KR101785910B1 - Sialidosis human cell model and use thereof - Google Patents

Abstract

The present invention relates to a method for producing a model cell based on a human cell model of sialidosis, human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived neural progenitor cells, The present invention relates to the use of hiPSCs derived from a fibroblast of a patient suffering from a sialoidosis in a neural progenitor cell (NPC), which reproduces characteristics of a patient suffering from sialoidosis. And neural cells, and these cells can effectively identify accumulation of intracellular sialidosyl lysosomes, which are symptoms of sialidosis as a model cell of sialidosis, and changes in gene expression patterns, It is useful for studies on developmental mechanism analysis and development of therapeutic agents There.

Description

≪ Desc / Clms Page number 2 > Mycoplasma human cell model and its use {

The present invention relates to a method of producing human-induced pluripotent stem cells (hiPSCs) derived from a patient suffering from sialoidosis (muccosis) and producing tissue-specific cells differentiated therefrom, ), And the use of the cell model for screening for a therapeutic agent for sialidosis (muccioangiosis).

Sialidosis is an autosomal recessive inherited by a deficiency of the lysosomal sialidase or neuraminidase enzyme caused by the NEU1 (HGNC: 7758) gene mutation, and due to sialidase enzyme deficiency, mucoid lipid carbohydrate Is classified as an abnormally accumulated lysosomal storage disease (LSD). The sialidase enzyme removes sialic acid having a sugar-like molecular structure in the sugar-protein complex (glycoprotein), thereby allowing the cell to function normally, When the enzyme is deficient, the sugar-like substance is not normally removed, which causes abnormal symptoms due to accumulation in various tissues and various cells in the body, especially cells protecting from external pathogenic infection including nerve and bone marrow.

Sialidosis is known to occur at birth or within 1 year of birth. Infants with the disease have excessive swelling at the time of birth and are often observed with flatulence, swollen eyelids, hypertrophy of the gums, enlargement of the tongue, It is also accompanied by musculoskeletal abnormalities such as the dislocation of the pelvic bone. Newborns have sudden shrinkage of the involuntary muscles, cherry red macules on the retina, gait disturbances, progression, vision problems, and convulsions.

Examination shows hepatomegaly, splenomegaly, and excessive abnormal swelling. In the case of infants, muscular strength is weakened (hypotonia) and mental retardation may be seen from the beginning or rapidly progressed. Many patients have growth disturbances and often have a respiratory infection leading to death before the first year of life.

Four types of human sialidase genes (NEU1, 2, 3 and 4) are known, and the intracellular location of the protein is known to be different from the specificity of the substrate. Asp-box motifs important for sialidase enzyme activity are well conserved among species. The NEU1 gene encodes a lysosomal sialidase composed of 415 amino acids, and a mutation in the NEU1 gene causes intracellular accumulation of sialylated oligosaccharides and glycoproteins. Although more than 30 genetic mutations have been reported in patients with sialidosis, studies on the clinical pathology and phenotypic relationship with mutant types have yet to be performed.

The molecular mechanisms for understanding the pathogenesis of human Alidocis have not been extensively known and are currently limited and conservative. Although a mouse model of sialidocis has been developed and can mimic many of the features of human alidosys similarly, it can not completely replace human disease, thus limiting research using the sialidocyte mouse model There is a disadvantage. Therefore, the development of a human-derived sialoid cell model that can reproduce the pathogenesis mechanism of sialidoxys can be useful for understanding the causes of diseases and developing effective therapies.

Stem cells can be obtained from embryonic, fetal, and adult tissues, which are the cells of the pre-differentiation stage of each cell that constitutes the tissue. They are capable of infinitely proliferating in the undifferentiated state, Refers to cells with differentiation potential, which is a potential potential to differentiate into cells of various tissues. Stem cells are differentiated into specific cells by differentiation stimuli (environment), and unlike the differentiated cells in which cell division is stopped, they can self-renew themselves by cell division, , expansion, and it can be differentiated into other cells by different environments or differentiation stimuli, and is characterized by plasticity in differentiation.

Human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) And has excellent ability to differentiate. In particular, patient-derived hiPSCs can produce tissue-specific differentiated cells with immune and genetic characteristics, such as patients, in an in vitro differentiation system, resulting in the development of patient- (2009) Epilepsy Behav 14 Suppl 1: 81-85; Marchetto, MC, and B. Winner, et al. (2010), who are known to be effective evaluators of the mechanisms of complex disease during the early development of organogenesis (Muotri, Hum MoI Genet 19 (R1): R71-76).

To date, hiPSCs derived from patients with various genetic diseases have been reported to exhibit disease-specific phenotypes when directly differentiated into disease-associated cell types (Park, IH et al. Cell 134 , 877-886 (2008); Tiscornia, G. et al., Nature medicine 17, 1570-1576 (2011)). These disease-specific hiPSCs can be differentiated into tissue cells that are associated with a direct cause or impairment of the disease, so that the differentiated tissue-cell having the disease characteristic is useful for developing a specific mechanism research or therapeutic agent for identifying the cause of the disease Can be utilized.

Therefore, the present inventors have made efforts to establish a human cell model for studying sialoidosis based on the patient-derived hiPSCs technology. As a result, the inventors of the present invention have found that, from fibroblasts of patients with sialidoxys, human induced pluripotent stem cells induced differentiation and differentiation of human induced pluripotent stem cells (hiPSCs), embryoid body (EB) and neural progenitor cells (NPCs). HiPSCs of the sheath between alrido patient-derived in vitro (in vitro ) and in vivo ( in In addition to having pluripotency in vivo , it was confirmed that the mutation of causative gene in sialoidosis patients resulted in the decrease of sialidase activity, and it was confirmed that the hiPSCs derived from sialidosis patients were differentiated into neural progenitor cells Induction results also confirmed that sialidase activity decreased. In addition, a novel gene mutation (R347Q / V275A) was identified by examining the nucleotide sequence of the NEU1 gene, which is a gene responsible for the aliotissism of somatic cells of different sialoids, and hiPSCs and neural progenitor cells from the mutant somatic cells Day activity was decreased. The sialoidosis-derived human induced pluripotent stem cells (hiPSCs) and hiPSC-derived neural progenitor cells of the present invention significantly preserve the disease-specific phenotype and are therefore useful in the study of the cause of sialoidosis The present invention has been completed by suggesting a human cell model of the above-mentioned sialidocys.

It is an object of the present invention to develop a human cell model capable of reflecting the disease characteristics of patients with sialidosis (mucciosis) using human induced pluripotent stem cells (hiPSCs) The present invention provides a method for the development of a therapeutic and therapeutic agent for psoriasis, and a method for detecting a candidate agent effective for treatment of psoriasis using the human cell model of the onset, thereby developing a symptom relief and therapeutic agent for psoriasis to provide.

In order to achieve the above object,

i) In vitro ( in deriving a hiPSCs); vitro) alrido sheath (sialidosis) induced human fibroblasts (fibroblast) iPS cells (induced pluripotent stem cells isolated from human patients range from; And

ii) obtaining the hiPSCs derived in step i) above in vitro. < RTI ID = 0.0 > hiPSCs < / RTI >

The present invention also provides a model of sialidosis hiPSCs prepared by the above method.

In addition,

i) In vitro ( in preparing human induced pluripotent stem cells (hiPSCs) from fibroblasts isolated from patients with sialidosis in vitro ;

ii) inducing hiPSCs prepared in step i) to neural progenitor cells (NPCs); And

iii) obtaining the NPCs derived in step ii) above in vitro. < Desc / Clms Page number 10 >

The present invention also provides a sialidosis neural progenitor cell model prepared by the above method.

In addition,

i) inducing differentiation from the hiPSCs into an embryoid body (EB), neural progenitor cells (NPCs) or neurospheres; And

ii) using hiPSCs, neural progenitor cells or nerve ganglia as a model of the alliotosis, including the step of analyzing the differentiation markers, neural progenitor cell characteristics or morphological characteristics of neural progenitor cells derived from said step i) . ≪ / RTI >

In addition,

i) treating the specimens to differentiate from the hiPSCs model, neural progenitor cells or neurons;

ii) analyzing the characteristics of said somatic cells, neural progenitor cells or nerve cells of said step i); And

iii) comparing the result of the analysis of step ii) with that of the untreated control, to screen candidate therapeutic substances of sialidoxys.

Human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients with sialoidosis (muccozygia) of the present invention can be used as neural progenitor cells cells, NPCs) and neural cells, confirming that it is possible to effectively identify gene mutation, accumulation of intracellular lysosomes, and hypoallergenic activity, which are disease symptoms of the sialidoxic neuronal progenitor cells in the cells , And the cell was developed as a model cell of the sialidoxic neuronal progenitor cell. The human cell model of the sialidoxic neuronal progenitor cell of the present invention is useful for analysis of pathogenic mechanism of sialidoxic neuronal progenitor cells and development of therapeutic agent Respectively.

Brief Description of the Drawings Fig. 1 is a schematic diagram of a process for producing hiPSCs derived from somatic cells of sialoidosis according to the present invention and its use.
Fig. 2 is a diagram showing mutation of the gene responsible for the sialidosis of fibroblasts derived from normal and sialoidosis patients. Fig.
a: gene mutation of G227R, a protein mutation responsible for the sialoidosis in fibroblasts derived from a patient with sialoidosis; And
b: V275A (Het), which is a sialidase-causative protein mutation, and R347Q (Het) mutation which was first identified in the present invention in fibroblasts derived from a patient suffering from sialoidosis.
Fig. 3 is a diagram showing the morphology and accumulation of lysosomes of fibroblasts derived from normal and sialoidosis patients. Fig.
a: Identification of a typical morphology of cultured fibroblasts from normal and sialidosis patients by phase contrast microscopy; And
b: Confirmation of accumulation of lysosomes specifically found in sialidosis in fibroblasts derived from normal and sialidosis patients.
Fig. 4 is a graph showing inhibition of sialidase enzyme activity specifically in sialidosis in fibroblasts derived from normal and sialoidosis patients. Fig.
Figure 5 shows the activity of sialidase enzymes in HEK 297T cells overexpressing the gene NEU1 with the normal and mutated nucleotide sequences (R347Q or V275A).
Figure 6 is a Western blot analysis of NEU1 protein expression in HEK 297T cells overexpressing the gene NEUl with the normal and mutated nucleotide sequences (R347Q or V275A).
Fig. 7 is a diagram showing the structural variation caused by NEU1 protein sequence variation. Fig.
a: alignment of partial NEU1 protein sequences in vertebrates;
Amino acid sequences are shown in red (G277 and V275) and purple (R347), and Asp-boxes in orange;
b: Homologous modeling of the NEU1 protein structure based on the crystal structure of the human NEU2 protein; And
c: R347 amino acid mutation part near the Asp-boxes sequence part of NEU1 protein.
FIG. 8A is a schematic diagram showing a process for producing induced pluripotent stem cells (hiPSCs) derived from patients suffering from sialoadisosis.
FIG. 8B is a graph showing the morphological characteristics of hiPSCs derived from patients suffering from sialoidosis and alkaline phosphatase staining (AP staining).
FIG. 8C shows the expression of a pluripotency maker protein in sialidosis-derived induced pluripotent stem cells (hiPSCs).
Figure 8d is, in between the sheath alrido patient-derived iPS cells (hiPSCs) In vitro , the ability to differentiate into ectoderm such as ectoderm, endoderm, and mesodermal lobe was confirmed.
9 is a diagram showing a result of short chain repetition (STR) analysis of induced pluripotent stem cells (hiPSCs) derived from patients with sialoidosis.
FIG. 10 is a diagram showing karyotype analysis results of induced allergic stem cells (hiPSCs) derived from patients suffering from sialoidosis.
Figure 11 is, in vivo (in (hiPSCs) derived from a patient suffering from a cirrhosis patient in vivo .
FIG. 12A shows the gene mutation of G227R in induced pluripotent stem cells (hiPSCs) established in a cell line of a patient with normal and G227R gene mutations.
12B is a graph showing the mutation of V275A (Het) and R347Q (Het) gene mutations in induced pluripotent stem cells (hiPSCs) established in normal and siRNA disease cell lines with V275A (Het) and R347Q (Het) gene mutations to be.
FIG. 13 shows the accumulation of lysosomes specifically found in sialidoxys in sialidoxys-hiPSCs.
Fig. 14 is a graph showing inhibition of sialidase enzyme activity, which is specifically found in sialidoxys in sialidoxys-hiPSCs.
15A is a diagram showing the morphology of neural progenitor cells (sialidoxis-NPCs) differentiated from sialidoxys-hiPSCs by a phase contrast microscope.
FIG. 15B shows the expression of neural marker proteins in neural progenitor cells differentiated from sialidoxys-hiPSCs (sialidoxys-NPCs).
16A shows the gene mutation of G227R, a protein mutation that is a sialidosis-causing protein in neural progenitor cells differentiated from normal and sialidosis-hiPSCs (sialidoxys-NPCs).
FIG. 16B shows the expression of V275A (Het), which is a sialidase-causative protein mutation in the neural progenitor cells differentiated from normal and sialoadisis-hiPSCs (sialidoxyl-NPCs), and the gene of R347Q (Het) The mutation was confirmed.
FIG. 17 shows the accumulation of lysosomes specifically found in sialidosis in neuronal progenitor cells (sialoidosis-NPCs) differentiated from sialidoxys-hiPSCs.
Fig. 18 is a graph showing inhibition of sialidase enzyme activity, which is specifically found in sialidosis in neuronal progenitor cells (sialidosis-NPCs) differentiated from sialidoxys-hiPSCs.

Hereinafter, the present invention will be described in detail.

The present invention

i) In vitro ( in deriving a hiPSCs); vitro) alrido sheath (sialidosis) induced human fibroblasts (fibroblast) iPS cells (induced pluripotent stem cells isolated from human patients range from; And

ii) obtaining the hiPSCs derived in step i) above in vitro. < RTI ID = 0.0 > hiPSCs < / RTI >

The induction of the step i) is preferably performed using an ectopic expression of a pluripotency marker. Specifically, the ectopic expression is expressed by the reprogramming factor OCT4 ((octamer -binding transcription factor 4), SOX2 (SRY (sex determining region Y) -box 17), C-MYC and Kruppel-like factor 4 (Son MY et. al., Stem cells 31, 2374-2387; 2013), or a method through transformation of an episome vector expressing the reprogramming factor can be used, as is known in the art to produce hiPSCs.

In a specific embodiment of the present invention, the present inventors have found that mutation genes (G227R and V275A (Het) / R347Q (Het)) of different aspects of sialoids specifically appear in fibroblast cells derived from different sialoadic patients, (See Fig. 2). And from fibroblasts of the patient making the hiPSCs through somatic cell reprogramming technique (see Fig. 8), between the results confirm the characteristics of the system between alrido -hiPSCs, alrido cis -hiPSCs is in vitro (in vitro ) and in vivo ( in Reference vivo) before multipotential having at the same time (8 to 11 a in), keeping the cause genetic mutation that appears between alrido cis patients (see FIG accumulation (Fig. 13 of the reference 12), lysosomes (lysosome) from thereof), and Indicating a decrease in the activity of sialidase (see Fig. 14).

Therefore, since the hiPSCs model derived from the somatic cell of the present invention exhibits the ability to differentiate while maintaining the disease phenotype of the sialidoxic somatic cells, the method for producing the hiPSCs model is not limited to the analysis of the pathogenic mechanism of sialidosis, Lt; RTI ID = 0.0 > cell < / RTI >

The present invention also provides a model of sialoadithi hiPSCs prepared by the above method.

The hiPSCs are preferably but not limited to a model of sialoadithi hiPSCs characterized by any one or more of i) and vi)

i) c.G679> A mutation or c.T824> C and c.G1040> A mutation of NEU1 gene;

ii) G227R mutations or V275A and R347Q mutations of proteins synthesized from the NEU1 gene;

iii) hiPSCs form of normal cells;

iv) a stemness maker (OCT4) comprising OCT4, NANOG, TRA-1-81, specific embryonic antigen 3 (SSEA3), specific embryonic antigen 4 (SSEA4) );

v) accumulation of lysosomes; And

vi) Reduced sialidase activity.

The NEU1 gene is preferably composed of SEQ ID NO: 8, and the protein synthesized from the NEU1 gene is preferably composed of SEQ ID NO:

Since the hiPSCs model derived from the sialidopsis of the present invention exhibits the ability to differentiate while maintaining the same characteristics as the somatic cells of the sialidoxic patient, the hiPSCs model can be usefully used for development of the pathogenesis mechanism and treatment of sialoidosis.

In addition,

i) In vitro ( in producing human induced pluripotent stem cells (hiPSCs) from a fibroblast isolated from a patient suffering from sialoidosis in vitro ;

ii) inducing hiPSCs prepared in step i) to neural progenitor cells (NPCs); And

iii) obtaining the NPCs derived in step ii) above in vitro. < Desc / Clms Page number 10 >

The present invention also provides a model of a sialidoxic neuronal progenitor cell prepared by the above method.

Preferably, the neuro progenitor cells are characterized by any one or more of the following i) to iii), but are not limited thereto.

i) expressing a neuronal marker protein comprising Nestin (type VI intermediate filament (IF) protein), TUJ1 (class III beta-tubulin) and MAP2 (Microtubule-associated protein 2);

ii) accumulation of lysosomes; And

iii) Reduced sialidase activity.

In another specific embodiment of the present invention, the present inventors have found that when hiPSCs derived from a patient suffering from sialoidosis (sialidoxys-hiPSCs) are induced to differentiate into embryoid bodies (EB) It was confirmed that all of the differentiation markers of the mesodermal and ectodermal lobes were expressed, thereby confirming that the sialidase-hiPSCs-derived somatic cells exhibit the ability to differentiate (see Fig. 8D).

In addition, induction of the differentiation of sialidoxyl-hiPSCs into neural progenitor cells (sialidase-NPCs) revealed that neural marker proteins were expressed (see Fig. 15b), maintaining the causative gene mutation of sialidoxys (See FIG. 16).

In addition, the present inventors have confirmed that sialidoxis-NPCs maintain a specific disease phenotype of sialidoxis. As a result, the accumulation of lysosomes in the cells increases (see FIG. 17) and the activity of sialidase is markedly decreased (See Fig. 18).

Therefore, the neural progenitor cells differentiated from the hiPSCs derived from the patient of sialoidosis according to the present invention maintain the characteristics appearing in the cells of the sialidoxic patient, so that they can be usefully used as a human-derived cell model for studying the onset of sialidosis .

In addition,

i) inducing differentiation from the hiPSCs into an embryoid body (EB), neural progenitor cells (NPCs) or neurospheres; And

ii) using hiPSCs, neural progenitor cells or nerve ganglia as a model of the alliotosis, including the step of analyzing the differentiation markers, neural progenitor cell characteristics or morphological characteristics of neural progenitor cells derived from said step i) . ≪ / RTI >

The differentiation markers of the above-mentioned epitope are NESTIN and TUJ1 which are ectoderm markers, SOX17 (sexy determining region Y) -box 17 which is an endoderm marker, alpha-FP (Alpha-fetoprotein) and mesoderm ) Smooth muscle actin (α-SMA) and DESMIN (Muscle Cell Marker).

Preferably, the neural progenitor cells are characterized by any one or more of the following i) to iii):

i) expressing a neuronal marker protein comprising Nestin (type VI intermediate filament (IF) protein), TUJ1 (class III beta-tubulin) and MAP2 (Microtubule-associated protein 2);

ii) accumulation of lysosomes; And

iii) Reduced sialidase activity.

In addition,

i) treating the specimens to differentiate from the hiPSCs model, neural progenitor cells or neurons;

ii) analyzing the characteristics of said somatic cells, neural progenitor cells or nerve cells of said step i); And

iii) comparing the result of the analysis of step ii) with that of the untreated control, to screen candidate therapeutic substances of sialidoxys.

The candidate substance for therapeutic treatment of sialidoxys can be selected as a substance which exhibits a similar level of characteristics to the hiPSC model of the present invention or a normal control in analyzing characteristics of the differentiated nerve cells or differentiated therefrom, Specifically, a substance that increases the expression level of the NEU1 gene in neurons differentiated from the hiPSC model of the present invention to a level similar to that of a normal control is the most preferable, but is not limited thereto.

The hiPSCs derived from a patient suffering from a sialoidosis according to the present invention, the differentiated specimens derived therefrom, and the neural progenitor cells maintain the characteristics appearing in the cells of a patient suffering from sialidosis, and thus are useful as a human-derived cell model for studying the onset of sialidosis Can be used.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example 1 > Sialidocys ( Mucciosis ) Human somatic cell-derived human All-purpose induction  Stem Cells( human induced pluripotent stem cells ; hiPSCs )

<1-1> Sialidocys  Identification of causative gene mutations in patient somatic cells

The present inventors cultured a fibroblast derived from a patient suffering from a sialidoxis to identify a gene mutation that causes sialidosis (mucciosis), and found that the sequence of NEU1 (SEQ ID NO: 8 ).

In particular, the fibroblasts derived from the sialoadicosis patients, GM02685 and GM02921 (Coriell Institute for Medical Research, USA), were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Invitrogen, USA) (Invitrogen, USA), 1 mM L-glutamine (Invitrogen, USA) and 0.1 mM beta-mercaptoethanol (Sigma, USA) (Dulbecco ' s modified Eagle &apos; s medium, DMEM). After the incubation, RNA was extracted from the cultured cells using RNAiso Plus (Takara, Japan) or RNeasy mini kit (Qiagen, USA) according to the manufacturer's protocol. Then, PCR was performed to synthesize the cDNA of NEU1 gene using the primers (SEQ ID NOS: 1 and 2) and Superscript III (Invitrogen, USA) shown in Table 1 below. The PCR products were separated on 1% agarose gel and full-length bands were cut out and purified using QIAquick Gel Extraction Kit (Qiagen, USA). The purified oligomers were analyzed for the entire sequence of the cDNA of the gene NEU1 using the primers (SEQ ID NOS: 3 to 7) shown in Table 1 below. As a control, the cDNA of the gene NEU1 was synthesized using the same method as described above for CRL-2097 (American Cell Line Bank, ATCC), a wild-type fibroblast derived from human skin, and its sequence was confirmed. As its wild type, .

Forward Reverse NEU1 cDNA synthesis 5'-gcttaagggtgacatctgcgc-3 ' SEQ ID NO: 1 5'-ctgtctttcaggcgtctctag-3 ' SEQ ID NO: 2 Whole sequence analysis of NEU1 cDNA 5'-gtctagctgccagggtcgcg-3 ' SEQ ID NO: 3 5'-gactccgtcccgctccagcg-3 ' SEQ ID NO: 4 5'-ggtatggagcaaggatgatgg-3 ' SEQ ID NO: 5 5'-cctgaaggcagaatacccct-3 ' SEQ ID NO: 6 5'-gccgaattgtcctccgcagc-3 ' SEQ ID NO: 7

As a result, as shown in Fig. 2, in one type of sialoadocytic fibroblast cell line, the nucleotide sequence variation of c.G679 > A in the NEU1 gene encoding sialidase was found to be different from that of the sialidase protein G227R mutation was observed. In addition, in another type of cyadylase fibroblast cell line, nucleotide sequence mutations of cT824> C and c.G1040> A in the NEU1 gene encoding sialidase were shown, and the sialidase protein synthesized from the gene showed V275A And R347Q mutations were observed. It was the first time that a cell line having the above sequence mutation was utilized in the invention (Fig. 2).

<1-2> Sialidocys  patient Fibroblast cell line  Derived human All-purpose induction  Stem Cells( hiPSCs Production

The present inventors have conducted reprogramming using a retrovirus expressing four reprogramming factors including OCT4, SOX2, C-MYC and KLF4 (Son MY et. ali, Stem cells 31, 2374-2387; 2013), hiPSCs (sialidoxys-hiPSCs) with differentiating ability were prepared from somatic cells of sialoidosis (Fig.

Specifically, retroviruses encoding OCT4, SOX2, C-MYC, and KLF4 were transfected into fibroblasts of patients with sialidoxys and then cultured in somatic cell medium for 5 days. After 5 days, the infected cells were transferred to a matrigel-coated plate containing human embryonic stem cell (hESCs) culture medium (hESCs-CM), and further cultured for 2 to 3 weeks And cultured to obtain a hiPSCs colony. The cDNA of the NEU1 gene was synthesized and analyzed for the hiPSCs derived from the sialidopsis using the method of Example <1-1>. As a control, the CRL-2097 cell line was subjected to the same procedure as above to induce the production of wild-type hiPSCs.

As a result, as shown in Fig. 12, the expression of sialidoxys-hiPSCs (c.G679 > A or c.T824 > C and c.G1040> A) from fibroblasts derived from a patient with a different base sequence mutation , And it was confirmed that it exhibited the same base sequence variation as that of the sialidoxylated fibroblast (Fig. 12).

As another reprogramming method for producing hiPSCs, hiPSCs having differentiating ability from somatic cells of sialidoxic patients were prepared by reprogramming culture using retrovirus using the somatic reprogramming method (Fig. 8A).

Specifically, as described in Fusaki et al. Proc. Jpn. Acad., Ser., B 85, 2009, the production of non-intercalating human induced pluripotent stem cells (or human degenerated stem cells, hiPSCs) We used the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (A16517, Invitrogen). For the generation of hiPSCs, fibroblasts of the patient with sialoidosis GM02685 (Coriell Institute) were infected into 6-well plates at a concentration of 2 X 10 ⁵ cells per well using CytoTune®-iPS 2.0 KOS, CytoTune®-iPS 2.0 hc-Myc, and CytoTune®-iPS 2.0 hKlf4. At this time, the amount of transfection (or titer) was determined by MOI (CIU / cell) according to the lot number. Seven days after transduction, the cells were re-inoculated to a 6-well plate at a concentration of 1 to 5 X 10 ⁴ cells in a gamma-mouse embryonic fibroblast (MEF) feeder condition at a concentration of 2.5 × 10 ⁵ cells. Were replaced once every two days with hiPSC culture medium supplemented with bFGF at a concentration of 20 ng / ml. After about 21 days of transduction, colonies similar in shape to hESCs were obtained, transferred to 6-well plates of the feeder conditions, and cultured continuously by the same culture method as hESC culture.

Sialidase-hiPSCs produced by different reprogramming techniques have similar phenotypic character and phenotype.

< Example  2> Sialidocys  origin hiPSCs ( Sialidocys - hiPSCs ) Identification of features

<2-1> Sialidocys - hiPSCs Morphological morphology ) Feature identification

To confirm the morphological characteristics of the sialidopsis-hiPSCs prepared in the above Example <1-2>, the present inventors confirmed the colony form and karyotype of the sialidosis-hiPSCs (Fig. 10) Short Tandem Repeat, STR) analysis (FIG. 9).

Specifically, reprography of sialidopsis-hiPSCs was induced in the same manner as in Example <1-2>, and then the phase of sialidopsis-hiPSCs colonies was confirmed by phase-contrast microscopy. In addition, a karyotyping analysis was carried out by Chromosomal G-banding analysis with reference to GenDix Inc., Korea. STR genotyping of Sialidosis-hiPSCs and Sialidosis-hiPSCs was performed by HumanPass Inc. (Korea), and compared with H9 hESCs, a normal human embryonic stem cell line. Respectively.

As a result, as shown in FIG. 8B and FIG. 10, it was confirmed that sialidoxis-hiPSCs having obtained the ability to differentiate from sialidoxyl-fibroblast were successfully derived (Fig. 8B), and the produced sialidoxys-hiPSCs Showed similar colony morphology and normal karyotype as human embryonic stem cells (hESCs) (Figs. 9 and 10).

<2-2> Sialidocys - hiPSCs of AP ( Alkaline 포스화제 ) Confirm dyeing

The inventors of the present invention used alkaline phosphatase staining (AP), which is a pre-differentiation marker, to identify the ability of sialidopsis-hiPSCs to differentiate from sialidoxys prepared in Example <1-2> staining).

Specifically, 1 ml of a citrate solution, 2.6 ml of acetone, and 320 쨉 l of 37% formaldehyde (Sigma Aldrich, USA) were mixed with an AP staining kit (Alkaline phosphatase kit, Sigma Aldrich, USA) To prepare a fixative solution. The fixative solution thus prepared was added to the sialidopsis-hiPSCs prepared in Example <1-2>, and left at room temperature for 15 minutes in the dark. 100 μl of sodium nitrate solution and 100 μl of FRV-alkaline solution were left for 2 minutes and then 100 μl of 4.5 ml of sterilized water and Naphthol AS-BI alkali solution was added And wrapped with foil to block the light. The immobilized cells were washed once with PBS, allowed to stand in the prepared AP staining mixture, washed twice with water or PBS for 2 minutes, and the AP-stained cells were observed with a phase contrast microscope.

As a result, as shown in FIG. 8B, it was confirmed that the sialidoxys-hiPSCs stained positive for the AP as a marker for the differentiation ability (FIG. 8B).

<2-3> Sialidocys - hiPSCs of Total differentiation ability ( pluripotency ) Confirm

In order to further confirm whether sialidase-hiPSCs in undifferentiated state derived from the patient of sialoidosis prepared in Example <1-2> exhibited the ability to differentiate, the inventors of the present invention conducted a study on the effect of sialidoxys-hiPSCs The expression of pluripotency maker protein was confirmed.

Specifically, the sialidosys-hiPSCs prepared in Example <1-2> were treated with 4% formaldehyde, fixed at room temperature for 10 minutes, and then treated with 0.1% Triton X-100 Was treated for 15 minutes to impart permeability to the cell membrane. After treatment, the treated cells were washed with PBS containing 4% bovine serum albumin (BSA) and then incubated with anti-OCT4 antibody (1: 100 dilution, sc-9081, Cruz Biotechnology Inc. Anti-TRA-1-81 antibody (1: 100 dilution, MAB4381, Chemicon, USA), anti-SSEA3 (USA), anti-NANOG antibody (1: 100 dilution, sc-33759, Cruz Biotechnology, USA) Treated with antibodies (1: 100 dilution, MAB1435, R & D Systems, USA), anti-SSEA4 antibody or anti-TRA-1-60 antibody (1: 300 dilution, Millipore, USA) , And washed. After washing, the secondary antibody conjugated with Alexa Fluor 488 (Alexa Fluor 488) or Alexa Fluor 594 (Alexa Fluor 594) (Invitrogen, USA) was treated and allowed to stand at room temperature for 2 hours to immobilize sialidocyte-hiPSCs And the expression of OCT4, NANOG, SOX2, SSEA4, Tra-1-80 and Tra-1-61 protein was observed by fluorescence microscope. In order to compare the degree of expression, 4'6-diamidino-2-phenylindole (DAPI) was treated to stain nuclei of cells and compared.

As a result, it was confirmed that OCA4, NANOG, TRA-1-81, SSEA3, SSEA4, and TRA-1-60 protein, which are the differentiation-inducing markers, expressed at the normal cell level in the sialidoxys -hiPSCs as shown in FIG. 8C 8c).

<2-4> In vitro ( in vitro )in Sialidocys - hiPSCs The possibility of differentiation of

The present inventors induced differentiation to form an embryoid body (EB) from sialidocis-hiPSCs in order to confirm whether hiPSCs derived from sialidopsis show pluripotency in vitro, The endoderm markers SOX17 and alpha-FP, the mesoderm markers alpha-smooth muscle actin (alpha-SMA) and DESMIN, and the ectoderm markers NESTIN and Expression of three germ layer markers of TUJ1 was confirmed.

Specifically, the sialidopsis-hiPSCs prepared by the method of Example 1-2 were cultured in DMED / F12 medium containing 10% serum replacement (SR) in an embryoid body differentiation medium for 7 days to induce differentiation into sialidase-hPSCs-derived soma (sialidocis-EB). Then, the expression of NESTIN, TUJ1, SOX17, α-FP, α-SMA or DESMIN protein was confirmed by immunofluorescence staining in the same manner as in Example <2-3. SMA antibody (1: 400 dilution; A5228, Sigma-Aldrich, USA) as anti-DESMIN antibody (1:50 dilution; AB907, Chemicon, USA) as the primary antibody for the immunofluorescent staining, (1: 100 dilution; 1: 100 dilution; MAB1924, R & D Systems, USA), anti-TUJ1 antibody (1: 500 dilution; (1: 100 dilution; MAB5326, Chemicon, USA) were used, respectively. To check the degree of expression, 4'6-diamidino-2-phenylindole (4'6-diamidino-2-phenylindole, DAPI) to stain nuclei of cells and compare them.

As a result, as shown in FIG. 8D, it was confirmed that the sialoid cells differentiated cells express all three types of germinal marrow markers of NESTIN, TUJ1, SOX17,? -FP,? -SMA or DESMIN protein 8d).

<2-5> In vivo in vivo )in Sialidocys  origin hiPSCs The possibility of differentiation of

In order to confirm whether the hiPSCs derived from the sialidopsis prepared in Example <1-2> exhibited pluripotency in vivo, the inventors of the present invention have found that in the immunodeficient nude mice, the teratoma type of sialidosis-hiPSCs (Teratoma formation).

Specifically, the sialidopsis-hiPSCs prepared by the same method as in Example <1-2> were counted as 1 × 10 ⁶ cells, and 4-week-old SPF / VAF immunodeficient nude mice (Orient Bios, ), And then mice were bred. After 12 weeks, the mice were sacrificed and teratomas were obtained, and 4% formaldehyde was added and embedded in paraffin. Then, treatment with Harris hematoxylin and Eosin is performed to form teratoma of endoderm, ectoderm and mesoderm through H & E staining (Hematoxylin & Eosin staining) Respectively.

As a result, as shown in Fig. 11, formation of ectodermal nerve tissues, mesodermal cartilage and muscles, and endodermial epithelium were observed in teratomas derived from sialidoxys-hiPSCs (Fig. 11) .

<2-6> Sialidocys - hiPSCs of Sialidocys  Identification of causative protein mutations

In order to characterize the mutant protein due to the mutation of the gene responsible for the sialidosis in the sialidoxys-hiPSCs prepared in the above Example <1-2>, the present inventors investigated the activity of the sialidase protein in the sialidase-hiPSCs Respectively.

Specifically, a fibroblast cell line and hiPSCs prepared by the same method as in Example <1-2> were obtained and analyzed by pH 4.5 analysis containing 20 mM citrate, 60 mM NaCl and 1 mM CaCl ₂ And suspended in a buffer solution. Then, the cells were disrupted by repeating 5 to 10 times through a sonicator to dissolve the cells, centrifuged at 13,000 rpm at 4 ° C for 10 minutes to remove cell lysates, and only proteins dissolved in the buffer solution were extracted. After removal, 2 μg of protein lysate was added to the assay buffer solution containing 0.2 mM 2 '- (4-methylumbelliferyl) -α-DN-acetylneuraminic acid (4-MUNANA, Sigma-Aldrich, USA) For 4 hours. To terminate the reaction, 0.1 M glycine-NaOH buffer (pH 10.5) was added. The fluorescence of lex = 365 nm / lem = 450 nm was measured by SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices) to confirm the activity of sialidase. As a control, the activity of sialidase was examined in the same manner as that of normal human-hiPSCs, and then the activity of sialidase-hiPSCs relative sialidase was compared.

As a result, as shown in Figs. 4 and 14, in contrast to normal fibroblasts, in contrast to normal fibroblast-derived hiPSCs, similar to sialidase activity (Fig. 4) Alidosyl-hPSCs was found to exhibit significantly lower interleaved activity (Fig. 14).

<2-7> Sialidocys - hiPSCs Confirmation of lysosomal accumulation

The present inventors used LysoTracker Red DND-99 (diluted 1: 20000; Invitrogen) in the cell culture medium in order to confirm the content of cialic acid accumulated in the lysosome in the sialidoxys-hiPSCs prepared in Example <1-2> USA) and incubated for 30 min at 37 ° C to observe intracellular lysosomes.

As a result, as shown in Fig. 3 and Fig. 13, in contrast to normal fibroblasts, in contrast to normal fibroblast-derived hiPSCs, similar to the case in which accumulation of lysosomes was significantly increased in somatic cells derived from sialoidosis patients (Fig. 3) The accumulation of significantly increased lysosomes in cis-hPSCs was confirmed (Fig. 13).

<2-8> Sialidocys  Originating from somatic somatic cells hiPSCs  Identification of causative gene mutation

The present inventors confirmed the mutation of the gene responsible for hiPSCs derived from somatic cell of sialoidosis in the same manner as in Example <1-1>.

As a result, as shown in Fig. 12, the hiPSCs derived from the sialoid fibril cell line produced in the somatic cells of the mutant nucleotide sequence of c.G679 > A in the NEU1 gene were the same as the bases of c.G679 > A in the NEU1 gene , Showing that the G227R mutation appeared in the sialidase protein synthesized in the gene (Fig. 12A). In addition, the hiPSCs derived from the cytochalasin fibroblast cell line produced in the somatic cells of the nucleotide sequence mutations of c.T824> C and c.G1040> A in the NEU1 gene showed that c.T824> C And c.G1040 > A, indicating that V275A and R347Q mutations appeared in the sialidase protein synthesized in the gene (Fig. 12B).

< Example  3> Sialidocys  Derived neural progenitor cells ( Neural progenitor cells , NPCs )

<3-1> Sialidocys - hiPSCs from Amphibian ( embryoid body , EB ) And neural progenitor cells ( NPCs ) Induction of differentiation and identification of mutant proteins

The inventors have in vitro (in In order to induce neuronal differentiation from sialoadithyses from sialidopsis-hiPSCs in vitro , sialidase-hiPSCs were used to differentiate into neural progenitor cells (NPCs) Lt; / RTI &gt;

Specifically, the colony of sialidopsis-hiPSCs induced reprogramming for 7 days in the same manner as in Example <1-2> was diluted with DMED / F12 containing 10% serum replacement (SR) And cultured in embryoid body differentiation medium for 7 days to induce differentiation into sialidase-hPSCs-derived soma (sialidocis-EB). Differentiated sialidocys-EBs were incubated with 1 x N2 / B27 (Invitrogen, USA), 20 ng / ml bFGF, 20 ng / ml EGF (Invitrogen, USA) and 10 ng / ml leukemia inhibitory factor Sigma-Aldrich, USA) for 2 weeks to obtain neural progenitor cells (sialidocis-NPCs) derived from the differentiated sialoids. During NPCs differentiation, subculture was performed weekly using McClain tissue chopper (Mickle Engineering, UK). The medium was replaced once every two days. In order to finally differentiate into neuronal cells and glial cells, the resulting sialidoxis-NPCs were attached to matrigel-coated cover slips and incubated for 3 to 4 weeks with NPCs without growth factors Lt; / RTI &gt; The differentiated sialoadicase-NPCs were sequenced in the same manner as in Example <1-1> to confirm the nucleotide sequence variation of NEU1, which is a gene for sialidoxys. As a control group, normal human-derived hiPSCs were cultured in the same manner as above to induce differentiation into amphibodies and NPCs derived from normal human-hiPSCs.

As a result, as shown in Figs. 15A and 16, the NPCs derived from sialidopsis maintained a spherical neurosphere morphology (Fig. 15A). In addition, it was confirmed that the G227R mutation appeared in the sialidase protein synthesized in the above gene (Fig. 16 (a)), showing the nucleotide sequence variation of c.G679 > A in the NEU1 gene encoding sialidase And c.G1040 > A, indicating that V275A and R347Q mutations appeared in the sialidase protein synthesized in the gene (Fig. 16B).

<3-2> Sialidocys - Amphibious Ability to distinguish  Confirm

In order to confirm the differentiation ability of the sialidocys-amphorae prepared in the above Example <3-1>, the inventors of the present invention found that the ectoderm markers NESTIN and TUJ1, the endoderm markers Expression of three germ layer markers of SOX17 and alpha-FP and mesoderm markers alpha-smooth muscle actin (alpha-SMA) and DESMIN was confirmed.

Specifically, Sialoids-EB induced differentiation in the same manner as in Example <3-1> was immunofluorescently stained in the same manner as in Example <2-3> to obtain NESTIN, TUJ1, SOX17, FP, alpha-SMA, or DESMIN protein. (1: 100 dilution; MAB5326, Chemicon, USA), anti-TUJ1 antibody (1: 500 dilution; PRB-435P, Covance, USA) as the primary antibody for the immunofluorescent staining, anti- Anti-FP antibody (1: 200 dilution; A8452, Sigma-Aldrich, USA), anti-α-SMA antibody (1: 400 dilution; A5228 (Sigma-Aldrich, USA) or anti-DISMIN antibody (1:50 dilution; AB907, Chemicon, USA) were used. To check the degree of expression, 4'6-diamidino-2-phenylindole (4'6-diamidino-2-phenylindole, DAPI) to stain nuclei of cells and compare them.

As a result, as shown in FIG. 8D, it was confirmed that the sialidosis-differentiated cells express all three types of germline marker of ESTIN, TUJ1, SOX17,? -FP,? -SMA or DESMIN protein 8d).

< Example  4> Sialidocys - hiPSCs  origin NPCs ( Sialidocys - NPCs ) Identification of features

<4-1> Sialidocys - NPCs  And the nerves of the differentiated cells therefrom Marker  Confirmation of protein expression

In order to confirm whether NPCs derived from the above-described sialoids-hiPSCs (sialidoxys-NPCs) and nerve cells were induced to differentiate normally, the present inventors have used NPCs derived from sialidoxys -hiPSCs and nerve cell- Expression of the proteins NESTIN, TUJ1 and MAP2 proteins was confirmed.

Specifically, the method of Example <3-1> was performed to induce the differentiation of sialidoxyl-NPCs and the differentiated neurons (sialidocyte-nerve cells). Then, the expression of NESTIN, TUJ1 and MAP2 proteins was confirmed by immunofluorescent staining in the same manner as in Example <1-3>. (1: 100 dilution; MAB5326, Millipore) for neurons, anti-TUJ1 (1: 500 dilution; PRB-435P, covance) and anti-MAP2 : 1000 dilution; M4403, Sigma-Aldrich) antibody was used. In order to compare the degree of expression, 4'6-diamidino-2-phenylindole (DAPI) was treated to stain nuclei of cells and compared. As a control group, normal nPCs and neurons differentiated therefrom were immunofluorescently stained under the same conditions as above to confirm the expression of neuron-specific marker proteins.

As a result, it was confirmed that NESTIN-positive neuronal progenitor cells and TUJ1-positive neuronal cells were finally differentiated from both normal NPCs and sialidoxyl-NPCs as shown in FIG. 15B (FIG. 15B).

< Example  5> Sialidocys - hiPSCs Of differentiated neurons Molecular  Identification of phenotype

<5-1> Sialidocys - NPCs of Sialidocys  Identification of causative protein mutations

In order to characterize the mutant protein due to the mutation of the gene responsible for the sialoidosis in the sialidoxyl-NPCs prepared in the above Example <3-1>, the present inventors investigated the activity of the sialidase protein in the sialidoxys-hiPSCs Respectively.

Specifically, the fibroblast cell line and the same method as in the above Example <3-1> were performed to obtain sialidoxys-NPCs, and a pH of 20 mM citrate, 60 mM NaCl and 1 mM CaCl ₂ 4.5 assay buffer. Then, the cells were disrupted by repeating 5 to 10 times through a sonicator to dissolve the cells, centrifuged at 13,000 rpm at 4 ° C for 10 minutes to remove cell lysates, and only proteins dissolved in the buffer solution were extracted. After removal, 2 μg of protein lysate was added to the assay buffer solution containing 0.2 mM 2 '- (4-methylumbelliferyl) -α-DN-acetylneuraminic acid (4-MUNANA, Sigma-Aldrich, USA) For 4 hours. To terminate the reaction, 0.1 M glycine-NaOH buffer (pH 10.5) was added. The fluorescence of lex = 365 nm / lem = 450 nm was measured by SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices) to confirm the activity of sialidase. As a control, the activity of the sialidase was examined in the same manner as that of the normal human-NPCs, and then the activity of the sialidase-NPSCs relative sialidase was compared.

As a result, as shown in Fig. 18, it was confirmed that the sialidoxyl-NPCs exhibited remarkably low sialidase activity when compared with the sialidase activity of the normal-NPCs (Fig. 18).

<5-2> Sialidocys - NPCs Confirmation of lysosomal accumulation

To confirm the content of lysosome in the sialidoxyl-NPCs prepared in Example <3-1>, the present inventors treated LysoTracker Red DND-99 (1: 20000 dilution; Invitrogen, USA) And reacted at 37 캜 for 30 minutes to observe intracellular lysosomes. As a control group, the accumulation of lysosomes was confirmed by performing the same procedure as above for the normal-NPCs.

As a result, as shown in FIG. 17, the accumulation of lysosomes markedly increased in the sialidoxyl-NPCs was observed in contrast to the NPCs derived from the normal hiPSCs (FIG. 17).

< Example  6> human embryonic kidney cells ( HEK293T  Cell) Sialidocys  Cause protein NEU1 Overexpression of Molecular  Identification of phenotype

The present inventors conducted the following experiments to confirm sialidase enzyme activity and protein expression in human embryonic kidney cells (HEK 293T cells) through overexpression of NEU1, which is a protein of sialoidosis.

Specifically, the NEU1 gene having the normal NEU1 gene and the mutant base sequence (R347Q or V275A) obtained in the above Example <1-1> was amplified to bind the fluorescent protein GFP (green fluorescent protein) to the NEU1 protein, and pEGFP-N3 vector (BD bioscience Clontech). The FLAG tag was inserted between the 3-terminus of the gene NEU1 and the 5-terminus of the GFP. The prepared NEU1-pEGFP plasmid was transformed into HEK 293T cells using lipofectamine 2000 (Invitrogen). Sialidase enzyme activity was measured in the same manner as in Example <2-6>. In addition, to perform Western blot for confirmation of protein expression, 30 μg of cell lysate was loaded on a 4-20% gradient gel (BIO-rad) and transferred to nitrocellulose membrane. The NEU1-GFP binding protein was detected by anti-GFP antibody (Abcam, ab290, 1: 5000) and β-actin was detected by anti-β-actin antibody (Abcam, ab6276, 1: 5000).

As a result, as shown in FIG. 5 and FIG. 6, it was confirmed that the NEU1-GFP binding protein prepared from the NEU1 gene having the mutant nucleotide sequence (R347Q or V275A) was expressed in HEK 293T cells (FIG. 6) (R347Q or V275A) in the case of NEU1-GFP binding protein (Fig. 5).

<110> Korea Research Institute Bioscience and Biotechnology <120> Sialidosis human cell model and use thereof <130> 2015P-02-050 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_F <400> 1 gcttaagggt gacatctgcg c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_R <400> 2 ctgtctttca ggcgtctcca g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_Full_F <400> 3 gtctagctgc cagggtcgcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_Full_2 <400> 4 gactccgtcc cgctccagcg 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_Full_3 <400> 5 ggtatggagc aaggatgatg g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_Full_4 <400> 6 cctgaaggca gaatacccct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NEU1_DNA_Full_5 <400> 7 gccgaattgt cctccgcagc 20 <210> 8 <211> 1248 <212> DNA <213> human <400> 8 atgactgggg agcgacccag cacggcgctc ccggacagac gctgggggcc gcggattctg 60 ggcttctggg gaggctgtag ggtttgggtg tttgccgcga tcttcctgct gctgtctctg 120 gcagcctcct ggtccaaggc tgagaacgac ttcggtctgg tgcagccgct ggtgaccatg 180 gagcaactgc tgtgggtgag cgggagacag atcggctcag tggacacctt ccgcatcccg 240 ctcatcacag ccactccgcg gggcactctt ctcgcctttg ctgaggcgag gaaaatgtcc 300 tcatccgatg agggggccaa gttcatcgcc ctgcggaggt ccatggacca gggcagcaca 360 tggtctccta cagcgttcat tgtcaatgat ggggatgtcc ccgatgggct gaaccttggg 420 gt; aaggccggct gccaggtggc ctctaccatg ttggtatgga gcaaggatga tggtgtttcc 540 tggagcacac cccggaatct ctccctggat attggcactg aagtgtttgc ccctggaccg 600 ggctctggta ttcagaaaca gcgggagcca cggaagggcc gcctcatcgt gtgtggccat 660 gggacgctgg agcgggacgg agtcttctgt ctcctcagcg atgatcatgg tgcctcctgg 720 cgctacggaa gtggggtcag cggcatcccc tacggtcagc ccaagcagga aaatgatttc 780 aatcctgatg aatgccagcc ctatgagctc ccagatggct cagtcgtcat caatgcccga 840 aaccagaaca actaccactg ccactgccga attgtcctcc gcagctatga tgcctgtgat 900 acctaaggc cccgtgatgt gaccttcgac cctgagctcg tggaccctgt ggtagctgca 960 ggagctgtag tcaccagctc cggcattgtc ttcttctcca acccagcaca tccagagttc 1020 cgagtgaacc tgaccctgcg atggagcttc agcaatggta cctcatggcg gaaagagaca 1080 gtccagctat ggccaggccc cagtggctat tcatccctgg caaccctgga gggcagcatg 1140 gatggagagg agcaggcccc ccagctctac gtcctgtatg agaaaggccg gaaccactac 1200 acagagagca tctccgtggc caaaatcagt gtctatggga cactctga 1248 <210> 9 <211> 415 <212> PRT <213> human <400> 9 Met Thr Gly Glu Arg Pro Ser Thr Ala Leu Pro Asp Arg Arg Trp Gly   1 5 10 15 Pro Arg Ile Leu Gly Phe Trp Gly Gly Cys Arg Val Trp Val Phe Ala              20 25 30 Ala Ile Phe Leu Leu Leu Ser Leu Ala Ala Ser Trp Ser Lys Ala Glu          35 40 45 Asn Asp Phe Gly Leu Val Gln Pro Leu Val Thr Met Glu Gln Leu Leu      50 55 60 Trp Val Ser Gly Arg Gln Ile Gly Ser Val Asp Thr Phe Arg Ile Pro  65 70 75 80 Leu Ile Thr Ala Thr Pro Arg Gly Thr Leu Leu Ala Phe Ala Glu Ala                  85 90 95 Arg Lys Met Ser Ser Ser Asp Glu Gly Ala Lys Phe Ile Ala Leu Arg             100 105 110 Arg Ser Met Asp Gln Gly Ser Thr Trp Ser Pro Thr Ala Phe Ile Val         115 120 125 Asn Asp Gly Asp Val Pro Asp Gly Leu Asn Leu Gly Ala Val Val Ser     130 135 140 Asp Val Glu Thr Gly Val Val Phe Leu Phe Tyr Ser Leu Cys Ala His 145 150 155 160 Lys Ala Gly Cys Gln Val Ala Ser Thr Met Leu Val Trp Ser Lys Asp                 165 170 175 Asp Gly Val Ser Trp Ser Thr Pro Arg Asn Leu Ser Leu Asp Ile Gly             180 185 190 Thr Glu Val Phe Ala Pro Gly Pro Gly Ser Gly Ile Gln Lys Gln Arg         195 200 205 Glu Pro Arg Lys Gly Arg Leu Ile Val Cys Gly His Gly Thr Leu Glu     210 215 220 Arg Asp Gly Val Phe Cys Leu Leu Ser Asp Asp His Gly Ala Ser Trp 225 230 235 240 Arg Tyr Gly Ser Gly Val Ser Gly Ile Pro Tyr Gly Gln Pro Lys Gln                 245 250 255 Glu Asn Phe Asn Pro Asp Glu Cys Gln Pro Tyr Glu Leu Pro Asp             260 265 270 Gly Ser Val Val Ile Asn Ala Arg Asn Gln Asn Asn Tyr His Cys His         275 280 285 Cys Arg Ile Val Leu Arg Ser Tyr Asp Ala Cys Asp Thr Leu Arg Pro     290 295 300 Arg Asp Val Thr Phe Asp Pro Glu Leu Val Asp Pro Val Val Ala 305 310 315 320 Gly Ala Val Val Thr Ser Ser Gly Ile Val Phe Phe Ser Asn Pro Ala                 325 330 335 His Pro Glu Phe Arg Val Asn Leu Thr Leu Arg Trp Ser Phe Ser Asn             340 345 350 Gly Thr Ser Trp Arg Lys Glu Thr Val Gln Leu Trp Pro Gly Pro Ser         355 360 365 Gly Tyr Ser Ser Leu Ala Thr Leu Glu Gly Ser Met Asp Gly Glu Glu     370 375 380 Gln Ala Pro Gln Leu Tyr Val Leu Tyr Glu Lys Gly Arg Asn His Tyr 385 390 395 400 Thr Glu Ser Ile Ser Val Ala Lys Ile Ser Val Tyr Gly Thr Leu                 405 410 415

Claims (11)

i) Fibroblasts isolated from patients with sialidosis in vitro were transfected with ectopic expression of pluripotency markers to induce human induced &lt; RTI ID = 0.0 > inducing pluripotent stem cells (hiPSCs);
ii) c.T824> C and c.G1040> A mutation of the NEU1 gene consisting of the above step i) to SEQ ID NO: 8; The V275A and R347Q mutations of the protein consisting of SEQ ID NO: 9 synthesized from the NEU1 gene; HiPSCs form of normal cells; A stemness maker comprising octamer-binding transcription factor 4 (OCT4), NANOG, TRA-1-81, specific embryonic antigen 3 (SSEA3), specific embryonic antigen 4 (SSEA4) Expression; Accumulation of lysosomes; &Lt; / RTI &gt; and sialidase activity;
Iii) culturing the hiPSCs of step ii) above in an ampoule differentiation medium containing serum replacement (SR) to produce ectoderm markers NESTIN and TUJ1, endoderm markers SOX17 (SRY Y-box 17), alpha-FP (Alpha-fetoprotein), and mesoderm markers such as alpha-smooth muscle actin (alpha-SMA) and DESMIN (Muscle Cell Marker) (EB), characterized in that the marker is expressed in a cell.
Iv) further inducing neuronal progenitor cells to a culture of step iii) in a neural progenitor cell medium containing bEGF, EGF and a leukemia inhibitory factor;
v) obtaining neural progenitor cells of step iv); And
Vi) the neural progenitor cells of step (v) include expression of a neuronal marker protein comprising Nestin (type VI intermediate filament (IF) protein), TUJ1 (class III beta-tubulin) and MAP2 (Microtubule-associated protein 2); Accumulation of lysosomes; Or reduction of sialidase activity, in a sample of the subject's skin.
The method according to claim 1, wherein the ectopic expression of the pluripotency marker is an OCT4 (octamer-binding transcription factor 4) reprogramming factor, a sex determining region (SRY) box 17), C-MYC and Kruppel-like factor 4 (KLF4), or using a transformation of the vector expressing the reprogramming factor. A method of preparing a precursor cell model.
delete delete delete A sialidosis neural progenitor cell model prepared by the method of claim 1.
delete delete delete delete i) c.T824> C and c.G1040> A mutations of the NEU1 gene consisting of SEQ ID NO: 8 of claim 1; Treating the specimen with a specimen differentiated from the hiPSCs model expressing V275A and R347Q mutations of the protein consisting of SEQ ID NO: 9 synthesized from the NEU1 gene;
ii) analyzing the characteristics of the somatic or neural progenitor cells of step i); And
iii) comparing the result of the analysis of step ii) with that of the untreated control.

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