IMPROVED IN VIVO REPROGRAMMING SYSTEM AND CELL CONVERSION METHOD USING SAME

Abstract

The present disclosure relates to an advanced in vivo reprogramming system and a cell conversion method using same. The reprogramming system of the present disclosure comprises a start cell marker promoter, a pluripotency-maintaining gene protein, an amino acid isolation peptide, Cre recombinase, a target cell marker promoter, LoxP, and a gene encoding a fluorescent protein, does not require cell fixation in order to confirm cell conversion, enables real-time monitoring in a living cell state, and may be used both in vitro and in vivo. Therefore, the present disclosure is expected to be widely used in the biological and medical fields.

Claims

1. An expression vector for converting cell A into cell B, the expression vector sequentially containing a cell A marker promoter, a pluripotency maintaining gene protein, Cre recombinase, a cell B marker promoter, LoxP, and a gene encoding a fluorescent protein.

2. The expression vector of claim 1, which enables to confirm that cell A is converted into cell B in a living cell state.

3. The expression vector of claim 1, wherein the gene encoding the fluorescent protein is contained in a direction of reverse transcription.

4. The expression vector of claim 1, further comprising an amino acid isolation peptide.

5. The expression vector of claim 4, wherein the amino acid isolation peptide is any one or more selected from the group consisting of T2A, F2A, and E2A.

6. The expression vector of claim 1, wherein the pluripotency maintaining gene protein is any one or more selected from the group consisting of a SOX gene family (sex determining region gene family), a Myc gene family (proto-oncogene gene family), a Klf gene family (Kruppel-like factors gene family), and an Oct gene family (octamer-binding transcription factor gene family).

7. The expression vector of claim 1, wherein the cell A is a stromal cell, and the cell B is a neuron.

8. The expression vector of claim 7, wherein the stromal cell is any one selected from the group consisting of fibroblasts, chondroblasts, osteoblasts, neuroglial cells, adipocytes, macrophages, and plasma cells.

9. The expression vector of claim 7, wherein the cell A marker promoter is any one promoter selected from the group consisting of Col1A2 (Collagen Type I Alpha 2 Chain), FAP (fibroblast-activation protein), FSP1 (fibroblast-specific protein 1), vimentin, ACTA (alpha smooth muscle actin), Hsp47 (heat shock protein 47), aggrecan, CD44, CD45, CD73, calcitonin, OSCAR (osteoclast-associated receptor), RANK (receptor activator of nuclear factor κ B), GFAP (glial fibrillary acidic protein), TREM2 (triggering receptor expressed on myeloid cells 2), HexB (beta-hexosaminidase subunit beta), S100 (calcium-binding protein), CD69, and Gpr34 (probable G-protein coupled receptor 34).

10. The expression vector of claim 7, wherein the cell B marker promoter is any one promoter selected from the group consisting of SYN (synapsin), Tuj1 (neuron-specific class HI beta-tubulin), MAP2 (microtubule-associated protein 2), and Neurofilament.

11. A pharmaceutical composition for treating nerve injury containing the expression vector of claim 1 as an active ingredient.

12. A lentiviral vector containing the expression vector of claim 1, VSV-G (fusiogenic envelope G glycoprotein of the vesicular stomatitis virus), and a GAG/Pol gene.

13. A non-human transformant containing the expression vector of claim 1.

14. A method for converting cell A into cell B, the method comprising steps of: (a) producing the expression vector of claim 1; and (b) introducing the expression vector into cell A.

15. The method of claim 14, further comprising, after step (b), a step of culturing the cell with a cell B culture medium.

16. The method of claim 14, further comprising, after step (b), a step of confirming fluorescence expression in the cell.

17. A live cell imaging method for confirming the conversion of cell A into cell B in a living cell state, the method comprising steps of: (a) producing the expression vector of claim 1; and (b) introducing the expression vector into cell A.

18. The live cell imaging method of claim 17, wherein the imaging is performed using a fluorescence microscope.

19. A method for producing an animal model in which cell A has been converted into cell B, the method comprising step of: (a) producing the expression vector of claim 1; and (b) introducing the expression vector into a non-human subject.

20. A method for screening cell A for conversion into cell B using a first expression vector and a second expression vector each according to claim 1, the method comprising steps of: (a) preparing a first subject and a second subject as a non-human disease animal model; (b) producing the first expression vector by selecting cell (i) as cell A and cell (iii) as cell B; (c) producing the second expression vector by selecting cell (ii) as cell A and cell (iii) as cell B; (d) introducing expression vector I into the first subject, and introducing expression vector II into the second subject; and (e) comparing a disease therapeutic effect between the first subject and the second subject, and selecting cell (i) as a cell for conversion into cell (iii) when the therapeutic effect on the first subject is better.

21. A pharmaceutical composition for treating a disease caused by damage to cell B containing the expression vector of claim 1 as an active ingredient.

22. The pharmaceutical composition of claim 21, wherein the cell B is a neuron, and the disease caused by damage to cell B is any one selected from the group consisting of epilepsy, amyotrophic lateral sclerosis (Lou Gehrig's disease), meningitis, encephalomeningitis, cerebral palsy, encephalitis, stroke, cerebral infarction, cerebral hemorrhage, ischemic brain attack, multiple sclerosis, headache, migraine, tension headache, chorea, Huntington's disease, Wilson's disease, concussion, brain contusion, subdural hematoma, traumatic subarachnoid hematoma, spinal cord injury, arteriovenous malformation, cerebral aneurysm, hydrocephalus, spina bifida, sleep apnea syndrome, syncope, nerve paralysis, severe asthenia, tremor, myelitis, Alzheimer's, Parkinson's disease, and motor dysfunction.

23. A method of preventing or treating a disease caused by damage to cell B by administering the vector of claim 1 as an active ingredient.

24. The method of claim 23, wherein the cell B is a neuron, and the disease caused by damage to cell B is any one selected from the group consisting of epilepsy, amyotrophic lateral sclerosis (Lou Gehrig's disease), meningitis, encephalomeningitis, cerebral palsy, encephalitis, stroke, cerebral infarction, cerebral hemorrhage, ischemic brain attack, multiple sclerosis, headache, migraine, tension headache, chorea, Huntington's disease, Wilson's disease, concussion, brain contusion, subdural hematoma, traumatic subarachnoid hematoma, spinal cord injury, arteriovenous malformation, cerebral aneurysm, hydrocephalus, spina bifida, sleep apnea syndrome, syncope, nerve paralysis, severe asthenia, tremor, myelitis, Alzheimer's, Parkinson's disease, and motor dysfunction.

25-26. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0067] FIG. 1 is a view showing the structure of an advanced in vivo reprogramming system (A-IVR), which is an expression vector for direct reprogramming according to one embodiment of the present disclosure.

[0068] FIG. 2 is a view showing the results of immunostaining of neuroglial cells, more specifically astrocytes, converted into neurons by A-IVR in vitro according to one embodiment of the present disclosure.

[0069] FIG. 3 shows optical micrographs and the results of immunostaining of mouse embryonic fibroblasts, converted into neurons by A-IVR in vitro according to one embodiment of the present disclosure.

[0070] FIG. 4 shows optical micrographs and the results of immunostaining of human fibroblasts, converted into neurons by A-IVR in vitro according to one embodiment of the present disclosure.

[0071] FIG. 5 shows results indicating that the spinal cord injury animal model transplanted with A-IVR according to one embodiment of the present disclosure has recovered the motor function thereof.

[0072] FIG. 6 shows immunostaining results for the spinal cord tissue of the spinal cord injury animal model transplanted with A-IVR according to one embodiment of the present disclosure.

BEST MODE

[0073] In one embodiment of the present disclosure, neuroglial cells and neurons are selected as starting cells and target cells, respectively, and the neuroglial cells are converted into the neurons. In this case, a GFAP (glial fibrillary acidic protein) promoter is selected as a marker promoter for the starting cells, and a SYN (synapsin) promoter is selected as a marker promoter for the target cells. In another embodiment of the present disclosure, fibroblasts cells and neurons are selected as starting cells and target cells, respectively, and the fibroblasts are converted into the neurons. In this case, a Col1A2 (Collagen Type I Alpha 2 Chain) promoter is selected as a marker promoter for the starting cells.

MODE FOR INVENTION

[0074] Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples serve merely to explain the present disclosure in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure according to the subject matter of the present disclosure is not limited by these examples.

Example 1. Construction of Lentiviral Vector for In Vivo Reprogramming

[0075] VSV-G (fusiogenic envelope G glycoprotein of the vesicular stomatitis virus), GAG/Pol gene and an expression vector were introduced into HEK293FT (human embryonic kidney 293FT) cells using a Viafect transfection reagent (E4981, Promega, USA). The expression vector was designed to sequentially transcribe a starting cell marker promoter, SOX2 (sex determining region Y-box 2), T2A (2A peptide), Cre recombinase, a target cell marker promoter, LoxP, and eGFP (enhanced green fluorescent protein), and the eGFP was designed in the direction of transcription so as to be reversed in the direction of transcription by LoxP expression. The gene structure of the expression vector is shown in FIG. 1.

[0076] For example, in one embodiment of the present disclosure, neuroglial cells and neurons are selected as starting cells and target cells, respectively, and the neuroglial cells are converted into the neurons. In this case, a GFAP (glial fibrillary acidic protein) promoter is selected as a marker promoter for the starting cells, and a SYN (synapsin) promoter is selected as a marker promoter for the target cells. In another embodiment of the present disclosure, fibroblasts cells and neurons are selected as starting cells and target cells, respectively, and the fibroblasts are converted into the neurons. In this case, a Col1 A2 (Collagen Type I Alpha 2 Chain) promoter is selected as a marker promoter for the starting cells.

[0077] After 72 hours, the medium was removed, the cells were concentrated for 72 hours by PEG-it Virus Precipitation Solution (LV810A-1, System Biosciences, USA) capable of concentrating a lentiviral vector, and then re-suspended in EMEM (Eagle's minimum essential medium), thereby constructing an advanced in vivo reprogramming system (A-IVR) which is a lentiviral vector for direct reprogramming according to the present disclosure.

Example 2. Production and Identification of Neurons Converted from Neuroglial Cells

[0078] Neuroglial cells were dispensed into a 6-well plate at a density of 1×10.sup.5 cells/well and cultured with EMEM containing 10% FBS and 1% penicillin-streptomycin (PS). The lentiviral vector (A-IVR) constructed according to the method of Example 1 by selecting fibroblasts as starting cells and neurons as target cells was introduced into the cultured cells together with 10 μg/ml of Polybrene transfection reagent (TR-1003-G, Merck Millipore, USA). Polybrene increases the binding affinity between the virus and the target cell by neutralizing the electrical repulsion between the virus and the target cell membrane. After 3 days, the medium was replaced with EMEM containing 10% FBS and 1 μg puromycin, and cells into which the gene was not introduced were selected using puromycin for 1 week. After 1 week, the medium was replaced with DMEM/F12: neurobasal (2:1) containing 0.8% N.sub.2, 0.4% B27 and 1% PS, and then conversion into neurons was induced for 5 weeks while the medium was replaced once every 2 days.

[0079] After 5 weeks, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 30 minutes. Next, the cells were washed three times with 0.3% Tween20 and then blocked using 10% normal donkey serum, followed by immunostaining with a neuroglial cell marker (GFAP) and neural markers (Tuj1, MAP2, Neurofilament). The specific process of immunostaining was performed in the same manner as in the prior literature (Lee H Y et al. Tissue Eng Part A. 2015 July; 21(13-14):2044-52). The results of the immunostaining are shown in FIG. 2.

[0080] As a result of the experiment, it was shown that, when the conversion of neuroglial cells, more specifically, astrocytes, into neurons, was induced using A-IVR in vitro, the expression of the neuroglial cell marker protein GFAP decreased and the neural marker proteins Tuj1, Map2 and Neurofilament were expressed together with GFP. Thereby, it could be confirmed that neuroglial cells were converted into neurons by A-IVR, and GFP was also expressed along with neural differentiation.

Example 3. Production and Identification of Neurons Converted from Fibroblasts

[0081] STO feeder cells (serving as a scaffold for cell growth) whose growth was inhibited by treatment with mitomycin C were seeded into a 12-well plate (coated with 0.1% gelatin) at a density of 2.5×10.sup.5 cells/well, and cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin (PS) for 1 day. On the next day, mouse embryonic fibroblasts (MEF) or human fibroblasts were seeded onto the feeder cells at a density of 4×10.sup.5 cells/well and cultured with a DMEM or IMDM containing 10% FBS and 1% P/S for 1 day. Next, the lentiviral vector (A-IVR) constructed according to the method of Example 1 by selecting skin cells as starting cells and neurons as target cells was introduced into the cultured cells together with 10 μg/ml of Polybrene transfection reagent (TR-1003-G, Merck Millipore, USA). After 24 hours, the medium was replaced with DMDM/F12 containing 1% B27, 2 mM L-glutamate, 1% P/S, 20 ng/ml FGF, 20 ng/ml EGF, and 2 μg/ml heparin, followed by additional culture for 6 to 7 days. Thereafter, when a small colony was formed, the cells were detached using accutase (cell detachment solution) and further cultured in a 6-well plate for 3 days. The resulting sphere was transferred to a 0.1% gelatin-coated 12-well plate and cultured for 14 days to induce conversion into neurons.

[0082] Immunostaining was performed in the same manner as in Example 2, except that a fibroblast marker (Col1 A2) and a neural marker (Tuj1) were used in the immunostaining. The results of the immunostaining are shown in FIGS. 3 and 4 together with optical micrographs of the cells.

[0083] As a result of the experiment, it was shown that, when the conversion of fibroblasts into neurons was induced using A-IVR in vitro, the expression of the fibroblast marker protein Col1A2 decreased, and the neural marker protein Tuj1 was expressed together with GFP. The above results were consistent regardless of the use of mouse embryonic fibroblasts or human fibroblasts as the starting cells. Thereby, it could be confirmed that the fibroblasts were converted into neurons by A-IVR, and GFP was also expressed along with neural differentiation.

Example 4. Evaluation of Nerve Regeneration Effect in Spinal Cord Injury Animal Model Transplanted with A-IVR

[0084] For construction of a spinal cord injury animal model, male C57BL/6 mice weighing about 20 g were used. Each mouse was anesthetized by intraperitoneal injection of a mixture of ketamine and Rompun, and thoracic vertebrae 10 (T10) was incised by laminectomy to expose the spinal cord, and then the spinal cord was injured by compressing the spinal cord using self-closed forceps (Germany) for 3 seconds. Thereafter, the muscles and skin were sutured. Two weeks after the construction of the spinal cord injury animal model, the skin and muscles of the injured site were opened again, and 4 μl of the lentiviral vector (A-IVR), constructed according to the method of Example 1 by selecting neuroglial cells (more specifically, astrocytes) as starting cells and neurons as target cells, was transplanted into the injured site by a 33G Hamilton syringe (n=10). Control mice were transplanted with EMEM instead of A-IVR (n=10). From one week after injury, Basso-Mouse-Scale (BMS) was measured weekly for motor function test. BMS is based on a 9-point scale and composed mainly of three steps. In the first step, the movement of the ankle is measured, and in second step, the weight-bearing gait and the recovery of the gait are measured, and in the last step, the stability of the gait and the recovery state of the tail are measured. The specific process of BMS was performed in the same manner as in the prior literature (Basso D M et al. J Neurotrauma. 2006 May; 23(5):635-59). The BMS measurement results are shown in FIG. 5. As a result of the experiment, it could be seen found that the motor function of the mice transplanted with A-IVR was significantly restored from 2 weeks after transplantation of the A-IVR compared to the control group.

[0085] Eight weeks after spinal cord injury (6 weeks after A-IVR transplantation), the mice were sacrificed, and spinal cord tissue was isolated and fixed with 4% paraformaldehyde, followed by dehydration and freeze-embedding. The injured site (thoracic vertebrae 10) of the tissue subjected to embedding was immunostained with markers of neurons (MAP2), activated neuroglial cells (GFAP), neuroblasts (SOX2), early neurons (Tuj1), mature neurons (neurofilament), and the nuclei of all nucleated cells were counterstained with 4′6′-diamidino-2-phenylindole (DAPI, 1 μg/ml) (vector, CA, USA). The specific process of the immunostaining was performed in the same manner as in the prior literature (Lee H L et al. J Control Release. 2016 Mar. 28; 226:21-34), and the results of the staining were visualized by laser scanning confocal microscopy (LSCM). The staining results are shown in FIG. 6. As a result of the experiment, it was confirmed that the neuroglial cell marker protein GFAP was expressed at and around the injured site. In addition, it was shown that the neuroblast marker protein Sox2 was not expressed together with GFP, but the early neural marker protein Tuj1 and the mature neural marker protein Neurofilament were expressed together with GFP. GFAP was not co-expressed. Thereby, it could be seen that neuroglial cells were converted (reprogrammed) into neurons by A-IVR, and 6 weeks after vector introduction, the cells differentiated into neurons via neuroblasts.

[0086] From the results of Examples 1 to 4 above, it could be seen that the advanced in vivo reprogramming system of the present disclosure can convert (reprogram) starting cells into target cells, and confirm this conversion a living cell state. This means that necessary cells may be produced from other cells in vivo.

[0087] Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

[0088] The present disclosure relates to an advanced in vivo reprogramming system and a cell conversion method using the same. The reprogramming system of the present disclosure does not require cell fixation to confirm cell conversion, enables real-time monitoring in a living cell state, and may be used both in vitro and in vivo. Thus, the reprogramming system of the present disclosure is expected to be widely used in the biological and medical fields.