Bank of stem cells for producing cells for transplantation having HLA antigens matching those of transplant recipients, and methods for making and using such a stem cell bank

• US 20040091936A1
• Filed: 05/27/2003
• Published: 05/13/2004
• Est. Priority Date: 05/24/2002
• Status: Abandoned Application

• Current Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: West, Michael
• Application Number:
  US10/445,195
• Publication Number:
  US 20040091936A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• 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 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer
• 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 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

Methods for producing stem cell banks, preferably human, which optionally may be transgenic, e.g., comprised of homozygous MHC allele cell lines are provided. These cells are produced preferably from parthenogenic, IVF, or same-species or cross-species nuclear transfer embryos or by de-differentiation of somatic cells by cytoplasm transfer. Methods for using these stem cell banks for producing stem and differentiated cells for therapy, especially acute therapies, and for screening for drugs for disease treatment are also provided.

Claims

1. A stem cell bank comprising a library or plurality of human or non-human animal stem cell lines, each of which is homozygous for at least one MHC allele present in a human or non-human animal population, wherein which each member of said plurality of stem cell lines is homozygous for a different set of MHC alleles relative to the remaining members of the plurality of stem cell lines.

2. The stem cell bank of claim 1 which is comprised of a plurality of human stem cell lines each member of which is homozygous for a different combination of MHC alleles than the other members of the library.

3. The stem cell bank of claim 1 which is comprised of at least five different stem cell lines, each member homozygous for a different combination of histocompatibility or MHC antigen alleles.

4. The human stem cell bank of claim 2 which is comprised of at least five different stem cell lines, each member homozygous for a different combination of histocompatibility or MHC antigen alleles.

5. The stem cell bank of claim 1 which is comprised of at least ten different stem cell lines each member homozygous for a different combination of histocompatibility or MHC antigen alleles.

6. The stem cell bank of claim 2 which is comprised of at least ten different stem cell lines each member homozygous for a different combination of histocompatibility or MHC antigen alleles.

7. The stem cell bank of claim 1 which comprises at least about 100 to 1000 different stem cell lines each homozygous for a different combination of histocompatibility or MHC antigen alleles.

8. The stem cell bank of claim 1 which is comprised of ES cell lines or inner cell mass derived stem cell lines.

9. The stem cell bank of claim 8 which is comprised of human ES cell lines.

10. The human stem cell bank of claim 2 which comprises human stem cell lines that are at least homozygous for the 10 most frequent MHC alleles in the human population.

11. The human stem cell bank of claim 2 which comprises human stem cell lines that are homozygous for at least the 15 most frequent MHC alleles in the human population.

12. The human stem cell bank of claim 2 wherein all of the stem cell lines are O-negative.

13. The human stem cell bank of claim 10 wherein the library includes human stem cells that are homozygous for one or more of the following MHC alleles:

HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A11, HLA-A28, HLA-A29, HLA-A32, HLA-B15, HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B14, HLA-B18, HLA-B35, and HLA-B40.

14. The human stem cell bank of claim 13 wherein all of said human stem cell lines are O-negative.

15. The human stem cell bank of claim 13 wherein said stem cell lines are derived from parthenogenic human embryos.

16. The human stem cell bank of claim 13 wherein said stem cell lines are derived from human nuclear transfer embryos.

17. The human stem cell bank of claim 13 wherein said stem cell lines are derived from human somatic cells which have been de-differentiated by contacting their DNA with cytoplasm from an oocyte, ES cell, EG cell or another embryonic cell type.

18. The human stem cell bank of claim 2 which is produced from human embryos derived from an artificially haploidized somatic cell.

19. The human cell bank of claim 2 which includes at least one human stem cell line which is rendered homozygous for an MHC allele by removal of one ore more MHC alleles knockout or RNA interference.

20. The human cell bank of claim 10 which includes stem cell lines which are homozygous for at least one of the following HLA-A, HLA-B and HLA-DR haplotype combinations:

1, 7, 2;

1, 8, 3;

2, 14, 1;

2, 35, 4;

2, 35, 8;

2, 44, 4;

3, 7, 2;

3, 7, 4;

3, 7, 8;

3, 35, 1;

31, 51, 4; and

32, 14, 7.

21. The human stem cell bank of claim 20 which contains cell lines homozygous for all of said HLA-A, HLA-B and HLA-DR haplotype combinations.

22. The human stem cell bank of claim 20 which is O-negative.

23. The human stem cell bank of claim 21 which is O-negative.

24. The human stem cell bank of claim 19 which the cells are rendered homozygous by the removal of one set of MHC alleles by knockout or RNA interference and the addition of another homozygous set of MHC alleles.

25. The stem cell bank of claim 1, which contains lines of stem cells selected from the group consisting of totipotent, nearly totipotent, and pluripotent stem cells.

26. The stem cell bank of claim 1, which contains a plurality of lines of embryonic stem cells.

27. The stem cell bank of claim 1, which contains a plurality of lines of stem cells that can differentiate into hematopoietic stem cells.

28. The stem cell bank of claim 1, which contains a plurality of lines of hematopoietic stem cells.

30. The stem cell bank of claim 1 which contain a plurality of stem cells that can differentiate into myocardial cells.

31. The stem cell bank of claim 1, which contains a plurality of stem cells that can differentiate into hepatic cells.

32. The stem cell bank of claim 1, which contains a plurality of stem cells that can differentiate into pancreatic beta cells.

33. The stem cell bank of claim 2, which contains a stem cell line homozygous for an MHC allele selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP, wherein the cells are of the o-negative blood group.

34. The stem cell bank of claim 2, which contains a stem cell line homozygous for the MHC alleles encoding HLA-A, HLA-B, and HLA-DR.

35. The stem cell bank of claim 2, which contains a stem cell line homozygous for the MHC alleles encoding HLA-A, HLA-B, and HLA-DR, wherein the cells are of the o-negative blood group.

36. The stem cell bank of claim 1, which contains lines of stem cells derived from embryos produced by in vitro fertilization or intracytoplasmic sperm injection.

37. The stem cell bank of claim 1, which contains lines of diploid stem cells derived from embryos produced by parthenogenesis.

38. The stem cell bank of claim 37, which contains lines of diploid stem cells in which all of the MHC alleles are homozygous.

39. The stem cell bank of claim 1, which contains lines of stem cells derived from embryos produced by cloning by same or cross-species nuclear transfer.

40. The stem cell bank of claim 39, which contains at least one line of rejuvenated stem cells.

41. The stem cell bank of claim 40, wherein the rejuvenated stem cells have telomeres that are on average at least as long as the telomeres of age-matched control cells of the same type that are not generated by nuclear transfer techniques.

42. The stem cell bank of claim 41, wherein the proliferative life-span of the rejuvenated stem cells is at least as long as the proliferative life-span of age-matched control cells of the same type that are not generated by nuclear transfer techniques.

43. The stem cell bank of claim 41, wherein the proliferative life-span of the rejuvenated stem cells is longer than the proliferative life-span of age-matched control cells of the same type that are not generated by nuclear transfer techniques.

44. The stem cell bank of claim 41, wherein EPC-1 activity in the rejuvenated stem cells is greater than EPC-1 activity in age-matched control cells of the same type that are not generated by nuclear transfer techniques.

45. The stem cell bank of claim 41, wherein telomerase activity in rejuvenated stem cells is greater than telomerase activity in age-matched control cells of the same type that are not generated by nuclear transfer techniques.

46. The stem cell bank of claim 40, which contains at least one line of stem cells comprising non-human mitochondria.

47. The stem cell bank of claim 1, which contains at least one stem cell line that compromises DNA that is genetically modified relative to the DNA of the non-human donor from which the cell line is derived.

48. The stem cell bank of claim 2, which contains at least one stem cell line that comprises DNA that is genetically modified relative to the human donor from which the cell line is derived.

49. The stem cell bank of claim 47, wherein the DNA of the genetically altered stem cell line is modified by adding, modifying, substituting, or deleting one or more DNA sequences.

50. The human stem cell bank of claim 48, which the DNA or the genetically altered stem cell line is modified by adding, modifying, substituting or deleting one or more DNA sequences.

51. The stem cell bank of claim 49, wherein the DNA of the genetically altered stem cell line is modified so as to obtain, increase, decrease, inhibit, or otherwise modify, the expression of a gene that is native to, or introduced into, cells of the at least one cell line, relative to expression of said gene in a control cell without the genetic modification.

52. The stem cell bank of claim 49, wherein the DNA of the genetically altered stem cell line is modified so as to obtain, increase, decrease, inhibit, or otherwise modify, the expression of a gene that is native to, or introduced into, cells of the at least one cell line, relative to expression of said gene in a control cell without the genetic modification.

53. The stem cell bank of claim 51, wherein the DNA of the genetically altered stem cell line is modified by homologous recombination.

54. The human stem cell bank of claim 52, wherein the DNA or the genetically altered stem cell line is modified by homozygous recombination.

55. The stem cell bank of claim 51, wherein the DNA of the genetically altered stem cell line is altered to prevent the expression of a gene encoding an antigenic protein that elicits an immune response contributing to rejection.

56. The stem cell bank of claim 51, wherein the DNA of the genetically altered stem cell line is altered to prevent the expression of a gene encoding an antigenic protein that elicits an immune response contributing to rejection.

57. The stem cell bank of claim 51, wherein the DNA of the genetically altered stem cell line is altered to express a gene that inhibits the immune rejection of that cell.

58. The stem cell bank of claim 51, wherein the DNA of the genetically altered stem cell line is altered to express a gene that inhibits the immune rejection of that cell.

59. The stem cell bank of claim 56, wherein the DNA of the genetically altered stem cell line is modified so as to inhibit production of at least one HLA antigen by cells of said cell line.

60. The stem cell bank of claim 59, wherein the DNA of the genetically altered stem cell line is modified so as to inhibit production of one or more HLA antigens selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.

61. The stem cell bank of claim 55, wherein the DNA of the genetically altered stem cell line is modified so as to inhibit production of β

2-microglobulin.

62. The stem cell bank of claim 47, wherein the DNA of the genetically altered stem cell line is altered by replacing a non-homozygous MHC allele with one that is homozygous.

29. The stem cell bank of clam 1, which contains a plurality of lines of stem cells that can differentiate into vascular endoethelial precursor cells.

63. A method for treatment, preferably acute treatment, comprising transplanting cells or tissue that are homozygous for at least HLA one allele in a person in need of such a transplant, comprising:

a. identifying the MHC alleles of a person in need of a transplant (the recipient);

b. obtaining from a stem cell bank comprising a plurality of stem cells homozygous for at least one MHC allele of the transplant recipient;

c. generating cells or tissue suitable for transplant from said stem cells; and

d. transplanting said cells or tissue suitable for transplant into said recipient.

64. The method of claim 63 wherein said stem cell bank comprises at least 10 different human stem cell lines, wherein each of said stem cell lines are homozygous for a different combination of HLA alleles relative to the other stem cell lines.

65. The method of claim 63 wherein said stem cell bank comprises at least 15 different human stem cell lines wherein each of said stem cell lines are homozygous for a different combination of HLA alleles relative to the other human stem cell lines in the cell bank.

66. The method of claim 63 wherein said stem cell bank comprises at least about 100 to 1000 stem cell lines each homozygous for a different combination of HLA alleles relative to the other human stem cell lines in the cell bank.

67. The method of claim 63 wherein one or more of said human stem cell lines are ES or inner cell mass-derived stem cells.

68. The method of claim 63 wherein one or more of said human stem cell lines is derived from a parthenogenetic human embryo.

69. The method of claim 63 wherein one or more of said human stem cell lines are produced by haploidization comprising the steps of

a) inserting or fusing a somatic donor cell or nucleus thereof into or with an oocyte which is treated to remove or destroy its endogenous genomic DNA before, during or after insertion or fusion;

b) activation of the reconstructed embryo to expel haploid genome into a pseudopolar body;

c) screening of the pseudopolar body for the genetype of the remaining pronuclear;

d) combination of the two pronuclei to generate a reconstructured diploid embryo by pronuclear transfer or alternatively producing a diploid embryo by transferral of a pronucleus to an activated haploid oocyte comprising desired haploid genome;

e) optionally injecting human morula stage embryo lysates into the reconstructed embryos; and

f) isolating human stem cell lines from said reconstructed diploid embryo.

70. The method of claim 63 wherein one or more of said human stem cell lines is produced by the insertion of first and second polar bodies into a recipient cell.

71. The method of claim 63 wherein at least one of said stem cell lines is produced by de-differentiation of a somatic cell by cytoplasmic transfer.

72. The method of claim 63 wherein said human stem cell bank comprises cells which are homozygous for one of the following HLA serotypes:

HLA-A1, HLA-A3, HLA-A11, HLA-A15, HLA-A22, HLA-A27, HLA-A28, HLA-A29, HLA-A32, HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B17, HLA-B18, HLA-B35 and HLA-B40.

73. The method of claim 63 wherein said human stem cell bank comprises stem cells which are homozygous for at least one of the following HLA-A, -B or -DR haplotypes:

1, 7, 2;

1, 8, 3;

2, 14, 1;

2, 35, 4;

2, 35, 8;

2, 44, 4;

3, 7, 2;

3, 7, 4;

3, 7, 8;

3, 35,1;

31, 51, 4; and

32, 14, 7.

74. The method of claim 72 wherein said cell lines are O-negative.

75. The method of claim 73 wherein said cell lines are O-negative.

76. The method of claim 63, wherein step b comprises obtaining stem cells selected from the group consisting of totipotent, nearly totipotent, and pluripotent stem cells.

77. The method of claim 63, wherein step b comprises obtaining embryonic stem cells.

78. The method of claim 63, wherein step b comprises obtaining stem cells that can differentiate into hematopoietic stem cells.

79. The method of claim 63, wherein step b comprises obtaining hematopoietic stem cells from the stem cell bank.

80. The method of claim 63, wherein step b comprises obtaining stem cells homozygous for an MHC allele selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.

81. The method of claim 63, wherein step b comprises obtaining stem cells homozygous for the MHC alleles encoding HLA-A, HLA-B, and HLA-DR.

82. The method of claim 63, wherein step b comprises obtaining stem cells derived from embryos produced by in vitro fertilization or intracytoplasmic sperm injection.

83. The method of claim 63, wherein step b comprises obtaining diploid stem cells derived from embryos produced by parthenogenesis.

84. The method of claim 83, comprising obtaining diploid stem cells in which all of the MHC alleles are homozygous.

85. The method of claim 63, wherein step b comprises obtaining stem cells derived from embryos produced by cloning by nuclear transfer.

86. The method of claim 85, comprising obtaining rejuvenated stem cells.

87. The method of claim 86, comprising obtaining rejuvenated stem cells having telomeres that are on average at least as long as the telomeres of age-matched control cells of the same type that are not generated by nuclear transfer techniques.

88. The method of claim 86, comprising obtaining rejuvenated stem cells for which the proliferative life-span is at least as long as the proliferative life-span of age-matched control cells of the same type that are not generated by nuclear transfer techniques.

89. The method of claim 86, comprising obtaining rejuvenated stem cells for which the proliferative life-span is longer than the proliferative life-span of age-matched control cells of the same type that are not generated by nuclear transfer techniques.

90. The method of claim 86, comprising obtaining rejuvenated stem cells having EPC-1 activity that is greater than EPC-1 activity in age-matched control cells of the same type that are not generated by nuclear transfer techniques.

91. The method of claim 86, comprising obtaining rejuvenated stem cells having telomerase activity that is greater than telomerase activity in age-matched control cells of the same type that are not generated by nuclear transfer techniques.

92. The method of claim 85, comprising obtaining stem cells comprising non-human mitochondria.

93. The method of claim 63, wherein step b comprises obtaining stem cells having DNA that is genetically modified relative to the DNA of the human donor from which the stem cells are derived.

94. The method of claim 92, comprising obtaining genetically altered stem cells, the DNA of which is modified by adding, modifying, substituting, or deleting one or more DNA sequences.

95. The method of claim 93, comprising obtaining genetically altered stem cells, the DNA of which is modified so as to obtain, increase, decrease, inhibit, or otherwise modify, the expression of a gene that is native to or introduced into said cells, relative to expression of said gene in a control cell without the genetic modification.

96. The method of claim 93, comprising obtaining genetically altered stem cells, the DNA of which is modified by homologous recombination.

97. The method of claim 93, comprising obtaining genetically altered stem cells, the DNA of which is altered to prevent the expression of a gene encoding an antigenic protein that elicits an immune response contributing to rejection.

98. The method of claim 96, comprising genetically altered stem cells, the DNA of which is modified so as to inhibit production of at least one HLA antigen by cells of said cell line.

99. The method of claim 96, comprising genetically altered stem cells, the DNA of which is modified so as to inhibit production of one or more HLA antigens selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.

100. The method of claim 96, comprising genetically altered stem cells, the DNA of which is modified so as to inhibit production of β

2-microglobulin.

101. The method of claim 96, comprising genetically altered stem cells, the DNA of which is altered by replacing a non-homozygous MHC allele with one that is homozygous.

104. The method of claim 101 wherein said method is repeated with oocytes from different donors and a bank or library of different stem cell lines which are homozygous for different MHC alleles relative to the other members in the group are obtained.

107. The method of claim 104 wherein said stem cell bank includes stem cell lines which are homozygous for one or more of the following HLA serotypes:

HLA-A1, HLA-A3, HLA-A11, HLA-A15, HLA-A22, HLA-A24, HLA-A28, HLA-A29, HLA-A32, HLA-B5, HLA-7, HLA-B8, HLA-B12, HLA-B17, HLA-B 18, HLA-B35 AND HLA-B40.

110. The method of claim 107 wherein said stem cell lines are O-negative.

102. A method for producing stem cells homozygous for at least one MHC allele present in a human population, comprising:

a) obtaining oocytes from a donor;

b) activating the oocytes;

c) exposing the activated oocytes to chemical treatment that inhibits extrusion of the second polar body;

d) culturing the embryo in vitro under conditions resulting in formation of a blastocyst;

e) culturing inner cell mass cells of the blastocyst in vitro under conditions resulting in production of stem cells; and

f) screening to identify stem cells homozygous in a particular MHC allele.

103. The method of claim 102 wherein said stem cells are human.

105. The method of claim 102 wherein said stem cell lines are human.

106. The method of claim 105 wherein said stem cell lines are O-negative.

108. The method of claim 105 wherein said stem cell bank includes stem cell lines which are homozygous for at least one of the following HLA-a, -B and -DR haplotypes:

1,7,2;

1, 8, 3;

2, 14, 1;

2, 35, 4;

2, 35, 8;

2, 44, 7;

3, 7, 2;

3, 7, 4;

3, 7, 8;

3, 35, 1;

31, 51, 4 and 32, 14, 7.

109. The method of claim 106 wherein said stem cell lines are O-negative.

111. The method of claim 102, wherein the stem cells are totipotent, nearly totipotent, or pluripotent stem cells.

112. The method of claim 102, wherein the stem cells can differentiate into hematopoietic stem cells.

113. The method of claim 102, wherein the stem cells are homozygous for an MHC allele selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.

114. The method of claim 102, wherein the stem cells are homozygous for the MHC alleles encoding HLA-A, HLA-B, and HLA-DR.

115. A diploid cell line that is parthenogenetically-derived and is homozygous for at least one recessive disease-causing allele.

116. The parthenogenetically-derived cell line of claim 115, which is a line of stem cells.

117. The diploid cell line of claim 116 which is human.

118. The diploid cell line of claim 116 which is murine.

119. The parthenogenetically-derived stem cell line of claim 116, which is a line of totipotent, nearly totipotent, or pluripotent stem cells.

120. The parthenogenetically-derived stem cell line of claim 116, which is a line of embryonic stem cells.

121. The parthenogenetically-derived cell line of claim 116, which is a line of cells that are homozygous for at least one recessive disease-causing allele causing a pathological condition selected from the group consisting of Adenosine deaminase deficiency, Albinism, Adenylosuccinate lyase deficiency, Alpha-1 antitrypsin deficiency, Cystic Fibrosis, Friedreich'"'"'s ataxia, Gaucher'"'"'s disease, hypercholesterolemia, Alzheimer'"'"'s Disease, Autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy. deficiency of activation-induced cytidine deaminase, Ataxia-telangiectasia, severe combined immunodeficieny (SCID), chronic granulomatous disease, Phenylketonuria, Tetrahydrobiopterin deficiency, Hereditary fructose intolerance, Porphyria (one of the six forms is caused by a recessive gene), Sickle Cell Anemia, Tay Sachs syndrome, Thalassemia, Wilson'"'"'s disease, and Xeroderma pigmentosum.

122. A method for producing stem cells homozygous for at least one recessive disease-causing allele, comprising:

a) obtaining oocytes from a donor whose DNA contains a recessive disease-causing allele;

b) activating the oocytes;

c) exposing the activated oocytes to chemical treatment that inhibits extrusion of the second polar body;

d) culturing the embryo in vitro under conditions resulting in formation of a blastocyst;

e) culturing inner cell mass cells of the blastocyst in vitro under conditions resulting in production of stem cells; and

a) screening the stem cells to identify stem cells that are homozygous for a recessive disease-causing allele.

123. The method of claim 122, wherein step e) results in production of totipotent, nearly totipotent, or pluripotent stem cells.

124. The method of claim 122, wherein step e) results in production of embryonic stem cells.

125. The method of claim 122, wherein the stem cells identified in step f) are homozygous for at least one recessive disease-causing allele causing a pathological condition selected from the group consisting of Adenosine deaminase deficiency, Albinism, Adenylosuccinate lyase deficiency, Alpha-1 antitrypsin deficiency, Cystic Fibrosis, Friedreich'"'"'s ataxia, Gaucher'"'"'s disease, hypercholesterolemia, Alzheimer'"'"'s Disease, Autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy. deficiency of activation-induced cytidine deaminase, Ataxia-telangiectasia, severe combined immunodeficieny (SCID), chronic granulomatous disease, Phenylketonuria, Tetrahydrobiopterin deficiency, Hereditary fructose intolerance, Porphyria (one of the six forms is caused by a recessive gene), Sickle Cell Anemia, Tay Sachs syndrome, Thalassemia, Wilson'"'"'s disease, and Xeroderma pigmentosum.

126. The method of claim 122 wherein the stem cells are human.

127. The method of claim 125 wherein the stem cells are human.

128. The method of claim 122 wherein said stem cells are used to produce differentiated cells which are used in screening assays to identify molecules that inhibit or block the expression of said recessive disease-causing gene.

129. The method of claim 128 wherein said screening assays are affected in vitro.

130. The method of claim 128 wherein said screening assays are affected in a non-human animal that composes said differentiated cells.

131. A non-human animal model for study of a disease that is caused by the expression of a recessive disease causing gene which is produced by introducing into a non-human animal stem cells according to claim 122 or differentiated cells derived therefrom.

132. The non-human animal model of claim 131 which is non-human primate.

133. The non-human animal model of claim 131 which is murine.