Method for separating pigmented focuses from cell masses with non-pigmented regions around pigmented focuses

Abstract

The invention provides a method for separating pigmented lesion from a cell mass with a non-pigmented region around the pigmented lesion, also provides a method for differentiating pluripotent stem cells into retinal pigment epithelial cells, and also provides application of the retinal pigment epithelial cells obtained by the method in manufacturing medicines for treating or preventing retinal degenerative diseases.

Description

[Technical field]

[0001] The present invention relates to a method for separating a pigmented focus from a cell cluster with a non-pigmented area surrounding the pigmented focus, a method for differentiating pluripotent stem cells into retinal pigment epithelial cells, and use of the retinal pigment epithelial cells obtained by the method in the manufacture of drugs for treating or preventing retinal degenerative diseases. [Background technology]

[0002] Retinal degenerative diseases, such as age-related macular degeneration (AMD), are one of the main causes of blindness. They are closely related to the damage or loss of retinal pigment epithelium (RPE) cells, and there is currently no effective drug or surgical treatment. Under physiological conditions, human RPE cannot proliferate, and the missing RPE cannot be obtained through cell regeneration. Therefore, RPE cell transplantation based on human pluripotent stem cells is considered to be the most promising method to delay or cure retinal degenerative diseases.

[0003] Human pluripotent stem cells are differentiated to produce pigmented foci (PF), and RPE cells are obtained by picking, inoculating, passage, and amplifying the pigmented foci. Among them, the picking of the pigmented foci is crucial, which directly determines the purity of the RPE cells finally obtained. However, regarding the picking of the pigmented foci, most of the literature only involves the use of microsurgical blades and other auxiliary separation of the pigmented foci, and does not involve the separation effect and the purity of the obtained RPE; quite a few literatures do not even mention the method of separating the pigmented foci. Existing methods for separating pigmented foci, such as "Following formation, pigmented foci were excised mechanically using the tip of a glass Pasteur pipette and microsurgical blades. This approach was only practical when foci had reached at least 1 mm in diameter. During this procedure, every effort was made to dissect away surrounding, non-pigmented material prior to placement of pigmented foci onto 35 mm tissue culture dishes coated with growth factor reduced Matrigel." (Vugler, A., et al., Elucidating the phenomenon of hESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp Neurol, 2008.214(2):p.347-61), the operation is cumbersome, time-consuming and labor-intensive, and usually miscellaneous cells other than RPE cells are obtained at the same time. If these cells are used in clinical practice, it will not only affect the treatment effect, but also bring unknown risks. Therefore, how to develop a simple and efficient method for picking pigment foci to achieve high-purity RPE cells differentiated from human pluripotent stem cells is a technical problem that needs to be solved urgently. [Summary of the invention]

[0004] The present invention includes the following contents:

[0005] 1. A method for separating a pigmented focus from a cell mass with a non-pigmented area surrounding the pigmented focus, comprising:

[0006] (a) for a cell cluster with a non-pigmented area surrounding a pigmented focus differentiated from a pluripotent stem cell, a completely black area as the pigmented focus is drilled out from the cell cluster using a tissue biopsy device; and

[0007] (b) Pick up the pigment focus drilled in step (a).

[0008] 2. The method according to the preceding embodiment, wherein the tissue biopsy device is a skin tissue biopsy device.

[0009] 3. The method according to the preceding embodiment, wherein the skin tissue biopsy device is a manual skin tissue biopsy device or a mechanical skin tissue biopsy device.

[0010] 4. The method according to the preceding embodiment, wherein the manual skin tissue biopsy device comprises a drilling portion and a holding portion.

[0011] 5. The method according to the preceding embodiment, wherein the mechanical skin tissue biopsy device comprises a drilling portion, a holding portion, and a drilling control portion.

[0012] 6. The method according to the preceding embodiment, wherein the drilling control part controls the drilling action of the drilling part by means of a spring member.

[0013] 7. The method according to the preceding embodiment, wherein the drilling portion is cylindrical.

[0014] 8. The method according to the preceding embodiment, wherein the inner diameter of the drilling portion is 1 to 5 mm.

[0015] 9. The method according to the preceding embodiment, wherein the drilling part is made of stainless steel.

[0016] 10. The method according to the preceding embodiment, wherein the tissue biopsy device is disposable.

[0017] 11. A method for differentiating pluripotent stem cells into retinal pigment epithelial cells, comprising:

[0018] (i) Differentiating pluripotent stem cells into cell clusters with non-pigmented areas surrounding pigmented foci;

[0019] (ii) separating the pigmented foci from the cell clusters with non-pigmented areas surrounding the pigmented foci, comprising:

[0020] (a) for a cell cluster with a non-pigmented area surrounding a pigmented focus differentiated from a pluripotent stem cell, a completely black area as the pigmented focus is drilled out from the cell cluster using a tissue biopsy device; and

[0021] (b) picking up the pigment focus drilled in step (a); and

[0022] (iii) further differentiating the pigmented foci obtained in step (ii) into retinal pigment epithelial cells.

[0023] 12. The method according to the preceding embodiment, wherein the tissue biopsy device is a skin tissue biopsy device.

[0024] 13. The method according to the preceding embodiment, wherein the skin tissue biopsy device is a manual skin tissue biopsy device or a mechanical skin tissue biopsy device.

[0025] 14. The method according to the preceding embodiment, wherein the manual skin tissue biopsy device comprises a drilling portion and a holding portion.

[0026] 15. The method according to the preceding embodiment, wherein the mechanical skin tissue biopsy device comprises a drilling portion, a holding portion, and a drilling control portion.

[0027] 16. The method according to the preceding embodiment, wherein the drilling control part controls the drilling action of the drilling part by means of a spring member.

[0028] 17. The method according to the preceding embodiment, wherein the drilling portion is cylindrical.

[0029] 18. The method according to the preceding embodiment, wherein the inner diameter of the drilling portion is 1 to 5 mm.

[0030] 19. The method according to the preceding embodiment, wherein the drilling part is made of stainless steel.

[0031] 20. The method according to the preceding embodiment, wherein the tissue biopsy device is disposable.

[0032] 21. Use of the retinal pigment epithelial cells obtained by the method described in the above embodiments in the manufacture of a drug for treating or preventing retinal degenerative diseases.

[0033] 22. The use according to the preceding embodiment, wherein the retinal degenerative disease is selected from age-related macular degeneration and retinitis pigmentosa.

[0034]

Technical Effect

[0035] In the process of differentiating pluripotent stem cells into RPE cells, the pigment focus picking method of the present invention can be used to simply and quickly obtain the pigment focus, and then further differentiate to obtain high-purity RPE cells.

Brief Description of the Drawings

[0037]

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[0041]

Figure 5

[0042]

Figure 6

[0043] [Obtaining RPE cells]

[0044] As long as it can be transplanted to RPE injury patients (humans and other animals) and have a curative effect, the source of RPE cells is not particularly limited. In a preferred embodiment, RPE cells are differentiated from pluripotent stem cells. In a preferred embodiment, pluripotent stem cells are embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). In a preferred embodiment, ESC is mammalian ESC. In a preferred embodiment, mammals are selected from people, cats, dogs, monkeys, pigs, cattle, horses, goats, sheep, rabbits, mice, rats, etc.

[0045] The process of differentiating RPE cells from pluripotent stem cells generally includes: culturing pluripotent stem cells to a super-fusion state, differentiating in an RPE cell culture medium to obtain a cell cluster with a non-pigmented area surrounding the pigmented focus, separating the pigmented focus from the cell cluster with a non-pigmented area surrounding the pigmented focus (also referred to as "picking the pigmented focus" or "picking of the pigmented focus" in this application) and inoculating, and culturing in an RPE cell culture medium to obtain RPE cells.

[0046] [Differentiation of cell clusters with non-pigmented areas around pigmented foci from pluripotent stem cells]

[0047] As long as the pluripotent stem cells can be differentiated to obtain cell clusters with non-pigmented areas around the pigmented foci, the composition of the culture medium, the frequency of culture fluid replacement and the length of culture are not particularly limited. In a preferred embodiment, the pluripotent stem cells are first cultured in a culture medium dedicated to the pluripotent stem cells (such as human embryonic stem cells (hESC)) or a universal culture medium to a super-fusion state, and then the culture medium is replaced with an RPE cell culture medium and continued to be cultured until differentiation obtains cell clusters with non-pigmented areas around the pigmented foci. In a preferred embodiment, the culture medium is regularly replaced with a fresh culture medium. In a preferred embodiment, the culture medium is replaced with a fresh culture medium every 1 to 3 days, preferably every 1 to 2 days, and more preferably every day. In a preferred embodiment, the culture time for obtaining cell clusters with non-pigmented areas around the pigmented foci differentiated from pluripotent stem cells is about 45 to 50 days.

[0048]

Pigmentation of pigment foci

[0049] In one embodiment, for a cell cluster with a non-pigmented area surrounding a pigmented focus differentiated from a pluripotent stem cell, a specific area is first drilled therefrom using a tissue biopsy device, and then the pigmented focus drilled as above is picked up. In a preferred embodiment, for a cell cluster with a non-pigmented area surrounding a pigmented focus differentiated from a pluripotent stem cell, a completely black area as a pigmented focus is first drilled therefrom using a tissue biopsy device, and then the pigmented focus drilled as above is picked up.

[0050] Picking out pigment foci in this way is very accurate and quick, which not only saves time and effort, but also greatly helps to keep the cells in the pigment foci in the best possible state, thereby significantly improving the yield rate of their further applications, and is especially suitable for obtaining high-purity RPE cells through further differentiation.

[0051]

Biopsy Punch

[0052] As long as the target pigment foci can be accurately drilled, there is no particular limitation on the tissue biopsy device used to drill the pigment foci. In a preferred embodiment, the tissue biopsy device is a skin tissue biopsy device. In a preferred embodiment, the skin tissue biopsy device is a manual skin tissue biopsy device or a mechanical skin tissue biopsy device. In a preferred embodiment, the manual skin tissue biopsy device includes a drilling portion and a holding portion. In a preferred embodiment, the mechanical skin tissue biopsy device includes a drilling portion, a holding portion and a drilling control portion. In a preferred embodiment, the drilling control portion controls the drilling action of the drilling portion by a spring component. In a preferred embodiment, the drilling portion is cylindrical. In a preferred embodiment, the drilling portion is made of stainless steel. In a preferred embodiment, the (skin) tissue biopsy device is disposable.

[0053] As long as the target pigment foci can be accurately drilled, the drilling volume of the drilling part of the skin tissue biopsy device is not particularly limited. In a preferred embodiment, the inner diameter of the cylindrical drilling part is about 1 to 5 mm, preferably about 1 to 4.5 mm, preferably about 1 to 4 mm, preferably about 1 to 3.5 mm, preferably about 1 to 3 mm, preferably about 1 to 2.5 mm, preferably about 1 to 2 mm, preferably about 1 to 1.5 mm, preferably 1 mm.

[0054]

Pigment spot picking device

[0055] As long as the drilled pigment lesion can be easily picked up, the device for picking up the pigment lesion is not particularly limited. In a preferred embodiment, the pigment lesion picking device is ophthalmic forceps.

[0056] Differentiation of RPE cells from the selected pigment foci

[0057] As long as the pigmented foci picked can be differentiated into RPE cells, the composition of culture medium, inoculation density and culture fluid replacement frequency are not particularly limited. In a preferred embodiment, the pigmented foci picked are inoculated into, for example, 6-well culture plates with 10 to 30 pigmented foci / well, preferably 15 to 25 pigmented foci / well, more preferably 20 pigmented foci / well, and are cultivated in RPE cell culture medium for about 15 to 25 days, preferably about 18 to 22 days, more preferably about 20 days and obtain RPE cells. In a preferred embodiment, every 1 to 3 days, preferably every 1 to 2 days, more preferably every day, culture medium is replaced with fresh culture medium.

[0058]

Identification of differentiated RPE cells

[0059] Any marker that can distinguish RPE cells from other cells can be used. In a preferred embodiment, the RPE cell-specific marker is selected from: ZO-1, RPE65, CRALBP and PMEL17. Any molecule that can specifically identify the above-mentioned RPE cell-specific marker can be used. In a preferred embodiment, the molecule that can specifically identify the above-mentioned RPE cell-specific marker is an antibody for the above-mentioned marker. In a preferred embodiment, the antibody for the above-mentioned marker is a monoclonal antibody. In one embodiment, the above-mentioned antibody can be a commercially available antibody or an antibody currently prepared or donated.

[0060] Any method capable of identifying differentiated RPE cells may be used. In a preferred embodiment, the method may be an immunofluorescence-based or flow cytometry-based method.

[0061]

Uses of differentiated RPE cells

[0062] Differentiated RPE cells can be used to treat or prevent diseases related to RPE damage. For example, by transplanting differentiated RPE cells to RPE damage patients, retinal degenerative diseases such as age-related macular degeneration (AMD), retinitis pigmentosa, etc. can be treated or prevented.

[0063] [Example]

[0064] 【1. Cell clusters with non-pigmented areas around pigmented foci were obtained by differentiation from human embryonic stem cells】

[0065] Human embryonic stem cells (hESC, Q-CTS-hESC-356, from the National Stem Cell Resource Bank) were cultured in CTS medium as a complete hESC medium. TM Essential 8 TM Culture medium (Cat.# A2656101, Gibco) was replaced with fresh culture medium every day at 37°C and 5% CO 2 The culture medium was replaced with RPE cell culture medium [containing CTS TM KnockOut TM DMEM (Cat. #A1286101, Gibco), 20% CTS TM KnockOut TM SR XenoFree Medium (Cat.#12618013, Gibco), 2 mM CTS TM GlutaMAX TM -I supplement (Cat.#A1286001, Gibco), 1×MEM non-essential amino acid solution (Cat.#11140050, Gibco) and 5μg / ml L-ascorbic acid (Cat.#A4403-100MG, Sigma-Aldrich)] and continue to culture, changing to fresh culture medium every day for the first 7 days, and then every 1 to 3 days. After about 20 days of culture, cell clusters with non-pigmented areas around the pigmented foci appear visible to the naked eye. Continue to culture for 25 to 30 days, and then the pigmented foci can be picked.

[0066] 【2. Picking out pigment foci】

[0067] 【2.1. Picking method 1】

[0068] For the cell clusters with non-pigmented areas around the pigmented foci differentiated from hESC, a disposable skin tissue biopsy device (Cat.#BPP-10F, KAI Medical) was used to drill out the completely black area as the pigmented foci, and then the drilled pigmented foci were picked up with ophthalmic forceps ( Figure 1 A).

[0069] 【2.2. Picking method 2】

[0070] For the cell clusters with non-pigmented areas around the pigmented foci differentiated from hESC, the pigmented foci were first cut off as much as possible with ophthalmic scissors, the surrounding non-pigmented areas were peeled off as much as possible, and then the pigmented foci were picked up with ophthalmic forceps in a manner similar to that described in Vugler, A., et al., Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp Neurol, 2008. 214(2): p. 347-61. Figure 1 B).

[0071] [2.3. Comparison of operating efficiency between picking method 1 and picking method 2]

[0072] A certain number of pigment foci were picked by picking method 1 and picking method 2 respectively, and the time taken was recorded. The comparison results of operation efficiency are shown in Table 1 below.

[0073] [Table 1: Comparison of operating efficiency of two picking methods]

[0074] Picking method Number of pigment foci Total time (min) Average time / pigment focus (min) Picking method 1 32 16 0.5 Picking method 2 35 70 2

[0075] The above results show that picking method 1 is significantly faster than picking method 2.

[0076] 【3. Differentiation of RPE cells from the selected pigment foci】

[0077] The pigment foci picked by picking method 1 and picking method 2 were inoculated into the CTS-coated wells at a rate of 20 pigment foci / well. TM Vitronectin (VTN-N) (Cat.# CTS279S3, Gibco) was added to 6-well culture plates and RPE cell culture medium [containing CTS TM KnockOut TM DMEM (Cat. #A1286101, Gibco), 20% CTS TM KnockOut TMSR XenoFree Medium (Cat.#12618013, Gibco), 2 mM CTS TM GlutaMAX TM -I supplement (Cat.#A1286001, Gibco), 1×MEM non-essential amino acid solution (Cat.#11140050, Gibco) and 5 μg / ml L-ascorbic acid (Cat.#A4403-100MG, Sigma-Aldrich)] were replaced with fresh medium every other day at 37°C and 5% CO 2 The culture was carried out under the conditions of . The microscopic photos (50×) on the 1st day and the 9th day after inoculation are shown in Figure 2 and 3 After about 20 days, a cobblestone-shaped monolayer of cells was obtained. The obtained cells were identified and tested for purity and function.

[0078] 【4. Identification of the obtained cells based on immunofluorescence method】

[0079] The cells differentiated from the pigmented foci picked by picking method 1 and picking method 2 were stained with Hoechst 33258 (Cat. # H3569, Invitrogen) and observed under a fluorescence microscope (400×). Figure 4 As shown in A, the cell nuclei are stained blue.

[0080] Mouse anti-human anti-ZO-1 monoclonal antibody (Cat.#33-9100, Invitrogen) was applied as the primary antibody to cells differentiated from pigment foci picked by picking method 1 and picking method 2, and Alexa Fluor 576-conjugated red fluorescent dye was further applied as the secondary antibody. TM Plus 555-conjugated goat anti-mouse IgG (H+L) Highly Cross-Adsorbed secondary antibody (Cat.#A32727, Invitrogen) was used for observation under a fluorescence microscope (400×). Figure 4 As shown in B, red fluorescence was observed, indicating that the cells differentiated from the pigmented foci picked by the two picking methods all expressed ZO-1, one of the RPE cell-specific molecular markers (ie, all were ZO-1 positive cells).

[0081] Rabbit anti-human anti-RPE65 polyclonal antibody (Cat.# GTX103472, GeneTex) was applied as the primary antibody to cells differentiated from pigment foci picked by picking method 1 and picking method 2, and Alexa Fluor 5764 was further applied as the secondary antibody in combination with green fluorescent dye. TM488-conjugated goat anti-rabbit IgG (H+L) Cross-Adsorbed secondary antibody (Cat.#A-11008, Invitrogen) was used for observation under a fluorescence microscope (400×). Figure 4 As shown in C, green fluorescence was observed, indicating that the cells differentiated from the pigmented foci picked by the two picking methods all expressed RPE65, one of the RPE cell-specific molecular markers (ie, all were RPE65-positive cells).

[0082] Figure 4 D is Figure 4 A. Figure 4 B and Figure 4 C The merged image.

[0083] Based on the fact that the obtained cells were double positive for ZO-1 and RPE65 (ZO-1 + RPE65 + ) results, it can be determined that the obtained cells are RPE cells.

[0084] 【5. Identification based on flow cytometry and purity detection of obtained cells】

[0085] For cells differentiated from pigment foci picked by picking method 1 and picking method 2,

[0086] The following primary antibodies were administered simultaneously:

[0087] Rabbit anti-human anti-CRALBP monoclonal antibody (Cat.# ab243664, Abcam), and

[0088] Mouse anti-human anti-PMEL17 monoclonal antibody (Cat.#MA1-34759, Invitrogen),

[0089] The following secondary antibodies were further administered simultaneously:

[0090] Donkey anti-rabbit IgG (minimal x-reactivity) coupled to phycoerythrin (PE) (Cat.# 406421, Biolegend), and

[0091] • Rat anti-mouse IgG1 conjugated to allocyanin (APC) (Cat. #406610, Biolegend).

[0092] The treated cells were passed through a flow cytometer (Cat. #CytoFLEX, Beckman).

[0093] from Figure 5As can be seen from the figure, the cells differentiated from the pigmented foci picked by the two picking methods all expressed CRALBP and PMEL17, which are RPE cell-specific molecular markers, that is, they were both CRALBP and PMEL17 double positive (CRALBP + PMEL17 + ) cells. It can also be determined that the obtained cells are RPE cells.

[0094] In addition, the purity of RPE cells differentiated from the pigmented foci picked by picking method 1 was 98.88% ( Figure 5 A), the purity of RPE cells differentiated from the pigmented foci picked by picking method 2 was 92.56% ( Figure 5 B), the former is significantly higher than the latter.

[0095] 【6. Detection of phagocytic function of obtained RPE cells】

[0096] The RPE cells differentiated from the pigmented foci picked by picking method 1 and picking method 2 were treated as follows:

[0097] · Application of latex yellow-green fluorescent microbeads (Cat.#L4655, Merck);

[0098] Mouse anti-human anti-ZO-1 monoclonal antibody (Cat.#33-9100, Invitrogen) was used as the primary antibody, and Alexa Fluor 5476 as a red fluorescent dye was further used as the secondary antibody. TM Plus555-conjugated goat anti-mouse IgG (H+L) Highly Cross-Adsorbed secondary antibody (Cat.#A32727, Invitrogen); and

[0099] • Cell nuclei were stained with Hoechst 33258 (Cat. # H3569, Invitrogen).

[0100] Observe under a fluorescence microscope (400×). Figure 6 It can be seen that the RPE cells differentiated from the pigment foci picked by the two picking methods have the ability to phagocytize fluorescent microbeads.

Claims

1. A method for separating a pigmented focus from a cell mass with a non-pigmented area surrounding the pigmented focus, comprising: (a) for a cell cluster with a non-pigmented area surrounding a pigmented focus differentiated from a pluripotent stem cell, a completely black area as the pigmented focus is drilled out from the cell cluster using a tissue biopsy device; and (b) Pick up the pigment focus drilled in step (a).

2. The method of claim 1, wherein the tissue biopsy device is a skin tissue biopsy device.

3. The method according to claim 2, wherein the skin tissue biopsy device is a manual skin tissue biopsy device or a mechanical skin tissue biopsy device. The method according to claim 3 , wherein the manual skin tissue biopsy device comprises a drilling portion and a holding portion. 5 . The method according to claim 3 , wherein the mechanical skin tissue biopsy device comprises a drilling portion, a holding portion, and a drilling control portion. 6 . The method according to claim 5 , wherein the drilling control part controls the drilling action of the drilling part by a spring member.

7. The method according to any one of claims 4 to 6, wherein the drilling portion is cylindrical.

8. The method according to claim 7, wherein the inner diameter of the drilling portion is 1 to 5 mm.

9. The method according to any one of claims 4 to 6, wherein the drilling part is made of stainless steel.

10. The method of claim 1, wherein the tissue biopsy device is single-use.

11. A method for differentiating pluripotent stem cells into retinal pigment epithelial cells, comprising: (i) Differentiating pluripotent stem cells into cell clusters with non-pigmented areas surrounding pigmented foci; (ii) separating the pigmented foci from the cell clusters with non-pigmented areas surrounding the pigmented foci, comprising: (a) for a cell cluster with a non-pigmented area surrounding a pigmented focus differentiated from a pluripotent stem cell, a completely black area as the pigmented focus is drilled out from the cell cluster using a tissue biopsy device; and (b) picking up the pigment focus drilled in step (a); and (iii) further differentiating the pigmented foci obtained in step (ii) into retinal pigment epithelial cells.

12. The method of claim 11, wherein the tissue biopsy device is a skin tissue biopsy device.

13. The method according to claim 12, wherein the skin tissue biopsy device is a manual skin tissue biopsy device or a mechanical skin tissue biopsy device.

14. The method of claim 13, wherein the manual skin tissue biopsy device comprises a drilling portion and a holding portion.

15. The method according to claim 13, wherein the mechanical skin tissue biopsy device comprises a drilling portion, a holding portion and a drilling control portion. 16 . The method according to claim 15 , wherein the drilling control part controls the drilling action of the drilling part by a spring member.

17. The method according to any one of claims 14 to 16, wherein the drilling portion is cylindrical.

18. The method according to claim 17, wherein the inner diameter of the drilling portion is 1 to 5 mm.

19. The method according to any one of claims 14 to 16, wherein the drilling portion is made of stainless steel.

20. The method of claim 1, wherein the tissue biopsy device is single-use.

21. Use of the retinal pigment epithelial cells obtained according to the method according to any one of claims 11 to 20 in the manufacture of a drug for treating or preventing retinal degenerative diseases.

22. The use according to claim 21, wherein the retinal degenerative disease is selected from age-related macular degeneration and retinitis pigmentosa.

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