 Pro Gln Pro Gly Gln Leu Gly Leu Thr Ala Ser Gln Val Ala Pro Pro 35 40 45 Thr Pro Gln Thr Pro Val Gln Gly Pro Pro Asn Asn Asn Asn Asn Thr 50 55 60 Gln Ser Thr Pro Thr Asn Gln Thr Thr Gln Ser Gln Ser Glu Lys Gln 65 70 75 80 Gln Pro Gln His Ile Glu Cys Val Val Cys Gly Asp Lys Ser Ser Gly 85 90 95 Lys His Tyr Gly Gln Phe Thr Cys Glu Gly Cys Lys Ser Phe Phe Lys 100 105 110 Arg Ser Val Arg Arg Asn Leu Thr Tyr Thr Cys Arg Ala Asn Arg Asn 115 120 125 Cys Pro Ile Asp Gln His His Arg Asn Gln Cys Gln Tyr Cys Arg Leu 130 135 140 Lys Lys Cys Leu Lys Val Gly Met Arg Arg Glu Val Ser Leu Phe Thr 145 150 155 160 Ala Ala Val Gln Arg Gly Arg Met Pro Pro Thr Gln Pro His His Gly 165 170 175 Gln Phe Ala Leu Thr Asn Gly Asp Pro Leu His Cys His Ser Tyr Leu 180 185 190 Ser Gly Tyr Ile Ser Leu Leu Leu Arg Ala Glu Pro Tyr Pro Thr Ser 195 200 205 Arg Tyr Gly Ser Gln Cys Met Gln Pro Asn Asn Ile Met Gly Ile Glu 210 215 220 Asn Ile Cys Glu Leu Ala Ala Arg Met Leu Phe Ser Ala Val Glu Trp 225 230 235 240 Ala Arg Asn Ile Pro Phe Phe Pro Asp Leu Gln Ile Thr Asp Gln Val 245 250 255 Ala Leu Leu Arg Leu Thr Trp Ser Glu Leu Phe Val Leu Asn Ala Ala 260 265 270 Gln Cys Ser Met Pro Leu His Val Ala Pro Leu Leu Ala Ala Ala Gly 275 280 285 Leu His Ala Ser Pro Met Ser Ala Asp Arg Val Val Ala Phe Met Asp 290 295 300 His Ile Arg Ile Phe Gln Glu Gln Val Glu Lys Leu Lys Ala Leu His 305 310 315 320 Val Asp Ser Ala Glu Tyr Ser Cys Leu Lys Ala Ile Val Leu Phe Thr 325 330 335 Ser Asp Ala Cys Gly Leu Ser Asp Val Ala His Val Glu Ser Leu Gln 340 345 350 Glu Lys Ser Gln Cys Ala Leu Glu Glu Tyr Val Arg Ser Gln Tyr Pro 355 360 365 Asn Gln Pro Thr Arg Phe Gly Lys Leu Leu Leu Arg Leu Pro Ser Leu 370 375 380 Arg Thr Val Ser Ser Ser Val Ile Glu Gln Leu Phe Phe Val Arg Leu 385 390 395 400 Val Gly Lys Thr Pro Ile Glu Thr Leu Ile Arg Asp Met Leu Leu Ser 405 410 415 Gly Ser Ser Phe Asn Trp Pro Tyr Met Ser Ile Gln 420 425 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 cattaaccct cataaaggga a 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 11 taatacgact cactataggg 20 <210> 12 <211> 125 <212> DNA <213> Danio rerio <400> 12 tgaagcttgt acctttcatt tcctttttgc tgagctttat tttctctaga attgccattg 60 tgtttccatt ctagcaaatc agcattttt tttcagctga aagaaaaata ccaggaactg 120 agagc 125 <210> 13 <211> 158 <212> DNA <213> Danio rerio <400> 13 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc tttcctttat aaacagagct gtagatatcc 120 acaggaaatg ggggtgtttt tgccattatt tcttcctg 158 <210> 14 <211> 820 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 14 ttgtgggtgc tatttctgta atgaggaagc tggtacaaca ctgagcaaac acactaaaca 60 gagtggcatc ctagaaggtt caaggtctca cgaaatttca taatgagaaa ttcctggaga 120 gcttaacaat aatggctttt aaacaaagca aatataatac caccctattt ttaaattcaa 180 ttaaatggta ttggacatat attattgtca aatacgtctt ctgatctaac gcttgttatt 240 ttggtatgag aaaattttag tttatggttt attttattaa cattttatgg gagtcatatg 300 cttatggcat gtttacatga tgttctcttt ccatgaaact gggaagatga aagcattagt 360 tgaattgtta ctggcaacat cttctctgta atgccccctg tgacccatat tgtctcgctc 420 tttcctttat aaacagagct gtagatatcc acaggaaatg ggggtgtttt tgccattatt 480 tcttcctgct aaagttaatc aggtggtcca aaaattggta taattcattc cttcatttgc 540 ttttcggctt agtccatcag ttaatcaggg gtcgccacag tggaatgaac ctatatccaa 600 tgtatgtttt acgcagcgga tgcccttcca gctgcaaccc aacactggga aacattttag 660 cttactcaat tcacctatac cacgtctttg ggcttgtggg ggaaaccaga gcacccggag 720 gaaacccatg caaacacggg gagaacatga aaactccaaa cagaaacgct aactgaccca 780 gccagggctc gaaccagcaa gcttcctgct gtgaggcgat 820 <210> 15 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 15 ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata 60 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg 120 ttatgagcca tattcattta tgaagcttgt acctttcatt tcctttttgc tgagctttat 180 tttctctaga attgccattg tgtttccatt ctagcaaatc agcatttttt tttcagctga 240 aagaaaaata ccaggaactg agagcgcggc tcctggcagc tctggcccgt gtctcaaaat 300 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct 360 tacataaaca gtaatacaag gggtgttatg agccatattc aacgg 405 <210> 16 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 16 cattaaccct cataaaggga agctccatag tatcgtctga acc 43 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 17 cattaaccct cataaaggga agggagttct tcgggtgact g 41 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 18 cattaaccct cataaaggga atgtgtgatc tcaactgcgc t 41 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 cattaaccct cataaaggga accagcaaac cccatggatc t 41 <210> 20 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 cattaaccct cataaaggga acgactggag aaccaaggga c 41 <210> 21 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 21 cattaaccct cataaaggga accatgcaac agaggaaggg t 41 <210> 22 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 22 cattaaccct cataaaggga aaagttccgc aggatggact g 41 <210> 23 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 cattaaccct cataaaggga aagttatgcc agctcagtgg g 41 <210> 24 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 cattaaccct cataaaggga acttcccaca gcagcacaaa c 41 <210> 25 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 cattaaccct cataaaggga agaatttgct cgcatgaggg g 41 <210> 26 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 cattaaccct cataaaggga aatggaggtc taccacaggc t 41 <210> 27 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 cattaaccct cataaaggga attcggcttt aacctgtgcg t 41 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 cattaaccct cataaaggga aagttagaat gggcgccacc 40 <210> 29 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 cattaaccct cataaaggga aaacttgagc caccgaggat tt 42 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 30 cattaaccct cataaaggga agctacagtc tgcgtagcat 40 <210> 31 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 31 cattaaccct cataaaggga atacccaatg gttcgagtgg c 41 <210> 32 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 32 cattaaccct cataaaggga agtgtcccct catcaatgcc a 41 <210> 33 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 33 cattaaccct cataaaggga atgcccagcc cttgataatc t 41 <210> 34 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 34 cattaaccct cataaaggga aaggcagcac tggactttag c 41 <210> 35 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 35 cattaaccct cataaaggga aacaaagaga tctgcattcc aagc 44 <210> 36 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 36 cattaaccct cataaaggga aaaggcgtac tatgtcctca ggc 43 <210> 37 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 37 cattaaccct cataaaggga attgtggatt acggggttcg g 41 <210> 38 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 38 cattaaccct cataaaggga agagctttac tctgctggcg a 41 <210> 39 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 39 cattaaccct cataaaggga actgttttcc cccaccagtg a 41 <210> 40 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 40 cattaaccct cataaaggga atgggattta tttgtaacta cacgga 46 <210> 41 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 41 cattaaccct cataaaggga accaggaagc gcaaagaaca g 41 <210> 42 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 42 cattaaccct cataaaggga agcagcaaac gactgctaca a 41 <210> 43 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 43 cattaaccct cataaaggga atcatccaca cctgagacgc a 41 <210> 44 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 44 cattaaccct cataaaggga aatccgactg ctggaatgga c 41 <210> 45 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 45 cattaaccct cataaaggga agtgctggtc aacatgtacg c 41 <210> 46 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 46 cattaaccct cataaaggga atccactggg aaaagctgga at 42 <210> 47 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 47 cattaaccct cataaaggga aatggggctg acggatacc 39 <210> 48 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 48 cattaaccct cataaaggga attgtccaga ctaccaaggc g 41 <210> 49 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 49 cattaaccct cataaaggga aatccaaact gaggccggaa g 41 <210> 50 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 50 cattaaccct cataaaggga aggcccaagc atggcagaaa c 41 <210> 51 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 51 cattaaccct cataaaggga aacatgggac atggagcgaa g 41 <210> 52 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 52 cattaaccct cataaaggga agactctctg cggatgtcag g 41 <210> 53 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 53 cattaaccct cataaaggga acatgtccac ccctgaagct g 41 <210> 54 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 54 cattaaccct cataaaggga aggcgtctct ttttctgctg c 41 <210> 55 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 55 cattaaccct cataaaggga accctagtgt ccgaggtctc a 41 <210> 56 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 56 cattaaccct cataaaggga agataccgtg ctgggcgatt a 41 <210> 57 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 57 cattaaccct cataaaggga atcccggtgc aaaatctgag g 41 <210> 58 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 58 cattaaccct cataaaggga agtttattgc tggcgcctac g 41 <210> 59 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 59 cattaaccct cataaaggga agctgccgaa caacgaaaca t 41 <210> 60 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 60 cattaaccct cataaaggga aagtacactc cggacccaag a 41 <210> 61 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 61 cattaaccct cataaaggga atccagagcg attcagcatc a 41 <210> 62 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 62 cattaaccct cataaaggga aagtctccta cttcgagtgg ct 42 <210> 63 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 63 cattaaccct cataaaggga aaactgttct ttctcccggt ccc 43 <210> 64 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 64 cattaaccct cataaaggga aactggtcaa ccacctcctc t 41 <210> 65 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 65 cattaaccct cataaaggga atgcctttca aagtggaccg t 41 <210> 66 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 66 cattaaccct cataaaggga aagcctgttc ctggaccatt g 41 <210> 67 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 67 cattaaccct cataaaggga aggttcgcca gaaggacaag a 41 <210> 68 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 68 cattaaccct cataaaggga aaagctgtca tcaaagccgg a 41 <210> 69 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 69 cattaaccct cataaaggga atgaaagccc tgaacgagac c 41 <210> 70 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 70 cattaaccct cataaaggga aaggagtttg gcttcgacca g 41 <210> 71 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 71 cattaaccct cataaaggga atcgctctga tgctcagctt g 41 <210> 72 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 72 cattaaccct cataaaggga atacacattt tctgccccac tga 43 <210> 73 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 73 cattaaccct cataaaggga actcaccctc ggtccagaac t 41 <210> 74 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 74 cattaaccct cataaaggga aacagccatc agttcctctg c 41 <210> 75 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 75 cattaaccct cataaaggga aggaagccac tcctgatacg g 41 <210> 76 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 76 cattaaccct cataaaggga aggtcacgca ccaaatgaac c 41 <210> 77 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 77 cattaaccct cataaaggga atctgcatct cgcaagctga t 41 <210> 78 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 78 cattaaccct cataaaggga aagaactacg acagcgactg c 41 <210> 79 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 79 cattaaccct cataaaggga aacagactct gtacgtttga atgcgt 46 <210> 80 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 80 cattaaccct cataaaggga atatgactgc agtggtgaag acc 43 <210> 81 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 81 cattaaccct cataaaggga acagacccgt ctctgtggtc 40 <210> 82 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 82 cattaaccct cataaaggga aacccccgaa caacaataac a 41 <210> 83 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 83 cattaaccct cataaaggga atatagccct tcgttccccc a 41 <210> 84 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 84 cattaaccct cataaaggga atccttggac gctgtggacc aac 43 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 85 taatacgact cactataggg aaaccattgc cattttgcca 40 <210> 86 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 86 taatacgact cactataggg gcttgcaaca aaaagcgcag 40 <210> 87 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 87 taatacgact cactataggg ggtgcaatct agtctgatcg gt 42 <210> 88 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 88 taatacgact cactataggg aaccgagtac aggacacacg 40 <210> 89 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 89 taatacgact cactataggg tggaggctaa tcgagtgtgc 40 <210> 90 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 90 taatacgact cactataggg tactgggcgg gtctccttta 40 <210> 91 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 91 taatacgact cactataggg tcgcttgtgt gatcaagtat gac 43 <210> 92 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 92 taatacgact cactataggg ggcctcccca agaaaccatt 40 <210> 93 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 93 taatacgact cactataggg gaaagttgtt gttgaagtga acg 43 <210> 94 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 94 taatacgact cactataggg cggcagtggc caacaaatag 40 <210> 95 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 95 taatacgact cactataggg agtgcaggta tgtgtccgtg 40 <210> 96 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 96 taatacgact cactataggg cctgacgcga gtagtgttgt 40 <210> 97 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 97 taatacgact cactataggg ggcaagacca ctggcatttg 40 <210> 98 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 98 taatacgact cactataggg ccctaactcc agcacacact 40 <210> 99 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 99 taatacgact cactataggg tggaagcagc tctaagtgac ag 42 <210> 100 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 100 taatacgact cactataggg gcttaggtga tcaattttgg gaca 44 <210> 101 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 101 taatacgact cactataggg acggcattcc acaaaccaga 40 <210> 102 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 102 taatacgact cactataggg acccaaactg actttatatg tgc 43 <210> 103 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 103 taatacgact cactataggg aggtacaaat gcaagtacaa cactg 45 <210> 104 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 104 taatacgact cactataggg agcctgtcag ctcactttat t 41 <210> 105 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 105 taatacgact cactataggg cgccgttcta taatgcaccg 40 <210> 106 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 106 taatacgact cactataggg aaagagagct gcaccgact 39 <210> 107 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 107 taatacgact cactataggg acaaatgatg tctgtctccg ct 42 <210> 108 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 108 taatacgact cactataggg caaaggattg gcagggacca 40 <210> 109 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 109 taatacgact cactataggg ttttaagcac atttctgaag caca 44 <210> 110 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 110 taatacgact cactataggg acattaggcg ggcaaaacaa a 41 <210> 111 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 111 taatacgact cactataggg cccacttcgc tgctctttac 40 <210> 112 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 112 taatacgact cactataggg acaccttaaa accgcagcc 39 <210> 113 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 113 taatacgact cactataggg gctttgagga gcttaagagg c 41 <210> 114 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 114 taatacgact cactataggg cgtcactttt cacccccaga 40 <210> 115 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 115 taatacgact cactataggg tttcagatgc agcacaggca 40 <210> 116 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 116 taatacgact cactataggg gcacaggaaa cgcacatgat 40 <210> 117 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 117 taatacgact cactataggg ttacttcact gccagtcggc 40 <210> 118 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 118 taatacgact cactataggg agaaaataca gtgcatacat gtcaa 45 <210> 119 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 119 taatacgact cactataggg cctacaagcc tcattcagac agt 43 <210> 120 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 120 taatacgact cactataggg acactagaaa accgatcgtg tca 43 <210> 121 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 121 taatacgact cactataggg ggtcatgccc ttttggcttg 40 <210> 122 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 122 taatacgact cactataggg gggttctgtt gtggagtgct 40 <210> 123 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 123 taatacgact cactataggg tgacgcttaa acagagcggt 40 <210> 124 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 124 taatacgact cactataggg tttcccctgt gtggatgagc 40 <210> 125 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 125 taatacgact cactataggg ccaccactca ctcattcaga ca 42 <210> 126 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 126 taatacgact cactataggg agtcatgcac aaaatctgcg g 41 <210> 127 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 127 taatacgact cactataggg ttcttggggt agttgcagcc 40 <210> 128 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 128 taatacgact cactataggg taggacgcgt cattgtgctt 40 <210> 129 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 129 taatacgact cactataggg caccagaaag aatgtcaccg t 41 <210> 130 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 130 taatacgact cactataggg cagtccctca gtattccccg 40 <210> 131 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 131 taatacgact cactataggg acgcctgaga ttcatcctgc 40 <210> 132 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 132 taatacgact cactataggg cctctggctg aacaggaagg 40 <210> 133 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 133 taatacgact cactataggg acacaatcag gcaaggtctc 40 <210> 134 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 134 taatacgact cactataggg accagtcaca ccagccattc 40 <210> 135 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 135 taatacgact cactataggg agctacaacc attgagggct 40 <210> 136 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 136 taatacgact cactataggg agcatccaaa agtactcggt gt 42 <210> 137 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 137 taatacgact cactataggg ttctcgacct gtcagctgtt 40 <210> 138 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 138 taatacgact cactataggg cgtcagcatc caaacgcaat 40 <210> 139 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 139 taatacgact cactataggg gtgacgctgg aatatcccgt 40 <210> 140 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 140 taatacgact cactataggg cactcggcga cagtattccc 40 <210> 141 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 141 taatacgact cactataggg aatggggcaa gagtccatct 40 <210> 142 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 142 taatacgact cactataggg acagacacac ttgccagtca 40 <210> 143 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 143 taatacgact cactataggg agctttgcat ccccatcact 40 <210> 144 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 144 taatacgact cactataggg gtacaacatt tacttgctgt cca 43 <210> 145 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 145 taatacgact cactataggg aacagttctg attggatttt gctca 45 <210> 146 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 146 taatacgact cactataggg catgtgtagt gcagacagaa ca 42 <210> 147 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 147 taatacgact cactataggg ggttttggat aagagctgtg tca 43 <210> 148 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 148 taatacgact cactataggg gtccagactt tactcgtccg tgtc 44 <210> 149 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 149 taatacgact cactataggg ctttcccgcc gttttgtgaa 40 <210> 150 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 150 taatacgact cactataggg ccagtatggg gttgtgggac 40 <210> 151 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 151 taatacgact cactataggg agagggcaag cgcagtaata 40 <210> 152 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 152 taatacgact cactataggg accgaaaccg gctaaactga 40 <210> 153 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 153 taatacgact cactataggg tcaaagcgct gctttcctcg c 41 <210> 154 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 154 cttttggcca ttactgccg 19 <210> 155 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 155 gctctcagtt cctggtattt ttct 24 <210> 156 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 156 tcgttactgc acttgaaagc gt 22 <210> 157 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 157 ccatgaaact gggaagatga a 21 <210> 158 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 158 gaagctctcc agcagctca 19 <210> 159 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 159 gctgtcagca cattcttttc c 21 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 160 actgctcctc accaatcgtc 20 <210> 161 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 161 tttatataat cggaaggaac cttttt 26 <210> 162 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 162 ggcaaaatgc ttagatgcag a 21 <210> 163 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 163 tagccttgtg caatgcttgt 20 <210> 164 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 164 cacttctggt accaaatgat caac 24 <210> 165 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 165 gcggcaaact ttttgagtgt 20 <210> 166 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 166 cgcgtgatga ggatctgatt 20 <210> 167 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 167 aaaattaaga gcgggcagac t 21 <210> 168 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 168 aaagcacttg attgagaatt gc 22 <210> 169 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 169 cagtttccca agcttcaagg 20 <210> 170 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 170 aatggttgca gcattgtgtt 20 <210> 171 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 171 gttacctggc aaccaccaac 20 <210> 172 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 172 acgttaacaa aggcgatgtt tt 22 <210> 173 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 173 tgacaggact catcagcacg 20 <210> 174 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 174 tgggaaaaat accaggaagc gt 22 <210> 175 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 175 agatcaatga gagcgaggcg 20 <210> 176 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 176 cggacagtaa tgtctggatg g 21 <210> 177 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 177 tcatcatggc caacagaatg 20 <210> 178 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 178 tgactcaacc aatcaatcag cct 23 <210> 179 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 179 ttctgtcttt taatcagcaa tcc 23 <210> 180 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 180 tgaagcttgt acctttcatt tcc 23 <210> 181 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 181 tatcagtgat gttctgcagt ggtc 24 <210> 182 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 182 caggaagaaa taatggcaaa aa 22 <210> 183 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 183 catttccacc agctgtctga t 21 <210> 184 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 184 ccctgctgat cacacatgac 20 <210> 185 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 185 tgcactaaat ctgtgccaag tc 22 <210> 186 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 186 tcctgtcagc tgttttcatc c 21 <210> 187 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 187 tgcgaggagg acataaacaa 20 <210> 188 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 188 tgctgaattc aaaagccact t 21 <210> 189 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 189 gagggttaaa cgtggcctta 20 <210> 190 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 190 gccagcctca aagtttgttc t 21 <210> 191 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 191 ggtgttgaaa ggtgatgctg 20 <210> 192 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 192 tggaaacaac aacagcctga 20 <210> 193 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 193 tgtttggttc agttacacgt ttt 23 <210> 194 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 194 tgtgattaca cattcccaca cat 23 <210> 195 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 195 gcttttgttt ggtgatgtgc 20 <210> 196 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 196 tggtcagaat aagcacgttt ca 22 <210> 197 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 197 tctaaacaat ttttaaggta aaccaaa 27 <210> 198 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 198 aatagtctct ggtctgctgt taaa 24 <210> 199 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 199 aagcagcgag ctctcataat aaa 23 <210> 200 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 200 ggagcagatg aggttaagtc ct 22 <210> 201 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 201 ccacaactcc atactgggaa a 21 <210> 202 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 202 gtgcaggaaa tgagcacaga 20 <210> 203 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 203 tagcaaagct ctcaggccc 19 <210> 204 <211> 158 <212> DNA <213> Danio rerio <400> 204 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc tttcctttat aaacagagct gtagatatcc 120 acaggaaatg ggggtgtttt tgccattatt tcttcctg 158 <210> 205 <211> 125 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 205 tgaagcttgt acctttcatt taaattttgc tgagctttat tttctctaga attgccattg 60 tgtttccatt ctagcaaatc agcatttttt tttcagctga aagaaaaata ccatttactg 120 agagc 125 <210> 206 <211> 124 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 206 tgaagcttgt acctttcatt tcctttttgc tgagcgggcg gggatctaga attgcacggt 60 ggtttccatt ctagcaaatc agccgggggg tttcagctaa agaaaaatac caggaactga 120 gagc 124 <210> 207 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 207 attagcagat ttaatttcat ttcctttttg cattaattta ttttctctag aattgccatt 60 gtgtttccat tctagcaaat cagcattttt ttttcagctg aaagaaaaat accaggaact 120 gagagc 126 <210> 208 <211> 125 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 208 tgaagcttgt acctttcatt tcctttttgc tgagctttat tttctctaga attgccattg 60 tgtttccatt ccggcaaatc agcatttttt tttcagctga ccgaaaaata ccaggaactg 120 agagc 125 <210> 209 <211> 158 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 209 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc tttaaattat aaacagagct gtagatatcc 120 acatttaatg ggggtgtttt tgccattatt tctaaatg 158 <210> 210 <211> 158 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 210 ccatgaaact gggaagatga aagcattagt tgaaggtgga ctggcaacat cttctctgta 60 atgccccctg tgacccatag gtgatcgctc tttcctttat aaacagagct gtagatatcc 120 acaggaaatg ggggtgtttt tgccattatt tcttcctg 158 <210> 211 <211> 158 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 211 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg attaacatat tgtctcgctc tttcctttat aaacagagct gtagatatcc 120 acaggaaatg ggggtgtttt tgccattatt tcttcctg 158 <210> 212 <211> 158 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 212 ccatgaaact gggaatctga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc tttcctttat aaacagagag gtagatatcc 120 acaggaaatg ggggactttt tgccattatt tcttcctg 158 <210> 213 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 213 tgaagcttgt acctttcatt tcctttttgc tgagctttat tttctctaga attgccattg 60 tgtttccatt ctag 74 <210> 214 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 214 tgaagcttgt acctttcatt taaattttgc tgagctttat tttctctaga attgccattg 60 tgtttccatt ctag 74 <210> 215 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 215 tgaagcttgt acctttcatt tcctttttgc tgagcgggcg gggatctaga attgcacggt 60 ggtttccatt ctag 74 <210> 216 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 216 attagcagat ttaatttcat ttcctttttg cattaattta ttttctctag aattgccatt 60 gtgtttccat tcta 74 <210> 217 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 217 tgaagcttgt acctttcatt tcctttttgc tgagctttat tttctctaga attgccattg 60 tgtttccatt ctag 74 <210> 218 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 218 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc t 91 <210> 219 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 219 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc t 91 <210> 220 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 220 ccatgaaact gggaagatga aagcattagt tgaaggtgga ctggcaacat cttctctgta 60 atgccccctg tgacccatag gtgatcgctc t 91 <210> 221 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 221 ccatgaaact gggaagatga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg attaacatat tgtctcgctc t 91 <210> 222 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 222 ccatgaaact gggaatctga aagcattagt tgaattgtta ctggcaacat cttctctgta 60 atgccccctg tgacccatat tgtctcgctc t 91 <210> 223 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 223 gctctcagtt cctggtattt ttctttcagc tgaaaaaaaa atgctgattt gctagaatgg 60 aaacacaatg gcaat 75 <210> 224 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 224 gctctcagta aatggtattt ttctttcagc tgaaaaaaaa atgctgattt gctagaatgg 60 aaacacaatg gcaat 75 <210> 225 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 225 gctctcagtt cctggtattt ttctttcagc tgaaaccccc cggctgattt gctagaatgg 60 aaaccaccgt gcaat 75 <210> 226 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 226 gctctcagtt cctggtattt ttctttcagc tgaaaaaaaa atgctgattt gctagaatgg 60 aaacacaatg gcaat 75 <210> 227 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 227 gctctcagtt cctggtattt ttcggtcagc tgaaaaaaaa atgctgattt gccggaatgg 60 aaacacaatg gcaat 75 <210> 228 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 228 caggaagaaa taatggcaaa aacaccccca tttcctgtgg atatctacag ctctgtttat 60 aaaggaaaga gcgagacaat atgggtcaca g 91 <210> 229 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 229 catttagaaa taatggcaaa aacaccccca ttaaatgtgg atatctacag ctctgtttat 60 aatttaaaga gcgagacaat atgggtcaca g 91 <210> 230 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 230 caggaagttt attaggcaaa aacaccccca tttcctgtgg atatctacag ctctgtttat 60 aaaggaaaga gcgatcacct atgggtcaca g 91 <210> 231 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 231 caggaagaaa taatggcaaa aacaccccca tttcctgtgg atatctacag ctctgtttat 60 aaaggaaaga gcgagacaat atgttaatca g 91 <210> 232 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 232 caggaagaaa taatggcaaa aagtccccca tttcctgtgg atatctacct ctctgtttat 60 aaaggaaaga gcgagacaat atgggtcaca g 91 <210> 233 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 233 cgggatccat ggcaatggta gtcagcacg 29 <210> 234 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 234 tttatgaagc ttgtaccttt catttccttt ttg 33 <210> 235 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 235 tttaattagc agatttaatt tcatttcctt tttg 34 <210> 236 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 236 ttcatttcct ttttgctgag ctttattttc 30 <210> 237 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 237 ttcatttcct ttttgcatta atttattttc 30 <210> 238 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 238 gtaatgcccc ctgtgaccca tattgtctcg ctctttcctt tata 44 <210> 239 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 239 gtaatgcccc ctgattaaca tattgtctcg ctctttcctt tata 44 <210> 240 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 240 ccggaattcc ggttgaattg ccatatatgg c 31 <210> 241 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 241 caaaaaggaa atgaaaggta caagcttcat aaa 33 <210> 242 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 242 caaaaaggaa atgaaattaa atctgctaat taaa 34 <210> 243 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 243 gaaaataaag ctcagcaaaa aggaaatgaa 30 <210> 244 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 244 gaaaataaat taatgcaaaa aggaaatgaa 30 <210> 245 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 245 tataaaggaa agagcgagac aatatgggtc acagggggca ttac 44 <210> 246 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 246 tataaaggaa agagcgagac aatatgttaa tcagggggca ttac 44

Claims

1. 1. A method for generating / modifying endothelial niche cells, comprising: expressing one or more transcription factors in an endothelial cell; the one or more transcription factors include at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family; The transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA, the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; method.

2. The method of claim 1, wherein the transcription factor from the Ets family is human transcription factor ETV2, FLI1 or ETS1, the transcription factor from the Sox family is human transcription factor SOX18 or SOX7, and the transcription factor from the nuclear hormone receptor family is human transcription factor RXRA or NR2F2.

3. The method according to claim 1, wherein the transcription factor from the Ets family is ETV2, FLI1 or ETS1, and the transcription factor from the Sox family is SOX18 or SOX7.

4. The method of claim 1 , wherein the transcription factor is expressed from at least one vector.

5. The method of claim 1, wherein the vector comprises an exogenous nucleic acid sequence encoding one or more transcription factors.

6. The method of claim 1, wherein the exogenous nucleic acid sequence is integrated into the genome of the endothelial cell.

7. A modified endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding one or more transcription factors, the one or more transcription factors include at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family; The transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA, the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; Modified endothelial niche cells.

8. A composition comprising the modified endothelial niche cells of claim 7.

9. 10. The composition of claim 8, further comprising a therapeutic agent or a pharma- ceutically acceptable carrier.

10. 10. The composition of claim 8, further comprising a culture dish, a 3D cell system, or a suspension system.

11. 10. The composition of claim 8, comprising a scaffold.

12. 1. A method for culturing HSPCs, comprising: Culturing the HSPCs in the presence of a population of modified endothelial niche cells; The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; method.

13. 13. The method of claim 12, which is carried out in vitro.

14. The method of claim 12, wherein the modified endothelial niche cells secrete factors that affect the growth and / or expansion of HSPC cells.

15. The method of claim 12, wherein HSPCs cultured in the presence of modified endothelial niche cells can be cultured for at least 3 (e.g., at least 4, at least 5, at least 6, at least 7) ​​days longer than HSPCs cultured in the absence of such modified endothelial niche cells.

16. The method of claim 12, wherein the cells are cultured on a biologically compatible scaffold.

17. The method of claim 12, wherein the HSPCs cultured in the presence of the modified endothelial niche cells exhibit enhanced engraftment when administered to a subject compared to the engraftment of substantially similar HSPCs that were not cultured with the modified endothelial niche cells.

18. 1. A composition for use in a method of treating a subject, comprising a population of HSPCs and modified endothelial niche cells, The method includes implanting a composition in a subject; The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; composition.

19. 1. A composition for use in a method for improving engraftment of HSPCs, comprising a population of HSPCs and modified endothelial niche cells, The method includes administering the composition to a subject in need thereof; The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; composition.

20. The composition of claim 19, wherein engraftment of HSPCs is improved by at least 10% compared to engraftment of substantially similar HSPCs in the absence of the modified endothelial niche cells.

21. A co-culture comprising modified endothelial niche cells and HSPCs, The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; Co-culture.

22. The co-culture of claim 21, wherein the endothelial cells are produced by the method of any one of claims 1 to 6.

23. A kit for culturing HSPCs comprising a population of modified endothelial niche cells, reagents, and instructions for use thereof, The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; Kit for culturing HSPCs.

24. 1. A composition for use in a method for generating an ectopic vascular niche comprising modified endothelial niche cells, comprising: The method includes administering a composition to a target site in a subject in need thereof; The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; composition.

25. 1. A composition for use in a method for extramedullary hematopoiesis comprising modified endothelial niche cells, The method includes implanting the composition into a site outside the bone marrow (e.g., the forearm) in a subject, thereby creating an artificial niche; The modified endothelial niche cells (i) expressing at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, where the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; or (ii) one or more exogenous nucleic acid sequences encoding at least one transcription factor from the Ets family, at least one transcription factor from the Sox family, and at least one transcription factor from the nuclear hormone receptor family, wherein the transcription factor from the nuclear hormone receptor family is NR2F2 or RXRA; the endothelial niche cells express one or more genes including Prcp, Cldn11, Tll1, Ctsh, Hexb, Hyal2, Cltc, Ifi30, Glu1, Lyve1, Gpr182, Dab2, Ctsl, Stab2, Npl, Stab1, Snx8, Mrc1, Ap1b1, Pxk, Sepp1, Lgmn, Man2b2, Sele, Slc16a9, or Il13ra2; and the endothelial cell is mammalian; composition.

26. The composition of claim 25, wherein the endothelial cells are produced by the method of any one of claims 1 to 6.

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