KR101559481B1 - Method for inducing differentiation of human pluripotent stem cells into nephron progenitor cells - Google Patents

Abstract

The present invention relates to a method for inducing the differentiation of nephron progenitor cells (NPCs) from human pluripotent stem cells (hPSCs) and a composition for inducing differentiation therefrom. More specifically, the present invention relates to human pluripotent stem cells By differentiating the differentiated kidney progenitor cells into the kidney, intermediate mesoderm and nephron precursor cells, which are the main steps in the normal kidney development process, the special kidney progenitor cells are treated with the special cultivation under the individual conditions and the mesodermal system tissues and structures And it is expected to be useful for cell replacement therapy.

Description

The present invention relates to a method for inducing differentiation of human pluripotent stem cells into nephron progenitor cells,

The present invention relates to a method for inducing differentiation of nephron progenitor cells (NPCs) from human pluripotent stem cells (hPSCs).

Stem cells are pre-stage cells that differentiate into the cells constituting the tissue. They are self-renewing ability capable of infinite proliferation in undifferentiated state, and pluripotent stem cells, which are potential potential for differentiation into various tissue cells by specific differentiation stimulus ≪ / RTI > That is, even if the culture is continued, the self-proliferating ability is maintained without deteriorating, and it is possible to differentiate into various kinds of cells.

Stem cells are divided into embryonic stem cells (ESCs) and adult stem cells (ASCs) depending on the possibility of differentiation. After the sperm and the egg meet, they are fertilized and the morphogenesis takes place. At that time, the cells undergo proliferation, migration and differentiation. Embryonic stem cells are stem cells isolated from the inner cell mass (ICM) of the embryo, which is a very early stage of blastocyst embryo formation before embryo implantation. Embryonic stem cells are divided into three types: endoderm, ectoderm and mesodermal It is a pluripotent cell that can be differentiated into cells of all tissues produced by the embryonic germ layer.

Adult stem cells, on the other hand, are adult stem cells that are specific for each organ or are obtained from the placenta at the stage where embryonic development proceeds and the organs of the embryo are formed, It is multipotent only to cells. These adult stem cells remain in most organs even after they become adults and serve to compensate for the loss of normal or pathologically occurring cells. Representative adult stem cells include hematopoietic stem cells present in bone marrow and mesenchymal stem cells differentiated into connective tissue cells other than hemocytes. Hematopoietic stem cells are differentiated into various hematopoietic cells such as red blood cells and white blood cells. Mesenchymal stem cells are differentiated into osteoblasts, chondroblasts, adipocytes and myoblasts.

Since the successful isolation of embryonic stem cells from humans, there has been a growing interest in its clinical application, and cell replacement therapy using stem cells as a complete source of cells has received attention. Cell replacement therapy includes diseases such as Parkinson's disease, intractable neurodegenerative diseases such as Alzheimer's disease, limb paralysis caused by spinal injuries, leukemia, stroke, childhood diabetes, myocardial infarction and liver cirrhosis, Which is destroyed by the disease caused by the disease, and supplies the deficient cells from the outside.

In addition, kidneys were generally considered to be non-regenerative organs due to irreversible damage, but recently, cell replacement therapy has been proposed as a new strategy for the treatment of renal failure in the field of regenerative medicine (Brodie, JC and HD Humes, Pharmacol Rev 57 (3): 299-313, 2005; Hopkins, C., J et. al., J Pathol 217 (2): 265-281, 2009). As bone marrow-derived mesenchymal stem cells (BM-MSCs) and adult renal progenitor cells (ARPCs), two sources of renal regeneration using cell- It has been reported.

BM-MSC protects and restores damaged kidney function in acute renal failure (ARF) and chronic renal failure (CRF) mouse models (Lange, C. et al., Kidney Int 68 Magnasco, A. et. Al., Cell Transplant 17 (10-11), < RTI ID = 0.0 > : 1157-1167, 2008). The advantage of BM-MSCs in actual kidney disease is due to paracrine effects of BM-MSCs, and direct differentiation into kidney cells is limited by differences in origin (Harari-Steinberg, O. et al., Organogenesis 7 (2): 123-134, 2011). Although the presence of adult renal progenitor cells (ARPCs), a type of adult stem cells, is still being discussed, ARPCs have been suggested as an alternative to cellular therapy for renal failure (Bussolati, B. et al. , Am J Pathol 166 (2), 545-555, 2005; Al-Awqati, Q. and JA Oliver, Stem Cell Rev 2 (3), 181-184, 2006; Sagrinati, C. et al., J Am Lindgren, D. et al., Am J Pathol 178 (2), pp. 243-2456, 2006; Lee, PT et al., Stem Cells 28 (3), 573-584 ), 828-837, 2011). In the ARF mouse model, human ARPCs have been reported to restore elevated levels of blood urea nitrogen (BUN) / creatinine and to regenerate damaged nephrons (Ronconi, E. et (J Am Soc Nephrol 20 (2), 322-332, 2009; Angelotti, ML et al., Stem Cells 30 (8), 1714-1725, 2012). Nonetheless, human ARPCs have limitations in clinical applications, such as ethical issues in separating ARPCs from human donors, low efficiency of isolation, and incomplete culture systems for the proliferation of ARPCs (Pleniceanu, O. et al. Stem Cells 28 (9), 1649-1660, 2010).

In addition to the above two materials, human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells (iPSCs), have the ability to differentiate into various specific cell types And is presented as a new alternative to cell therapy. To avoid tumorigenesis by undifferentiated cells, the cells must undergo differentiation into specific cell types prior to transplantation in vivo. To date, methods for differentiation from embryonic stem cells into kidney-derived cells have been reported, and most methods have been reported in mouse models (Kim, D. and GR Dressler, J Am Soc Nephrol 16 (12) , Batchelder, CA et al., Differentiation 78 (1), 45-56, 2005, pp. 3527-3534, 2005; Agarwal, S., KL Holton and R. Lanza, Stem Cells 26 Nat Biotechnol 28 (11), 1187-1194, 2010; Pleniceanu, O. et al., 2009; Lin et al., Stem Cells Dev 19 (10), 1637-1648, 2010; Acta Biochim Biophys Sin (Shanghai) 42 (7), 464-471, 2010; Uosaki, H. et. al., Stem Cells 28 (9), 1649-1660, 2010; al., PLoS One 6 (8), e23657, 2011).

Recently, a method of effectively inducing intermediate mesoderm (IM) cells from human pluripotent stem cells has been reported. The mesodermal cells showed the ability to differentiate into kidney-derived cells as well as external-renal cell lines such as gonad and adrenal gland cells when transplanted into the epididymal fat pad ( Mae, S. et al., Nat Commun 4: 1367, 2013). However, in the future clinical application through in vivo transplantation, it is thought that a precursor cell differentiated into the kidney line rather than the intermediate mesenchymal cell in the early stage is needed.

Accordingly, the inventors of the present invention have been studying a method of inducing differentiation from human pluripotent stem cells (hPSCs) into kidney cells, Nephron progenitor cells (NPCs) were induced by inducing differentiation into mesenchymal, mesenchymal, and posterior mesenchymal cells. Specifically, the kidney progenitor cells were formed into neuron-forming cells such as renal tubular epithelial cells and glomeruli Confirming that differentiation is possible, thereby completing the present invention.

The object of the present invention is to cultivate human pluripotent stem cells (hPSCs) with Act A (Activin A) and Wnt3a-containing serum-free medium, and then treat BMP4 and bFGF (RA), bFGF and BMP7 to induce an intermediate mesoderm (IM), followed by treatment with bFGF and BMP7 to produce nephron progenitor cells (NPCs) ) Of the present invention.

Another object of the present invention is to provide a human pluripotent stem cell derived line-derived composition comprising Act A, Wnt3a, BMP4, and bFGF.

It is still another object of the present invention to provide a composition for inducing mesenchymal stem cells derived from human pluripotent stem cells comprising RA, bFGF and BMP7.

It is still another object of the present invention to provide a composition for inducing a nephron progenitor cell from a human pluripotent stem cell-derived mesenchymal stem cell comprising bFGF and BMP7.

In order to achieve the above object, the present invention relates to a method for culturing human pluripotent stem cells (hPSCs) in a serum-free medium containing Act A (A) and Wnt3a, bone morphogenetic protein 4), and bFGF (basic fibroblast growth factor) to induce a line of sight from human pluripotent stem cells and treat RA (retinoic acid), bFGF, and BMP7 in a medium containing differentiated raw line To induce the differentiation of nephron progenitor cells (NPCs) from human pluripotent stem cells by treating bFGF and BMP7 in medium containing differentiated mesenchymal stem cells.

The present invention also provides a composition for inducing human pluripotent stem cell origin derived from Act A, Wnt3a, BMP4, and bFGF.

The present invention also provides a composition for inducing mesenchymal stem cells derived from human pluripotent stem cells comprising RA, bFGF and BMP7.

In addition, the present invention provides a composition for inducing a nephron progenitor cell from a human pluripotent stem cell-derived mesenchymal stem cell comprising bFGF and BMP7.

The method for inducing the differentiation of nephron progenitor cells (NPCs) from human pluripotent stem cells (hPSCs) of the present invention is characterized in that human pluripotent stem cells are cultured under special conditions in individual conditions, Can be used as a new material for cell therapy by demonstrating the therapeutic effect of differentiated nephron progenitor cells in a future renal failure animal model by easily differentiating them into primary endodermic medium, middle mesoderm and nephron precursor cells, Or genetic disease modeling studies, as well as a platform for new drug development.

Brief Description of the Drawings Figure 1 is a diagram showing the entire process of differentiation from human pluripotent stem cells (hPSCs) into nephron progenitors (Nephron progenitors)
1A: a schematic diagram showing the process of differentiation;
1B: medium composition used for differentiation;
REGM: renal epithelial growth medium;
RTECs: renal tubular epithelial cells;
1C: Representative marker gene for each step of differentiation;
1D: morphological changes at different stages of differentiation from hESCs (human embryonic stem cells) and hiPSCs (human induced pluripotent stem cells) into nephron precursor cells;
Scale bar: 250 μm.
FIG. 2 is a schematic view showing a primitive streak from hESCs (human embryonic stem cell). PS) cells: < RTI ID = 0.0 >
2A: Comparison of the expression levels of T in the cells before and after differentiation;
2B: Expression of T , MIXL1 , EOMES expression levels were compared;
2C: AW or the processing result of the BF confirming the effect on other strains endoderm markers (Endodermal marker, SOX17, FOXA2) and ectoderm markers (Ectodermal marker, PAX6, SOX1) expression;
Relative expression (y-axis): Mean ± SEM (n = 3) with values of fold changes for the control (GAPDH);
C: control group;
AW: Activin A (ActA) and Wnt3a;
BF: BMP4 and FGF2;
2D: The expression level of T (red) and TRA1-81 (pluripotency marker, green) in primitive streak cells derived from hESCs (0 day after induction) (3 days after induction) ; And
Scale bar: 200 μm.
FIG. 3 shows induction of differentiation from hESCs-derived primitive streak cells into intermediate mesoderm (IM)
3A: Comparing the expression levels of OSR1 in different cells before and after differentiation;
3B: Comparison of expression levels of OSR1 according to exogenous factors in the mesenchymal stem cells derived from the raw line;
3C: Comparison of the expression level of OSR1 according to the throughput of retinoic acid (RA);
Relative expression (relative expression, y axis): mean ± SEM (n = 3) as a multiple of the control (GAPDH);
C: control group;
B7: BMP7;
F2: FGF2;
3D: The expression level of OSR1 (red) in the primitive and mesodermal cells was confirmed by immunofluorescence;
Scale bar: 50 μm;
3E: RT-PCR of the expression levels of OSR1 , PAX2 , SALL1 , EYA1 and WT1 ;
HFK: Human fetal kidney;
GAPDH: control group;
3F: Immunofluorescence of PAX2 (green) and SALL1 (red) expression in mesenchymal cells; And
Scale bar: 100 μm.
Figure 4 shows the induction of differentiation from mesenchymal (IM) into nephron progenitors (Nephron progenitors)
4A: Comparison of the relative expression levels of SIX2 , GDNF , HOXD11, and WT1 in the cells before and after differentiation;
Relative expression (relative expression, y axis): mean ± SEM (n = 3) as a multiple of the control (GAPDH);
4B: SIX2 , CITED1 , OSR1 , PAX2 , SALL1 And EYA1 expression by RT-PCR;
4C: Immunofluorescence of SAX2 (red) expression in mesenchymal (IM) and nephron progenitor cells;
Scale bar: 200 μm;
4D: The expression levels of PAX2, WT1 (green) and SALL1 (red) in differentiated nephron progenitor cells were confirmed by immunofluorescence;
Scale bar: 100 μm; And
4E: In the differentiated nephron progenitor cells (NPCs), organ specific markers (metanephric stroma / ureteric bud, FOXD1 and HOXB7 ), bone ( RUNX2 and COL1A1 ), vascular endothelium, PECAM1 And TIE2 ), smooth muscle ( MYH11 and CALPONIN ), liver ( ALB and AAT ), and neurons (neuron, TUJ1 and MAP2 )).
FIG. 5 shows in vivo and in vitro confirmation of complete differentiation of nephron progenitor cells (NPCs) to nephron:
5A: To confirm mesenchymal-epithelial transitions in the differentiation from nephron progenitor cells (NPCs) to renal tubular epithelial cells (RTECs), epithelial cell markers E-CADHERIN (red), ZO1 (Red) and KRT18 (green) were detected by immunofluorescence;
Scale bar: 50 μm;
5B: Expression of proximal renal tubular markers ALP (alkaline phosphatase) and CD13 (green) and distal renal tubular marker MUC1 (red) in nephron progenitor cells (NPCs) and renal tubular epithelial cells (RTECs) The results were confirmed by immunofluorescence;
Scale bar: 100 μm;
5C: It was confirmed that when three-dimensional culture of nephron precursor cells (NPCs) was mixed with Matrigel or Matrigel, it had the ability to form a tube-like structure in vitro (Phase contrast ) And the expression of KRT18 (green), an epithelial cell marker, was confirmed by immunofluorescence in order to confirm that this tube structure was composed of epithelial cells.
MG coated: cells were cultured in a matrigel-coated culture dish;
MG mixed: culture by mixing cells with Matrigel;
Scale bar: 100 μm;
5D: The expression levels of SYN (green) and PODOCXL (red), which are glomerular cell specific markers, in the cells differentiated into glomeruli from nephron progenitor cells (NPCs) were examined by immunofluorescence;
Scale bar: 100 μm;
5E: In vivo transplantation of nephron progenitor cells (NPCs) into subcutaneous parts of mice was analyzed by tissue immunochemistry (H & E staining and immunofluorescence);
LAMININ (normal, green), CD13 (proximal ductal cell, green), MUC1 (distal ductal cell, red): renal tubular cell marker;
White arrow: tube-like structure observed in Matrigel plug;
hNA: human nuclear antigen, red;
Rabbit IgG (green) and mouse IgG (red): negative control; And
Scale bar: 100 μm.
FIG. 6 shows induction of differentiation from hiPSCs (human induced pluripotent stem cells) to nephron progenitors into cells:
6A: Comparing the expression levels of T in different cells before and after differentiation;
6B: The expression levels of T (red) and TRA1-81 (pluripotency marker, green) in primitive streak cells derived from hiPSCs (3 days after induction) were confirmed by immunofluorescence;
Scale bar: 200 μm;
6C: T, MIXL1 , EOMES , SOX17 and PAX6 expression levels were determined according to BMP4 concentration (0, 20 and 50 ng / ml);
Relative expression (relative expression, y axis): mean ± SEM (n = 3) as a multiple of the control (GAPDH);
AW: Activin A (ActA) and Wnt3a; And
BF: BMP4 and FGF2.
Figure 7 shows the induction of differentiation from hiPSCs primitive streak cells into intermediate mesoderm (IM)
7A: Comparing the expression levels of OSR1 in cells before and after differentiation;
7B: Comparison of the expression level of OSR1 according to the throughput of retinoic acid (RA);
Relative expression (relative expression, y axis): mean ± SEM (n = 3) as a multiple of the control (GAPDH);
7C: Immunofluorescence of OSR1 (red), PAX2 (green), SALL1 (red) and DAPI (blue) in mesodermal cells was confirmed; And
Scale bar: 100 μm.
FIG. 8 is an immunofluorescence analysis of the expression levels of WT1 (red) (D0, D6, D10, D15) during the differentiation from hESCs-derived mesenchymal (IM) to nephron progenitor cells (NPCs)
Scale bar: 100 μm.
Figure 9 shows the differentiation from hiPSC-derived mesenchymal (IM) to nephron progenitor cells (NPCs)
9A: Immunofluorescence of SIX2 (red) expression in mesenchymal (IM) cells and nephron progenitor cells (NPCs);
Scale bar: 100 μm;
9B: The expression levels of PAX2 (green), SALL1 (red) and WT1 (green) in nephron progenitor cells (NPCs) were confirmed by immunofluorescence;
100 μm.
FIG. 10 shows in vivo and in vitro confirmation of complete differentiation of neurons from neurons derived from hiPSCs into nephrons:
10A: To confirm mesenchymal-epithelial transitions in the differentiation of neurotubule epithelial cells (RTECs) from nephron progenitor cells (NPCs), epithelial cell markers E-CADHERIN (red), ZO1 (Red) and KRT18 (green) were detected by immunofluorescence;
Scale bar: 50 μm;
10B: Expression of proximal renal tubular markers ALP (alkaline phosphatase) and CD13 (green) and distal renal tubular marker MUC1 (red) in nephron progenitor cells (NPCs) and renal tubular epithelial cells (RTECs) The results were confirmed by immunofluorescence;
Scale bar: 100 μm;
10C: In order to confirm that the neuron precursor cells (NPCs) were mixed with Matrigel and cultured three-dimensionally, they were capable of forming a tubular structure in vitro, and in order to confirm that the tubular structure was composed of epithelial cells, The expression level of the marker KRT18 (red) was confirmed by immunofluorescence;
Scale bar: 100 μm;
10D: The expression levels of SYN (green) and PODOCXL (red), which are glomerular cell specific markers, in the cells differentiated from glomeruli of glomeruli of nephron progenitor cells (NPCs) derived from hiPSC were examined by immunofluorescence;
Scale bar: 100 μm;
10E: Matrigel mixture in hippocampal neuronal progenitor cells (NPCs) derived from hiPSC was inoculated in vivo and analyzed by H & E staining;
White arrow: tube-like structure observed in Matrigel plug; And
Scale bar: 100 μm.
FIG. 11 is a diagram showing glomerular podocytes derived from hPSC as immunofluorescence at a high magnification for structural observation in a single cell unit:
11a: glomerular cell derived from hESC;
11b: glomerular cells derived from hiPSC;
SYNAPTOPODIN (green): glomeruli cell specific marker; And
Scale bar: 50 μm.

Hereinafter, the terminology of the present invention will be described in detail.

In the present invention, the term "embryonic stem cell" refers to an embryonic stem cell obtained by extracting an inner cell mass from a blastocyst embryo immediately before embryo implantation into the uterus of a mother, Refers to a cell that can be pluripotent or totipotent capable of differentiating into cells, and broadly includes embryoid bodies derived from embryonic stem cells. The embryoid body is an intermediate structure formed by stem cells in spontaneous differentiation of embryonic stem cells into various tissue forms, and is an aggregate form formed during culturing of embryonic stem cells. On the other hand, the embryonic stem cells of the present invention can be derived from mammals including humans, and are preferably human embryonic stem cells.

The term "differentiation" in the present invention means a phenomenon in which the structure or function of a cell is specialized while the cells proliferate and grow. Pluripotent embryonic stem cells can be further differentiated into different types of progenitor cells (for example, angiostatic cells, etc.) after differentiation into progenitor cells with limited phylogeny (e.g., exocrine cells, mesodermal cells or endodermal cells) , And then differentiated into terminal differentiated cells (such as vascular endothelial cells and vascular smooth muscle cells) that perform a characteristic role in certain tissues (e.g., blood vessels, etc.).

Hereinafter, the present invention will be described in detail.

The present invention provides a method for inducing differentiation of nephron progenitor cells (NPCs) from human pluripotent stem cells (hPSCs) comprising the steps of:

1) human pluripotent stem cells (hPSCs) were cultured in serum-free medium containing Act A (A) and Wnt3a, and bone morphogenetic protein 4 (BMP4) and bFGF (PS) from hPSCs by treating a basic fibroblast growth factor;

2) inducing intermediate mesoderm (IM) by treating retinoic acid (RA), bFGF, and BMP7 in medium containing the differentiated line in step 1); And

3) treating bFGF and BMP7 in medium containing mesenchymal stem cells differentiated in step 2).

The kidney precursor cells are preferably nephron progenitor cells (NPCs), but are not limited thereto.

The human pluripotent stem cells of step 1) are preferably embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), but are not limited thereto.

The culture in step 1) is preferably, but not limited to, culturing in a matrigel-coated culture dish.

The serum-free medium of step 1) is constituted to be present at a concentration of 80 to 120 ng / ml of Act A and Wnt3, respectively, and is preferably constituted to be present at a concentration of 100 ng / ml each.

BMP4 and bFGF of step 1) were treated at a concentration of 10 to 60 ng / ml and 5 to 15 ng / ml, respectively, and when the human pluripotent stem cells were hESCs, 20 ng / ml and 10 ng / ml, and when the human pluripotent stem cells are hiPSCs, it is more preferable to treat them at a concentration of 50 ng / ml and 10 ng / ml, respectively.

The BMP4 and bFGF of step 1) are preferably treated for 2 days, but are not limited thereto.

The RA, bFGF and BMP7 of the step 2) are treated at a concentration of 5 to 20 μM, 5 to 15 ng / ml and 30 to 70 ng / ml, respectively, and preferably 10 μM, 10 ng / ng / ml.

The RA, bFGF and BMP7 of step 2) are preferably treated for 6 to 8 days, but are not limited thereto.

BFGF and BMP7 in step 3) above are treated at a concentration of 30 to 70 ng / ml and 130 to 170 ng / ml, respectively, and preferably at a concentration of 50 ng / ml, and 150 ng / ml.

The bFGF and BMP7 in step 3) are preferably treated for 13 to 17 days, but are not limited thereto.

The medium of step 1) to 3) is preferably RPMI-1640 medium containing 2% B27, but is not limited thereto.

In a specific embodiment of the present invention, human pluripotent stem cells (hPSCs) are cultured in a medium containing actin A (ActA) and Wnt3a in one step, followed by bone morphogenetic protein 4 (BMP4) And FGF2 (fibroblast growth factor 2), induced mesenchymal differentiation in a medium containing retinoic acid (RA), BMP7 and FGF2 in two steps, And then cultured in a medium containing BMP7 and FGF2 to induce differentiation into nephron progenitor cells (NPCs) (see Fig. 1). Quantitative RT-PCR and immunohistochemistry were used to determine whether the differentiation into each step was correct at the gene expression and cell stage.

In addition, nephron progenitor cells (NPCs) formed in hPSCs were induced into cells constituting nephron, and as a result, renal epithelial cell growth media (REGM) containing hepatocyte growth factor (HGF) ) Formed glomerular epithelial cells and formed glomerular cells in medium containing vitamin D3 (vitamin D3), retinoic acid (RA), and fetal bovine serum (FBS) Immunohistochemistry confirmed the formation of renal tubules in the cells.

Therefore, the method of inducing the differentiation of nephron progenitor cells (NPCs) from human pluripotent stem cells of the present invention confirms that embryonic stem cells or human induced pluripotent stem cells obtained from adult subjects can differentiate into cells constituting nephron, By demonstrating the therapeutic effect of differentiated nephron progenitor cells in renal failure animal models, they can be used as new materials for cell therapy and are expected to be useful as a platform for normal kidney development or genetic disease modeling studies, .

The present invention also provides a composition for inducing human pluripotent stem cell origin derived from Act A, Wnt3a, BMP4 and bFGF.

Preferably, the Act A, Wnt3a, BMP4, and bFGF are contained at a concentration of 100 ng / ml, 100 ng / ml, 20 to 50 ng / ml and 10 ng / ml, More preferably 20 ng / ml when the cells are ESCs, and 50 ng / ml when the human pluripotent stem cells are hiPSCs.

The composition may be used in a culture medium when hESCs or hiPSCs are cultured for differentiation into a raw line. Act A, Wnt3a, BMP4 and bFGF may be used in a stock solution solution, and may be diluted in the culture medium.

The present invention also provides a composition for inducing mesenchymal stem cells derived from human pluripotent stem cells comprising RA, bFGF and BMP7.

The RA, bFGF, and BMP7 are preferably at a concentration of 5 to 20 μM, 5 to 15 ng / ml, and 30 to 70 ng / ml, respectively, and a concentration of 10 μM, 10 ng / ml, and 50 ng / ml, Is more preferable.

The RA, bFGF, and BMP7 are each provided as a stock solution concentrated to 1 to 100 times the concentration of hESCs or hiPSCs for differentiation into intermediate mesoderm, , And diluted in the above culture medium.

The present invention also provides a composition for inducing a nephron progenitor cell from a human pluripotent stem cell-derived mesenchymal stem cell comprising bFGF and BMP7.

Preferably, bFGF and BMP7 have a concentration of 30 to 70 ng / ml and 130 to 170 ng / ml, respectively, and more preferably 50 ng / ml and 150 ng / ml, respectively.

The composition may be used in a culture medium when hESCs or hiPSCs are cultured for differentiation into nephron progenitor cells. The bFGF and BMP7 may be provided in the form of a stock solution concentrated to 1 to 100 times the concentration, It may be diluted in the above culture medium and used.

Therefore, the composition for inducing a nephron progenitor cell from a human pluripotent stem cell-derived source line-derived composition, a composition for inducing an intermediate mesoderm from a human pluripotent stem cell-derived source line, and a mesodermal stem derived from a human pluripotent stem cell, By confirming that the embryonic stem cells or human induced pluripotent stem cells obtained from the subject are capable of differentiating into cells constituting nephrons, it is possible to prove the therapeutic effect of the nephron progenitor cells differentiated in a future renal failure animal model, And can be used as a platform for normal renal development or genetic disease modeling studies and further development of new drugs.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

However, the following examples and preparation examples illustrate the present invention specifically, and the content of the present invention is not limited by the examples and the production examples.

< Example  1> Human Versatility  Stem Cells( human pluripotent stem cells ; hPSCs ) To induce differentiation into kidney cells

<1-1> Human Versatility  Stem Cells( hPSCs ) Culture

H9 human embryonic stem cells (hESCs) cell lines were purchased from Wi-Cell (Madison, WI, www.wicell.org). CRL-2097 human induced pluripotent stem cells (hiPSCs) cell lines were obtained from a previous study (Park et al., 2010 Blood 116 (25): 5762-5772). The two cell lines were cultured in a stem cell culture medium using mouse embryonic fibroblasts treated with mitomycin C (MMC) as feeder cells, respectively. The composition of CRL-2097 fibroblast-derived human induced pluripotent stem cell culture was as follows: DMEM / F12, 20% knockout serum replacement, 1% penicillin-streptomycin (PenStrep) % Non essential amino acids (NEAA), 2 mM L-glutamate, 0.1 mM beta-mercaptoethanol, and basic fibroblast growth factor (bFGF) : 4 ng / ml hESCs or 10 ng / mL hiPSCs containing 4 ng / ml for hESCs and 10 ng / mL for hiPSCs). All of the compositions of the culture were purchased from Invitrogen (Carlsbad, Calif.). The passage of human embryonic stem cells (hESCs) and hiPSCs (human induced pluripotent stem cells) was performed every 5 days. Specifically, a 1 cc insulin syringe was used to cut into 16 to 25 small pieces of stem cell colonies 10 mg / mL collagenase IV (Gibco, Carlsbad, Calif.) Was treated for 5 minutes, then removed and transferred to a new support cell culture dish.

<1-2> Human Versatility  Stem Cells( hPSCs )from Nephron  Progenitor cells ( nephron  progenitor cells ; NPCs )

In order to induce differentiation into human neural progenitor cells (NPCs) from human pluripotent stem cells (hPSCs), primitive streaks (PS), midend mesenchymal cells (PS), which are the main stage cells in normal kidney development, Sequential differentiation induction was performed with intermediate mesoderm (IM) and nephron progenitor cells (Fig. 1).

Specifically, Matrigel (1:40 dilution, BD Biosciences, Bedford, Mass., USA) was coated on a 4-well culture dish the day before induction of differentiation and cultured on DMEM / F12 medium The dish was washed and mTeSR medium (Stemcell Technologies, Vancouver, BC, Canada) containing 10 ng / ml bFGF was added. Each of the undifferentiated hESCs or hiPSCs prepared in <Example 1> was cut into small pieces, transferred to the prepared culture dish, and cultured for 4 days to stabilize the cells. In addition, RPMI-1640 medium, a serum-free medium, was used as a basal medium for the following differentiation. The composition was as follows: 2% B27, 2 mM L-glutamine and 1 % PenStrep (Invitrogen). For induction of differentiation, the mTeSR medium was washed once with PBS to induce differentiation to the raw line on the fourth day and in the first stage, followed by 100 ng / mL of Actin A and 100 ng / mL of Wnt3a ml of bFGF (Peprotech, Rocky Hill, Calif.) were cultured for 1 day in a serum-free basal medium containing 10 ng / ml BMP4 (bone morphogenetic protein 4) (50 ng / ml for hiPSCs) NJ, USA) for 2 days. Then, in step 2, 10 μM retinoic acid (RA, R & D Systems), and 10 ng / mL bFGF were added to the basal medium for induction into the medium mesoderm, followed by addition of 50 ng / ml of BMP4 BMP7 (Peprotech) for 6 to 8 days. In three steps, 150 ng / ml BMP7 and 50 ng / ml bFGF without retinoic acid were treated for 15 days to induce neuronal progenitor cells (Fig. 1B). The differentiation medium was changed once every two days during the period of differentiation into nephron progenitor cells.

<1-3> Nephron  Progenitor cells ( NPCs ) To induce differentiation into fully differentiated kidney cells

In order to determine whether the nephron precursor cells obtained by differentiation in the above Example <1-2> have differentiation potential into kidney epithelial cells, differentiation was induced in various media compositions.

Specifically, the nephron progenitor cells (NPCs) differentiated in the above Example <1-2> were transferred to a fibronectin-coated plate and BMP7 (150 ng / ml) and bFGF (50 ng / ml) 1 < / RTI > in the same differentiation medium as in Example &lt; RTI ID = 0.0 &gt; 1-2 &lt; / RTI &gt; They were then differentiated into renal tubular epithelial cells (RTECs) in kidney epithelial cell growth medium (REGM; Lonza, Allendale, NJ) supplemented with 50 ng / ml HGF (Peprotech). In order to differentiate glomerular podocytes, nephron progenitor cells (NPCs) were cultured in VRAD medium (DMEM / F12, 100 nM vitamin D3 (Sigma-Aldrich, Saint Louis, Missouri) 10% fetal bovine serum (FBS)) (Lasagni et al., Stem Cells, 28 : 1674-1685, 2010). In addition, the medium was replaced every two days during induction of differentiation (Fig. 1B).

<1-4> Human Versatility  Stem Cells( hPSCs )from Nephron  Progenitor cells ( NPCs ) Morphological confirmation of cell differentiation

Microscopic observations were performed to confirm the differentiation of human pluripotent stem cells (hPSCs) into nephron progenitor cells (NPCs).

Specifically, morphological changes of cultured cells were observed using an optical microscope (Olympus, Tokyo, Japan) in each differentiation stage (raw line, mesodermal and nephron precursor cells).

As a result, as shown in FIG. 1D, as the cell differentiated, the distance between the cells gradually became distant, and as each cell shape changed into a triangular or ovoid shape including a small nucleus, To a mesenchymal cell form (Fig. 1D).

< Example  2> human Versatility  Stem Cells( hPSCs )from Raw line ( Primitive streak ; PS ) Identification of differentiation into cells

<2-1> Raw line ( PS ) Confirmation of the cell's free distribution

To determine the optimal culture conditions for the induction of differentiation from human pluripotent stem cells (hPSCs) to the source line (PS), q-PCR was performed using the primer (PS) cell-specific marker gene of FIG. 1A .

Specifically, human pluripotent stem cells (hPSCs) were treated with actin A (A) and WNT3a (AW) and then cultured in medium containing BMP4 and FGF2 (BF) for 1 day. Total RNA was extracted from the derivative of the cell induced by differentiation using TRIzol (Invitrogen), and the remaining DNA was then removed by treatment with DNase I (Invitrogen). CDNA was synthesized from 1 ug of the obtained RNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Enzynomics, Seoul, Korea) and oligo-dT (oligo-dT). Quantitative real-time RT-PCR (q-PCR) was performed using Bio-Rad CFX manager (Bio-Rad Laboratories, Hercules, Calif.) Using CyberGreen to confirm the expression levels of specific genes at each step, CA) machine. After the treatment at 95 ° C for 10 minutes, the reaction was repeated 40 times at 95 ° C for 10 seconds, 60 ° C for 10 seconds, and 72 ° C for 30 seconds. Primers of the target gene were designed using PubMed's primer design tool (Table 1), and each primer was used at a concentration of 0.25 to 0.5 pmole. All reactions were repeated twice to minimize experimental error, and the difference between the sample and the control group was analyzed by the formula Ct method. The cells were normalized using an internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the relative expression level of the target gene was compared with the negative control Respectively. The results of gene expression by all q-PCR were repeated three times, and the relative expression results were expressed as mean ± S.E.M (n = 3).

As a result, BMP4 and FGF2 were added to human pluripotent stem cells (hPSCs) and hiPSCs, as shown in Fig. 2 and Fig. 6, and it was confirmed that the expression level of all T transcription steps increased rapidly after two days. Further treatment of the exogenous elements over 3 days had no effect on T activation. or BF compared to the AW-only treatment in hESCs, a series of processes (AW / BF) is T, MIXL1 and bring the original eye expression improvement in specific genes, such as EOMES, hESCs- derivative in complete endoderm, such as SOX17, GATA4 Gene and genes such as PAX6 and SOX1 were not expressed. In addition, it was confirmed that high concentration (50 ng / ml) of BMP4 is essential for improving the PS gene transfer rate in hiPSCs.

<2-2> Immunocytochemistry ( immunocytochemistry )through Raw line ( PS ) Identification of cell differentiation

Immunostaining was performed to determine whether the differentiation from the cell level to the source cell stage was well induced.

Specifically, a sample washed with PBS to perform immunological staining was fixed with 4% formaldehyde (Sigma-Aldrich) at room temperature for 20 minutes. Then, PBST (PBS containing 0.1% Tween 20 ) And permeabilized to the cell membrane with 0.1% triton X-100 (Sigma-Aldrich). After washing three times with PBST, the samples were blocked with 4% donkey serum (Jackson ImmunoResearch Laboratories) for 1 hour at room temperature. The primary antibody (Table 2) was then added and incubated overnight at 4 &lt; 0 &gt; C. The next day, the samples were rinsed three times with PBST and the samples were incubated with secondary antibodies (Dunkin anti-mouse IgG-, Dunkin anti-goat IgG-, and Dunkin anti-rabbit IgG-Alexa 488 or 594; Invitrogen) for 1 hour. After washing 6 times with PBST, samples were incubated in VECTASHIELD mounting medium containing DAPI for 5 minutes to stain intracellular nuclei. Immunologically stained cells were stained with fluorescence microscope or Zeiss LSM 510 confocal ) Microscope.

As a result, as shown in Fig. 2, the immunostaining of human pluripotent stem cells (hPSCs) resulted in the treatment of four exogenous factors with the mesendodermal marker T and the plutipotent marker TRA1-81 Respectively.

< Example  3> Raw line ( PS ) To the middle mesoderm ( intermediate mesoderm ; IM )

The most important step to differentiate into the kidney line from the PS line to the intermediate mesoderm (IM) was to perform q-PCR in order to confirm whether the differentiation into this line was successful. Cellular changes were observed through staining.

Specifically, raw PS cells derived from human pluripotent stem cells (hPSCs) were cultured in a basal medium supplemented with RAB7F2 (retinoic acid (RA), BMP7, and FGF2). Quantitative RT-PCR was performed on the expression level of OSR1 (Odd skipped related 1), which is a superior molecule of renal development, in the same manner as in Example <2-1>. Cells were immunostained as in Example < 2-2 >.

As a result, as shown in FIG. 3 and FIG. 7, it was confirmed that the transcription level of OSR1 was improved on PS cells derived from hESCs and PS cells derived from hiPSCs on days 6 and 8. In addition, when RAB7F2 was treated in hESCs-derived PS and hiPSCs-derived PS cells, different combinations (retinoic acid alone, BMP7 alone, FGF2 alone, or retinoic acid and BMP7 treatment) ) cDNA, it was confirmed that the transcription activity of the IM gene was most effective. hESC and hiPSCs, the expression level of OSR1 was increased in proportion to the RA throughput. In addition, immunohistochemical staining revealed that OSR1 was expressed in the nucleus in both hESCs and hiPSCs-derived mesenchymal (IM) cells, whereas OSR1 was not expressed in PS cells. We also confirmed that other intermediate mesodermal markers, PAX2 and SALL1, are expressed in the nucleus. The percentage of OSR1-positive cells in intermediate mesodermal (IM) cells derived from hESC and hiPSCs was 35.6% and 25.6%, respectively.

< Example  4> middle mesoderm ( IM ) Cells in the nephron progenitor cells (N PCs )

To determine whether mesenchymal stem cells (IM) derived from human pluripotent stem cells (hPSCs) were differentiated into nephron progenitor cells (NPCs), specifically nephron progenitor cells (NPCs), belonging to the late mesenchymal , were verified not only by molecular genetic testing by q-PCR but also by immunohistochemistry at the cellular level.

Specifically, the cells were treated with high concentrations of BMP7 (150 ng / ml) and FGF2 (50 ng / ml) in which mesenchymal (IM) cells were differentiated into nephron progenitor cells (NPCs) And cultured in a basal medium. After completion of the differentiation, q-PCR and immunological staining were carried out in the same manner as in <Example 2>. In particular, GDNF (glial cell-derived neurotrophic factor) and SIX2 (SIX homeobox 2), HOXD11, and WT1 were selected as the major genes of CM (cap mesenchymal) cells known to contain nephron progenitor cells (NPCs). The expression levels of PAX2, WT1 and SALL1, the major transcription factors involved in differentiation from mesenchymal (IM) to nephron progenitor cells (NPCs), were also confirmed.

As a result, as shown in FIGS. 4 and 9, the relative expression of all of the marker genes (SIX2, GDNF, HOXD11, WT1) of neuron precursor cells (NPC) in the hESC-derived cells was significantly increased. As a result, it was confirmed that the ratio of SIX2-positive cells was 30.2% and the other markers of nephron progenitor cells (NPCs), PAX2, SALL1 and WT1 were also positive.

< Example  5> Nephron  Progenitor cells ( NPCs )of Nephron  Identification of differentiation into the constituting cells

&Lt; 5-1 > In vitro ) Nephron  Progenitor cells ( NPCs )of Multiparameter  Confirm

The pluripotency of nephron progenitor cells derived from human pluripotent stem cells (hPSCs) of the present invention was confirmed in vitro.

Specifically, in order to confirm that the nephron progenitor cells (NPCs) differentiate into renal tubular epithelial cells (RTECs) and glomeruli cells constituting the nephron, E-CADHERIN, ZO1 and KRT18 which are specific markers of renal tubular epithelial cells (RTECs) , And the expression levels of SYNAPTOPODIN and PODOCALYXIN, which are glomerular cell specific markers, were measured. Quantitative RT-PCR and immunohistochemical staining were carried out as in Example <2-1> and <2-2>, respectively.

As a result, as shown in FIGS. 5 and 10, in a limited culture condition, nephron progenitor cells (NPCs) can differentiate into renal tubular epithelial cells (RTECs) and glomeruli cells, Respectively. (RTECs) developed in nephron progenitor cells (NPCs) express E-CADHERIN, ZO1 and KRT18, thus confirming the occurrence of mesenchymal-epithelial metastasis (MET). In addition, it was confirmed that AP (alkaline phosphatase) and CD13, which are marker genes expressed in renal tubular epithelial cells (RTECs), are expressed in proximal duct cells and MUC1 in distal duct cells, respectively. In addition, SYNAPTOPODIN and PODOCALYXIN, which are cell-specific markers, were expressed in hESC-derived podocytes but not in nephron progenitor cells, confirming that neoplastic progenitor cells derived from hESC can differentiate into glomeruli Respectively. In addition, neuronal progenitor cells derived from hiPSC can develop RTECs and glomerular cells under the same culture conditions, respectively.

<5-2> In vitro tube formation under 3D culture conditions tubulogenic ) Confirm

Tubulogenic tests were performed to determine whether neuronal progenitor cells differentiated into extra-tubular epithelial cells outside the cell.

Specifically, nephron progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) were treated with Accutase ™ (Innovative cell Technologies, San Diego, Calif.) For 10 minutes and then transferred to a culture medium. The cells were then filtered through a 40 μm filter mesh and centrifuged at 30 × g for 3 min. Cell pellets were suspended in kidney epithelial cell growth medium (REGM) to a concentration of 2 x 10 ⁵ cells / ml and mixed with 90% matrigel. 500 μl of the mixture of cells and Matrigel was placed on a 4-well dish and incubated at 37 ° C for 20 minutes, then 500 μl of REGM containing 50 ng / ml of HGF was added and further cultured for 14 days. 100% Martrigel was incubated in a 4-well dish at 37 ° C for 1 hour in a 3-D culture condition for confirming the production of a tube in an experimental tube, and the cells were coated with Matrigel at a concentration of 2 × 10 ⁵ cells / ml Seeded and incubated for 14 days in REGM containing 50 ng / ml HGF. The medium was replaced every 2 days during cell culture.

As a result, as shown in Fig. 11, when the cells were cultured in a culture dish coated with Matrigel and a medium containing Matrigel, all of the 3D tube-like structures were formed. The tube-like structure was confirmed by staining with KRT18, a marker of epithelial cells. In addition, SYNAPTOPODIN and PODOCALYXIN, which are podocyte-specific markers, were expressed in hESC-derived podocytes but not in RPCs, confirming that hESC-derived RPCs could differentiate into glomerular cells. When the F-actin was stained together with SYNAPTOPODIN and WT1, it was confirmed that the pod cells were aborized shapes. Cells treated with angiotensin II were found to have contractility of fluidity. In addition, neuronal progenitor cells derived from hiPSC can develop RTECs and glomerular cells under the same culture conditions, respectively.

< Example  6> Nephron  Intracellularly in progenitor cells Performance  Confirm

Immunohistochemistry and intracellular performance tests were performed to evaluate intracellular differentiation potential.

Specifically, Matrigel plugs were added to nephron progenitor cells derived from human pluripotent stem cells (hPSCs) and then injected subcutaneously into mice. After 15 days, the plug was cut and reacted for histological and immunohistochemical examination. Histopathological examination was performed using an embedded sample in a paraffin block. Antigen retrieval was performed with sodium citrate solution (pH 6.0) for 20 minutes followed by addition of PBS-T (0.03% Triton X-100 and PBS) containing 5% Dunki's serum (Jackson ImmunoResearch Laboratories) (4 mm thickness) was incubated at room temperature for 1 hour. When mouse Fab blocking was required, 10% Fab fragment anti-mouse IgG was added to the same blocking agent and incubated for an additional 2 hours. After washing three times with PBS-T, the samples were further incubated at room temperature for 3 hours to bind a fluorescent antibody-conjugated secondary antibody (1: 1,000). After washing several times with PBS-T, DAPI staining for nuclear staining was performed for 10 minutes at room temperature. Fluorescence signals were observed on a mounting medium (Dako, Carpinteria, CA) using a confocal microscope. All primary antibodies used in the present invention are shown in Table 2.

In addition, a 6-week-old female Crl: Nu-Foxn1 nude mouse with nephron progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) was used to assess the differentiation into nephron constitutive cells in vivo Respectively. All experiments using mice were approved by the KAIST Animal Protection Committee. 1 × 10 ⁷ nephron progenitor cells (NPCs) were mixed with 90% matrigel to a total volume of 100 μl. The Martrigel plug was implanted into the right lateral area of the subcutis of the mouse. After 21 days, Matrigel plugs were observed by H & E staining and immunohistochemistry in the tissues of the mice.

As a result, as shown in FIG. 5 and FIG. 10, the similar tubular structure was confirmed by H & E staining in cells differentiated from nephron progenitor cells. In addition, immunohistochemical analysis revealed that the constructs identified the renal tubular cell markers CD13 (proximal) and MUC1 (distal). At this time, an isotype control group was used as a negative control group. The above structure confirmed that the structure was derived from a human derivative by expressing a human nuclear antigen (hNA). Therefore, it was confirmed that the nephron progenitor cells derived from hPSC were completely differentiated into renal tubular epithelial cells and had a renal tubular tubule production activity in vivo.

<110> Korea Advanced Institute of Science and Technology <120> Method for inducing differentiation of human pluripotent stem          cells into nephron progenitor cells <130> 13p-06-33 <160> 58 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F <400> 1 cttcgctctc tgctcctcct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R <400> 2 gttaaaagca gccctggtga 20 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> T F <400> 3 cagtggcagt ctcaggttaa gaagga 26 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> T R <400> 4 cgctactgca ggtgtgagca a 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MIXL1 F <400> 5 tccaggatcc aggtatggtt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MIXL1 R <400> 6 cgtttcagtt ccaggagcac 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EOMES F <400> 7 atgctgaaga gtatagtaaa gaca 24 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> EOMES R <400> 8 aacaccacca agtccatc 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX17 F <400> 9 aaagacccag ggtacctaaa 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SOX17 R <400> 10 aggaagacaa attctcacag cag 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FOXA2 F <400> 11 ctgagcgaga tctaccagtg ga 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FOXA2 R <400> 12 agtcgttgaa ggagagcgag t 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX6 F <400> 13 agcccagtat aagcgggagt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX6 R <400> 14 ctagccaggt tgcgaagaac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX1 F <400> 15 cacaactcgg agatcagcaa 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX1 R <400> 16 ggtacttgta atccgggtgc 20 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> OSR1 F <400> 17 cggagagtga gtggagag 18 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OSR1 R <400> 18 tgaagcagat acagggatta ca 22 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PAX2 F <400> 19 acgcccatta aagcacag 18 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PAX2 R <400> 20 ttacagagaa agagccaaca aa 22 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SALL1 F <400> 21 agcgaagcct caacatttcc aatcc 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SALL1 R <400> 22 aattcaaaga actcggcaca gcacc 25 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EYA1 F <400> 23 gatgtcaagt gtcagtaagg at 22 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EYA1 R <400> 24 aagtgaggtg gtaggagag 19 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> WT1 F <400> 25 tgtgtgttgt gttgtgttt 19 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> WT1 R <400> 26 tgtcaaagag caaatcatta tca 23 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SIX2 F <400> 27 cttgccaccg ttcattct 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SIX2 R <400> 28 ggaccaggac acagagta 18 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GDNF F <400> 29 caagaagcag cagttacca 19 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GDNF R <400> 30 gagcagaaag gacagagaag 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HOXD11 F <400> 31 tggaacgcga gtttttcttt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HOXD11 R <400> 32 ctgcagacgg tctctgttca 20 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CITED1 F <400> 33 gctgctaatg ccaagtgt 18 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CITED1 R <400> 34 gcccgtctct ttgtaagtaa c 21 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXD1 F <400> 35 tgcgggtccc tctatttatg 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXD1 R <400> 36 taacgcctgg acctgagaat 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HOXB7 F <400> 37 gtggactgtg ggtctggact 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HOXB7 R <400> 38 gaacacgcga gtggtaggtt 20 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RUNX2 F <400> 39 gacagcccca acttcctgt 19 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RUNX2 R <400> 40 ccggagctca gcagaataat 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1 F <400> 41 acttgcttga agacccatgc 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1 R <400> 42 ggtgtttgag cattgccttt 20 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PECAM1 F <400> 43 tgcgaatcga tcagtgga 18 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PECAM1 R <400> 44 accggggcta tcaccttc 18 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TIE2 F <400> 45 ccttagtgac attcttcc 18 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TIE2 R <400> 46 gcaaaaatgt ccacctgg 18 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH11 F <400> 47 cagttcgaaa gggatctcca 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH11 R <400> 48 gtagctgctt gatggcttcc 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CALPONIN F <400> 49 aggctccgtg aagaagatca 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CALPONIN R <400> 50 ccacgttcac cttgtttcct 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALBUMIN F <400> 51 tgcacagaat ccttggtgaa 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALBUMIN R <400> 52 ttcacgagct caacaagtgc 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AAT F <400> 53 gaagtcaagg acaccgagga 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AAT R <400> 54 gctggcagac cttctgtctt 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TUJ1 F <400> 55 gggcctttgg acatctcttc 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TUJ1 R <400> 56 cctccgtgta gtgacccttg 20 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MAP2 F <400> 57 gtggcggacg tgtgaaaatt gag 23 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MAP2 R <400> 58 ctggatctgc ctggggactg tg 22

Claims (16)

1) Human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) were cultured in serum-free medium containing Act A (A) and Wnt3a (Mixed paired-like homeobox) and EOMES (human embryonic stem cell) or human induced pluripotent stem cells (BMP4) and bFGF (basic fibroblast growth factor) inducing primitive streak (PS) cells in which eomesodermin is expressed;
2) Treatment of retinoic acid (RA), bFGF, and BMP7 with media containing the differentiated ROS cells in step 1) resulted in OSR1 (odd skipped related 1), PAX2 (paired box 2) and SALL1 inducing intermediate mesoderm (IM) cells in which transcription factor 1 is expressed; And
3) The medium containing mesenchymal stem cells differentiated in step 2) was treated with bFGF and BMP7 to induce SIX2 (SIX homeobox 2), GDNF (glial cell derived neurotrophic factor), HOXD11 (Homeobox protein Hox-D11) and WT1 Inducing differentiation into nephron progenitor cells (NPCs) in which Wilms tumor 1) is expressed.
A method for inducing differentiation of nephron progenitor cells (NPCs) from human embryonic stem cells or human induced pluripotent stem cells.
delete The method according to claim 1, wherein the culture in step 1) is cultured in a matrigel-coated culture dish. 2. The method according to claim 1, wherein the culture in step 1) is cultured in a matrigel-coated culture dish.
The method according to claim 1, wherein the serum-free medium of step 1) comprises Act A and Wnt3 in an amount of 80 to 120 ng / ml, respectively, and a method for inducing nephron progenitor differentiation from human embryonic stem cells or human induced pluripotent stem cells .
The method according to claim 1, wherein BMP4 and bFGF in step 1) are treated at 10 to 60 ng / ml and 5 to 15 ng / ml, respectively, from human embryonic stem cells or human induced pluripotent stem cells, Induction method.
The method according to claim 1, wherein the BMP4 and bFGF of step 1) are treated for 2 days, and the method for inducing nephron progenitor cell differentiation from human embryonic stem or human induced pluripotent stem cells.
The method according to claim 1, wherein the RA, bFGF and BMP7 of step 2) are treated with 5 to 20 μM, 30 to 70 ng / ml and 5 to 15 ng / ml, respectively, Induction of neuronal progenitor cell differentiation from induced pluripotent stem cells.
The method according to claim 1, wherein the RA, bFGF and BMP7 of step 2) are treated for 6 days to 8 days, and the induction of nephron progenitor cell differentiation from human embryonic stem or human induced pluripotent stem cells.
The method according to claim 1, wherein bFGF and BMP7 in step 3) are treated at 30 to 70 ng / ml and 130 to 170 ng / ml, respectively, from human embryonic stem cells or human induced pluripotent stem cells, Cell differentiation induction method.
The method according to claim 1, wherein the bFGF and BMP7 in step 3) are treated for 13 to 17 days.
(Brachyury) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) containing Act A, Wnt3a, BMP4, and bFGF, Mix paired- like homeobox) and EOMES (eomesodermin).
12. The method according to claim 11, wherein Act A, Wnt3a, BMP4, and bFGF are contained in an amount of 80 to 120 ng / ml, 80 to 120 ng / ml, 10 to 60 ng / ml, and 5 to 15 ng / ml, Wherein the composition comprises at least one member selected from the group consisting of:
T (brachyury) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) including RA, bFGF and BMP7, Mix paired-like homeobox (MIXL1) A composition for inducing mesenchymal cells expressing OSR1 (Odd skipped related 1), PAX2 (paired box 2), and SALL1 (spalt-like transcription factor 1) from ROS cells expressing EOMES (eomesodermin).
14. The composition according to claim 13, wherein RA, bFGF and BMP7 are contained in an amount of 5 to 20 [mu] M, 5 to 15 ng / ml and 30 to 70 ng / ml, respectively.
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