Lineage Reprogramming to Induced Cardiac Progenitor Cells (iCPC) By Defined Factors
Animal cells, notably adult fibroblasts, are advantageously reprogrammed in direct lineage reprogramming methods using defined factors to produce proliferative and multipotent induced cardiac progenitor cells (iCPC). The iCPC thus produced can be differentiated under suitable differentiation conditions to cardiac lineage cells including cardiomyocytes, smooth muscle cells, and endothelial cells, as evidenced by expression of lineage specific markers. Sets of factors effective in combination to reprogram the fibroblasts can include a set that includes some or all of 5 factors (Mespl, Baf60c, Nkx2.5, Gata4, Tbx5), a set that includes some or all of 11 factors (Mespl, Mesp2, Gata4, Gata6, Baf60c, SRF, Isll, Nkx2.5, Irx4, Tbx5, Tbx20), a set that includes some or all of 18 factors (T, Mespl, Mesp2, Tbx5, Tbx20, Isll, Gata4, Gata6, Irx4, Nkx2.5, Handl, Hand2, Tbx20, Tbx18, Tip60, Baf60c, SRF, Hey2), and a set that includes some or all of 22 factors (T, Mespl, Mesp2, Tbx5, Tbx20, Isll, Gata4, Gata6, Irx4, Nkx2.5, Hand1, Hand2, Tbx20, Tbx18, Tip60, Baf60c, SRF, Hey2, Oct4, Klf4, Sox2, L-myc).
1 . A method for producing an induced cardiac progenitor cell (iCPC) from somatic cells, the method comprising the steps of:
expressing in the somatic cells a set of factors sufficient to induce reprogramming of the cells to produce iCPC, the set of factors comprising transcription factors and at least one chromatin remodeling factor, whereby iCPCs are obtained.
2 . The method of claim 1 , further comprising separating the iCPCs from non-reprogrammed cells.
3 . The method of claim 1 , wherein the expressing step comprises introducing into the mammalian somatic cell the set of factors.
4 . The method of claim 3 , wherein the set of factors is introduced into the mammalian somatic cell using one or more vectors selected from the group consisting of a nonviral episomal vector, a synthetic RNA, and an engineered membrane-permeable biomolecule.
5 . The method of claim 4 , wherein introducing comprises introducing the set of factors by a method selected from the group consisting of infection, transfection, electroporation, and application of an engineered membrane-permeable biomolecule.
6 . The method of claim 1 , wherein the set of factors comprises a plurality of early cardiac transcription factors.
7 . The method of claim 1 , wherein the plurality of early cardiac transcription factors includes some or all of T, Mesp1, Mesp2, Tbx5, Tbx20, Isl1, Gata4, Gata6, Irx4, Nkx2.5, Hand 1, Hand2, Tbx20, Tbx18, Tip60, Baf60c, SRF, and Hey2.
8 . The method of claim 1 , wherein the plurality of early cardiac transcription factors comprises T, Mesp1, Mesp2, Tbx5, Tbx20, Isl1, Gata4, Gata6, Irx4, Nkx2.5, Hand1, Hand2, Tbx20, Tbx18, Tip60, Baf60c, SRF, Hey2, Oct4, Klf4, Sox2, and L-myc.
9 . The method of claim 1 , wherein the plurality of early cardiac transcription factors comprises Mesp1, Mesp2, Gata4, Gata6, Baf60c, SRF, Isl1, Nkx2.5, Irx4, Tbx5, and Tbx20.
10 . The method of claim 1 , wherein the plurality of early cardiac transcription factors comprises Mesp1, Baf60c, Nkx2.5, Gata4, and Tbx5.
11 . The method of claim 1 , wherein the factors are encoded by nucleic acid obtained from at least one of a human animal and a non-human animal.
12 . The method of claim 1 , wherein the somatic cells are mammalian.
13 . The method of claim 1 , wherein the expressing step comprises induction of expression by exposure to an inducing agent.
14 . The method of claim 13 , wherein the inducing agent is doxycycline.
15 . The method of claim 1 , further comprising the step of maintaining the produced iCPC in a proliferative state by culturing the produced iCPC in a medium that comprises an activator of canonical Wnt signaling and, optionally, an activator of Jak/Stat signaling, each in an amount sufficient to maintain proliferating iCPC.
16 . The method of claim 15 , wherein the medium comprises the activator of canonical Wnt signaling and the activator of Jak/Stat signaling.
17 . The method of claim 15 , wherein the activator of canonical Wnt signaling is selected from the group consisting of 6-bromoindirubin-3′-oxime (BIO), CHIR 99021, CHIR 98014, BIO-acetoxime, LiCl, SB 216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.
18 . The method of claim 15 , wherein the activator of Jak/Stat signaling is selected from the group consisting of Leukemia Inhibitory Factor (LIF), L-2, IL-6, IL-11, leptin, and ciliary neurotrophic factor (CNTF).
19 . The method of claim 1 , wherein the iCPC are separated from the non-reprogrammed cells by at least one of cell sorting, splitting, or manual dissection.
20 . The method of claim 1 , wherein the somatic cell comprises a heterologous sequence for inducing expression of nucleic acids encoding the set of factors and wherein the expressing step comprises inducing the expression.
21 . The method of claim 20 , wherein inducing comprises exposing the somatic cell comprising the heterologous sequence to an inducing agent.
22 . The method of claim 21 , wherein the inducing agent is doxycycline.
23 . The method of claim 1 , wherein the somatic cell comprises a heterologous fluorescent marker that indicates a developmental cell stage characteristic of a cardiac progenitor cell.
24 . The method of claim 1 , wherein the somatic cell comprises a heterologous sequence for inducing expression of nucleic acids encoding the set of factors and comprises a heterologous sequence encoding a fluorescent marker that is expressed only in cells at a developmental stage characteristic of cardiac progenitor cells, and wherein the expressing step comprises inducing the expression such that the cell is reprogrammed to produce the iCPC, such that the fluorescent marker indicates the presence of a cell at the developmental stage characteristic of cardiac progenitor cells.
25 . The method of claim 24 , wherein inducing comprises exposing the somatic cell comprising the heterologous sequence to an inducing agent.
26 . The method of claim 25 , wherein the inducing agent is doxycycline.
27 . An in vitro population of induced cardiac progenitor cells produced according to the method of claim 1 .
28 . A culture comprising an in vitro population of induced cardiac progenitor cells produced according to the method of claim 1 .