Method for Differentiating Epithelial Stem Cells

Patent metadata

Field	Value
Publication number	US-2022340881-A1
Application number	US-202017632939-A
Country	US
Kind code	A1
Filing date	Aug 7, 2020
Priority date	Aug 9, 2019
Publication date	Oct 27, 2022
Grant date	—

Abstract

The subject matter of the present invention is a method for differentiating epithelial stem cells, comprising culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of an expansion medium, a bovine pituitary extract, a receptor tyrosine kinase ligand, a supernatant of primary fibroblasts and optionally, a Rho kinase inhibitor.

First claim

1 . A method for differentiating bladder epithelial stem cells, wherein said method comprises: culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of an expansion medium, the expansion medium comprising a basal medium for animal or human cells to which is added a bovine pituitary extract (BPE), a receptor tyrosine kinase ligand, a supernatant of bladder primary fibroblasts, and optionally, a Rho kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor).

2 . A method according to claim 1 , wherein, in the expansion medium, the receptor tyrosine kinase ligand is selected from the group consisting of FGF, HGF and EGF.

3 . A method according to claim 1 , wherein, in the expansion medium, the receptor tyrosine kinase ligand is EGF.

4 . The method according to claim 1 , wherein, in the expansion medium, the ROCK inhibitor is selected from the group consisting of Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, C21H16F4N4O2 or Y-30141.

5 . The method according to claim 1 , wherein, in the expansion medium, the ROCK inhibitor is Y-27632.

6 . The method according to claim 1 , wherein the supernatant of bladder primary fibroblasts is a supernatant of bladder primary fibroblast originating from human bladder tissue.

7 . The method according to claim 1 , wherein the epithelial stem cells are from human bladder.

8 . An expansion medium, comprising a basal medium for animal or human cells to which is added a bovine pituitary extract (BPE), a receptor tyrosine kinase ligand, a supernatant of bladder primary fibroblasts.

9 . The expansion medium of claim 8 , wherein the ROCK inhibitor is selected from the group consisting of Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, C21H16F4N4O2 or Y-30141.

10 . The expansion medium of claim 9 , wherein the ROCK inhibitor is Y-27632.

11 . The expansion medium of claim 8 , wherein the receptor tyrosine kinase ligand is selected from the group consisting of FGF, HGF and EGF or their combinations.

12 . The expansion medium of any one of claim 8 , wherein the supernatant of primary fibroblasts is a supernatant of bladder fibroblasts.

13 . (canceled)

14 . (canceled)

15 . (canceled)

16 . (canceled)

17 . (canceled)

18 . (canceled)

19 . (canceled)

20 . (canceled)

21 . (canceled)

22 . (canceled)

23 . A method of preparation of an organoid culture of human bladder tissue, in particular of preparation of an organoid culture of normal bladder tissue or of cancer bladder tissue, comprising the steps of:
a. Providing a suspension of cells comprising urothelial cells including epithelial adult stem cells originating from said human bladder tissue in a suspension medium which comprises a basal medium for culture of animal or human cells, a ROCK inhibitor and optionally bovine pituitary extract (BPE) and/or a receptor tyrosine kinase ligand selected among the group of EGF, FGF and HGF and wherein said suspension medium is devoid of supernatant of primary fibroblast and, b. Culturing the cells provided in step above (a.) in an extracellular matrix and an expansion medium suitable for differentiation of said human epithelial adult stem cells, in conditions allowing cell expansion and cell differentiation of the epithelial adult stem cells originating from said human bladder tissue until a three dimensional tissue is obtained, wherein such step of culturing encompasses one or multiple passages or the cells, advantageously 2, 3, 4, or 5 passages, in particular up to 10 or up to 15 passages, c. Allowing the three dimensional tissue obtained in step above (b.) to grow as an organoid culture and optionally performing cellular and/or molecular characterization of the obtained organoids, wherein the expansion medium comprises at least a basal medium for culture of animal or human cells and a supernatant of primary fibroblast and wherein the composition of the expansion medium is adjusted during cell culture step as disclosed in b. so that in the each passage of the cell culture the expansion medium initially additionally comprises bovine pituitary extract (BPE) and/or a receptor tyrosine kinase ligand selected among the group of EGF, FGF and HGF and, optionally a ROCK inhibitor, when the expansion medium in said each passage is changed or replenished it is devoid of the ROCK inhibitor, in particular the expansion medium is as defined in any one of claims 1 to 12 for replenishment during successive cell passages.

24 . The method according to claim 23 , wherein the organoid culture is prepared from a bladder tissue previously obtained from a patient bladder tumour wherein the method comprises the following additional steps before first step (a.) of claim 23 :
i. providing a sample of bladder tumour tissue of a patient and dissecting said tissue in order to recover urothelium separated from submucosa, and recovering a suspension enriched with urothelial cells in particular as a supernatant following agitation of the medium comprising the dissociated cells; ii. centrifugating the suspension enriched with urothelial cells of i. and recovering the centrifugation pellet that contains urothelial cells.

25 . The method according to claim 23 , wherein the organoid culture is prepared from a patient derived tumour xenograft (PDX) of a bladder and wherein the method comprises the following additional steps before first step (a.) of claim 23 :
i. Providing bladder tumour tissue previously obtained from the xenograft, dissecting said bladder tumour tissue into pieces that may allow cells to be in contact with a dissociation solution, in particular bladder tumour tissue pieces of about 5 mm3; ii. Optionally incubating the obtained bladder tumour tissue of i. with a stripping solution to break intercellular junctions between urothelial cells and submucosal cells and recovering a suspension enriched with urothelial cells in particular as a supernatant following agitation of the suspension comprising the dissociated cells; iii. centrifugating the suspension enriched with urothelial cells of ii. and recovering the centrifugation pellet that contains urothelial cells.

26 . The method according to claim 23 , wherein the organoid culture is prepared from a bladder tissue previously obtained from a healthy bladder tissue of a human subject wherein the method comprises the following additional steps before first step (a.) of claim 23 :
i. Providing bladder tissue of a human subject and dissecting said tissue in order to recover urothelium separated from submucosa, and optionally incubating the bladder tumour tissue with a stripping solution to break intercellular junctions between urothelial cells and submucosal cells and recovering a suspension enriched with urothelial cells in particular as a supernatant following agitation of the suspension comprising the dissociated cells; ii. centrifugating the suspension enriched with urothelial cells of i. and recovering the centrifugation pellet that contains urothelial cells.

27 . The method according to claim 23 wherein in step a. the expansion medium contains 80% to 40% of volume of a basal medium or of a basal medium supplemented with BPE and receptor tyrosine kinase ligand and receptor tyrosine kinase ligand in particular complete KSFM medium and 20% to 60% of volume of primary fibroblasts supernatant (PFS), and wherein in cu

Assignees

• Urosphere
• Institute National De La Sante Et De La Rech Medicale
• Univ De Toulouse Iii Paul Sabatier
• Ecole Nat Veterinaire De Toulouse
• Ct Hospitalier Universitaire Toulouse
• Institut National De Rech Pour Lagriculture Lalimentation Et Lenvironnement

Inventors

• Lluel Philippe
• Vergnolle Nathalie
• Game Xavier
• Rouget Celine
• Sébert Morgane

Classifications

• C12N2500/84

  from mammals · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• C12N2503/02

  Drug screening · CPC title

• C12N2501/12

  Hepatocyte growth factor [HGF] · CPC title

• C12N2506/25

  from renal cells, from cells of the urinary tract · CPC title