Multipotent adult stem cells and methods for isolation

• US 20050181502A1
• Filed: 03/21/2005
• Published: 08/18/2005
• Est. Priority Date: 08/05/1999
• Status: Active Grant

• Current Assignee: Athersys, Inc.
• Original Assignee: Athersys, Inc.
• Inventors: Verfaillie, Catherine M., Furcht, Leo T., Reyes, Morayma
• Granted Patent: US 10,226,485 B2
• Time in Patent Office: Days
• US Class Current: 435/354
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/5156 expressing foreign proteins
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• 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: A61K 39/461 characterised by the cell type used
• CPC Class Codes: A61K 39/46432 Nervous system antigens
• 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 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Abstract

The invention provides isolated stem cells of non-embryonic origin that can be maintained in culture in the undifferentiated state or differentiated to form cells of multiple tissue types. Also provided are methods of isolation and culture, as well as therapeutic uses for the isolated cells.

Claims

1-63. -63. (canceled)

64. A substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor.

65. The substantially homogenous cell population of claim 64, further including co-expression of telomerase.

66. The substantially homogenous cell population of claim 64, wherein the cells are derived from human bone marrow cells.

67. The substantially homogenous cell population of claim 64, wherein the cardiac-related transcription factor is selected from the group consisting of GATA4, Irx4 and Nkx2.5.

68. The substantially homogenous cell population of claim 64, further including a label.

69. The substantially homogenous cell population of claim 64, wherein the cell population differentiates into cardiac muscle cells.

70. The substantially homogenous cell population of claim 64, wherein the cells express at least one trophic factor selected from the group consisting of IL-6, VEGF, MCP1 and BDNF.

71. A substantially homogenous cell population which co-expresses CD49c, CD90, and at least one cardiac-related transcription factor, but does not express bone sialoprotein.

72. The substantially homogenous cell population of claim 71, wherein the cardiac-related transcription factor is selected from the group consisting of GATA4, Irx4 and Nkx2.5.

73. A substantially homogenous cell population which co-expresses CD49c, CD90, GATA4, Irx4 and Nkx2.5.

74. A substantially homogenous cell population which co-expresses CD49c, CD90, telomerase, GATA4, Irx4 and Nkx2.5.

75. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor, comprising the steps of:

a) culturing a source of the cell population under a low oxygen condition; and

b) treating the cultured source of the cell population with a protein kinase C inhibitor and a DNA methylation inhibitor.

76. The method of claim 75, wherein the source of the cell population includes a bone marrow source.

77. The method of claim 75, wherein the protein kinase C inhibitor is chelerythrine.

78. The method of claim 77, wherein the DNA methylation inhibitor is 5-azacytidine.

79. The method of claim 75, wherein the treated cell population co-expresses a cardiac-related transcription factor selected from the group consisting of GATA4, Irx4 and Nkx2.5.

80. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase and at least one cardiac-related transcription factor, comprising the steps of:

a) culturing a source of the cell population under a low oxygen condition; and

b) treating the cultured source of the cell population with a protein kinase C inhibitor and a DNA methylation inhibitor.

81. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase, GATA4, lrx4 and Nkx2.5, comprising the steps of:

a) culturing a source of the cell population under a low oxygen condition; and

b) treating the cultured source of the cell population with a protein kinase C inhibitor and a DNA methylation inhibitor.

82. The method of claim 81, wherein the source of the cell population includes a bone marrow source.

83. The method of claim 81, wherein the protein kinase C inhibitor is chelerythrine.

84. The method of claim 83, wherein the DNA methylation inhibitor is 5azacytidine.

85. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90, GATA4, Irx4 and Nkx2.5, comprising the steps of:

a) culturing a source of the cell population under a low oxygen condition; and

b) treating the cultured source of the cell population with a protein kinase A inhibitor and a DNA methylation inhibitor.

86. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor, comprising the step of treating a cell population which co-expresses CD49c and CD90 with a protein kinase C inhibitor and a DNA methylation inhibitor.

87. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase and at least one cardiac-related transcription factor, comprising the step of treating a cell population which co-expresses CD49c, CD90 and telomerase with a protein kinase C inhibitor and a DNA methylation inhibitor.

88. A method of making a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase, GATA4, Irx4 and Nkx2.5, comprising the step of treating a cell population which co-expresses CD49c, CD90 and telomerase with a protein kinase C inhibitor and a DNA methylation inhibitor.

89. A method of treating a myocardial infarction in a human, comprising the step of administering a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor to the human.

90. The method of claim 89, wherein the cardiac-related transcription factor is selected from the group consisting of GATA4, Irx4 and Nkx2.5.

91. A method of treating a myocardial infarction in a human comprising the step of administering a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor to the human.

92. A method of treating a myocardial infarction in a human comprising the step of administering to the human a substantially homogenous cell population which co-expresses CD49c, CD90, GATA4, Irx4 and Nkx2.5.

93. A method of treating a myocardial infarction in a human, comprising the step of administering to the human a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase, GATA4, Irx4 and Nkx2.5.

94. A method of treating a myocardial infarction in a human, comprising the steps of:

a) culturing a source of a cell population under a low oxygen condition;

b) treating the cultured source of the cell population with a protein kinase C inhibitor and a DNA methylation inhibitor; and

d) administering the treated cell population to the human.

95. The method of claim 94, wherein the treated cell population is administered proximate to the myocardial infarction.

96. The method of claim 95, wherein the treated cell population is administered into a cardiac muscle.

97. The method of claim 94, further including selecting from the treated cell population, a population of cells which co-expresses CD49c, CD90, and at least one cardiac-specific marker.

98. The method of claim 97, wherein the selected cell population further includes cells which express telomerase.

99. The method of claim 97, wherein the cardiac-specific marker is selected from the group consisting of GATA4, Irx4 and Nkx2.5.

100. The method of claim 94, wherein the source of the cell population includes a bone marrow source.

101. A method of treating a myocardial infarction in a human, comprising the steps of:

a) treating a cell population which co-expresses CD49c and CD90 with a protein kinase C inhibitor and a DNA methylation inhibitor; and

b) administering the treated cells to the human.

102. The method of claim 101, wherein the cell population is derived from bone marrow.

103. The method of claim 101, wherein the cell population further includes cells which co-express telomerase.

104. The method of claim 101, wherein the cell population expresses at least one cardiac-related transcription factor selected from the group consisting of GATA4, Irx4 and Nkx2.5.

105. A method of treating a congestive heart failure in a human, comprising the step of administering a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor to the human.

106. A method of treating a congestive heart failure in a human comprising the step of administering a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor to the human.

107. A method of treating a congestive heart failure in a human comprising the step of administering a substantially homogenous cell population which co-expresses CD49c, CD90, GATA4, Irx4 and Nkx2.5 to the human.

108. A method of treating a congestive heart failure in a human, comprising the step of administering to the human a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase, GATA4, Irx4 and Nkx2.5.

109. A method of treating a congestive heart failure in a human, comprising the steps of:

a) culturing a source of a cell population under a low oxygen condition;

b) treating the cultured source of the cell population with a protein kinase C inhibitor and a DNA methylation inhibitor; and

d) administering the treated cell population to the human.

110. A method of forming a committed progenitor cell-type, comprising the step of combining a substantially homogenous population of cells that co-expresses CD49c and CD90 with a population of cells that includes at least one committed progenitor cell type.

111. The method of claim 110, wherein said population of cells is selected from the group consisting of a population of nerve cells and a population of cardiac muscle cells.

112. The method of claim 110, wherein the population of cells expresses telomerase.

113. A substantially homogenous cell population which co-expresses CD49c, CD90 and has a doubling time of less that about 144 hours when cultured under a low oxygen condition.

114. The substantially homogenous cell population of claim 113, wherein the doubling time is less than about 72 hours.

115. The substantially homogenous cell population of claim 113, wherein the doubling time is less than about 48 hours.

116. The substantially homogenous cell population of claim 113, wherein the doubling time is less than about 65 hours.

117. The substantially homogenous cell population of claim 113, wherein the doubling time is less than about 35 hours.

118. The substantially homogenous cell population of claim 113, wherein the low oxygen condition is less than about 5% oxygen.

119. A substantially homogenous cell population which co-expresses CD49c, CD90 and has a doubling time less than about 144 hours when cultured under a low oxygen condition, wherein the substantially homogenous cell population is formed by a method, comprising the step of culturing a cell population source at a seeding density of about 100 cells/cm.sup.2 under the low oxygen condition.

120. A pharmaceutical composition comprising a substantially homogenous cell population which co-expresses CD49c, CD90 and at least one cardiac-related transcription factor.

121. A pharmaceutical composition comprising a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase and at least one cardiac-related transcription factor.

122. A pharmaceutical composition comprising a substantially homogenous cell population which co-expresses CD49c, CD90, telomerase, GATA4, Irx4 and Nkx2.5.

123. A pharmaceutical composition comprising a substantially homogenous cell population which co-expresses CD49c, CD90, GATA4, Irx4 and Nkx2.5.