Embryonal cardiac muscle cells, their preparation and their use

Details Embryonal cardiac muscle cells, their preparation and their use Embryonal cardiac muscle cells, their preparation and their use

1997-08-22

1999-07-27

Degen, Nancy

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Primate cell, per se

435 29, 435 34, 435325, 435366, 435375, C12N 506, C12N 510, C12N 516, C12Q 102

Patent

active

059289430

DESCRIPTION:

BRIEF SUMMARY
The invention relates to embryonal (cardiomyocytary and cardiomyoblastary) cardiac muscle cells, their preparation and their use, in particular for cell-mediated gene transplant. The areas of application of the invention are medicine and genetic engineering.
There are already several works which deal with cardiac muscle cell cultures of mammals. The first investigations were performed with primary cultures of embryonal, neonatal, or adult cardiac tissue. The use of permanent cell lines of cardiomyocytary tissue is more advantageous since in this case one can make available a larger amount of a homogeneous cell population of a defined level of development. For this reason, there existed a series of experiments for the immortalization of cardiac muscle cells (amongst others, A. Sen et al., J. Biol. Chem. 263/1988/, 19132-19136). However, all of the hitherto existing cell lines exhibit the disadvantage that functional defects become noticeable in case of a long-term cultivation.
It is the goal of the invention to obtain embryonal (cardiomyocytary and cardiomyoblastary, respectively) cardiac muscle cells starting from pluripotent embryonal stem cells (ESC) or primordial germ cells (EGC, Steward et al., Dev. Biol. 161/1994, 626-628) after their differentiation into spontaneously pulsatile cardiac cells, where said embryonal cardiac muscle cells exhibit substantially identical properties to the cardiac muscle tissue. These cells are to be suitable for a therapeutic application, possible after additional genetic-engineering change. The object of the invention is to construct a vector system for the modification of the stem cells and to develop a selection method for the transfected cells.
The invention is realized with modified embryonal stem cells according to claim 1 to 4, the vector systems according to claim 5 and 6, and the selection method according to claim 7. The use of the modified embryonal stem cells according to claim 8 to 10 also belongs to the scope of the protection of the invention.
The invention vectors comprise the following components: myosin light-chain-2(MLC-2v) as promoter, the selectable marker gene .beta.-galactosidase and the reporter gene neomycin as fusion gene ".beta.geo" and the SV40-PolyA-Tail (pAA) and possibly a position for the receiving of immortalizing genes. promoters (HSV-Tk), the selectable marker gene hygromycin, and the SV40-PolyA-Tail (pAA).
Pluripotent embryonal stem cells are transfected in vitro with these vectors. The successfully transfected cells are selected in a first step with the aid of hygromycin. Hygromycin-resistant cells are then differentiated to so-called embryoid bodies. Thereupon, a selection of the hygromycin-resistant embryoid bodies is made with the cytotoxin Geneticin (G418). The resulting cells are further cultured and investigated as to their composition (gene expression, proteins), their function, and their contractile properties.
Embryonal stem cells (ESC) or primordial germ cells (EGC) of different origin, amongst others, of a mouse, rat, pig, cattle, dog, rabbit, hamster, including human cells, can be employed as starting material.
The invention vectors and the course of the cell selection method are illustrated in FIG. 1.
A further object of the invention are cells, which contain in addition to the recited vectors a) and b) also therapeutic genes such as, for example, the angiogenesis factors VEGF or bFGF, which therapeutic genes are obtained by viral or non-viral gene transfer. The thus obtained cell lines can be employed--with or without viral sequences--for the cell-mediated gene transplant, in particular for constructing healthy tissue and assisting contractile functions.
A further important use of the invention cell lines is the in-vitro testing of biologically active substances, in particular for the investigation of pharmacologically relevant substances or for the determination of toxic effects of exogenic agents on cardiac cells in culture. Animal testing is thereby spared in particular in screening programs and, as a result, the urgent

REFERENCES:
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Orkin et al. "Report & Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy", 1995.
Wobus et al. Retinoic acid induces expression of the ventricular 2.1 kb myosin-light-chain-2 promoter during in vitro cardiogenesis of embryonic stem cells. Circulation. 92 (8 Suppl.): 1114, 1995.
Engelmann et al. Formation of fetal rat cardiac cell clones by retroviral transformation: Retention of select myocyte characteristics. J. Mol. Cell. Cardiol. 25 (2): 197-213, Feb. 1993.
Hunter et al. Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice. J. Biol. Chem. 270 (39): 23173-23178, 1995.
Lee et al. Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene. J. Biol. Chem. 267 (22): 15875-15885, 1992.
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