Patient-specific stem cell lines derived from human parthenogenetic blastocysts

a technology of parthenogenetic blastocysts and stem cells, applied in the field of embryonic stem cells, can solve problems such as teratoma formation, and achieve the effect of minimizing the use of animal-derived cells and high levels of alkaline phosphatase and telomerase activity

Inactive Publication Date: 2008-12-04
INT STEM CELL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0014]The present invention discloses methods for generating HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines from both HLA homozygous and HLA heterozygous donors. These hpSC-Hhom lines demonstrate human embryonic stem cell morphology, expressing typical stem cell markers (i.e., SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-4) and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. SNP data analysis demonstrates that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome. The protocol as disclosed minimizes the use of animal-derived components, which makes these stem cells ideally suited for clinical application.

Problems solved by technology

Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation.

Method used

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  • Patient-specific stem cell lines derived from human parthenogenetic blastocysts
  • Patient-specific stem cell lines derived from human parthenogenetic blastocysts
  • Patient-specific stem cell lines derived from human parthenogenetic blastocysts

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example 1

Generation of Parthenotes

[0203]Five oocyte donors, all over 31 years of age, participated in this study. Oocytes were obtained using hormonal stimulation with the primary intent of in vitro fertilization (IVF). A total of 46 cumulus-oocyte complexes (COCs) were taken from five donors and used for this study (Table 1).

TABLE 1Generation of parthenotes and parthenogenetic embryonic stem cell lines.Blastocystsderived6withoutOocytesOocytesOocytesParthenoteswithvisibleDonorDonorderiveddonatedactivatedcreated5ICMICMLines generateddestiny18 4 442—phESC-1pregnantimmunosurgery215 8 8833phESC-3pregnantphESC-4twinsphESC-5all from wholeblastocysts32714141,a1132phESC-6pregnantfrom wholeblastocyst422111131023phESC-7 frompregnantwholeblastocyst520  94 7714no cell linenotgeneratedpregnant1two oocytes were not activated;2one oocyte degenerated after activation;3one oocyte was not activated;4two oocytes were at metaphase I stage and were discarded;5total pathenogenetically activated oocytes = 40;6tota...

example 2

Derivation of an hpSC-Hhom Line from an HLA Homozygous Donor

[0221]With an initial goal of isolating an HLA homozygous parthenogenetic human stem cell line, oocytes from an HLA homozygous donor were used. HLA genotyping of both the donor (donor 1) and her parents demonstrated that both parents were heterozygous. The same haplotype A*25, B*18, DRB1*15 was inherited from each parent, with the donor having an HLA homozygous genotype A*25, A*25, B*18, B*18, DRB1*15, DRB1*15 (Table 5, Case 1).

[0222]Nineteen cumulus-oocyte complexes (COCs) were taken from donor 1, of which seven were used for research (Table 4).

TABLE 4Origin of parthenotes andHLA homozygous parthenogenetic human stem cell lines.DonorOocytesOocytesBlasto-NumberharvesteddonatedcystsCell linesIVF Result11974hpSC-Hhom-1Successful21873hpSC-Hhom-2SuccessfulhpSC-Hhom-3(Twinpregnancy)3201000Unsuccessful427142hpSC-Hhom-4SuccessfulTotal843894NA

[0223]Parthenogenetic activation was performed using a previously described protocol with ...

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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.

Description

RELATED APPLICATIONS[0001]This application claims benefit of U.S. Provisional Application No. 60 / 922,244, filed Apr. 6, 2007, which is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates generally to embryonic stems cells, and more specifically to a process for obtaining HLA homozygous parthenogenetic human stem cell lines for cell-based therapy.[0004]2. Background Information[0005]The first human embryonic stem cells (ESC) were derived from a blastocyst inner cell mass (ICM) obtained from a fertilized oocyte, capable of infinite division and differentiation into cells of all tissues types. The embryonic stem cell is a potentially limitless source of pluripotent cells for transplant-based cell therapies.[0006]Human embryonic stem cells (ES) cells are pluripotent cells that can differentiate into a large array of cell types. When injected into immune-deficient mice, embryonic stem cells form di...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12C12N5/08C40B40/02C12N5/02A61P43/00C12N5/0735C12N5/075
CPCC12N5/0606C12N5/0609C12N2500/02C12N2500/14C12N2501/115C12N2501/235C12N2502/1323A61P1/16A61P11/00A61P13/02A61P17/02A61P19/08A61P21/04A61P25/00A61P25/14A61P25/16A61P25/28A61P27/02A61P31/18A61P35/00A61P37/00A61P37/02A61P43/00A61P9/00A61P3/10A61K35/545
Inventor REVAZOVA, ELENA S.KUZMICHEV, LEONID N.TUROVETS, NICKOLAY A.JANUS, JEFFREY D.
Owner INT STEM CELL CORP
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