Epithelial Stem Cells and Production and Uses thereof
AssigneeStemson Therapeutics Corp.
InventorsYulia Shamis, Antonella Pinto, Alexey Terskikh
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Abstract
Provided herein is method for producing epithelial stem cells (EPSCs) from human induced pluripotent stem cells. Provided herein are also methods for culturing the EPSCs, and methods of producing or growing organoids, hair follicles, interfollicular epidermis, or skin grafts using the EPSCs.
Claims59
A method of producing epithelial stem cells (EPSCs) from human induced pluripotent stem cells (hiPSCs), wherein the method comprises:
- (a) culturing hiPSCs in a medium comprising retinoic acid (RA);
- (b) culturing the cells of step (a) in a medium comprising RA, bone morphogenetic protein 4 (BMP4), and epidermal growth factor (EGF);
- (c) culturing the cells of step (b) in a medium comprising a keratinocyte serum-free medium, RA, BMP4, EGF, and ROCK inhibitor;
- and
- (d) culturing the cells of step (c) in a medium comprising a keratinocyte serum-free medium, BMP4, EGF, and Rock inhibitor, thereby producing EPSCs.
A method of producing epithelial stem cells (EPSCs) from human induced pluripotent stem cells (hiPSCs), wherein the method comprises:
- (a) culturing hiPSCs in a medium comprising retinoic acid (RA) for 2 days;
- (b) culturing the cells of step (a) in a medium comprising RA, bone morphogenetic protein 4 (BMP4), and epidermal growth factor (EGF) for 2 days;
- (c) culturing the cells of step (b) in a medium comprising RA, BMP4, EGF, and ROCK inhibitor for 2 days;
- and
- (d) culturing the cells of step (c) in a medium comprising BMP4, EGF, and Rock inhibitor for 2 days.
The method of any one of the preceding claims, wherein the medium of culturing step (a) does not include BMP4.
The method of any one of the preceding claims, wherein the medium of culturing step (a) further comprises a keratinocyte serum-free medium and a matrix comprising a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and human Collagen Type I (BMM/Coll1 matrix).
The method of any one of the preceding claims, wherein the medium of culturing step (b) further comprises the BMM/Coll1 matrix.
The method of any one of the preceding claims, wherein the medium of culturing step (c) further comprises human Collagen type I, fibronectin, and laminin-521 (Coll1/FN/LN521).
The method of any one of the preceding claims, wherein the medium of culturing step (d) further comprises Coll1/FN/LN521.
The method of any one of the preceding claims, wherein the medium used in culturing steps (a)-(d) is feeder-free and serum-free.
A method of producing epithelial stem cells (EPSCs) from human induced pluripotent stem cells (hiPSCs) comprising:
- differentiating hiPSCs into EPSCs using a feeder-free and serum-free system comprising retinoic acid (RA), bone morphogenetic protein 4 (BMP4), epidermal growth factor (EGF), and ROCK inhibitor.
The method of any one of the preceding claims, wherein the EPSCs express tumor protein p63 (TP63) and keratin 15 (KRT15).
The method of any one of the preceding claims, further comprising culturing the hiPSCs for one or more days with:
- i. a BMM/Coll1 matrix;
- and
- ii. a serum-free medium comprising basal medium, recombinant human basic fibroblast growth factor (rh bFGF), and recombinant human transforming growth factor β (rh TGFβ).
The method of any one of the preceding claims, wherein the method further comprises expanding the EPSCs by maintaining the cells on a feeder-free medium.
The method of the immediately preceding claim, wherein the expanding step is for 6 passages.
The method of the immediately preceding claim, wherein feeder medium further comprises ROCK inhibitor.
The method of the immediately preceding claim, wherein the keratinocyte growth medium comprises DMEM/F12, dipeptide L-alanine-L-glutamine, keratinocyte growth medium 2 (KG2) basal medium, fetal bovine serum (FBS), adenine, an effective amount of one or more antibiotics and/or antimycotics, hydrocortisone, cholera toxin (CT), epidermal growth factor (EGF), and insulin.
The method of any one of the preceding claims, wherein the EPSCs have increased expression of markers TP63 and KRT15 after/during expansion as compared to iPSC controls.
The method of any one of the proceeding claims, wherein the method further comprises culturing the EPSCs with a perturbagen.
The method of any one of the preceding claims, wherein the EPSC expresses at least two, three, four, or each of CD59, CD99, LEF1, CTNNB1, FZD1, LGR5, DCT, TCF4, and LGR6.
The method of any one of the preceding claims, wherein the EPSC expresses at least two, three, four, five, six, seven, eight, nine, or each of CD59, CD99, KRT15, KRT19, DKK3, FZD1, FZD2, CTNNB1, TGFB2, FST, DCN, SOX9, NFATC1, ITGA6, ITGB1, LEF1, TCF4, TP63, and LGR6.
An epithelial stem cell (EPSC) produced by the method of any one of the preceding claims.
The EPSC of the immediately preceding claim, wherein the EPSC expresses one or more of CD59, CD200, KRT15, KRT19, DKK3, FZD1, FZD2, CTNNB1, TCF4, LEF1, LGR6, TGFB2, SOX9, NFATC1, and DCN at an increased level relative to a naturally occurring EPSC, and/or one or more of ITGA6, ITGB1, FST, KRT14, and TP63 at a decreased level relative to a naturally occurring EPSC.
An EPSC differentiated from an induced pluripotent stem cell, wherein the EPSC expresses one or more of CD59, CD200, KRT15, KRT19, DKK3, FZD1, FZD2, CTNNB1, TCF4, LEF1, LGR6, TGFB2, and DCN at an increased level relative to a naturally occurring EPSC, and/or one or more of ITGA6, ITGB1, FST, KRT14, and TP63 at a decreased level relative to a naturally occurring EPSC.
The organoid of the immediately preceding claim, wherein the organoid is a folliculogenic organoid.
The method of the immediately preceding claim, further comprising piercing the dermal tissue with a needle prior to implanting the organoid.
Dependencies are inferred from the claim text.