Methods of Screening Embryonic Progenitor Cell Lines

Abstract

Aspects of the present invention include methods and compositions related to the production and use of numerous clonal lineages of embryonic progenitor cell lines derived from differentiating cultures of primordial stem cells with diverse molecular markers and having been cultured for >21 doublings of clonal expansion. The robustness of these clonally-purified lines, their ability to expand for >40 passages while maintaining their pattern of gene expression, lack of tumorigenicity, and their embryonic pattern of gene expression offers novel compositions and methods for modeling numerous differentiation pathways for the first time in vitro, and for the manufacture of purified product not existing in such a purified state in nature or using other manufacturing modalities. Representative progenitor cell lines described herein are capable of development into cutaneous adipocytes, blood-brain barrier cells, neuronal cells, or smooth muscle cells each with therapeutic potential.

1 . An isolated clonal progenitor cell line expressing EYA4, wherein the clonal progenitor cell line is an embryonic cutaneous progenitor cell. 
     
     
         2 . The isolated clonal progenitor cell line of  claim 1 , wherein the cell line also expresses ADH1A, ADH1B, FABP4, CD36, PPARG, ANGPT2, EBF2 AND DBC1. 
     
     
         3 . The isolated clonal progenitor cell line of  claim 1 , wherein the cell line is encapsulated in a biomaterial. 
     
     
         4 . The isolated clonal progenitor cell line of  claim 3 , wherein the biomaterial comprises a hydrogel. 
     
     
         5 . The isolated clonal progenitor cell line of  claim 3 , wherein the biomaterial comprises hyaluronic acid. 
     
     
         6 . The isolated clonal progenitor cell line of  claim 3 , wherein the biomaterial is chosen from calcium alginate, agarose, polylactic acid/poly-glycolic acid derivatives, and fibrin. 
     
     
         7 . A kit comprising the cell line of  claims 1 - 6 . 
     
     
         8 . A method of differentiating a clonal progenitor cell line into cutaneous adipocytes comprising 1) obtaining a clonal progenitor cell line of  claim 1 ; 2) contacting the clonal progenitor cell line with MDI Induction Media; and 3) contacting the clonal progenitor cell line of 2) with Insulin Media thereby differentiating a clonal progenitor cell line into a cutaneous adipocyte. 
     
     
         9 . The method of  claim 8 , wherein the cell line also expresses ADH1A, ADH1B, FABP4, CD36, PPARG, ANGPT2, EBF2 AND DBC1. 
     
     
         10 . The method of  claim 8 , wherein the cell line is grown to confluence prior to step 2). 
     
     
         11 . The method of  claim 8 , wherein the cells are contacted with the MDI Induction Media with for two days. 
     
     
         12 . The method of  claim 8 , further comprising encapsulating the cutaneous adipocyte in a biomaterial. 
     
     
         13 . The method of  claim 12 , wherein the biomaterial comprises a hydrogel. 
     
     
         14 . The method of  claim 12 , wherein the biomaterial comprises hyaluronic acid. 
     
     
         15 . A method of differentiating a clonal progenitor cell line into cutaneous adipocytes comprising 1) obtaining a clonal progenitor cell line of  claim 1 ; 2) contacting the clonal progenitor cell line with serum free differentiation media; and 3) contacting the clonal progenitor cell line of 2) with a differentiation media without rosiglitazone thereby differentiating a clonal progenitor cell line into a cutaneous adipocyte. 
     
     
         16 . The method of  claim 15 , wherein the cell line also expresses ADH1A, ADH1B, FABP4, CD36, PPARG, ANGPT2, EBF2 AND DBC1. 
     
     
         17 . The method of  claim 15 , further comprising encapsulating the cutaneous adipocyte in a biomaterial. 
     
     
         18 . The method of  claim 17 , wherein the biomaterial comprises a hydrogel. 
     
     
         19 . The method of  claim 17 , wherein the biomaterial comprises hyaluronic acid. 
     
     
         20 . The method of  claim 15 , wherein the clonal progenitor cell line in step 2 is cultured in a serum free differentiation media for 3 days and the clonal progenitor cell line in step 3 is cultured in differentiation media without rosiglitazone for 5 days.