Serum-free induction method for sensory neuron cell

Abstract

Provided is a human-derived sensory neuron induction culture system. A combination of a small molecule inhibitor LY2157299 and a growth factor is added into a serum-free basal medium. Compared with an induction method involving serum, not only is the efficiency of inducing pluripotent stem cells into sensory neurons greatly improved, but the expression of a variety of ion channel proteins is also significantly improved, thereby achieving successful induction of multiple induced pluripotent stem cells from different sources into sensory neurons.

1 . Use of LY2157299, RepSox, SB5253334 or TEW7179 in the induction of pluripotent stem cells to differentiate into sensory nerve precursor cells and sensory neuron cells. 
     
     
         2 . The use of  claim 1 , wherein the dosage of LY2157299, RepSox, SB5253334 or TEW7179 is 55 nM-25 μM, preferably 100 nM, 300 nM, 500 nM, 700 nM, 900 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM or 22 μM. 
     
     
         3 . Use of human-NT3 or CHIR98014 in culturing sensory neurons. 
     
     
         4 . The use of  claim 3 , wherein the dosage of human-NT3 is 0.1-49 ng/ml, or the dosage of CHIR98014 is 0.5 nM-3 μM. 
     
     
         5 . A sensory neuron cell induction medium, characterized in that the sensory neuron cell induction medium is prepared as follows: a basal medium is formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then the basal medium is supplemented with 0.5%-2% (v/v) N2 supplement (preferably 0.7%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7% (v/v)), 0.2%-3.1% (v/v) GS21 supplement or B27 supplement (preferably 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0% (v/v)), 0.1-15 mM HEPES buffer (preferably 1, 5, 10 mM), 5-22 mM glycine-glutamine monohydrate (preferably 10, 15, 20 mM), 12-180 nM LDN-193189 or LDN-193189 2HCl (preferably 20, 50, 70, 80, 90, 100, 110, 120, 150, 170 nM), and 55 nM-25 μM LY2157299, RepSox, SB5253334 or TEW7179 (preferably 100 nM, 300 nM, 500 nM, 700 nM, 900 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 20 μM or 22 μM). 
     
     
         6 . The sensory neuron cell induction medium of  claim 5 , characterized in that the sensory neuron cell induction medium is prepared as follows: a basal medium is formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then the basal medium is supplemented with 1% (v/v) N2 supplement, 2% (v/v) GS21 supplement or B27 supplement, 0.5 mM HEPES buffer, 20 mM glycine-glutamine monohydrate, 100 nM LDN-193189 or LDN-193189 2HCl, and LY2157299, RepSox, SB5253334 or TEW7179 selected from 5 μM, 7.5 μM or 12.5 μM. 
     
     
         7 . The sensory neuron cell induction medium of  claim 5 , wherein the induction medium is prepared as follows: a basal medium is formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then supplemented with 1% (v/v) N2 supplement, 2% (v/v) GS21 supplement or B27 supplement, 0.5 mM HEPES buffer, 20 mM glycine-glutamine monohydrate, 100 nM LDN-193189 or LDN-193189 2HCl, and LY2157299 selected from 5 μM, 7.5 μM or 12.5 μM; preferably, the sensory neuron cell induction medium is further supplemented with serum replacement (such as serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form. 
     
     
         8 . A sensory neuron cell medium, characterized in that the sensory neuron cell medium is prepared as follows: the sensory neuron cell induction medium of  claim 5  is supplemented with 3-10 μM SU5402 (preferably 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 3-10 μM DAPT (4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 1-3 μM CHIR99021 (preferably 1.3 μM, 1.5 μM, 1.7 μM, 1.9 μM, 2.0 μM, 2.2 μM, 2.5 μM, 2.7 μM) or 0.5 nM-3 μM CHIR98014 (preferably 5 nM, 50 nM, 100 nM, 200 nM, 300 mM, 500 mM, 700 mM, 900 mM, 1 μM, 1.4 μM, 1.8 μM, 2.0 μM, 2.5 μM, 2.7 μM), and 0.1-49 ng/ml human-NT3 (preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 ng/ml), preferably, the sensory neuron cell medium is further supplemented with serum replacement (e.g., serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form. 
     
     
         9 . A method for inducing pluripotent stem cells to differentiate into sensory neuron cells, characterized in that the pluripotent stem cells are cultured in the induction medium of  claim 5  to obtain sensory nerve precursor cells, and then the sensory nerve precursor cells are cultured in a sensory neuron cell medium of  claim 8  to obtain sensory neuron cells, preferably, the culture is performed in the presence of a basal membrane, more preferably, the basal membrane is composed of one or more of Matrigel (STEMCELL Technologies), Laminin and Vitronectin,
 wherein the sensory neuron cell medium is prepared as follows: the sensory neuron cell induction medium of  claim 5  is supplemented with 3-10 μM SU5402 (preferably 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 3-10 μM DAPT (4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 1-3 μM CHIR99021 (preferably 1.3 μM, 1.5 μM, 1.7 μM, 1.9 μM, 2.0 μM, 2.2 μM, 2.5 μM, 2.7 μN) or 0.5 nM-3 μM CHIR98014 (preferably 5 nM, 50 nM, 100 nM, 200 nM, 300 mM, 500 mM, 700 mM, 900 mM, 1 μM, 1.4 μM, 1.8 μM, 2.0 μM, 2.5 μM, 2.7 μM), and 0.1-49 ng/ml human-NT3 (preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 ng/ml), preferably, the sensory neuron cell medium is further supplemented with serum replacement (e.g., serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form. 
 
     
     
         10 . Sensory nerve precursor cells, which are prepared by culturing pluripotent stem cells by using the induction medium of  claim 5 . 
     
     
         11 . Sensory neuron cells, which are prepared by culturing the sensory nerve precursor cells of  claim 10  by using a sensory neuron cell medium,
 wherein the sensory neuron cell medium is prepared as follows: a sensory neuron cell induction medium is supplemented with 3-10 μM SU5402 (preferably 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 3-10 μM DAPT (4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 1-3 μM CHIR99021 (preferably 1.3 μM, 1.5 μM, 1.7 μM, 1.9 μM, 2.0 μM, 2.2 μM, 2.5 μM, 2.7 μM) or 0.5 nM-3 μM CHIR98014 (preferably 5 nM, 50 nM, 100 nM, 200 nM, 300 mM, 500 mM, 700 mM, 900 mM, 1 μM, 1.4 μM, 1.8 μM, 2.0 μM, 2.5 μM, 2.7 μM), and 0.1-49 ng/ml human-NT3 (preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 ng/ml), preferably, the sensory neuron cell medium is further supplemented with serum replacement (e.g., serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form; and 
 wherein the sensory neuron cell induction medium is prepared as follows: a basal medium is formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then the basal medium is supplemented with 0.5%-2% (v/v) N2 supplement (preferably 0.7%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7% (v/v)), 0.2%-3.1% (v/v) GS21 supplement or B27 supplement (preferably 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0% (v/v)), 0.1-15 mM HEPES buffer (preferably 1, 5, 10 mM), 5-22 mM glycine-glutamine monohydrate (preferably 10, 15, 20 mM), 12-180 nM LDN-193189 or LDN-193189 2HCl (preferably 20, 50, 70, 80, 90, 100, 110, 120, 150, 170 nM), and 55 nM-25 μM LY2157299, RepSox, SB5253334 or TEW7179 (preferably 100 nM, 300 nM, 500 nM, 700 nM, 900 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 20 μM or 22 μM. 
 
     
     
         12 . A method for inducing sensory neuron cells comprising, culturing pluripotent stem cells by the sensory neuron cell induction medium of  claim 5  to obtain sensory nerve precursor cells, and then culturing the sensory nerve precursor cells by a sensory neuron cell medium,
 wherein the sensory neuron cell medium is prepared as follows: the sensory neuron cell induction medium of  claim 5  is supplemented with 3-10 μM SU5402 (preferably 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 3-10 μM DAPT (4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 1-3 μM CHIR99021 (preferably 1.3 μM, 1.5 μM, 1.7 μM, 1.9 μM, 2.0 μM, 2.2 μM, 2.5 μM, 2.7 μM) or 0.5 nM-3 μM CHIR98014 (preferably 5 nM, 50 nM, 100 nM, 200 nM, 300 mM, 500 mM, 700 mM, 900 mM, 1 μM, 1.4 μM, 1.8 μM, 2.0 μM,  2 . 5 μM, 2.7 μM), and 0.1-49 ng/ml human-NT3 (preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 ng/ml), preferably, the sensory neuron cell medium is further supplemented with serum replacement (e.g., serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form. 
 
     
     
         13 . The method of  claim 9  or  12 , wherein the pluripotent stem cells are prepared from somatic cells by using a reprogramming medium, preferably, wherein the reprogramming medium consists of a DMEM-F12 basal medium and supplementary components, and the supplementary components include 60 μg/mL-180 μg/mL L-ascorbic acid, 5.3 μmol/L-74 μmol/L hydrocortisone, 3 ng/mL-89 ng/mL sodium selenite, 8 μmol/L-23 Optiferrin, 0.5 μmol/L-7.4 retinyl acetate, 40 ng/mL-60 ng/mL plant-derived recombinant human basic growth factor, 8 μg/mL-12 μg/mL IGF, 0.2 μmol/L-0.6 μg/mL A-83, 2 μmol/L-6 CHIR99021, and 100 μmol/L-450 μmol/L sodium butyrate. 
     
     
         14 . The method of  claim 13 , characterized in that the supplementary components include 70 μg/mL-90 μg/mL L-ascorbic acid, 40 μmol/L-60 μmol/L hydrocortisone, 10 ng/mL-30 ng/mL sodium selenite, 8 μmol/L-12 μmol/L Optiferrin, 3 μmol/L-6 retinyl acetate, 45 ng/mL-55 ng/mL plant-derived recombinant human basic growth factor, 8 μg/mL-12 μg/mL IGF, 0.3 μmol/L-0.5 μg/mL A-83, 3 μmol/L-5 CHIR99021 and 280 μmol/L-410 μmol/L sodium butyrate. 
     
     
         15 . The method of  claim 13 , characterized in that the supplementary components comprise 80 μg/mL L-ascorbic acid, 50 μmol/L hydrocortisone, 20 ng/mL sodium selenite, 10 μmol/L Optiferrin, 4 μmol/L retinyl acetate, 50 ng/mL plant-derived recombinant human basic growth factor, 10 μg/mL IGF, 0.4 μg/mL A-83, 4 μmol/L CHIR99021 and 400 μmol/L sodium butyrate. 
     
     
         16 . The method of  claim 13 , wherein the culturing is performed in the presence of a basal membrane, preferably, the basal membrane is composed of one or more of Matrigel (STEMCELL Technologies), Laminin and Vitronectin. 
     
     
         17 . The method of  claim 13 , wherein the somatic cells are human mesenchymal cells, human CD34+ cells or human fibroblasts (e.g. skin fibroblasts, lung fibroblasts, bladder fibroblasts, foreskin fibroblasts, uterine fibroblasts). 
     
     
         18 . Use of the sensory neuron of  claim 11  in screening a medicine for treating or alleviating pain or preparing a model for screening a medicine for treating or alleviating pain. 
     
     
         19 . The method of  claim 12 , wherein the pluripotent stem cells are prepared from somatic cells by using a reprogramming medium, preferably, wherein the reprogramming medium consists of a DMEM-F12 basal medium and supplementary components, and the supplementary components include 60 μg/mL-180 μg/mL L-ascorbic acid, 5.3 μmol/L-74 μmol/L hydrocortisone, 3 ng/mL-89 ng/mL sodium selenite, 8 μmol/L-23 Optiferrin, 0.5 μmol/L-7.4 retinyl acetate, 40 ng/mL-60 ng/mL plant-derived recombinant human basic growth factor, 8 μg/mL-12 μg/mL IGF, 0.2 μmol/L-0.6 μg/mL A-83, 2 μmol/L-6 CHIR99021, and 100 μmol/L-450 μmol/L sodium butyrate.