Method of obtaining pigmented cells in vitro by the differentiation of human induced pluripotent stem cells

Abstract

The invention discloses the method of differentiating human induced Pluripotent Stem cells (iPS cells) to obtain pigmented cells which produce melanin. The protocol described is much more efficient than any other method used to obtain melanin in vitro.

Claims

1. The method of obtaining pigmented cells in vitro by differentiating human induced pluripotent stem (iPS) cells, comprising: a) plating a suspension of detached confluent iPS cells on a non-adherent cell culture dish in iPS cell medium that does not comprise bFGF and does comprise inhibitor Y27632, where the density is 2-2.5×10.sup.4 cells/cm.sup.2 and culturing to form embryoid bodies (EB), b) harvesting and seeding the embryoid bodies on an adherent cell culture dish and then culturing in iMEF culture media for at least 18 hours to generate progenitor cells, c) selecting the progenitor cells for 10 days with periodic removal of dead cells in medium N1 comprising supplement N2 at 1× concentration fibronectin in the concentration of 250 ng/ml, a solution of antibiotics including penicillin and streptomycin (P/S) in the concentration of 100 U/ml penicillin / 100 μg/ml streptomycin, diluted in medium DMEM/F12, d) dissociating the progenitor cells and placing the progenitor cells at a density of 0.5- 2×10.sup.5/cm.sup.2 onto dishes covered with laminin and polyornithine, and expanding the progenitor cells for 7 days in medium N2 comprising supplement N2 at 1× concentration, laminin in the concentration of 1 mg/ml, bFGF in the concentration 20 μg/ml, FGF8 in the concentration of 100 ng/ml and P/S in the concentration of 100 U/ml penicillin / 100 μg/ml streptomycin, diluted in medium DMEM/F12, and e) differentiating the expanded progenitor cells for 7-16 days by culturing them in medium N3 comprising supplement N2 at 1× concentration, laminin in the concentration of 1 mg/ml, dibutyryl-cAMP in the concentration of 0.5 mM, ascorbic acid in the concentration of 200 μM and P/S in the concentration of 100 U/ml penicillin / 100 μg/ml streptomycin, diluted in medium DMEM/F12, in order to obtain human cells pigmented with melanin.

2. The method according to the claim 1 characterized in that media N1, N2 and N3 contain the indicated components only.

3. The method of claim 2, further comprising isolating the melanin from the cells.

4. The method of claim 1, further comprising isolating the melanin from the cells.

5. The method of claim 4, further comprising administering the melanin to a patient.

6. The method of claim 4, further comprising making a cream comprising the melanin.

7. The method of claim 1, further comprising administering the cells obtained according to the method to a patient.

Description

EXAMPLE 1

The Method of Obtaining Human Cells Extensively Pigmented with Melanin From Human Induced Pluripotent Stem Cells

(1) The cells used were confluent piPS cells (protein-iPS cells) purchased from System Bioscience (cat no.SC801A-1, SC802A-1) (Kim et al. 2009) or the cell lines derived at the Department of Transplantation of the Jagiellonian University Medical College by reprogramming the somatic cells from donors.

(2) In the first step, the confluent iPS cells (FIG. 1A) cultured on the feeder layer made of iMEF cells were harvested using Accutase and plated as a suspension of single cells on a non-adherent cell culture dish in the medium for iPS cells without bFGF with inhibitor Y27632 in the density of 2-2.5×10.sup.4/cm.sup.2. Next, iPS cells were cultured in the suspension for 4 days until embryoid cells (EB) were formed (FIG. 1B).

(3) The composition of the medium for iPS cells:

(4) TABLE-US-00001 Component Concentration KSR 20% (ThermoFisher Scientific) β-Mercaptoethanol 0.1 mM (Sigma-Aldrich) Non-essential Amino Acids 1× (ThermoFisher Scientific) Penicillin/ 100 U/ml/100 μg/ml Streptomycin (P/S) (ThermoFisher Scientific) bFGF   10 ng/ml (ThermoFisher Scientific) DMEM/F12 (ThermoFisher Scientific)

(5) In the next step, the embryoid cells were harvested, centrifuged (300 rpm, 5 minutes) and seeded on an adherent dish with the same surface as the non-adherent dish in the medium for iMEF cells.

(6) The composition of the medium for iMEF cells:

(7) TABLE-US-00002 Component Concentration Foetal Bovine Serum (EurX) 10% L-glutamine 2 mM (ThermoFisher Scientific) P/S 100 U/ml/100 μg/ml DMEM 4.5 g/l (Lonza)

(8) After about 18 hours, the selection of progenitors (FIG. 1C) was initiated by changing the medium to medium N1 with the following composition:

(9) TABLE-US-00003 Component Concentration Supplement N2 1× (ThermoFisher Scientific) Fibronectin 250 ng/ml (ThermoFisher Scientific) P/S 100 U/ml/100 μg/ml DMEM/F12 (ThermoFisher Scientific)

(10) The selection in serum-free media was carried out for 10 days by removing dead cells and providing the fresh medium N1 every second day. Supplement N2 provided by Life Technologies was used. It contains human insulin (0.1 mg/ml), holo-Transferrin (5 μg/ml), progesterone (20 μM), putrescine (0.1 mM), sodium selenite (30 nM) (final concentration, after dilution in the medium).

(11) The next step was the expansion of selected progenitors (FIG. 1D). The cells were dissociated into single cells using trypsin and plated in the density of 0.5-2×10.sup.5/cm.sup.2 onto dishes covered with laminin and poly-ornithine.

(12) Cell expansion was carried out for 7 days in medium N2 with the following composition:

(13) TABLE-US-00004 Component Concentration N2 supplement 1× Laminin 1 mg/ml (ThermoFisher Scientific) bFGF 20 μg/ml FGF8 100 ng/ml (ThermoFisher Scientific) P/S 100 U/ml/100 μg/ml DMEM/F12

(14) Progenitors were frozen in medium N2 with 10% of DMSO in the density of 2×10.sup.6 cells/ml. After the expansion was completed, the cells were terminally differentiated by changing the medium from N2 to N3. The composition of medium N3 can be found in the table below:

(15) TABLE-US-00005 Component Concentration N2 supplement 1× Laminin 1 mg/ml Dibutyryl cAMP 0.5 mM (Sigma-Aldrich) Ascorbic acid 200 μM (Sigma-Aldrich) P/S 100 U/ml/100 μg/ml DMEM/F12

(16) As a result of the differentiation method described extensively pigmented cells (FIG. 1E-I) were obtained in a highly repeatable manner. The efficiency of the differentiation process was always similar and made it possible to obtain a large number (>3×10.sup.6) of highly pigmented cells.

(17) FIG. 1 presents the results of the consecutive stages of iPS cell differentiation to obtain pigmented cells. A—piPS cells cultured on the feeder layer. B—multi-cellullar embryoid bodies (EB) in a suspension. C—selected NPC cells subjected to expansion (D). In the final stage of differentiation, the cells acquired a black pigmentation (E-I).

EXAMPLE 2

The Characteristics of the Cells Obtained and the Pigment Isolated From Them

(18) The pigment obtained was unambiguously identified as melanin using electron paramagnetic resonance (EPR) (FIG. 2A) and Fontana-Masson staining (FIG. 2B). The obtained pigmented cells were characterised with respect to their gene expression using a RT-PCR reaction (FIG. 3) and immunocytochemical staining (ICC) (FIG. 4).

(19) FIG. 3 shows the expression of markers at the RNA level. A—pigmented cells which, unlike iPS cells, express melanocyte markers (MITF-M, TRP1, TYR) B—in the next steps of cell differentiation (A-F) the expression of embryoid markers is reduced (OCT-4, NANOG, TERT) and the expression of neuroectodermal markers (NURR1, TUJ-1, TH) grows. TERT—telomerase, TUJ-1—βIII-tubulin, TH—tyrosine hydroxylase NURR1—nuclear-receptor related protein, the constitutive gene GAPDH was used as a positive control of the PCR reaction.

(20) FIG. 4 shows the expression of markers at the level of protein. The cells obtained express melanocyte markers—TRP1 and MITF (A and B). In the heterogenic population of cells obtained, there are also dopaminergic neurons which express TUJ-1 and TH (C and D). The subcellular location of melanin was defined using transmission electron microscopy (TEM) (FIG. 5).

(21) FIG. 5 shows the subcellular location of melanin in the cells obtained. Melanin is organised in organelles resembling the consecutive stages of melanosome development (I-IV in A). The organelles are surrounded with a dual cellular membrane (B).

(22) The results obtained make it possible to confirm unambiguously that the pigment obtained is melanin. The differentiated pigmented cells resemble melanocytes in many aspects (marker expression, gene expression profile, the presence of membranous intracellular organelles filled with the pigment), but the number of pigmented cells and the level of pigmentation for the cells obtained is unexpectedly high as compared with other available technologies. The method described can be employed to provide very large numbers of extensively pigmented cells that may be used for the isolation of pure, non-degraded melanin in large amounts and at a relatively low cost.