Pancreatic cells for treating diabetes and methods of generating the same

Abstract

The present disclosure provides cell-based compositions for treating diabetes, methods for identifying cells that preferentially differentiate into endoderm cells, and methods for preparing insulin-producing pancreatic cells, as well as related methods of use for treating diseases related to insulin deficiency.

1 . A reprogrammed cell that was reprogrammed by expressing reprogramming genes without incorporating the reprogramming genes into the genome of the cell, wherein after a 4 day exposure to Activin-A and Wortmannin the reprogrammed cell (i) does not express Brachyury, (ii) expresses BHMT2, Cox7A1, and HSPB2 at lower levels compared to a control pluripotent cell, and (iii) expresses NAP1L1 at a higher level compared to the control pluripotent cell. 
     
     
         2 . The reprogrammed cell of  claim 1 , wherein BHMT2 expression is down-regulated at least 2 logs relative to the control pluripotent cell and NAP1L1 expression is up-regulated at least 2 logs relative to the control pluripotent cell. 
     
     
         3 . The reprogrammed cell of  claim 1 , wherein both Cox7A1 and HSPB2 expression are down-regulated at least 2 logs relative to the control pluripotent cell. 
     
     
         4 . The reprogrammed cell of  claim 1 , wherein GLIS2, CCDC58, and MTX3 expression is up-regulated relative to the control pluripotent cell and C7orf29 expression is down-regulated relative to the control pluripotent cell. 
     
     
         5 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell is multipotent. 
     
     
         6 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell can differentiate into endoderm or ectoderm cell types, but not mesoderm cell types. 
     
     
         7 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell is not tumorogenic. 
     
     
         8 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell does not form a teratoma when implanted in an animal. 
     
     
         9 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell expresses Oct4, Tra-1-81, Tra-1-60, Sox2, and SSEA. 
     
     
         10 . The reprogrammed cell of  claim 1 , wherein the reprogramming genes are Oct4, Sox2, Klf4, and L-Myc. 
     
     
         11 . The reprogrammed cell of  claim 1 , wherein the reprogramming genes were expressed from an episomal plasmid. 
     
     
         12 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell possesses a normal karyotype. 
     
     
         13 . The reprogrammed cell of  claim 1 , wherein the reprogrammed cell possesses a population doubling time of about 30 hours. 
     
     
         14 . A method of producing insulin-secreting cells, comprising:
 (a) culturing human stem cells on an adhesive substrate in a first medium comprising Activin-A and Wortmannin, wherein the human stem cells are not exposed to Wnt3a, wherein the human stem cells are capable of differentiating into at least endodermal lineages;   (b) further culturing the human stem cells in at least one additional medium comprising retinoic acid and cyclopamine; and   (c) transferring the human stem cells to a suspension culture when the cells form three-dimensional cell structures;   wherein the human stem cells are exposed to retinoic acid and cyclopamine for at least 20 days.   
     
     
         15 . The method of  claim 14 , wherein the human stem cells form three-dimensional structures when cultured on the adhesive substrate. 
     
     
         16 . A method of producing insulin-secreting cells, comprising:
 (a) culturing human stem cells capable of differentiating into endodermal lineages in a first medium comprising Activin-A and Wortmannin, thereby differentiating the human stem cells into endoderm cells, wherein the human stem cells were not exposed to keratinocyte growth factors (KGF) prior to differentiation into endoderm cells;   (b) culturing the endoderm cells from (a) in a second medium comprising retinoic acid and cyclopamine, thereby differentiating the endoderm cells into endocrine cells;   (c) culturing the endocrine cells from (b) in a third medium comprising KGF, noggin, and EGF, thereby differentiating the endocrine cells into pancreatic progenitor cells;   (d) culturing the pancreatic progenitor cells from (c) in a fourth medium comprising noggin, EGF, γ-secretase inhibitor XXI, and Alk5i II, thereby differentiating the pancreatic progenitor cells into insulin-producing cells.   
     
     
         17 . The method of  claim 16 , wherein the second culture medium further comprises KGF. 
     
     
         18 . The method of  claim 16 , wherein the third and fourth mediums further comprise retinoic acid and cyclopamine. 
     
     
         19 . The method of  claim 16 , wherein the insulin producing cells from (d) are further cultured in a fifth medium comprising Alk5I II and retinoic acid, and optionally cyclopamine. 
     
     
         20 . The method of  claim 19 , further comprising further culturing the cells in a sixth medium comprising Alk5i II, nicotinamine, IGF-I, and optionally retinoic acid and cyclopamine. 
     
     
         21 . The method of  claim 16 , wherein the total culture time is less than 30 days. 
     
     
         22 . The method of  claim 16 , wherein the human stem cells are derived from pancreatic primary tissue.