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

一种造血干细胞、干细胞的技术，应用在治疗眼血管疾病,造血干细胞群领域，能够解决机制不清楚、无法确定细胞数目等问题

Inactive Publication Date: 2008-12-03
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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Effect test

Embodiment 1

[0066] Example 1. Cell Separation and Enrichment: Mouse Lin - Preparation of HSC populations A and B

[0067] General steps. 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 Scripps Research Institute (TSRI, La Jolla, CA) Animal Care and Use Committee. 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).

[0068] by using 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 HSC (Lin + ) cells and rem...

Embodiment 2

[0075] Example 2. Intravitreal Administration of Cells in a Murine Model

[0076] 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

[0077] Example 3. EPC transfection

[0078] 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 (SEQID NO: 1, Figure 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 as Figure 8 Shown in SEQ ID NO:2.

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Abstract

An isolated mammalian lineage-negative hematopoietic stem cell population derived from mature bone marrow containing endothelial progenitor cells (EPCs) (Lin - HSCs) can repair retinal blood vessels and neural networks in the eye. Preferably, the isolated Lin - At least about 20% of cells in HSCs express the cell surface antigen CD31. Separated Lin - HSC populations are useful for the treatment of ocular vascular diseases. In a preferred embodiment, Lin - HSC populations were obtained by: aspirating the bone marrow of an adult mammal; isolating a variety of monocytes from the bone marrow; Lineage surface antigen-positive monocytes were depleted from monocytes, followed by recovery of Lin containing EPCs - HSCs populations, to isolate. Also provided is an isolated Lin transfected with a therapeutically useful gene. - HSCs 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 (E...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12N5/078A61K35/12A61K35/28A61K48/00A61P9/10A61P27/02A61P27/12C12N5/071C12N5/074C12N5/0789C12N5/10C12N15/09G01N33/48G01N33/53
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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