Method for suppressing teratoma formation via selective cell death induction in undifferentiated human-induced pluripotent stem cells

Abstract

The present invention relates to a method for preparing differentiated cells derived from induced pluripotent stem cell, wherein undifferentiated induced pluripotent stem cells (iPS) are removed, the method comprising steps of: (a) preparing a cell sample including undifferentiated induced pluripotent stem cells and differentiated cells by differentiating induced pluripotent stem cells; and (b) causing selective apoptosis of the undifferentiated induced pluripotent stem cells by treating the resultant in step (a) with quercetin of Formula 1 below or with YM-155 of Formula 2 below. According to the present invention, the present invention makes it possible to effectively selectively cause apoptosis only of undifferentiated induced pluripotent stem cells by causing induced pluripotent stem cells to differentiate into specific differentiated cells and then carrying out culturing in a differentiating culture medium comprising quercetin or YM-155, and, in the induced pluripotent stem cell differentiation method according to the present invention, only undifferentiated induced pluripotent stem cells that are a cause of teratoma formation are selectively caused to die, and thus differentiated differentiating cells are completely unaffected. In other words, the invention can be expected to ensure safety as the possibility of tumor formation during clinical use as a cell therapeutic agent is eliminated since the survival and functioning of the differentiated cells is maintained unchanged.

Claims

1. A method for preparing a composition of differentiated cells derived from induced pluripotent stem cells (iPS), from which undifferentiated induced pluripotent stem cells have been removed, comprising: (a) preparing a cell sample including undifferentiated induced pluripotent stem cells and differentiated cells obtained by differentiating induced pluripotent stem cells; and (b) treating the cell sample of step (a) with quercetin of Formula 1 below or YM-155 of Formula 2 below, to cause selective apoptosis of the undifferentiated induced pluripotent stem cells of the cell sample: ##STR00003##

2. The method of claim 1, wherein the induced pluripotent stem cells are derived from humans, cattle, horses, goats, sheep, dogs, cats, mice, rats, or birds.

3. The method of claim 1, wherein the concentration of quercetin or YM-155 is in the range of 20 M to 100 M, or 2.5 nM to 80 nM, respectively.

4. A method for selective apoptosis of undifferentiated induced pluripotent stem cells, the method comprising contacting the cells with quercetin of Formula 1 below or YM-155 of Formula 2 below: ##STR00004##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1a shows the images of the degree of apoptosis in an apoptosis-specific cell type of human induced pluripotent stem cells (iPS) derived from human aortic smooth muscle cells (HASMC), HASMC, smooth muscle cell no. 1 differentiated-derived from iPS (iPS-SMC-1), and smooth muscle cell no. 3 differentiated-derived from iPS (iPS-SMC-3) observed under microscope, after treating them with 50 M quercetin, respectively, for 24 hours. As a result, the human induced pluripotent stem cells treated with quercetin exhibited a distinct apoptosis-specific cell phenotype. FIG. 1b shows the results of confirming the apoptosis marker proteins by a cell lysate, treated according to the same method as in FIG. 1a, using an immunoblotting method. As a result, the human induced pluripotent stem cells treated with quercetin exhibited the expression of Parp-1 cleavage (T type), cleavage types of caspase-3 and caspase-9 (cleaved caspase)

(2) FIG. 2a shows the images of the degree of apoptosis in an apoptosis-specific cell phenotype of concentration-dependent pluripotent induced stem cells observed under microscope, after treating them with various concentrations of quercetin (QC). FIG. 2b shows the results of the degree of apoptosis of iPS and HASMC treated with various concentrations of quercetin in FIG. 2a, confirmed by Annexin V method. FIG. 2c is a graph showing the results of the degree of apoptosis obtained in FIG. 2b, wherein the rate of live cells in each of iPS and HASMC.

(3) FIG. 3a shows the results of expression of -Smooth muscle actin (-SMA), which is known to specifically express in smooth muscle cells, confirmed by immunofluorescence method, after treating HASMC and iPS-SMC3 with 50 M quercetin for 24 hours. FIG. 3b shows the results of mRNA expression level of intracellular -SMA of iPS-SMC1 and iPS-SMC3, treated by the same method as in FIG. 3a, confirmed by RT-PCR. FIG. 3c shows the results of smooth muscle cells differentiated-derived from human induced pluripotent stem cells by measuring the intracellular calcium concentration of iPS-SMC1 and iPS-SMC3, after treating iPS-SMC1 and iPS-SMC3 with 50 M quercetin or DMSO for 24 hours, followed by treating with ATP or 75 mM potassium solution (75K.sup.+). FIG. 3d is a graph showing the results of interior calcium concentrations measured in FIG. 3c.

(4) FIG. 4a shows the comparison result of teratoma formation capability between undifferentiated induced pluripotent stem cells, 10 weeks after treating them with 50 M quercetin for 24 hours followed by injection into a mouse testis, and control group. FIG. 4b shows the results of PAS staining and H&E staining of the secretory epithelium and neural rosette structure, respectively, in order to confirm the teratoma formation in FIG. 4a.

(5) FIG. 5 shows the images of the degree of apoptosis in an apoptosis-specific cell type of iPS, HASMC, and iPS-SMC-3, after treating them with various concentrations of YM-155 for 24 hours, confirmed by microscope. As a result, the human induced pluripotent stem cells treated with YM-155 exhibited a distinct concentration-dependent apoptosis-specific phenotypic cell type.

(6) FIG. 6a shows the results of confirming the degree of apoptosis of concentration-dependent iPS, HASMC, and iPS-SMC3 by various YM-155 concentrations using Annexin V method, and measured by the FACS method. FIG. 6b is a graph showing the degree of apoptosis obtained in FIG. 6a, wherein the rate of live cells in each of iPS, HASMC, and iPS-SMC3.

(7) FIG. 7a shows the results of smooth muscle cells differentiated-derived from human induced pluripotent stem cells by measuring the intracellular calcium concentration of iPS-SMC1 and iPS-SMC3, after treating iPS-SMC1 and iPS-SMC3 with 50 nM YM-155 or DMSO for 24 hours, followed by treating with ATP or 75 mM potassium solution (75K.sup.+). FIG. 7b is a graph showing the results of interior calcium concentrations measured in FIG. 7a.

(8) FIG. 8a shows the analysis results of immunostaining by SSEA-4 (green) and cleavage type-3 (red) (upper panel), or SMA (green) and cleavage type caspase-3 (red) (lower panel), after mixing human induced pluripotent stem cells and smooth muscle cells derived from human induced pluripotent stem cells (SMC3) at a 1:1 ratio, followed by treating with 50 M quercetin for 24 hours. The scale bar denotes 100 m. FIG. 8b shows a percentage of cleavage type caspase-3 positive population in SMA-positive population or SSEA-4-positive population. ** denotes a significance of P<0.01.

(9) FIG. 9a shows the results of injection of a 1:1 mixture of human induced pluripotent stem cells and smooth muscle cells into a mouse. It shows the images of teratoma formed by injecting human induced pluripotent stem cells untreated or pretreated with 10 mM YM155 into a mouse. NC refers to a mouse testis into which cells were not injected. FIG. 9b shows the results of staining teratoma, formed in a mouse injected with human induced pluripotent stem cells, by H&E, Masson's trichrome, and Acian Blue staining. Teratoma produced intestinal epithelium, cartilage, secretory epithelium, and neural rosette structure.

MODE FOR CARRYING OUT THE INVENTION

(10) Hereinafter, the present invention will be described in greater details with reference to the accompanying examples. However, these examples disclosed herein are only for illustrative purposes of the present invention, and it shall be obvious to a skilled person in the art that they should not be construed as limiting the scope of the present invention.

EXAMPLES

(11) Materials and Methods

(12) Reagents

(13) Quercetin (Cat # Q0125) was purchased from Sigma-Aldrich (St. Louis, Mo., USA), and YM-155 (Cat # S1130) was purchased from Selleck Chemicals.

(14) Culturing and Differentiation of Human Induced Pluripotent Stem Cells (iPS)

(15) Human induced pluripotent stem cells (iPS) derived from human aortic smooth muscle cells (HASMC) was subcultured in an ESC medium (DMEM/F/12 supplemented with a 20% KnockOut serum replacement, 0.1% gentamycin, 1% non-essential amino acids, 0.1% -mercaptoethanol and 4 ng/ml bFGF) containing mouse embryonic fibroblasts (MEF) treated with mitomycin-C as supporting cells Lee, T. H., et al., (2010). Functional recapitulation of smooth muscle cells via induced pluripotent stem cells from human aortic smooth muscle cells. Circ Res 106, 120-128).

(16) For the pretreatment of quercetin and YM-155 of the present invention, iPS was cultured in a matrigel-coated 60 mm dish containing mIeSR1 medium (29106, Stem Cell Technology) to induce them into smooth muscle cells (SMC).

(17) Culturing of Human Aortic Smooth Muscle Cells (HASMC) and Human Induced Pluripotent Stem Cells-Derived Differentiated Cells (iPS-SMC)

(18) Human aortic smooth muscle cells (HASMC) and human induced pluripotent stem cells-derived differentiated cells, i.e., human induced pluripotent stem cells-derived smooth muscle cells (iPS-SMC) were cultured in an SMCM medium (ScienCell research laboratories, cat #1101) as described in the reference (Lee et al., 2010).

(19) Immunoblotting and Immuno Fluorescence Cytochemistry (IFC)

(20) The antibodies used in the immunoblotting assay and anti-PARP1 (SC-7150) and anti--actin (SC-47778) were purchased from Santa Cruz Biotech Inc. (CA, USA). Additionally, anti-caspase-9 (Cat#9502) and anti-cleaved caspase-3 (Cat#9661) purchased from Cell Signaling Technology (Danvers, Mass., USA).

(21) As the first antibody used in IFA was anti--smooth muscle actin (-SMA) (Dako Inc, Carpinteria, Calif.). Images were obtained using Axioscope A1 microscope (Carl Zeiss) and analyzed.

(22) Real Time PCR

(23) RNA extraction and cDNA synthesis were performed according to a general protocol (VanGuilder H D, et al (2008). Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44 (5): 619-626).

(24) The gene-specific primer sequences used in the Examples of the present invention are as follows:

(25) TABLE-US-00001 -actin: 5-GTCCTCTCCCAAGTCCACAC-3, (SEQIDNO:1) 5-GGGAGACCAAAAGCCTTCAT-3 (SEQIDNO:2) -SMA: 5-AGAACATGGCATCATCACCA-3, (SEQIDNO:3) 5-TACATGGCTGGGACATTGAA-3. (SEQIDNO:4)
FACS Assay

(26) In all FACS assays were used FACS Calibur (BD Biosciences) and CellQuest. Cells were stained using PE Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to the protocol described in the envelope of the product.

(27) Teratoma Formation and Immunohistochemistry

(28) Since the differentiated cells lack of teratoma forming ability, in order to confirm the inhibitory effect of quercetin or YM-155 against teratoma formation, a teratoma forming experiment was performed by creating a condition including cells in an undifferentiated state. That is, cells (about 510.sup.6 cells), where the embryoid body (EB) formed from iPS was treated with quercetin (100 M) or YM-155 (10 nM), or untreated cells (about 510.sup.6 cells) obtained were injected into testis of an NOD/SCID mouse (Charles River Laboratories, Yokohama, Japan) thereby forming teratoma. 10 weeks after the injection, the xenografted cells (xenograft mass) were obtained and fixed in 4% PFA for 2 weeks, and embedded in paraffin using Tissue-Tek VIP embedding machine (Miles Scientific, Naperville, Ill.) and Thermo Shandon Histocenter2 (Thermo Fisher Scientific, Waltham, Mass.). The thus prepared paraffin block was prepared into slices (2 m thick) using a Leica RN2065 microtome (Leica, Wetzlar, Germany). In order to confirm teratoma formation, the slices were stained with Hematoxylin and eosin stain (H&E) and PAS (Periodic-Acid-Schiff), and analyzed by an experienced pathologist.

(29) All the experiments of the present invention were approved by the Institutional Animal Care and Use Committee of CHA University, and all the procedures of the present invention were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the U.S. NIH (NIH publication no. 85-23, revised 1996).

(30) Measurement of Intracellular Calcium (Ca.sup.2+)

(31) In order to confirm the functions of the differentiated cells which were completely differentiated into smooth muscle cells, an intracellular calcium reaction was used. As described in the reference article (Lee et al., 2006), intracellular or extracellular calcium transients were induced using pharmacological reagents, ATP or 75 mM K.sup.+ solution, and the values were numerized in fluorescence using a calcium indicator Fura-2 (Molecular Probes, Eugene, Oreg.), and imaged.

(32) Experimental Results

(33) Apoptosis of Undifferentiated Human Induced Pluripotent Stem Cells by Quercetin and Confirmation of Human Induced Pluripotent Stem Cells-Derived Smooth Muscle Cells (iPS-SMC)

(34) Apoptosis by Quercetin

(35) In order to confirm the selective apoptosis of undifferentiated human induced pluripotent stem cells by quercetin, human induced pluripotent stem cells (iPS) derived from human aortic smooth muscle cells (HASMC), HASMC, smooth muscle cell no. 1 differentiated-derived from iPS (iPS-SMC-1), and smooth muscle cell no. 3 differentiated-derived from iPS were treated with 50 M quercetin for 24 hours, and apoptosis-specific phenotypic cell types were examined, as a result, a distinctive apoptosis-specific phenotypic cell type was discovered in the human induced pluripotent stem cells treated with quercetin (FIG. 1a). Additionally, when apoptosis-associated marker proteins were examined via immunoblotting assay, cleavage of Parp-1 (T type), cleavage types of caspase-3 and caspase-9 (cleaved caspase), which occur at the time of apoptosis, were expressed (FIG. 1b).

(36) When treated with various concentrations of quercetin (QC) the degree of apoptosis of concentration-dependent induced pluripotent stem cells were confirmed in an apoptosis-specific phenotypic cell type (FIG. 2a). Additionally, the degree of apoptosis of iPS and HASMC by various concentrations of quercetin was confirmed via Annexin V method, and the results are shown in graphs, and the ratio of the respective live cells of iPS and HASMC were confirmed (FIGS. 2b and 2c).

(37) Confirmation of Functions of Human Induced Pluripotent Stem Cells-Derived Smooth Muscle Cells (iPS-SMC)

(38) In order to confirm the functions of smooth muscle cells differentiated-derived from human induced pluripotent stem cells, HASMC and iPS-SMC3 were treated with 50 M quercetin for 24 hours, and examined the expression of -Smooth muscle actin (-SMA), which is known to be specifically expressed in smooth muscle cells, via immuno fluorescence method, and as a result, the expression of -SMA was confirmed. Additionally, the expression of -SMA mRNA in the cells of iPS-SMC1 and iPS-SMC3 was confirmed via RT-PCR (FIGS. 3a and 3b).

(39) Additionally, in order to confirm the functions of smooth muscle cells differentiated-derived from human induced pluripotent stem cells, iPS-SMC1 and iPS-SMC3 were treated with 50 M quercetin or DMSO for 24 hours, followed by treatment with ATP or 75 mM potassium solution (75K.sup.+), and measured the concentration of intracellular calcium of iPS-SMC1 and iPS-SMC3, and thereby confirmed the functions as smooth muscle cells (FIGS. 3c and 3d).

(40) Teratoma Forming Ability

(41) In order to confirm the teratoma formation when treated with quercetin, undifferentiated induced pluripotent stem cells treated with 50 M quercetin for 24 hours were injected into testis, and in 10 weeks thereafter, their teratoma forming ability was compared with the control group (induced pluripotent stem cells not treated with quercetin). As a result, secretory eipthelium and neural rosette structure were observed in the control group, and they were respectively stained with PAS stain and H&E stain, thereby confirming teratoma formation.

(42) Apoptosis of Undifferentiated Human Induced Pluripotent Stem Cells by YM-155 and Confirmation of Human Induced Pluripotent Stem Cells-Derived Smooth Muscle Cells (iPS-SMC)

(43) Apoptosis by YM-155

(44) In order to confirm the selective apoptosis of undifferentiated human induced pluripotent stem cells by YM-155, human induced pluripotent stem cells (iPS) derived from human aortic smooth muscle cells (HASMC), HASMC, and smooth muscle cell no. 3 differentiated-derived from iPS were treated with YM-155 for 24 hours, and apoptosis-specific phenotypic cell types were examined, as a result, a distinctive apoptosis-specific phenotypic cell type was discovered in the human induced pluripotent stem cells treated with concentration-dependent YM-155 (FIG. 5).

(45) Additionally, the degree of apoptosis of iPS, HASMC, and iPS-SMC3 by various concentrations of YM-155 was confirmed via Annexin V method, and the results are shown in graphs, and the ratio of the respective live cells of iPS, HASMC, and iPS-SMC3 were confirmed (FIGS. 6a and 6b).

(46) Confirmation of Functions of Human Induced Pluripotent Stem Cells-Derived Smooth Muscle Cells (iPS-SMC)

(47) In order to confirm the functions of smooth muscle cells differentiated-derived from human induced pluripotent stem cells, iPS-SMC1 and iPS-SMC3 were treated with 50 nM YM-155 or DMSO for 24 hours, followed by treatment with ATP or mM potassium solution (75K.sup.+), and measured the concentration of intracellular calcium of iPS-SMC1 and iPS-SMC3, and thereby confirmed the functions as smooth muscle cells (FIGS. 7a and 7b).

(48) Confirmation of the Effect of Quercetin Treatment on the Functions of Differentiated Cells

(49) In order to confirm the functions of smooth muscle cells differentiated-derived from human induced pluripotent stem cells, the population, which was cultured by mixing human induced pluripotent stem cells and the smooth muscle cells differentiated-derived from the human induced pluripotent stem cells in a 1:1 ratio, was treated with 50 M quercetin for 24 hours, and examined the cleavage type caspase-3 positive population in the population of SMA-positive population or SSEA-4-positive population. As a result, the mixed cell population of human induced pluripotent stem cells-smooth muscle cells, after quercetin treatment, was shown to have an excellent cleavage type caspase-3 positive population within the SSEA-4 positive population (FIGS. 8a and 8b).

(50) Inducing Apoptosis of Human Induced Pluripotent Stem Cells by YM-155, a Survivin Inhibitor

(51) In order to confirm the selective apoptosis of human induced pluripotent stem cells by YM-155, a Survivin inhibitor, the result of injection of a 1:1 mixture of human induced pluripotent stem cells and smooth muscle cells is shown. Human induced pluripotent stem cells, either untreated or pretreated with 10 mM YM155, were injected into a mouse to induce teratoma (FIG. 9a). When the teratoma formed in the mouse injected with the human induced pluripotent stem cells was stained with H&E, Masson's trichrome, and Acian Blue, a single treatment with YM-155 for 24 hours was shown to inhibit teratoma formation.

(52) The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated by those skilled in the art that the specific technical features are merely preferred embodiments of the present invention and they should not be construed as limiting the scope of the present invention, and thus the substantial scope of the present invention shall be defined in the appended claims and their equivalents