METHOD FOR DIFFERENTIATING MESENCHYMAL STEM CELLS FROM PLURIPOTENT STEM CELLS

Abstract

The present disclosure relates to a method for producing mesenchymal stem cells from pluripotent stem cells and mesenchymal stem cells prepared by the method. The present disclosure enables mesenchymal stem cells having superior proliferation rate while having superior intrinsic biological activity to be obtained with high yield by sequentially performing three-dimensional suspension culture using pluripotent stem cells, particularly induced pluripotent stem cells (iPSCs), as starting cells and adherent culture of cell aggregates formed therethrough. The mesenchymal stem cells produced by the method of the present disclosure can be usefully used in compositions for treating bone diseases, cartilage diseases or inflammatory and autoimmune diseases owing to high differentiation efficiency into bone and cartilage and superior anti-inflammatory activity.

Claims

1. A method for producing mesenchymal stem cells from pluripotent stem cells, comprising: (a) a step of forming embryoid bodies (EBs) by culturing pluripotent stem cells isolated from a subject; (b) a step of forming spheroids by three-dimensionally culturing the embryoid bodies in a bioreactor under microgravity; and (c) a step of differentiating the spheroids into mesenchymal stem cells by adherent-culturing in a culture vessel coated with an adhesive polymer.

2. The method according to claim 1, wherein the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

3. The method according to claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells.

4. The method according to claim 1, wherein the step (a) is performed by three-dimensionally culturing the pluripotent stem cells in a multi-well culture plate.

5. The method according to claim 4, wherein the step (a) further includes a step of inducing cell aggregation during the three-dimensional culture through centrifugation.

6. The method according to claim 1, wherein the microgravity in the step (b) is induced by a microgravity simulator which offsets the gravity applied to the bioreactor by rotating the bioreactor.

7. The method according to claim 6, wherein the step (b) is performed by culturing the embryoid bodies for 3-8 days while rotating the microgravity simulator at 40-80 rpm.

8. The method according to claim 7, wherein the step (b) is performed by rotating the microgravity simulator first at 40-60 rpm and increasing the rotation speed by 5 rpm every day.

9. The method according to claim 1, wherein the adhesive polymer is selected from a group consisting of hyaluronic acid, alginate, heparin, fucoidan, cellulose, dextran, chitosan, albumin, fibrin, collagen and gelatin.

10. The method according to claim 9, wherein the adhesive polymer is gelatin.

11. Mesenchymal stem cells produced by the method according to claim 1.

12. A composition for treating a bone or cartilage disease, comprising the mesenchymal stem cells according to claim 11 as an active ingredient.

13. A composition for treating an inflammatory or autoimmune disease, comprising the mesenchymal stem cells according to claim 11 as an active ingredient.

14. The composition according to claim 13, wherein the inflammatory or autoimmune disease is rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, cryptogenic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD) or Crohn's disease.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0059] FIG. 1 schematically illustrates a protocol of the present disclosure whereby MSCs are differentiated from iPSCs.

[0060] FIG. 2 shows the images of embryoid bodies (EBs) formed on AggreWell.

[0061] FIG. 3 shows spheroids formed using the microgravity bioreactor BAM (Bio Array Matrix) (top) and a result of staining them with OCT4 and DAPI antibodies (bottom).

[0062] FIG. 4 shows the morphology of mesenchymal stem cells derived from spheroids. It can be seen that the cells exhibit a spindle shape after passaging (right).

[0063] FIG. 5 shows the morphology of MSCs differentiated by a method of the present disclosure depending on passages.

[0064] FIGS. 6a-6c show the cumulative cell proliferation of mesenchymal stem cells differentiated by a method of the present disclosure. Cumulative population doubling (CPD) (FIG. 6a), doubling time (FIG. 6b) and logarithmic cell number (FIG. 6c) depending on passages are shown.

[0065] FIG. 7 shows a result of confirming the expression of cell surface markers in mesenchymal stem cells differentiated by a method of the present disclosure by FACS analysis.

[0066] FIG. 8 shows a result of confirming that mesenchymal stem cells differentiated by a method of the present disclosure differentiated into fat, bone and cartilage through Alizarin Red S, Oil Red O and Alcian blue staining.

[0067] FIG. 9 shows a result of confirming that pluripotent stem cells induced by a method of the present disclosure lose pluripotency and differentiate into cells expressing the markers of mesenchymal stem cells by immunocytochemistry.

[0068] FIG. 10 schematically illustrates an experimental procedure of confirming the anti-inflammatory effect of stem cells differentiated from LPS-induced inflammatory cells by a method of the present disclosure.

[0069] FIG. 11 shows a result of confirming the expression of inflammatory markers by RT-PCR.

BEST MODE

[0070] Hereinafter, the present disclosure will be described in more detail through examples. The examples are only for describing he present disclosure specifically, and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.

Examples

Experimental Method

[0071] Culturing of Single Colonies

[0072] Single colonies were formed by culturing single iPSCs adhered onto a Matrigel (354234, Corning, USA)-coated 96-well plate for a week using an iPSC medium. Each colony was passaged using Matrigel-coated 24-well dish and 6-well dish, subsequently. When the cell number was increased to about 1×10.sup.5, the cells were used for spheroid experiments (FIG. 1).

[0073] Formation of Spheroids Using BAM System

[0074] After seeding about 1×10.sup.5 iPSCs onto an AggreWell plate (34460, StemCell, Canada), the cells were aggregated by centrifuging at 300 g for 5 minutes and then cultured in a 5% CO.sub.2 incubator for 24 hours to form embryoid bodies (EBs). The EBs were cautiously transferred into a bioreactor (CelVivo, Denmark) and rotated for 5 days using the microgravity device BAM (CelVivo, Denmark). The rotation was started at 50 rpm and the rotation speed was increased by 5 rpm every day (FIG. 1).

[0075] Production of iPSC-MSCs

[0076] The spheroids grown in the BAM system were transferred to a 0.1% gelatin-coated 6-well culture dish and cultured using DMEM/F12 supplemented with 10% FBS and 1% P/S while replacing the medium every 2-3 days. When the cells grew from the spheroids attached to the coated surface to 70-80% confluency, they were passaged using TrypLE. The first passage was denoted as P0, and the passaging was continued until homogenous cell morphology was achieved.

[0077] Investigation of Pluripotency of Mesenchymal Stem Cells by Immunohistochemical (ICC) Staining

[0078] The spheroids formed from the iPSCs were taken out of the bioreactor and fixed with 4% paraformaldehyde (PFA). 1×10.sup.5 iPSC-MSCs formed from the spheroids of the iPSCs were seeded onto a confocal dish (101350, SPL, Korea) and then fixed with 4% PFA at 60-70% confluency. The fixed spheroids and iPSC-MSCs were washed 3 times with DPBS for 5 minutes and the surface was permeabilized by treating with 0.3% Triton X-100. Then, after washing 3 times with DPBS for 5 minutes, the washed spheroids were cultured with 3% BSA/PBS at room temperature for 1 hour for blocking. After removing 3% BSA/PSB, the spheroids were reacted with the primary antibodies (1:200) anti-OCT4, anti-SSEA4 and anti-PDGFRβ for 12 hours in a refrigerator. Then, the spheroids were taken out and washed 3 times with DPBS for 5 minutes. After the washing, the spheroids were incubated with the secondary antibody (1:200) goat anti-mouse 488 at room temperature for 1 hour. Then, after removing the secondary antibody and staining the nuclei with DAPI or Topro3 for 20 minutes, the spheroids were washed 3 times with DPBS for 5 minutes. The washed sample was stored in an antifade mounting medium (H-1000, Vector Laboratory, UK) to prevent loss of fluorescence.

[0079] Passaging of iPSC-MSCs and Cell Growth Curves

[0080] When the morphology of the iPSC-MSCs became homogenous, the cell number was counted to create a growth curve. After seeding 2×10.sup.5 iPSC-MSCs onto a 60-mm culture dish from P5, the cells were cultured for 5 days while replacing the medium once in 5 days. The cell proliferation was investigated with three growth curves. First, cumulative population doubling (CPD) was calculated by “CPD=log(number of harvested cells/number of seeded cells)/log(2)”. Then, doubling time, i.e., the time spent until the cell number was doubled, was calculated by “doubling time=days of culturing×log(2)/(log(number of harvested cells)−log(number of seeded cells))”. And, the logarithm of the number of passaged cells was calculated by “cell number=log(number of cells)”. The calculated values were plotted as graphs. AD-MSCs and hWJ-MSCs were used as control groups.

[0081] Immunophenotype Testing by Flow Cytometry Analysis (FACS)

[0082] After detaching cells during culturing using TrypLE™ Express (10624013, GIBCO, USA) and centrifuging at 1,500 rpm for 5 minutes, the supernatant was removed and suspended in a FACS buffer (D-PBS supplemented with 2% FBS), and then reacted primarily with mouse anti-CD34, mouse anti-CD45, mouse anti-CD73 and sheep anti-CD90. The cells were incubated at 4° C. for 30 minutes after adding 200 μL of these primary antibodies diluted to 1:500. Then, after washing with D-PBS and centrifuging at 1,500 rpm for 5 minutes, the supernatant was removed and incubated at 4° C. for 20 minutes after adding 200 μL of rabbit anti-mouse 488 or donkey anti-sheep PE, as a secondary antibody, diluted to 1:500. Then, the stained cells were subjected to flow cytometry analysis (FACS Calibur; Becton Dickinson, Heidelberg, Germany) by suspending in 500 μL of a FACS buffer, by using the Cell Quest pro software.

[0083] Induction of Differentiation into Bone, Fat and Cartilage

[0084] In order to induce differentiation into three lineages, the cells were adhered onto a 24-well plate at 2×10.sup.4 cells/well and differentiation was started when the density reached 80%.

[0085] For osteogenic differentiation, 10% FBS, 1% penicillin/streptomycin (P/S), 100 nM dexamethasone (Sigma-Aldrich, MO, USA), 50 μg/mL ascorbate-2-phosphate (Sigma-Aldrich, MO, USA) and 10 mM β-glycerophosphate (Sigma-Aldrich, MO, USA) were added to DMEM (Dulbecco's modified Eagle's medium)-low glucose (Invitrogen, CA, USA). The differentiation medium was replaced every 2 days for 2 weeks. When the differentiation was completed, the cells were fixed with 4% PFA for 15 minutes and then washed with sterile water. The differentiation was confirmed by staining calcium phosphate deposits with Alizarin Red S.

[0086] For adipocyte differentiation, 10% FBS, 1% P/S, 500 μM isobutylmethylxanthine, 1 μM dexamethasone, 100 μM indomethacin and 10 μg/mL insulin were added to DMEM-high glucose. The differentiation medium was replaced every 3 days for 2 weeks. When the differentiation was completed, the cells were fixed with 4% PFA for 15 minutes and then washed with sterile water and then with 60% isopropanol. The differentiation was confirmed by staining lipid deposits in the cells with 5% (wt/vol) Oil Red O diluted in isopropanol.

[0087] For chondrogenic differentiation, 2% FBS, 1% P/S, 50 μg/mL ascorbate-2-phosphate, 100 μg/mL sodium pyruvate, 1% insulin-transferrin-selenium (ITS, GIBCO), 100 nM dexamethasone, 40 μg/mL L-proline and 10 ng/mL TGF-β3 (Prospec, East Brunswick, N.J., USA) were added to DMEM-high glucose. The differentiation medium was replaced every 2 days for 2 weeks. When the differentiation was completed, the cells were fixed with 4% PFA for 15 minutes and then washed with sterile water. The differentiation was confirmed by staining acidic mucopolysaccharides such as glycosaminoglycan with Alcian blue.

[0088] Inflammatory Cell Models

[0089] Raw 264.7 cells, which are macrophages used in inflammatory cell models, were used to evaluate the anti-inflammatory activity of the iPSC-MSCs of the present disclosure. The Raw 264.7 cells were cultured in an α-MEM (minimum essential medium) supplemented with 10% FBS and 1% P/S. First, after culturing the iPSC-MSCs in a culture vessel for 48 hours, the conditioned medium was collected, filtered through a 0.20-μm syringe filter and then kept in a refrigerator at 4° C. The Raw 264.7 cells were inoculated onto a 6-well culture dish at 3.75×10.sup.5 cells per well. 12 hours later, when the cells adhered to the bottom, the medium was replaced with the conditioned medium prepared above. 12 hours later, all the groups including the conditioned medium group, except for a control group, were treated with 200 ng/mL LPS (lipopolysaccharide, Sigma, USA) as shown in FIG. 10. A positive control groups were treated with LPS and 1 μM DEX (dexamethasone, Peprotech, USA). 7 hours later, images were recorded and total RNAs were isolated. Then, RT-PCR was conducted using the inflammatory marker IL-6.

[0090] Isolation of Total RNAs and RT-PCR

[0091] The total RNAs of the Raw 264.7 cells were extracted using a Labo Pass kit and TRIzol (Cosmo Genetech, Seoul, Korea) according to the manufacturer's instructions. The concentration of the total RNAs was measured using a Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington Del., USA). Then, cDNAs were synthesized using 2 μg of the total RNAs and M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. The RT-PCR reaction was analyzed on 2% agarose gel. The sequences of the used primers are given in Table 1:

TABLE-US-00001 TABLE 1 Sequences of primers used in RT-PCR Genes Forward Reverse IL-6 GTC CTT CCT ACC CCA TAA CGC ACT AGG TTT GCC ATT TCC A GA GAPDH CTC ACT CAA GAT TGT GTC ATC ATA CTT GGC AGG CAG CA TT

Experimental Result

[0092] Formation of Spheroids Using BAM System

[0093] The iPSCs placed on the AggreWell plate were identified to form spherical EBs with homogenous morphology and size 24 hours later (FIG. 2).

[0094] Investigation of Pluripotency of Spheroids by Immunohistochemical Staining

[0095] The spheroids were stained green when stained with the pluripotency marker OCT4, and the nuclei were stained green and blue specifically when stained with DAPI. Therefore, it was confirmed that OCT4 is expressed in the nuclei and the spheroids derived from the iPSCs retain pluripotency (FIG. 3).

[0096] Production of iPSC-MSCs

[0097] It was confirmed that the spheroids (black arrows) adhere to the bottom of the 0.1% gelatin-coated culture dish and cells protrude therefrom (white arrows). The protruding cells were increased with time. When the cells were passaged at 70-80%, the cells showed a uniform spindle shape (white broken arrows) as the passaging proceeded (FIG. 4).

[0098] Passaging of iPSC-MSCs and Cell Growth Curves

[0099] The iPSC-MSCs showed a spindle shape from P5 and could be passaged until P13 (FIG. 5). The cells were prepared into a stock solution and stored in LN.sub.2. The cell growth curves were compared with hWJ-MSCs and AD-MSCs as control groups. As a result, the AD-MSCs showed increase in cell number up to P9 followed by decrease, whereas the hWJ-MSCs showed consistent increase even at P13. The iPSC-MSCs showed remarkably higher CPD and a larger number of cumulative cells than the control groups, and also showed a faster doubling time than the control groups (FIG. 6).

[0100] Immunophenotype Testing by Flow Cytometry Analysis (FACS)

[0101] As a result of FACS analysis, the iPSC-MSCs were confirmed to be positive for anti-CD73 and anti-CD90 and negative for anti-CD34 and anti-CD45, unlike the control group AD-MSCs (FIG. 7). Through this, it was confirmed that the iPSC-MSCs produced using the BAM system have the distinct characteristics of mesenchymal stem cell.

[0102] Induction of Differentiation into Bone, Fat and Cartilage

[0103] The AD-MSCs and iPSC-MSCs were induced to differentiate into bone, fat and cartilage. As a result of staining with Alizarin Red S, Oil Red O and Alcian blue after 2 weeks of differentiation, it was confirmed that the iPSC-MSCs were differentiated into bone, fat and cartilage similarly to the AD-MSCs used as a control group. Especially, bone formation was achieved effectively (FIG. 8).

[0104] Identification of iPSC-MSCs as Mesenchymal Stem Cells by Immunohistochemical Staining

[0105] The iPSC-MSCs were subjected to ICC using the pluripotency markers OCT4 and SSEA4 and the mesenchymal stem cell marker PDGFRβ. The iPSC-MSCs showed a small number of the cells stained green with the OCT4 and SSEA4 markers but a large number of the cells stained green with the PDFGRβ marker. Through this, it was confirmed that the iPSC-MSCs lost pluripotency and were converted to mesenchymal stem cells.

[0106] Inflammatory Cell Models

[0107] Round cells were observed in a group not treated with LPS, whereas large multi-nucleated cells were observed in the group in which inflammation was induced by treating with LPS. Meanwhile, multi-nucleated cells were not observed in the positive control group treated with LPS and DEX, and the large multi-nucleated cells were also nonexistent in the group treated with the conditioned medium of the iPSC-MSCs. As a result of investigating the expression of IL-6 by RT-PCR, the expression level was high as 98% in the LPS-treated group and low as 14% in the group treated with LPS and DEX. The expression level was 65% in the group treated with the conditioned medium of the iPSC-MSCs. Accordingly, it was confirmed that IL-6 is expressed at a lower level in the group treated with the conditioned medium of the iPSC-MSCs than the group treated with LPS (FIG. 11)

[0108] While specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are mere specific exemplary embodiments and the scope of the present disclosure is not limited by them. It is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.