Methods for differentiating pluripotent cells

Abstract

Methods are provided, in some aspects, for differentiating pluripotent cells into midbrain dopaminergic (DA) neurons using a mono-SMAD inhibition or inhibition of SMAD signaling with only one SMAD inhibitor. In some embodiments, mono-SMAD inhibition utilizes a single inhibitor of bone morphogenic protein (BMP) for differentiating pluripotent cells into midbrain DA neurons.

What is claimed is: 
     
         1 . An in vitro method for preparing a cell composition comprising human cells that express both forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 (LMX1) (FOXA2 + /LMX1 +  cells) comprising culturing human pluripotent cells in the presence of the following signaling modulators:
 (a) a single inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, 
 (b) at least one activator of Sonic hedgehog (SHH) signaling, and 
 (c) at least one activator of wingless (Wnt) signaling; 
 
       and culturing said cells in the presence of said modulators for a period of time sufficient to provide a cell composition comprising said FOXA2 + /LMX1 +  cells. 
     
     
         2 . The method of  claim 1 , wherein the inhibitor of SMAD signaling is a BMP inhibitor. 
     
     
         3 . The method of  claim 2 , wherein the BMP inhibitor is LDN-193189, dorsomorphin, DMH-1, or noggin. 
     
     
         4 . The method of  claim 3 , wherein the BMP inhibitor is LDN-193189. 
     
     
         5 . The method of  claim 4 , wherein the LDN-193189 is present at a concentration of from about 0.2 μM to about 4 μM. 
     
     
         6 . The method of  claim 5 , wherein the LDN-193189 is present at a concentration of from about 1 μM to about 3 μM. 
     
     
         7 . The method of  claim 1 , wherein the SMAD signaling inhibitor is a TGFβ inhibitor. 
     
     
         8 . The method of  claim 7 , wherein the TGFβ inhibitor is SB431542. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the pluripotent cells are cultured with the inhibitor of SMAD on culture days 1-15, 1-16, or 1-17. 
     
     
         10 . The method of  claim 9 , wherein the pluripotent cells are cultured with the inhibitor of SMAD on culture days 1-17. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the pluripotent cells are cultured with the inhibitor of SMAD substantially continuously or on a daily basis for 15, 16, or 17 days. 
     
     
         12 . The method of  claim 11 , wherein the pluripotent cells are cultured with the inhibitor of SMAD substantially continuously or on a daily basis for 17 days. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the inhibitor of SMAD is present at a concentration of about 50-2000 or 50-500 nM. 
     
     
         14 . The method of  claim 13 , wherein the inhibitor of SMAD is present at a concentration of about 180-240 nM. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the method further comprises contacting the pluripotent cells with a MEK inhibitor. 
     
     
         16 . The method of  claim 15 , wherein the MEK inhibitor is PD0325901. 
     
     
         17 . The method of  claim 16 , where the PD0325901 is present at a concentration of about 0.25-2.5 μM. 
     
     
         18 . The method of any one of  claims 14 - 17 , wherein the MEK inhibitor is contacted to the pluripotent cells for about 1-3 days, or on days 1-3, 2-4, 3-5, or on days 1, 2, 3, 4, or 5, after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         19 . The method of  claim 18 , wherein the MEK inhibitor is contacted to the pluripotent cells from about 24 to about 48 hours after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         20 . The method of any one of  claims 15 - 19 , wherein the MEK inhibitor is contacted to the pluripotent cells on a daily or substantially continual basis for about 3-4 days beginning about 1-2 days after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         21 . The method of  claim 20 , wherein the MEK inhibitor is contacted to the pluripotent cells on days 2-5 or days 3-6 after initiation of contact with the inhibitor of SMAD signaling on day 1. 
     
     
         22 . The method of any one of  claims 1 - 19 , wherein the activator of Wnt signaling is a GSK3 inhibitor. 
     
     
         23 . The method of  claim 22 , wherein the GSK3 inhibitor is CHIR99021. 
     
     
         24 . The method of  claim 23 , wherein the CHIR99021 is present at a concentration of about 0.5-3 μM. 
     
     
         25 . The method of  claim 24 , wherein the CHIR99021 is present at a concentration of from greater than about 1.25 μM to about 2 μM. 
     
     
         26 . The method of  claim 23 , wherein the CHIR99021 is present at a concentration of about 4-7 μM on days 9-17 after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         27 . The method of any one of  claims 1 - 26 , wherein the activator of Wnt signaling is contacted to the pluripotent cells 1-3 days after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         28 . The method of  claim 27 , wherein the activator of Wnt signaling is contacted to the pluripotent cells within 24-48 hours after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         29 . The method of any one of  claims 1 - 28 , wherein the pluripotent cells are cultured with the activator of Wnt signaling substantially continuously or on a daily basis for 14, 15, or about 16 days. 
     
     
         30 . The method of any one of  claims 1 - 29 , wherein the activator of Wnt signaling is contacted to the pluripotent cells on days 2-17 after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         31 . The method of any one of  claims 1 - 29 , wherein the activator of SHH signaling is purmorphamine or C25II Shh. 
     
     
         32 . The method of  claim 31 , wherein the method further comprises contacting the pluripotent cells with two activators of SHH signaling. 
     
     
         33 . The method of  claim 32 , wherein the two activators of SHH signaling are purmorphamine and C25II Shh. 
     
     
         34 . The method of any one of  claims 1 - 33 , wherein the at least one activator of SHH signaling is contacted to the pluripotent cells on the same day as initiation of contact with the inhibitor of SMAD signaling or within 24-48 hours after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         35 . The method of  claim 34 , wherein the at least one activator of SHH signaling is contacted to the pluripotent cells on days 1-7 with or after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         36 . The method of any one of  claims 1 - 35 , wherein the method further comprises contacting the pluripotent cells with FGF-8. 
     
     
         37 . The method of  claim 36 , wherein the FGF-8 is not contacted to the pluripotent cells on the same day as the initiation of contact with the inhibitor of SMAD signaling. 
     
     
         38 . The method of any one of  claims 36 - 37 , wherein the FGF-8 is contacted with the pluripotent cells on days 9-17 or 11-17 after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         39 . The method of any one of  claims 36 - 38 , wherein the FGF-8 is present at a concentration of about 50-200 ng/mL. 
     
     
         40 . The method of any one of  claims 1 - 39 , wherein the pluripotent cells comprise an antibiotic resistance transgene under the control of a neuronal promoter. 
     
     
         41 . The method of any one of  claims 1 - 40 , wherein the method further comprises selecting for neural cells or midbrain DA neurons derived from the pluripotent cells by contacting cells with an antibiotic, a chemotherapeutic, a DNA crosslinker, a DNA synthesis inhibitors, or a mitotic inhibitor. 
     
     
         42 . The method of any one of  claims 1 - 40 , wherein the method further comprises contacting the pluripotent cells with an antibiotic or a chemotherapeutic. 
     
     
         43 . The method of any one of  claims 41 - 42 , wherein the chemotherapeutic is mitomycin C. 
     
     
         44 . The method of  claim 43 , wherein the mitomycin C is contacted with the pluripotent cells on days 27, 28, 28, and/or 29 after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         45 . The method of any one of  claims 41 - 42 , wherein the antibiotic is G418 (geneticin). 
     
     
         46 . The method of any one of  claims 1 - 45 , wherein the method further comprises culturing or incubating the pluripotent cells in a media comprising a ROCK inhibitor prior to initiation of contact with the inhibitor of SMAD signaling. 
     
     
         47 . The method of any one of  claims 1 - 46 , wherein the method further comprises contacting the pluripotent cells with blebbistatin. 
     
     
         48 . The method of any one of  claims 1 - 47 , wherein the blebbistatin is contacted with the cells on day 5 and day 17 of differentiation. 
     
     
         49 . The method of any one of  claim 1 - 6  or  9 - 48 , wherein at least 40% of cells differentiate and express both FOXA2 and LMX1. 
     
     
         50 . The method of  claim 49 , wherein at least 60% of cells differentiate and express both FOXA2 and LMX1. 
     
     
         51 . The method of  claim 50 , wherein at least 80% of cells differentiate and express both FOXA2 and LMX1. 
     
     
         52 . The method of  claim 50 , wherein at least 85% of cells differentiate and express both FOXA2 and LMX1. 
     
     
         53 . The method of any one of  claim 1 - 6  or  9 - 48 , wherein at least 50% of cells differentiate and express both FOXA2 and tyrosine hydroxylase (TH). 
     
     
         54 . The method of any one of  claim 53 , wherein at least 70% of cells differentiate and express both FOXA2 and tyrosine hydroxylase (TH). 
     
     
         55 . The method of any one of  claims 1 - 54 , wherein the pluripotent cells are human induced pluripotent stem (iPS) cells. 
     
     
         56 . The method of any one of  claims 1 - 55 , wherein the LMX1 is LIM homeobox transcription factor 1 alpha (LMX1A). 
     
     
         57 . The method of any one of  claims 49 - 56 , wherein the differentiated cells expressing FOXA2 and LMX1, or FOXA2 and TH, further express at least one marker selected from the group consisting of orthodenticle homeobox 2 (OTX2), nuclear receptor related 1 protein (NURR1), Neuron-specific class III beta-tubulin (Tuj1), TTF3, paired-like homeodomain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), and G-protein coupled, inwardly rectifying potassium channel (Kir3.2/GIRK2), CD142, DCSM1, CD63 and CD99. 
     
     
         58 . The method of any one of  claims 1 - 57 , wherein the method further comprises incubating human pluripotent cells in the presence of a DNase or an endonuclease. 
     
     
         59 . The method of  claim 58 , wherein the endonuclease is DNase I or Benzonase®. 
     
     
         60 . The method of  claim 59 , wherein the DNase I or Benzonase® is present at a concentration of about 100 U/mL. 
     
     
         61 . The method of any one of  claims 58 - 60 , wherein the human pluripotent cells are cultured in the presence of an endonuclease on at least one of days 4-6 after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         62 . The method of any one of  claims 58 - 60 , wherein the human pluripotent cells in the presence of an endonuclease on day 5 after initiation of contact with the inhibitor of SMAD signaling. 
     
     
         63 . A culture of midbrain dopaminergic (DA) neurons generated by the method of any one of  claims 1 - 57 . 
     
     
         64 . The culture of  claim 63 , where the culture is comprised in a container means. 
     
     
         65 . The culture of any one of  claims 63 - 64 , wherein the neurons are comprised in a pharmaceutical preparation. 
     
     
         66 . The culture of  claim 65 , wherein the pharmaceutical preparation is formulated for injection. 
     
     
         67 . A method of treating a disease in a mammalian subject comprising administering to the subject a therapeutically effective amount of the culture of any one of  claims 63 - 66 . 
     
     
         68 . The method of  claim 67 , wherein the mammalian subject is a human. 
     
     
         69 . The method of  claim 68 , wherein the disease is a disease of the central nervous system (CNS). 
     
     
         70 . The method of  claim 69 , wherein the disease is Parkinson's disease (PD) or a Parkinson-plus syndrome (PPS). 
     
     
         71 . The method of any one of  claims 67 - 70 , wherein the culture comprises dopaminergic neurons that are not fully differentiated or are at day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or day 17-24 of differentiation. 
     
     
         72 . The method of  claim 71 , wherein the culture comprises a hyaluronic acid matrix. 
     
     
         73 . A method of screening a test compound comprising:
 (a) contacting the test subject with FOXA2 + /LMX1A +  cells differentiated by the method of any one of  claims 1 - 57 , and   (b) measuring the function, physiology, or viability of the cells.   
     
     
         74 . The method of  claim 73 , wherein said measuring comprises testing for a toxicological response or altered electrophysiological responses of the cells. 
     
     
         75 . The method of any one of  claims 73 - 74 , wherein the cells are midbrain dopaminergic (DA) cells.