US20220403341A1

US20220403341A1 - Exosomes from clonal progenitor cells

Info

Publication number: US20220403341A1
Application number: US17/592,200
Authority: US
Inventors: Dana Larocca; Mohammad Hassanipour; Paola A. Bignone
Original assignee: Recyte Therapeutics Inc
Current assignee: Recyte Therapeutics Inc
Priority date: 2014-07-03
Filing date: 2022-02-03
Publication date: 2022-12-22
Also published as: US11274281B2; US20160108368A1; US10240127B2; US20190241873A1

Abstract

The invention provides methods, compositions, uses and kits relating to exosomes isolated from progenitor cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/270,295, filed Feb. 7, 2019, which is a divisional application that claims priority to U.S. application Ser. No. 14/748,215, filed Jun. 23, 2015, which claims priority to U.S. Provisional Application No. 62/020,869, filed on Jul. 3, 2014. The entire contents of the foregoing applications are hereby incorporated by reference.

FIELD OF THE INVENTION

The field of the invention relates to exosomes isolated from progenitor cells.

BACKGROUND

Exosomes are believed to contain important signaling molecules that may provide the source of trophic factors responsible for some regenerative benefits seen in cell replacement therapy. As such they would provide an alternative to some cell based therapies that would be easier to manufacture on a large scale and potentially safer to administer to a subject in need of cell therapy. In particular, the risk associated with transmission of infectious agents such as viruses may be lower compared to transplanting whole cells. Moreover, the risk of immune rejection of the exosomes relative to transplanted cells may also be lower. Accordingly, exosomes may provide an attractive alternative or adjunct to cell based therapies and cell based regenerative medicine.
Exosomes are 30 to 120 nm vesicles secreted by a wide range of mammalian cell types. Keller et al. (2006) Immunol Lett. 107(2):102; Camussi et al. (2010) Kidney International 78:838. The vesicles are enclosed by a lipid bilayer and are larger than LDL which has a size of 22 nm, but smaller than a red blood cell, which is 6000 to 8000 nm in diameter and has a thickness of 2000 nm. Keller et al. (2006) Immunol Lett. 107(2):102.
Exosomes are found both in cells growing in vitro as well as in vivo. They can be isolated from tissue culture media as well as bodily fluids such as plasma, urine, milk and cerebrospinal fluid. George et al. (1982) Blood 60:834; Martinez et al. (2005) Am J Physiol Health Cir Physiol 288:H1004. Exosomes originate from the endosomal membrane compartment. They are stored in intraluminal vesicles within multivesicular bodies of the late endosome. Multivesicular bodies are derived from the early endosome compartment and contain within them smaller vesicular bodies that include exosomes. Exosomes are released from the cell when multivesicular bodies fuse with the plasma membrane. Methods of isolating exosomes from cells has been described, see e.g. US Patent Application Publication No. 20120093885
Exosomes contain a variety of molecules including proteins, lipids and nucleic acids such as DNA, mRNA and miRNA. Their contents are believed to play a part in cell to cell communication involving the release of the exosome from one cell and the binding/fusion of the exosome with a second cell, wherein the contents of the exosomal compartment are released within the second cell.
It has been reported that exosomes derived from endothelial progenitor cells may act as vehicle for mRNA transport among cells. These exosomes were shown to incorporate into normal endothelial cells by interacting with the α4β1 integrin. Once incorporated into the endothelial cells, the exosomes stimulated an angiogenic program. Deregibus et al. (2007) Blood 110:2440. Similar results were obtained in vivo using severe combined immunodeficient mice. Exosome stimulated endothelial cells implanted subcutaneously in Matrigel (a murine sarcoma extract) organized into a patent vessel network connected with the murine vasculature. Deregibus, supra. Bruno et al. (2009) J Am Soc Nephrol 20:1053; Herrera et al. (2010) J Cell Mol Med 14:1605.
Of the various molecular cargo of exosomes, miRNAs have recently attracted a lot of attention due to their regulatory roles in gene expression. MiRNAs are small, non-coding regulatory RNAs that can have a wide range of effects on multiple RNA targets, thus having the potential to have greater phenotypic influence than coding RNAs. MiRNA profiles of exosomes often differ from those of the parent cells. Profiling studies have demonstrated that miRNAs are not randomly incorporated into exosomes but rather a subset of miRNAs is preferentially packaged into exosomes, suggesting an active sorting mechanism of exosomal miRNAs. Guduric-Fuchs et al. (2014) Nucleic Acid Res. 42:9195; Ohshima et al. (2010) PloS One 5(10):e13247.
Because exosomes contain a variety of molecules, many believed to play an important role in cell signaling, exosomes would prove useful in research and industry and would have applications as therapeutics, diagnostics and in screening assays. Frequently, however, the availability of reproducible, essentially identical populations of exosomes is limited by the fact that most sources of exosomes are cells that senesce and thus have limited replicative capacity. Accordingly, there is a need for exosomes that are derived from a clonal source that has an extended replicative capacity that is greater than most adult or fetal derived cells. The invention described infra meets this need and as well as other needs in the field.

SUMMARY OF THE INVENTION

In various embodiments described herein the invention provides compositions comprising exosomes obtained from progenitor cell lines, as well as methods of making and using exosomes obtained from progenitor cell lines.
The isolation of embryonic progenitor cells has been described. See West et al. (2008) Regen Med 3:287; US Patent Application Publication Nos. 20080070303 20100184033. Embryonic progenitors are cell lines derived under a variety of culture conditions from pluripotent stem cells, such as human embryonic stem (hES) cells or induced pluripotent stem (iPS) cells. The progenitor cell lines are clonal and while they do, in most instances, senesce, they also possess longer telomeres compared to adult or fetal derived tissue or cells (such as adult stem cells) and accordingly have enhanced replicative capacity relative to those cell types. Because of their clonality and their enhanced replicative capacity they provide a suitable source of exosomes that will offer the benefit of uniformity with regard to the exosome composition and abundance relative to exosomes derived from their typical sources such as adult cells or adult stem cells.
In certain embodiments the invention provides an exosome isolated from a progenitor cell line, such as clonal progenitor cell line.
In certain embodiments the invention provides an exosome isolated from a human progenitor cell line, such as a clonal human progenitor cell line.
In some embodiments the invention provides an exosome isolated from endothelial progenitor cell.
In some embodiments the invention provides an exosome isolated from a clonal human endothelial progenitor cell.
In other embodiments the invention provides an exosome isolated from the 30-MV2-6 human clonal progenitor cell line.
In further embodiments the invention provides an exosome isolated from a human clonal progenitor cell that expresses CD31 and CD34.
In certain embodiments the invention provides an exosome isolated from a human progenitor cell line, wherein the human progenitor cell is not an adult stem cell.
In further embodiments the invention provides an exosome isolated from a human progenitor cell line, wherein the human progenitor cell is not a mesenchymal stem cell (MSC).
In certain embodiments the invention provides an exosome isolated from a cell that has not been transfected with an exogenous gene.
In certain other embodiments the invention provides an exosome isolated from a cell that has been transfected with an exogenous gene, wherein the gene is not c-myc.
In yet other embodiments the invention provides an exosome isolated from a cell that does not overexpress c-myc.
In other embodiments the invention provides an exosome isolated from the 30-MV2-6 clonal human progenitor cell line.
In still other embodiments the invention provides an exosome isolated from a cell expressing one or more genes chosen from the genes listed in Table 1.
In further embodiments the invention provides an exosome isolated from a cell expressing a plurality of the genes chosen from the genes listed in Table 1.
In yet other embodiments the invention provides an exosome isolated from a cell expressing the genes listed in Table 1.
In some embodiments, the invention provides an exosome containing CD63.
In other embodiments, the invention provides an exosome containing one or more miRNAs listed in Table 2 or Table 4.
In further embodiments, the invention provides an exosome containing one or more angiogenic miRNAs.
In yet further embodiments, the invention provides an exosome containing miR-126.
In some embodiments the invention provides an exosome isolated from a human clonal progenitor cell, wherein the exosome contains one or more miRNAs listed in Table 2 or Table 4.
In other embodiments the invention provides an exosome isolated from a human clonal progenitor cell, wherein the exosome contains one or more angiogenic miRNAs.
In yet other embodiments the invention provides an exosome isolated from a human clonal progenitor cell, wherein the exosome contains miR-126.
In further embodiments the invention provides an exosome isolated from a human clonal progenitor cell, wherein the exosome contains CD63.
In some embodiments the invention provides an exosome that induces a cell to form vascular tube like structures.
In other embodiments the invention provides an exosome that induces a cell to form branching vascular tube like structures.
In yet other embodiments the invention provides a cell culture comprising an exosome isolated from a progenitor cell and a cell which was not the source of the isolated exosome.
In certain embodiments the invention provides a cell culture comprising an exosome isolated from a progenitor cell and a cell which was not the source of the isolated exosome, wherein the cell has the ability to form vascular tube like structures.
In further embodiments the invention provides a cell culture comprising an exosome isolated from a progenitor cell and a cell which was not the source of the isolated exosome, wherein the cell is an endothelial cell.
In still further embodiments the invention provides a cell culture comprising an exosome isolated from a progenitor cell and a cell which was not the source of the isolated exosome, wherein the cell is a human umbilical vein endothelial cell (HUVEC).
In the cell culture embodiments described above the progenitor cell may be a human progenitor cell, such as a human embryonic progenitor cell. One example of a human embryonic progenitor cell is the 30-MV2-6 cell line.
In the cell culture embodiments described above the progenitor cell may be, for example, a clonal progenitor cell line, an oligoprogenitor cell line. The progenitor cell may express one or more genes listed in Table 1. The progenitor cell may express a plurality of the genes listed in Table 1. The progenitor cell line may express the genes listed in Table 1. The progenitor cell line may express CD31 and CD34.
In some embodiments the invention provides a method of isolating an exosome from a progenitor cell, such as a clonal progenitor cell comprising 1) culturing the progenitor cell in a suitable media or buffer for a time sufficient to allow the cells to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a clonal progenitor cell.
In some embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell comprising 1) culturing the human clonal progenitor cell in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a clonal progenitor cell.
In other embodiments the invention provides a method of isolating an exosome from a 30-MV2-6 human clonal progenitor cell line comprising 1) culturing the 30-MV2-6 human clonal progenitor cell line in a suitable media or buffer for a time sufficient to allow the 30-MV2-6 human clonal progenitor cell line to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a 30-MV2-6 human clonal progenitor cell line.
In still other embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell line expressing CD31 and CD34 comprising 1) culturing the human clonal progenitor cell line expressing CD31 and CD34 in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line expressing CD31 and CD34 to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line expressing CD31 and CD34.
In some embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell line expressing one or more of the genes listed in Table 1 comprising 1) culturing the human clonal progenitor cell line expressing one or more of the genes listed in Table 1 in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line expressing one or more of the genes listed in Table 1 to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line expressing one or more of the genes listed in Table 1.
In still other embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell line expressing a plurality of the genes listed in Table 1 comprising 1) culturing the human clonal progenitor cell line expressing a plurality of the genes listed in Table 1 in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line expressing a plurality of the genes listed in Table 1 to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line expressing a plurality of the genes listed in Table 1.
In further embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell line expressing the genes listed in Table 1 comprising 1) culturing the human clonal progenitor cell line expressing the genes listed in Table 1 in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line expressing the genes listed in Table 1 to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line expressing the genes listed in Table 1.
In some embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell wherein the human clonal progenitor cell line has not been transfected with an exogenous gene comprising 1) culturing the human clonal progenitor cell line that has not been transfected with an exogenous gene in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line that has not been transfected with an exogenous gene to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line that has not been transfected with an exogenous gene.
In other embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell, wherein the human clonal progenitor cell line has been transfected with an exogenous gene, wherein the gene is not c-myc, comprising 1) culturing the human clonal progenitor cell line that has been transfected with an exogenous gene, wherein the exogenous gene is not c-myc, in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line that has been transfected with an exogenous gene, wherein the exogenous gene is not c-myc, to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line that has been transfected with an exogenous gene, wherein the exogenous gene is not c-myc.
In still other embodiments the invention provides a method of isolating an exosome from a human clonal progenitor cell wherein the human clonal progenitor cell line does not overexpress c-myc comprising 1) culturing the human clonal progenitor cell line that does not overexpress c-myc in a suitable media or buffer for a time sufficient to allow the human clonal progenitor cell line that has not been transfected with an exogenous gene to exocytose exosomes into the culture media; 2) harvesting the media from the cell culture of step 1; and 3) isolating the exosomes from the media of step 2, thereby isolating an exosome from a human clonal progenitor cell line that does not overexpress c-myc.
In further embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a progenitor cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
In still further embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting an endothelial cell with an exosome isolated from a progenitor cell thereby inducing or enhancing an endothelial cells ability to form vascular tube like structures.
In other embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a clonal progenitor cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
In further embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a human clonal progenitor cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
In certain embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a human endothelial progenitor cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
In yet other embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a human clonal endothelial progenitor cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
In further embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a cell expressing one or more genes listed in Table 1 thereby inducing or enhancing a cells ability to form vascular tube like structures.
In other embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a cell expressing a plurality of genes listed in Table 1 thereby inducing or enhancing a cells ability to form vascular tube like structures.
In yet other embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a cell expressing the genes listed in Table 1 thereby inducing or enhancing a cells ability to form vascular tube like structures.
In still other embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a cell expressing the markers CD31 and CD34 thereby inducing or enhancing a cells ability to form vascular tube like structures.
In further embodiments the invention provides a method of inducing or enhancing a cells ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a 30-MV2-6 cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
In still other embodiments the invention provides a method of regeneration a tissue or an organ comprising contacting one or more cells capable of regenerating a tissue or an organ with an exosome isolated from a progenitor cell.
In yet other embodiments the invention provides a method of regenerating a vascular tissue or organ comprising contacting a cell capable of regenerating a vascular tissue or organ with an exosome isolated from a progenitor cell.
In some embodiments the invention provides a method of regenerating a vascular tissue or organ comprising contacting a cell capable of regenerating a vascular tissue or organ with an exosome isolated from a human clonal endothelial progenitor cell.
In further embodiments the invention provides a method of regenerating a vascular tissue or organ comprising contacting a cell capable of regenerating a vascular tissue or organ with an exosome isolated from a 30-MV2-6 cell.
In certain embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a progenitor cell.
In some embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a human clonal progenitor cell.
In further embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from an endothelial progenitor cell.
In certain embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from human clonal endothelial progenitor cell.
In yet other embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a 30-MV2-6 human endothelial progenitor cell.
In still other embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a human clonal progenitor cell expressing CD31 and CD34.
In further embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a human clonal progenitor cell expressing one or more genes listed in Table 1.
In some embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a human clonal progenitor cell expressing a plurality of genes listed in Table 1.
In yet other embodiments the invention provides a method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a human clonal progenitor cell expressing the markers listed in Table 1.
In further embodiments the invention provides a kit comprising an exosome isolated from a progenitor cell and at least one container.

BRIEF DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings, in which:

FIG. 1 shows a graph of the size and concentration of exosomes isolated from

- a) human embryonic progenitor cell line 30-MV2-6; and b) the HT1080 cell line.

FIG. 2A shows three photomicrographs, the first showing the effects on vascular tube like formation in HUVECs grown in the presence of basal media supplemented with exosomes isolated from human embryonic progenitor cell line 30-MV2-6 (top); the second showing the effects on vascular tube formation in HUVECs grown in base media supplemented with PBS, but without exposure to exosomes isolated from human embryonic progenitor cell line 30-MV-2-6 (middle); and the third showing the effects on vascular tube like formation in HUVECs grown in complete medium, but without exposure to exosomes isolated from human embryonic progenitor cell line 30-MV2-6 (bottom).

FIG. 2B is a graph quantifying four parameters: cell covered area; total tube length; total number of branching points and total number of loops in HUVECs grown in the presence of exosomes isolated from human embryonic progenitor cell line 30-MV2-6 (“MV2-6 EXO”); HUVECs grown in basal media+PBS, but without exposure to exosomes isolated from human embryonic progenitor cell line 30-MV2-6 (“Basal PBS”); and HUVECs grown in complete media, but without exposure to exosomes isolated from human embryonic progenitor cell line 30-MV2-6 (“Complete”).

FIG. 3A is photomicrograph showing that hES derived perivascular cells form aggregates when cultured in the presence of complete EGM-MV2 media with serum and growth factors.

FIG. 3B is a photomicrograph showing that hES derived perivascular cells form incomplete tubes when cultured in EGM-MV2 basal media.

FIGS. 3C-E are photomicrographs showing that increasing doses of exosomes isolated from the human clonal endothelial progenitor cell line 30-MV2-6 resulted in increasing tube formation in hES derived perivascular cells.

FIG. 3F is a graph showing the cell covered area of hES derived perivascular cells grown either in complete media (complete); basal media (base) or basal media supplemented with increasing doses (2.5×10⁷, 5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonal endothelial progenitor cell line 30-MV2-6.

FIG. 3G is a graph showing the total number of branching points in hES derived perivascular cells grown either in complete media (complete); basal media (base) or basal media supplemented with increasing doses (2.5×10⁷, 5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonal endothelial progenitor cell line 30-MV2-6.

FIG. 3H is a graph showing the total tube length of vascular tube like structures formed by hES derived perivascular cells grown either in complete media (complete); basal media (base) or basal media supplemented with increasing doses (2.5×10⁷, 5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonal endothelial progenitor cell line 30-MV2-6.

FIG. 3I is a graph showing the total number of loops formed by hES derived perivascular cells grown either in complete media (complete); basal media (base) or basal media supplement with increasing doses (2.5×10⁷, 5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonal endothelial progenitor cell line 30-MV2-6.

FIG. 4 shows comparison of in vitro angiogenic activity of exosomes isolated from the human clonal endothelial cell line 30-MV2-6 versus exosomes isolated from bone marrow mesenchymal stem cells (BM-MSC). Two different commercially available sources of BM-MSCs were used, Promocell (panel A) and Lonza (panel B). 30-MV2-6: exosomes derived from 30-MV2-6 cell line; MSC: exosomes derived from BM-MSC; BM: basal medium, negative control; CM: complete growth medium. Results were normalized to tube length obtained using complete growth medium (CM).

FIG. 5 shows that the angiogenic activity of exosomes isolated from the 30-MV2-6 cell line is dose dependent (panel A) and at least six times more potent than the angiogenic activity of exosomes isolated from BM-MSCs (panel B).

FIG. 6A depicts analysis of miRNA content in 30-MV2-6 derived exosomes versus BM-MSC derived exosomes. 30-MV2-6 exosome RNA was compared to BM-MSC RNA for miRNA content using an 84 miRNA PCR array. The scatter plot indicates miRNAs with greater than 6-fold differences. The upper circled dot represents miRNA miR-126-3p, which is expressed at 77.6-fold higher in 30-MV2-6 exosomes than in BM-MSC exosomes. The lower circled dot represents miRNA miR-376c-3p which is expressed at 752-fold lower in 30-MV2-6 exosomes than in MB-MSC exosomes.

FIG. 6B is a bar graph illustrating the differences (measured as fold-change) in miRNA expression profile between 30-MV2-6-derived exosomes versus BM-MSC-derived exosomes.

FIG. 7 consists of 5 photomicrographs showing in vivo angiogenic activity of human clonal endothelial cell line 30-MV2-6 in the Matrigel plug assay in immunocompromised mice. Blood vessel-like structures are seen in the exosome treated plugs (panels A and C) but not in the control plug (panel E). Endothelial cell content is confirmed by staining with anti-Von Willebrand factor antibody (panels B and D).

FIG. 8 shows that the in vitro angiogenic activity of exosomes derived from 30-MV2-6 cells grown in T-flasks is comparable to the in vitro angiogenic activity of exosomes derived from 30-MV2-6 cells grown in the Quantum cell expansion bioreactor. Panel A depicts tube network formation and panel B depicts quantitative analysis of tube length using Image J angiogenesis analyzer software. BM: basal medium, negative control; CM: complete growth medium.

FIG. 9 is a bar graph illustrating the effect of oxygen level and conditioning medium used on the angiogenic activity of exosomes derived from 30-MV2-6 cells. Use of PBS versus basal medium as the conditioning medium had no significant effect on the angiogenic activity of the 30-MV2-6 exosomes. Similarly, no significant effect on the angiogenic activity was observed in conditioning the cells in 1% versus 5% oxygen.

FIG. 10 shows ELISA detection of CD63 on intact exosomes to determine exosome concentration. The standard curve was prepared using exosomes (from HT1080 cells) of known concentration as determined by nanoparticle analysis (NTA). Quantitation of exosomes in samples of unknown concentration was calculated from OD value at 450 nm. The assay assumes CD63 content of exosomes derived from various different cells remains relatively constant.

FIG. 11 depicts the high proliferative capacity and stable angiogenic exosome production of the clonal human embryonic progenitor cell line 30-MV2-6. The 30-MV2-6 cells continue to proliferate in cell culture past 50 population doublings (panel A). Similarly, exosomes that retain their angiogenic activity as measured by the in vitro tube formation assay may be prepared from 30-MV2-6 cells that have been cultured for at least 50 population doublings (panel B).

FIG. 12 is a heat map of Illumina gene expression array (Illumina, Hayward, Calif.) data showing that the clonal embryonic epithelial progenitor cell lines derived from the embryonic stem cell line ESI-017 have similar endothelial specific gene expression pattern as various adult endothelial cells from different sources.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.
The invention provides exosomes isolated from clonal progenitor cells, such as human clonal progenitor cells derived from a human pluripotent stem cell. Because the cells are clonal and have enhanced replicative capacity in vitro, the invention provides a means of producing the same exosomes over and over again. This provides for a consistent product allowing either the researcher or clinician to alleviate any concerns regarding both the quality and the consistency of the exosomes in any application. Accordingly, the invention also provides methods of making the progenitor cells from which the exosomes are derived and methods of isolating the exosomes from these cells. The invention also contemplates uses, cell cultures and kits comprising the exosomes all of which are described infra.

Definitions

As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “therapeutic” is a reference to one or more therapeutics and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45% to 55%.
“CD31”, also known as PECAM-1 (platelet endothelial cell adhesion molecule) is a protein in the immunoglobulin superfamily found on the surface of platelets, monocytes, neutrophils, and some types of T-cells. CD31 makes up a large portion of endothelial cell intercellular junctions. CD31 is encoded in humans by the PECAM-1 gene and is commonly used as a marker for endothelial cells.
“CD34” is a cell surface glycoprotein that functions as a cell-cell adhesion factor. The CD34 protein is a member of a family of single-pass transmembrane sialomucin proteins that are expressed on early hematopoietic and vascular-associated tissue. CD34 is encoded in humans by the CD34 gene. It is commonly used as a marker for hematopoietic and/or vascular endothelial cells.
As used herein, the term “clonal” refers to a population of cells obtained by the expansion of a single cell into a population of cells all derived from that original single cell and not containing other cells. The terms “clonal progenitor cell”, “embryonic clonal progenitor cell”, “clonal progenitor cell line” and “embryonic clonal progenitor cell line” each refer to progenitor cell lines that are derived clonally, i.e., derived by the expansion of a single cell into a population of cells all derived from that original single cell and not containing other cells.
The term “embryonic stem cell” as used herein refers to a pluripotent cell that is derived from a blastocysts, such as an in vitro fertilized blastocyst. Embryonic stem cells include human embryonic stem cells, which are available as established cell lines. The established cell lines are available commercially from numerous public cell banks, e.g. WiCell and private corporations, e.g. ESI BIO.
The term “human pluripotent cell” or “human pluripotent stem cell” as used herein refers to a human cell which is capable of differentiating into at least one cell type found in or derived from each of the three primary germ layers. Some human pluripotent stem cells have the ability to differentiate into all cells found in or derived from each of the three primary germ layers. Examples of human pluripotent stem cells include human embryonic stem cells (Thomson (1998) Science 282:1145), human embryonic germ cells (Shamblott et al. (2001) PNAS 98:113 and induced pluripotent cells (Takahashi et al. (2007) Cell 131:861.
The term “induced pluripotent stem cell” as used herein, refers to a pluripotent cell that has been genetically reprogrammed using any technique known in the art from an adult somatic cell back to the developmentally less mature pluripotent state.
The term “miRNA,” as used herein, refers to microRNA which includes RNA species that are 21-25 nt long and may be single- or double-stranded. MicroRNAs are short, non-coding RNA molecules that have been found in animals, including humans, and in plants. The term encompasses small interfering RNA (siRNA) and small temporal RNA (stRNA), as well as miRNA proper. miRNAs are transcribed as parts of longer RNA molecules and processed in the nucleus by the dsRNA ribonuclease Drosha to hairpin structures 70-100 nucleotides long. These are transported to the cytoplasm where they are digested to 21-23-mers by the dsRNA ribonuclease Dicer. Single-stranded miRNAs bind to complementary sequences in mRNA thereby inhibiting translation.
“miR-126” is a human microRNA that is specifically expressed in endothelial cells, throughout capillaries and in larger blood vessels. miR-126 plays a role in angiogenesis by regulating the expression levels of various genes by pre- and post-transcription mechanisms. As used herein, the term “miR-126” refers to all of the following: the stem-loop miR-126, miR-126-3p (3′ arm of the hairpin precursor) and miR-126-5p (5′ arm of the hairpin precursor). miRNA naming conventions are described in Kozomara and Griffiths-Jones, (2014) Nucleic Acids Res. 42(Database issue):D68. The terms “miR-126-3p” and “hsa-miR-126-3p” are also used interchangeably throughout this application.
The use of “nucleic acid,” “polynucleotide” or “oligonucleotide” or equivalents herein means at least two nucleotides covalently linked together. In some embodiments, an oligonucleotide is an oligomer of 6, 8, 10, 12, 20, 30 or up to 100 nucleotides. In some embodiments, an oligonucleotide is an oligomer of at least 6, 8, 10, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides. A “polynucleotide” or “oligonucleotide” may comprise DNA, RNA, cDNA, PNA or a polymer of nucleotides linked by phosphodiester and/or any alternate bonds.
The term “peptide,” as used herein, refers to two or more amino acids joined by a peptide bond. A peptide can, in some instances, be a portion of a full length protein.
The term “protein” as used herein, refers to a full length protein, i.e. one having all of the amino acids coded for by the mRNA that encodes the particular protein. Also included in the definition are modified proteins where one or more amino acids have been cleaved (e.g. a signal sequence) as a result of the protein being secreted from a cell.
By “pharmaceutically acceptable”, it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The term “pluripotent cell” or “pluripotent stem cell” as used herein, refers to a cell which is capable of differentiating into at least one cell type found in or derived from each of the three primary germ layers. Some pluripotent stem cells have the ability to differentiate into all cells found in or derived from each of the three primary germ layers.
The term “progenitor cell line” as used herein refers to a line of cells that is more differentiated (developed) compared to a pluripotent cell, such as iPS cell or an hES cell, but is not terminally differentiated. Progenitor cells will have enhanced replicative capacity compared to a terminally differentiated cell which typically has senesced. Progenitor cells may also have longer telomere lengths compared to a cell that has terminally differentiated. Progenitor cell lines, when cultured, may be able double in population size at least 5, at least 10, at least 20, at least 30, at least 40, at least 50 times. In some instances progenitor cell lines may be able to double in population size 5-400 times, 10-300 times, 20-200 times, 30-80 times, 40-60 times. One example of a progenitor cell line is an embryonic progenitor cell. Embryonic progenitor cell is obtained from a pluripotent cell such as an iPS cell or a hES as previously described. See West et al. (2008) Regen Med 3:287; US Patent Application Publication Nos. 20080070303 20100184033.
The term “subject,” as used herein includes, but is not limited to, humans, non-human primates and non-human vertebrates such as wild, domestic and farm animals including any mammal, such as cats, dogs, cows, sheep, pigs, horses, rabbits, rodents such as mice and rats. In some embodiments, the term “subject,” refers to a male. In some embodiments, the term “subject,” refers to a female.
The term “suitable media,” as used herein, refers to a solution that can be used to grow cells in culture. A suitable media may include a formulation of salts and/or buffering reagents. A suitable media may include any or all of the following: salts, sugars, amino acids, proteins, growth factors, cytokines, and hormones, additives such as serum, albumin, antibiotics, insulin, selenium and transferrin. Suitable culture media includes for example commercially available culture media such as DMEM, MEM Stem Pro and the like.
A “therapeutically effective amount” of a composition such as a therapeutic agent described infra, e.g. an exosome, is a predetermined amount calculated to achieve the desired effect. In some embodiments, the effective amount is a prophylactic amount. In some embodiments, the effective amount is an amount used to medically treat the disease or condition. The specific dose of a composition administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the composition administered, the route of administration, and the condition being treated. It will be understood that the effective amount administered will be determined by the physician in the light of the relevant circumstances including the condition to be treated, the choice of composition to be administered, and the chosen route of administration. A therapeutically effective amount of composition of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the targeted tissue.
The terms “treat,” “treated,” or “treating,” as used herein, can refer to both therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, symptom, disorder or disease, or to obtain beneficial or desired clinical results. In some embodiments, the term may refer to both treating and preventing. For the purposes of this disclosure, beneficial or desired clinical results may include, but are not limited to one or more of the following: alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

Exosomes

Exosomes of the invention are double membrane bound vesicles secreted from cells of plants and animals, such as mammals including humans, non-human primates, dogs, cats, sheep, cows, pigs, horses, rabbits, mice, rats and guinea pigs to name but a few. Thus exosomes may be isolated from any cell type from any source. In some embodiments of the invention the exosomes of the invention may be secreted from a human cell, such as a human clonal progenitor cell. In some embodiments the exosomes may be secreted from an endothelial human clonal progenitor cell.
The exosomes may contain one or more markers expressed by their cell of origin. In some embodiments the exosomes contain CD63.
The exosomes may contain one or more miRNAs. In some embodiments, the exosomes of the invention contain one or more miRNAs chosen from Table 2 or 4. In some embodiments, the exosomes of the invention contain one or more angiogenic miRNAs. In some embodiments, the exosomes of the invention contain miR-126.
Where the exosomes are derived from a clonal progenitor cell, the exosomes will be of uniform quality and composition. Thus the exosomes isolated from a clonal progenitor cell will not vary as a result of genetic variation of the source cell. The molecular composition of the contents and the bio-physical characteristics of the vesicles will be consistent and reproducible. Moreover, because of the replicative capacity of the human embryonic progenitor cells, the invention provides an overabundance of the exosomes of the invention. This is in direct contrast with exosomes obtained from other sources known in the art where the paucity of the cell type or the problem of senescence limits the availability of a reproducible exosome. Moreover, in certain embodiments the cells giving rise to the exosomes of the invention, are neither transformed nor malignant, thus avoiding any possible concern regarding carcinogenesis of the exosomes.
The exosomes of the invention may have diameter ranging from about 20 nm-130 nm; from about 30 nm-120 nm; about 40 nm-110 nm; about 50 nm-100 nm; about 85 nm-95 nm. In some embodiments the exosomes of the invention have a diameter of about 90 nm. In some embodiments the exosomes of the invention have a diameter of about 88 nm.
The exosomes may be comprised of a lipid bilayer containing transmembrane proteins and may contain hydrophilic components within the vesicle of the exosome. The contents of the vesicle may be derived from the cytoplasm of the cell or from other vesicle structures within the cell, e.g., endosomes. The vesicle may contain nucleic acids, such as DNA, RNA including mRNA, miRNA as well as proteins and peptides.
The exosomes of the invention may serve as depots for the delivery of therapeutic molecules of any kind. The exosomes of the invention can be engineered to contain therapeutic molecules such as nucleic acids, proteins, peptides, small molecules such as drugs and the like. Any technique known in the art can be used to load the exosomes of the invention with a desired therapeutic molecule. For example cationic lipids could be used to transfect the exosomes with a desired nucleic acid such as DNA, RNA, include mRNA and miRNA. HIV tat protein could be used to transport protein or peptide therapeutics into the exosomes of the invention. The therapeutic molecules can be chosen, engineered or designed to have any desired therapeutic effect. For example molecules associated with enhanced angiogenesis could be loaded into the exosomes of the invention, e.g. VEGF.
The secreted exosomes of the invention can be contacted with a target cell (e.g. a cell that is not the same as the cell of origin for the exosome) such that the exosome is taken up by the target cell, e.g. endocytosed. Once inside the cell, the contents of the vesicle may be released into the cytoplasm where the molecules contained within the vesicle may act as signaling molecules in one or more signaling pathways thereby inhibiting or enhancing gene expression. The signaling molecules may act at the level of transcription or translation for example. In some instances, where the vesicles contain RNA, the RNA can be transcribed by the target cell. In some instances where the RNA is a miRNA the miRNA can inhibit gene expression.

Methods of Isolating Exosomes

Exosomes may be isolated from any suitable cell that contains exosomes. Described infra are several exemplary cell and cell types that may be used to implement this method. The method may involve seeding the cell at an appropriate density in a tissue culture vessel and then incubating the cells in a suitable media or buffer for a suitable period of time. In some embodiments the cells may be permitted to attach to the culture vessel before the exosomes are isolated. In other embodiments the cells may be kept in suspension while the exosomes are isolated. The cells may be permitted to replicate in culture before the exosomes are isolated. Alternatively, the exosomes may be isolated from the cells that have not replicated, or replicated minimally (e.g. less than 1 doubling).
To initiate the method the cells are seeded in a tissue culture method at a suitable cell density. The cell density (cells per unit area) may range from about 5 k/cm², about 10 k/cm², about 15 k/cm², about 20 k/cm², about 25 k/cm², about 30 k/cm², about 35 k/cm², about 40 k/cm², about 45 k/cm², about 50 k/cm², about 55 k/cm², about 60 k/cm², about 70 k/cm², about 75 k/cm². In some embodiments the cell density (cells per unit area) may range from about 1 k/cm²-100 k/cm², 10 k/cm²-90 k/cm², 20 k/cm²-80 k/cm², 30 k/cm²-70 k/cm², 40 k/cm²-60 k/cm². In one embodiment the cells are seeded at a density (cells per unit area) of 40 k/cm².
The cells may be seeded in any isotonic solution. In one embodiment a suitable solution may include a suitable buffer. Examples of suitable buffers may include phosphate buffered saline (PBS), HEPES and the like. In other embodiments the cells may be seeded in any suitable cell culture media, many of which are commercially available. Exemplary media include DMEM, RPMI, MEM, Media 199, HAMS and the like. In one embodiment the media is EGM-MV2. The media may be supplemented with one or more of the following: growth factors, cytokines, hormones, serum, such as fetal calf serum, serum substitutes such as knock out replacement serum or B27, antibiotics, vitamins and/or small molecule drugs. In one embodiment the media is supplemented with a TGFβ inhibitor, e.g. SB43154).
The method may be practiced by placing the cells in a suitable environment, such as a cell incubator heated to about 37° C. In some embodiments the cells may be incubated at room temperature. The incubator may be humidified and have an atmosphere that is about 5% CO₂and about 1% 02. In some embodiments the CO₂concentration may range from about 1-20%, 2-10%, 3-5%. In some embodiments the O₂concentration may range from about 1-20%, 2-10%, 3-5%.
The method may be practiced by incubating the cells in the media or buffer for about 1-72 hours, 1-48 hours, 2-24 hours, 3-18 hours, 4-16 hours, 5-10 hours. In some embodiments the cells are incubated for about 16 hours.
Incubation of the cells as described above allows for the exocytosis of the exosomes by the cells into the isotonic solution. After incubation of the cells in the isotonic solution as described above, the isotonic solution may be harvested. For example the isotonic solution may be pipetted or decanted into another vessel such as a centrifuge tube. A precipitating agent may be added to the isotonic solution at this time to facilitate the precipitation of the exosomes in the solution. Examples of precipitating agents include a solution that is about 15% polyethylene glycol. Alternatively, a commercially prepared precipitating agent may be used, e.g., Total Exosome Isolation Reagent (Life Technologies, Carlsbad, Calif.). The cells may then be incubated for a suitable time period e.g., 1-48 hours, 2-24 hours, 3-18 hours, 4-16 hours, 5-10 hours. In some embodiments the cells are incubated for about 16 hours. The cells may be incubated at a temperature of about 1° C., 5° C., 10° C., 15° C., 20° C., 25° C., 30° C. In one embodiment the cells are incubate at about 4° C.
After incubating the harvested cell conditioned isotonic solution with the precipitating reagent described above the harvested cell conditioned isotonic solution may be centrifuged at about 1,000×g, 2,000×g, 4,000×g, 6,000×g, 8,000×g; 10,0000×g; 12,000×g, 14,000×g, 16,000×g; 18,000×g. In one embodiment the harvested cell conditioned isotonic solution is centrifuged at about 10,000×g. The harvested cell conditioned isotonic solution may be centrifuged at about a temperature of 2° C., 4° C., 6° C., 8° C., 10° C., 12° C., 14° C., 16° C., 18° C., 20° C., 22° C., 24° C., 26° C. In one embodiment the harvested cell conditioned isotonic solution are centrifuged at about a temperature of 4° C.
After centrifugation the isotonic solution is removed and the exosomes are resuspended in a suitable buffer such as PBS. The volume of buffer may be about 0.01 volumes-about 0.09 volumes, about 0.02 volumes to about 0.08 volumes; about 0.03 volumes to about 0.07 volumes of the precipitating solution. In one embodiment the harvested cell conditioned isotonic solution is resuspended in PBS at a volume equivalent to about 0.01 volumes of the precipitating solution. The harvested exosomes may be used immediately or frozen and stored, e.g., at −20° C., for later use.

Progenitor Cells

In certain embodiments of the invention progenitor cells serve as the source of the exosomes described infra. The progenitor cell may be from any animal or plant. For example the exosome may be from a mammal, such as a human, a non-human primate, a horse, a cow, a sheep, a goat, a pig, a cat, a dog, a rabbit, a guinea pig, a rodent such as a mouse or a rat. Typically a progenitor cell will not have an essentially unlimited replicative capacity as typically found in embryonic stem cells, but will nonetheless have, a result of their longer telomeres, a greater replicative capacity compared to adult primary cells or tissues (e.g. primary cells) or adult stem cells.
The progenitor cell may be derived from a pluripotent stem cell, such as an embryonic stem cell or an induced pluripotent stem cell. The progenitor cell may be a clonal cell or an oligoclonal cell. An oligoclonal cell would include a population of cells similar cells, e.g. phenotypically or genetically. The progenitor cell may be a clonal human embryonic progenitor cell. The progenitor cell may be a clonal human embryonic endothelial progenitor cell. The progenitor cell may be a clonal embryonic progenitor cell that expresses CD31 and CD36. The progenitor cell may be a clonal embryonic progenitor cell expressing one or more genes listed in Table 1. The progenitor cell may be a clonal embryonic progenitor cell expressing a plurality of the genes listed in Table 1. The progenitor cell may be a clonal embryonic progenitor cell expressing the genes listed in Table 1.
Where the progenitor cells are clonal cells obtained from pluripotent stem cells they will provide an almost unlimited source of the same exosomes. This is due to two factors: the genetic identity of the original cellular source material and the enhanced telomere lengths found in early progenitors which provide for enhanced replicative capacity relative to adult tissue or cells or adult stem cells. Moreover, unlike adult stem cells which are typically available in very small numbers and are difficult to expand in culture, the clonal embryonic progenitors described infra are available in large numbers and are relatively easy to expand in culture.
In some embodiments the progenitor cell is not an adult stem cell. In some embodiments of the invention the progenitor cell is not an MSC. In some embodiments the clonal progenitor cell is not transfected or engineered to express an exogenous gene. In some embodiments the clonal progenitor cell is not transfected to express an oncogene. In some embodiments the clonal progenitor does not express c-myc. In other embodiments the clonal progenitor cell is transfected or engineered to express an exogenous gene. Examples of suitable exogenous genes include the catalytic component of human telomerase, e.g. hTERT.

Uses of Exosomes

The exosomes described herein may be used in therapeutic, research and diagnostic applications. For example the exosomes described infra may be added to a cell culture to enhance one or more phenotypic traits of the cells. The exosomes of the invention may be added to a cell culture to inhibit one or more phenotypic traits of the cells. The exosomes of the invention may be added to a cell culture to provide a new phenotypic trait of the cells.
The exosomes of the invention may be added to a culture of endothelial cells to enhance the ability of the cells to form vascular tube like structures. The exosomes of the invention may be added to any cell having the ability to form vascular tube like structures to enhance the cells ability to form tube like structures.
In some embodiments the exosomes of the invention are contacted with a cell thereby providing at least one new phenotypic trait to the cell. For example, the exosomes of the invention may confer the ability to form vascular tube like structures to cell lacking the ability to form vascular tube like structures before it was contacted with the exosomes of the invention.
In certain embodiments the exosomes of the invention may be added to a culture of perivascular cells to enhance the ability of the perivascular cells to form vascular tube like structures.
In some embodiments the invention provides a method of increasing the length of a vascular tube like structure formed by a cell such as an endothelial relative to an endothelial cell that has not been treated with the exosomes of the invention comprising contacting the endothelial cell with an exosome isolated from a progenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6 cells. In some embodiments the invention provides a method of increasing the length of a vascular tube like structure formed by a cell such as a perivascular cell relative to a perivascular cell that has not been treated with the exosomes of the invention comprising contacting the perivascular cell with an exosome isolated from a progenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6 cells. In some embodiments the invention provides a method of increasing the branching of a vascular tube like structure formed by an endothelial cell relative to an endothelial cell that has not been treated with the exosomes of the invention comprising contacting the endothelial cell with an exosome isolated from a progenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6 cells. In some embodiments the invention provides a method of increasing the branching of a vascular tube like structure formed by a perivascular cell relative to a perivascular cell that has not been treated with the exosomes of the invention comprising contacting the perivascular cell with an exosome isolated from a progenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6 cells. In still other embodiments the invention provides a method of increasing the number of loops in the vascular tube like structures formed by an endothelial cell relative to an endothelial cell that has not been treated with the exosomes of the invention comprising contacting the endothelial cell with an exosome isolated from a progenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6 cells. In yet other embodiments the invention provides a method of increasing the number of loops in the vascular tube like structures formed by a perivascular cell relative to a perivascular cell that has not been treated with the exosomes of the invention comprising contacting the perivascular cell with an exosome isolated from a progenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6 cells.
The exosomes of the invention may be administered therapeutically to a subject in need of treatment. For example the exosomes of the invention may be administered to a subject in need of treatment for any disease requiring the enhanced ability to form vascular tube like structures. The exosomes of the invention may be used to treat a subject suffering from cardiovascular disease, heart failure, infarction, chronic wounds, ulcer, clogged vessels or arteries, damaged vessels, stenotic vessels, arteriosclerosis, angina, peripheral vascular disease, Alzheimer's disease, ischemia, diabetes, cancer, cell replacement transplant or therapy, tissue and cell regenerative therapy and Parkinson's disease. The exosomes may be used as depot to deliver therapeutic molecules such as small molecules, nucleic acids, proteins and peptides.
The exosomes of the invention may be directly administered to a subject in need of treatment or an in vitro cell culture. Alternatively the exosomes can be provided enclosed within a matrix or scaffold. Suitable matrices or scaffolds may include a matrix or scaffold comprised of one or more extracellular matrix proteins, e.g. laminin, fibronectin and the like. Other suitable matrices or scaffolds include Matrigel® which is a murine sarcoma extract. The matrix or scaffold may be a hydrogel. The hydrogel may be comprised of hylauronate and gelatin (see U.S. Pat. Nos. 8,324,184; 7,928,069). In one embodiment the exosomes of the invention may be delivered in HyStem (Biotime, Inc., Alameda Calif.).
Using the methods described infra along with routine chromatographic techniques known in the art the exosomes of the invention may be used to isolate one or more nucleic acids, proteins or peptides expressed by a progenitor cell serving as the source of the exosome. Once isolated, the proteins or peptides isolated from the exosomes of the invention can be used to make antibodies to the isolated proteins or peptides (See Harlow et al. Antibodies: A Lab Manual 2^ndEdition; Cold Spring Harbor Press 2013).
The exosomes of the invention may be used in drug screening assays. For example where the exosomes described infra enhance vascular tube formation in vitro, the exosomes can be used to screen for drugs that enhance or inhibit this capability. A cell culture comprising cells having the ability to form vascular tube like structures may be contacted with the exosomes of the invention and a drug candidate may be applied to the same cell culture either before, after or simultaneously with the exosomes to determine the effect of the drug the ability of the exosomes to enhance vascular tube formation in the cell culture. The effects can be compared to untreated cells and cells treated only with the exosomes of the invention.

Pharmaceutical Compositions

Modes of administration for a therapeutic (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.
Specific modes of administration will depend on the indication. The selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response. The amount of therapeutic to be administered is that amount which is therapeutically effective. The dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular animal treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).
Pharmaceutical formulations containing the therapeutic of the present disclosure and a suitable carrier can be solid dosage forms which include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules; topical dosage forms which include, but are not limited to, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments, pastes, creams, gels and jellies, and foams; and parenteral dosage forms which include, but are not limited to, solutions, suspensions, emulsions, and dry powder; comprising an effective amount of a polymer or copolymer of the present disclosure. It is also known in the art that the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) can be consulted.
The compositions of the present disclosure can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. The compositions can be administered by continuous infusion subcutaneously over a period of about 15 minutes to about 24 hours. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
For oral administration, the compositions can be formulated readily by combining the therapeutic with pharmaceutically acceptable carriers well known in the art. Such carriers enable the therapeutic of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinyl pyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic doses.
Pharmaceutical preparations which can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as, e.g., lactose, binders such as, e.g., starches, and/or lubricants such as, e.g., talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active therapeutic can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the pharmaceutical compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.
For administration by inhalation, the therapeutic for use according to the present disclosure is conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the therapeutic and a suitable powder base such as lactose or starch.
The compositions of the present disclosure can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the therapeutic of the present disclosure can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
Depot injections can be administered at about 1 to about 6 months or longer intervals. Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
In transdermal administration, the compositions of the present disclosure, for example, can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism.
Pharmaceutical compositions can include suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.
The compositions of the present disclosure can also be administered in combination with other active ingredients, such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.
In some embodiments, the disintegrant component comprises one or more of croscarmellose sodium, carmellose calcium, crospovidone, alginic acid, sodium alginate, potassium alginate, calcium alginate, an ion exchange resin, an effervescent system based on food acids and an alkaline carbonate component, clay, talc, starch, pregelatinized starch, sodium starch glycolate, cellulose floc, carboxymethylcellulose, hydroxypropylcellulose, calcium silicate, a metal carbonate, sodium bicarbonate, calcium citrate, or calcium phosphate.
In some embodiments, the diluent component may include one or more of mannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powdered cellulose, microcrystalline cellulose, carboxymethylcellulose, carboxyethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodium starch glycolate, pregelatinized starch, a calcium phosphate, a metal carbonate, a metal oxide, or a metal aluminosilicate.
In some embodiments, the optional lubricant component, when present, comprises one or more of stearic acid, metallic stearate, sodium stearylfumarate, fatty acid, fatty alcohol, fatty acid ester, glycerylbehenate, mineral oil, vegetable oil, paraffin, leucine, silica, silicic acid, talc, propylene glycol fatty acid ester, polyethoxylated castor oil, polyethylene glycol, polypropylene glycol, polyalkylene glycol, polyoxyethylene-glycerol fatty ester, polyoxyethylene fatty alcohol ether, polyethoxylated sterol, polyethoxylated castor oil, polyethoxylated vegetable oil, or sodium chloride.

Kits

In some embodiments the invention provides a kit comprising exosomes isolated from a progenitor cell, such as a human clonal progenitor cell. The progenitor cell may be an endothelial progenitor cell, such as human clonal embryonic progenitor cell, e.g. 30MV2-6. The exosomes may be provided in one or more containers. The exosomes may be provided in a suitable buffer, e.g. PBS or a suitable media, such as a commercially available cell culture media, e.g. DMEM. The kit may further contain a cell having the ability to form vascular tube like structures. The cell may be an endothelial cell, e.g. HUVEC and/or a perivascular cell. The cells may be provided in a suitable media, e.g. DMEM or the like or alternatively the cells may be provided in a buffer such as PBS. In some embodiments the cells may be provided frozen in a suitable freezing media such as a commercially available media supplemented with DMSO. The kit may optionally include instructions as to how to reconstitute the exosomes, culture the cells and/or contact the cells with exosomes so as to enhance vascular tube like formation.
In other embodiments the invention provides a kit comprising a human clonal embryonic progenitor cell, such as 30-MV2-6. The cell may be provided in at least one container in suitable media or buffer. The kit may include buffers and/or media for isolating exosomes from the cells. The kit may contain one or more vessels, e.g. a multi-well plate for culturing the cells. The kit may further contain a cell line capable of forming vascular tube like structures such as endothelial cells. Suitable cells include endothelial cells such as HUVEC and/or a a perivascular cell. Any or all of the cells may be provided frozen in a suitable media, e.g. freezing media such as a commercially available media supplemented with DMSO. The kit may optionally include instructions as to how to culture the cells and/or contact the endothelial cells with exosomes isolated from the progenitor cells so as to enhance or induce vascular tube like formation.

Additional Embodiments of the Invention

1. An exosome isolated from a progenitor cell line.
2. The exosome of 1, wherein the progenitor cell line is a human progenitor cell line.
3. The exosome of 1, wherein the progenitor cell line is a clonal progenitor cell line.
4. The exosome of 1, wherein the progenitor cell line is an endothelial progenitor cell line.
5. The exosome of 1, wherein the exosome contains CD63.
6. The exosome of 1, wherein the exosome contains one or more miRNAs listed in Table 2 or Table 4.
7. The exosome of 1, wherein the exosome contains one or more angiogenic miRNAs.
8. The exosome of 1, wherein the exosome contains miR-126.
9. The exosome of 1, wherein the progenitor cell line expresses CD31 and
CD34.
10. The exosome of 1, wherein the progenitor cell line expresses one or more genes listed in Table 1.
11. The exosome of 1, wherein the progenitor cell line is 30-MV2-6.
12. The exosome of 1, wherein the exosome enhances the formation of vascular tube like formations when contacted with an endothelial cell.
13. The exosome of 1 further comprising a pharmaceutical carrier.
14. A method of isolating an exosome from a clonal progenitor cell comprising 1) culturing the clonal progenitor cell in a suitable media or buffer for a time sufficient to allow the clonal progenitor cell to exocytose exosomes into the culture media or buffer; 2) harvesting the media or buffer from the cell culture of step 1; and 3) isolating the exosomes from the media or buffer of step 2, thereby isolating an exosome from a clonal progenitor cell.
15. The method of 14, wherein the suitable media or buffer is PBS.
16. The method of 14, wherein the suitable media or buffer is EGM-MV2.
17. The method of 14, wherein after step 2 a precipitating agent is added to the media or buffer.
18. The method of 14, wherein the precipitating agent comprises polyethylene glycol.
19. The method of 14, wherein step 3 comprises centrifuging the harvested media of buffer.
20. The method of 14, wherein the suitable time of step 1 is about 16 hours.
21. The method of 14, wherein after step 2, the method further comprises a step of incubating harvested media or buffer.
22. The method of 21, wherein the incubation step is performed for about 16 hours.
23. The method of 21, wherein the incubation step is performed at about 4° C.
24. A cell culture comprising an exosome isolated from a progenitor cell and a cell which was not the source of the isolated exosome.
25. The exosome of 24, wherein the progenitor cell line is a human progenitor cell line.
26. The exosome of 24, wherein the progenitor cell line is a clonal progenitor cell line.
27. The exosome of 24, wherein the progenitor cell line is an endothelial progenitor cell line.
28. The exosome of 24, wherein the exosome contains CD63.
29. The exosome of 24, wherein the exosome contains one or more miRNAs listed in Table 2 or Table 4.
30. The exosome of 24, wherein the exosome contains one or more angiogenic miRNAs.
31. The exosome of 24, wherein the exosome contains miR-126.
32. The exosome of 24, wherein the progenitor cell line expresses CD31 and CD34.
33. The exosome of 24, wherein the progenitor cell line expresses one or more genes listed in Table 1.
34. The exosome of 24, wherein the progenitor cell line is 30-MV2-6.
35. The exosome of 24, wherein the exosome enhances the formation of vascular tube like formations when contacted with an endothelial cell.
36. A method of inducing or enhancing a cell's ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a progenitor cell thereby inducing or enhancing a cells ability to form vascular tube like structures.
37. The method of 36, wherein the cell capable of making vascular tube like structures is an endothelial cell.
38. The method of 37, wherein the endothelial cell is a HUVEC.
39. A method of treating a subject in need of vascular therapy comprising administering an exosome isolated from a progenitor cell.
40. The method of 39, wherein the subject is human.
41. The method of 39, wherein the subject exosome is administered to the subject to treat a condition chosen from cardiovascular disease, heart failure, infarction, chronic wounds, ulcer, clogged vessels or arteries, damaged vessels, stenotic vessels, arteriosclerosis, angina, peripheral vascular disease, Alzheimer's disease, ischemia, diabetes, cancer, cell replacement transplant or therapy, tissue and cell regenerative therapy and Parkinson's disease.
42. The method of 39, wherein the progenitor cell line is a human progenitor cell line.
43. The method of 39, wherein the progenitor cell line is a clonal progenitor cell line.
44. The method of 39, wherein the progenitor cell line is an endothelial progenitor cell line.
45. The method of 39, wherein the exosome contains CD63.
46. The method of 39, wherein the exosome contains one or more miRNAs listed in Table 2 or Table 4.
47. The method of 39, wherein the exosome contains one or more angiogenic miRNAs.
48. The method of 39, wherein the exosome contains miR-126.
49. The method of 39, wherein the progenitor cell line expresses CD31 and CD34.
50. The method of 39, wherein the progenitor cell line expresses one or more genes listed in Table 1.
51. The method of 39, wherein the progenitor cell line is 30-MV2-6.
52. The method of 39, wherein the exosome enhances the formation of vascular tube like formations when contacted with an endothelial cell.
53. The method of 39 further comprising a pharmaceutical carrier.

EXAMPLES

Example 1: Preparation of Exosomes Derived from a Human Embryonic Progenitor Cell Line

Exosomes were prepared from a human embryonic progenitor cell line (PureStem® cell line, ESI Bio, Alameda, Calif.). PureStem® cell lines are scalable clonally pure embryonic progenitor cell lines derived from human embryonic stem (hES) cells (West et al. (2008) Regen Med. 3(3):287). The 30-MV2-6 PureStem® cell line is a CD31 positive, CD34 positive, endothelial progenitor line derived from the ESI-017 embryonic stem cell line. A gene expression profile of the 30-MV2-6 cells as analyzed by microarray is provided herein in Table 1, and includes genes yielding relative fluorescence units >1000 rfu.

	TABLE 1

		30-MV2-6 P6,
	Gene symbol	MBA_3877

	EEF1A1	29470.94
	EEF1A1	28581.27
	EEF1A1	28032.06
	TMSB4X	27627.77
	GNB2L1	27295.94
	TPT1	26985.77
	LOC100129758	26697.4
	LAIR1	26461.51
	F2R	26252
	LOC285176	26055.83
	RPL41	25872.51
	NAG18	25689.47
	LOC649150	25513.84
	FTL	25344.95
	LOC91561	25177.92
	LOC100132593	25023.94
	RPLP2	24889.1
	LOC388474	24755.51
	MGC16703	24611.59
	UBC	24480.02
	LOC389342	24350.84
	CLUAP1	24238.08
	RPS27	24117.06
	ZNF674	23992.58
	IMAA	23881.17
	RRP7B	23769.37
	C19orf31	23655.06
	ANXA2P2	23547.03
	LOC401206	23440.4
	LOC100133876	23333.52
	GGA1	23233.85
	MSH3	23140.74
	LOC729439	23047.74
	LOC440589	22950.21
	UBC	22847.36
	FTL	22745.35
	LOC100130553	22658.54
	LOC728658	22562.38
	ACTG1	22481.09
	LOC644604	22386.22
	RPL38	22292.94
	LOC642210	22201
	LOC148430	22112.95
	LOC100133465	22026.54
	LOC400963	21937.41
	TMSB10	21854.66
	LOC284393	21771.97
	RN7SL1	21682.27
	RPS29	21599.25
	LOC100133931	21519.28
	RPS12	21431.97
	ITIH5	21348.51
	LOC341457	21263.76
	LOC642250	21185.09
	KIAA0101	21102.53
	LOC389435	21019.57
	LOC100132488	20946.02
	ANXA2	20864.81
	LOC441034	20786.82
	RPS19	20710.05
	ACTB	20640.19
	LOC440589	20572.73
	LOC388720	20496.3
	LOC728658	20419.73
	FAM177A1	20346.14
	LOC387930	20271.08
	LOC440595	20196.18
	LOC653232	20127
	ORC6L	20058.03
	RPS25	19986.5
	ACTB	19922.52
	LOC100130980	19848.39
	PDCD7	19772.26
	RPL18A	19703.81
	LOC642892	19628.75
	LOC727808	19558.66
	LOC389223	19478.59
	ROCK2	19416.46
	CCR6	19350.04
	RPS15A	19290.37
	RPS11	19219.96
	RPL18	19149.27
	RPL6	19086.13
	RPS29	19022.98
	RPL38	18959.29
	RPL27A	18894.7
	RPS27	18834.63
	LOC387841	18770.86
	TUBA1A	18706.36
	LOC647361	18639.92
	RPL11	18576.44
	LOC100130446	18517.91
	LOC728553	18455.8
	UBA52	18391.57
	LOC728576	18328.88
	LOC100129553	18269.35
	MYL6	18203.91
	ACTG1	18140.81
	FTHL16	18076.95
	LOC441876	18016.04
	LOC343184	17954.72
	LOC391777	17893.68
	RPL9	17830.68
	RPS27A	17768.43
	LOC643863	17711.83
	RPS6	17652.27
	PSMD12	17594.21
	MYL6	17535.65
	LOC646195	17476.44
	RPL32	17418.94
	RPLP0	17356.64
	LOC729402	17294.36
	RPS17	17236.98
	RPL35A	17181.16
	RPL11	17118.82
	RPL18A	17060.66
	LOC100129158	17003.18
	LOC401019	16944.5
	LOC100133607	16889.64
	LOC729324	16834.91
	LOC645895	16775.01
	LOC649076	16721.19
	RPS16	16662.93
	PPIAL4A	16607.66
	RPL17	16550.82
	RPS10	16491.53
	LOC440733	16435.98
	RPL18A	16384.7
	LOC645899	16331.75
	LOC644029	16276.86
	VIM	16220.75
	LOC100129902	16168.98
	LOC440027	16117.11
	LOC728517	16063.24
	C10orf58	16009.07
	LOC728368	15951.03
	LOC439953	15892.32
	LOC651894	15837.98
	LOC644745	15784.83
	FARSLB	15733.5
	LOC388524	15675.03
	FTHL7	15621.63
	RPL27	15573.05
	RPL12	15524.71
	RPL37A	15473.36
	RPLP1	15423.13
	RPS20	15371.34
	RPS14	15315.83
	XPNPEP3	15265.39
	LOC389101	15209.69
	LOC402057	15164.27
	LOC643509	15114.24
	LOC284230	15063.66
	LOC642357	15015.66
	LOC652071	14963.6
	RPL39	14914.66
	LOC728576	14864.7
	LOC644464	14816.74
	ACTB	14769.08
	RPL30	14718.38
	RPS3	14668.78
	IFITM2	14620.77
	LOC644039	14568.97
	LOC645683	14518.14
	FAM115A	14471.98
	RPL19	14423.17
	LOC653162	14375.34
	LOC441775	14323.48
	LGALS1	14273.15
	RPS24	14227.73
	RPL3	14179.84
	RPS15	14130.03
	GNG11	14079.57
	CAV1	14033.15
	LOC729090	13979.81
	TM4SF1	13927.96
	NACA	13881.36
	ATP5EP2	13834.41
	LOC653881	13785.78
	RPL17	13738.14
	OAZ1	13689.32
	LOC645296	13641.2
	S100A10	13595.26
	RPL31	13545.91
	RPL24	13494.67
	LOC100128505	13442.05
	LOC645174	13394.88
	LOC648729	13345.11
	RPL8	13298.89
	LOC731096	13253.78
	LOC642741	13211.35
	LOC647276	13169.46
	LOC729903	13121.3
	LOC401019	13076.43
	LOC728128	13029.34
	CD81	12985.19
	LOC731542	12939.34
	LOC651436	12894.54
	RPL10A	12850.49
	LOC441013	12803.59
	RPS4X	12755.92
	GJC1	12710.73
	LDB2	12664.08
	RPS8	12614.56
	BTF3	12567.71
	WBP5	12522.85
	LOC650276	12477.88
	PFN1	12433.05
	RPL6	12389.93
	LOC100129141	12346.77
	ATP5B	12304.98
	CD93	12259.89
	VIM	12217.76
	LOC730754	12172.71
	HSPB1	12127.76
	LOC728453	12085.1
	EIF4A1	12041.45
	LOC389404	11999.19
	CD151	11957.83
	LOC647276	11912.95
	LOC729789	11868.46
	LOC728937	11828.98
	IFITM3	11788.35
	LILRB3	11744.43
	LOC646294	11705.26
	RPS2	11661.18
	FSCN1	11621.16
	LOC441246	11578.21
	LOC645387	11538.01
	LOC647099	11499.43
	PRCP	11460.34
	SOX18	11422.95
	LOC440575	11382.54
	LOC641814	11344.01
	KCNH6	11306.56
	LOC653314	11266.4
	LOC100133649	11225.89
	LOC729603	11184.33
	TUBA1C	11147.33
	LOC286444	11110.15
	LOC643531	11068.19
	LOC643284	11030.72
	AP2S1	10993.52
	PTRF	10952.09
	H3F3A	10913.04
	LOC100132742	10876.23
	LOC648210	10835.95
	EIF3E	10798.77
	RPL3	10762.44
	TXN	10725.63
	RPS29	10688.26
	MYL12A	10651.34
	GABPB2	10615.8
	RPS9	10581.37
	ATP5EP2	10543.17
	LOC647000	10509.38
	UBB	10473.09
	LOC388556	10436.98
	LOC728693	10403.82
	NGFRAP1	10370.85
	COX7C	10337.94
	GAPDH	10305.26
	GNAS	10271.86
	LOC400721	10233.35
	ICAM2	10200.27
	RPS3A	10164.91
	LOC100131713	10128.06
	B2M	10097.39
	UBB	10066.7
	CLEC2D	10033.68
	MGC26356	10004.77
	LOC100133273	9971.113
	TPT1	9939.753
	RPSA	9911.836
	RPS13	9882.49
	ENO1	9852.533
	LOC100132742	9822.571
	LOC729617	9790.951
	S100A10	9759.989
	LOC730187	9729.674
	LOC648000	9697.648
	LOC644464	9669.355
	RPS5	9640.032
	RPL14L	9608.673
	RPL36AL	9578.568
	NEDD8	9548.513
	RPS6	9520.098
	TGFBR2	9492.364
	RPLP1	9463.913
	LOC440926	9432.986
	TUBA1B	9407.654
	LOC284821	9377.909
	BTF3	9350.254
	LOC730246	9325.183
	LOC731365	9295.564
	LOC729466	9265.775
	LOC646200	9235.456
	ADAM15	9210.81
	RPS27L	9182.401
	AKR1D1	9154.34
	CYB5R3	9128.27
	RPS3A	9101.282
	RPS4X	9076.407
	CREB1	9049.403
	PDE4C	9022.809
	TFPI	8997.954
	LOC728782	8971.264
	LOC645387	8944.967
	SEPT2	8917.304
	GLTSCR2	8894.782
	SLC25A5	8867.191
	LOC646294	8843.528
	PECAM1	8815.434
	H3F3A	8791.886
	LOC649548	8767.153
	POTEF	8740.947
	TGFBR2	8714.794
	VWF	8689.385
	ITGB1	8666.613
	LOC729301	8641.795
	LOC100133812	8618.085
	EIF3L	8594.98
	LOC642947	8573.153
	DNCL1	8550.102
	TFPI	8526.81
	CDKN2AIPNL	8504.479
	VAMP5	8479.908
	CDH5	8455.61
	LRAP	8433.076
	RHOC	8409.996
	CDKN1A	8386.336
	S100A6	8366.138
	LOC645385	8343.072
	SNRPD2	8321.468
	ATP5A1	8298.988
	LDHA	8275.572
	EEF2	8253.723
	LOC389141	8231.352
	COX4I1	8212.02
	RPS9	8190.523
	LOC650152	8168.617
	CDC37	8145.008
	LOC643358	8121.837
	LOC100133233	8102.312
	YWHAQ	8082.7
	SNX3	8061.547
	AV762104	8040.466
	H3F3A	8017.827
	PFN1	7996.922
	GAPDH	7973.344
	YWHAZ	7953.511
	RPS14	7934.452
	RPL15	7915.846
	FAU	7885.26
	GPX1	7885.26
	LOC728620	7854.277
	RPL12	7833.73
	LOC648294	7816.09
	SLC16A12	7795.425
	LOC645715	7775.663
	RPS3A	7756.622
	ALDOA	7737.847
	CDK2AP1	7718.065
	TCEAL4	7699.396
	EEF1G	7679.361
	LOC646766	7659.651
	LOC100190938	7640.343
	C21orf55	7621.212
	EIF4G2	7602.998
	LOC100128731	7583.863
	LOC100133177	7566.641
	ZNF430	7547.497
	CCNI	7529.735
	RPL36	7510.359
	SERPINB6	7494.061
	LOC285053	7475.076
	EEF1B2	7455.902
	C11orf10	7436.825
	CALM3	7420.417
	LOC441087	7403.128
	TUBA1A	7386.972
	ZMAT3	7370.062
	KLF6	7351.817
	LOC643031	7336.027
	PABPC1	7319.402
	FKTN	7301.646
	CFL1	7282.935
	LOC644863	7265.614
	RPS27A	7248.343
	RPS17	7229.865
	COX7A2	7213.416
	RPS15A	7198.388
	LOC100134134	7180.453
	ATP5G2	7162.671
	IL18	7144.369
	LOC283412	7127.359
	NUCB1	7110.135
	LOC729798	7092.872
	LOC387867	7076.424
	PCBP1	7060.549
	MRLC2	7043.793
	LOC389517	7027.69
	LOC399900	7013.314
	SERF2	6997.182
	EEF1B2	6981.873
	MARCKS	6967.221
	NACA	6951.604
	LOC100129424	6936.077
	NPC2	6920.058
	RPL35	6904.67
	GPX4	6889.884
	C10orf58	6874.525
	TOMM7	6860.304
	SLC25A3	6845.603
	MDH2	6832.719
	RPL26	6817.137
	ATP5I	6800.876
	CTNNA1	6785.87
	RALA	6771.026
	FAM69B	6754.661
	CALM1	6738.351
	SHCBP1	6723.184
	SRP14	6708.369
	LOC646531	6693.502
	TACC1	6679.065
	DPYSL2	6663.472
	LOC100127993	6648.476
	LOC100130168	6634.481
	LOC285900	6620.755
	RPL7L1	6604.924
	PCBP2	6590.635
	PNPT1	6575.888
	HCG2P7	6562.18
	GPR116	6548.164
	H2AFZ	6534.282
	COX6C	6521.256
	ANXA5	6508.408
	NQO1	6495.337
	DAD1	6480.547
	COL4A1	6468.127
	ATP5A1	6454.348
	ZNF549	6440.91
	MYH9	6426.936
	SEC61G	6412.935
	FKBP1A	6399.774
	ARPC2	6387.262
	EIF4A2	6373.784
	EMP1	6360.16
	LOC729102	6346.829
	DDX5	6333.568
	HINT1	6318.728
	LOC645436	6305.671
	NOP10	6292.858
	PMP22	6279.687
	PSMB1	6265.957
	SQSTM1	6253.051
	LOC653737	6240.21
	HSPA8	6226.94
	TUBB	6214.39
	SHANK3	6201.642
	UQCRH	6188.65
	LOC730313	6176.263
	ATP5L	6163.733
	LOC100133372	6150.941
	LOC550643	6139.155
	TXN	6126.585
	DBI	6113.103
	TMBIM6	6100.574
	SEPT2	6088.34
	LOC642489	6075.764
	CDAN1	6062.012
	PLSCR3	6050.002
	LOC649049	6038.353
	JUND	6026.033
	YWHAH	6013.875
	GSTP1	6002.531
	HSP90AA1	5991.17
	SLC44A4	5979.221
	PSAP	5967.178
	CLDN5	5954.744
	LOC100130445	5943.334
	HNRNPD	5931.25
	SOD1	5919.922
	EEF1AL7	5909.133
	LOC647856	5897.997
	TM4SF18	5886.761
	PTBP1	5875.687
	RAN	5864.145
	RPL4	5852.968
	RAC1	5840.897
	CSTB	5829.299
	C14orf156	5817.476
	NME1-NME2	5807.022
	ITM2B	5796.124
	BGN	5785.701
	SCD	5776.214
	LOC645317	5764.427
	CMTM7	5753.074
	TOMM7	5741.794
	SEC61G	5730.923
	PPM1F	5719.127
	SLC25A3	5709.5
	ACVRL1	5699.607
	COMMD6	5689.162
	CLIC1	5677.854
	C17orf45	5667.08
	PRDX1	5656.452
	SAT1	5645.519
	SEPT9	5634.972
	ATP6AP2	5624.128
	CSDA	5613.424
	PRDX1	5602.954
	BTG1	5592.841
	MTCH1	5582.283
	LOC134997	5573.499
	LOC286444	5563.255
	RPS18	5552.792
	HLA-E	5543.25
	EDF1	5532.461
	ITGB1	5522.71
	MGST2	5511.287
	EIF3L	5500.726
	TM4SF18	5490.636
	NONO	5480.652
	ECSCR	5470.367
	PSAP	5460.726
	PSMA6	5451.676
	MARCKSL1	5442.01
	LOC729742	5432.988
	LOC100131387	5423.154
	NGFRAP1	5413.279
	MAP4K2	5402.065
	BEXL1	5392.217
	TBCA	5382.14
	EIF1	5371.946
	MCART1	5360.794
	MCM8	5351.053
	PSMB6	5340.972
	DAZAP2	5331.065
	QARS	5320.504
	LOC440055	5310.703
	APLNR	5301.342
	RPL13A	5290.971
	C14orf85	5281.173
	CNN3	5271.19
	LOC100132795	5260.873
	LAMA5	5251.119
	SLC44A1	5241.342
	LOC100131609	5232.272
	ARL16	5222.225
	LOC100129362	5212.558
	WSB1	5203.553
	TSPO	5193.677
	LOC645173	5184.411
	PRCP	5175.037
	ESD	5165.838
	HNRNPD	5156.636
	LOC648771	5148.032
	CST3	5139.557
	PRKAR1A	5130.244
	EPAS1	5121.617
	HSPA8	5112.767
	TPI1	5103.14
	NFIB	5094.79
	LOC646942	5086.126
	NCOA4	5077.186
	FLOT2	5069.23
	LOC729978	5060.045
	IGFBP4	5051.913
	LOC650646	5043.719
	ANP32B	5034.827
	CCND1	5026.154
	ATP5J	5017.307
	SHFM1	5008.422
	ATP5O	5000.003
	LOC440927	4992.161
	RHOA	4983.717
	H2AFY	4975.798
	CTGF	4966.956
	LOC728809	4957.689
	RALB	4949.349
	FABP5L2	4940.152
	NDUFB2	4932.081
	LOC646483	4923.669
	GNAI2	4916.183
	MRPL33	4908.738
	DDB1	4900.69
	LOC441073	4892.921
	LOC649447	4885.438
	WDR1	4877.46
	LOC400948	4868.795
	TIMP1	4860.48
	LOC402251	4852.983
	GNB1	4845.2
	RPL36AL	4837.16
	NOP10	4828.969
	CHCHD2	4821.331
	HIST1H4C	4813.682
	FLJ46309	4805.906
	ANGPT2	4798.277
	C20orf52	4790.903
	HOXB5	4782.823
	NDUFS5	4774.766
	UQCRQ	4766.396
	LOC402694	4758.111
	LOC644914	4750.406
	CXCR4	4742.489
	WBP2	4733.885
	COX5B	4725.663
	LOC646630	4717.094
	PRDX5	4704.898
	RPS26P11	4704.898
	NFKBIA	4694.165
	LOC728481	4686.756
	LOC100128084	4679.009
	SRGN	4670.712
	LOC645452	4663.261
	ARHGDIB	4655.951
	SUMO2	4648.817
	TRAM1	4641.106
	LDHA	4633.259
	MYH9	4625.853
	CTGLF7	4618.362
	LOC388654	4611.233
	CALM2	4604.143
	POFUT1	4596.614
	HDAC1	4588.697
	ROMO1	4581.606
	SHANK3	4574.51
	RPL5	4566.714
	NDUFAF3	4559.808
	GSTO1	4551.681
	SRP9	4544.382
	CCDC72	4537.007
	EIF3F	4529.952
	SRGN	4518.68
	RPS6P1	4518.68
	PFDN5	4508.432
	SCARB2	4500.946
	ESAM	4494.812
	HNRNPAB	4487.656
	EGLN2	4480.514
	LOC401537	4473.142
	EMP3	4466.904
	COX5A	4459.584
	SHC1	4452.944
	LOC648249	4445.896
	ANXA1	4438.55
	LOC728428	4431.064
	COMMD7	4424.43
	LOC392437	4417.459
	CSNK1E	4410.571
	GNS	4403.982
	LAMP1	4397.37
	DNAJA1	4390.667
	SPARC	4384.334
	SNRPG	4377.093
	RNF7	4367.271
	LOC729679	4367.271
	CAP1	4356.992
	LOC613037	4350.417
	FAM129B	4343.163
	PRDX5	4336.744
	SNHG5	4330.19
	LILRB1	4323.835
	ATP5J	4317.569
	CCT7	4311.306
	VDAC3	4304.92
	GIMAP8	4298.598
	PTTG1IP	4291.833
	PEA15	4285.228
	MDK	4279.023
	LOC728672	4272.423
	LTA4H	4265.782
	ARPC3	4259.694
	SFRS5	4253.734
	FABP5	4247.866
	B2M	4241.581
	JAM3	4235.323
	ATP5H	4229.135
	ZFAND5	4223.899
	UBE2E1	4217.975
	LOC100128266	4211.829
	NDUFA1	4206.047
	FKSG30	4199.931
	TUBB	4194.265
	LOC389168	4188.835
	C21orf24	4182.646
	PAM	4176.248
	LOC647340	4170.147
	ZNF14	4164.111
	MIF	4158.17
	COX6B1	4151.975
	NDUFS8	4145.751
	SF3B14	4139.69
	LOC389168	4132.783
	PSMD10	4126.234
	ATP5H	4119.575
	LOC644315	4113.929
	LOC643357	4107.532
	COX8A	4101.995
	HSP90B1	4095.739
	SAE1	4090.083
	YWHAB	4084.048
	LOC390345	4077.921
	RPS26L	4072.481
	SFRS6	4066.232
	CMTM3	4060.137
	NDUFB8	4054.134
	RPL7A	4048.417
	LASP1	4042.834
	LOC730029	4037.351
	GSTO1	4031.487
	HMGN1	4026.092
	HBXIP	4020.532
	LOC390557	4014.99
	KLF6	4009.341
	RTN4	4003.429
	AP2S1	3997.423
	TMEM17	3991.647
	LOC100132795	3986.28
	DYNLL1	3980.465
	UBE2D3	3973.772
	LOC92755	3968.05
	GPX4	3962.772
	APLN	3956.977
	LYVE1	3951.747
	AHNAK	3946.472
	LOC652624	3940.562
	ATP6V0E1	3934.799
	EIF4G2	3929.242
	FAM43A	3923.254
	LOC728873	3917.709
	PFDN5	3912.337
	LOC440737	3906.923
	HNRPM	3898.701
	CYB5B	3898.701
	LOC728126	3890.699
	RALGDS	3885.374
	GIMAP4	3880.145
	PPP2CA	3874.804
	CIRBP	3869.011
	C9orf80	3863.877
	CD34	3858.429
	ATP6AP1	3852.803
	MRFAP1	3847.485
	LOC649821	3842.339
	EZR	3837.019
	C20orf24	3831.573
	CD99L2	3825.978
	AIRE	3820.74
	PSMC1	3815.655
	C10orf10	3810.431
	LOC23117	3805.025
	ATP1A1	3799.669
	TKT	3794.411
	PSMB7	3789.396
	JUP	3784.532
	LOC643433	3779.673
	TMEM66	3774.548
	PSMC1	3769.582
	NDUFA3	3764.494
	MAGED1	3759.579
	C20orf24	3754.453
	LOC646785	3746.863
	LOC653226	3746.863
	SET	3739.565
	CRIP2	3734.717
	GLRX5	3730.108
	LOC100131196	3725.29
	PGD	3720.447
	TCEB2	3715.75
	BX537698	3711.047
	TMEM59	3706.181
	C8orf37	3701.639
	ZNF428	3696.942
	PHLDA1	3692.216
	TUBA1C	3687.356
	ERP29	3682.382
	RPL21	3677.297
	ESM1	3672.187
	LOC728139	3666.905
	FAM50A	3661.865
	LAMC1	3657.38
	UBE2I	3652.873
	ACTR2	3648.318
	RPS15A	3643.939
	C8orf45	3639.256
	B4GALT5	3635.08
	ADD1	3628.227
	FAM119A	3628.227
	LOC646819	3621.287
	EIF3B	3617.007
	C6orf48	3611.933
	HLA-A	3607.342
	ALKBH5	3602.68
	KHDRBS1	3598.262
	LOC100134648	3593.131
	SNRK	3588.844
	MAPRE1	3584.246
	APP	3579.695
	ATP5F1	3574.877
	DYNLRB1	3570.567
	RASIP1	3565.849
	LOC729926	3561.556
	CS	3556.809
	NUCKS1	3552.277
	C20orf100	3547.784
	SFRS5	3543.466
	FCGRT	3539.09
	ALDH9A1	3534.536
	JTB	3530.451
	DCTN2	3525.898
	FAM127A	3521.184
	EPN1	3516.76
	LOC402112	3512.346
	RRAGA	3507.74
	ARHGEF2	3503.294
	ITGB1	3498.718
	FKBP1A	3493.963
	NDUFA12	3489.713
	VEGFB	3485.396
	FEZ2	3480.915
	FKBP1A	3476.397
	TRMT112	3471.984
	PRKCH	3467.297
	LOC391370	3462.874
	RAB11A	3458.473
	S100A16	3454.14
	ROBLD3	3449.96
	TALDO1	3445.991
	RPL22	3441.584
	LOC644511	3437.516
	LOC127295	3431.188
	ANXA2	3431.188
	ARF4	3424.504
	AHR	3420.322
	TXNDC5	3415.883
	LOC646688	3411.785
	NDUFB3	3407.559
	AP1S2	3403.573
	DNAJC8	3399.217
	SFRS2	3394.83
	MATR3	3390.652
	ATP6V0C	3386.519
	C3orf34	3382.759
	NDUFB3	3378.886
	LOC728553	3374.77
	HNRPA2B1	3370.461
	OCIAD1	3366.672
	TMEM14C	3362.56
	IGFBP2	3358.728
	VAMP8	3354.294
	UQCRFS1	3350.384
	EIF3D	3346.006
	CTNNA1	3341.683
	SQLE	3337.535
	TSPAN18	3333.603
	RNASE1	3329.575
	CD99	3325.701
	ATP6V1E1	3321.748
	OSTC	3318.133
	PRR13	3313.96
	HNRPA1P4	3310.016
	LOC440353	3306.287
	ERGIC3	3301.974
	C2orf69	3297.976
	LOC100133477	3293.898
	LOC728698	3289.942
	LOC648390	3286.072
	HK1	3282.036
	PDHB	3278.294
	FLJ44124	3274.603
	TUG1	3270.77
	MORF4L2	3266.977
	AL15748	3263.189
	MYADM	3259.222
	DEGS1	3255.744
	LOC727865	3251.966
	LOC729236	3248.111
	LOC158345	3244.281
	PARK7	3240.659
	CS	3236.723
	BMS1P5	3233.004
	LOC390354	3229.485
	SNRPB2	3226.027
	PCBP2	3222.358
	LOC440043	3218.805
	LOC402175	3215.17
	TMBIM4	3211.304
	LOC730004	3207.415
	LOC374395	3203.745
	ZDHHC8	3200.006
	MFNG	3196.474
	AMY1C	3192.522
	VCL	3188.645
	GABARAPL2	3185.102
	TUBB2B	3181.562
	RCN1	3176.229
	PTK2	3176.229
	C14orf173	3170.849
	LOC399804	3166.942
	VKORC1	3163.391
	CCNY	3159.724
	PRNP	3156.361
	PTP4A2	3152.617
	NDUFB5	3148.956
	LOC100131801	3145.122
	NDUFA4	3141.587
	GAPDH	3137.906
	MRPS21	3134.596
	HSPD1	3131.109
	DARS	3127.618
	PLOD1	3124.315
	LOC347544	3120.537
	DUXAP3	3116.969
	POMP	3113.4
	GPIHBP1	3109.723
	PLS3	3106.332
	PGK1	3101.494
	ITPR3	3101.494
	HIATL2	3096.506
	ZNF486	3092.927
	MFSD10	3089.188
	PON2	3085.436
	NME1	3082.096
	LOC731985	3078.831
	ILF2	3075.404
	DSTN	3071.802
	SFRS9	3068.507
	DUSP19	3064.885
	GHITM	3061.454
	FAM124B	3057.985
	ATP6V1E1	3054.703
	PTTG1IP	3051.21
	HPCAL1	3047.698
	ZNF394	3042.981
	RAB7A	3042.981
	CAV1	3037.999
	DYNC1LI2	3033.362
	FASN	3033.362
	PTBP1	3028.638
	CCDC130	3025.44
	PSMA4	3022.079
	HMGN1	3018.673
	TMED3	3015.24
	CCT8	3011.764
	IL10	3008.282
	LOC645058	3005.028
	MORF4L1	3001.861
	SLC44A2	2998.583
	TMEM123	2995.415
	MAT2A	2992.155
	ADM	2988.951
	PRND	2985.625
	HNRNPK	2982.658
	NOL7	2979.434
	YBX1	2976.279
	LOC391656	2973.021
	CAMLG	2969.814
	FLNB	2966.365
	ARL6IP1	2963.044
	LOC399988	2959.773
	LOC100130562	2956.718
	RWDD1	2953.291
	LOC650518	2949.922
	TCEAL3	2947.075
	S100A4	2944.066
	EIF2S3	2940.487
	PRDX6	2937.178
	TSPAN9	2934.115
	GLO1	2931.327
	PSMD6	2928.123
	ILK	2924.745
	ACADVL	2921.625
	RHOC	2918.659
	PSME1	2915.555
	LOC387820	2912.308
	LDLR	2909.147
	TPM2	2904.888
	LOC728888	2904.888
	SEC11A	2900.347
	TEAD2	2897.152
	SLC25A6	2894.058
	BTBD2	2890.722
	NCL	2887.878
	LOC100132391	2884.602
	RPN1	2881.471
	TRIM8	2878.486
	HEXB	2875.395
	ZMAT3	2872.252
	MGST3	2869.117
	APP	2866.02
	LOC728244	2863.085
	ARGLU1	2860.002
	LEPROT	2857.123
	DDX51	2854.073
	CXXC5	2850.969
	AP1S2	2847.996
	LOC653314	2845.126
	SRP14P1	2842.19
	ACP1	2838.992
	C14orf153	2836.086
	C20orf30	2832.782
	UBA1	2829.927
	SNRPB	2826.874
	TXNIP	2823.728
	NUDT14	2820.921
	LOC642817	2818.102
	ATP1B1	2815.268
	CSNK2B	2812.341
	SNRPF	2809.605
	UXT	2806.481
	EIF3M	2803.284
	ALDOA	2800.582
	EFEMP1	2797.68
	STAU1	2794.503
	ANAPC13	2791.876
	DMC1	2788.981
	HNRNPH1	2785.906
	LPP	2783.35
	KRTCAP2	2780.536
	RPL14L	2777.793
	RPRC1	2774.822
	DKK3	2771.896
	BUB3	2769.109
	CAPZA2	2766.271
	MGC16121	2763.458
	EIF4B	2760.512
	MYH10	2757.669
	LOC100134159	2754.867
	ARL2	2751.979
	COLEC12	2749.382
	RHOJ	2746.622
	LOC401115	2743.952
	TIMM23	2741.358
	CARM1	2738.743
	PJA2	2736.055
	CMIP	2733.33
	TINP1	2730.681
	COPA	2728.078
	SSR4	2725.157
	LOC645688	2722.435
	PALM	2719.394
	UBE2D3	2716.675
	TMSL3	2714.115
	EID2B	2711.423
	TGM2	2708.749
	P4HB	2706.261
	NAT5	2703.492
	LOC653079	2700.971
	STX16	2698.241
	PUF60	2695.483
	SEC61B	2692.776
	KLF2	2690.108
	LOC441506	2687.565
	PSMA1	2684.755
	DAPP1	2682.076
	RAB10	2679.581
	TIMP2	2677.038
	NDUFA8	2674.402
	PRDX5	2671.801
	PSMA5	2668.989
	PIGY	2666.487
	PRSS23	2663.739
	ATP6V1F	2661.253
	C2orf28	2658.504
	PLS3	2655.947
	STARD7	2653.165
	FDFT1	2649.481
	LOC100130003	2649.481
	NUP62	2645.797
	PSMB3	2643.409
	FAM39E	2640.664
	LOC653505	2637.95
	TOMM6	2635.176
	AK095855	2631.407
	CAPNS1	2631.407
	LOC649553	2627.467
	RPL17	2625.058
	RBX1	2622.313
	CYBA	2619.898
	ARPC1A	2617.495
	VAMP3	2614.954
	LOC100133772	2612.487
	SUMO3	2609.9
	CD34	2607.309
	PRMT1	2605.064
	CD63	2602.476
	TPI1	2599.884
	BRI3	2597.245
	LMNA	2594.722
	SNRNP70	2592.098
	ID3	2589.513
	LOC442454	2587.179
	CAV2	2584.557
	POLR2G	2582.077
	LOC388707	2579.665
	ATP6V0E1	2577.161
	LOC654194	2574.714
	PHPT1	2572.017
	POLR2F	2569.517
	APEX1	2567.108
	EIF3K	2564.684
	LOC653226	2562.343
	C15orf24	2559.838
	IMPDH2	2557.397
	DUSP3	2553.815
	KPNB1	2553.815
	NDUFA11	2549.928
	CTGF	2547.565
	NDUFS4	2544.957
	BLZF1	2542.604
	RHEB	2540.184
	PRICKLE4	2537.998
	PTPLAD1	2535.534
	HSP90AA1	2533.024
	BANF1	2530.587
	COL4A5	2528.138
	SDCBP	2525.598
	LRRC37B2	2523.156
	LRRC32	2520.757
	GSTM1	2518.524
	TTC3	2516.152
	DYSF	2513.921
	ETS1	2511.662
	PON2	2509.463
	PDCD6	2507.209
	TOMM20	2504.969
	REPIN1	2502.723
	BOLA2	2499.166
	LOC391126	2499.166
	SIVA1	2495.534
	HPRT1	2493.245
	PRDX3	2491.116
	CDC16	2488.933
	ATOX1	2486.616
	RBM22	2484.209
	NUAK1	2481.962
	VPS29	2479.728
	VDAC1	2477.395
	EVL	2475.158
	TAGLN2	2473.013
	LY6E	2470.802
	GPR56	2468.456
	REEP5	2466.211
	ZNF69	2464.034
	LOC728590	2461.808
	EEF1D	2459.394
	POLR2H	2457.249
	PPP2R1A	2454.819
	PSMB5	2452.568
	C20orf43	2450.369
	SPCS1	2448.261
	ATF4	2444.895
	EIF4A1	2444.895
	OSTC	2441.645
	RASGRP3	2438.988
	LOC100128353	2436.823
	LOC648024	2434.574
	EHD4	2432.192
	VPS26A	2429.896
	ARAP3	2427.613
	SDHB	2425.444
	RPS6KA2	2423.269
	MRPS6	2420.95
	LOC648210	2418.767
	EIF3G	2416.711
	CNBP	2414.27
	GPR56	2412.085
	SH2B3	2409.804
	REXO2	2407.484
	RNF181	2405.349
	TSPAN3	2403.161
	ADCY4	2401.073
	HNRPUL1	2398.99
	LOC388339	2397.042
	DDX3X	2394.857
	GALK1	2392.643
	PPP1CC	2390.58
	HSP90AB1	2388.513
	FAM107B	2386.169
	CREB1	2384.065
	VIL2	2381.946
	SNRPF	2379.903
	TST	2377.882
	LOC730534	2375.733
	MKNK2	2373.554
	STC1	2371.352
	EIF3I	2369.413
	PPP1R11	2367.293
	MYLIP	2365.426
	SNRPB	2363.423
	SDCBP	2361.481
	PSMB4	2359.383
	YY1	2357.516
	NDUFS3	2355.493
	H1F0	2353.539
	THBS1	2351.476
	SMS	2349.239
	LOC391075	2347
	ARF1	2345.002
	ZMIZ1	2343.074
	RHOG	2341.097
	EIF4H	2339.187
	RAC2	2336.985
	PPA2	2334.924
	MSN	2332.827
	RPL23	2330.874
	ITGB4BP	2328.691
	MYL6B	2326.759
	MFGE8	2324.612
	ADSL	2322.313
	RPL10A	2320.322
	SHISA5	2318.275
	SGSM2	2316.145
	ARL5A	2314.145
	LOC644063	2312.122
	DHX15	2310.085
	LOC642956	2307.807
	DDOST	2305.894
	SDHALP1	2303.914
	EIF4H	2302.04
	MRPL22	2300.216
	LOC100132528	2298.284
	LOC653658	2296.237
	DYNC1I2	2294.276
	C20orf30	2292.387
	HNRNPR	2290.423
	G3BP2	2288.484
	ZNF682	2286.593
	PGRMC1	2284.582
	LOC728492	2282.552
	BAX	2280.554
	MGC4677	2278.547
	NNAT	2276.699
	SRRM2	2274.895
	GUK1	2272.81
	S100A4	2270.843
	TMEM14C	2268.872
	TIE1	2267.041
	IL32	2265.217
	RPS27	2263.252
	GSTM2	2261.182
	SUMF2	2259.346
	DDT	2257.489
	C20orf199	2255.455
	ARCN1	2253.645
	CSNK1G2	2251.65
	UCHL1	2249.632
	MDH1	2247.564
	ARL2BP	2245.594
	TEK	2243.715
	TCEB1	2241.851
	M6PRBP1	2239.955
	CAV2	2238.078
	HYAL2	2236.148
	PRKCDBP	2234.444
	NUDC	2232.664
	NBPF10	2230.705
	NDUFA2	2228.844
	LXN	2227.013
	LOC647000	2225.325
	C20orf160	2223.387
	PPM1G	2220.516
	UBL5	2220.516
	URM1	2217.626
	VASH1	2215.82
	XRCC6	2213.876
	PSMB2	2212.134
	TCEAL8	2210.404
	ZNHIT1	2208.435
	SETD3	2206.564
	NHP2	2204.765
	LOC100128288	2202.999
	SNRK	2200.977
	SDPR	2199.231
	ESYT1	2197.376
	GDI2	2195.532
	LOC100130561	2193.647
	CUTA	2191.759
	GJA4	2189.946
	SFRS4	2187.892
	TMED9	2186.194
	CATSPER2	2184.456
	RAI14	2182.639
	PSMD10	2180.94
	AB074172	2179.276
	UQCRHL	2177.506
	ARMET	2175.753
	MAP1LC3A	2173.799
	ZNF652	2172.067
	TPM1	2170.117
	LMO2	2168.384
	WDR18	2166.626
	PODXL	2164.78
	PCMT1	2163.02
	FERMT2	2161.297
	SNHG6	2158.576
	ACLY	2158.576
	TMEM98	2155.966
	GYPC	2154.276
	HNRNPM	2152.428
	FAM171A1	2150.789
	PXDN	2148.125
	NGRN	2148.125
	LSM2	2145.52
	CIB1	2143.783
	NDUFB7	2142.01
	ANKS1A	2140.256
	NPTN	2138.669
	EFNB2	2137.024
	C12orf57	2135.199
	PRDX4	2133.33
	BEX4	2131.534
	RDX	2129.679
	TPM1	2128.18
	LOC391811	2126.614
	HNRNPA0	2124.898
	RAB5B	2123.318
	AARS	2121.396
	RBM10	2119.742
	CKLF	2117.921
	C15orf63	2116.23
	ARPC5	2114.352
	DAB2	2112.622
	HLX	2110.92
	CD46	2109.263
	ARHGAP23	2107.661
	ERH	2105.924
	GLTP	2104.153
	OXA1L	2102.456
	ADAMTS9	2100.784
	TUBB6	2098.927
	PRPSAP1	2097.122
	LOC728533	2095.487
	CETN2	2092.925
	COMT	2092.925
	MIR1978	2090.444
	ATP1B3	2088.676
	TCF25	2086.963
	GSPT1	2085.414
	LOC728031	2083.839
	HOXB7	2082.164
	LOC644907	2080.516
	XPNPEP1	2078.884
	ZNF22	2077.23
	DPY30	2075.5
	LOC653773	2073.876
	LOC100128410	2072.218
	CCL14	2070.749
	LOC648622	2069.157
	CRCP	2067.613
	COMMD3	2066.041
	LOC728661	2064.404
	FEZ2	2062.769
	QRFPR	2061.197
	CD2BP2	2059.596
	LOC645138	2057.918
	ANXA2P1	2056.255
	ACTN1	2054.614
	SASH1	2052.96
	TPM2	2051.325
	RBM5	2049.709
	CTSL1	2048.076
	HSPD1	2046.443
	LOC728554	2044.892
	TEK	2043.162
	LOC148430	2041.445
	CLTA	2039.745
	DDX1	2038.117
	MGC87895	2036.414
	ENY2	2034.704
	C21orf58	2033.058
	EIF3H	2031.374
	PTOV1	2029.659
	C19orf10	2027.986
	NSA2	2026.356
	SLC2A3	2024.764
	PRDX3	2023.325
	GTF2A2	2021.712
	BCKDK	2019.916
	SPTLC1	2018.472
	SPCS2	2016.07
	C17orf61	2016.07
	C13orf15	2013.725
	GRAP	2012.201
	TXNDC17	2010.558
	GLG1	2009.077
	RING1	2007.525
	CDC16	2005.976
	FTHL12	2004.417
	JMJD8	2002.931
	DYNLRB1	2001.493
	LOC730740	2000
	GTPBP6	1998.578
	ADAM19	1997.024
	OCIAD1	1995.423
	ALPP	1993.895
	LOC645452	1992.252
	LOC730455	1990.712
	CIP29	1989.102
	HDHD1A	1987.7
	PRDX2	1986.069
	SSU72	1984.546
	TBCB	1983.009
	UBXN4	1981.49
	LAMP2	1979.835
	SOX7	1978.374
	TSPAN4	1977.045
	SH3BGRL	1975.596
	JAG2	1974.152
	LDHB	1972.673
	ANKRD30B	1971.064
	STOML2	1969.434
	MT2A	1967.846
	CKAP4	1966.368
	PABPC4	1965.115
	COX7A2L	1963.582
	BCAP31	1962.06
	VPS35	1960.581
	LOC730316	1959.032
	HSPE1	1957.463
	DECR1	1956.08
	NBPF20	1954.56
	HDAC2	1953.03
	DYNLT1	1951.625
	MTPN	1950.182
	ATP5E	1948.728
	CLCN7	1947.253
	KDELR1	1944.949
	GARS	1944.949
	FNTA	1941.981
	NME4	1941.981
	ADAR	1939.685
	SS18L2	1938.165
	SNHG7	1936.806
	CLIC4	1935.304
	MRPL22	1933.809
	PLIN2	1932.481
	NDEL1	1930.949
	LOC100131531	1929.469
	BC036485	1928.086
	UNC84B	1926.666
	SEC61A1	1925.247
	PPP2R2A	1923.752
	CCT2	1922.433
	HGS	1921.051
	RNASEK	1919.6
	EIF4A3	1918.241
	TUBB2C	1916.747
	APEX1	1915.376
	CCND3	1913.884
	RPAIN	1912.433
	LOC729841	1911.051
	UNC50	1909.61
	RPP21	1908.262
	LZTR1	1907.012
	ABCA1	1905.602
	NDUFV2	1904.214
	TAF15	1902.111
	F2R	1902.111
	GSTK1	1900.015
	LSM1	1898.52
	GRK5	1897.066
	RPS23	1895.565
	RPLP0	1894.206
	SRPX	1892.791
	SRP14	1891.423
	LOC642590	1890.132
	MRPL51	1888.738
	MTSS1	1887.426
	SPG7	1886.074
	ETFA	1884.622
	NCSTN	1883.33
	ARS2	1882.034
	LOC90586	1880.692
	TPD52L2	1879.422
	RHBDF1	1878.106
	MAPK3	1876.805
	RSL24D1	1875.555
	PIN1	1874.261
	CTSB	1872.918
	CCDC90B	1871.493
	NAP1L4	1870.159
	ATP9A	1868.852
	EVI1	1866.765
	POLR1D	1866.765
	SSTR1	1864.751
	PCID2	1863.457
	HIGD1A	1861.98
	MGC10997	1860.583
	STRAP	1859.285
	MRPL36	1857.963
	ARHGEF7	1856.522
	ATP5D	1855.212
	NR2F2	1853.965
	FNBP1	1852.702
	LMBRD1	1851.216
	LOC392437	1849.863
	DNAJB11	1847.944
	ELTD1	1847.944
	ZNF358	1846.034
	PPP6C	1844.683
	LOC646567	1843.41
	GOLGA7	1842.093
	ID1	1840.693
	UBXN1	1839.336
	IPO11	1838.036
	TNFRSF10B	1836.729
	C7orf59	1835.465
	SYT11	1834.199
	OSTF1	1832.859
	ZNHIT3	1831.481
	GOT2	1828.835
	LOC399748	1827.599
	HSBP1	1826.317
	EFNA1	1825.115
	IDH1	1823.88
	HNRPK	1822.669
	ANGPTL2	1821.346
	HOXB8	1820.111
	TMEM85	1818.718
	SIAH1	1817.475
	LOC285741	1816.02
	CFDP1	1814.757
	LOC652489	1813.434
	BANP	1812.163
	C2orf25	1810.833
	ARHGAP17	1809.513
	SMS	1808.21
	ATP6V1G1	1806.998
	TMED2	1805.879
	CTDSP2	1804.656
	PPP2CB	1803.45
	PSMD4	1802.198
	FKBP14	1800.996
	LUZP1	1799.739
	CTSL1	1798.517
	GLCE	1797.266
	DCTPP1	1795.899
	PCNP	1794.674
	MRPL37	1793.311
	SSBP1	1792.031
	BZW2	1790.698
	GLB1	1789.417
	STOM	1788.173
	ZYX	1786.884
	EIF5A	1785.669
	NUMB	1784.498
	PSMD7	1783.367
	FXR1	1782.152
	PARP4	1780.857
	RARS	1779.561
	RBMS1	1778.275
	FAM175A	1776.427
	SF3B1	1776.427
	LOC100128936	1774.579
	LOC644191	1773.38
	CHRNA5	1772.196
	EIF2AK1	1771.002
	MGC71993	1769.83
	LOC255167	1768.607
	CAB39	1767.443
	FGD5	1766.278
	HNRPR	1765.174
	RPS26	1763.91
	TAX1BP3	1762.65
	PSMC5	1761.404
	LOC642755	1760.256
	LOC202781	1758.944
	DKK3	1757.831
	ZMYND11	1756.637
	C19orf70	1755.48
	SVIL	1754.196
	SELS	1753.02
	NDUFB11	1751.891
	CPNE3	1750.703
	MRI1	1749.53
	LOC401397	1748.318
	TPRG1L	1747.141
	VAT1	1746.025
	TNFRSF1B	1744.824
	C5orf28	1743.673
	NOSIP	1742.474
	FER1L3	1741.29
	RPS28	1740.056
	TCEAL4	1738.878
	FAHD1	1737.738
	PQLC1	1736.625
	ATP6V1A	1734.904
	TAX1BP1	1734.904
	WDR6	1733.152
	GPS1	1731.944
	DAB2	1730.771
	LOC338870	1729.516
	HNRNPM	1728.303
	PUM1	1727.184
	SLC9A1	1725.915
	TRMT5	1724.831
	ATP1B1	1723.685
	EMD	1722.585
	PSMG2	1720.345
	CCDC23	1720.345
	C20orf24	1720.345
	UROD	1718.176
	PPP2R2B	1717.048
	COPB1	1715.868
	PUM1	1714.76
	CASP2	1713.646
	TFG	1712.523
	LOC728820	1711.473
	CHCHD9	1710.374
	CSE1L	1709.265
	LOC100131785	1708.063
	FAM120A	1706.955
	TMEM111	1705.855
	HMGN2	1704.702
	TNK2	1703.603
	PTPRF	1702.506
	C19orf56	1701.464
	C1orf85	1700.401
	CLDND1	1699.33
	SF3B5	1698.141
	CCDC56	1696.976
	HIGD2A	1695.904
	SRP54	1694.673
	MRPS18C	1693.505
	SLC38A2	1692.344
	SYPL1	1691.19
	PWP1	1690.03
	CYC1	1688.812
	VPS28	1687.69
	BSG	1686.67
	TRIOBP	1685.549
	IDH3B	1684.483
	FAM65A	1683.363
	IMP3	1682.299
	SCRN1	1681.224
	PLOD3	1680.136
	TSC22D1	1679.005
	UCKL1	1677.916
	C3orf54	1676.807
	TCEAL3	1675.69
	ZRANB2	1674.65
	SFRS18	1673.475
	PSMC2	1672.436
	TMEM147	1671.356
	CNDP2	1670.377
	BAT1	1669.32
	PSMD4	1667.617
	SYPL1	1667.617
	FAM96A	1666.04
	PFDN1	1664.983
	MRPS12	1663.866
	MXD4	1662.821
	PSMA6	1661.627
	LOC729279	1660.481
	LRP10	1659.336
	MRPL17	1658.299
	WSB1	1657.178
	ARL6IP5	1656.15
	HSD17B7	1655.049
	CYP1A1	1654.03
	RGL1	1653.014
	ARHGDIA	1651.931
	LSM3	1650.846
	LOC440359	1649.8
	LOC730744	1648.793
	SFRS14	1647.759
	LOC653566	1646.763
	LOC651894	1645.664
	CLDND1	1644.57
	PTDSS1	1643.545
	SSB	1642.473
	GRIPAP1	1641.337
	RRAS	1640.329
	PRPF8	1639.153
	CRTAP	1638.192
	AV737317	1637.094
	PDIA5	1635.993
	GYPC	1634.996
	LOC653086	1633.937
	SULT1A1	1632.887
	EXOSC10	1631.823
	SEC14L1	1630.73
	CMTM7	1629.708
	CDK4	1628.643
	SLC12A2	1627.639
	LOC100128062	1626.109
	DUSP22	1626.109
	LOC100129379	1624.099
	TMEM158	1624.099
	SH3GLB1	1622.482
	TGFBR3	1621.398
	SFRS1	1620.42
	C1QBP	1619.375
	MMS19L	1618.318
	FABP4	1617.363
	SYF2	1615.805
	LOC647285	1615.805
	POM121C	1614.33
	CTXN1	1613.323
	PMP22	1612.342
	DCTN3	1611.336
	EI24	1610.259
	SNURF	1609.211
	PFKP	1608.135
	AK3	1607.133
	NAP1L1	1606.141
	PSMB10	1605.125
	FIS1	1604.15
	RCN2	1603.109
	COPS5	1602.015
	UBE1	1600.951
	METAP2	1599.995
	DEGS1	1598.954
	TPP1	1597.891
	TCF4	1596.551
	PLD3	1596.551
	SLC20A1	1595.085
	BRD2	1593.958
	GTF2E2	1592.892
	CARHSP1	1591.833
	KIAA1949	1590.829
	PSMF1	1589.926
	DCTN1	1588.867
	LOC643668	1587.862
	C11orf59	1586.41
	CDC42EP4	1586.41
	LOC729317	1584.87
	ATXN2	1584.024
	ZDHHC16	1582.96
	KLHL3	1581.971
	FBXO11	1581.021
	HSD17B12	1579.986
	WDR54	1578.996
	THOC7	1578.022
	LOC286157	1577.048
	PGAM1	1576.049
	RRBP1	1575.086
	COPS3	1574.042
	TGOLN2	1572.574
	ATP5J2	1572.574
	PAICS	1571.114
	MYCT1	1570.123
	CCNG1	1569.125
	EIF1B	1568.22
	PTPLAD1	1567.188
	GLUD1	1566.225
	SRRM1	1565.277
	BCL2L1	1564.333
	SDHAF2	1563.342
	TMEM126B	1562.454
	COX6A1	1561.487
	LOC651198	1560.508
	TSC22D3	1559.555
	ACAT1	1558.552
	LOC389787	1557.626
	RALY	1556.622
	SSR2	1555.669
	MTUS1	1554.711
	RNF144	1553.795
	LOC100132727	1552.848
	JUN	1551.928
	NDUFAB1	1550.977
	MCM3	1550.039
	MRFAP1L1	1549.157
	PRNP	1548.212
	COL18A1	1547.275
	ECH1	1546.324
	LOC650369	1545.353
	CDC42EP5	1543.892
	ZNF738	1543.892
	HSPA9	1542.471
	KCTD12	1541.497
	SUZ12	1540.117
	RABGAP1	1540.117
	GTF2H5	1538.645
	DDA1	1537.81
	BIN1	1536.906
	DUSP1	1535.978
	STK24	1535.023
	ITGA5	1534.085
	DDX47	1533.17
	SEC31A	1532.248
	PNPT1	1531.378
	SPTBN1	1530.478
	GPSM1	1529.602
	EXOC7	1528.681
	PSMC6	1527.768
	GTPBP4	1526.921
	DBN1	1525.956
	GLT25D1	1525.037
	EIF2A	1523.676
	LIMCH1	1523.676
	CCNDBP1	1522.32
	TROVE2	1521.457
	RPS26L	1520.508
	AURKAIP1	1519.568
	WBP5	1518.619
	RPAIN	1517.721
	TPM3	1516.876
	KIAA1671	1516.034
	LOC653994	1515.152
	DBNL	1514.297
	NTAN1	1513.386
	BOLA2	1512.461
	TMEM44	1511.551
	EDN1	1510.177
	ITGB1BP1	1510.177
	CCDC109B	1508.421
	ZNF22	1508.421
	C4orf18	1507.078
	APOA1BP	1506.23
	SH3BGRL3	1505.32
	UBE2M	1504.415
	CAPNS1	1503.518
	SPOP	1502.701
	GNG10	1501.888
	PLSCR4	1500.944
	TMEM181	1500.131
	LOC388275	1499.324
	FEZ1	1498.408
	COX17	1497.494
	LMNA	1496.663
	C21orf33	1495.672
	ITM2C	1494.223
	DNMT1	1494.223
	ITM2C	1492.824
	PRR14	1492.013
	CYR61	1491.16
	BNIP3	1490.33
	IGF2R	1489.489
	SON	1488.169
	GFOD1	1488.169
	LOC641700	1486.891
	MRI1	1486.039
	PROCR	1485.086
	LOC732007	1484.229
	CTTN	1483.402
	LOC644799	1482.539
	RUSC1	1481.565
	GRN	1480.712
	ITGB5	1479.823
	GYG1	1478.946
	MRPL23	1478.119
	ASAP1	1477.166
	KPNA4	1476.338
	PSME2	1475.46
	PRAGMIN	1474.691
	RCC2	1473.898
	INPP1	1472.989
	LOC100132291	1472.102
	NHP2L1	1471.226
	ANAPC11	1470.023
	CGNL1	1470.023
	NFATC2IP	1468.795
	IARS2	1467.883
	TJP1	1467.001
	LOC729768	1466.205
	ATP6V1B2	1465.338
	LOC644774	1464.489
	CSF2RA	1463.664
	ANGPT2	1462.786
	ATP5J2	1462.035
	ANAPC5	1461.197
	BRMS1	1460.376
	ADRM1	1459.571
	C2orf28	1458.684
	DGUOK	1457.91
	SLC25A39	1456.678
	CHCHD10	1456.678
	DGUOK	1455.463
	PEBP1	1454.59
	PPP1R14B	1453.763
	TMEM205	1453.072
	GNL2	1452.202
	LOC646723	1451.384
	BCLAF1	1450.526
	PAPSS2	1449.722
	ROD1	1448.848
	C8orf59	1448.044
	CLNS1A	1447.207
	TAX1BP1	1446.445
	BNIP3L	1445.532
	NFKB1	1444.768
	LOC644934	1443.99
	ADAM15	1443.15
	PPP1CA	1442.281
	C17orf49	1441.421
	CGGBP1	1440.555
	AP3B1	1439.754
	ARPC4	1439.019
	TMEM87A	1438.202
	LOC440093	1437.389
	PRDX2	1436.503
	CENPB	1435.284
	RIOK3	1435.284
	PEPD	1434.051
	C7orf30	1433.277
	SF3B4	1432.503
	C7orf50	1431.741
	PAICS	1430.9
	DHRS7	1430.109
	SCHIP1	1429.311
	SMARCA4	1428.443
	LOC391833	1427.659
	CD151	1426.918
	C14orf112	1426.134
	CCT3	1425.327
	TSPAN17	1424.433
	HEBP1	1423.707
	PALMD	1422.87
	PSMA3	1422.059
	HCFC1R1	1420.912
	FAM96B	1420.912
	LOC728532	1419.695
	TRAPPC2L	1418.904
	BASP1	1418.075
	ZFYVE21	1416.821
	EDF1	1416.821
	TMEM43	1415.619
	KIAA1191	1414.827
	COMMD1	1414.003
	VEZF1	1413.213
	TMCO1	1412.417
	PSMD6	1411.674
	PAFAH1B3	1410.928
	C19orf43	1410.128
	CWC15	1409.341
	PHB2	1408.51
	FAM45A	1407.765
	SPTLC1	1407.045
	C2orf29	1406.196
	PGLS	1405.486
	GNPDA1	1404.659
	AIDA	1403.945
	FNBP1L	1403.256
	TCEAL8	1402.467
	WFS1	1401.669
	CYTSA	1400.85
	IFNGR2	1400.118
	MRPS24	1399.314
	SASH1	1398.612
	LOC728590	1397.854
	LSM5	1397.054
	NDUFB10	1396.327
	PTPRM	1395.602
	BIN1	1394.867
	MLLT11	1394.156
	KLHL5	1393.377
	CAMK2N1	1392.62
	IFI16	1391.929
	RAB2B	1391.157
	TSG101	1390.367
	ARHGAP21	1389.62
	TXNL2	1388.918
	EIF2B4	1388.222
	AKR7A2	1387.016
	PPP4C	1387.016
	MARCH7	1385.806
	EWSR1	1385.086
	MGEA5	1383.891
	JMJD8	1383.891
	TSPAN3	1382.76
	FAM62B	1381.996
	PLVAP	1381.302
	ATP1B1	1380.575
	LOC220433	1379.767
	KIAA1751	1378.533
	NOX4	1378.533
	BCAT1	1377.397
	TRAPPC5	1376.299
	DIABLO	1376.299
	ATG4A	1375.226
	EWSR1	1374.548
	LOC100130919	1373.854
	EIF6	1373.121
	SERF1B	1372.435
	C2orf25	1371.71
	ISCU	1370.6
	EMCN	1370.6
	NAE1	1369.468
	CBX2	1368.713
	GTF3A	1367.917
	FXYD5	1367.183
	RNU6-1	1366.377
	CIAO1	1365.645
	SF3A2	1364.954
	LOC729217	1364.264
	SMARCD1	1363.514
	YPEL5	1362.416
	MAT2B	1362.416
	CD9	1361.367
	CLK1	1360.635
	SHE	1359.905
	ARPC4	1359.19
	ENG	1358.525
	DPM1	1357.848
	RPS26L	1356.814
	SEC24C	1356.814
	WDFY1	1355.688
	ABCF1	1354.999
	SEPN1	1354.331
	CTPS2	1353.527
	JAK1	1352.825
	IGF2BP2	1352.016
	CALU	1351.325
	NSUN2	1350.603
	ETS2	1349.889
	PSMA4	1349.159
	NOTCH4	1348.37
	PPIL3	1347.714
	EBPL	1346.925
	UBE2A	1346.266
	TMEM14D	1345.427
	TSPO	1344.7
	UBE2E3	1344.022
	SNX17	1343.293
	AHCY	1342.54
	APH1A	1341.847
	CTTN	1341.154
	SH3GLB2	1340.142
	LOC646347	1340.142
	TUBB3	1339.084
	LSM4	1338.417
	MCM7	1337.661
	UBE2G2	1336.937
	CCL15	1336.199
	VPS37C	1335.507
	MRPL43	1334.784
	AKT1	1334.044
	TSC22D1	1333.376
	GLRX3	1332.713
	C3orf21	1332.065
	UFC1	1331.382
	DDEF2	1330.627
	HNRPDL	1329.926
	OAT	1329.173
	SAMM50	1328.509
	DDX42	1327.8
	TMEM189-UBE2V1	1327.136
	BRD9	1326.459
	TRABD	1325.811
	LYN	1325.106
	LOC100130516	1324.409
	UBAP2L	1323.751
	LYL1	1323.043
	NAT5	1322.032
	C19orf43	1322.032
	C22orf13	1320.994
	PAFAH1B1	1320.273
	HECW2	1319.607
	DDX39	1318.904
	NSMCE4A	1318.229
	NRP1	1317.531
	NLRP8	1316.868
	IFFO1	1316.188
	SERPINH1	1315.531
	TOMM20	1314.873
	SLC35B1	1314.205
	BMP6	1313.502
	ACTR10	1312.85
	AIMP2	1312.181
	PLRG1	1311.423
	TUBB6	1310.078
	SWAP70	1310.078
	LOC345041	1310.078
	PPA1	1308.715
	CUTA	1308.027
	MAGT1	1307.382
	LOC388621	1306.728
	TSPO	1306.076
	CKLF	1305.451
	ACTR1A	1304.807
	HSPA1B	1304.1
	DYNLRB1	1303.429
	LOC651149	1302.787
	TINF2	1302.134
	ACTL6A	1301.494
	CNIH4	1300.809
	NECAP2	1300.112
	AKT1	1299.402
	FLOT1	1298.767
	C6orf153	1298.085
	CUEDC2	1297.413
	AK90694	1296.433
	LOC728903	1296.433
	TXNRD1	1295.501
	AFAP1L1	1294.547
	CAPN11	1294.547
	UBE2L6	1293.604
	KIAA0355	1292.622
	LAPTM4B	1292.622
	RNU6-15	1291.31
	TGFB1I1	1291.31
	TNFRSF21	1290.303
	HNRNPAB	1289.261
	C14orf166	1289.261
	C3orf10	1288.225
	MAPK3	1287.591
	BUD31	1286.944
	CCDC50	1286.227
	DPM1	1285.552
	TSEN34	1284.894
	FAM32A	1284.298
	PTGR1	1283.698
	BTBD6	1283.024
	COQ5	1282.389
	DNAJA2	1281.734
	YTHDC1	1281.127
	CXCR4	1280.516
	SNCA	1279.908
	C19orf53	1279.337
	TMED10P	1278.664
	PIP5K2B	1278.034
	CTDSPL	1277.381
	CSE1L	1276.724
	LOC728973	1276.059
	ITM2A	1275.44
	SEPT15	1274.779
	DERA	1274.102
	THOC4	1273.114
	SNRPN	1273.114
	ATG12	1272.123
	SUPT16H	1271.505
	NINJ1	1270.853
	TRAF3IP2	1270.277
	LOC100132247	1269.647
	MMP1	1269.005
	GPN1	1268.349
	C16orf61	1267.703
	ZFP91	1267.065
	CLTA	1266.103
	RBM3	1266.103
	STK25	1265.098
	CD99L2	1264.439
	SEMA3E	1263.804
	MMRN1	1263.233
	FAM38A	1262.61
	CXXC5	1261.953
	FAM125A	1261.062
	COPE	1261.062
	CNRIP1	1260.09
	NDUFB6	1259.195
	AKR1A1	1259.195
	ATP2B4	1257.931
	SF3A3	1257.931
	ACSS2	1256.642
	SGSH	1256.642
	MRPL32	1255.638
	FBLN1	1254.976
	CKAP5	1254.373
	PPP1R15A	1253.721
	PCDHB2	1253.122
	FBXO21	1252.554
	TMEM183B	1251.959
	TYK2	1251.349
	EIF2B4	1250.723
	KIAA1310	1250.124
	UBE3C	1249.545
	ZNF207	1248.609
	TOMM22	1248.609
	AP2M1	1247.656
	RBM9	1247.018
	NAGK	1246.158
	SIVA	1246.158
	PGAM4	1245.267
	BRPF1	1244.707
	LOC653232	1244.138
	MRPL24	1243.487
	ITPRIPL2	1242.837
	RANBP1	1242.19
	PIR	1241.579
	NDUFS7	1241.024
	MRPL33	1240.402
	LOC731777	1239.449
	ACSL3	1239.449
	SYNCRIP	1238.21
	SCAMP1	1238.21
	HSPH1	1237.276
	SNORD13	1236.367
	ATP5C1	1236.367
	RNPS1	1235.512
	YRDC	1234.929
	FNBP4	1234.016
	SLC27A3	1234.016
	SNTB2	1233.115
	AK2	1232.525
	C9orf78	1231.887
	SHROOM4	1231.273
	CHMP5	1230.644
	KLHDC3	1229.734
	COL5A2	1229.734
	MKRN1	1228.856
	CLPTM1L	1228.232
	FZD4	1227.591
	AHCYL1	1226.997
	C11orf2	1226.102
	TCEA2	1226.102
	SERTAD2	1225.225
	ZNF581	1224.638
	TXNRD1	1224.021
	MRPS22	1223.439
	COPB2	1222.825
	EIF2B2	1222.166
	MPDZ	1221.614
	RABAC1	1220.997
	LRRFIP1	1220.45
	CCT7	1219.851
	LOC643336	1219.27
	EIF4E2	1218.658
	SNRNP70	1218.084
	UNC45A	1217.535
	EPRS	1216.981
	LOC653147	1216.359
	EIF4G2	1215.815
	CHIC2	1215.238
	RALY	1214.672
	COMMD4	1214.132
	HAGH	1213.555
	ATIC	1212.981
	SEMA6A	1212.362
	SDAD1	1211.776
	TMEM173	1211.176
	WDR61	1210.555
	UQCRC1	1209.94
	ERGIC3	1209.292
	C16orf58	1208.466
	FTHL12	1208.466
	TIGA1	1207.301
	ITGB5	1207.301
	ATP2B4	1206.435
	HSPC268	1205.868
	ACP5	1205.3
	CHST7	1204.734
	LOC728643	1204.165
	TMEM93	1203.577
	RNF5P1	1203.002
	IMMT	1202.47
	NOP56	1201.878
	STX5	1201.322
	TXNDC5	1200.716
	LOC100131905	1200.21
	PLEKHM2	1199.673
	LSM7	1199.112
	SPRY1	1198.584
	C19orf60	1198.021
	LSM14A	1197.449
	SRP54	1196.849
	AMZ2	1196.268
	FKBP9L	1195.389
	RAB8A	1195.389
	SPSB3	1194.239
	GIPC1	1194.239
	SLC29A1	1193.425
	MRPL3	1192.832
	CNIH	1192.266
	FAM127B	1191.741
	ATP6V0B	1191.148
	ATP1B3	1190.628
	IDH3B	1190.099
	TFDP1	1189.245
	TECR	1189.245
	GAR1	1188.402
	CDR2L	1187.799
	KIAA1147	1187.189
	IGFBP7	1186.642
	HRASLS3	1186.117
	PFN2	1185.528
	RPL7L1	1184.957
	TDP1	1184.364
	RASA1	1183.793
	BMS1	1183.25
	DRAP1	1182.717
	POLE3	1182.185
	NARF	1181.617
	EBNA1BP2	1181.081
	LOC644563	1180.512
	HECTD1	1179.933
	ATG4A	1179.092
	IRAK1	1179.092
	CCDC92	1178.273
	SNRPA1	1177.46
	CAPZB	1177.46
	SCAMP3	1176.606
	LOC642975	1176.084
	CNIH	1175.454
	TRAPPC4	1174.829
	NISCH	1174.275
	ADARB1	1173.705
	ECHS1	1173.163
	GSN	1172.576
	GOLGA3	1171.965
	TMEM183A	1171.472
	PREI3	1170.618
	COPZ1	1170.618
	RNF149	1169.794
	PRKRIR	1169.219
	KLHL9	1168.706
	RPL9	1168.134
	ANKRD9	1167.583
	MRPL14	1167.011
	CCBE1	1166.449
	VBP1	1165.62
	LAMB1	1165.62
	C12orf10	1164.491
	MRPS10	1164.491
	TWSG1	1163.691
	LOC100132585	1163.139
	MED6	1162.643
	GAK	1162.143
	HPS6	1161.618
	SOX4	1161.072
	CLSTN1	1160.531
	TAF1C	1159.974
	LIMS1	1159.434
	TRIM44	1158.63
	TNPO2	1158.63
	CHMP1B	1157.806
	ATP5G1	1157.298
	TUBG1	1156.749
	NDUFV1	1156.212
	MAP2K1	1155.693
	NOTCH1	1155.126
	UBE2F	1154.596
	POLR2I	1154.025
	RSL1D1	1153.5
	MRPL21	1152.919
	LOC648695	1152.101
	POLR2J3	1152.101
	DNAJB6	1151.286
	TMEM131	1150.796
	CHMP5	1150.222
	UNC84A	1149.634
	FAM84B	1149.095
	SOX7	1148.576
	NEDD8	1148.045
	CRELD2	1147.203
	EEF2K	1147.203
	SH2D3C	1146.37
	ACSS2	1145.794
	CNIH	1145.304
	RPL13	1144.794
	ARF4	1144.238
	ASAP2	1143.712
	HCFC1	1143.163
	SLC41A3	1142.617
	ARID1A	1142.087
	MRPL54	1141.562
	SNRPB2	1140.987
	PAIP2	1140.448
	ULK1	1139.961
	CALD1	1139.447
	MAPBPIP	1138.939
	HARS	1138.45
	HCLS1	1137.98
	SFRS17A	1137.448
	ASH2L	1136.926
	LOC441131	1136.194
	AIF1L	1136.194
	METAP1	1135.416
	TTC37	1134.883
	RNASET2	1134.356
	CARD10	1133.855
	ATP5SL	1133.309
	CTSC	1132.787
	GDPD5	1132.295
	C5orf15	1131.76
	C1orf123	1131.216
	MED28	1130.738
	ADD3	1130.209
	HES4	1129.346
	VPS28	1129.346
	SIRPA	1128.332
	PPP1R16B	1128.332
	ATP1A1	1127.569
	TOP2B	1127.102
	CLDN14	1126.581
	BRIX1	1126.127
	GLRX	1125.649
	PHRF1	1125.135
	ANXA7	1124.618
	PEX11B	1124.07
	LOC390466	1123.317
	FTHL8	1123.317
	RBM4	1122.305
	ACO1	1122.305
	FAF2	1121.286
	ZSCAN18	1121.286
	USO1	1120.278
	PDCD4	1120.278
	BOLA3	1119.499
	GIMAP6	1118.759
	EXOSC1	1118.759
	TNPO1	1117.953
	MRPL21	1117.431
	ETF1	1116.964
	TMEM109	1116.22
	PICALM	1116.22
	PPP2R5E	1115.468
	DHX15	1115.038
	RANGAP1	1114.519
	NUAK1	1114.015
	RAPGEF1	1113.275
	C1orf43	1113.275
	FAM38B	1112.526
	ATP1B3	1111.986
	AW276479	1111.51
	CNPY2	1111.025
	CORO1B	1110.552
	AV737943	1110.036
	ARL6IP6	1109.565
	SRF	1109.065
	GALNT11	1108.587
	DIMT1L	1108.078
	SEC14L1	1107.615
	PTS	1107.107
	PHF5A	1106.657
	NIPA2	1106.208
	LOC728564	1105.495
	LOC728666	1105.495
	CCM2	1104.829
	PLDN	1104.368
	TMEM189	1103.863
	LOC647302	1103.405
	ACOT9	1102.912
	EHD1	1102.392
	TMEM88	1101.651
	RIOK3	1101.651
	CHMP2A	1100.961
	LOC729495	1100.251
	LOC100129211	1100.251
	C11orf74	1099.23
	DHRS4	1099.23
	ALDH7A1	1098.483
	RYBP	1098.015
	CISD1	1097.296
	NCBP2	1097.296
	PLCG1	1096.586
	FBXW11	1096.097
	LAMP2	1095.627
	C11orf67	1095.123
	TXLNA	1094.675
	ST13	1094.182
	ASNSD1	1093.703
	THAP11	1093.181
	SCYL1	1092.677
	C20orf20	1092.201
	ANKRD11	1091.664
	KIAA0494	1090.984
	ATP6V1D	1090.984
	MAGED1	1090.273
	TIA1	1089.785
	HPRT1	1089.321
	C1orf128	1088.849
	STIP1	1088.359
	LAPTM4B	1087.642
	MED16	1087.642
	SLC16A3	1086.895
	EFCAB4A	1086.425
	ERAL1	1085.952
	AKR1B1	1085.496
	GLIPR2	1085.028
	SNRPC	1084.522
	SLC41A3	1084.067
	C12orf35	1083.54
	SMTN	1083.064
	SCAP	1082.62
	UBAC1	1082.171
	CLINT1	1081.704
	TNFRSF25	1081.222
	MRPL45	1080.789
	C4orf32	1080.313
	LOC100128196	1079.856
	CHFR	1079.43
	FUBP3	1078.945
	FLJ35390	1078.417
	ARSD	1077.77
	IMPDH1	1077.77
	TSPAN6	1077.097
	GLB1	1076.624
	SFRS2IP	1076.142
	RHOJ	1075.704
	TIMM22	1075.007
	ARAP3	1075.007
	MYC	1074.047
	PRICKLE1	1074.047
	LOC728620	1073.366
	ZNF467	1072.903
	FAM160B1	1072.44
	PRPF31	1071.962
	LOC100129742	1071.49
	FOXO1	1071.02
	P4HA2	1070.563
	C19orf2	1070.108
	XLKD1	1069.626
	NOS3	1069.22
	FXYD5	1068.745
	RAB32	1068.311
	CPNE1	1067.883
	ARPC1B	1067.446
	LOC729406	1067.012
	LSM3	1066.554
	MYOF	1066.106
	POGK	1065.674
	SFRS2B	1065.215
	GPR137	1064.777
	FAM189B	1064.313
	TOMM40	1063.822
	CRK	1063.404
	DSTN	1062.951
	UGP2	1062.271
	ORMDL1	1062.271
	LOC653566	1061.591
	RBMX	1061.134
	ASAP1	1060.679
	CDC25B	1059.996
	UTP11L	1059.996
	U2AF2	1059.358
	ARF5	1058.938
	ERCC1	1058.498
	VGLL4	1058.016
	CREB3L2	1057.327
	NSL1	1057.327
	AKR1A1	1056.706
	NUDT5	1056.246
	UBQLN1	1055.836
	VPS41	1055.356
	PDIA6	1054.718
	LOC100133516	1054.718
	PELO	1054.057
	LACTB	1053.587
	XRCC2	1053.146
	HIGD1A	1052.714
	SEC22C	1052.285
	CARD8	1051.832
	MAP1B	1051.378
	DRG1	1050.899
	STMN1	1050.489
	LOC440345	1050.027
	DCAF7	1049.575
	BOLA3	1049.18
	APRT	1048.483
	ZDHHC9	1048.483
	SFT2D1	1047.81
	ZNF207	1047.343
	FLJ36131	1046.928
	C6orf125	1046.454
	YIPF3	1045.998
	LOC729992	1045.539
	IGF2BP3	1045.127
	TM9SF2	1044.687
	DCTN6	1044.225
	FXR1	1043.764
	RPL34	1043.313
	AP2M1	1042.912
	LOC644330	1042.479
	TXNDC12	1042.033
	BEX1	1041.572
	PGM1	1041.145
	NRBP2	1040.679
	IRF2BP2	1040.249
	ITFG1	1039.802
	MRPL20	1039.355
	MRPS17	1038.91
	FAM3A	1038.477
	MAN2B2	1037.795
	S100A13	1037.795
	PTPN11	1037.141
	NPEPL1	1036.503
	DPAGT1	1036.503
	STUB1	1035.88
	CDK5RAP3	1035.203
	LOC100128266	1035.203
	LRRC41	1034.576
	RPS21	1034.106
	PIAS4	1033.669
	CHP	1033.24
	CPSF4	1032.79
	FRMD4A	1032.13
	CDK10	1032.13
	LOC650157	1031.44
	LOC100132717	1030.745
	G6PD	1030.745
	UROS	1030.116
	BC035081	1029.698
	LOC730255	1029.265
	ENPP2	1028.837
	CNN2	1028.433
	OSBPL9	1027.99
	TRPT1	1027.561
	RN7SK	1027.133
	COPS7A	1026.742
	NHP2	1026.284
	PAPSS1	1025.863
	MACF1	1025.215
	ACOT7	1025.215
	SERINC3	1024.519
	LAMA4	1024.067
	MRPS15	1023.652
	TM9SF4	1023.053
	ACAT2	1023.053
	LOC645166	1022.43
	NCOA7	1022.016
	TBC1D4	1021.59
	RHOQ	1021.206
	FAM39DP	1020.8
	TNFRSF1A	1020.394
	FKBP5	1019.963
	FAM120B	1019.554
	LCMT1	1019.166
	CCDC59	1018.55
	AK022936	1018.55
	RPUSD4	1017.934
	IGFBP3	1017.538
	SLC35E1	1017.154
	CCDC125	1016.336
	RIN2	1016.336
	MBTPS1	1016.336
	TMEM126B	1015.509
	GPR177	1015.087
	LOC728661	1014.67
	XPO1	1014.219
	ATG4B	1013.813
	DAP3	1013.397
	CISD1	1012.933
	STK19	1012.524
	AES	1011.867
	NDUFA13	1011.867
	NDRG4	1011.251
	FIBP	1010.835
	VHL	1010.439
	RNF38	1009.799
	PRMT1	1009.799
	VPS4B	1009.162
	SHMT2	1008.756
	MRPL34	1008.353
	OCIAD2	1007.943
	PSMD1	1007.326
	HSD17B4	1007.326
	VBP1	1006.484
	MCRS1	1006.484
	TNFAIP1	1005.872
	TNRC6B	1005.446
	COASY	1005.033
	ST3GAL1	1004.617
	RHBDD2	1003.993
	SURF4	1003.993
	KLHDC8B	1003.384
	TSPAN4	1002.961
	KDM5B	1002.552
	STK4	1002.151
	LPHN2	1001.764
	POLR2A	1001.35
	CD59	1000.938
	DNAL4	1000.492
	RHBDF2	1000.062

The 30-MV2-6 cells were maintained in EGM-MV2 media (PromoCell) plus TGFβ inhibitor (SB43154) (Cayman Chemical Co., Ann Arbor, Mich.) in a 5% CO₂, 5% O₂humidified cell culture incubator. The 30-MV2-6 cells were seeded at a density of 40 k/cm². The culture media was removed and after two washes with phosphate buffered saline (PBS), (PBS) was added at 0.1 ml/cm²to produce conditioned medium from which exosomes were isolated. Alternatively, basal EGM-MV2 medium (PromoCell, Heidelberg, Germany) without fetal calf serum or growth factor additives was substituted for PBS. The media was conditioned by the cells in a humidified tissue culture incubator for 16 hours at 37° C. at 5% CO₂and 1% O₂. The conditioned medium was collected and 0.5 volumes of Total Exosome Isolation Reagent (Life Technologies) was added and mixed well by vortexing until there was a homogenous solution. Alternatively, a solution of 15% polyethylene glycol (Hampton Research, Aliso Viejo, Calif.), 1.5 M NaCl (Sigma, St Louis, Mo.) was substituted for the Total Exosome Isolation Reagent. The sample was incubated at 4° C. for at least 16 hours to precipitate the exosomes, followed by centrifugation at 10,000×g for 1 hour at 4° C. The supernatant was removed and the pellet is resuspended in 0.01 volume of PBS.
Exosome particle size and concentration were measured using nanoparticle tracking analysis (NTA; Nanosight, Malvern Instrument, Ltd, Malvern Worcestershire, UK) and by ELISA. The experiments were repeated using commercially available HT1080 cells (ATCC) as a comparison. HT1080 cells are a human fibrosarcoma cell line known to form exosomes with vesicle forming ability (see, e.g. Kim et al. (2002) Cancer Res. 62:6312).
The results of the Nanosight NTA (triplicates) for exosome preparations derived from 30-MV2-6 and HT1080 cells (ATCC, Manassas, Va.) are shown in FIG. 1 . The results indicate that particles prepared from 30-MV2-6 are from 80 to 110 nm with predominant peak at 88 nm+/−2.9 nm. The particles prepared from a HT1080 human fibrosarcoma cells were larger by comparison with a mode of 120 nm+/−7.4 nm. The concentration of exosomes bearing the exosome marker CD63 was measured by ELISA, using samples of known concentrations of HT1080 exosomes as a standard curve. Samples were adsorbed to the ELISA plate by incubation overnight in PBS. The PBS was removed and wells were washed 3 times in ELISA wash buffer (Thermo Scientific, Waltham, Mass.) followed by incubation with primary anti-CD63 antibody (BD Pharmingen, Franklin Lakes, N.J.) for 1 hour at room temperature. The primary antibody was removed followed by washing 3 times in wash buffer and incubation with secondary antibody (HRP conjugated anti-mouse) (Invitrogen, Grand Island, N.Y.)) at 1:3000 dilution for 1 hour at room temperature. The wells were washed 3 additional times with wash buffer and incubated in Super sensitive TMB ELISA substrate (Sigma, St Louis, Mo.) for 0.5 hour followed by addition of ELISA stop solution (InVitrogen, Grand Island, N.Y.). The concentration of exosomes was determined by reading optical density in a standard plate reader at wavelength of 450 nm.

Example 2: Angiogenic Activity of Exosomes Prepared from a Human Embryonic Progenitor Cell Line

Angiogenic activity of exosomes was assayed using an in-vitro endothelial tube forming assay. The assay was performed in triplicate in a μ well slide (Ibidi, Verona, Wis.) or in single wells of a 96-well plate. The wells were coated with reduced growth factor Matrigel (BD, Franklin Lakes, N.J.). Human umbilical cord vascular endothelial cells (HUVEC) that were grown to 70-80% confluence were plated at 5000-7000 cells per well in a μ well slide in 50 ul of EGM-MV2 basal medium (Promocell, Heidelberg, Germany) (no supplements) containing up to 10 μl of exosomes in PBS or equivalent volume of PBS without exosomes as a negative control or in 50 μl of complete EGM-MV2 medium with growth factor supplements as a positive control. Alternatively, the assay was performed in a 96-well plate using 60,000 to 90,000 cells per well in 280 μl of medium and 20 μl of exosomes or PBS. The cells are incubated at 37° C. in a 5% CO₂incubator for 16-18 hours. The cells were photographed under phase contrast at low power or stained with calcein and photographed using a fluorescence microscope. The images were scored for cell covered area, total tube length, number of branch points, and number of loops using Wimasis image analysis (Ibidi, Verona, Wis.). At least 3 random images were quantified per well.
FIG. 2A shows an increase in HUVEC endothelial tube formation when grown in the presence of 30-MV2-6 derived exosomes (in PBS) compared to basal medium with an equivalent amount of PBS (with no exosomes) added (negative control). The quantified results (FIG. 2B) indicate that total tube length, cell covered area, branch points and the number of loops were all increased by the addition of exosomes compared to basal medium indicating that the 30-MV2-6 exosomes are angiogenic.
Angiogenic activity of exosomes was also assessed by their ability to stimulate in vitro tube formation using human embryonic stem (hES) cell derived perivascular embryonic progenitor cells (PEPCs) (also called 017-PC-A) cells bearing pericyte and stemness markers (CD146, CD133, Podoplanin)(U.S. patent application Ser. No. 14/625,621, filed on Feb. 18, 2015). The assay was performed as described for HUVECs except that hES cell derived PEPCs were used instead of HUVECs. The assay was performed in triplicate using μ well slides (Ibidi, Verona, Wis.). The hES pericytes that were grown in defined medium differ from HUVECs in their response to complete medium. HUVECs respond to complete EGM-MV2 medium in the tube forming assay with robust tube formation (FIG. 2A). In contrast, hES PEPCs migrate to form foci consisting of cellular aggregates (FIG. 3A) when grown on Matrigel in complete EGM-MV2 medium and thus exhibited reduced tube formation. The hES PEPCs grown in defined medium form incomplete tubes (FIG. 3B) similar to HUVEC in basal medium (FIG. 2A). Like HUVECs, the hES PEPCs grown in the presence of 30-MV2-6 exosomes displayed an increase in tube formation as shown in FIG. 3C-3E. The tube formation was dose responsive and quantitative analysis indicated an increase in all 4 tube formation parameters (FIG. 3F-3I). Unlike HUVECs which respond to both exosomes and complete medium with increased tube formation, hES derived PEPCs respond to exosomes with increased tube formation but respond to complete medium with reduced tube formation compared to basal medium. Thus, 30-MV2-6 exosomes induce angiogenesis in a non-angiogenic cell type that does not respond to the angiogenic factors present in EGM-MV2 complete medium. These data indicate that 30-MV2-6 derived exosomes do not simply mimic the factors in complete medium but instead are capable of stimulating hES derived PEPC tube formation by a mechanism that is distinct from the action of complete medium on these cells.

Example 3: Comparison of Angiogenic Activity of Exosomes Derived from a Human Embryonic Progenitor Cell Line and Exosomes Derived from Adult Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs)

Exosomes were prepared from an embryonic stem cell derived PureStem® cell line, 30-MV2-6, and from adult bone marrow-derived mesenchymal stem cells (BM-MSCs) from two different commercial sources (Lonza and Promocell), according to methods described in Example 1. The angiogenic activity was assessed using the in vitro endothelial tube formation assay described in Example 2. Briefly, the exosomes (2×10⁸particles/50 μl) were incubated with human umbilical cord vascular endothelial (HUVEC) cells for 12-16 hours on low growth factor Matrigel using a μ-well slide (Ibidi, Verona, Wis.). Tube length was assessed by image capture and analyzed using Angiogenesis Analyzer in ImageJ (http://rsb.info.nih.gov/ij/) image processing program. Total tube length formed per image/μ-well was calculated relative to total tube length formed for HUVEC in complete EGM-MV2 medium (CM) containing angiogenic growth factors (VEGF and FGF2) and fetal bovine serum (FBS).
Exosomes derived from the 30-MV2-6 cell line were compared to early passage BM-MSCs from the two different sources, Promocell (Heidelberg, Germany) (FIG. 4 , panel A) and from Lonza (Basel, Switzerland) (FIG. 4 , panel B). In both cases the angiogenic activity of 30-MV2-6 derived exosomes was greater than the angiogenic activity of BM-MSC derived exosomes. The total tube length of HUVECs incubated in basal medium with 30-MV2-6 derived exosomes (in PBS) was similar to HUVECs incubated in complete EGM-MV2 medium and significantly greater than BM-MSC derived exosomes (from either source) or HUVEC incubated in basal EGM-MV2 medium and PBS alone. The total tube length resulting from BM-MSC derived exosomes was on average slightly higher than PBS in basal medium (BM) but the difference was not statistically significant (p<0.05). These results indicate that embryonic endothelial progenitor stem cell line derived exosomes are advantageous over adult BM-MSC derived exosomes for inducing angiogenic activity in endothelial cells.
BM-MSC and 30-MV2-6 derived exosomes were further compared in a dose response experiment to determine differences in their potency. Exosomes were prepared and their concentration determined using nanoparticle tracking analysis (NTA; Nanosight, Malvern Instruments Ltd, Malvern, Worcestershire, UK). ELISA was used to confirm the presence of transpanins CD63, CD81 and CD9 that are typically expressed on exosomes. The 30-MV2-6 exosomes were tested at doses ranging from 50 million to 400 million exosomes per well. The BM-MSC exosomes were tested at doses ranging from 400 to 1200 million exosomes per well, because no significant activity was observed at 200 million exosomes per well. The results, shown in FIG. 5 indicate that the angiogenic response of HUVEC cells to 30-MV2-6 exosomes is dose responsive starting at 50 million per well and saturating at doses of ≥200 million exosomes per well (FIG. 5 , panel A). In contrast, the BM-MSC exosomes showed a dose response of increasing angiogenic activity at doses from 400 million to 1200 million exosomes per well (FIG. 5 , panel B). The potency of 30-MV2-6 derived exosomes was at least 6-fold greater than that of BM-MSC derived exosomes, having the equivalent activity at 200 million exosomes per well as BM-MSC derived exosomes at 1200 million exosomes per well (FIG. 5 , panel B).

Example 4: Comparison of miRNA Content in Exosomes Derived from a Human Embryonic Progenitor Cell Line Versus Exosomes Derived from Adult BM-MSCs

The miRNA content of the 30-MV2-6 derived exosomes was analyzed and compared to the miRNA content of the less angiogenic BM-MSC exosomes. RNA was extracted and purified from exosomes using the miRNeasy mini kit according to the manufacturer's recommended protocol (Qiagen, Hilden, Germany). The exosome RNA was quantified using a Nanodrop spectrophotometer and cDNA was prepared from the RNA using the miScript II RT kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended protocol. The exosome cDNA was amplified by polymerase chain reaction (PCR) using the miScript pre-AMP PCR kit (Qiagen) and miScript pre-AMP pathway primer mix (human miFinder MBHS-001Z; Qiagen) according to the manufacturer's recommended protocol. Relative miRNA levels were assessed for 84 human miRNAs by quantitative PCR using the human miFinder miScript miRNA PCR array (#331221; Qiagen) according to the manufacturer's recommended protocol. The results were analyzed using the ΔΔCT method of relative quantitation available at (http://perdataanalysis.sabiosciences.com/mirna).
There were substantial differences in the miRNA content of the 30-MV2-6 derived exosomes and BM-MSC derived exosomes. The miRNAs with greater than 6-fold difference between 30-MV2-6 exosomes and BM-MSC exosomes are shown in FIG. 6A scatter plot. Table 2 lists miRNAs that are more than 2-fold overexpressed in 30-MV2-6 exosomes relative to BM-MSC exosomes and Table 3 lists miRNAs that are more than 2-fold underexpressed in 30-MV2-6 exosomes relative to BM-MSC exosomes.

TABLE 2

miRNAs overexpressed in 30-MV2-6 exosomes
compared to BM-MSC exosomes

		Fold
	miRNA Mature ID	Difference

	hsa-miR-155-5p	3.98
	hsa-miR-18a-5p	2.54
	hsa-miR-374a-5p	2.69
	hsa-miR-126-3p	77.60

TABLE 3

miRNAs underexpressed in 30-MV2-6 exosomes
compared to BM-MSC exosomes

		Fold
	miRNA Mature ID	Difference

	hsa-miR-142-5p	−56.29
	hsa-miR-9-5p	−14.69
	hsa-miR-27b-3p	−6.92
	hsa-miR-101-3p	−4.82
	hsa-let-7d-5p	−3.12
	hsa-miR-16-5p	−10.56
	hsa-let-7g-5p	−7.60
	hsa-miR-30c-5p	−3.26
	hsa-miR-96-5p	−11.14
	hsa-miR-185-5p	−3.44
	hsa-miR-142-3p	−17.38
	hsa-miR-24-3p	−9.55
	hsa-miR-181b-5p	−2.03
	hsa-miR-302b-3p	−42.52
	hsa-miR-30b-5p	−3.34
	hsa-miR-21-5p	−16.16
	hsa-miR-15b-5p	−4.08
	hsa-miR-223-3p	−18.36
	hsa-miR-194-5p	−2.69
	hsa-miR-15a-5p	−4.25
	hsa-miR-125b-5p	−38.36
	hsa-miR-99a-5p	−10.43
	hsa-miR-29b-3p	−15.10
	hsa-miR-29a-3p	−35.08
	hsa-miR-141-3p	−4.30
	hsa-let-7a-5p	−4.03
	hsa-miR-124-3p	−13.56
	hsa-miR-92a-3p	−2.28
	hsa-miR-23a-3p	−5.74
	hsa-miR-25-3p	−3.16
	hsa-let-7e-5p	−2.35
	hsa-miR-376c-3p	−752.81
	hsa-miR-144-3p	−57.26
	hsa-miR-195-5p	−10.03
	hsa-miR-143-3p	−82.38
	hsa-miR-191-5p	−4.88
	hsa-let-7i-5p	−9.62
	hsa-miR-302a-3p	−17.39
	hsa-miR-222-3p	−2.31
	hsa-let-7b-5p	−35.56
	hsa-miR-186-5p	−3.40
	hsa-miR-196b-5p	−71.33
	hsa-miR-27a-3p	−3.42
	hsa-miR-22-3p	−4.58
	hsa-miR-130a-3p	−2.68
	hsa-let-7c-5p	−10.92
	hsa-miR-29c-3p	−24.99
	hsa-miR-140-3p	−2.98
	hsa-miR-128-3p	−2.77
	hsa-let-7f-5p	−2.89
	hsa-miR-122-5p	−9.20
	hsa-miR-100-5p	−10.00
	hsa-miR-302c-3p	−144.85

The miRNA with the highest relative expression in 30-MV2-6 exosomes compared to BM-MSC exosomes is miR-126-3p (77.6-fold difference, Table 2). MiR-126 is a known angiogenic miRNA (“angiomiR”) that is endothelial cell-specific and has been shown to regulate both vascular integrity and developmental angiogenesis. Fish et al. (2008) Dev. Cell 15(2):272; Zou et al. (2011) Circ. Res. 108 (2):201; Jakob and Landmesser (2012) Cardiovasc. Res. 93(4):614; Nicoli et al. (2010) Nature 464(7292): 1196. Induction of miR-126 in endothelial cells and transport of miR-126 via exosomes has been shown to be important for the effective treatment of myocardial infarction (MI) using transplanted cardiosphere derived cells in a mouse model. Ong et al. (2014) Circulation 130 (11 Suppl 1):S60.
Accordingly, the clonal human embryonic progenitor cell line derived, miR-126-containing exosomes of the instant invention can be used in in vitro angiogenesis studies as well as in treatment of myocardial infarction and other ischemic conditions, either by themselves or in combination with transplanted cells.
RNA from 30-MV2-6 exosomes was also used to compare the miRNA content of angiogenic versus non-angiogenic exosomes. Exosomes derived from HT1080 cells (a human sarcoma cell line) are not angiogenic in the HUVEC in vitro angiogenesis assay at 2.0×10⁸exosomes, a dose at which 30-MV2-6 exosomes show maximum angiogenic activity. HT1080 exosome RNA was analyzed on the miFinder miScript PCR array of 84 human miRNAs as described above and compared to 30-MV2-6 and BM-MSC exosome RNA (Table 4).

TABLE 4

Exosomal miRNAs in BM-MSC and 30-
MV2-6 Relative to HT1080 exosomes

	BM-MSC/	30-MV2-6/
	HT1080	HT1080
	Fold	Fold
miRNA Mature ID	Difference	Difference

hsa-miR-142-5p	13.3253	0.2367
hsa-miR-9-5p	19.9437	1.3579
hsa-miR-150-5p	4.3783	3.3457
hsa-miR-27b-3p	100.911	14.5862
hsa-miR-101-3p	14.0298	2.9096
hsa-let-7d-5p	26.3588	8.441
hsa-miR-103a-3p	4.5649	5.7031
hsa-miR-16-5p	38.0552	3.605
hsa-miR-26a-5p	41.3044	24.3186
hsa-miR-32-5p	15.9192	14.3894
hsa-miR-26b-5p	52.2677	31.9112
hsa-let-7g-5p	76.0374	10.0027
hsa-miR-30c-5p	17.5335	5.3714
hsa-miR-96-5p	1.1148	0.1
hsa-miR-185-5p	29.375	8.5466
hsa-miR-142-3p	0.5468	0.0315
hsa-miR-24-3p	40.061	4.1927
hsa-miR-155-5p	1.0346	4.118
hsa-miR-146a-5p	1.4501	1.0803
hsa-miR-425-5p	3.3003	2.8192
hsa-miR-181b-5p	16.2276	8.0058
hsa-miR-302b-3p	45.0196	1.0589
hsa-miR-30b-5p	19.9194	5.9589
hsa-miR-21-5p	52.4899	3.2475
hsa-miR-30e-5p	4.4078	3.3143
hsa-miR-200c-3p	2.2547	1.3309
hsa-miR-15b-5p	22.3822	5.4881
hsa-miR-223-3p	202.2804	11.0146
hsa-miR-194-5p	8.5348	3.1718
hsa-miR-210-3p	1.8074	0.957
hsa-miR-15a-5p	17.2702	4.0614
hsa-miR-181a-5p	28.6581	16.7792
hsa-miR-125b-5p	31.2942	0.8158
hsa-miR-99a-5p	13.0061	1.2466
hsa-miR-28-5p	11.1092	8.1182
hsa-miR-320a	17.2154	13.7088
hsa-miR-125a-5p	13.0238	8.8833
hsa-miR-29b-3p	9.9121	0.6563
hsa-miR-29a-3p	51.1831	1.4592
hsa-miR-141-3p	7.9244	1.841
hsa-miR-19a-3p	4.5159	3.3341
hsa-miR-18a-5p	7.7906	19.7947
hsa-miR-374a-5p	45.91	123.3183
hsa-miR-423-5p	20.4698	18.6725
hsa-let-7a-5p	25.3493	6.294
hsa-miR-124-3p	20.2226	1.4916
hsa-miR-92a-3p	14.4072	6.3169
hsa-miR-23a-3p	52.5584	9.1492
hsa-miR-25-3p	23.7331	7.5106
hsa-let-7e-5p	22.9181	9.7439
hsa-miR-376c-3p	2411.7739	3.2037
hsa-miR-126-3p	123.9456	9618.223
hsa-miR-144-3p	114.87	2.0061
hsa-miR-424-5p	57.34	92.8881
hsa-miR-30a-5p	3.966	3.4279
hsa-miR-23b-3p	19.0703	13.0079
hsa-miR-151a-5p	12.8604	25.4071
hsa-miR-195-5p	40.2617	4.0149
hsa-miR-143-3p	273.1733	3.316
hsa-miR-30d-5p	3.9436	4.0508
hsa-miR-191-5p	21.2256	4.3493
hsa-let-7i-5p	62.9552	6.545
hsa-miR-302a-3p	37.209	2.14
hsa-miR-222-3p	22.8182	9.8699
hsa-let-7b-5p	129.1842	3.6327
hsa-miR-19b-3p	5.758	3.3451
hsa-miR-17-5p	4.6186	7.3351
hsa-miR-93-5p	16.2937	16.7172
hsa-miR-186-5p	14.8015	4.3526
hsa-miR-196b-5p	15.3129	0.2147
hsa-miR-27a-3p	48.4243	14.148
hsa-miR-22-3p	13.8229	3.0157
hsa-miR-130a-3p	11.5632	4.3074
hsa-let-7c-5p	93.5201	8.5615
hsa-miR-29c-3p	39.8245	1.5935
hsa-miR-140-3p	9.8899	3.3169
hsa-miR-128-3p	21.7273	7.8472
hsa-let-7f-5p	30.5392	10.5782
hsa-miR-122-5p	71.7561	7.8014
hsa-miR-20a-5p	4.6119	9.1362
hsa-miR-106b-5p	5.093	8.4218
hsa-miR-7-5p	4.8173	6.4945
hsa-miR-100-5p	14.7509	1.4748
hsa-miR-302c-3p	120.5214	0.832

The data in Table 4 and FIG. 6B illustrate the dramatic differences in the miRNA content of the three types of exosomes. These data show that HT1080 exosomes contain the lowest amount of miRNA for all miRNAs tested, except for miR-96-5p and miR-142-3p, which are lowest in 30-MV2-6 exosomes. The miRNA miR-142-3p is expressed at highest levels in HT1080 exosomes and is known to repress several inhibitors of oncogenic transformation. It is mimicked by Kaposi sarcoma viral miRNA, miR-K10a. Forte et al. (2015) J Virol 89(4): 2333. The miR-96-5p miRNA is present in highest levels in BM-MSC exosomes and is higher in HT1080 exosomes than 30-MV2-6 exosomes. This miRNA is thought to be involved in osteogenic and adipogenic differentiation in BM-MSCs (Laine et al. (2012) J Cell Biochem. 113(8):2687) but is also involved in tumor cell proliferation (Lin et al. (2010) Plos One 5(12):e15797; Haflidadottir et al. (2013) Plos One 8(8):e72400). Strikingly the miRNA with the highest levels in 30-MV2-6 exosomes relative to HT1080 exosomes is miR-126-3p. This known angiogenic miRNA is present at a 9618-fold higher level in 30-MV2-6 exosomes than HT1080 exosomes. MiR-155 is 4-fold higher in 30-MV2-6 exosomes than BM-MSC or HT1080 exosomes and is anti-angiogenic but pro-arteriogenic. Pankrtaz et al. (2015) Circulation 131(18):1575. Of the 9 remaining miRNAs that are highest in 30-MV2-6 exosomes, 6 are known to be involved in angiogenesis. The miRNAs miR-18a-5p, miR-20a-5p, miR-424-5p, miR-17-5p, and miR-7-5p miRNAs have anti-angiogenic activity. However, none of these anti-angiogenic miRNAs are more than 2.5-fold enriched in 30-MV2-6 exosomes compared to BM-MSC exosomes. The pro-angiogenic miR-106b is 9-fold enriched in 30-MV2-6 but only 4-fold enriched in BM-MSC exosomes compared to HT1080 exosomes. It is needed for neovascularization after hind limb ischemia. Semo et al. (2014) Eur Heart J. 35(45):3212.
Notably, several anti-angiogenic miRNAs, including miR-143-3p (Climent et al. (2015) Circ Res. 116(11):1753), miR-223-3p (Dai et al. (2014) Plos One 9(10):e108468), miR-222-3p (Suarez and Sessa (2009) Circ Res. 104(4):442), miR-15a, miR-15b and miR-16 (Spinetti et al. (2013) Circ Res. 112(2):335; Liu et al. (2012) Cell Physiol Biochem. 29(5-6):851)) were enriched for and/or present at highest level in the BM-MSC-derived exosomes.

Example 5: Comparison of Angiogenic Activity of Exosomes Derived from Various Clonal Embryonic Stem Cell Lines and Exosomes Derived from the Parental Pluriopotent Stem Cell Lines

Clonal embryonic progenitor cell lines were previously established from human pluripotent stem (hPS) cell lines using methods previously described. West et al. (2008) Regen Med. 3(3):287. The resulting cell lines are not immortalized but have higher replicative potential than primary cell lines because of their long telomere length that is near that of the parental hPS cell line from which they are derived. A wide diversity of cell types was produced by exposing hPS cells to an array of cell culture medium, cell matrix, and growth conditions followed by selective pressure for clonal growth and scalability. Over 140 such cell types have been determined to be distinct by analysis of total transcribed RNA using standard Illumina microarrays. The in vitro angiogenesis assay (described in detail in Example 2) was used to screen clonal embryonic progenitor cells for production of angiogenic exosomes. As shown in Table 5, most embryonic endothelial progenitor cell-derived exosomes have angiogenic activity in the range of 30-MV2-6 derived exosomes (+; relative tube length (RTL)>0.75 and <1.25). The 30-MV2-9 exosomes scored highest (++; RTL>1.25). Two endothelial progenitor lines scored negative (−; RTL<0.75). Exosomes from an osteochondral line, primary fibroblasts (BJ), BM-MSCs, and a human sarcoma cell line (HT1080) were also negative. The two clonal smooth muscle cell progenitor cell lines and one clonal pericyte line tested were positive in the in vitro vascular tube formation assay. Exosomes prepared from conditioned medium of the parental human embryonic stem cell lines H9 (WA09) and ESI-017 were also positive in the in vitro vascular tube formation assay.

TABLE 5

TABLE 5: Angiogenic activity of PureStem vascular
progenitors and other cell lines.

			Relative tube	Angiogenic
Cell Line	Source	Cell Type	length	Index

30-MV2-9	PureStem	Endothelial	1.67	++
30-MV2-6	PureStem	Endothelial	1.07	+
30-MV2-7	PureStem	Endothelial	0.94	+
30-MV2-17	PureStem	Endothelial	0.76	+
30-MV2-19	PureStem	Endothelial	0.84	+
RP1-MV2-8	PureStem	Endothelial	1.07	+
30-MV2-14	PureStem	Endothelial	1.00	+
30-MV2-15	PureStem	Endothelial	1.00	+
RP1-MV2-8	PureStem	Endothelial	1.07	+
30-MV2-24	PureStem	Endothelial	0.41	−
30-MV2-4	PureStem	Endothelial	0.72	−
W10	PureStem	Smooth Muscle	0.83	+
Z11	PureStem	Smooth Muscle	0.92	+
E164	PureStem	Pericyte	0.86	+
PC-M	hES	Pericyte	0.47	−
BJ	Primary	Foreskin	0.61	−
		Fibroblast
SM30	PureStem	MSC-like	0.71	−
MSCs	Primary	Adult	0.68	−
		BM-MSC
HT1080	Transformed	Sarcoma	0.71	−
H9	Embryonic	Pluripotent	1.06	+
ESI-017	Embryonic	Pluripotent	1.27	+

Example 6: In Vivo Angiogenic Activity of Embryonic Progenitor Stem Cell Derived Exosomes

Angiogenic activity of exosomes was assessed in vivo using the Matrigel plug assay in mice as previously described. Sahoo et al. (2011) Circ Res. 109(7):724. Immunocompromised mice (Female Nu/J mice aged 6-8 weeks; 2 plugs/mouse; 2 mice/group) were injected subcutaneously with approximately 300 μl of Matrigel containing PBS, 4×10⁸/ml exosomes, or 150 ng/ml bFGF plus 60 ng/ml VEGF (positive control). The plugs were removed at day 14 after implant followed by fixation and paraffin embedding. The sections were stained with hematoxylin and Eosin (H&E) for histological examination and stained with von Willabrand factor antibody for detection of endothelial cells.
The data indicate that 30-MV2-6 exosomes are angiogenic in the Matrigel plug assay (FIG. 7 ). The exosome containing plugs show regions of infiltration of cells into the plug with vessel formation (FIG. 7 , panels A and C). The positive control plugs containing growth factors have regions of vessel formation (not shown) Immunostaining with antibody against von Willabrand factor (FIG. 7 , panels B and D) confirmed the endothelial identity of cells lining the vessel structures observed by H&E staining. The PBS control plugs show less cell infiltration and no vessel formation (FIG. 7 , panel E).

Example 7: Scale-up of Clonal Embryonic Progenitor Stem Cells for Exosome Production

Clonal embryonic progenitor cell lines described here are advantageous over other sources of biologically active exosomes because of their scalability. The parental pluripotent stem cell line to 30-MV2-6 which also produces angiogenic exosomes is costly to scale up because of the requirements for specialized medium and cell matrix (e.g. Matrigel). Primary endothelial stem cells or mesenchymal stromal cells rapidly lose differentiation and proliferative capacity upon culture in vitro. Typically MSCs begin to senesce in culture after 7-12 passages (approximately 10 population doublings) and show multiple changes including altered surface marker expression and increased autofluorescence. Wagner et al. (2008) Plos One 3(5):e2213. In contrast, human embryonic clonal progenitor lines such as the cell lines of the instant invention are grown under standard tissue culture conditions and medium and are highly scalable with typical replicative lifespans of 60 to 100 population doublings.
The Terumo Quantum Cell Expansion system (the bioreactor used in the instant example) is an automated hollow fiber cell culture platform designed for GMP compatible production of cells for use in cell therapy. The bioreactor was seeded at a density of approximately 900 cells/cm²with approximately 4.0×10⁷30-MV2-6 cells (passage 9) and the cells were cultured for 13 days under their standard growth conditions of EGM-MV2 medium and 5% oxygen. The exosomes were collected by exchanging the complete medium for conditioning medium (basal EGM-MV2 medium without serum added; alternatively PBS may be also used). The conditioning medium was left in the bioreactor for 16 hours and collected for exosome purification. The cells were harvested by exchanging medium with a 0.25% trypsin solution to remove cells for the reactor, tested for viability and counted. Cells were scaled over 10-fold from the initial 40 million to approximately 440 million. The purified exosomes were quantified using CD63 detection ELISA (alternatively, nanoparticle tracking analysis as described in Example 1 may be used to quantify the exosomes). The yield of exosomes from one bioreactor run is at least 2.3×10¹⁰, which is equivalent to the approximate exosome yield from 72 T-225 flasks of 30-MV2-6 cells.
The purified exosomes were tested for angiogenic activity at a dose of 2.0×10⁶exosomes per well in the in vitro tube formation assay (described in detail in Example 2). As shown in FIG. 8 , the angiogenic activity of exosomes prepared from media conditioned by 30-MV2-6 cells grown in T-flasks was equivalent to the angiogenic activity of exosomes prepared from medium conditioned by 30-MV2-6 cells grown in the Quantum Cell Expansion system.

Example 8: Effect of Oxygen Concentration and Conditioning Medium on Exosome Activity

Hypoxia has been reported to increase exosome production from mammalian cells (Tadokoro et al. (2013) J Biol Chem. 288(48):34343; King et al. (2012) BMC Cancer 12:241). Furthermore, clonal embryonic progenitor cell lines are derived and maintained under low oxygen (5%). West et al. (2008) Regen Med. 3(3): 287. Therefore, 1% oxygen was tested for exosome production to determine if increasing hypoxia will increase exosome production or angiogenic activity.
Other stress conditions can also have an effect on exosome yield or activity. Serum starvation is used to induce exosome production. Nutrient deprivation was tested by using PBS as the conditioning medium. The use of PBS versus basal EGM-MV2 medium for conditioning the cells was also tested.
The results shown in FIG. 9 indicate that there is no significant difference in angiogenic activity of isolated exosomes when the medium was conditioned by cells incubated in 1% or 5% oxygen. These data also indicate the exosome angiogenic activity is not significantly different when PBS is used as the conditioning media compared to when the basal medium is used as the conditioning medium, although there is a trend toward higher activity when PBS is used.

Example 9: Quantitation of Exosome Concentration by ELISA Detection of CD63 on Intact Exosomes

There is a need for simple and convenient method to measure the concentration of exosome particles in a purified preparation of exosomes. Currently available ELISA kits for measuring exosome concentration (System Biosciences, Inc., Mountain View, Calif.) require lysing exosomes and have a lower limit of detection of approximately 2.0×10⁸exosomes. It is advantageous to measure low concentration samples directly without diluting the sample in a lysis buffer. Moreover, lysing the exosomes releases other proteins and nucleic acids that potentially interfere with the assay. The method described herein takes advantage of markers commonly presented on the surface of exosomes, such as transpanins CD63, CD9 and CD81 and allows for quantitation of intact exosomes.
A standard curve is prepared from exosome samples of known concentration (ranging from 5×10⁸to 8×10⁷exosomes/mL). The unknown samples are prepared in PBS or a buffer exchanged into PBS. Samples of intact exosomes are bound to 96 well ELISA plate wells in PBS at 50 μl/well for at least 16 hours at 37° C. The wells are washed 3×5 minutes in wash buffer (e.g. TBS-Tween). The wells are incubated with a mouse monoclonal antibody prepared in a suitable blocking buffer (e.g. PBS containing exosome depleted FBS and 0.05% Tween 20) that recognizes the extracellular domain of CD63 on intact exosomes for 1 hour at room temperature. The wells are washed again 3×5 minutes at room temperature. The wells are incubated with a suitable secondary antibody in a blocking buffer for detection of mouse anti-CD63 antibody bound to exosomes on the plate surface (e.g. HRP conjugated goat anti-mouse IgG) for 1 hour at room temperature. The wells are washed again 3×5 minutes at room temperature and the wells incubated with 50 μl of HRP substrate (e.g. Supersensitive TMB ELISA substrate) for 30 minutes at room temperature. The wells are washed 3×5 minutes at room temperature and 50 μl of stop buffer (0.16M sulfuric acid) is added to provide a fixed endpoint. The concentration of exosomes is quantitated by measuring the absorbance of each well at 450 nm.
An example of a standard curve and quantitation of samples is shown in FIG. 10 .

Claims

1.-10. (canceled)

11. A method of inducing or enhancing a cell's ability to form vascular tube like structures comprising contacting a cell capable of making vascular tube like structures with an exosome isolated from a progenitor cell thereby inducing or enhancing a cell's ability to form vascular tube like structures.

12. The method of claim 11, wherein the cell capable of making vascular tube like structures is an endothelial cell.

13. The method of claim 12, wherein the endothelial cell is a HUVEC.

14. A method of providing or sustaining vasculature in a natural or an artificial organ for transplantation, the method comprising contacting the natural or artificial organ with an exosome isolated from a clonal progenitor cell.

15. The method of claim 14, wherein the clonal progenitor cell is positive for the expression of PCDHB2.

16. The method of claim 14, wherein the exosome comprises CD63.

17. A method of treating a degenerative disease associated with vascular aging in a subject in need of treatment, the method comprising administering to the subject an exosome isolated from a clonal progenitor cell.

18. The method of claim 17, wherein the degenerative disease associated with vascular aging is sarcopenia, dementia, chronic wounds, osteoporosis, cardiovascular disease, heart failure, infarction, chronic wounds, ulcer, clogged vessels or arteries, damaged vessels, stenotic vessels, arteriosclerosis, angina, peripheral vascular disease, Alzheimer's disease, ischemia, diabetes, cancer, a disease associated with cell replacement transplant or therapy, a disease associated with tissue and cell regenerative therapy, Parkinson's disease, stroke, or any combination thereof.

19. The method of claim 17, wherein the clonal progenitor cell line is positive for the expression of PCDHB2.

20. The method of claim 17, wherein the exosome comprises CD63.