Patient-specific stem cell lines derived from human parthenogenetic blastocysts

• US 9,920,299 B2
• Filed: 11/24/2015
• Issued: 03/20/2018
• Est. Priority Date: 04/06/2007
• Status: Active Grant

• Current Assignee: International Stem Cell Corporation
• Original Assignee: International Stem Cell Corporation
• Inventors: Revazova, Elena S., Kuzmichev, Leonid N., Turovets, Nickolay A., Janus, Jeffrey D.
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US14/951,199
• Publication Number:
  US 20160186133A1
• Time in Patent Office: 847 Days
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/14 : Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/115 : Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 : Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/1323 : Adult fibroblasts
• 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
• CPC Class Codes: C12N 5/0609 : Oocytes, oogonia fertilised oocytes C12N5/0604

Abstract

Methods are disclosed for generating HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines from both HLA homozygous and HLA heterozygous donors. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor'"'"'s parents. SNP data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by single-nucleotide polymorphism (SNP) analysis. The protocol as disclosed minimizes the use of animal-derived components, which makes the stem cells more practical for clinical application.

Claims

1. A method of generating HLA homozygous stem cells comprising:

a) screening oocyte donors for HLA-haplotypes found commonly in a given population group;

b) parthenogenetically activating a human oocyte from (a), by;

i) contacting the oocyte with an ionophore at high O₂ tension; and

ii) contacting the oocyte with a serine-threonine kinase inhibitor under low O₂ tension;

c) cultivating the activated oocyte of (b) at low O₂ tension until blastocyst formation;

d) transferring the blastocyst to a layer of feeder cells and culturing the transferred blastocyst under high O₂ tension;

e) isolating an inner cell mass (ICM) from the blastocyst of (c); and

f) culturing the cells of the ICM of (d) on a layer of feeder cells under high O₂ tension, thereby producing HLA homozygous stem cells.

2. The method of claim 1, further comprising HLA-typing of the biological parents of the oocyte donor or step (a).

3. The method of claim 1, wherein the ionophore is selected from the group consisting of ionomycin or A23187.

4. The method of claim 1, wherein the serine-threonine kinase inhibitor is selected from the group consisting of staurosporine, 2-aminopurine, sphingosine, and 6-dimethylaminopurine (DMAP).

5. The method of claim 1, wherein low O₂ tension is maintained by incubation in a gas mixture environment comprising an O₂ concentration of about 2% O₂ to about 5% O₂.

6. The method of claim 1, wherein high O₂ tension is maintained by incubation in a gas mixture environment comprising an O₂ concentration of about 5% O₂ to about 20% O₂.

7. The method of claim 1, wherein the layer of feeder cells comprises human fibroblasts.

8. The method of claim 7, wherein the fibroblasts are postnatal human dermal fibroblasts.