WO2016167392A1

WO2016167392A1 - Sialidosis human cell model and use thereof

Info

Publication number: WO2016167392A1
Application number: PCT/KR2015/003969
Authority: WO
Inventors: Yee Sook Cho; Binna SEOL; Jae Eun Kwak; Mi Young Son
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2015-04-16
Filing date: 2015-04-21
Publication date: 2016-10-20
Also published as: KR20160123528A; KR101785910B1

Abstract

The present invention relates to a sialidosis human cell model, human induced pluripotent stem cells (hiPSCs) thereof, and a method for preparing the model cell based on hiPSCs derived neural progenitor cells, and also a use of the sialidosis model cell for the development of a sialidosis treating agent and cell therapy. The human induced pluripotent stem cells (hiPSCs) originated from sialidosis patient fibroblasts can be differentiated into neural progenitor cells (NPCs) and neurosphere cells that can reproduce the characteristics of sialidosis. The hiPSCs were successfully developed as the model cells of sialidosis neural progenitor cells, confirmed by the gene mutation, the accumulation of intracellular lysosome, and the decrease of sialidase activity, which are the symptoms of sialidosis neural progenitor cell disease, in the cells. Therefore, the human cell model of the present invention can be effectively used for the study of the mechanism to explain the outbreak of sialidosis and the study for the development of a therapeutic agent for the disease.

Description

[DESCRIPTION]

[invention Title]

SIALIDOSIS HUMAN CELL MODEL AND USE THEREOF

[Technical Field]

The present invention relates to a method for preparing a sialidosis human cell model by constructing human induced pluripotent stem cells (hiPSCs) derived from sialidosis (mucolipidosis) patients and tissue-specific cells differentiated from the hiPSCs, and a use of the said cell model for the screening of a sialidosis (mucolipidosis) treating agent and development of cell therapy.

[Background Art]

Sialidosis is a very rare genetic disease caused by the deficiency of lysosome sialidase or neuraminidase resulted from the mutation of NEU1 (HGNC: 7758) gene, which is the autosomal recessive inherited disease. This disease is classified as a lysosomal storage disease (LSD) characterized by the abnormal accumulation of mucolipid caused by the deficiency of sialidase. Sialidase plays a role in eliminating sialic acid having the molecular structure that is similar to one of glucose of glycoprotein, by which cells are functioning normally. When this enzyme is deficient, the glucose like material is not eliminated and rather accumulated in various tissues and cells including neuron and bone marrow which are usually- functioning to protect from foreign pathogen infection, resulting in the development of abnormal symptoms.

The symptoms of sialidosis are detected from the birth or within a year from the birth. An infant with sialidosis displays an excessive swelling on the whole body, and more precisely, a flat nose, swollen eyelid, gingival hypertrophy, and pachyglossia are often observed. Sometimes, this disease accompanies musculoskeletal abnormality such as pelvic bone dislocation. New born babies with this disease might display sudden contraction of involuntary muscle, cherry red macules, ataxia or tremor, eyesight abnormality, and convulsion, etc.

When examined, patients often display hepatomegaly, splenomegaly, and excessive edema. In infant patients, muscles are weaken (hypotonia) , and mental retardation is observed from the beginning or progresses fast. Many of those patients display growth trouble and might die within a year from the birth because of repeated respiratory infections.

Four kinds of human sialidase genes have been identified so far (NEU1, 2, 3, and 4), whose corresponding proteins are all different in intracellular position and substrate specificity. Asp-box motifs

an important role in sialidase activity are well preserved across species. NEUl gene encodes the lysosome sialidase composed of 415 amino acids. The mutation of NEUl gene induces the intracellular accumulation of sialylated oligosaccharides and glycoproteins. At least 30 kinds of gene mutation have been found in sialidosis patients, but studies on mutation type and clinicopathologic relations with that and the phenotype thereof are still short.

The molecular mechanism to cause human sialidosis has not been disclosed, yet. Accordingly, the treatment of sialidosis has been limited and stays at conservative manner. Even though many of characteristics of human sialidosis might have been copied now since the sialidosis mouse model was established, this attempt cannot fit in human treatment. That is, the studies using the sialidosis mouse model are limited. Therefore, the development of human originated sialidosis cell model that is able to reproduce the mechanism of human sialidosis outbreak is useful for understanding the disease and thereby establishing an efficient treatment method of the disease.

Stem cells are the cells in the pre-differentiation phase before being differentiated into each tissue forming cells, which can be obtained from embryo, fetus, and adult tissues. Stem cells have unlimited auto-reproductivity in non-differentiation stage and differentiation potency, so that they can be proliferated into the various tissue forming cells by a specific stimulus. Again, stem cells are differentiated into a specific cell type by a differentiation stimulus (environment) and have proliferation (expansion) capacity making self -renewal possible by cell division, unlike the differentiated cells whose cell division is finished. They can also be differentiated into another cell type when a different environment or a different differentiation stimulus is given, indicating the stem cells have plasticity for differentiation as well.

Human pluripotent stem cells (hPSCs) including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) display excellent pluripotency, by which hPSCs can be differentiated almost every tissue cells that form a human body. Particularly, patient-derived human induced pluripotent stem cells (hiPSCs) can produce in vitro tissue-specific differentiated cells having immunologically and genetically same characteristics as the patient. So, they are considered to be useful not only for the development of an agent for the patient-customized cell therapy but also for understanding the complicated mechanism of disease developed in the early organogenesis stage (Muotri, A. R. (2009) Epilepsy Behav 14 Suppl 1: 81- 85; Marchetto, M. C. , B. Winner, et al . (2010) Hum Mol Genet 19(R1): R71-76). It has been reported that hiPSCs derived from patients with different genetic diseases display disease- specific phenotypes when they are differentiated directly into the cells involved in the disease (Park, I. H. et al . Cell 134, 877-886 (2008); Tiscornia, G. et al . Nature medicine 17, 1570-1576 (2011)). The disease-specific hiPSCs can be differentiated into the tissue cells related to the immediate cause or the lesion. The differentiated tissue cells having the characteristics of disease can be efficiently used for the study on a specific mechanism to explain a cause of disease or for the development of a therapeutic agent and cell therapy.

The present inventors tried to establish a human cell model for the study of sialidosis based on patient-derived hiPSCs. First, the inventors induced the development and differentiation of human induced pluripotent stem cells (hiPSCs) , embryoid body (EB) , and neural progenitor cells (NPCs) derived from sialidosis patient fibroblasts. The said hiPSCs derived from sialidosis patient fibroblasts (sialidosis-hiPSCs) were confirmed to have pluripotency both in vitro and in vivo, and at the same time confirmed to have the causing gene mutation observed in sialidosis patients and lower sialidase activity as well. The inventors also induced the differentiation of sialidosis- hiPSCs into NPCs . As a result, the inventors confirmed that the sialidase activity was reduced in sialidosis- hiPSCs derived NPCs (sialidosis-NPCs). The inventors further investigated the nucleotide sequence of NEU1 gene, the causing gene of sialidosis, in different sialidosis patients. As a result, the inventors identified the novel gene mutation (R347Q/V275A) and thereafter confirmed that the sialidase activity was reduced in sialidosis-hiPSCs and sialidosis-NPCs derived from the patient somatic cells having the gene mutation. Therefore, the present inventors confirmed that the sialidosis-hiPSCs and sialidosis-NPCs of the present invention can be efficiently used for the study to explain the reason of sialidosis outbreak and for the development of a therapeutic agent for the disease since they have preserved disease -specific phenotypes significantly well, leading to the completion of this invention.

[Disclosure]

[Technical Problem]

It is an object of the present invention to provide an improved study method to disclose the mechanism of sialidosis outbreak and a therapeutic agent for the same by establishing a human cell model reflecting all the disease characteristics observed in sialidosis patient by using human induced pluripotent stem cells (hiPSCs) and hiPSC- derived NPCs . It is also an object of the present invention to provide a method for relieving sialidosis and a therapeutic agent for the same using the human cell model by providing a method for screening a therapeutic agent candidate that is efficient for the treatment of sialidosis

[Technical Solution]

To achieve the above objects, the present invention provides a method for preparing sialidosis -hiPSCs model in vitro comprising the following steps:

i) inducing human induced pluripotent stem cells (hiPSCs) from fibroblasts separated from sialidosis patient in vitro; and

ii) collecting the hiPSCs induced in step i) .

The present invention also provides a method for preparing sialidosis neural progenitor cell model in vitro comprising the following steps:

i) preparing human induced pluripotent stem cells (hiPSCs) from fibroblasts separated from sialidosis patient in vitro; ii) inducing neural progenitor cells from the hiPSCs prepared in step ij ; and

iii) collecting the NPCs induced in step ii) .

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

The present invention also provides a use of hiPSCs, neural progenitor cells, or neurosphere as the sialidosis model, which includes the following steps:

i) inducing the differentiation of embryoid body (EB) , neural progenitor cells, or neurosphere from the hiPSCs prepared above; and

ii) analyzing the differentiation marker of the embryoid body, the characteristics of the neural progenitor cells, or the morphological characteristics of the neural progenitor cells induced in step i) .

In addition, the present invention provides a method for screening a sialidosis treating agent candidate comprising the following steps:

i) treating the embryoid body, neural progenitor cells, or neurosphere differentiated from the hiPSCs model with a sample material;

ii) analyzing the characteristics of the embryoid body, neural progenitor cells, or neurosphere of step i) ; and

iii) comparing the result of the analysis of step ii) with that of the non- treated control group.

[Advantageous Effect]

The human induced pluripotent stem cells derived from fibroblasts of sialidosis (mucolipidosis) patient can be differentiated into neural progenitor cells and neurosphere cells that can reproduce the characteristics of sialidosis. The hiPSCs were developed as the model cells of sialidosis neural progenitor cells by confirming the gene mutation, the accumulation of intracellular lysosome, and the decrease of sialidase activity, which are the symptoms of sialidosis neural progenitor cell disease, in the cells. The human cell model of the present invention can be effectively used for the study of the mechanism to explain the outbreak of sialidosis and the study for the development of a therapeutic agent for the disease.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 is a schematic diagram illustrating the preparation process of the hiPSCs originated from sialidosis patient fibroblasts and a use of the same. Figure 2 is a diagram illustrating the mutation of a sialidosis causing gene in the fibroblasts originated from a normal subject or a sialidosis patient.

a: mutation of G227R gene, that is the mutation of a sialidosis causing protein in the fibroblasts originated from a sialidosis patient; and

b: V275A(Het) , the mutation of a sialidosis causing protein in the fibroblasts originated from a sialidosis patient, and R347Q(Het), the mutation which was first identified in this invention.

Figure 3 is a diagram illustrating the morphology of the fibroblasts originated from a normal subject or a sialidosis patient and the lysosome accumulation therein. a: typical morphology of the cultured fibroblasts originated from a normal subject or a sialidosis patient, observed under phase-contrast microscope; and

b: sialidosis specific lysosome accumulation in the fibroblasts originated from a normal subject or a sialidosis patient.

Figure 4 is a diagram illustrating the sialidosis- specific inhibition of sialidase activity in the fibroblasts originated from a normal subject or a sialidosis patient.

Figure 5 is a diagram illustrating the enzyme activity of sialidase in HEK 297T cells over-expressing NEUl gene having either normal or mutant nucleotide sequence (R347Q, or V275A) .

Figure 6 is a diagram illustrating the expression of NEUl protein in HEK 297T cells over-expressing NEUl gene having either normal or mutant nucleotide sequence (R347Q, or V275A) , confirmed by Western blotting.

Figure 7 is a diagram illustrating the structural variation caused by the mutation of NEUl protein sequence. a: partial array of NEUl protein sequence in a vertebrate;

the mutation of the amino acid sequence is presented by red (G277 and V275) and purple (R347) and Asp-boxes sequence is presented by orange line;

b: homology modeling of the NEUl protein structure based on the crystal structure of human NEU2 protein; and c: mutated region of R347 amino acid which is close to the Asp-boxes sequence of NEUl protein.

Figure 8a is a schematic diagram illustrating the preparation process of sialidosis patient originated human induced pluripotent stem cells.

Figure 8b is a diagram illustrating the morphological characteristics of sialidosis patient originated human induced pluripotent stem cells and the differentiation or non-differentiation of the hiPSCs investigated by alkaline phosphatase staining (AP staining) . Figure 8c is a diagram illustrating the expression of the pluripotency marker protein in sialidosis patient originated human induced pluripotent stem cells.

Figure 8d is a diagram illustrating the in vitro differentiation of sialidosis patient originated human induced pluripotent stem cells into ectoderm, endoderm, and mesoderm.

Figure 9 is a diagram illustrating the results of the analysis of short tandem repeat (STR) of sialidosis patient originated human induced pluripotent stem cells.

Figure 10 is a diagram illustrating the result of the analysis of karyotype of sialidosis patient originated human induced pluripotent stem cells.

Figure 11 is a diagram illustrating the teratoma differentiated from sialidosis patient originated human induced pluripotent stem cells in vivo.

Figure 12a is a diagram illustrating the mutation of G227R gene in human induced pluripotent stem cells established from the sialidosis patient cell line either normal or having G227R mutation.

Figure 12b is a diagram illustrating the mutations of V275A(Het) and R347Q (Het) genes in human induced pluripotent stem cells established from the sialidosis patient cell line either normal or having V275A or R347Q mutation. Figure 13 is a diagram illustrating the sialidosis- specific lysosome accumulation in sialidosis-hiPSCs .

Figure 14 is a diagram illustrating the sialidosis- specific inhibition of sialidase activity in sialidosis- hiPSCs.

Figure 15 is a diagram illustrating the morphology of the neural progenitor cells (sialidosis-NPCs) differentiated from sialidosis-hiPSCs, observed under phase-contrast microscope.

Figure 15b is a diagram illustrating the expression of the neural marker protein in the neural progenitor cells (sialidosis-NPCs) differentiated from sialidosis-hiPSCs.

Figure 16a is a diagram illustrating the mutation of G227R, which is the mutation of a sialidosis causing protein, in the neural progenitor cells (sialidosis-NPCs) differentiated from normal or sialidosis-hiPSCs.

Figure 16b is a diagram illustrating the mutation of V275A(Het) , which is the mutation of a sialidosis causing protein, and the mutation of R347Q (Het) which is first identified in this invention, in the neural progenitor cells (sialidosis-NPCs) differentiated from normal or sialidosis-hiPSCs .

Figure 17 is a diagram illustrating the sialidosis specific lysosome accumulation in the neural progenitor cells (sialidosis-NPCs) differentiated from sialidosis- hiPSCs.

Figure 18 is a diagram illustrating the sialidosis specific inhibition of sialidase enzyme activity in the neural progenitor cells (sialidosis-NPCs) differentiated from sialidosis-hiPSCs .

[Best Mode]

Hereinafter, the present invention is described in detail.

The present invention provides a method for preparing sialidosis hiPSCs model in vitro comprising the following steps :

i) inducing human induced pluripotent stem cells from fibroblasts separated from sialidosis patient in vitro; and ii) collecting the hiPSCs induced in step i) .

In step (1) , the induction of step i) is preferably achieved by the ectopic expression of a pluripotency marker More precisely, the ectopic expression is achieved by the method using retrovirus including reprogramming factors such as OCT4 (octamer-binding transcription factor 4), S0X2 (SRY (sex determining region Y) -box 17), C-MYC, and KLF4 (Kruppel-like factor 4) or the method based on the transfection of episome vector expressing the said reprogramming factors, or by any other method for preparing hiPSCs well known to those in the art.

In a preferred embodiment of the present invention, the inventors confirmed different mutant genes causing sialidosis (G227R and V275A (Het) /R347Q (Het) ) specifically observed in fibroblasts originated from different sialidosis patients (see Figure 2) . By somatic cell reprogramming technique, hiPSCs were prepared from fibroblasts of the different sialidosis patients (see Figure 8) . Then, characteristics of the sialidosis-hiPSCs were investigated. As a result, it was confirmed that the sialidosis-hiPSCs had pluripotency in vitro and in vivo as well (see Figures 8 - 11) and preserved the mutation of the sialidosis causing gene shown in sialidosis patients (see Figure 12) , and had the activity to inhibit lysosome accumulation (see Figure 13) and sialidase activity (see Figure 14) .

The hiPSCs model of the present invention originated from sialidosis patient somatic cells maintains the disease phenotype of sialidosis patient somatic cells and has pluripotency, so that they can be efficiently used as a human cell model for the study of mechanism to cause sialidosis outbreak and for the development of a therapeutic agent for the same. The present invention also provides the sialidosis hiPSCs model prepared by the above method.

The said hiPSCs are preferably the sialidosis hiPSCs model characterized by at least one of the following i) ~ vi) , but not always limited thereto:

i) the mutation of NEUl gene, that is c.G679>A or c.T824>C and c.G1040>A;

ii) the mutation of G227R or V275A and R347Q in the protein synthesized from NEUl gene;

iii) hiPSCs form of normal cells;

iv) the expression of a sternness maker including OCT4 (octamer-binding transcription factor 4), NANOG, TRA-1-81, SSEA3 (specific embryonic antigen 3) , SSEA4 (specific embryonic antigen 4), and TRA-1-60;

v) the accumulation of lysosome; and

vi) the decrease of sialidase activity.

The said NEUl gene is preferably composed of the sequence represented by SEQ. ID. NO: 8, and the protein synthesized therefrom is preferably composed of the sequence represented by SEQ. ID. NO: 9.

The hiPSCs model of the present invention originated from sialidosis patient displays the characteristics of sialidosis patient somatic cells and at the same time shows pluripotency, so that it can be efficiently used for the study to understand the mechanism of sialidosis outbreak and for the development of a therapeutic agent for the disease .

i) preparing human induced pluripotent stem cells (hiPSCs) from fibroblasts separated from sialidosis patient in vitro;

ii) inducing neural progenitor cells from the hiPSCs prepared in step i) ; and

iii) collecting the NPCs induced in step ii) .

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

The said neural progenitor cells are preferably characterized by at least one of the following i) ~ iii) , but not always limited thereto.

i) the expression of neural marker protein including Nestin (type VI intermediate filament (IF) protein) , TUJ1 (class III beta- tubulin) , and MAP2 (Microtubule-associated protein 2) ;

ii) the accumulation of lysosome; and

iii) the decrease of sialidase activity.

In a preferred embodiment of the present invention, the present inventors confirmed that when embryoid body (EB) was induced from sialidosis-hiPSCs (see Figure 8a) , all the differentiation markers of endoderm, mesoderm, and ectoderm were all expressed, suggesting that sialidosis- hiPSCs derived embryoid body had pluripotency (see Figure 8d) .

The differentiation of neural progenitor cells (sialidosis-NPCs) from sialidosis-hiPSCs was also induced. As a result, it was confirmed that neural marker protein was expressed (see Figure 15b) and sialidosis causing gene mutation was preserved (see Figure 16) .

The present inventors also investigated whether or not the sialidosis specific disease phenotype was maintained in sialidosis-NPCs. As a result, it was confirmed that the intracellular lysosome accumulation was increased (see Figure 17) but the sialidase activity was significantly reduced (see Figure 18) .

Therefore, the neural progenitor cells differentiated from sialidosis-hiPSCs displayed the characteristics shown in sialidosis patient cells, so that they can be efficiently used as a human cell model for the study of the mechanism of sialidosis outbreak.

The present invention also provides a use of hiPSCs, neural progenitor cells, or neurosphere as the sialidosis model, which includes the following steps: i) inducing the differentiation of embryoid body, neural progenitor cells, or neurosphere from the hiPSCs prepared above; and

The differentiation marker of the embryoid body is preferably selected from the group consisting of ectoderm markers such as NESTIN and TUJ1, endoderm markers such as

SOX17 (SRY (sex determining region Y) -box 17) and a-FP

(Alpha-fetoprotein) , and mesoderm markers such as oi-smooth muscle actin (a-SMA) and DESMIN (Muscle Cell Marker) , but not always limited thereto.

The said neural progenitor cells are preferably characterized by. at least one of the following i) ~ iii) , but not always limited thereto.

i) the expression of neural marker protein including Nestin (type VI intermediate filament (IF) protein) , TUJ1 (class III beta- tubulin) , and MAP2 (Microtubule-associated protein 2 ) ;

ii) the accumulation of lysosome; and

iii) the decrease of sialidase activity. In addition, the present invention provides a method for screening a sialidosis treating agent candidate comprising the following steps:

iii) comparing the result of the analysis of step ii) with that of the non-treated control group.

The sialidosis treating agent candidate can be a material that is able to recover the cell characteristics to the normal cell like standard when the characteristics were compared with those of the hiPSC model of the present invention, or the embryoid body or neurons differentiated therefrom. Particularly, the material that is able to increase the expression of NEU1 gene in neurons differentiated from the hiPSC model of the invention similarly to the level of the normal control is most preferred, but not always limited thereto.

The hiPSCs of the present invention derived from sialidosis patient somatic cells, the embryoid body or neural progenitor cells differentiated therefrom displayed the characteristics shown in sialidosis patient cells, so that they can be efficiently used as a human cell model for the study of the mechanism of sialidosis outbreak.

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1 : Construction of human induced pluripotent stem cells originated from sialidosis (mucolipidosis) patient somatic cells

<!-!> Confirmation of mutation of sialidosis causing gene in sialidosis patient somatic cells

To confirm the mutation of the causing gene of sialidosis (mucolipidosis) , the present inventors cultured fibroblasts obtained from sialidosis patient. As a result, the sialidosis causing gene NEU1 sequence (SEQ. ID. NO: 8) was identified.

Particularly, GM02685 and GM02921 (Coriell Institute for Medical Research, USA) , which were the fibroblast cell lines originated from sialidosis patient, were purchased and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) , 1% non-essential amino acid (NEAA; Invitrogen, USA) , 1 mM L-glutamine (Invitrogen, USA), and 0.1 mM β- mercaptoethanol (Sigma, USA) . Upon completion of the culture, RNA was extracted from the cultured cells by using RNAiso Plus (Takara, Japan) or RNeasy mini kit (Qiagen, USA) according to the manufacturer's protocol. Then, PCR was performed with the primers (SEQ. ID. NO: 1 and NO: 2) listed in Table 1 and Superscript III (Invitrogen, USA) in order to synthesize cDNA of NEUl gene. The PCR product was separated on 1% agarose gel. The full-length band was cut off and purified by QIAquick Gel Extraction Kit (Qiagen, USA) . Whole sequence of NEUl cDNA was analyzed by using the primers (SEQ. ID. NO: 3 ~ NO: 7) listed in Table 1. CRL-2097 (ATCC) , the human skin derived wild-type fibroblast cell line, was used for the control. The control cDNA of NEUl gene was also synthesized by the same manner as the above, whose sequence was identified later on This identified sequence was the wild-type, the control, which was then compared with the sialidosis cell line.

[Table l]

Forward Backward 5 ' - 5 ' -

NEUl cDNA SEQ. ID. SEQ. ID.

gcttaagggtga ctgtctttcaggcgt synthesis NO: 1 NO: 2 catctgcgc-3¹ ctccag-3 '

5 ' - 5 ' -

SEQ. ID. SEQ . ID . gtctagctgcca gactccgtcccgctc

NO: 3 NO: 4 gggtcgcg-3 ' cagcg-3¹

NEUl cDNA 5 ' - 5 ' -

SEQ . ID . SEQ. ID. whole ggtatggagcaa cctgaaggcagaata

NO: 5 NO: 6 sequence ggatgatgg-3¹ cccct-3¹

5 ' -

SEQ. ID.

gccgaattgtcc

NO: 7

tccgcagc-3 '

As a result, as shown in Figure 2, one of those sialidosis fibroblast cell lines displayed the c.G679>A sequence mutation in the NEUl gene encoding sialidase, and accordingly the mutation of G227R in the sialidase protein synthesized from the above gene was confirmed. Another sialidosis fibroblast cell line showed the mutations of c.G1040>A sequence and c/T824>C sequence in the NEUl gene encoding sialidase, and accordingly the mutation of V275A and R347Q in the sialidase protein synthesized from the above gene was confirmed as well. The present invention is the first to use the cell line having such sequence mutations (Figure 2) .

<l-2> Construction of hiPSCs derived from sialidosis patient fibroblasts

To generate the induced pluripotent stem cells, the present inventors produced hiPSCs ( sialidosis-hiPSCs) that is the cell model derived from sialidosis patient somatic cells and having pluripotency, via reprogramming culture (Son MY et. al, Stem cells 31, 2374-2387; 2013) using the retrovirus expressing 4 reprogramming factors such as OCT , SOX2, C-MYC, and KLF4 (Figure 8a) .

Particularly, sialidosis patient fibroblasts were transfected with the retrovirus encoding OCT4 , SOX2 , C-MYC, and KLF4 , followed by culture in somatic cell medium for 5 days. 5 days later, the transfected cells were transferred in the matrigel-coated plate containing human embryonic stem cell culture medium, followed by further culture for 2 ~ 3 weeks. Then, hiPSCs colony was collected. cDNA of NEU1 gene was synthesized with the obtained sialidosis-hiPSCs by the same manner as described in Example <l-2>, followed by sequencing. For the control, wild-type hiPSCs were constructed with CRL-2097 cell line by the same manner as described above .

As a result, as shown in Figure 12, sialidosis-hiPSCs having different mutations each other in nucleotide sequence were constructed (c.G679>A, or c.T824>C and c.G1040>A) . These mutations were confirmed to be same as those of sialidosis derived fibroblasts (Figure 12) .

In this invention, hiPSCs having pluripotency were constructed from sialidosis patient somatic cells via reprogramming culture using retrovirus (Figure 8a) .

Particularly, as described in a paper (Fusaki et al . Proc. Jpn. Acad., Ser., B 85, 2009), non-inserted human induced pluripotent stem cells were constructed by using cytoTune-iPS 2.0 sendal reprogramming kit (A16517, Invitrogen) . To produce hiPSCs, fibroblasts derived from the sialidosis patient GM02685 (Coriell Institute) were loaded in a 6 -well plate at the density of 2 X 10^s cells/well, followed by infection with CytoTune- iPS 2.0 KOS, CytoTune -iPS 2.0 hc-Myc, and CytoTune-iPS 2.0 hklf4 of the said reprogramming kit. At this time, the transfection amount (titer) was determined by the amount of MOI (ClU/cell) according to the lot number. 7 days after the transfection, gamma-mouse embryonic fibroblasts (MEF) were inoculated in the 6-well plate at the density of 1 ~ 5 X 10⁴ cells/well under feeder condition. The medium was replaced with hiPSC culture medium supplemented with 20 ng/ mi of bFGF every other day. About 21 days after the transfection, colony displaying hESC like morphology was collected and transferred in the 6-well plate under feeder condition, followed by sub-culture by the same manner as applied for hESC culture.

As a result, it was confirmed that the sialidosis- hiPSCs constructed by different reprogramming methods had all similar pluripotency and disease phenotypes.

Example 2: Characteristics of sialidosis-hiPSCs

<2-l> Morphological characteristics of sialidosis-hiPSCs

To confirm the morphological characteristics of the sialidosis-hiPSCs constructed in Example <l-2>, the inventors first investigated morphology and karyotype of the sialidosis-hiPSCs colony (Figure 10) and also performed short tandem repeat (STR) assay (Figure 9) .

Particularly, reprogramming of the sialidosis-hiPSCs was induced by the same manner as described in Example <1- 2>, followed by observation of sialidosis-hiPSCs colony under phase-contrast microscopy. Karyotyping was performed by chromosomal G-banding analysis at GenDix Inc., Korea. Also, STR genotyping of sialidosis-hiPSCs and sialidosis patient derived fibroblasts was performed at HumanPass Inc., Korea. The results were compared with those of the normal human embryonic stem cell line H9 hESCs .

As a result, as shown in Figure 8b and Figure 10, it was confirmed that the sialidosis-hiPSCs having pluripotency was successfully differentiated from sialidosis-fibroblasts (Figure 8b) and the prepared sialidosis-hiPSCs displayed the colony morphology similar to human embryonic stem cells and the normal karyotype (Figure 9 and Figure 10) .

<2-2> AP (Alkaline phosphatase) staining of sialidosis- hiPSCs

To confirm the pluripotency of the sialidosis-hiPSCs originated from sialidosis patient which had been constructed in Example <l-2>, the inventors performed AP staining using the pluripotency marker alkaline phosphatase

Particularly, 1 mi of citrate solution, 2.6 ml of acetone, and 320 βΐ of 37% formaldehyde (Sigma Aldrich, USA) were mixed by using AP staining kit (alkaline phosphatase staining kit, Sigma Aldrich, USA) to prepare a fixative solution. The fixative solution was added to the sialidosis-hiPSCs prepared in Example <l-2>, which stood at room temperature under the dark for 15 minutes. 100 βί of sodium nitrate solution and 100 μ of FRV-alkaline solution were mixed, which stood for 2 minutes, to which 4.5 ml of sterilized water and 100 μί of Naphthol AS-BI alkaline solution were added. Light was blocked by rapping the mixture with aluminum foil. The fixed cells were washed with PBS once and then placed in the prepared AP staining mixture. The cells were washed with water or PBS twice for 2 minutes each time. Then, the AP stained cells were observed under phase-contrast microscope.

As a result, as shown in Figure 8b, the sialidosis- hiPSCs could be stained with AP, the pluripotency marker (Figure 8b) .

<2-3> Confirmation of pluripotency of sialidosis-hiPSCs

To confirm whether or not the non-differentiated sialidosis-hiPSCs derived from sialidosis patient fibroblasts, that had been constructed in Example <l-2>, had pluripotency, the present inventors investigated the expression of the pluripotency marker protein in the sialidosis-hiPSCs.

Precisely, 4% formaldehyde was treated to the sialidosis-hiPSCs prepared in Example <l-2>, followed by fixation at room temperature for 10 minutes. PBS containing 0.1% triton X-100 was treated thereto for 15 minutes to give permeability to the cell membrane. The cells were washed with PBS containing 4% bovine serum albumin (BSA) , and then treated with the primary antibody which was one of anti-OCT4 antibody (1:100, sc-9081, Cruz Biotechnology, USA), anti-NANOG antibody (1:100, sc-33759, Cruz Biotechnology, USA), anti-TRA-1-81 antibody (1:100, MAB4381, Chemicon, USA), anti-SSEA3 antibody (1:100, AB1435, R&D Systems, USA), anti-SSEA4 antibody, and anti- TRA-1-60 antibody (1:300, Millipore, USA) . The reaction mixture stood at 4^°C for overnight, followed by washing. After washing, the cells were treated with the secondary antibody (Invitrogen, USA) conjugated with Alexa Fluor 488 or Alexa Fluor 594, which stood at room temperature for 2 hours. Immunofluorescent staining was performed with the sialidosis-hiPSCs , followed by observation under fluorescence microscope to confirm the expressions of OCT4 , NANOG, SOX2, SSEA4 , Tra-1-80, and Tra-1-61. To compare the expression levels, 4¹6-diamidino-2-phenylindole (DAPI) was treated to the cells to stain the nuclei.

As a result, as shown in Figure 8c, the expressions of those proteins OCT4, NANOG, TRA-1-81, SSEA3 , SSEA4 , .and TRA-1-60, the pluripotency markers, were confirmed as the normal levels as shown in normal cells (Figure 8c) .

<2-4> in vitro differentiation potential of sialidosis- hiPSCs

To investigate whether or not the sialidosis-hiPSCs could be differentiated in vitro and had pluripotency, the inventors induced the differentiation of embryoid body from sialidosis-hiPSCs. Then, the expressions of three types of germ layer markers, which are the endoderm markers such as SOX17 and α-FP, the mesoderm markers such as -smooth muscle actin ( -SMA) and DESMIN, and the ectoderm markers such as NESTIN and TUJ1, were confirmed in the sialidosis- embryoid body.

Particularly, the sialidosis-hiPSCs constructed by the method of Example <l-2> were cultured in embryoid body differentiation medium (DMED/F12) supplemented with 10% serum replacement (SR) for 7 days to induce the differentiation of sialidosis-hiPSCs derived embryoid body (sialidosis-EB) . Then, immunofluorescent staining was performed by the same manner as described in Example <2-3>, followed by observation under fluorescence microscope to confirm the expressions of NESTIN, TUJ1, SOX17, a-FP, a-SMA, and DESMIN. For the immunofluorescent staining, the primary antibody such as anti-DESMIN antibody (1:50; AB907, Chemicon, USA) , anti-a-SMA antibody (1:400; A5228, Sigma- Aldrich, USA), anti-FP antibody (1:200; A8452 , Sigma- Aldrich, USA), anti-SOX17 antibody (1:100; MAB1924, R&D Systems, USA), anti-TUJl antibody (1:500; PRB-435P, Covance, USA), or anti-NESTIN antibody (1:100; MAB5326, Chemicon, USA) was used. To compare the expression levels, 4'6- diamidino-2-phenylindole (DAPI) was treated to the cells to stain the nuclei.

As a result, as shown in Figure 8d, the expressions of those proteins NESTIN, TUJ1, S0X17, a-FP, a-SMA, and DESMIN were confirmed in sialidosis-EB, suggesting that pluripotency was confirmed (Figure 8d) .

<2-5> in vivo differentiation potential of sialidosis- hiPSCs

To investigate whether or not the sialidosis derived hiPSCs constructed in Example <l-2> had pluripotency in vivo, the inventors first performed teratoma formation of sialidosis-hiPSCs in immunodeficient nude mouse.

Particularly, lxlO⁶ cells of the sialidosis-hiPSCs prepared by the same manner as described in Example <l-2> was injected to 4 weeks old SPF/VAF immunodeficient nude mouse (OrientBio, Korea) and the mouse was raised for 12 weeks. 12 weeks later, the mouse was sacrificed. Tetroma was extracted from the mouse and added with 4% formaldehyde, followed by embedding in paraffin. The embedded tetroma was stained with hematoxylin & eosin to confirm the formation of endoderm, ectoderm, and mesoderm.

As a result, as shown in Figure 11, the formations of neural tissue (ectoderm) , cartilage and muscle (mesoderm) , and epithelium (endoderm) were confirmed in the tetroma originated from sialidosis-hiPSCs (Figure 11) .

<2-6> Mutation of sialidosis causing protein in sialidosis- hiPSCs To investigate the characteristics of a mutant protein resulted from the mutation of a^' sialidosis causing gene in the sialidosis-hiPSCs prepared in Example <l-2>, the inventors first investigated the activity of sialidase protein in the sialidosis-hiPSCs.

Particularly, fibroblasts and hiPSCs were obtained by the same manner as described in Example <l-2>, which were then suspended in analysis buffer (pH 4.5) supplemented with 20 mM citrate, 60 mM NaCl, and 1 mM CaCl₂. The cells were lysed by using a sonicator 5 ~ 10 times repeatedly, followed by centrifugation at 13,000 rpm for 10 minutes at 4^°C to eliminate cell debris. The protein dissolved in the buffer was extracted. Then, 2 of the protein lysate was added to analysis buffer containing 0.2 mM 2'- (4- methylumbelliferyl) -a-D-N-acetylneuraminic acid (4-MUNA A, Sigma-Aldrich, USA) , followed by culture at 37^°C for 4 hours to induce reaction. To terminate the reaction, 0.1 M glycine-NaOH buffer (pH 10.5) was added thereto. To confirm the sialidase activity, fluorescence (lex = 365 nm/lem = 450 nm) was measured by using SpectraMax© M3 Multi-Mode Microplate Reader (Molecular Devices) . The sialidase activity in the normal human fibroblast-derived hiPSCs, the control, was also measured and compared with the above .

As a result, as shown in Figure 4 and Figure 14, unlike the control normal fibroblasts, the sialidase activity in the sialidosis patient derived somatic cells was significantly reduced (Figure 4) . And the sialidase activity in sialidosis-hiPSCs was also significantly reduced, compared with the hiPSCs originated from the normal fibroblasts (Figure 14) .

<2-7> Accumulation of lysosome in sialidosis-hiPSCs

To measure the amount of sialic acid accumulated in lysosome in the sialidosis-hiPSCs prepared in Example <l-2>, the cell culture medium was treated with LysoTracker Red DND-99 (1:20000; Invitrogen, USA), followed by reaction at

37^°C for 30 minutes. Then, Intracellular lysosome was observed.

As a result, as shown in Figure 3 and Figure 13, the accumulation of lysosome was significantly increased in the sialidosis patient derived somatic cells, compared with the normal fibroblasts (Figure 3) , and similarly the accumulation of lysosome was also significantly increased in sialidosis-hiPSCs, compared with that of the normal fibroblast derived hiPSCs (Figure 13) .

<2-8> Mutation of sialidosis causing gene in sialidosis- hiPSCs

The present inventors investigated the mutation of a sialidosis causing gene in hiPSCs originated from sialidosis patient somatic cells by the same manner as described in Example <1-1>.

As a result, as shown in Figure 12, the hiPSCs originated from sialidosis patient somatic cells having the mutation of c/G679>A in NEUl gene displayed the same mutation of c.G679>A in NEUl gene, suggesting that the sialidase protein synthesized by the above gene had the mutation of G227R (Figure 12a) . The hiPSCs originated from sialidosis patient somatic cells having the mutation of c.T824>C and c.G1040>A in NEUl gene displayed the mutation of c.T824>C and c.G1040>A in NEUl encoding sialidase, indicating that the sialidase protein synthesized by the above gene had the mutation of V275A and R347Q (Figure 12b) .

Example 3 : Differentiation of sialidosis derived neural progenitor cells

<3-l> Confirmation of the differentiation of embryoid body and neural progenitor cells and the mutant gene therein

To induce the in vitro differentiation of sialidosis originated neuronal cells from sialidosis-hiPSCs , the present inventors first induced the differentiation of embryoid body from sialidosis-hiPSCs to induce the generation of neural progenitor cells eventually.

Precisely, the colony of sialidosis-hiPSCs induced by reprogramming for 7 days by the same manner as described in Example <l-2> was cultured in embryoid body differentiation medium (DMEM/F12) supplemented with 10% serum replacement (SR) for 7 days to induce the differentiation of sialidosis-EB from sialidosis-hiPSCs . The differentiated sialidosis-EB was further cultured for 2 more weeks in NPCs medium (DMEM/F12) supplemented with lxN2/B27 (Invitrogen, USA) , 20 ng/mi bFGF, 20 ng/ i EGF (Invitrogen, USA) , and 10 ng/mi leukemia inhibitory factor (Sigma-Aldrich, USA) . As a result, the differentiated sialidosis derived neural progenitor cells (sialidosis-NPCs) were obtained. In the course of the differentiation of NPCs, sub-culture was performed every week by using McClain tissue chopper (Mickle Engineering, England) , during which the medium was replaced every other day. To induce the differentiation into neuronal cells and glial cells, the obtained sialidosis-NPCs were attached on the cover slip coated with matrigel, followed by culture in growth factor- free NPCs medium for 3 ~ 4 weeks. The mutation of NEU1, the sialidosis causing gene, was investigated by the same manner as described in Example <1-1>. The normal cell derived hiPSCs were cultured by the same manner as described above to prepare the control, so that the normal hiPSCs derived embryoid body and NPCs were differentiated as the controls. As a result, as shown in Figure 15a and Figure 16, the sialidosis originated NPCs displayed round- shaped neurosphere (Figure 15a). The mutation of c.G679>A in NEU1 gene encoding sialidase was also confirmed with showing the mutation of G227R in the sialidase protein synthesized by the gene (Figure 16a). The mutation of c.T824>C and c.G1040>A was also confirmed with showing the mutation of V275A and R347Q in the sialidase protein synthesized thereby (Figure 16b) .

<3-2> Differentiation potential of sialidosis-embryoid body To investigate the differentiation potential of the sialidosis-EB prepared in Example <3-l>, the present inventors investigated the expressions of three types of germ layer markers, which were the ectoderm markers NESTI and TUJ1, the endoderm markers S0X17 and -FP, and the mesoderm markers a- smooth muscle actin (a-SMA) and DESMIN.

Particularly, the sialidosis-EB differentiated by the same manner as described in Example <3-2> proceeded to immunofluorescent staining by the same manner as described in Example <2-3> and then the expressions of those proteins, NESTIN, TUJ1, S0X17, a-FP, a-SMA, and DESMIN were confirmed. For the immunofluorescent staining, the primary antibody such as anti-NESTIN antibody (1:100; MAB5326, Chemicon, USA), anti-TUJl antibody (1:500; PRB-435P, Covance, USA), anti-SOX17 antibody (1:100; MAB1924, R&D Systems, USA), anti-FP antibody (1:200; A8452 , Sigma-Aldrich, USA), anti- a-SMA antibody (1:400; A5228, Sigma-Aldrich, USA), or anti- DESMIN antibody (1:50; AB907, Chemicon, USA) was used. To compare the expression levels, 4 ' 6 -diamidino-2 -phenylindole (DAPI) was treated to the cells to stain the nuclei.

As a result, as shown in Figure 8a, the expressions of all the three types of germ layer markers of ESTIN, TUJ1, S0X17, a-FP, a-SMA, and DESMIN, were confirmed in the sialidosis-EB, indicating that the differentiated cells had pluripotency (Figure 8d) .

Example 4: Characteristics of sialidosis-NPCs

<4-l> Expression of neural marker protein in sialidosis- NPCs and the cells differentiated therefrom

To investigate whether or not the sialidosis-hiPSCs derived NPCs (sialidosis-NPCs) and neuronal cells were normally differentiated, the present inventors first examined the expressions of the neuronal cell specific marker proteins, NESTIN, TUJ1, and MAP2 , in sialidosis-NPCs and neuronal cells.

Particularly, the present inventors induced the differentiation of sialidosis-NPCs and neuronal cells differentiated therefrom (sialidosis-neuronal cells) by the same manner as described in Example <3-l>. Then, immunofluorescent staining was performed by the same manner as described in Example <3-l> to confirm the expressions of NESTIN, TUJ1, and MAP2 proteins. For the immunofluorescent staining, the primary antibody such as anti-NESTIN antibody (1:100; MAB5326, Millipore) was used, for NPCs , and the primary antibody such as anti-TUJl antibody (1:500; PRB- 435P, covance) or anti-MAP2 antibody (1:1000; M4403, Sigma- Aldrich) was used for neuronal cells. To compare the expression levels, ' 6-diamidino-2-phenylindole (DAPI) was treated to the cells to stain the nuclei. For the controls, immunofluorescent staining was performed with normal-NPCs and neuronal cells differentiated therefrom under the same conditions as the above, and the expressions of neuronal cell specific marker proteins were confirmed.

As a result, as shown in Figure 15b, it was confirmed that NESTIN positive neural progenitor cells and TUJ1 positive neuronal cells were successfully differentiated in both normal-NPCs and sialidosis-NPCs (Figure 15b) . Example 5 : Molecular phenotype of neuronal cells differentiated from sialidosis-hiPSCs

<5-l> Mutation of sialidosis causing gene in sialidosis- NPCs

To investigate the characteristics of a mutant protein resulted from the mutation of a sialidosis causing gene in the sialidosis-NPCs prepared in Example <3-l>, the present inventors examined the sialidase protein activity in sialidosis-hiPSCs .

Particularly, fibroblasts and sialidosis-NPCs were obtained by the same manner as described in Example <3-l>, which were suspended in analysis buffer containing 20 mM citrate, 60 mM NaCl, and 1 mM CaCl₂ (pH 4.5) . The cells were lysed by using a sonicator 5 - 10 times repeatedly, followed by centrifugation at 13,000 rpm for 10 minutes at 4^°C to eliminate cell debris. The protein dissolved in the buffer was extracted. Then, 2 /!g of the protein lysate was added to analysis buffer containing 0.2 mM 2'-(4- methylumbelliferyl) -a-D-N-acetylneuraminic acid (4-MUNANA, Sigma-Aldrich, USA) , followed by culture at 37°C for 4 hours to induce reaction. To terminate the reaction, 0.1 M glycine-NaOH buffer (pH 10.5) was added thereto. To confirm the sialidase activity, fluorescence (lex = 365 nm/lem = 450 nm) was measured by using SpectraMax© M3 Multi-Mode Microplate Reader (Molecular Devices) . The sialidase activity in the normal human fibroblast-derived hiPSCs, the control, was also measured and compared with the above .

As a result, as shown in Figure 18, when compared with the sialidase activity of normal-NPCs, the sialidase activity in sialidosis-NPCs was significantly reduced (Figure 18) .

<5-2> Accumulation of lysosome in sialidosis-NPCs

To measure the amount of lysosome in the sialidosis- NPCs prepared in Example <3-l>, the culture medium was treated with LysoTracker Red DND-99 (1:20000; Invitrogen, USA), followed by reaction at 37^°C for 30 minutes. Then, Intracellular lysosome was observed. For the control, normal-NPCs were prepared and the accumulation of lysosome therein was examined by the same manner as described in the above .

As a result, as shown in Figure 17, unlike the NPCs originated from normal-hiPSCs , the accumulation of lysosome in sialidosis-NPCs was significantly increased (Figure 17) .

Exam le 6 : Confirmation of the over-expression of NEU1, the sialidosis causing gene, in human embryonic kidney cells (HEK293T cells) and the molecular phenotype thereof

The following experiment was performed to investigate the enzyme activity of sialidase and the protein expression by measuring the over-expression of NEU1, the sialidosis causing gene, in human embryonic kidney cells (HE 293T cells) .

Particularly, the normal NEU1 gene and the mutant NEU1 gene having the mutation of R347Q or V275A, obtained in Example <1-1>, were amplified, and the amplified products were cloned in pEGFP-N3 vector (BD bioscience Clonetech) to conjugate the green fluorescent protein to NEU1 protein. FLAG tag was inserted in between 3 ' -end of NEU1 and 5 ' -end of GFP. The prepared NEUl-pEGFP plasmid was transfected in HEK 293T cells by using lipofectamine 2000 (Invitrogen) . The enzyme activity of sialidase was measured by the same manner as described in Example <2-6>. Western blotting was also performed to investigate the protein expression. Precisely, 30 of cell lysate was loaded in 4 ~ 20% gradient gel (BIO-rad) , which was transferred onto nitrocellulose membrane. The NEUl-GFP conjugated protein was detected by anti-GFP antibody (Abeam, ab290, 1:5000). β-actin was detected by anti- -actin antibody (Abeam, ab6276, 1:5000).

As a result, as shown in Figure 5 and Figure 6, the NEUl-GFP conjugated protein constructed with the mutant NEU1 gene having the mutation of R347Q or V275A was expressed in HEK 293T cells (Figure 6) . The enzyme activity of sialidase was inhibited more significantly by the NEUl-GFP conjugated protein having the mutant sequence of R347Q or V275A (Figure 5) .

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims

Claims

[CLAIMS ]

[Claim l]

A method for preparing sialidosis hiPSCs model in vitro comprising the following steps:

ii) collecting the hiPSCs induced in step i) .

[Claim 2]

The method for preparing sialidosis hiPSCs model in vitro according to claim 1, wherein the induction of step i) is preferably achieved by the ectopic expression of a pluripotency marker.

[Claim 3]

A sialidosis hiPSCs model prepared by the method of claim 1.

[Claim 4]

The sialidosis hiPSCs model according to claim 3, wherein the hiPSCs have at least one of the following characteristics of i) ~ vi) :

i) the mutation of NEU1 gene, that is c. G679>A or c.T824>C and c.G1040>A ; ii) the mutation of G227R or V275A and R347Q in the protein synthesized from NEU1 gene;

iii) hiPSCs form of normal cells;

iv) the expression of a sternness maker including OCT4 (octamer-binding transcription factor 4) , NA OG, TRA-1-81, SSEA3 (specific embryonic antigen 3), SSEA4 (specific embryonic antigen 4), and TRA-1-60;

v) the accumulation of lysosome; and

vi) the decrease of sialidase activity.

[Claim 5]

A method for preparing sialidosis neural progenitor cell model in vitro comprising the following steps:

ii) inducing neural progenitor cells (NPCs) from the hiPSCs prepared in step i) ; and

iii) collecting the NPCs induced in step ii) .

[Claim 6]

A sialidosis neural progenitor cell model prepared by the method of claim 5.

[Claim 7] The sialidosis neural progenitor cell model according to claim 6, wherein the neural progenitor cells have at least one of the following characteristics of i) ~ iii) :

i) the expression of neural marker protein including Nestin (type VI intermediate filament (IF) protein) , TUJ1

(class III beta- tubulin) , and MAP2 (Microtubule-associated protein 2) ;

ii) the accumulation of lysosome; and

iii) the decrease of sialidase activity.

[Claim 8]

A use of hiPSCs, neural progenitor cells, or neurosphere as the sialidosis model, which includes the following steps:

i) inducing the differentiation of embryoid body (EB) , neural progenitor cells (NPCs) , or neurosphere from the hiPSCs of claim 3; and

[Claim 9]

The use of hiPSCs, neural progenitor cells, or neurosphere as the sialidosis model according to claim 8, wherein the EB differentiation marker is characteristically confirmed by the expression of one of those markers selected from the group consisting of the ectoderm markers NESTIN and TU 1 , the endoderm markers SOX17 (SRY (sex determining region Y) -box 17) and a-FP (alpha- fetoprotein) , and the mesoderm markers a-sraooth muscle actin (a-SMA) and DESMIN (Muscle Cell Marker) .

[Claim 10]

The use of hiPSCs, neural progenitor cells, or neurosphere as the sialidosis model according to claim 8, wherein the neural progenitor cells are characterized by one of those characteristics of i) ~ iii) :

i) the expression of neural marker protein including Nestin (type VI intermediate filament (IF) protein), TUJ1

(class III beta-tubulin) , and MAP2 (Microtubule-associated protein 2) ;

ii) the accumulation of lysosome; and

iii) the decrease of sialidase activity.

[Claim ll]

A method for screening a sialidosis treating agent candidate comprising the following steps:

i) treating the embryoid body, neural progenitor cells, or neurosphere differentiated from the hiPSCs model of claim 3 with a sample material;