Methods of controlling proliferation and differentiation of stem and progenitor cells

Abstract

A method of expanding a population of cells, while at the same time inhibiting differentiation of the cells, the method includes the step of providing the cells with conditions for cell proliferation and, at the same time, for reducing a capacity of the cells in utilizing copper. The method can be executed both in-vivo and ex-vivo. A method of inducing differentiation in a population of cells, the method includes the step of providing the cells with a transition metal chelator which binds copper and which is effective in inducing cell differentiation.

Description

[0001] This is a continuation of PCT / IL99 / 00444, filed Aug. 17, 1999, which claims priority from U.S. patent application Ser. No. 09 / 161,659, filed Sep. 29, 1998, which is a continuation in part of U.S. patent application Ser. No. 09 / 130,367, filed Aug. 7, 1998, which is a continuation in part of U.S. patent application Ser. No. 09 / 024,195, filed Feb. 17, 1998. In addition, PCT / IL99 / 00444 claims priority from PCT / US99 / 02664, filed Feb. 8, 1999, which claims priority from U.S. patent application Ser. Nos. 09 / 024,195 and 09 / 30,367.FIELD AND BACKGROUND OF THE INVENTION[0002] The present invention relates to methods of controlling proliferation and differentiation of stem and progenitor cells. In one aspect, the present invention relates to a method of imposing proliferation yet restricting differentiation of stem and progenitor cells by treating the cells with chelators of transitional metals, resulting in reduction if transitional metals availability. In another aspect, the present in...

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Benefits of technology

0020] The Effect of Copper in Cell Lines:

0021] The effect of Copper was also studied in-vitro established cell lines (31-34). One such line (HL-60) was derived from a patient with acute promyelocytic leukemia. These cells, that have the characteristics of myeloblasts and promyelocytes, can grow indefinitely in culture. Upon addition of various agents, such as retinoic acid (RA), to the culture medium, the cells undergo differentiation, which results in cells which demonstrate some, but not all, features of mature granulocytes.

0022] The study of Copper status in these cells has shown that although the cytosolic Copper content per cell was not significantly different in RA-treated cells compared to untreated cells, the Copper content per protein content was doubled. This is due to the fact that RA-treated cells have about half the protein content as compared to their untreated counterpart. Using .sup.67Cu, it has been shown that the rate of Copper uptake was significantly faster during the two first days of RA treatment, but not at later times. The intracellular distribution of .sup.67Cu was found predominantly in high molecular weight (MW) fractions (>100 kD) and a lower MW fraction of about 20 kD, with a higher proportion of Copper present in the high MW fractions in RA-treated cells.

0023] Addition of excess Copper to regular serum-supplemented growth medium modestly increased RA-induced differentiation. Although RA-treated HL-60 cells do not necessarily represent normal cell development, these results point to the possibility that neutrophilic differentiation may require Copper.

0024] In other experiments it has been shown that HL-60 cells can be made Copper deficient by treatment with Copper chelators, and that following such treatment their viability and growth rate were unaffected.

0025] Although all these phenomena have been attributed to Copper, it has been reported that some clinical and biological effects are shared by Copper and other transition metals:

Problems solved by technology

Thus, the process of differentiation limits, and eventually halts cell division.

It may be caused by Copper non-supplemented total parenteral nutrition (e.g., following intestinal resection), by consumption of high levels of Zinc, which interferes with Copper utilization, in underweight and / or cow milk (poor source of Copper) fed new-borns, which may result in severe cases in Shwanchman syndrome.

Unbalanced treatment with Copper chelators in Copper overload cases such as in Wilson's disease may also lead to Copper deficiency.

The limiting factor for PB transplantation is the low number of circulating pluripotent stem / progenitor cells.

Such treatment is obviously not suitable for normal donors.

Clinical studies have indicated that transplantation of ex-vivo expanded cells derived from a small number of PB CD.sub.34.sup.+ cells can restore hematopoiesis in patients treated with high doses of chemotherapy, although the results do not allow yet firm conclusion about the long term in-vivo hematopoietic capabilities of these cultured cells (3-4).

However, being very scarce, such cells should undergo cell expansion prior to analysis.

For these reasons gene transfer into fresh BM stem cells is very inefficient.

The use of chemotherapy is, of course, not suitable for normal donors.

Following cessation of administration of the drug and its removal from the body, this early population then might undergo accelerated maturation which may result in elevated production of fetal hemoglobin.

Indeed, this approach is limited by the toxicity of the treatment to a variety of normal tissues.

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