COMPOSITIONS AND PROCESS FOR INTEGRATING CELLS INTO EPITHELIUM

Abstract

The invention provides a combination of compositions comprising in a first composition 17β-estradiol as the active ingredient and, as a second composition, a suspension of cells for use in the treatment of functional defects of an epithelium, e.g. of an epithelium of a tissue, which tissue may be part of an organ.

Claims

1. Combination of compositions for use in the treatment of epithelium, comprising a first composition containing 17β-estradiol as the active ingredient, and a second composition comprising a suspension of cells, wherein the second composition is for contacting the epithelium at a time after the contact with the first composition.

2. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the epithelium is an endothelium contained in a tissue or organ that is e.g. selected from lung, a blood vessel, kidney or urinary tract.

3. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the second composition is for contacting the epithelium separate from the first composition contacting the epithelium.

4. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the first composition and the second composition are for contacting the epithelium by perfusing, flushing, or contacting with an aerosol the tissue or organ containing the epithelium separate from the blood circulation of a patient from whom the tissue or organ containing the epithelium originates.

5. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the first composition contains 17β-estradiol in a concentration suitable for adjusting the concentration of 17β-estradiol in a medium contacting the epithelium to at least 1 μM 17β-estradiol.

6. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the first composition is for contacting the epithelium for 5 min to 3 h and the second composition is for subsequently contacting the epithelium for at least 15 min.

7. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the cells of the second composition are epithelial and/or endothelial cells or progenitor cells of epithelial and/or of endothelial cells, stem cells or progenitor cells, pluripotent stem cells (PSC), induced PSC (iPSC), homologous cells generated from a biopsy of the patient, and/or heterologous stem cells, in each case optionally with a genetic alteration introduced.

8. Combination of compositions for use in the treatment of epithelium according to claim 1, comprising a third composition which is a medium free from 17β-estradiol and free from cells, for contacting the tissue containing the epithelium subsequent to contacting the tissue containing the epithelium with the first composition and before contacting the tissue containing the epithelium with the second composition.

9. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the time after the contact with the first composition is at least 1 min.

10. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the epithelium is an endothelium.

11. Combination of compositions for use in the treatment of epithelium according to claim 1, wherein the epithelium is the epithelial and/or endothelial layer of a blood vessel, of a lung, of a kidney, or of the urinary tract.

12. Process for treatment of an extracorporeal epithelium or of intracorpareal epithelium, comprising contacting the epithelium with a first composition containing 17β-estradiol as the active ingredient, and after contacting the epithelium with the first composition, contacting the epithelium with a second composition comprising a suspension of cells.

13. Process according to claim 12, wherein the extracorporeal endothelium is comprised in an extracorporeal tissue or organ, which is located outside of the body of a patient and artificially perfusing the extracorporeal tissue or organ separate from the blood circulation of the patient.

14. Process according to claim 12, wherein the epithelium is contacted with the first composition for at least 5 min and is subsequently contacted with the second composition for at least 15 min.

15. Process according to claim 12, wherein subsequent to being contacted with the first composition and prior to being contacted with the second composition, the epithelium is contacted with a third composition that is free from 17β-estradiol and free from added cells.

16. Process according to claim 12, wherein the extracorporeal tissue or organ is isolated from a lung or is a lung, a portion of the respiratory tract including trachea, mouth and nose, a kidney, a part of the urinary tract, a part of the small intestine, colon, stomach, esophagus, bile duct, or pancreas.

17. Method of treatment of epithelium by the treatment of epithelium, comprising a first composition containing 17β-estradiol as the active ingredient, and a second composition comprising a suspension of cells, wherein the second composition is for contacting the epithelium at a time after the contact with the first composition

18. Method according to claim 17, wherein the epithelium is defective epithelium or defective endothelium.

19. Method according to claim 17, wherein the epithelium is located in or at a patient undergoing the treatment.

20. Method according to claim 17, wherein the epithelium is perfused separately from the blood circulation of the patient undergoing the treatment.

21. Method according to claim 17, wherein the first composition is for contacting the epithelium for at least 5 min up to at maximum 3 h, and the second composition is for subsequently contacting the epithelium for at least 15 min.

22. Method according to claim 17, wherein the epithelium is contacted with the first composition and at a time after the contact with the first composition, the epithelium is contacted with the second composition, wherein the first composition and/or the second composition is contacted with the epithelium by perfusing, flushing, or contacting with an aerosol the tissue or organ while the epithelium is separate from the blood circulation of a patient from whom the tissue or organ containing the epithelium originates.

Description

[0027] The invention is also described in greater detail with reference to the figures, which show in

[0028] FIG. 1 microscopic pictures of monolayers of cultivated endothelial cells following contact with 17β-estradiol at different concentrations after 30 min, in column A in phase contrast and in column B after anti-VE-cadherin (green) and DAPI (blue) staining after 4 h regeneration in medium without 17β-estradiol subsequent to the incubation in the medium containing 17β-estradiol. C shows phase contrast, D shows anti-VE-cadherin and DAPI staining.

[0029] FIG. 2 shows microscopic pictures of monolayers, in column A after 30 min in presence of 17β-estradiol, and in column B subsequent to additional contacting with cells in suspension, Upper row: phase contrast; middle row: anti-VE-cadherin staining (red) and DAPI staining (blue); lower row: anti-VE-cadherin staining (red), DAPI staining (blue) and detection newly integrated eGFP expressing endothelial cells (green).

[0030] In the figures, the scale bar is 100 μm.

Example: Integration of Human Primary Venous Endothelial Cells into a Venous Endothelial Tissue

[0031] As an example for an endothelium, human primary venous endothelial cells were cultivated in EGM-medium under cell culture conditions until reaching confluence. Then, the medium was removed from this exemplary epithelium and replaced by fresh EGM-medium containing 17β-estradiol in a concentration of 1 μM, 10 μM, 30 μM, 100 μM, or 300 μM. The concentrations of 17β-estradiol are also indicated in the rows of FIG. 1. FIG. 1 shows microscopic pictures of monolayers of the cultivated endothelial cells following the contact with 17β-estradiol at the concentrations after 30 min, in column A in phase contrast and in column B after immunological staining for anti-VE-cadherin and DAPI staining. This shows that the endothelial layer of venous endothelial cells was loosened, respectively shows disintegration after presence of at least 10 μM, preferably at least 30 μM 17β-estradiol, and at 100 μM and 300 μM 17β-estradiol for 30 min.

[0032] Subsequent to the presence of the 17β-estradiol in medium, this first composition was removed and replaced by EGM-medium without added 17β-estradiol, with subsequent incubation for 4 h under cell culture conditions. The result is depicted in FIG. 1 in column C in phase contrast, and in column D after immunological staining for VE-cadherin as an essential component of adherens junctions between cells, and DAPI staining for nuclei. The results demonstrate that that after removal of 17β-estradiol a closed layer of the endothelial cells is re-established.

[0033] A confluent layer of human primary venous endothelial cells cultivated in EGM-medium was used as a representative for endothelium. This endothelial cell layer was contacted with a first composition containing 17β-estradiol in a concentration yielding 100 μM 17β-estradiol in fresh cell culture medium. After incubating this endothelial cell layer under cell culture conditions for 30 min in the presence of 100 μM 17β-estradiol, the medium was removed and replaced by a suspension of iPSC-derived endothelial cells that were genetically manipulated to constitutively express enhanced green fluorescent green protein (eGFP) in EGM-medium without added 17β-estradiol.

[0034] The results are depicted in FIG. 2, showing in column A that contacting the endothelial cell layer with 17β-estradiol within 30 min results in loosening of the cell layer and loss of VE-cadherin expression. The result of the subsequent 4 h of incubation of the endothelial cell layer with eGFP-labelled endothelial cells in suspension is depicted in FIG. 2 in the right column B. This result shows that during reestablishment of VE-cadherin expression and adherens junctions, eGFP-labelled endothelial cells of the cell suspension efficiently integrate between the original endothelial cells of the epithelium, which here is an endothelium, and form a closed endothelial cell layer comprising both endothelial cells of the original endothelium formed by the endothelial cell layer and eGFP-labelled cells of the cell suspension.