COMPOSITIONS AND METHODS FOR PRODUCING SMOOTH MUSCLE CELLS
Methods and compositions are provided for producing a smooth muscle cell (SMC) from a pluripotent stem cell, e.g., an induced pluripotent stem cell (iPSC), via MEOX1 overexpression. MEOX1 expression can be induced using any convenient method, e.g., by introducing an exogenous nucleic acid encoding MEOX1 or by using a tool such as CRISPRa to stimulate expression from the MEOX1 endogenous locus.
1 . A method producing a smooth muscle cell (SMC), the method comprising:
overexpressing MEOX1 expression in a pluripotent stem cell (PSC), thereby producing an SMC.
2 . The method of claim 1 , wherein said overexpressing MEOX1 comprises expressing, in the PSC, a CRISPRa fusion protein that, in the presence of a guide RNA, stimulates expression of MEOX1.
3 . The method of claim 2 , wherein MEOX1 expression is stimulated from its endogenous locus.
4 . The method of claim 2 , wherein a nucleotide sequence encoding the CRISPRa fusion protein is operably linked to an inducible promoter, and wherein said overexpressing MEOX1 comprises stimulating expression from the inducible promoter.
5 . The method of claim 4 , wherein the nucleotide sequence encoding the CRISPRa fusion protein is integrated into the genome of the PSC.
6 . The method of claim 2 , wherein said CRISPRa fusion protein comprises dCas9 fused to VPR.
7 . The method of claim 2 , wherein the method comprises introducing the guide RNA or a nucleic acid encoding the guide RNA into the PSC.
8 . The method of claim 1 , wherein said overexpressing MEOX1 comprises introducing a nucleic acid encoding MEOX1 into the PSC.
9 . The method of claim 8 , wherein the nucleic acid encoding MEOX1 is a recombinant expression vector.
10 . The method of claim 8 , wherein said overexpressing MEOX1 comprises contacting the PSC with a virus comprising the nucleic acid encoding MEOX1.
11 . The method of claim 8 , wherein the nucleic acid encoding MEOX1 is an RNA.
12 . The method of claim 1 , further comprising measuring expression of one or more SMC markers after overexpressing MEOX1 begins to determine whether an SMC has been produced.
13 . The method of claim 12 , wherein said one or more SMC markers comprises TAGLN or CNN1, or both.
14 . The method of claim 12 , wherein said one or more SMC markers comprises: CNN1, TAGLN, TAGLN2, ZEB1, NCAM1, CALD1, FN1, SNA12, MYH11, ACTA2, or any combination thereof.
15 . The method of claim 1 , wherein the method further comprises an analysis of cell morphology to facilitate determination of whether an SMC was produced.
16 . The method of claim 1 , wherein MEOX1 is overexpressed in a population of PSCs, and said method further comprises, isolating SMCs and/or enriching the PSC population for SMCs.
17 . The method of claim 16 , wherein said isolating and/or enriching comprises an analysis of cell morphology.
18 . The method of claim 1 , wherein the PSC is a human cell.
19 . The method of claim 1 , wherein the PSC was derived from a human patient.
20 . The method of claim 1 , wherein the PSC is an induced pluripotent stem cell (iPSC).