Double-fusion human embryonic stem cells, methods of making double-fusion human embryonic stem cells, triple-fusion human embryonic stem cells, methods of making triple-fusion human embryonic stem cells, and methods of monitoring double-fusion human embryonic stem cells and triple-fusion human embryonic stem cells

Abstract

Embodiments of the present disclosure include double-fusion human embryonic stem cells, methods of imaging double-fusion human embryonic stem cells, double-fusion polynucleotides, double-fusion proteins, triple-fusion human embryonic stem cells, methods of imaging triple-fusion human embryonic stem cells, triple-fusion polynucleotides, triple-fusion proteins, methods of monitoring the progression of human embryonic stem cells, methods of making isolated double-fusion human embryonic stem cells, methods of making isolated triple-fusion human embryonic stem cells, and the like.

1 . An isolated double-fusion human embryonic stem cell (hESC) comprising hESC that:
 expresses a firefly luciferase reporter gene encoding a firefly luciferase protein; and   expresses an enhanced green fluorescent reporter gene encoding an enhanced green fluorescent protein.   
     
     
         2 . The isolated double-fusion human embryonic stem cell of  claim 1 , wherein the hESC is an H9 human embryonic stem cell. 
     
     
         3 . The isolated double-fusion human embryonic stem cell of  claim 1 , wherein the firefly luciferase reporter gene has a polynucleotide sequence of SEQ ID NO: 1. 
     
     
         4 . The isolated double-fusion human embryonic stem cell of  claim 1 , wherein the enhanced green fluorescent reporter gene has a polynucleotide sequence of SEQ ID NO: 2. 
     
     
         5 . The isolated double-fusion human embryonic stem cell of  claim 1 , wherein each of the firefly luciferase reporter gene and the enhanced green fluorescent reporter gene are not silenced after 44 passages. 
     
     
         6 . A method of preparing an isolated double-fusion human embryonic stem cell (hESC), comprising:
 providing a human embryonic stem cell;   transducing the human embryonic stem cell with an expression vector including Fluc and eGFP, Fluc is a firefly luciferase reporter gene and eGFP is enhanced green fluorescent reporter gene.   
     
     
         7 . The method of  claim 6 , wherein the expression vector is LV-pUP-Fluc-eGFP, wherein LV is lentivirus, pUP is constituitve human ubiquitin promoter, Fluc is firefly luciferase reporter gene, and eGFP is enhanced green fluorescent reporter gene. 
     
     
         8 . A method of monitoring double-fusion human embryonic stem cell, comprising:
 providing an isolated double-fusion human embryonic stem cell (hESC) of  claims 1  to  5 ; and   detecting fluorescence, bioluminescence, or both fluorescence and bioluminescence emitted from the double-fusion hESC and progeny thereof.   
     
     
         9 . An isolated double-fusion human embryonic stem cell (hESC) comprising an hESC that includes a double-fusion gene that includes a firefly luciferase reporter gene encoding a firefly luciferase protein and an enhanced green fluorescent reporter gene encoding an enhanced green fluorescent protein. 
     
     
         10 . The isolated double-fusion human embryonic stem cell hESC of  claim 9 , wherein the double-fusion gene includes a LV-pUP-Fluc-eGFP, wherein LV is lentivirus, pUP is constituitve human ubiquitin promoter, Fluc is firefly luciferase reporter gene, and eGFP is enhanced green fluorescent reporter gene. 
     
     
         11 . The isolated double-fusion human embryonic stem cells hESC of  claim 10 , wherein the LV-pUP-Fluc-eGFP has a SEQ ID No; 10. 
     
     
         12 . An isolated triple-fusion human embryonic stem cells (hESC) comprising hESC that:
 expresses a firefly luciferase reporter gene encoding a firefly luciferase protein,   expresses a red fluorescent reporter gene encoding a red fluorescent protein, and   expresses a Herpes Simplex Virus 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene encoding an enzyme protein (HSV1-TK).   
     
     
         13 . The isolated triple-fusion human embryonic stem cell (hESC) of  claim 12 , wherein the HSV1-tk is a truncated HSV1-tk (HSV1-ttk), and the HSV1-TK is a truncated HSV1-TK (HSV1-tTK). 
     
     
         14 . A method of preparing isolated double-fusion human embryonic stem cells (hESC), comprising:
 providing a human embryonic stem cell;   transducing the human embryonic stem cell with an expression vector including Fluc, RFP, and tTK, Fluc is a firefly luciferase reporter gene, mRFP is the red fluorescent reporter gene, and tTK is the truncated HSV1-tk PET reporter gene.   
     
     
         15 . The method of  claim 14 , wherein the expression vector is LV-pUP-Fluc-RFP-tTK, where LV is lentivirus, pUP is constituitve human ubiquitin promoter, Fluc is firefly luciferase reporter gene, mRFP is the red fluorescent reporter gene, and tTK is the truncated HSV1-tk PET reporter gene. 
     
     
         16 . A method of monitoring triple-fusion human embryonic stem cells, comprising:
 providing an isolated triple-fusion human embryonic stem cells (hESC) of  claims 12  to  13 ; and   detecting fluorescence, bioluminescence, or both fluorescence, and bioluminescence emitted from the triple-fusion hESC and progeny thereof.