Cell lines expressing inserted secretable reporter genes at multiple stages of differentiation

Abstract

A composition of matter comprises one or more cell lines configured to inducibly differentiate to at least a first stage of differentiation and a second, subsequent stage of differentiation. Each of the one or more cell lines are genetically edited to express one or more first stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed during the first stage of differentiation. The cell lines are further genetically edited to express one or more second stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed during the second stage of differentiation, but not during the first stage of differentiation, wherein the one or more second stage inserted secretable reporter genes are different than the one or more first stage inserted secretable reporter genes.

1 . A composition of matter, comprising:
 one or more cell lines configured to inducibly differentiate to at least a first stage of differentiation and a second, subsequent stage of differentiation, the one or more cell lines genetically edited to express:
 one or more first stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed during the first stage of differentiation; and 
 one or more second stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed during the second stage of differentiation, but not during the first stage of differentiation, wherein the one or more second stage inserted secretable reporter genes are different than the one or more first stage inserted secretable reporter genes. 
   
     
     
         2 . The composition of matter of  claim 1 , wherein the one or more first stage inserted secretable reported genes and one or more second stage inserted secretable reporter genes are configured to be expressed and secreted into culture media used to culture the genetically edited cell lines during differentiation, such that the culture media may be collected and assayed for the one or more first stage inserted secretable reported genes and one or more second stage inserted secretable reporter genes. 
     
     
         3 . The composition of matter of  claim 2 , wherein culturing the genetically edited cell lines with a perturbing agent that impacts differentiation results in a change in a detected ratio of one or more first stage inserted secretable reporter genes compared to one or more second stage secretable reporter genes in the culture media collected from the differentiating edited cell lines. 
     
     
         4 . The composition of matter of  claim 1 , wherein the one or more cell lines comprise partially differentiated, immortalized cells. 
     
     
         5 . The composition of matter of  claim 4 , wherein inducing differentiation of the genetically edited cell lines to the first stage of differentiation includes inducing differentiation of the one or more cell lines to edited neural progenitor cells. 
     
     
         6 . The composition of matter of  claim 5 , wherein the one or more first stage inserted secretable reporter genes are placed under control of a promoter for a gene encoding Nestin. 
     
     
         7 . The composition of matter of  claim 5 , wherein inducing differentiation of the genetically edited cell lines to the second stage of differentiation comprises inducing differentiation of the edited neural progenitor cells to neurons and glial cells. 
     
     
         8 . The composition of matter of  claim 7 , wherein the one or more second stage inserted secretable reporter genes are placed under control of promoters for genes encoding TUJ1 and GFAP. 
     
     
         9 . The composition of matter of  claim 1 , wherein one or more secretable reporter genes includes a 2A family self-cleaving peptide sequence. 
     
     
         10 . The composition of matter of  claim 1 , wherein one or more of the one or more first stage inserted secretable reporter genes is downregulated upon differentiation to the second stage of differentiation. 
     
     
         11 . The composition of matter of  claim 1 , wherein the one or more cell lines are genetically edited via targeted insertion of one or more known genetic mutations into the genome of the one or more cell lines. 
     
     
         12 . A composition of matter, comprising:
 neuronal progenitor cells edited to express:
 one or more inserted secretable reporter genes placed under control of promoters for genes canonically expressed by the neuronal progenitor cells; and 
 one or more inserted secretable reporter genes placed under control of promoters for genes canonically expressed upon differentiation of the neuronal progenitor cells to neurons, but not by neuronal progenitor cells; and 
 one or more inserted secretable reporter genes placed under control of promoters for genes canonically expressed upon differentiation of the neuronal progenitor cells to glial cells, but not by neuronal progenitor cells. 
   
     
     
         13 . The composition of matter of  claim 12 , wherein the neuronal progenitor cells are further edited to include one or more known genetic mutations. 
     
     
         14 . The composition of matter of  claim 12 , wherein the neuronal progenitor cells are derived from edited human induced pluripotent stem cells. 
     
     
         15 . The composition of matter of  claim 12 , wherein each of the inserted secretable reporter genes are configured to be expressed and secreted into culture media used to culture the neuronal progenitor cells, such that the culture media may be collected and assayed for the inserted secretable reporter genes. 
     
     
         16 . The composition of matter of  claim 12 , wherein one or more of the one or more inserted secretable reporter genes placed under control of promoters for genes canonically expressed by the neuronal progenitor cells is downregulated upon differentiation of the neuronal progenitor cells to neurons or glial cells. 
     
     
         17 . A composition of matter, comprising:
 one or more cell lines configured to inducibly differentiate to at least a first stage of differentiation and a second, subsequent stage of differentiation, the one or more cell lines genetically edited to express:
 one or more first stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed during the first stage of differentiation; 
 a first set of one or more second stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed by a first differentiated cell type during the second stage of differentiation, but not during the first stage of differentiation, 
 a second set of one or more second stage inserted secretable reporter genes placed under control of promoters for genes canonically expressed by a second differentiated cell type during the second stage of differentiation, but not during the first stage of differentiation, such that none of the one or more first stage inserted secretable reporter genes are represented in the first set of one or more second stage inserted secretable reporter genes or the second set of one or more second stage inserted secretable reporter genes. 
   
     
     
         18 . The composition of matter of  claim 17 , wherein the first set of one or more second stage inserted secretable reporter genes and the second set of one or more second stage inserted secretable reporter genes are different. 
     
     
         19 . The composition of matter of  claim 17 , wherein one or more of the one or more cell lines include partially differentiated, immortalized cells. 
     
     
         20 . The composition of matter of  claim 19 , wherein
 one or more of the first stage inserted secretable reporter genes are placed under control of a promoter for a gene encoding Nestin;   one or more of the first set of one or more second stage inserted secretable reporter genes placed under control of promoter for a gene encoding TUJ1; and   one or more of the second set of one or more second stage inserted secretable reporter genes placed under control of promoter for a gene encoding GFAP.