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Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

A technology of hematopoietic stem cells and stem cells, which is applied in the field of hematopoietic stem cell groups to treat ocular vascular diseases, and can solve problems such as unclear mechanisms and uncertain cell numbers

Inactive Publication Date: 2006-08-09
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these cells have been used in several experimental models of angiogenesis, the mechanism by which EPCs act on neovascularization remains unclear, and strategies to effectively increase the number of cells targeting specific vessels have not been identified

Method used

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  • Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith
  • Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith
  • Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

Examples

Experimental program
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Embodiment 1

[0065] Example 1. Cell Separation and Enrichment: Mouse Lin - Preparation of HSC populations A and B

[0066] General Procedures. All in vivo evaluations were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all evaluation procedures were approved by the Animal Care and Use Committee of the Scripps Research Institute (TSRI, La Jolla, CA). Bone marrow cells were extracted from B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB / NJ (rd / rd mice) or Balb / cBYJ adult mice (The Jackson Laboratory, ME).

[0067] By using HISTOPAQUE  Density gradient centrifugation of sucrose gradients (Sigma, St.Louis, MO) to isolate mononuclear cells was used in mice for Lin - Selected biotin-conjugated lineage population antibodies (CD45, CD3, Ly-6G, CD 11, TER-119, Pharmingen, San Diego, CA) were used to label these monocytes. Using magnetic separation equipment (AUTOMACS TM sorter, Miltenyi Biotech, Auburn, CA) from Lin - Positive lineage segregation in HS...

Embodiment 2

[0072] Example 2. Intravitreal Administration of Cells in a Murine Model

[0073] A portion of eyelid tissue was cut in the eyelid of the mouse using a sharp blade to expose the eyeball from P2 to P6. The lineage-negative HSC population A of the present invention was then injected intravitreally using a 33-gauge (Hamilton, Reno, NV) needle syringe (about 10 in about 0.5 μl to about 1 μl of cell culture medium). 5 cells).

Embodiment 3

[0074] Example 3. EPC transfection

[0075] According to the manufacturer's manual, using FuGENE TM 6 Transfection reagent (Roche, Indianapolis, IN), using the T2 fragment encoding TrpRS and containing His 6 Tag (SEQ ID NO: 1, Fig. 7) DNA transfection mouse Lin - HSC (group A). Lin - HSC cells (approximately 10 per ml 6 cells) were suspended in opti-MEM medium (Invitrogen, Carlsbad, CA) containing stem cell factor (PeproTech, Rocky Hill, NJ). Then DNA (about 1 μg) and FuGENE reagent (about 3 μl) were added, and the mixture was incubated at about 37° C. for about 18 hours. After incubation, cells are washed and harvested. The transfection efficiency of this system was confirmed to be about 17% by FACS analysis. T2 products were confirmed by Western blot. with His 6 The amino acid sequence of the tagged T2-TrpRS is shown in SEQ ID NO: 2 in FIG. 8 .

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Abstract

Isolated, mammalian, mature bone marrow-derived, lineage-negative hematopoietic stem cell populations (Lin-HSCs) containing endothelial progenitor cells (EPCs) are capable of repairing retinal vascular and neural networks in the eye. Preferably, at least about 20% of the cells in the isolated Lin-HSCs express the cell surface antigen CD31. The isolated Lin-HSC population has utility for the treatment of ocular vascular diseases. In a preferred embodiment, the Lin-HSC population is obtained by: extracting the bone marrow of an adult mammal, isolating multiple monocytes from the bone marrow, using biotin-conjugated lineage populations directed against one or more lineage surface antigens These monocytes were labeled with antibodies; monocytes positive for lineage surface antigens were removed from various monocytes, and then the Lin-HSCs population containing EPCs were recovered for isolation. Also provided are isolated Lin-HSCs transfected with a therapeutically useful gene for gene delivery into the eye in cell-based gene therapy. Also described are methods of making the isolated stem cell populations of the invention, and methods of treating eye diseases and eye injuries.

Description

[0001] Cross References to Related Applications [0002] This application is a continuation-in-part of U.S. Patent Application 10 / 628,783, filed July 25, 2003, a provisional application claiming 60 / 398,522, filed July 25, 2002, and 60 / 467,051, filed May 2, 2003 The entire disclosure content of the above-mentioned application is incorporated into this application by reference. [0003] Statement of Government Interest [0004] Part of the work described herein was supported by National Cancer Institute grant CA92577 and National Institutes of Health grants EY11254, EY12598, and EY125998. The US Government has certain rights in this invention. technical field [0005] The present invention relates to isolated, mammalian, lineage-negative hematopoietic stem cells derived from bone marrow (Lin - HSC) and its applications. More particularly, the present invention relates to isolated, mammalian, lineage-negative hematopoietic stem cells containing endothelial progenitor cells (...

Claims

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Application Information

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IPC IPC(8): A01N63/00A01N65/00C12N5/08C12N5/078A61K35/12A61K35/28A61K48/00A61P9/10A61P27/02A61P27/12C12N5/071C12N5/074C12N5/0789C12N5/10C12N15/09G01N33/48G01N33/53
CPCA61K2035/124A61K35/12A61K48/00C12N5/0692C12N5/0647A61P7/02A61P7/06A61P9/10A61P9/14A61P25/00A61P27/02A61P27/12A61P41/00A61P43/00C12N5/10
Inventor M·弗里德兰德A·奥塔尼K·达西尔瓦S·哈内坎普
Owner THE SCRIPPS RES INST
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