Compositions and methods to derive mesodermal lineage cells and mixed tissue organoids from embryonic stem cells
The disclosure relates to methods for producing mesodermal lineage cells, cardiac lineage cells, hematopoietic lineage cells, retinal lineage cells, and combinations thereof. In some aspects, the disclosure relates to methods of producing mixed tissue organoids comprising retinal lineage cells and cardiac lineage cells. When interaction of WDR5 protein at the binding pocket/interaction surface with RBBP5/MYC/KANSL2 is disrupted in the embryonic stem cell for a set period of time after differentiation of the embryonic stem cell has begun, and the disruption of that interaction is subsequently removed, differentiation of the embryonic stem cell to a mesodermal lineage cell is obtained. Such mesodermal lineage cells may, in some methods of the disclosure, be subsequently differentiated into cardiac lineage cells and hematopoietic lineage cells. The disclosure also relates to method for producing mixed lineage organoids, comprising retinal and mesodermal, including cardiac, lineage cells in a single organoid. The disclosure also relates to cells and organoids obtained by the methods, uses of the cells and organoids, and kits comprising the cells and/or reagents for producing them.
1. A method for producing in culture a mesodermal lineage cell from an embryonic stem cell, wherein the embryonic stem cell comprises recombinant WD repeat domain 5 (WDR5) protein and wherein endogenous wdr5 gene expression in the embryonic stem cell is knocked out, the method comprising:
a) transfecting the embryonic stem cell, wherein endogenous wdr5 gene expression is knocked out with a recombinant wdr5 gene and expressing recombinant WDR5 protein in the embryonic stem cell;
b) disrupting interaction of recombinant WDR5 protein with retinoblastoma-binding protein 5 (RBBP5), MYC, or KAT8 Regulatory NSL Complex Subunit 2 (KANSL2) protein in the embryonic stem cell for a period of time greater than 24 hours and less than 60 hours by inhibiting expression of recombinant WDR5 protein by the embryonic stem cell by using an inducible gene expression system, and
c) removing the disruption of the interaction of recombinant WDR5 protein with RBBP5, MYC, or KANSL2 in the embryonic stem cell to allow the mesodermal lineage cell to differentiate from the embryonic stem cell.
2. The method of claim 1 , wherein inhibiting expression of recombinant WDR5 protein comprises:
i) introducing a polynucleotide encoding a recombinant WDR5 protein under control of a transactivator protein into the embryonic stem cell; and
ii) limiting the expression of the recombinant WDR5 protein in the embryonic stem cell by controlling the transactivator protein.
3. The method of claim 1 , wherein the removing step comprises turning on the expression of the transactivator protein allowing expression of the recombinant WDR5 protein in the embryonic stem cell.
4. The method of claim 1 , wherein the period of time starts when the embryonic stem cell is allowed to begin differentiation.
5. The method of claim 1 , wherein the period of time is about 36 to about 48 hours.