Genetically altered mammalian embryonic stem cells, their living progeny, and their therapeutic application for improving cardiac function after myocardial infarction

• US 6,607,720 B1
• Filed: 10/07/2000
• Issued: 08/19/2003
• Est. Priority Date: 09/05/2000
• Status: Expired due to Fees

• Current Assignee: Polystem Inc.
• Original Assignee: Polystem Inc.
• Inventors: Xiao, Yong-Fu, Morgan, James P.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): TON, THAIAN N
• Application Number:
  US09/684,679
• Time in Patent Office: 1,046 Days
• Field of Search: 536/23.1, 435/325, 435/455, 435/377, 800/8, 424/93.21, 514/44
• US Class Current: 424/93.21
• CPC Class Codes: A61B 2017/00243 cardiac
• CPC Class Codes: A61B 90/00 Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges protective face masks A41D13/11; surgeon's or patient's gowns or dresses A41D13/12; devices for carrying-off, for treatment of, or for carrying-over, body liquids A61M1/00
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention provides genetically altered mammalian embryonic stem cells, their living descendent progeny having an altered genomic DNA, and therapeutic methods using these cells for improving cardiac function in a living subject after myocardial infarction. The genetically altered embryonic stem and progenitor cells may be maintained in-vitro as a stable cell line; and transplanted as active, mitotic cells to an infarcted area of the myocardial using any surgical procedure. After transplantation at a chosen anatomic site within the heart of the subject, these genetically altered cells will differentiate in-site, cause a regeneration of myocardocytes, and will effect a marked improvement in cardiac function for the subject.

Claims

1. A method for improving cardiac function in a mammal after a myocardial infarct, said method comprising the steps of:

maintaining a plurality of undifferentiated, genetically altered mammalian embryonic stem cells in vitro in a culture medium containing at least one selected from the group consisting of leukemia inhibitory factor and fibroblast feeder cells;

said undifferentiated, genetically altered mammalian embryonic stem cells comprising;

a mammalian embryonic stem cell which

(i) remains uncommitted and undifferentiated while passed in vitro,

(ii) is implantable in vivo at a chosen anatomic site as an uncommitted cell, and

(iii) engrafts in situ after implantation in a mammal at a local anatomic site, and,

(iv) contains a vector comprising a DNA sequence operably linked to a promoter, wherein the DNA sequence encodes an angiogenic factor which is expressed in situ;

subsequently culturing said undifferentiated, genetically altered mammalian embryonic stem cells for a predetermined time in a culture media in the absence of leukemia inhibitory factor and fibroblast feeder cells to yield a cellular inoculum comprising mammalian cells in which differentiation has been initiated;

introducing said cellular inoculum to at least a portion of the previously infarcted area of the heart tissue; and

allowing said introduced cellular inoculum to engraft in situ as viable cells situated within the previously infarcted area of the heart tissue, wherein the engraftment results in improved cardiac function in said mammal.

3. The method for improving cardiac function as recited in claim 1 or 2, wherein said DNA sequence in said genetically altered mammalian embryonic stem cells is an endogeneous nucleotide sequence.

4. The method for improving cardiac function as recited in claim 1 or 2, wherein said DNA sequence in said genetically altered mammalian embryonic stem cells is a heterologous nucleotide sequence.

5. The method as recited in claims 1 or 2, wherein said DNA sequence encodes an angiogenic factor selected from the group consisting of vascular endothelial growth factor, fibroblast growth factor 1, fibroblast growth factor 2, transforming growth factor α

, transforming growth factor β

_1-5, insulin-like growth factor 1 and insulin-like growth factor 2.

2. A method for improving cardiac function in a mammal after a myocardial infarct, said method comprising the steps of:

obtaining a plurality of progeny cells of undifferentiated, genetically altered mammalian embryonic stem cells,

maintaining said undifferentiated, genetically altered progeny cells in vitro in a culture medium containing at least one selected from the group consisting of leukemia inhibitory factor and fibroblast feeder cells, said undifferentiated, genetically altered progeny cells comprising;

progeny cells of mammalian embryonic stem cells which

(i) remain undifferentiated while passed in vitro,

(ii) are implantable in vivo at a chosen anatomic site as viable cells,

(iii) engraft in situ after implantation in a mammal at a local anatomic site, and,

(iv) contain a vector comprising a DNA sequence operably linked to a promoter, wherein the DNA sequence encodes an angiogenic factor, which is expressed in situ;

subsequently culturing said undifferentiated, genetically altered progeny cells for a predetermined time in a culture media in the absence of leukemia inhibitory factor and fibroblast feeder cells to yield a cellular inoculum comprising mammalian cells in which differentiation has been initiated;

introducing said cellular inoculum to at least a portion of the previously infarcted area of the heart tissue; and

allowing said introduced cellular inoculum to engraft in situ as viable cells situated within the previously infarcted area of the heart tissue, wherein the engraftment results in improved cardiac function in said mammal.