US 11,987,808 B2
Method of human periferic sensory neurons differenciation from human stem cells and uses thereof
Rodrigo De Vecchi, Rio de Janeiro (BR); Stevens Kastrup Rehen, Rio de Janeiro (BR); Marilia Zaluar, Rio de Janeiro (BR); and Lionel Breton, Aulnay-sous-bois (FR)
Assigned to L'OREAL, Paris (FR)
Appl. No. 16/624,663
Filed by L'OREAL, Paris (FR)
PCT Filed Jun. 20, 2017, PCT No. PCT/BR2017/050157
§ 371(c)(1), (2) Date Dec. 19, 2019,
PCT Pub. No. WO2018/232476, PCT Pub. Date Dec. 27, 2018.
Prior Publication US 2020/0216803 A1, Jul. 9, 2020
Int. Cl. C12N 5/0793 (2010.01)
CPC C12N 5/062 (2013.01) [C12N 2500/38 (2013.01); C12N 2501/01 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2501/13 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2502/094 (2013.01); C12N 2506/45 (2013.01)] 10 Claims
 
1. A method for inducing differentiation of human induced pluripotent stem cells to peripheral sensory neuronal (PSN) cells, comprising:
(a) the provision of:
(i) a cell culture comprising human induced pluripotent stem cells (HiPSCs);
(ii) a 3N medium;
(iii) Small Mothers Against Decapentaplegic (SMAD) pathway inhibitors LDN193189, SB431542, and CHIR;
(b) contacting said stem cells of (i) with (ii) and (iii) on a first culture vessel from 2 to 22 days in vitro to obtain primarily differentiated cells from the HiPSCs;
(c) contacting, in a second culture vessel, the primarily differentiated cells with 3N medium, supplemented with fibroblast growth factor (FGF-2) and epidermal growth factor (EGF), coated with polyornithine/laminin, wherein the primarily differentiated cells are maintained in said culture for up to 12 days;
(d) immediately following step (c), contacting the primarily differentiated cells with 3N medium, supplemented brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell line derived neurotrophic factor (GDNF), nerve growth factor (NGF), ascorbic acid, and cyclic adenosine monophosphate (cAMP), and maintaining the primarily differentiated cells from 5 to 25 days in culture to obtain secondary differentiated cells that are peripheral sensory neuronal (PSN) cells; and
(e) culturing the secondary differentiated PSN cells from 2 to 22 days in conditioned human epidermal keratinocytes neonatal (HEKn) medium.