Preparation and Expansion Methods for Human Pluripotent Stem Cell-Derived Human Retinal Pigment Epithelial Cells

Abstract

Preparation and expansion methods for human pluripotent stem cell-derived human retinal pigment epithelial cells are provided. The preparation method includes the following steps: collecting 3D-PRE spheroids derived from human pluripotent stem cells, performing mechanical separation to remove non-RPE cells and clusters which are non-pigmented and retain a pigmented RPE cell sheet, enzymatically digesting the pigmented RPE cell sheet to obtain an RPE single cell suspension, and thereby the human pluripotent stem cell-derived human retinal pigment epithelial cells are obtained. The expansion method includes centrifuging the RPE single cell suspension and removing a supernatant, resuspending in an RPE cell medium and seeding into a cell culture container precoated with extracellular matrix to allow primary culture, and subculturing the cells after cells spread out.

Claims

1. A preparation method for human pluripotent stem cell-derived human retinal pigment epithelial cells, comprising following steps: collecting 3D-PRE spheroids derived from human pluripotent stem cells, performing mechanical separation to remove non-pigmented non-RPE cells and clusters and retain a pigmented RPE cell sheet, enzymatically digesting the pigmented RPE cell sheet to obtain an RPE single cell suspension, and thereby obtaining the human pluripotent stem cell-derived human retinal pigment epithelial cells.

2. The preparation method according to claim 1, wherein, the 3D-PRE spheroids are prepared by inducing directed differentiation of the human pluripotent stem cells into retinal cells including RPE cells, collecting adherent cells including the RPE cells by scraping, and performing suspension culture to obtain the 3D-RPE spheroids; the 3D-RPE spheroids are free-floating, adherent to one side of a neural retinal cup, or adherent to one side of another cell cluster.

3. The preparation method according to claim 1, wherein, the step of performing mechanical separation to remove the non-pigmented non-RPE cells and clusters and retain a pigmented RPE cell sheet, comprises: transferring all the 3D-RPE spheroids into a cell culture container, digesting in a 37 C. water bath for 8-15 minutes using a digestive reagent, washing with 1PBS for a plurality of times after the digestive reagent was aspirated, separating the pigmented RPE cell sheet from the non-pigmented non-RPE cells and clusters using a tungsten needle, and retaining the pigmented RPE cell sheet.

4. The preparation method according to claim 1, wherein, the step of enzymatically digesting the pigmented RPE cell sheet to obtain the RPE single cell suspension, comprises: digesting the pigmented RPE cell sheet with a TrypLE Express solution in a 37 C. water bath for 7-10 minutes, terminating digestion, filtering the cells through a 70-100 m strainer, centrifuging to remove the digestive reagent, and resuspending in an RPE cell medium to obtain the RPE single cell suspension.

5. An expansion method for human pluripotent stem cell-derived human retinal pigment epithelial cells, comprising: centrifuging the RPE single cell suspension of claim 1 and removing a supernatant; resuspending in an RPE cell medium and seeding into a cell culture container precoated with extracellular matrix to allow primary culture; after cells spread out, subculturing the cells to obtain the human pluripotent stem cell-derived human retinal pigment epithelial cells.

6. The expansion method according to claim 5, wherein, the step of seeding for primary culture is performed at a cell density greater than or equal to 510.sup.4 cells/cm.sup.2.

7. The expansion method according to claim 5, wherein, the step of subculturing the cells after the cells spread out comprises: when the cells of the primary culture reach a confluence of 90%-100%, removing the medium, washing the cells with PBS, performing digestion in a 37 C. incubator for 7-10 minutes using a TrypLE Express solution, terminating the digestion with an RPE cell medium, gently blowing the cells off with a pipette, centrifuging to remove the digestive reagent, resuspending the cells in an RPE cell medium, seeding the cells into a cell culture container precoated with extracellular matrix to allow subculture; when the cells reach a confluence of 90%-100%, repeating the above steps to allow repetitive subcultures; wherein, the step of seeding for subculture is performed at a cell density greater than or equal to 210.sup.4 cells/cm.sup.2.

8. The expansion method according to claim 5, wherein the extracellular matrix is Matrigel or Gelatin.

9. The expansion method according to claim 5, wherein, a formula of the cell medium used in the primary culture and the subculture is as follows: 100 mL of the cell medium comprises 10 mL of fetal bovine serum, 2 mL of 50 B-27 supplement, 1 mL of 100 penicillin-streptomycin solution, 1 mL of 100 non-essential amino acids solution, 1 mL of 100 glutamine, 0.1 mL of 1000 taurine, and the balance is a DMEM/F12 mixed medium; the DMEM/F12 mixed medium is prepared by blending a DMEM/F12 (1:1) medium and a DMEM medium in a volume ratio of 3:2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is an inverted microscope image of hPSCs.

[0031] FIG. 2 is an inverted microscope image (40) of RPE cells formed after 26 days of induced differentiation of hPSCs.

[0032] FIG. 3 shows inverted microscope images (40) of 3D-RPE spheroids formed by induced differentiation of hPSCs in suspension. The 3D-RPE spheroids are adherent to (A) one side of neural retina (NR), or (B) one side of a cell cluster.

[0033] FIG. 4 is an image (100) of single RPE cells which were obtained by mechanical separating, purifying and enzymatically digesting the 3D-RPE spheroids.

[0034] FIG. 5 is an image (200) taken after a seven-day primary culture of the RPE cells prepared in embodiment 6.

[0035] FIG. 6 is an image (100) taken after a seven-day culture of the fifth passage of the RPE cells prepared in embodiment 6.

[0036] FIG. 7 shows the result of immunofluorescence.

[0037] FIG. 8 shows the result of transepithelial electrical resistance (TEER) test.

[0038] FIG. 9 shows the result of PEDF measurement by ELISA.

[0039] FIG. 10 shows the result of flow cytometry.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The following embodiments are for further describing this invention rather than limiting the invention.

Embodiment 1: Expansion of Human Pluripotent Stem Cells

[0041] Three human pluripotent stem cell (hPSC) lines were used in the study. BC1-GFP-hiPSC line and BC1-hiPSC line were obtained from friends while a third hiPSC line was purchased from Life Technologies Corporation (Gibco Episomal hiPSC Line, A18945). The cells were seeded onto 6-well plates coated with extracellular matrix MatriGel (Corning, 354277) and expanded in mTeSR1 media. When the cells reached a confluence of 80%-90%, they were digested with 0.5 mM EDTA (Life, 15575-038), and passaged in a ratio of 1:8 to 1:12. The inverted microscope image showed that the cells were sheet-shaped and exhibited colony-like growth wherein the cells in the colony were compactly arranged with unclear boundaries (FIG. 1). Accordingly, human pluripotent stem cells (hPSCs) were obtained by expansion.

Embodiment 2: Induced Differentiation of hPSCs Towards Retinal Cells Including Pigment Epithelium Cells

[0042] Induced differentiation of hPSCs towards retinal cells including pigment epithelium cells was conducted according to a reported protocol (Xiufeng Zhong, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun. 2014 Jun. 10; 5: 4047), and the day of initiation of the differentiation (when the expansion medium of the hPSCs was replaced with a differentiation medium, or when embryoid bodies were prepared) was regarded as day 0 of the differentiation.

Embodiment 3: Preparing and Culturing hPSC-Derived 3D-RPE Spheroids

[0043] The retinal cells including pigment epithelium cells which were obtained at the fourth week of the induced differentiation of hPSCs were observed under microscope. Neural retina (NR) and RPE, which slightly rose up, shaped like a ring, and had high refractivity, could both be identified. RPE could grow around the NR, or grow individually in the form of dots or sheets (FIG. 2). The NR and its surrounding RPE were scraped using a self-made tungsten needle or a 1 mL syringe, and then transferred to a low attachment culture dish to allow suspension culture in an RPE cell medium. The formula of the cell medium was as follows: 100 mL of the cell medium comprises 10 mL of fetal bovine serum (Gibco, 10099-141), 2 mL of 50 B-27 supplement (Gibco, 12587-010), 1 mL of 100 penicillin-streptomycin solution (Gibco, 15240), 1 mL of 100 non-essential amino acids solution (Gibco, 11140-050), 1 mL of 100 glutamine (Gibco, 35050-061), 0.1 mL of 1000 taurine (Sigma, T-0652), and the balance is a DMEM/F12 (3:1) mixed medium; the DMEM/F12 (3:1) mixed medium is prepared by blending a DMEM/F12 (1:1) medium (Gibco, C11330500BT) and a DMEM medium (Gibco, C11995500BT) in a volume ratio of 3:2. The rest of the cells, at any time from the fourth week to the sixth week, could be scraped by a cell scraper and transferred to low attachment culture dishes to allow suspension culture in a conventional incubator using a RPE cell medium. The suspension-cultured RPE usually curled into spheroids (i.e. the 3D-RPE spheroids), which could be adherent to one side of NR or another cell cluster (FIG. 3), or could be free-floating.

Embodiment 4: Collecting, Mechanically Separating and Purifying the hPSC-Derived 3D-RPE Spheroids

[0044] After six weeks of differentiation of hPSCs, all suspension-cultured 3D-RPE spheroids (including free-floating spheroids and those adherent to one side of NR or another cell cluster) were collected and placed in 60 mm culture dishes. Under a dissecting microscope, the NR and the non-RPE clusters were removed from the RPE spheroids, and the pigmented 3D-RPE spheroids were retained.

Embodiment 5: Separating and Purifying the hPSC-Derived Human RPE Sheets

[0045] The 3D-RPE spheroids purified in embodiment 4 were digested at 37 C. for 8-15 minutes in a Dispase II solution (Sigma, D4693-1G) having a mass fraction of 1%, and then washed with 1PBS for three times after the digestive reagent was removed. On the surface of 3D-RPE spheroids were RPE which were black while inside were non-RPE cell clusters. Under a dissecting microscope, the RPE were separated from the non-RPE cell clusters using a tungsten needle and then RPE cell sheets were collected.

Embodiment 6: Preparing a Single Cell Suspension of hPSC-Derived Human RPE Cells

[0046] The purified RPE cell sheets were digested in a TrypLE Express solution (Gibco, 12604-013) at 37 C. for 7-10 minutes, and then a same volume of RPE cell medium was added into the solution to terminate the digestion. The cells were then dispersed by blowing with a 1 mL pipette tip until there was no visible cell cluster, filtered through a 70 m strainer, and centrifuged at 1000 rpm at room temperature. The supernatants were removed and the cell pellets were retained. The cells were then resuspended in a RPE cell medium (identical to the medium in embodiment 3) to obtain a single cell suspension, and thereby hPSC-derived RPE cells were obtained. Through the above protocols, RPE cells were obtained from all the three hPSC lines in embodiment 1.

Embodiment 7: Primary Culture of hPSC-Derived Human RPE Cells

[0047] The total number of cells in a single cell suspension of hPSC-derived human RPE cells, which is obtained by the method of the present invention, was counted on a hemocytometer. The cells were seeded onto 6-well plates precoated with Matrigel at a cell density of 510.sup.4 cells/cm.sup.2, and primary culture was performed at 37 C. under 5% CO.sub.2 and saturated humidity with an RPE cell medium. Thirty minutes after the cells were seeded, they began to attach to the plate. The cells were circular, bright, and showed high refractivity, and most of the cells carried pigment particles (FIG. 4). At the seventh day of the primary culture, the RPE cells prepared by the method reached a confluence; they were polygonal in shape and filled with pigment (FIG. 5). Primary cultures of the PRE cells derived from the three hPSC lines showed the similar results.

Embodiment 8: Subculture of hPSC-Derived Human RPE Cells

[0048] Primary culture of RPE cells derived from hiPSCs (Gibco Episomal hiPSC Line, purchased from Life Technologies Corporation) was performed for 7-8 days to reach a 100% confluence when they could be passaged. The PRE cells to be passaged were collected, and washed with PBS twice after the medium was removed. The RPE cells were then digested in a TrypLE Express solution at 37 C. for 7-10 minutes, and then an RPE cell medium (identical to that in embodiment 3) was added to neutralize the digestive reagent. The digested PRE cells were collected and counted. The digestive solution was removed by centrifugation, and the cells were resuspended in an RPE cell medium. The cells were then seeded onto culture plates coated with Matrigel at a cell density of 2-510.sup.4 cells/cm.sup.2, and subculture was performed at 37 C. under 5% CO.sub.2 in an incubator. The RPE cells prepared by this method had high purity and proliferative capacity such that they could be subcultured once every week for at least five passages; the subcultured cells retained PRE cell morphology that they were polygonal in shape, arranged like pebbles and slightly pigmented (FIG. 6). The pigment particles in the cells gradually decreased as the number of passages increased, which is similar to the culturing characteristics of human fetal RPE cells. In addition, RPE cells derived from the BC1-GFP-hiPSC line and the BC1-hiPSC line, which were subcultured and expanded by the above protocols, also presented the same results.

Embodiment 9: Cryopreservation and Recovery of hPSC-Derived Human RPE Cells

[0049] After RPE cells derived from hiPSCs (Gibco Episomal hiPSC Line, purchased from Life Technologies Corporation) grew to confluence, they were digested by the protocol in embodiment 8, centrifuged and then subjected to conventional cell cryopreservation. The cryoprotectant was an RPE cell medium supplied with 10% by volume of DMSO (the RPE cell medium was same as used in embodiment 3). The cryopreserved cells could be recovered and cultured under the same conditions of the subculture process. The recovered cells had high proliferative capacity such that they could reach a confluence of 100% in 7-8 days and exhibit the similar typical characteristics of RPE cells. In addition, RPE cells derived from the BC1-GFP-hiPSC line and the BC1-hiPSC line, which were cryopreserved and recovered by the above protocols, also presented the same results.

Embodiment 10: Growth Dynamics Analysis of hPSC-Derived Human RPE Cells

[0050] Growth potential of RPE cells: Passage 5 of the cells obtained by induced differentiation of BC1-GFP-hiPSC were seeded onto culture plates coated with Matrigel at a density of 510.sup.4 cells/cm.sup.2 at 37 C. under 5% CO.sub.2 and saturated humidity with an RPE cell medium, and subcultured every seven days. Successive subcultures were performed until passage 10, and the total number of cells obtained in every passage was counted on a hemocytometer. The BC1-GFP-RPE cells (i.e. the RPE cells obtained by induced differentiation of BC1-GFP-hiPSC) exhibited stable expansion, and could be subcultured for more than 5 passages. When seeded on a Matrigel-coated culture plate at a density greater than 210.sup.4 cells/cm.sup.2, the cells were able to reach a confluence of more than 90% after about 7 days of growth. The RPE cells prepared by the method of the invention can be expanded to achieve a nearly 15-fold expansion, allowing a 3000-fold expansion in three successive subcultures.

[0051] Growth curve of RPE cells: Passage 5 of the cells obtained by induced differentiation of BC1-GFP-hiPSC were seeded onto a 96-well plate with an RPE cell medium. Three wells of cells were collected, digested and counted every day for seven successive days. A growth curve was plotted with cell number as ordinate against days as abscissa. The RPE cells obtained by the method of the invention have a doubling time of about 1.52 days such that it takes only 7 days for the cells to expand in every passage.

Embodiment 11: Immunofluorescence Identification of Specific Molecular Markers Expression of hPSC-Derived Human RPE Cells

[0052] A portion of RPE cells prepared by the present invention (the RPE cells obtained by induced differentiation of BC1-GFP-hiPSC), when subcultured, were seeded onto coverslips coated with Matrigel and cultured with an RPE cell medium containing 10% of FBS. When the cells reached a confluence of 100%, the FBS was removed, and the culture proceeded in a serum-free RPE cell culture solution. Formula of the serum-free RPE cell medium is as follows: 100 mL of the cell medium comprises 2 mL of 50 B-27 supplement (Gibco, 12587-010), 1 mL of 100 penicillin-streptomycin solution (Gibco, 15240), 1 mL of 100 non-essential amino acids solution (Gibco, 11140-050), 1 mL of 100 glutamine (Gibco, 35050-061), 0.1 mL of 1000 taurine (Sigma, T-0652), and the balance is a DMEM/F12 (3:1) mixed medium; the DMEM/F12 (3:1) mixed medium is prepared by blending a DMEM/F12 (1:1, Gibco, C11330500BT) medium and a DMEM medium (Gibco, C11995500BT) in a volume ratio of 3:2. At different time intervals after the culture began, the coverslips were taken out, washed with PBS once, and placed on ice to allow cell fixation using a 4% formaldehyde solution for 5-10 minutes. Then the coverslips were washed with PBS for three times, and added into a blocking solution (PBS containing 10% of normal donkey serum and 0.25% of Triton X-100) to allow blocking at room temperature for 1 hour. The samples were then incubated with primary antibodies at 4 C. overnight. The primary antibodies were: PAX6 (mouse, 1:50, DSHB), OTX2 (rabbit, 1:200, abcam), RPE65 (mouse, 1:500, abcam), ZO-1 (mouse, 1:400, Life Technologies), and CHX10 (sheep, 1:200, Millipore). The next day, the cells were washed with PBS for three times and then incubated with fluorescence-labeled secondary antibodies (1:500, Life Technologies) at room temperature for 1 hour. After incubated with secondary antibodies, the cells were washed with PBS and staining was performed with DAPI for 10 minutes. After washing with PBS for three times, observation and photographing were performed with an Olympus fluorescence microscope. The RPE cells, which were prepared by the method of the invention, expressed RPE cell specific molecular markers PAX6, OTX2, RPE65 and ZO-1 (FIG. 7), but did not express retinal neural precursor cell marker CHX10. The RPE cells which were obtained from induced differentiation of the hiPSC line (Gibco Episomal hiPSC Line, A18945, purchased from Life Technologies) and the BC1-hiPSC line also presented the same results.

Embodiment 12: Function Analysis of the RPE Cells Prepared by the Method of the Present Invention

[0053] Transepithelial electrical resistance test: A portion of the RPE cells prepared by the present invention (the RPE cells obtained by induced differentiation of BC1-GFP-hiPSC), when subcultured, were seeded onto Transwell (Corning, 0.4 m transparent polyester membrane, product number: 3470) precoated with Matrigel, and cultured using an RPE cell medium containing 10% of FBS (identical to the RPE cell medium in embodiment 3). When the cells reached a confluence of 100% (about 7-8 days later), the FBS was removed, and the culture proceeded in a serum-free RPE cell medium (identical to the medium in embodiment 11). 6-8 weeks after the culture began, a transepithelial electrical resistance (TER) measurement was carried out with an epithelial voltohmmeter (WPI, EVOM2). The electrodes were sterilized by soaking in 75% alcohol and rinsed with Hank's balanced salt solution before the measurement. The TER value of the Transwell coated with Matrigel was measured as the background value. The actual TER value was calculated by subtracting the background value from the display value. The TER measurement of each well was performed at three spots to obtain an average value, and three replicates were performed. An RPE cell line ARPE-19 was taken as a control group. The RPE cells (hPSC-RPE) prepared by the invention exhibited high electrical impedance (520.323.6 *cm.sup.2), significantly higher than that of ARPE-19 cells (210.710.5 *cm.sup.2) (FIG. 8), suggesting that the RPE cells prepared by the present invention exhibit an electrical impedance function similar to RPE cells in vivo and have high electrical impedance better than that of ARPE-19 cell line. The RPE cells which were obtained from induced differentiation of the hiPSC line (Gibco Episomal hiPSC Line, A18945, purchased from Life Technologies) and the BC1-hiPSC line also presented the same results.

[0054] Measurement of PEDF Secretion:

[0055] The RPE cells prepared by the present invention (the RPE cells obtained by induced differentiation of BC1-GFP-hiPSC) were seeded onto Transwell at a density of 510.sup.4 cells/cm.sup.2 and cultured under a condition identical to that in the transepithelial electrical resistance test section. When the TER value was determined to be greater than 200 /cm.sup.2, the cells were washed with PBS for three times, and then the medium was replaced with a serum-free RPE cell culture solution (identical to that in embodiment 11), with 120 L in upper compartment and 1 mL in the lower compartment. The cells were cultured in an incubator at 37 C. under 5% CO.sub.2 and saturated humidity for 24 hours, and then the media in the upper and lower compartments were collected respectively. Human pigment epithelium-derived factor (PEDF) levels in the culture solutions in the upper and lower compartments were measured by ELISA. The ELISA kit was purchased from Cusabio Technology LLC (product number: CSB-EO8818h). The measurement was conducted according to the manufacturer's specification. The RPE cells prepared by the present invention exhibited polarized secretion of PEDF wherein the PEDF level in the Transwell upper compartment (25.33.5 ng/mL) was higher than that in the lower compartment (7.30.8 ng/mL) (FIG. 9), which was similar to that of human RPE cells in vivo. The RPE cells which were obtained from induced differentiation of the hiPSC line (Gibco Episomal hiPSC Line, A18945, purchased from Life Technologies) and the BC1-hiPSC line also presented the similar results.

Embodiment 13: Flow Cytometry Analysis on Purity of the RPE Cells Prepared by the Present Invention

[0056] After the RPE cells prepared by the present invention (the RPE cells obtained by induced differentiation of BC1-GFP-hiPSC) was subcultured for 7-8 days to reach a confluence of 100%, the serum-containing RPE cell culture solution was replaced with a serum-free RPE cell culture solution (identical to that in embodiment 11) and then the culture proceeded for 6-8 weeks. The attached RPE cells were digested with a TrypLE Express solution to give a single cell suspension. The suspension was then centrifuged at 1000 rpm for 5 minutes, then the pellet was resuspended in 2 ml of 1% formaldehyde solution for cell fixation for 15 minutes. Then the suspension was centrifuged at 1000 rpm for 5 minutes and the cells were washed with a PBS solution containing 0.04% of triton-X-100 and 2% of donkey serum. This step was repeated twice. Primary antibody RPE65 (mouse, abcam, cat. AB78036) was diluted using a PBS solution containing 0.25% of triton-X-100 and 2% of donkey serum, then the cells were incubated with the primary antibody for 1 hour wherein the concentration of the primary antibody was 2 g/110.sup.6 cells. The cells were washed according the aforementioned method and then incubated with Alexa 555-labeled donkey anti-mouse secondary antibody (1:500, Life Technologies) at room temperature for 30 minutes. After washing, the cells were resuspended in 500 L PBS for analysis. The cells without primary antibody were taken as parallel negative control. The flow cytometry was purchased from BD company (model: LSRFortessa). The RPE cells prepared by the present invention expressed specific molecular marker RPE65, and positive cells accounted for 98.1% (FIG. 10), indicating that the RPE cells prepared by the present invention have a high purity and thus can be applied to related researches. The RPE cells which were obtained from induced differentiation of the hiPSC line (Gibco Episomal hiPSC Line, A18945, purchased from Life Technologies) and the BC1-hiPSC line also presented the same results.