Isolation, expansion and use of clonogenic endothelial progenitor cells

Abstract

A hierarchy of endothelial colony forming cells (EPCs) was identified from mammalian cord blood, umbilical vein and aorta. A newly isolated cell named high proliferative potential—endothelial colony forming cell (HPP-ECFC) was isolated and characterized. Single cell assays were developed that test the proliferative and clonogenic potential of endothelial cells derived from cord blood, or from HUVECs and HAECs. EPCs were found to reside in vessel walls. Use of a feeder layer of cells derived from high proliferative potential-endothelial colony forming cells (HPP-ECPCS) from human umbilical cord blood, stimulates growth and survival of repopulating hematopoietic stem and progenitor cells. Stimulation of growth and survival was determined by increased numbers of progenitor cells in in vitro cultures and increased levels of human cell engraftment in the NOD / SCID immunodeficient mouse transplant system.

Description

[0001] This application claims priority from U.S. Ser. No. 60 / 543,114 filed Feb. 9, 2004, U.S. Ser. No. 60 / 542,949 filed Feb. 9, 2004, U.S. Ser. No. 60 / 573,052 filed May 21, 2004 and U.S. Ser. No. 60 / 637,095 filed Dec. 17, 2004.BACKGROUND OF THE INVENTION [0002] Models for stem cell differentiation leading to endothelial and hematopoietic cells are of interest because of the clinical value of stem cells and their progeny. A hallmark of stem and progenitor cells is their ability to proliferate and give rise to functional progeny, and progenitor cells are identified by their clonogenic potential. Methods previously used do not guarantee that single endothelial cells have been isolated and characterized to identify the progenitors. [0003] Endothelial cell proliferation in vivo in normal, mature arterial, venous, and capillary vessels in most mammals is reported to be extremely low, if not nonexistent. In some experimental animals, such as pigs and dogs, radiolabeling studies have demon...

AI Technical Summary

Benefits of technology

[0043] Cord blood high proliferative potential—endothelial colony forming cells (HPP-ECFCs) in co-culture with autologous or unrelated cord blood, mobilized adult peripheral blood, or marrow-derived HSC expands the number of HSC cells and results in an increase in HSC and an increase in HSC repopulating activity leading to higher levels of engraftment in a recipient subject.

[0044] Use of a feeder layer of cells derived from high proliferative potential-endothelial colony forming cells (HPP-ECPCs) from human umbilical cord blood, stimulates growth and survival of repopulating hematopoietic stem and progenitor cells. Stimulation of growth and survival was determined by increased numbers of progenitor cells in in vitro culture and increased levels of human cell engraftment in the NOD / SCID immunodeficient mouse transplant system.

Problems solved by technology

Methods previously used do not guarantee that single endothelial cells have been isolated and characterized to identify the progenitors.

However, there is no uniform definition of an EPC, which makes interpretation of these studies problematic and prohibits reproduction of cell types suitable for clinical use.

Sole dependence on cell surface expression of molecules can be problematic because the expression may vary with the physiologic state of the cell.

No assay is reported to assess the proliferative potential (an intrinsic response) in individual endothelial cells or EPCs and thus, no comparative analysis is available.

Although genetic studies clearly show that the origin of endothelial cells is closely linked to hematopoietic cell development, evidence to support a similar hierarchy of stem and progenitor endothelial cells based on differences in proliferative potential has not been established.

Limitations to a more widespread use of cord blood for transplant include the fact that only a limited number of HSC and progenitor cells are present in a graft.

Because most patients do not have a matched sibling donor, most cord blood grafts are transplanted into mismatched recipients.

Approaches to cord blood HSC expansion have not been impressive.

However, few approaches have been effective in increasing the number of HSC as measured by SCID repopulating cells (SRC) frequency in NOD / SCID mice or long-term engraftment in fetal sheep.

The results of using expanded cord blood HSC in human patients have been disappointing.

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