Patient-specific stem cell lines derived from human parthenogenetic blastocysts

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.

1 . An isolated human stem cell line derived from parthenogeneic blastocysts, wherein at least one cell comprising the cell line is
 a) heterozygous for one or more single nucleotide polymorphisms (SNPs); or   b) homozygous for one or more HLA alleles; or   c) a combination of (a) and (b).   
     
     
         2 . The isolated cell line of  claim 1 , wherein the at least one cell is homozygous for HLA alleles. 
     
     
         3 . The isolated cell line of  claim 1 , wherein the at least one cell forms a teratoma when transplanted into an immunocompromised mouse. 
     
     
         4 . The isolated cell line of  claim 1 , wherein the at least one cell is MHC compatible with the unfertilized oocyte donor. 
     
     
         5 . The isolated cell line of  claim 4 , wherein the at least one cell is MHC compatible with a first degree blood relative of the blastocyst donor. 
     
     
         6 . The isolated cell line of  claim 1 , wherein the at least one cell is substantially genetically imprinted according to donor origin. 
     
     
         7 . The isolated cell line of  claim 1 , wherein the at least one cell (i) will proliferate in an in vitro culture for over one year, (ii) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iii) is inhibited from differentiation when cultured on a fibroblast feeder layer. 
     
     
         8 . The isolated cell line of  claim 7 , wherein the at least one cell differentiates into a cell selected from the group consisting of a neuronal cell, a cardiac cell, a smooth muscle cell, a striated muscle cell, an endothelial cell, an osteoblast, and oligodendrocyte, a hematopoietic cell, an adipose cell, a stromal cell, a chondrocyte, an astrocyte, a keratinocyte, a pancreatic islet cell, a lymphoid precursor cell, a mast cell, a mesodermal cell, and an endodermal cell. 
     
     
         9 . The isolated cell line of  claim 1 , wherein the at least one cell does not have the capacity to form a viable organism. 
     
     
         10 . The isolated cell line of  claim 1 , wherein the at least one cell expresses one or more markers selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-4. 
     
     
         11 . A method of treating a subject in need thereof, comprising administering a cellular composition comprising differentiated cells, wherein the differentiated cells are derived from the stem cell line of  claim 1 . 
     
     
         12 . The method according to  claim 11 , wherein the subject presents a disease selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, spinal cord defects or injuries, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, macular degeneration, cartilage defects or injuries, burns, foot ulcers, vascular disease, urinary tract disease, AIDS, and cancer. 
     
     
         13 . A library of stem cells comprising autologous or allogenic stem cells, wherein the stem cells are derived from parthenogenetically activated oocytes from one or more human donors, and wherein the stem cells are HLA homozygous stem cells. 
     
     
         14 . The library of  claim 13 , wherein each library member is identified as a full sibling, half sibling, or unrelated according to single nucleotide polymorphism (SNP) markers. 
     
     
         15 . The library of  claim 13 , wherein the oocyte donor is histocompatible with a member of the library. 
     
     
         16 . The library of  claim 13 , wherein a member of the library is genomically imprinted according to the oocyte donor origin. 
     
     
         17 . The library of  claim 13 , wherein stem cells are derived from an HLA heterozygous or HLA-homozygous oocyte donor. 
     
     
         18 . The library of  claim 13 , wherein each member is homozygous for a different combination of MHC alleles than the other members of the library. 
     
     
         19 . The library of  claim 13 , wherein each member is at least homozygous for one or more HLA class I genes and HLA class II genes. 
     
     
         20 . The library of  claim 13 , wherein the HLA class I genes are selected from HLA A*, HLA B*, HLA and Cw* haplotype combinations. 
     
     
         21 . The library of  claim 13 , wherein the HLA class II genes are selected from HLA DRB1*, DRB3*, DRB4*, DRB5*, DQA1*, and DQB1* haplotype combinations. 
     
     
         22 . The library of  claim 13 , wherein each member (i) will proliferate in an in vitro culture for over one year, (ii) maintains the potential to differentiate to derivatives of one or all of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iii) is inhibited from differentiation when cultured on a fibroblast feeder layer. 
     
     
         23 . The library of  claim 22 , wherein each member maintains a karyotype in which the chromosomes are euploid and not altered through prolonged culture. 
     
     
         24 . The library of  claim 13 , wherein each member can differentiate into to ectoderm, mesoderm, and endoderm germ line cells. 
     
     
         25 . The library of  claim 13 , wherein each library member is characterized according to HLA-type, and each member is HLA-matched to potential recipients. 
     
     
         26 . A method of generating HLA homozygous stem cells comprising:
 a) screening oocyte donors for HLA-haplotypes found commonly in a given population group;   b) incubating human metaphase II oocytes in in vitro fertilization (IVF) media;   c) incubating the cells of step (b) in IVF media comprising an ionophore;   d) incubating the cells of step (c) in IVF media comprising a puromycin; and   e) incubating the cells of step (d) in fresh IVF medium under low O 2  tension,   wherein the incubating steps (b)-(d) are carried out under high O 2  tension and wherein inner cell masses (ICM) obtained from cells at step (d) produce culturable stem cells.   
     
     
         27 . The method of  claim 26 , further comprising HLA-typing of the biological parents of the oocyte donor or step (a). 
     
     
         28 . A cell bank comprising HLA-homozygous stem cells, wherein the stem cells are derived from parthenogenetically activated oocytes from HLA-homozygous or HLA-heterozygous human oocyte donors. 
     
     
         29 . The cell bank of  claim 28 , wherein the stem cells are assorted by identity to HLA-haplotypes found commonly in given population groups. 
     
     
         30 . The cell bank of  claim 28 , wherein the cell bank comprises cryopreserved autologous or allogeneic stem cells.