Precursor Cells Of Induced Pluripotent Stem Cell (IPSC)-Derived Mesenchymal Stem Cells And Method For Preparing The Same

Abstract

The present disclosure relates to precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells and a preparation method therefor. The precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells of the present disclosure have enhanced functionality and excellent proliferative capacity compared with typical mesenchymal stem cells or induced pluripotent stem cell-derived mesenchymal stem cells.

Claims

1-10. (canceled)

11. A method for preparing cells of induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells, the method comprising: culturing induced pluripotent stem cells in a medium containing FBS and bFGF for 1 to 10 days; and isolating SSEA-4 (−) cells and culturing the SSEA-4 (−) cells in a medium containing FBS and bFGF for 1 to 10 days.

12. The method of claim 11, wherein the medium contains 5 to 20% (v/v) FBS.

13. The method of claim 11, wherein the medium contains 1 to 20 ng/ml bFGF.

14. A method for producing exosomes, the method comprising: culturing induced pluripotent stem cells in a medium containing FBS and bFGF for 1 to 10 days; isolating SSEA-4 (−) cells and culturing the SSEA-4 (−) cells in a medium containing FBS and bFGF for 1 to 10 days; further culturing the cultured SSEA-4(−) cells in a cell culture medium; and separating exosomes from the cell culture medium.

15. The method of claim 14, wherein the cell culture medium contains exosome-depleted bovine fetal serum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIGS. 1a and 1b show the expression rates of the cell surface expression marker SSEA-4 in the induced pluripotent stem cells (iPSC) on day 0 and after 7-day culture and microscopic images of the cells.

[0073] FIG. 1c shows the expression rate of the cell surface expression marker SSEA-4 in the cells (BxC) of the present disclosure obtained by culturing the induced pluripotent stem cells for 7 days, isolating cells corresponding to SSEA-4 (−), and then culturing the isolated cells for 7 days, and microscopic images of the cells.

[0074] FIG. 2 is a graph showing the types of cell surface markers in the induced pluripotent stem cells on day 0 (iPSC), the induced pluripotent stem cells after 7-day culture (pre-BxC), the cells after SSEA-4 (−) isolation and then 7-day culture (BxC), and mesenchymal stem cells (MSC).

[0075] FIG. 3 shows karyotyping results of the precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells (BxC) of the present disclosure and the same kind of tissue-derived mesenchymal stem cells (MSC).

[0076] FIG. 4a shows the number of cells through passage culture in the precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells (BxC) of the present disclosure and the same kind of tissue-derived mesenchymal stem cells (MSC).

[0077] FIG. 4b shows the comparison of expression rate of the cell proliferative factor Ki-67 between the precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells (BxC) of the present disclosure and the same kind of tissue-derived mesenchymal stem cells (MSC).

[0078] FIGS. 5a, 5b, 5c and 5d shows the comparison of the secretion amounts of functional proteins expressed in the precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells (BxC) of the present disclosure. (5a: endostatin, endothelin-1; 5b: VEGF-A, thrombospondin-2; 5c: PIGF, PDGF-AA; 5d: beta-NGF, HB-EGF)

[0079] FIGS. 6a and 6b show CFU-F assay results to compare the proliferative capacity between the precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells (BxC) of the present disclosure and the same kind of tissue-derived mesenchymal stem cells (MSC).

[0080] FIG. 7a shows a schematic view of the results of RNA-seq analysis performed to investigate genes showing a difference in expression level between the precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells (BxC) and the same kind of tissue-derived mesenchymal stem cells (MSC).

[0081] FIG. 7b shows genes expressed at higher levels in BxC compared with MSC.

[0082] FIG. 7c shows genes expressed at lower levels in BxC compared with MSC.

[0083] FIG. 8a is a graph confirming the number and size distribution of exosomes separated from BxC of the present disclosure.

[0084] FIG. 8b is a graph identifying the expression of the exosome-specific surface antigens TSG101 and CD63.

DETAILED DESCRIPTION

[0085] Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples are only for illustrating the present disclosure more specifically, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these examples.

EXAMPLES

[0086] Throughout the present specification, the term “%” used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt)% for solid/solid, (wt/vol)% for solid/liquid, and (vol/vol)% for liquid/liquid.

Example 1: Isolation and Culture of Precursor Cells of Induced Pluripotent Stem Cell (iPSC)-Derived Mesenchymal Stem Cells (BxC)

[0087] First, induced pluripotent stem cells (iPSC) were cultured in DMEM containing 10% FBS and 10 ng/ml bFGF for 7 days. Then, the stage-specific embryonic antigen 4 (SSEA-4) (−) cells without SSEA-4 protein expression on the cell surface were isolated from the cultured induced pluripotent stem cells through FACS. The isolated SSEA-4 (−) cells were passaged, and further cultured in the same medium as above for 7 days, thereby preparing the precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells of the present disclosure. The present inventors named the precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells as Brexogen stem cells (BxC) and the BxC is deposited under Accession number “KCTC14019BP” to the “Korean Collection for Type Culture (KCTC)”.

[0088] The precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells named BxC were further cultured in a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100×) (Gibco, Cat no. 11140-050)].

[0089] The proportions of the cell surface marker SSEA-4 for respective cell types in the induced pluripotent stem cells on day 0 (iPSC) (DO), the induced pluripotent stem cells after 7-day culture (Pre-BxC) (D7), and the precursor cells of induced pluripotent stem cell-derived precursor cells of the present disclosure (BxC) are shown in Table 1 below and FIGS. 1a to 1c and 2.

[0090] As a result, 96.6% of the initial induced pluripotent stem cell population expressed SSEA-4 protein on the cell surface (FIG. 1a), but after 7-day culture, the SSEA-4 protein-expressing SSEA-4 (+) cell population was reduced to 35.8% of the total cells (FIG. 1b). SSEA-4 (−) cells, which account for the remaining 64.2% of the total cells, were isolated, passaged, and cultured for 7 days (BxC cells). As a result, it was confirmed that SSEA-4 protein-expressing cells were 3% or less of the total cell population (FIG. 1c).

[0091] It can be therefore seen that the expression rate of SSEA-4 protein was 5% or less in the cell population of the induced pluripotent stem cell (iPSC)-derived functionally enhanced mesenchymal precursor cells (BxC).

Example 2: Expression of Cell Surface Marker in Induced Pluripotent Stem Cell (iPSC)-Derived Functionally Enhanced Mesenchymal Precursor Cells (BxC)

[0092] The cell surface markers (SSEA-4, CD34, CD45, CD73, CD90, CD105) for respective cell types in the induced pluripotent stem cells on day 0 (iPSC) (DO), the induced pluripotent stem cell-derived differentiated cells after 7-day culture (Pre-BxC) (D7), the precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells (BxC), and the mesenchymal stem cells (MSC) were analyzed as below.

[0093] The cells on each stage were made into a single cell suspension by treatment with TrypLE™ Express (Gibco, 12604-021), and then washed with PBS. The single cell suspension was divisionally placed in tubes such that the wavelengths of antibodies specifically binding to SSEA-4 (BD560128), CD73 (BD550257), CD90 (BD5555950), CD105 (BD560839), CD45 (BD555483), and CD34 (BD348053) do not overlap, and then centrifuged. The cell pellets were resuspended in 100 μL of FACS buffer, and 6 μL of antibodies were placed together two each such that the wavelengths thereof do not overlap, allowed to stand at room temperature in a dark state for 30 minutes, washed with PBS, resuspended in 300 μL of FACS buffer, and then analyzed by the FACS machine.

[0094] The expression rates of the cell surface markers (SSEA-4, CD34, CD45, CD73, CD90, and CD105) in the stem cells are shown in Table 1 and FIG. 2.

[0095] It can be seen from the above results that the expression rates of CD73, CD90, and CD105 proteins were 90% or more in the cell population of the induced pluripotent stem cell (iPSC)-derived functionally enhanced mesenchymal stem cells (BxC) of the present disclosure.

TABLE-US-00001 TABLE 1 Differentiation SSEA-4 CD34 CD45 CD73 CD90 CD105 Stage (%) (%) (%) (%) (%) (%) iPSC 96.7 0.1 0.7 0.3 0.6 0.9 Pre-BxC 38.8 0.4 0.9 1.3 1.7 3.8 BxC 2.7 1.4 1.2 99.0 96.9 98.6 MSC 0.2 0.3 0.5 99.7 99.2 98.6

Example 3: Analysis of Chromosomes of Induced Pluripotent Stem Cell (iPSC)-derived Functionally Enhanced Mesenchymal Precursor Cells (BxC)

[0096] In order to investigate whether the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure and the induced pluripotent stem cell-derived mesenchymal stem cells (MSC) had chromosome abnormality, BxC and MSC that have been passaged six times were karyotyped.

[0097] It was confirmed as shown in FIG. 3 that the BxC of the present disclosure and MSC had no abnormalities in the number and structures of chromosomes.

Example 4: Comparison of Proliferative Capacity of Induced Pluripotent Stem Cell (iPSC)-derived Functionally Enhanced Mesenchymal Precursor Cells (BxC)

[0098] In order to compare proliferative capacity between the induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells (BxC) of the present disclosure and the induced pluripotent stem cell-derived mesenchymal stem cells (MSC), the following experiment was conducted.

[0099] The induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells (BxC) and the same kind of tissue (umbilical cord)-derived mesenchymal stem cells (MSC) with the same passage number (P4) were seeded in 6-well culture plates at 1×105 cells per well. After the culture media were removed every 3.5 days (twice a week), the cells were washed with PBS, and then detached by treatment with TrypLE™ Express. The cells were centrifuged at 300×g for 5 minutes, and then resuspended in 1 mL of a culture medium, followed by cell counting. After cell counting, the cells were again seeded at 1×105 cells per well, and then an increase in cell count for each passage was investigated while culture was repeated every 3.5 days.

[0100] In order to investigate the expression rate of the cell proliferation indicator Ki67 in each cell type, the following experiment was conducted.

[0101] The induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) and the same kind of tissue (umbilical cord)-derived mesenchymal stem cells (MSC) with the same passage number (P4) were made into a single cell suspension by treatment with TrypLE™ Express (Gibco, 12604-021), and then washed with PBS. After cell washing, the cells were placed in 80% cold ethanol to be fixed at −20° C. for 2 hours or longer, and then washed three times with PBS. Then, 20 μL of Ki-67-specific antibody per 100 μL of buffer, which were contained in the PE Mouse Anti-Ki-67 kit (BD556027), was transferred into a new tube, allowed to stand at room temperature for 30 minutes, washed with PBS, and incubated with an indicator-attached secondary antibody, allowed to stand at room temperature in a dark state for 30 minutes, and analyzed by the FACS machine.

[0102] As shown in FIG. 4a, the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure showed a 10-fold or higher difference in proliferative capacity from the 9th passage, compared with the mesenchymal stem cells (MSC). In addition, the proliferative capacity of the mesenchymal stem cells (MSC) was reduced from the 12th passage, but the proliferative capacity of the induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells (BxC) of the present disclosure was not reduced in spite of 12 or more passages.

[0103] As shown in FIG. 4b, the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure showed at least 2-fold higher in the expression level of Ki67 as a cell proliferation indicator, compared with the mesenchymal stem cells (MSC).

[0104] It can be therefore seen that the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure had a very excellent effect in view of proliferative capacity compared with the mesenchymal stem cells (MSC).

Example 5: Comparison of Protein Secretory Capacity of Induced Pluripotent Stem Cell (iPSC)-derived Functionally Enhanced Mesenchymal Precursor Cells (BxC)

[0105] In order to compare the secretary capacity of functionality-associated proteins between the induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells (BxC) of the present disclosure and the induced pluripotent stem cell-derived mesenchymal stem cells (MSC), the following experiment was conducted.

[0106] The induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells (BxC) and the mesenchymal stem cells (MSC) with the same passage number were seeded in 6-well culture plates at 1×105 cells per well, and after 24 hours, the supernatants were removed. The wells were washed with DPBS, and 1 mL of serum-free medium was added thereto, and after 48 hours, the medium was removed. The dead cells or debris were precipitated by centrifugation at 300×g for 5 minutes, and then only the supernatants were taken, and stored at −80° C. The array kit was fabricated using custom service of Luminex High performance assay by R&D systems, and then Luminex assay was carried out.

TABLE-US-00002 TABLE 2 Protein MSC BxC Endostatin 3516.6 pg/ml 6682.0 pg/ml Endothelin-1 17.0 pg/ml 79.4 pg/ml VEGF-A 32.2 pg/ml 78.5 pg/ml Thrombospondin-2 21802.9 pg/ml 36912.7 pg/ml PIGF 5.8 pg/ml 9.4 pg/ml PDGF-AA 29.2 pg/ml 328.4 pg/ml beta-NGF 10.5 pg/ml 17.3 pg/ml HB-EGF 3.9 pg/ml 15.7 pg/ml

[0107] As shown in FIGS. 5a to 5d, the induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells (BxC) of the present disclosure secreted relatively large amounts of proteins associated with functionality of mesenchymal stem cells (MSC), compared with the induced pluripotent stem cell-derived mesenchymal stem cells (MSC). It can be therefore seen that the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure had enhanced functionality compared with the mesenchymal stem cells (MSC).

Example 6: Investigation on Sternness of Induced Pluripotent Stern Cell (iPSC)-derived Functionally Enhanced Mesenchymal Precursor Cells (BxC)

[0108] In order to compare stemness between the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure and the mesenchymal stem cells (MSC), the following experiment was conducted.

[0109] The mesenchymal stem cells and the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) were seeded in 100 mm-cell culture dishes containing 10 mL of culture medium [high-glucose DMEM (Gibco, Cat no. 11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100×) (Gibco, Cat no. 11140-050)] at 1000 cells per dish, and cultured for 21 days. The cultured cell-attached dishes were washed two times with PBS, and fixed with 95% methanol at room temperature for about 2 minutes. After the fixed cells were washed three times with PBS, a solution of 0.5% crystal violet [5 g of crystal violet (Sigma, C-3886, USA), 100 mL of methanol] was added, and then the cells were stained for 5 minutes, washed with wash, and then dried at room temperature. The number of colonies composed of 50 or more cells was counted in the respective dishes, and compared.

[0110] As shown in FIGS. 6a and 6b, the number of colony forming unit-fibroblasts (CFU-F) in the induced pluripotent stem cell-derived mesenchymal stem cells (MSC) was about three times or greater than that in the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure. It was therefore conformed that the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure had excellent stemness compared with the mesenchymal stem cells (MSC).

Example 7: Investigation on Characteristic Gene Expression Pattern of Induced Pluripotent Stem Cell (iPSC)-derived Functionally Enhanced Mesenchymal Precursor Cells (BxC)

[0111] In order to investigate a difference in gene expression pattern between the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure and the mesenchymal stem cells (MSC), mRNA was extracted from each cell population, and the gene expression level according to the cell type was compared by RNA-seq analysis.

[0112] The kind of gene showing a difference in gene expression level and the difference in gene expression level (gene expression level in BxC/gene expression level in MSC) are shown in Table 2 and FIGS. 7a to 7c.

[0113] As shown in FIGS. 7a to 7c, the gene expression levels of ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4, and ACKR3 were about at least 10-fold and about at most 55-fold higher in the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure compared with an equivalent number of mesenchymal stem cells (MSC).

[0114] Whereas, DHRS3, BMPER, IF16, PRSS12, RDH10, and KCNE4 were at least 10-fold and at most 20-fold less expressed in the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present disclosure.

TABLE-US-00003 TABLE 3 No. Gene Description Fold Change 1 ANKRD1 ankyrin repeat domain 1 55.1 2 CPE carboxypeptidase E 38.8 3 NKAIN4 sodium/potassium transporting ATPase interacting 4 34.3 4 LCP1 lymphocyte cytosolic protein 1 28.8 5 CCDC3 coiled-coil domain containing 3 25.4 6 MAMDC2 MAM domain containing 2 19.9 7 CLSTN2 calsyntenin 2 19.1 8 SFTA1P surfactant associated 1, pseudogene 16.4 9 EPB41L3 erythrocyte membrane protein band 4.1 like 3 16.1 10 PDE1C phosphodiesterase 1C 15.4 11 EMILIN2 elastin microfibril interfacer 2 15.1 12 SULT1C4 sulfotransferase family 1C member 4 13.8 13 TRIM58 tripartite motif containing 58 13.2 14 DENND2A DENN domain containing 2A 12.5 15 CADM4 cell adhesion molecule 4 12.2 16 AIF1L allograft inflammatory factor 1 like 11.6 17 NTM neurotrimin 11.5 18 SHISA2 shisa family member 2 11.0 19 RASSF4 Ras association domain family member 4 10.9 20 ACKR3 atypical chemokine receptor 3 10.1

Example 8: Isolation and Identification of Exosomes Derived from Induced Pluripotent Stem Cell (iPSC)-derived Functionally Enhanced Mesenchymal Stem Cells (BxC)

[0115] The induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC), prepared in Example 1, were further cultured in a culture medium containing 10% exosome-depleted FBS. After cell culture for 72 hours, the BxC cell culture medium was collected, and centrifuged at 300×g for 10 minutes to remove remaining cells and cell debris. The cell culture medium was centrifuged at 10000×g and 4° C. for 70 minutes using a high-speed centrifuge. The centrifuged supernatant was centrifuged at 100,000×g and 4° C. for 90 minutes using an ultracentrifuge to remove the supernatant, and the exosomes remaining below were diluted with phosphate buffered saline (PBS) before use. The separated exosomes were subjected to nanoparticle tracking assay (NanoSight NS300, Malvern), thereby investigating the number and size distribution of exosomes (FIG. 8a). The exosome-specific surface antigens TSG101 and CD63 were identified by western blot (FIG. 8b).

[0116] [Depository Authority] Korean Collection for Type Culture (KCTC)

[0117] [Accession Number] KCTC14019BP

[0118] [Date of Deposition] Nov. 11, 2019