Engineering a heterogeneous tissue from pluripotent stem cells
Aspects of the present disclosure are directed to methods and compositions for the production of heterogeneous tissue from human induced pluripotent stem (hiPS) cells.
1. A method comprising: culturing, in cell culture media under inducing conditions, human induced pluripotent stem (hiPS) cells genetically modified to comprise a nucleic acid comprising an inducible promoter operably linked to a nucleic acid encoding GATA6 protein, whereby upon expression of the nucleic acid, the hiPS cells differentiate into a heterogeneous cell population comprising mesodermal cells, endodermal cells, and ectodermal cells, wherein the induced expression of GATA6 results in an increased subpopulation of endoderm cells compared to that of a cell population derived in the absence of said GATA6 expression.
2. The method of claim 1 , further comprising culturing the heterogeneous cell population under conditions sufficient to result in differentiation of the heterogeneous cell population into heterogeneous tissue.
3. The method of claim 1 , wherein the hiPS cells are cultured for at least 7 to 15 days.
4. The method of claim 2 , wherein the heterogeneous tissue comprises hepatic endodermal cells, hepatoblast-like cells, cholangiocyte-like cells, endothelial progenitor cells, endothelial-like cells, hemogenic endothelial cells, erythrocyte-like cells, hematopoietic progenitor-like cells, mesenchymal progenitor cells, stellate-like cells, neurectodermal cells, neural plate-like cells, neural fold-like cells, or any combination thereof.
5. The method of claim 2 , wherein cells of the heterogeneous tissue are:
FOXA2 + and/or SOX17 + ; CEBPA + , HNF4A + , FOXA2 + and/or HHEX + ; CD133 + , AAT + , CEBPA + , FOXA2 + , AFP + , LGR5 + , CK19 + , DLK1 + and/or ALB + ; CK7 + and/or AQP1 + ; T + ; CD51 + , NES + and/or PDGFRA + ; CD34 + and/or CEBPA − ; NES + and/or PDGFRA + ; DES + ; CD31 + ; CD34 + , TAL1 + , FLK1 + and/or CD93 + ; hemoglobin + ; CD45 + ; OCT4 + , SOX10 + and/or NES + ; OCT4 + , SOX10 + and/or AP2A + ; OCT4 − , SOX10 + and/or AP2A − ; or PAX7 + , DLX5 + , FOXG1 + and/or PAX6 + .
6. The method of claim 1 , wherein cytokines and/or growth factors are not added to the cell culture media.
7. The method of claim 1 , wherein the cell culture media does not contain serum.
8. A heterogeneous tissue produced by the method of claim 2 .
9. A method comprising expressing a nucleic acid encoding GATA6 protein in human induced pluripotent stem (hiPS) cells, wherein the method comprises:
(i) introducing into hiPS cells (a) a first nucleic acid encoding a constitutively expressed doxycycline-activatable transactivator (rtTa) and (b) a second nucleic acid encoding GATA6 protein operably linked to a promoter that comprises a binding site for rtTA; and
(ii) culturing the hiPS cells in the presence of doxycycline to activate expression of the nucleic acid encoding GATA6 protein to result in differentiation of the hiPS cells into a heterogeneous cell population comprising mesodermal cells, endodermal cells, and ectodermal cells, wherein the induced expression of GATA6 results in an increased subpopulation of endoderm cells compared to that of a cell population derived in the absence of said GATA6 expression.
10. The method of claim 1 wherein the hiPS cells are obtained by isolating somatic cells from a human and inducing pluripotency in the isolated somatic cells to produce the hiPS cells.
11. The method of claim 2 , wherein the heterogeneous tissue comprises cells that are positive for fibrinogen (Fib), AAT, CEBPα, LGR5, HNF4α, CK19 and DLK1 expression.
12. A heterogeneous tissue produced by the method of claim 11 .