FIELD OF THE INVENTION
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This invention relates to entrapped cells, such as stem cells. The entrapped cells, when cultured in the entrapment material, produce a product which, when it is in contact with other non-entrapped, freely growing cells in vitro or in vivo, inhibits their proliferation. Further, the entrapment of the stem cells acts to inhibit the proliferation of at least some of the entrapped stem cells, and may inhibit the differentiation of at least a portion of the entrapped stem cells.
BACKGROUND AND PRIOR ART
Entrapment of biological materials, such as cells, is a technique that has been used for various ends. Exemplary of the patent literature in this area are U.S. Pat. Nos. 6,303,151 (Asina, et al.); 6,224,912 (Asina, et al.); 5,888,497 (Jain, et al.); 5,643,569 (Jain, et al.), and RE38,027 (Jain, et al.), all of which are incorporated by reference in their entirety. This family of related patents shows that cancer cells and islets can be entrapped in a biocompatible matrix, such as agarose, agarose/collagen mixtures, and agarose/gelatin mixtures, and then be coated with agarose. The resulting, entrapped cells produce materials which, inter alia, diffuse out of the permeable biocompatible matrices in which they are retained, and have useful biological properties. In the case of islets, insulin is produced. In the case of cancer cells, material diffuses from the matrix, and this material has an effect on the growth and proliferation of cancer cells. As review of the '912 and '151 patents, cited supra, will show, this effect crosses species, i.e., entrapped or encapsulated cancer cells from a given species produce material that inhibits the growth and/or proliferation of cancer cells from other species, as well as the species from which the cancer cells originated.
Additional examples of entrapment techniques include, e.g., U.S. Pat. Nos. 5,227,298 (Weber, et al.); 5,053,332 (Cook, et al.); 4,997,443 (Walthall, et al.); 4,971,833 (Larsson, et al.); 4,902,295 (Walthall, et al.); 4,798,786 (Tice, et al.); 4,673,566 (Goosen, et al.); 4,647,536 (Mosbach, et al.); 4,409,331 (Lim); 4,392,909 (Lim); 4,352,883 (Lim); and, 4,663,286 (Tsang, et al.). All of these references are incorporated by reference.
Entrapment does not always result in a positive impact on the entrapped cells. For example, see Lloyd-George, et al., Biomat. Art. Cells & Immob. Biotech., 21(3):323-333 (1993); Schinstine, et al., Cell Transplant, 41(1):93-102 (1995); Chicheportiche, et al., Diabetologica, 31:54-57 (1988); Jaeger, et al., Progress In Brain Research, 82:41-46 (1990); Zekorn, et al., Diabetologica, 29:99-106 (1992); Zhou, et al., Am. J. Physiol., 274 : C1356-1362 (1998); Darquy, et al., Diabetologica, 28:776-780 (1985); Tse, et al., Biotech. & Bioeng., 51:271-280 (1996): Jaeger, et al., J. Neurol., 21-469-480 (1992); Hortelano, et al., Blood, 87(12):5095-5103 (1996): Gardiner, et al., Transp. Proc., 29:2019-2020 (1997). All of these references are incorporated by reference.
None of the references discussed supra deals with the class of cells known as stem cells, including embryonic stem cells.
One definition of stem cells, advanced by Reya, et al., Nature, 414:105-111 (2001), incorporated by reference, refers to stems cells as cells which have the ability to perpetuate themselves through self renewal and to generate mature cells of particular tissues via differentiation. One can obtain different types of stem cells, including neural, hematolymphoid, myeloid, and other types of stem cells from various organs. These all have potential to develop into specific organs or tissues. Certain stem cells, such as embryonic stem cells, are pluripotent, in that their differentiation path has not been determined at all, and they can develop into various organs and tissues.
The discussions of the various therapeutic uses to which stem cells may be put are well known, and need not be discussed here. It is worth mentioning, as it bears on the invention described herein, that stem cells are very uncommon, their purification and separation from other cell types is laborious and difficult, and stem cells will differentiate into mature cell unless treated in some way to prevent this.
It has now been found that entrapment procedures, in line with those disclosed by Jain et al. and Iwata et al., Journ. Biomedical Material and Res., 26:967 (1992) affect stem cells in a very desirable way. To elaborate, entrapped stem cells produce materials which inhibit proliferation of various cell types, including stem cells and cancer cells. The effect of this material crosses species lines. Further, it has been found that stem cells, when entrapped as is described herein, retain their differentiating abilities, including their pluripotentiality, for an indefinite period of time.
These features, as well as others, will be seen in the disclosure which now follows.
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OF THE PREFERRED EMBODIMENTS
Two different murine embryonic stem (ES) cell lines (i.e., ES-D3 and SCC-PSA1, which are both publicly available) were obtained from the American Type Culture Collection (“ATCC”).
Both lines were grown under standard culture conditions, which included growth as a monolayer, atop “STO” embryonic fibroblast feeder cells. These were also obtained from the ATCC. The stem cells were cultured in DMEM medium that had been supplemented with 100% ES-Qualified fetal bovine serum, leukemia inhibitory factor (LIP), and β-mercaptoethanol (collectively, “Medium A”). The cells, which were cryopreserved when received, were thawed, and established as cultures after at least 3 passages before being cultured as described, supra.
After three days, the ES cells were 70-80% confluent, and were trypsinized and then entrapped in agarose beads, coated with agarose, in accordance with U.S. Pat. Nos. 6,303,151; 6,224,912; and, 5,888,497, all of which are incorporated by reference. In brief, however, Sigma XII agarose was used, at an initial concentration of about 1.0%. A 100:1 aliquot of this agarose solution was added to 34:1 of cell suspension. The resulting beads contained 2.0×105∀1.5×104 murine embryonic stem cells. The beads were given a second coat of agarose, at a concentration of about 5.0%. The beads were cultured in medium as described supra, except no LIF or viable STO feeder cells were present (“Medium B”).
The viability of cells in the beads over time was assessed, via standard histochemical and microscopic examination, as well as standard MTT assays, using cells removed from beads or maintained in the beads, at various points in time.
It was observed that entrapped stem cells increase their metabolic activity when first coated. This is followed by a decrease in activity, as cells die via apoptosis, reaching their lowest point of metabolic activity around day 21. After this low point, however, surviving cells slowly proliferate, and total metabolic activity was seen to gradually increase up to day 35 post entrapment and beyond. This parallels observations on entrapped cancer cells.
Morphologically, there was a significant difference between the colonies formed within the inner layer of agarose of the bead by the cancer cells and those formed by the stem cells. Although both types of colonies are ovoid in shape, those formed by the cancer cells are characterized by an outer zone of viable cells (generally two to three cells in thickness) with a central zone of eosiniphilic cellular debris. The colonies formed by the stem cells, on the other hand, are fully occupied by viable cells and there is no central zone of cellular debris.
In these experiments, the inhibitory effect of stem cells on the proliferation of other stem cells was tested.
Ten-week-old agarose/agarose beads containing stem cells (SCC-PSA1 cells) were tested for viability using the MTT assay, discussed supra, and were cultured in Medium B discussed in example 1, for 6 days. After 6 days, the medium had been conditioned by the entrapped stem cells. It is therefore called the Stem-cell Conditioned Medium (SCM).
After these 6 days, the SCM was transferred to 6 well plates that contained fresh SCC-PSA1 cells. These plates each contained 9×105 STO feeder cells, which were covered with 1.5×104 SCC-PSA1 cells. The STO cells had been treated with mitomycin C to prevent proliferation. There were three controls, i.e., wells which contained Medium B (an unconditioned medium), and three wells that contained the SCM.
After 3 days, the contents of all wells were trypsinized, and total cells were counted, using standard methods. The raw count was adjusted to account for the 9×105 feeder cells. The results follow:
(of SCC cells)
1.43 × 106
∀9.9 × 104
5.27 × 105
1.19 × 106
∀3.6 × 104
2.90 × 105
A similar experiment was carried out, with the following results: