Process for producing normal parenchymal cells tissue or organ by bioincubator

• US 20040072350A1
• Filed: 03/14/2003
• Published: 04/15/2004
• Est. Priority Date: 09/14/2000
• Status: Abandoned Application

• Current Assignee: Paul Diamond
• Original Assignee: Paul Diamond
• Inventors: Yuki, Atsushi
• Application Number:
  US10/380,525
• Publication Number:
  US 20040072350A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals

Abstract

Methods of producing cells, tissues and organs as close as possible to homoplasy have been developed to provide nonhuman mammals having normal parenchymal cells of a foreign mammal, a tissue made up of normal cells of a foreign mammal and their secretion products thereof, and/or an organ of a foreign mammal.

Claims

1. A method of producing normal parenchymal cells, tissues or organs (hereinafter referred to as “

organ or the like”

) originating in human or nonhuman mammal which comprises the following steps;

(1) preparing cells which are obtained from a mammal of the same species as the target organ or the like to be produced and capable of differentiating at least into the target organ or the like (hereinafter referred to as “

cells for organ”

);

(2) preparing cells from a nonhuman mammal of a species different from the species of the target organ or the like to be produced and have pluripotency or totipotency (hereinafter referred to as “

cells for live-culture device”

);

(3) introducing a DNA fragment containing a promoter region functioning specifically in the target organ or the like to be produced and a structural gene encoding an active cytotoxin or an enzyme to activate a cytotoxin precursor which is ligated under the control of said promoter region Into the genome of the cells for live-culture device as described in (2);

(4) culturing the cells for organ as described in (1) together with the cells for culture device as described in (3) to form a chimeric cell mass;

(5) culturing the chimeric cell mass under conditions adequat for the development thereof to provide a live-culture device made of a mammal mainly having the characters originating in the cells for live-culture device, or its embryo or embryoid body, while specifically expressing the active cytotoxin or the cytotoxin precursor activating enzyme in the cells originating in the cells for culture device of the target organ or the like formed in the course of the development of the live-culture device and, in the case of expressing the cytotoxin precursor activating enzyme, adding the cytotoxin precursor; and

(6) allowing the live-culture device to produce the target organ or the like which is made up of the calls originating in the cells for organ and substantially free from cells originating in the cells for culture device.

2. A method according to claim 1 wherein said cytotoxin precursor is ganciclovir, said enzyme converting ganciclovir into a cytotoxin is HSVtk (herpes symplex virus thymidine kinase), and said promoter region is the promoter of rat myosin light chain 2V gene which functions exclusively in cardiomyocytes.

3. A method according to claim 1 or 2 which aims at producing cardiomyocytes or a heart originating in a human or a nonhuman mammal.

4. A method according to claim 1 or 3 for producing an organ selected from the group consisting of organs made up of cells characteristic of said organ.

5. A method according to any of claims 1 to 4 wherein said cells for live-culture device are selected from the group consisting of cells of mammals usable in embryo manipulations such as pig, chimpanzee, gorilla, orangutan, monkey, dog, cattle, horse, sheep, goat, rat and mouse.

6. A recombinant DNA to be introduced into the genome of the cells for culture device in the method according to claim 1, comprising a promoter region functioning specifically in the target organ or the like and a gene encoding an enzyme for converting a cytotoxin precursor into the cytotoxln, wherein the expression of the enzyme gene is under the control of said promoter region.

7. A recombinant DNA according to claim 6 wherein said cytotoxln precursor is ganciclovir, said enzyme for converting ganciclovir into a cytotoxin is HSVtk (herpes symplex virus thymidine kinase), and said promoter region is the promoter of rat myosin light chain 2v gene which functions exclusively in cardiomyocytes.

10. A live-culture device according to claim 9 which is an embryo of a nonhuman mammal carrying a recombinant DNA according to claim 6 or 7 introduced thereinto.

11. A live-culture device according to claim 9 which comprises cells (for example, embryonic stem cells) carrying a recombinant DNA according to claim 6 or 7 introduced thereinto, wherein the cells can form a construct (for example, an embryoid body) which is necessarily and sufficiently capable of supporting the proliferation, differentiation and maturation of the target normal parenchymal cells and undergoes changes under a definite law.

8. A nonhuman mammal having normal parenchymal cells of a foreign mammal, which is either a human or nonhuman mammal, a tissue made up of normal cells of the foreign mammal and secretion products thereof, and/or an organ of the foreign mammal.

9. A live-culture device wherein a culture device for producing normal parenchymal cells, a tissue and/or an organ of a human or nonhuman mammal undergoes morphological and biochemical changes under a definite law to thereby support the proliferation of target cells and, at the same time, continuously and three-dimensionally induce the differentiation and maturation of the cells, thereby forming the tissue or organ.

12. A method of constructing a chimeric embryo or a chimeric embryoid body between a non-human mammalian embryo or calls of a mammal including a human which are capable of forming an embryoid body and undifferentiated cells of a foreign nonhuman mammal.

13. Cells and a cell-like construct wherein undifferentiated cells of a foreign animal such as human being or a nonhuman mammal are cells capable of differentiating into target cells, tissue or organ which are selected from the group consisting of female/male germ cells and precursor cells thereof, embryonic cells, embryo-derived pluripotent cells, embryonic stem cells, embryonic stem cell-derived pluripotent cells, stem cells of various tissues and dedifferntiated somatic cells, and whereby these cells can be developed into a structure having the whole functions or a part of the functions of the target cell, tissue or organ, for example, artificially treated cells such as genetically modified cells, modified cells by artificial treatment of the part of the cell functions, cells partly substituted by an artificial substance and artificially synthesized cells.

14. A method of promoting the proliferation/differentiation of foreign mammalian cells (for example, human cells) in a live-culture device into target cells or a tissue or-organ while removing specific cells, which comprises adding a cytotoxln precursor into the live-culture device and exclusively removing the cells in which a gene of an enzyme capable of converting the cytotoxin precursor into the cytotoxin is expressed.