KR101768581B1 - Generation of Reprogramming Cell Line from Single Cells of spontaneously Immortalized Somatic cell - Google Patents

Abstract

The present invention relates to a method for producing single cell-derived pluripotent stem cells from spontaneous immortalized cells. The present invention also relates to spontaneously immortalized somatic cells and single cell-derived induced pluripotent stem cells produced by the above method.
The monoclonal-derived pluripotent stem cells prepared according to the present invention are capable of reprogramming by spontaneous immortalization and are expected to be used as a stable cell therapy agent because they have homogeneous characteristics derived from a single cell .

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a reprogramming cell line from a single cell of a spontaneously immortalized somatic cell,

The present invention relates to a method for producing single cell-derived pluripotent stem cells from spontaneous immortalized cells.

The present invention also relates to spontaneously immortalized somatic cells and single cell-derived induced pluripotent stem cells produced by the above method.

It is known that 24 genes involved in the maintenance of embryonic stem cells are selected and induced to differentiate into four genes (Oct4, Sox2, c-Myc, and Klf4) involved in the differentiation among them using viral vector transfer method (Yamanaka, Cell 126: 633-678, 2006). These four degenerative transcription factors delivered by retrovirus were expressed in rat fibroblasts, and induced somatic cells to induce induced pluripotent stem cells. In addition, another study succeeded in the reprogramming of human somatic cells by transferring Oct4, Sox2 and novel factors Lin28 and Nanog among these four degenerated transcription factors to lentivirus (Yu J, Science 318 (5858): 1917 -20, 2007). Therefore, Oct4 and Sox2 are commonly used in six types of transcription factors (Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28) used for the differentiation and c-Myc, Klf4, Nanog, Establishes cells and plays an important role in the process of reprogramming. In addition, the gene is essential for maintaining pluripotency, self renewal, and undifferentiation of embryonic stem cells. Transcription factors expressed by these genes are required to activate each other, induce differentiation, and inhibit self-proliferation It plays a role of inhibiting.

The Oct-4 transcription factor is one of the octamer transcription factors and a member of the Class V POU domain of the POU (Pit-Oct-Unc) family. Oct4 is expressed only in embryonic stem cells and embryonic tumor cells, which are undifferentiated cells, and plays an important role in maintaining the pluripotency of stem cells. When the expression level of Oct-4 is normal, the embryonic stem cell maintains its pluripotency and pluripotency. However, when it is increased beyond the normal level, it is differentiated into primitive endoderm and primitive mesoderm, Oct-4 is an important factor that regulates the pluripotency and differentiation of embryonic stem cells, and Oct-4 interacts with Sox2 transcription factor to produce Oct-4 And the expression of Fgf4, Utf-1, Nanog, and Sox2 genes, which are subgeneric genes, in synergistic effect.

The genes of the Sox family are also associated with multipotent stem cells or unipotent stem cells, of which the Sox2 (SRY-type high mobility group box 2) transcription factor is an HMG box DNA binding motif , Which plays an important role in maintaining the pluripotency of embryonic stem cells among the 20 Sox family proteins. Similar to Oct-4, inhibition of Sox2 expression in mouse embryonic stem cells induces differentiation, and Sox2 binding sites found in the promoter region of several Sox2 subgenomes are often present adjacent to Oct-4 and Nanog binding sites. Therefore, the interaction between the Sox2 transcription factor and the Oct-4 transcription factor maintains the undifferentiated stem cell undifferentiated state and possesses the characteristics of embryonic stem cells.

The Helix-loop-helix / leucine zipper transcription factor, c-Myc, is a carcinogenic gene that performs various intracellular functions such as cell growth, differentiation, proliferation, transformation into cells and cancer cells, Mechanism of LIF (leukemia inhibitory factor) / STAT3 and Wnt signaling mechanism. The c-Myc transcription factor plays a role in inhibiting cell proliferation by activating the p21 gene in the degenerated stem cell by the Klf4 gene. In addition, c-Myc binds to the Myc recognition site present in the genome, and also induces the histone acetylase complex to change the chromatin structure so that Oct-4 and Sox2 bind well to the target gene do.

Klf4, a member of the Klf family, was used by the Yamanaka group and the Jaenisch group to produce degenerated stem cells of mice and humans. However, the Thomson group produced human degener- ated stem cells without Klf4, suggesting that Klf4 is not a necessary gene for the production of human degener- ated stem cells. Klf4 is a Kruppel-type zinc-finger transcription

As a factor, it plays a role in regulating growth and cell cycle. c-Myc, which acts as a sub-gene of STAT3 in embryonic stem cells and maintains Oct-4 expression in overexpression, inhibits the differentiation of mouse embryonic stem cells. In addition, Klf4 is involved in the regulation of Lefty1 expression, known as the Oct-4 subgenus, in embryonic stem cells, and Sox2 and Klf4 are involved and suppress the function of the p53 anti-cancer gene. Therefore, Klf4 is expected to increase the activity of Nanog, an embryo-specific gene, indirectly by inducing apoptosis induced by c-Myc. Klf4 as well as Klf2 can be used for the production of human degenerative stem cells, and related genes Klf1 and Klf5 can also be used for the production of human degenerative stem cells, but they are reported to be less efficient than Klf4 or Klf2.

Although various methods have been tried to promote reprogramming of cell reprogramming, the efficiency is still low. In addition, heterogeneity of somatic cells can also cause significant problems in cell reprogramming. Accordingly, drug-induced reprogramming systems have been used to solve these problems.

The present inventors have solved the problems of conventional cell reprogramming systems and established a single-cell expansion strategy. Specifically, a Dox-inducing somatic reprogramming cell line was successfully prepared from MEF single cells, and the present invention was completed.

Therefore, one aspect of the present invention is to provide a method for producing single cell-derived pluripotent stem cells from spontaneous immortalized cells.

In addition, one aspect of the present invention is to provide a spontaneously immortalized somatic cell or single cell-derived induced pluripotent stem cell produced by the above method.

One aspect of the present invention is a method for producing monoclonal-derived pluripotent stem cells from spontaneous immortalized cells, the method comprising the steps of:

(1) culturing somatic cells for 5 to 20 weeks;

(2) selecting cells except for the dead or aged cells in the step (1);

(3) transducing a cell of step (2) with a de-differentiation factor; And

(4) seeding the transduced cells of step (3) in a single cell unit on a plate.

As used herein, the term " stem cells " is undifferentiated cells capable of differentiating into various body tissues, including totipotent stem cells, pluripotent stem cells, multipotential stem cells multipotent stem cells). The stem cells may be used in combination with terms such as precursor cell progenitor cells and the like.

The term " induced pluripotent stem cells " used herein refers to artificially derived pluripotent stem cells obtained by inserting a specific gene from non-totally differentiable cells (for example, somatic cells). The inducible pluripotent stem cells are capable of expressing pluripotent stem cells in the presence of stem cell gene and protein expression, chromosome methylation, doubling time, embryoid body formation, teratoma formation, viable chimera formation, For example, embryonic stem cells).

According to a preferred embodiment of the present invention, the induced pluripotent stem cells used in the present invention are derived from human, bovine, horse, goat, sheep, dog, cat, mouse, rat or alga, It is a cell.

The induced pluripotent stem cells used in the method of the present invention may be autologous or allogenic to the subject to be derived.

In the present invention, the step of culturing somatic cells in step (1) for 5 to 20 weeks spontaneously immobilizes the cells. The cells can be cultured for 5 to 20 weeks, more preferably 6 to 15 weeks, and most preferably 6 to 8 weeks.

The step (1)

(1-1) culturing somatic cells without changing medium for 2-4 weeks; And

(1-2) exchanging the medium with the cultured somatic cells at intervals of 5 to 10 days.

Cells other than the killed or senescent cells of step (2) may be free from apoptosis, cell senescence or morphological changes at 30 to 50 passages.

In a preferred embodiment of the present invention, 5 passaged somatic cells were seeded on a 60 mm dish and cultured in MEF medium without medium change for 3 weeks. The MEF medium was then replaced every 7 days. Two months later, the cells were transferred to a 100 mm dish. Most of the cells in the cells are aged or killed, whereas some cells exhibit the property of increasing their growth again. In this invention, the immortalized cells without this separate treatment are referred to as spontaneously immortalized cells.

In the present invention, the method of introducing the dedifferentiation factor of step (3) can be easily constructed through various methods known in the art (Takahashi K, Yamanaka S (August 2006). Induction of pluripotent stem cells cell mouse embryonic and adult fibroblast cultures by defined factors ". Cell 126 (4): 66376). For example, mouse embryonic fibroblasts (MEFs) treated with mitomycin-C to inhibit proliferation are included as support cells and supplemented with fetal bovine serum (FBS), beta-mercaptoethanol and non-essential amino acids After culturing in a suitable medium such as Dulbecco's Modified Eagle's Medium (DMEM), induced pluripotent stem cells can be obtained by passaging in serum-free, β-mercaptoethanol, non-essential amino acids and Knockout-DMEM supplemented with bFGF2. (Tae-Hee Lee et al., &Quot; Functional Recapitulation of Smooth Muscle Cells Via Induced Pluripotent Stem Cells From Human Aortic Smooth Muscle Cells " Circulation Research (2010) (106: 120-128)) as known inducible pluripotent stem cell lines. But the present invention is not limited thereto.

In the above step (3), the kind of the reprogramming factor is not limited, but may be preferably Oct4, Sox2, KLF4 and C-Myc.

In addition, the dedifferentiation factor of step (3) may be included in a toxicycline or tetracycline inducible vector.

This is an expression system capable of determining the expression of a dedifferentiating factor depending on the presence or absence of tetracycline or doxycycline, whereby the method further comprises the step of (5) treating the cell-seeded plate with doxycycline or tetracycline .

In addition, the cell seeded plate may be further treated with a MEK inhibitor, a GSK-3 beta inhibitor, or a HDAC (Histone deacetylase) inhibitor.

The MEK inhibitor means a compound or medicament that inhibits MEK1 and / or MEK2 (mitogen-activated protein kinase kinase enzymes), and includes, for example, Trametinib, Selumetinib, Binimetinib, PD-325901, Cobimetinib , CI-1040, PD035901, and the like.

In addition, the GSK-3 beta (Glycogen synthase kinase 3 beta) inhibitor is a substance including compounds, medicines, etc. inhibiting the serine / threonine protein kinase GSK-3? A metal such as beryllium, copper, lithium, mercury or tungsten, 6-BIO, Dibromocantharelline, Hymenialdehyde, Indirubins, Meridianins, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR- , Cazpaullone, Kenpaullone, Manzamine A, Palinurine, Tricantine, TDZD-8, NP00111, NP031115, and the like.

In addition, the HDAC (Histone deacetylase) inhibitor is a substance including a compound or a drug that interferes with the function of HDAC,

HDAC inhibitors include, but are not limited to, (1) short chain fatty acids such as butyrate, 4-phenylbutyrate or valproic acid; (2) hydroxamic acids such as suroyl anilide hydroxamic acid (SAHA), bialyl hydroxamate A-161906, bicyclic aryl-N-hydroxycarboxamide, CG-1521, PXD- 101, sulphonamide hydroxamic acid, LAQ-824, oxfutlatin, scriptide, m-carboxycinnamic acid bishydroxamic acid, trypsin-hydroxamic acid analog, trichostatin A (TSA) , m-carboxy cinnamic acid bis-hydroxamidoxyoplatin (CBHA), ABHA, sulptide, piroxamide, and propenamide; (3) Epoxy ketone-containing cyclic tetrapeptides such as, for example, trypsin, apidicine, depsipeptide, HC-toxin, clamidosine, diheteropeptin, WF-3161, Cyl-1 and Cyl-2; (4) benzamide or non-epoxy ketone-containing cyclic tetrapeptides such as FR901228; (CHAP), benzamide, MS-275 (MS-27-275), and CI-994; (5) depudesin; (6) PXD101; And (7) organic sulfur compounds. Additional exemplary HDAC inhibitors include TSA, TPXA and B, oxampellatin, FR901228 (FK228), traxin B, CHAPl, aroyl-pyrolyl hydroxy-amide (APHA), apicidine, .

In addition, one aspect of the present invention relates to spontaneously immortalized somatic cells or single cell-derived induced pluripotent stem cells produced by the method.

The spontaneously immortalized somatic cells may be those produced by steps (1) and (2) of the method, preferably with the deposit number KCLRF-BP-00348.

In addition, the inducible pluripotent stem cells may be those prepared by the above-described method, preferably those derived from the spontaneous immortalized somatic cells of the deposit No. KCLRF-BP-00348.

The monoclonal-derived pluripotent stem cells prepared according to the present invention are capable of reprogramming by spontaneous immortalization and are expected to be used as a stable cell therapy agent because they have homogeneous characteristics derived from a single cell .

Brief Description of the Drawings Figure 1 shows the reprogramming of siOG2-MEF.
(A) Shows the morphology of Dox-induced MET in siOG-MEFs and siOG-MEFs.
(B) iPSCs prepared from siOG-MEFs by inducible OSKM, AP, alkaline phosphatase.
(C) Teratoma formed from iPSCs. Through H & E staining for the teratoma section, mouse iPSCs are differentiated into various tissues from all three germ layers.
(D) Embryonic development and X-gal staining of iPSCs.
Figure 2 shows a method for preparing a Dox-inducible reprogramming clone from siOG-MEFs.
(A) is a schematic diagram of a method for producing a Dox-inducible mouse reprogramming clone.
(B and F). ≪ / RTI >
(C and G) Dox treatment. Cells grown on MEF medium were treated with Dox for 3 days.
Lt; RTI ID = 0.0 > (D < / RTI > and H) GC5 cells. Cells grown in MEF medium were treated with Dox for 2 days and permeated with methanol for Oct4 staining.
(E and I) colony formation assays. 400 cells were seeded on MEF feeder cells in a 6-well plate. The next day, cells were treated with Dox and maintained in MEF for 3 days. Subsequently, the MEF medium was replaced with KSR / LIF / Dox medium, and the cells were further incubated for 11 days.
Fig. 3 shows the result of measuring the verification and reprogramming efficiency derived from a single clone of the reprogramming clone.
(A) shows the results of PCR analysis on genomic DNA. Clone specific primers were determined by chromosome gait analysis.
(B) Single-cell PCR analysis. Single cells were isolated and PCR was performed directly without DNA purification.
(C) Dox-inducible AP expression in GC5 cells. Cells (4 × 10 ² ) were seeded onto MEF feeder cells and incubated in the presence of Dox (4 μg / ml). Two weeks later, cells were stained with an AP staining kit. The AP-expression colonies were counted using an optical microscope. Three independent results were expressed as mean ± SD.
(D) Dox-inducible GFP expression in GC5 cells. Cells (4 × 10 ² ) were seeded onto MEF feeder cells and incubated in the presence of Dox (4 μg / ml). After 2 weeks, GFP-expressing colonies were counted under a fluorescence microscope. Three independent results were expressed as mean ± SD.
Fig. 4 is a graph showing reprogramming efficiency by chemical treatment on GC5 cells. Fig.
(A and B) flow cytometry. Three independent results were expressed as mean ± SD. The chemicals treated were PD (PD0325901; MEK inhibitor), CHIR (CHIR99021; GSK-3 inhibitor), TSA (Trichostatin A; Histone deacetylase inhibitor), VPA (Valproic Acid; Histone deacetylase inhibitor) inhibitor), Aza-C (5-Azacytidine, DNA methyltransferase inhibitor), RG108 (DNA methyltransferase inhibitor), BIX (BIX01294, Histone lysine methyltransferase inhibitor), SB431542 (TGF- -specific demethylase 1 inhibitor), and SB202190 (P38 inhibitor).
5 is a view showing signal transmission of the cell reprogramming.
(A) the result of a phospho-kinase antibody array. GC5 cells were incubated in MEF medium containing Dox (4 [mu] g / ml) for 3 days and analyzed using a human phospho-kinase antibody array.
(B) shows the relative intensity of a spot. Membranes containing phospho-kinase antibodies were analyzed using ImageJ software.
(C) Western blot assay results. Cells were incubated in MEF medium containing Dox (4 [mu] g / ml) for 3 days and Western blot analysis was performed. J1 mESCs and iPSCs were cultured in fresh mES / LIF medium 1 day before elution and Western blotting was performed. Cells differentiated from siOG-MEFs and iPSCs (iPS-DCs) were cultured in fresh MEF medium 1 day before elution and Western blotting was performed. P-2: passage-2.
(D) GC5 cells grown in 6-well plates were treated with PD for 16 h on MEF medium and eluted for Western blotting. Alternatively, PD-treated cells were washed with fresh E6 medium without removal of trypsin to remove PD and MEF medium and incubated in E6 / LIF / Dox medium for 8 days. Three independent results were expressed as mean ± SD. PD: PD0325901.
Figure 6 is a schematic diagram of a Dox-inducible system. (SF1)
Figure 7 shows the preparation and Western blotting of a Dox-inducible reprogramming clone from siOG-MEFs. NC means unchanged cells.
Fig. 8 shows the results of promoting E-cadherin expression and Dox-inducible growth.
(A) Cells grown in a 24-well plate were treated with Dox (4 ㎍ / ml) for 3 days in MEF medium, permeabilized with methanol, and stained with E-cadherin antibody (Green). The nuclei were stained with DAPI (Blue).
(B) Cells were infected with retrovirus for GFP. 400 cells were seeded into MEF feeder cells as single cells and maintained in MEF medium in the presence or absence of Dox (4 [mu] g / ml). After 2 days of incubation, the number of grown cells was counted under an optical microscope at 400x magnification. Results are expressed as mean ± SD (n = 15).
FIG. 9 is a diagram showing the genomic insertion site of the lentivirus transfer gene in GC5 and JC5 cells. The primers for confirming the insertion were indicated as red (mouse genome sequence) and blue (insertion lentivirus sequence).
Figure 10 shows a profile of a human phospho-kinase antibody array. R & D Systems, Inc. (# ARY003B) human phospho-kinase antibody array.
Fig. 11 shows a human iPSCs phospho-kinase antibody array.
(A) Human iPSCs phospho-kinase antibody arrays were performed by the methods described in the Examples. 1: p-Stat3, 2: p-ERK1 / 2, 3: p-
(B) shows the relative intensity of a spot. Membranes containing phospho-kinase antibodies were analyzed using ImageJ software.
(C) Western blot analysis.
Figure 12 shows signaling in the reprogramming of GC5 cells.
GC5 cells were maintained in the presence or absence of Dox (4 ug / ml) or in non-existing MEF medium and the vesicles were changed daily in fresh medium. After a defined period, cells were eluted and Western blot was performed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Preparation of reagents

Mouse LIF ProSpec Ltd. (Ness-Ziona, Israel). E-cadherin (# sc-7870), ERK1 (# sc-93), c-Myc (# The first day for P-ERK (# sc-7383), P-Akt / Thr 308 (# sc-16646), Akt (# sc-8312), P- Antibodies were obtained from Santa Cruz Biotechnology, Inc. (San Diego, CA). P-Akt / Ser 473 (# 4058), P-P38 (# 4511), P38 (# 9212), P-JNK (# 4668), JNK (# 9252), P- ≪ / RTI ># 9363) was purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Example  2: Cell culture

Feeder MEFs and i4F-MEFs with CF1 and R26 ^rt TA; Col1al DMEM containing syringe Life Technologies, Inc., Carlsbad, CA ), 1% Waist and streptomycin; ^4F2A mouse iPS separated from (Jackson Laboratory, Sacramento, CA), and this MEF media (10% FBS (# 16000-044 (# 10-017-CVR; Corning, Inc., Manassas, Va.). 293-FT cells were also cultured on MEF medium. J1 mouse ESCs were purchased from ATCC (# SCRC-1010; Manassas, Va.) And cultured in standard mES medium (15% FBS, 1% panicillin and streptomycin, 1 mM sodium tartrate, 2 mM glutamine, , 1 mM [beta] -mercaptoethanol and 10 ³ units / ml LIF supplemented DMEM). Mouse iPSCs were cultured on mitomycin C-treated MEF feeder cells in mES medium or KSR medium (mES medium supplemented with 15% KSR (# 10828-028; Life Technologies, Inc. instead of FBS)).

Example  3: OG - MEFs Voluntary immortalization

OG-MEFs were derived from OG2 / ROSA26 heterozygous double transgenic mice. These mice were treated with neo / lacZ < RTI ID = 0.0 > Is produced by crossing the ROSA26 strain carrying the transgene gene and the OG2 transgenic strain carrying the GFP. 5 Passage OG-MEFs were seeded on 60 mm dishes and cultured in MEF medium without medium exchange for 3 weeks. The MEF medium was then replaced every 7 days. Two months later, the cells were transferred to a 100 mm dish. After confluence, some cells were stored in liquid nitrogen at # 7 passages and the other cells were transferred to a three-dimensional dish at a 1: 5 split ratio. Cell culture was stopped at 40 passages where no changes in growth or survival or any morphological changes could be observed. OG-MEFs, which exhibit this potential by long-term culture, have been spontaneously immortalized as OG-MEFs (siOG-MEFs).

Example  4: Lentivirus  Produce

A transcriptional activator (FUW-M2rtTA) (# 20342), a Dox-inducible polycistronic cassette (Tet-O-FUW-OSKM) (# 20321) containing four repetitive and reprogramming factors, psPAX2 (# 12260), and pMD2.G (# 12259) were obtained from Addgene (Cambridge, MA). Three plasmids (FUW-M2rtTA, psPAX2, and pMD2.G or Tet-O-FUW-OSKM, psPAX2, and pMD2.G) were added to 200 μl of Opti-MEM at a ratio of 2: 1.5: Mu] l of polyethylenimine (1 mg / ml). After 30 minutes of incubation, a partial sample of the mixture containing 4 ㎍ plasmid was added to 293FT cells grown in a 60 mm dish. Two hours after the transfer, the medium was replaced with fresh MEF medium and incubated for 2 days. The supernatant was collected, passed through a filter having a pore size of 0.4 mu m, and the titer of lentivirus was determined using a Lentivirus Titration Kit (# LV900; Abm, Inc., Richmond, BC, Canada).

Example  5: siog - MEFs from Reprogramming  Production of cell lines

9 passages, 2 × 10 ⁴ siOG-MEFs were seeded into 6-well plates and the following day in the presence of polybrene (8 μg / ml) for 12 hours at a ratio of 1:10 (cell / virus) A lentiviral supernatant containing -M2rtTA and Tet-O-FUW-OSKM was added to the cells. The medium was replaced with fresh MEF medium and the cells were incubated for 12 hours. The cells were then infected with a second fraction of the lentiviral supernatant at the same rate for 12 hours and maintained in fresh MEF medium for 3 days. After trypsin treatment, cells were counted using a hemocytometer, seeded into 96-well plates with single cells per well, and maintained for 3-4 weeks without medium exchange. Wells containing a single colony were observed with an optical microscope and marked on the plate. Single colonies of the marked wells were trypsinized and replated at 2: 3 split ratios in the presence of 4 [mu] g / ml Dox (25% cells) or no Dox (75% cells) in 2 wells of 24- . After 3 days of incubation, untreated cells from the clones of the cells exhibiting Dox-induced morphological changes (e.g., MET and cell death) were recovered and replated in a 60 mm dish, and after confluence, Was transferred to a 100 mm dish. Some shades were kept in # 1 passages and the other cells were transferred to a three-dish at 1: 4 split ratios for subculture and reprogramming. This clone was named JC. To prepare another Dox-inducible stable reprogramming clone, a Dox-inducible polysystron cassette OKSM (# LVP-359) containing EF1α-TetR (# LVP-459-puro) and OKSM was purchased from GeneTarget, Inc . (San Diego, CA). LVP-359 does not contain selection markers. 9 passages of siOG-MEFs were infected with lentivirus against EF1α-TetR at a ratio of 1:10 (cell / virus) and selected by treatment with puromycin (1 μg / ml). SiOG-MEFs stably expressing TetR were infected twice with OKSM lentivirus at a ratio of 1: 10 (cell / virus), and Dox-inducible stable reprogram clones were prepared as described above. The clone was named GC.

In addition, GC5 cells were deposited with the Korean Cell Line Research Foundation with a deposit number KCLRF-BP-00348 on Aug. 10, 2015.

Example  6: Reprogramming  Efficiency and iPSC Manufacturing

To calculate reprogramming efficiency using a flow cytometer, GC5 cells were seeded onto a 100 mm dish in the presence of Dox (4 ug / ml). After incubation for 3 days, trypsinized cells, followed by grinding of retries on a 24-well plate to 1 × 10 ⁴ cells per well under Dox present. The following day, MEF medium was replaced with E6 medium (# A15165-01; Life Technologies, Inc.) containing LIF / Dox and chemo inhibitor. The medium was exchanged with fresh medium every two days. After 8 days of incubation, cells were trypsinized and GFP-positive cells were counted using flow cytometry. For colony formation assays, 400 GC5 cells were seeded on MEF feeder cells in 6-well plates. The next day, cells were treated with Dox and maintained in MEF medium for 3 days. Subsequently, the MEFs were exchanged with mES / LIF / Dox, KSR / LIF / Dox, or E6 / LIF / Dox medium, and the cells were further incubated for 11 days. AP-positive colonies were detected using an AP detection kit (ScienCell Research Laboratories, Inc., Calsbard, Calif.) And GFP-positive colonies counted by fluorescence microscopy. Growing GFP-positive colonies (GC5, GC8, JC5, JC8, and JC17) in 6-well plates were reseeded onto MEF feeder cells for the production of iPSCs. iPSCs were cultured in KSR / LIF or mES / LIF medium.

Example  7: Lentivirus  Gene insertion site determination and single-cell PCR  analysis

The transgene insertion site (Clontech Laboratories, Inc., Mountain View, Calif.) And confirmed by PCR on the genomic sequence of the plaque insertion site (GC5 cells, primer 5'-AAGCAATAGGTCCAGCCAGCTTGATCCG-3` for epidermal autoregulatory factor 1 homozygous isoform 1, and primer 5'-TTGTATGTCTTTCTCCCCAGCTGACCTC-3` for KRR small subunit of JC5 cells). PCR was also performed on the lentiviral gene (5'-TGATGAAATGCTAGGCGGCTGTCAAACC-3 ').

For single cell PCR, cells were trypsinized and suspended in PBS at low density. Single cells were picked up using a 10 μl micropipette under an optical microscope and transferred to PCR tubes. For cell elution and PCR reactions, single cells were incubated in 2X Direct MasterMix (# K0568010 ; Koma Biotech, Inc., Seoul, Korea) containing insert site amplification and GAPDHdp specific primers. The PCR was performed for 45 cycles and the annealing temperature was 60 ° C.

Example  8: Force - Kinase  Antibody array

GC5 cells were incubated for 3 days in MEF medium containing Dox (4 [mu] g / ml) and eluted using kinase elution buffer (# ARY003B; R & D Systems, Inc., Minneapolis, MN). Alternatively, human iPSCs (SES-8) were prepared from human aortic smooth muscle cells (HASMCs) and differentiated into SMCs (SES8-SMCs). Human H9 ESCs and SES-8 iPSCs were cultured in human standard ES medium containing 20% KSR and basic fibroblast growth factor (bFGF) (5 ng / ml). The eluate (200 μg) was analyzed via a human phospho-kinase antibody array (# ARY003B; R & D Systems, Inc.). Membranes were analyzed using ImageJ software (NIH, Bethesda, MD).

Example  9: Immunocytochemical analysis and Western Blot

Immunocytochemistry was performed by the method described above. For Western blots, cells were incubated with extraction buffer (# FNN0011; Life Technologies, Inc.) to obtain total cell eluate and centrifuged at 16,000 x g for 20 minutes. Western blot analysis was performed as described previously.

Example 10 :  In vitro  differentiation

For embryoid body (EB) formation, iPSCs were trypsinized, suspended in sterile, unattached dishes, suspended and maintained in LIF-deficient mES medium. After 7 days of incubation, EBs were attached to gelatin-coated dishes and kept in MEF medium for an additional 7 days.

Example  11: Teratoma  formation

1 × 10 ⁶ were injected subcutaneously in the mouse iPSCs of 6-week-old BalB / c mice (n = 4) of the. After 9 weeks of injection, the teratoma was collected at the injection site, incised, and fixed in 4% formaldehyde overnight. The teratoma was placed in paraffin and cut to a thickness of 6 μm and analyzed by H & E (hematoxylin and eosin) staining.

Example  12: shingles -free through embryo iPSCs And X-gal staining

The blastocyst injection of iPSCs and X-gal staining were performed by conventional methods.

Example  13: Immortalized MEFs (mouse embryonic fibroblasts) OSKM Verify that it can be reprogrammed by

First, to immortalize normal Oct4-GFP-MEFs (OG-MEFs), the MEF immortalization protocol was designed. In the case of other MEFs, growth ceases after several passages in culture. Senescent OG-MEFs were maintained in cultures for about 6-8 weeks, with some of the cells starting to grow vigorously (Figure 1A, top panel), while most cells were maintained in a dead or senescent state. Newly growing cells continued to grow beyond 40 passages without aging (Figure 1A, lower left panel), and these growing OG-MEFs were resistant to freezing / thawing stress.

We next confirmed that spontaneously immortalized OG-MEFs (siOG-MEFs) could be converted to iPSCs by the expression of OSKM (Oct4, Sox2, KLF4 and C-Myc).

As shown in Figure 1A (lower right panel), at the initial passages of siOG-MEFs

When infected with lentivirus encoding rtTA (FUW-M2rtTA) and OSKM (Tet-O-FUW-OSKM), some cells exhibited mesenchymal-epithelial transition (MET), which is characteristic of the early stages of reprogramming. Virus-infected siOG-MEFs were seeded into mitomycin C-treated MEF feeder cells and cultured in mES (mouse ES) medium / LIF (leukemia inhibitory factor) in the presence of Dox, Some colonies were identified, but did not express GFP in prolonged culture (4 weeks) (FIGS. 3C and D). However, when the mES medium was replaced with serum-free KSR / LIF (serum-free knockout serum replacement medium and LIF), some colonies expressed GFP 2 days after medium exchange. Isolated GFP-like colonies continued to express GFP under mES medium / LIF culture conditions and expressed ESC marker alkaline phosphatase (AP). These iPSCs also formed teratomas in vivo and contributed to the development of embryos in utero (Fig. 1D). Thus, this data indicates that siOG-MEFs can be fully reprogrammed with the expression of OSKM.

Example  14: Dox - inductive Reprogramming  Production of cell line

Two different Tet-On inducible vector systems were used for the expression of OSKM vectors (Supplementary Figure 1) to generate repaggramming clones from siOG-MEFs. As described in FIG. 2A, the initial passages of siOG-MEFs were infected with lentivirus encoding rtTA / OSKM or TetR / OKSM and seeded into 96 well plates at the density provided by single cells per well. Based on changes in morphology and survival rate induced by Dox (4 / / ml), a number of colonies were selected for each vector system and Dox-induced protein expression was confirmed by western blot analysis (Fig. 7). Some colonies representing the expression of Dox-induced OSKM were expanded and confirmed again by western blotting (Fig. 2B and F). When the reprogramming ability of these clones was confirmed, GC5 and JC5 cells exerted the MET phenotype within 3 days in the MEF / Dox medium (FIGS. 2C and G), and the medium was changed from MEF / Dox to KSR / LIF / Dox And within one week ES-like morphological development and GFP expression were confirmed (FIGS. 2E and I). On the other hand, GC8, JC8, and JC17 cells showed severe apoptosis within 3 days after Dox treatment and formed very few colonies, but surviving cells showed ES-like morphology and GFP expression (Fig. 2E and I).

The decrease in Dox concentration completely inhibited apoptosis, and the MET phenotype and significant GFP (0.1 μg / ml of Dox), JC8 (0.01 μg / ml of Dox), and JC17 - expression colonies. GC2 cells showed a clear MET phenotype induced by Dox (4 [mu] g / ml), similar to GC5 cells (Fig. 2C). However, most of these cells rapidly lost these traits, including Dox-induced MET and growth stimulation, after several passages. With these characteristics of each colony, except for GC2 cells, cells were preserved after general freezing / thawing, and then the experiment was carried out using cells (in 10 passages) after preservation in liquid nitrogen.

In addition, GC5 and JC5 were used as representative colonies to test whether the reprogrammed colonies were derived from a single cell, indicating that this clone has a clear MET phenotype by E-cadherin up-regulation (FIGS. 8A and 12) And showed minimal apoptosis in high concentrations of Dox. To examine whether such clones are derived from a single cell, the insertion site of an external gene in the GC5 and JC5 genomes through chromosomal walking analysis and specific primers for external and host genome flanking sequences (Figure 9). When PCR was performed on the genomic DNA, it was confirmed that the primer was a clone-specificity (Fig. 3A). In a single-cell PCR reaction, PCR products predicted from nine primer-matched single cells were generated, whereas single-cell or primer-mismatched clones from parent siOG-MEFs produced the expected PCR No product was produced (Fig. 3B). Insertion of external DNA is probabilistic, and this data indicates that GC5 and JC5 cells are derived from a single cell. As expected, all cells in both clones expressed Oct-4 by Dox stimulation (Figures 2D and H). These data indicate that GC5 and JC5 clones (and other clones) also maintain cellular homogeneity and maintain expanded growth and conserved reprogramming potential.

Example  15: GC5  Cell Reprogramming  Identify features

Depending on the growth and morphology, homogeneity for resistance to Oct4 expression, and Dox-induced cell death, the reprogramming efficiency of GC5 cells may be very high or very low.

To confirm this, GC5 cells were seeded in MEF feeder cells and cultured in MEF medium containing Dox (4 / / ml) for 3 days. On day 4, the medium was replaced with a standard mES medium containing LIF / Dox and the cells were incubated for a maximum of 4 weeks. Most of the cells formed ES-like colonies on the MEF monolayer and expressed the ES-specific marker AP (Fig. 3C). However, as described above, there were no cells expressing GFP, indicating that these colonies were not fully reprogrammed under these culture conditions. Serum was shown to inhibit cell reprogramming, and mES medium was replaced with serum-free KSR medium or serum-free E6 (Essential 6) medium. KSR and E6 medium induced GFP expression (FIG. 3D) and E6 medium induced more dense colony formation as compared to mES medium (FIG. 3C). However, such reprogramming efficiency as predicted by GFP-like colony formation was less than 1-3% of the seeded cells.

Most of the GC5 cells exhibit the MET phenotype (Fig. 2C) within 3 days in the presence of Dox and exhibit growth promotion (Fig. 8B). These results indicate that the MET repetition signal as an initial reprogramming signal is essential but induction of somatic reprogramming Lt; / RTI >

Some chemical inhibitors involved in the posterior and cellular signal pathways may increase reprogramming efficiency. Therefore, the reprogramming efficiency of GC5 cells was increased using a small molecule inhibitor. In the E6 / LIF / Dox medium, the reprogramming efficiency of GC5 cells was significantly increased when the MEK inhibitor PD0325901, the GSK-3 beta inhibitor CHIR99021, and the histone deacetylase inhibitor Trichostatin A (TSA) were treated (FIG. Reprogrammability reached about 80% efficiency when exposed to all three compounds (1 μM CHIR99021, 0.1 μM PD0325901, and 0.01 μM TSA (CPT)), indicating that GFP expression using flow cytometry (Fig. 4B). In the colony forming assay, CPT compounds did not significantly affect colony formation and AP expression, but induced GFP expression in all colonies in E6 / LIF / Dox medium (Fig. 3D) (Supplementary Video 1). In contrast, most colonies expressed AP regardless of culture conditions and chemical treatments (FIG. 3C), indicating that AP does not exhibit complete reprogramming.

Taken together, these results indicate that synchronized metabolic or epigenetic remodeling may be required after the appearance of the MET phenotype for complete reprogramming.

Example  16: cells In reprogramming  Initial signaling confirmation

GC5 cells in MEF medium were treated with Dox for 3 days, then eluted, and a phospho-specific antibody array covering 43 signal substances was performed (FIG. 10). This analysis showed that the phosphorylation of ERK, P38, and JNK was significantly down-regulated relative to cells not treated with Dox-treated cells (FIGS. 5A and B).

These results were confirmed by Western blot for GC5 cells (Fig. 5C, left) and normal i4F-MEFs isolated from iPS mice (Fig. 5C, center). Among the tested MAP kinases, down-regulation of ERK phosphorylation was more pronounced than P38 and JNK. Since the chemical inhibitor of the ERK signal significantly increases the reprogramming efficiency of GC5 cells (Fig. 4A), down-regulation of the ERK signal can be a positive driving force for cell reprogramming. To confirm this possibility, GC5 cells were pre-treated with the MEK inhibitor PD0325901 16 hours before Dox-addition. As expected, PD0325901 increased the efficiency of reprogramming in a concentration-proportional manner (Fig. 5D), indicating that down-regulation of the ERK signal can promote cell reprogramming. In addition, mouse iPSCs showed a reduced ERK signal relative to their parental somatic cells siOG-MEFs, or cells differentiated from iPSCs (Fig. 5C, right). Similarly, down-regulation of the ERK signal was demonstrated in human cell reprogramming (Figure 11). This data indicates that the ERK signal is down-regulated for successful cell reprogramming.

Korea Cell Line Research Foundation KCLRFBP00348 20150810

Claims (11)

A method for producing spontaneously immortalized somatic cell-derived derived pluripotent stem cells comprising the steps of:
(1) preparing mouse embryonic fibroblast (MEF) -spontaneously spontaneously immortalized somatic cells,
The spontaneously immortalized somatic cell is deposited with accession number KCLRFBP 00348;
(2) producing inducible pluripotent stem cells by transfecting the spontaneous immortalized somatic cells of step (1) with the demethylation factors Oct4, Sox2, KLF4 and C-Myc,
Wherein said dedifferentiation factor is comprised in a Doxycycline or tetracycline inducible vector;
(3) seeding the induced pluripotent stem cells of step (2) on a plate;
(4) treating the seeded plate of the inducible pluripotent stem cell of step (3) with doxycycline or tetracycline; And
(5) treating the induced pluripotent stem cell-seeded plate of step (4) with a MEK inhibitor, a GSK-3? Inhibitor and a HDAC (Histone deacetylase) inhibitor. delete The method according to claim 1, wherein the spontaneously immortalized somatic cell in step (1) does not cause apoptosis, cell senescence or morphological change in 30 to 50 passages. delete delete delete delete delete delete delete delete

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