Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells

Abstract

Methods for differentiating human pluripotent stem cells to dorsal neuroectoderm progenitors and further to glial progenitor cells and oligodendrocyte progenitor cells (OPCs) using inhibitors of BMP signaling and MAPK/ERK signaling are provided. Also provided are cells and cellular compositions obtained by such methods, and uses of such cells. Further provided are methods and protocols for efficiently differentiating human pluripotent stem cells to OPCs in the absence of the ventralizing morphogen SHH or a SHH signaling activator. The methods of the present disclosure reproducibly produce dorsal neuroectoderm progenitor cells by day 7 of the differentiation process, glial progenitor cells by day 21 of the differentiation process and OPCs by day 42 of the differentiation process.

1 - 18 . (canceled) 
     
     
         19 . A differentiated cell population comprising paired box 6 (PAX6) positive dNPCs obtained according to a method comprising: 
 a) obtaining a culture of undifferentiated human pluripotent stem cells;   b) culturing the undifferentiated human pluripotent stem cells adherently in the presence of at least one inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), at least one inhibitor of bone morphogenetic protein (BMP) signaling and retinoic acid for a first time period, thereby inducing differentiation to neuroectoderm; and   c) culturing the cells from b) adherently in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) or a SHH signaling activator for a second time period, thereby obtaining dorsal neural progenitor cells.   
     
     
         20 . The differentiated cell population of  claim 19 , wherein the dNPCs express one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7) and activating protein 2 (AP2). 
     
     
         21 - 29 . (canceled) 
     
     
         30 . A differentiated cell population comprising OPCs obtained according to a method comprising:
 a) obtaining dorsal neural progenitor cells (dNPCs) according to a method comprising: 
 i) obtaining a culture of undifferentiated human pluripotent stem cells; 
 ii) culturing the undifferentiated human pluripotent stem cells adherently in the presence of at least one inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), at least one inhibitor of bone morphogenetic protein (BMP) signaling and retinoic acid for a first time period, thereby inducing differentiation to neuroectoderm; and 
 iii) culturing the cells from ii) adherently in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) or a SHH signaling activator for a second time period, thereby obtaining dorsal neural progenitor cells; 
   b) harvesting the cells from a), replating them on a substrate and culturing the cells adherently in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for a further time period, thereby expanding the neural progenitor cells; 
 c) harvesting the cells from b) and further culturing the cells as aggregates in suspension in the presence of bFGF and EGF for a further time period, until the cells have matured into dorsal glial progenitor cells; and 
   d) plating down the aggregates from c) on a substrate and culturing the cells adherently for a further time period in the presence of epidermal growth factor (EGF),   optionally splitting the cells from time to time, until the cells have matured into OPCs; wherein the OPCs express one or more markers selected from neural/glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3).   
     
     
         31 . The cell population of  claim 19 , wherein the cells are cryopreserved at a stage in the method and are subsequently thawed to continue with the method. 
     
     
         32 . The cell population of  claim 19 , wherein the human pluripotent stem cells are human embryonic stem cells (hESCs). 
     
     
         33 . The cell population of  claim 19 , wherein the human pluripotent stem cells are human induced pluripotent stem cells (hiPSCs). 
     
     
         34 . The cell population of  claim 19 , wherein the at least one inhibitor of MAPK/ERK is selected from the group consisting of PD0325901, AZD6244, GSK1120212, PD184352, and Cobimetinib. 
     
     
         35 . The cell population of  claim 34 , wherein the at least one inhibitor of MAPK/ERK is PD0325901. 
     
     
         36 . The cell population of  claim 19 , wherein the at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2). 
     
     
         37 . The cell population of  claim 19 , wherein the at least one inhibitor of BMP signaling is selected from the group consisting of Dorsomorphin, DMH-1, K02288, ML347, LDN193189, and Noggin protein. 
     
     
         38 . The cell population of  claim 37 , wherein the at least one inhibitor of BMP signaling is Dorsomorpin. 
     
     
         39 . The cell population of  claim 30 , wherein the adherent culturing is performed on a substrate selected from (i) a cell adhesion peptide and (ii) an extracellular matrix selected from laminin and vitronectin. 
     
     
         40 . The cell population of  claim 30 , wherein the adherent culturing is performed on recombinant human laminin-521. 
     
     
         41 . The cell population of  claim 30 , wherein the adherent culturing is performed on laminin-511 E8 fragment. 
     
     
         42 . The cell population of  claim 30 , wherein step c) is performed in dynamic suspension. 
     
     
         43 . The cell population of  claim 30 , wherein during step d), the media additionally comprises platelet-derived growth factor AA (PDGF-AA). 
     
     
         44 . The cell population of  claim 30 , wherein the human pluripotent stem cells are hESCs. 
     
     
         45 . The cell population of  claim 30 , wherein the human pluripotent stem cells are hiPSCs. 
     
     
         46 . The cell population of  claim 30 , wherein the OPCs are cryopreserved and ready for administration to a subject upon thawing. 
     
     
         47 . A pharmaceutical composition comprising a differentiated cell population according to  claim 19 , wherein the composition is formulated as a thaw and inject (TAI) composition. 
     
     
         48 . A method of formulating the cell population of  claim 19  for administration to a subject in need thereof, comprising cyropreserving the cell population in a medium suitable for storage and shipping, wherein the cryopreserved cells are ready to be administered by injection directly after thawing. 
     
     
         49 . The method of  claim 48 , wherein the medium is selected from 90% Human Serum/10% DMSO, Media 3 10% (CS10), Media 2 5% (CS5), Media 1 2% (CS2), Stem Cell Banker, PRIME XV® FREEZIS, HYPOTHERMASOL®, CSB, and Trehalose. 
     
     
         50 . A method for formulating the cell population of  claim 30  comprising cyropreserving the cell population in a medium suitable for storage and shipping, wherein the cryopreserved cells are ready to be administered by injection directly after thawing. 
     
     
         51 . The method of  claim 50 , wherein the medium is selected from adenosine, dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl) piperazine-N′- (2- ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), and water. 
     
     
         52 . The method of  claim 50  wherein cryopreservation occurs at or below -80° C. to -195° C. 
     
     
         53 . A method for treating spinal cord injury in a subject in need thereof comprising administering the composition of  claim 50  to the subject by injection subsequent to thawing, without further processing. 
     
     
         54 . The method of  claim 53 , wherein the concentration of live cells administered to the subject is 100,000,000 live cells per ml. 
     
     
         55 . The method of  claim 53 , wherein the volume of injection comprises 100 microliters.