Methods for differentiating cells

Abstract

The invention provides a differentiation protocol to generate retinal pigmented epithelial cells (RPECs) for the in vitro differentiation of mammalian pluripotent stem cells to mature, functional retinal pigment epithelial cells (RPEC). A culture system is described that uses defined medium comprising xeno-free optimal combinations of small molecule agonists and antagonists where: (a) pluripotent stem cells are cultured in the absence of feeder cells, serum, or conditioned medium; (b) defined culture conditions is used to obtain an eye-field cell (EFC) population; (c) whereby EFC are subsequently differentiated to RPEC when exposed to culture medium containing small molecule agonists thus producing highly homogenous RPEC; and (d) cultured under optimal conditions until maturation, through stimulation via activin-like signalling through addition of either cyclic AMP or cyclic AMP inducers to produce complete monolayer formation within 21+7 days. An alternate differentiation procedure also takes EFC intermediates prepared above into photoreceptors.

1 . An in vitro method for the rapid and efficient generation of mammalian eye field progenitor cells (EFPCs) comprising: (a) culturing mammalian SCs under defined, feeder-free, and/or xeno-free culture conditions, and (b) culturing said mammalian SCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian EFPCs. 
     
     
         2 . An in vitro method for the rapid and efficient generation of mammalian eye field progenitor cells (EFPCs) comprising: (a) culturing mammalian PSCs under defined, feeder-free, and/or xeno-free culture conditions, and (b) culturing said mammalian PSCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian EFPCs. In one embodiment, the PSCs are ESCs or IPSCs. 
     
     
         3 . An in vitro method for the rapid and efficient generation of mammalian retinal pigment epithelial cells (RPECs) from mammalian SCs comprising: (a) culturing said mammalian SCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said mammalian SCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian EFPCs, and (c) culturing said mammalian EFPCs in the presence of agonists of one or more of Activin-A like and WNT signalling agonists, under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian SC-derived RPECs. 
     
     
         4 . An in vitro method for the rapid and efficient generation of mammalian retinal pigment epithelial cells (RPECs) from mammalian PSCs comprising: (a) culturing said mammalian PSCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said mammalian PSCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian EFPCs, and (c) culturing said mammalian EFPCs in the presence of agonists of one or more of Activin-A like and WNT signalling agonists, under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian PSC-derived RPECs. In one embodiment, the PSCs are ESCs or IPSCs. 
     
     
         5 . An in vitro method for the rapid and efficient generation of mammalian photoreceptors (PhRs) from mammalian SCs comprising: (a) culturing mammalian SCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said mammalian SCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian EFPCs, (c) culturing said mammalian EFPCs in the presence of one or more antagonists of one or more of Notch, Wnt, CKI, and activators of Retinoic Acid (RA) and Hedgehog signalling, whereby the culturing of said EFPCs is towards photoreceptor progenitor cells and (d) culturing the cells generated from step (c) in the presence of one or more agonists of RA and Hedgehog signalling, and taurine, to generate mammalian SC-derived PhRs. 
     
     
         6 . An in vitro method for the rapid and efficient generation of mammalian PhRs from mammalian PSCs comprising: (a) culturing said mammalian PSCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said mammalian PSCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to mammalian EFPCs, (c) culturing said mammalian EFPCs in the presence of antagonists of one or more of Notch, Wnt, CKI signalling, and activators of RA and Hedgehog signalling, towards photoreceptor progenitors and (d) culturing the cells generated from step (c) in the presence of one or more agonists of RA and Hedgehog signalling, and taurine, to generate mammalian PSC-derived PhRs. In one embodiment, the PSCs are ESCs or IPSCs. 
     
     
         7 . An in vitro method for the rapid and efficient generation of human eye field progenitor cells (hEFPCs) comprising: (a) culturing hSCs under defined, feeder-free, and/or xeno-free culture conditions, and (b) culturing said hSCs in the presence of one or more antagonists of each pf the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to hSC-derived EFPCs. 
     
     
         8 . An in vitro method for the rapid and efficient generation of human eye field progenitor cells (hEFPCs) comprising: (a) culturing hPSCs (for instance, hESCs or hIPSCs) under defined, feeder-free, and/or xeno-free culture conditions, and (b) culturing said hPSCs in the presence of one or more antagonists of each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to hPSC-derived EFPCs. 
     
     
         9 . An in vitro method for the rapid and efficient generation of human eye field progenitor cells (hEFPs) comprising: (a) culturing human embryonic stem cells (hESCs) under defined, feeder-free, and/or xeno-free culture conditions, and (b) culturing said hESCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to EFPCs. 
     
     
         10 . An in vitro method for the rapid and efficient generation of human eye field progenitor cells (hEFPs) comprising: (a) culturing human induced pluripotent stem cells (hIPSCs) under defined, feeder-free, and/or xeno-free culture conditions, and (b) culturing said hIPSCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to EFPCs. 
     
     
         11 . An in vitro method for the rapid and efficient generation of human retinal pigment epithelial cells (RPECs) from hPSCs comprising: (a) culturing hPSCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said hPSCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to human EFPCs, and (c) culturing said human EFPCs in the presence of one or more agonists from the family of Activin-A like agonists, WNT signalling agonists, or a combination thereof, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to human RPECs. 
     
     
         12 . An in vitro method for the rapid and efficient generation of human retinal pigment epithelial cells (RPECs) from hESCs comprising: (a) culturing hESCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said hESCs in the presence of more of more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to human EFPCs, and (c) culturing said human EFPCs in the presence of one or more agonists from the family of Activin-A like agonists or WNT signalling agonists, or a combination thereof, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to human RPECs. 
     
     
         13 . An in vitro method for the rapid and efficient generation of human retinal pigment epithelial cells (RPECs) from human IPSCs (hIPSCs) comprising: (a) culturing hIPSCs under defined, feeder-free, and/or xeno-free culture conditions, (b) culturing said hIPSCs in the presence of one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to human EFPCs, and (c) culturing said human EFPCs in the presence of one or more agonists from the family of Activin-A like agonists, or WNT signalling agonists, or a combination thereof, and under defined, feeder-free, and/or xeno-free culture conditions, such that they differentiate to human RPECs. 
     
     
         14 . The method according to any one of  claims 1  to  13  wherein the one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways is at least SB and either LDN or CKI. 
     
     
         15 . The method according to  claim 14 , wherein the one or more antagonists to each of the TGF-β, BMP and WNT signalling pathways are SB, LDN and CKI. 
     
     
         16 . The method according to  claim 15 , wherein CKI is CKI-7. 
     
     
         17 . An in vitro method for the generation of RPECs from PSCs comprises:
 (a) providing a cell culture of PSCs;   (b) culturing the PSCs within said cell culture in a culture system comprising base media supplemented with at least SB and either LDN or CKI, but in the absence of exogenous growth factors or animal-derived sera, wherein the stem cells are differentiated to EFPCs;   (c) culturing said EFPCs in a suitable culture system comprising a base media supplemented with at least IDE or CHIR, or both;   (d) dissociating the differentiated cells obtained from step (c) into single cells and seeding said cells in a suitable culture system comprising a suitable growth surface and base media to obtain PSC-derived RPE cells; and   (e) expanding the PSC-derived RPE cells in the absence of exogenous growth factors.   
     
     
         18 . The method according to  claim 17 , wherein the culture system in step (b) comprises base media that is supplemented with at least SB, LDN and CKI. 
     
     
         19 . The method according to  claim 17 , wherein the base media is further supplemented with nicotinamide (NIC) and/or iROCK. 
     
     
         20 . The method according to  claim 17 , wherein the base media defined in step (c) is further supplemented with iROCK. 
     
     
         21 . The method according to  claim 17 , wherein the base media defined in (d) is supplemented by one or both of iROCK and IDE. 
     
     
         22 . The method according to  claim 17 , further comprising the following step: (f) maturing the PSC-RPEs in a media to achieve a terminally differentiated functionality. 
     
     
         23 . The method according to any one of  claims 1  to  13  or  17 , wherein the method does not involve use of conditioned medium. 
     
     
         24 . The method according to any one of  claims 1  to  13  or  17 , wherein one or more of the following supplements is used in said method: forskolin, rolipram, and SU5402 which is 2-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propanoic acid, or any derivate or substitute of the components described herein, or any combination thereof. 
     
     
         25 . The method according to any one of  claims 17  to  22 , wherein the time to complete step (b) is about 6 days+/−3 days. 
     
     
         26 . The method according to any one of  claims 17  to  22  and  25 , wherein the time to complete step (c) is about 7 days+/−3 days. 
     
     
         27 . The method according to any one of  claims 17  to  22 ,  25  and  26 , wherein the time to complete steps (a), (b), (c), and (d) is about 14 days+/−7 days. 
     
     
         28 . A kit comprising one or more antagonists as defined in any one of the methods according to  claims 1  to  13  or  17 . 
     
     
         29 . The kit of  claim 28  wherein the one or more antagonists is to each of the TGF-β, BMP and WNT signalling pathways. 
     
     
         30 . The kit of  claim 28 , comprising one or more agonists of the Activin-A like agonists, or WNT signalling agonists, or a combination thereof. 
     
     
         31 . The kit of  claim 28  further comprising one or more antagonists of one or more of Notch, Wnt, CKI, and activators of Retinoic Acid (RA) and Hedgehog signalling. 
     
     
         31 . The kit of  claim 29  wherein the one or more antagonists is at least SB and either LDN or CKI. 
     
     
         32 . The kit of  claim 31  wherein the one or more antagonists are SB, LDN and CKI. 
     
     
         33 . The kit of  claim 31  wherein CKI is CKI-7. 
     
     
         34 . The kit of  claim 28  further comprising IDE or CHIR, or both. 
     
     
         35 . The kit of  claim 28  further comprising nicotinamide (NIC) and/or iROCK and/or Pur. 
     
     
         36 . The kit of  claim 28  further comprising one or more of the following supplements: forskolin, rolipram, and SU5402 which is 2-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propanoic acid, or any derivate or substitute of the components described herein, or any combination thereof. 
     
     
         37 . The kit of  claim 28 , containing instructions to perform the method according to any one of  claims 1  to  13  or  17 .