Methods for forming three-dimensional human retinal tissue in vitro

Patent metadata

Field	Value
Publication number	US-2016333312-A1
Application number	US-201515111883-A
Country	US
Kind code	A1
Filing date	Jan 16, 2015
Priority date	Jan 16, 2014
Publication date	Nov 17, 2016
Grant date	—

Abstract

The present invention relates to the field of stem cells. More specifically, the invention provides methods and compositions useful for forming three-dimensional human retinal tissue in vitro. In a specific embodiment, an in vitro method for differentiating hiPSCs into three-dimensional retinal tissue comprising functional photoreceptors comprises the steps of (a) culturing the hiPSCs to form aggregates; (b) transitioning the aggregates into a neural induction medium; (c) seeding the aggregates on to extracellular matrix coated cell culture substrates; (d) replacing NIM with a chemically-defined differentiation medium; (e) detaching NR domains; (f) culturing in suspension; and (g) adding animal serum or plasma component and retinoic acid.

First claim

1 . An in vitro method for differentiating human induced pluripotent stem cells (hiPSCs) into three-dimensional retinal tissue comprising functional photoreceptors, the method comprising the steps of:
a. on day 0 of differentiation:
i. enzymatically detaching hiPSCs cultured on extracellular matrix-coated cell culture substrates with feeder-free cell culture medium, and
ii. culturing the hiPSCs in suspension to induce formation of aggregates;
b. during days 1-3 of differentiation, transitioning the aggregates into neural induction medium (NIM); c. during day 6 or 7, seeding the aggregates on to extracellular matrix-coated cell culture substrates; d. at any time between day 14 and day 17, replacing NIM with a chemically-defined differentiation medium; e. during the fourth week of differentiation:
i. detaching neural retina (NR) domains, and
ii. culturing in suspension;
f. during the fifth or sixth week of differentiation, adding animal serum or plasma component to promote cell survival; and g. at any time between week 5 and week 14, adding all-trans retinoic acid to induce photoreceptor maturation.

2 . The method of claim 1 , wherein the all-trans retinoic acid of step (g) is added at a concentration of about 1 μM.

3 . The method of claim 2 , wherein the all-trans retinoic acid of step (g) is added for a period of about 30 days.

4 . The method of claim 3 , further comprising the step of (h) decreasing the concentration of all-trans retinoic acid to about 0.5 μM.

5 . The method of claim 4 , wherein the all-trans retinoic acid is added at any time between week 9 and week 10 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between week 13 and week 14.

6 . The method of claim 4 , wherein the all-trans retinoic acid is added on day 63 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between day 90 and day 98.

7 . The method of claim 1 , wherein the all-trans retinoic acid is added at any time between week 5 and week 6 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between week 13 and week 14.

8 . The method of claim 1 , wherein the all-trans retinoic acid is added on day 42 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between day 90 and day 98.

9 . The method of claim 1 , wherein the enzymatic detachment of step (a)(i) is accomplished using dispase.

10 . The method of claim 1 , wherein the extracellular matrix is Matrigel™.

11 . The method of claim 1 , wherein the cell culture substrate is a flask, plate or petri dish.

12 . The method of claim 1 , wherein the feeder-free cell culture medium is mTeSR™ 1 medium.

13 . The method of claim 1 , wherein the NIM of step (b) comprises Dulbecco's modified eagle medium (DMEM)/F12 (1:1), 1% N2 supplement, lx minimum essential media-non essential amino acids (NEAA), and 2 μg ml −1 heparin.

14 . The method of claim 1 , wherein the chemically-defined differentiation medium of step (d) comprises DMEM/F12 (3:1), 2% B27 (without vitamin A), 1× minimum essential media-non essential amino acids (NEAA), and 1% antibiotic-antimycotic.

15 . The method of claim 1 , wherein the detachment of step (e) comprises manual detachment.

16 . The method of claim 1 , wherein the animal serum or plasma component is fetal bovine serum.

17 . The method of claim 1 , wherein the hiPSCs are selected from the group consisting of CB-iPSC6.2, KA.1 and IMR90-4.

18 . The three-dimensional retinal tissue produced by the method of claim 1 .

19 . An in vitro method for differentiating human induced pluripotent stem cells (hiPSCs) into three-dimensional retinal tissue comprising functional photoreceptors, the method comprising the steps of:
a. culturing the hiPSCs to form aggregates; b. transitioning the aggregates into a neural induction medium; c. seeding the aggregates on to extracellular matrix coated cell culture substrates; d. replacing NIM with a chemically-defined differentiation medium; e. detaching NR domains; f. culturing in suspension; and g. adding animal serum or plasma component and retinoic acid.

20 . The method of claim 19 , wherein the hiPSCs were cultured on extracellular matrix coated cell culture substrates with feeder-free cell culture medium and enzymatically detached prior to step (a).

21 . The method of claim 20 , wherein the extracellular matrix is Matrigel™.

22 . The method of claim 20 , wherein the feeder-free cell culture medium is mTeSR™ 1 medium.

23 . The method of claim 20 , wherein the cell culture substrate is a flask, plate or petri dish.

24 . The method of claim 20 , wherein the enzymatic detachment step is accomplished using dispase.

25 . The method of claim 19 , wherein step (b) is performed during days 1-3 of differentiation.

26 . The method of claim 19 , wherein the NIM of step (b) comprises Dulbecco's modified eagle medium (DMEM)/F12 (1:1), 1% N2 supplement, lx minimum essential media-non essential amino acids (NEAA), and 2 μg ml −1 heparin.

27 . The method of claim 19 , wherein step (c) is performed during day 6 or 7.

28 . The method of claim 19 , wherein step (d) is performed at any time between day 14 and day 17.

29 . The method of claim 19 , wherein the chemically-defined differentiation medium of step (d) comprises DMEM/F12 (3:1), 2% B27 (without vitamin A), 1× minimum essential media-non essential amino acids (NEAA), and 1% antibiotic-antimycotic.

30 . The method of claim 19 , wherein the detachment of step (e) comprises manual detachment.

31 . The method of claim 19 , wherein the animal serum or plasma component is fetal bovine serum.

32 . The method of claim 19 , wherein the all-trans retinoic acid of step (g) is added at a concentration of about 1 μM.

33 . The method of claim 32 , wherein the all-trans retinoic acid of step (g) is added for a period of about 30 days.

34 . The method of claim 33 , further comprising the step of (h) decreasing the concentration of all-trans retinoic acid to about 0.5 μM.

35 . The method of claim 34 , wherein the all-trans retinoic acid is added at any time between week 9 and week 10 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between week 13 and week 14.

36 . The method of claim 34 , wherein the all-trans retinoic acid is added on day 63 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between day 90 and day 98.

37 . The method of claim 19 , wherein the all-trans retinoic acid is added at any time between week 5 and week 6 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between week 13 and week 14.

38 . The method of claim 19 , wherein the all-trans retinoic acid is added on day 42 at a concentration of about 1 μM and then decreased to about 0.5 μM at any time between day 90 and day 98.

39 . The method of claim 19 , wherein the hiPSCs are selected from the group consisting of CB-iPSC6.2, KA.1 and IMR90-4.

40 . The three-dimensional retinal tissue produced by the method of claim 19 .

41 . An in vitro method

Assignees

• Univ Johns Hopkins

Inventors

• Canto-Soler Maria Valeria
• Zhong Xiufeng

Classifications

• C12N2501/385

  of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR] · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N5/062 Primary

  Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain · CPC title

• C12N2533/90

  Substrates of biological origin, e.g. extracellular matrix, decellularised tissue · CPC title

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title