LU506249B1 - BARTH SYNDROME INDUCABLE PLURIPOTENT STEM CELLS AND THEIR PRODUCTION METHODS, DIFFERENTIATION CULTURE MEDIA AND APPLICATIONS - Google Patents

Abstract

The present invention discloses inducible pluripotent stem cells for Barth syndrome as well as their production methods, differentiation culture media and applications. 150-200 ml of urine from a patient with Barth syndrome is collected and urine cells are obtained by centrifugation at 2000 rpm for 10 minutes. After cell counting, 3.5-4×10^5 cells are seeded into a 6-well culture plate coated with Matrigel and incubated at 37 °C for more than one hour. After passage and cultivation of the third generation of urine cells to 80% confluence, the cells are infected with a Sendai virus containing the four transcription factors OCT4, SOX2, KLF4 and c-MYC to induce reprogramming. 15 days after viral infection, clonal cell groups begin to appear, which are manually selected and transferred to a new 24-well culture plate incubated with Matrigel. After several passages and fulfilling several criteria, a purified cell line of iPS cells (TAZ-UiPSC) is thus obtained from urine cells of patients with Barth syndrome. These TAZ-UiPSC can be specifically induced to differentiate into cardiomyocytes to establish an in vitro cell model used to study the pathogenesis of Barth syndrome and to select targeted drugs.

Description

Barth syndrome inducible pluripotent stem cells and their LU506249

Production methods, differentiation culture media and applications

Technical area

This invention belongs to the field of biotechnology and relates specifically to inducible pluripotent stem cells for Barth syndrome and their production methods,

Differentiation culture media and applications.

Technology in the background

Barth syndrome (BTHS) was first discovered by Dr. Peter Barth in 1983 and is generally considered a rare, X-linked, autosomal recessive genetic disorder. The disease includes skeletal muscle diseases, cardiomyopathy,

Growth retardation and 3-methylglutaconic aciduria, with an incidence of only 1/300,000-400,000. The clinical features of Barth syndrome include hypertrophic cardiomyopathy, dilated cardiomyopathy, left ventricular noncompaction, ventricular arrhythmias,

Endocardial fibroelastosis, proximal myopathy, sudden cardiac death, neutropenia, drowsiness and

Fatigue, hypoglycemia, lactic acidosis, difficulty eating, etc.

In 1996, the potential Barth syndrome mutation on the tafazzin (TAZ) gene was

Gene located on the Xq28 chromosome, mainly including the deletion mutation (c.517delG) and the missense mutation (c.328T>C) of the TAZ gene. The TAZ gene is mainly involved in the biosynthesis of cardiolipin (CL) in cardiac muscle cells, a process that takes place mainly in the mitochondrial membrane. Cardiolipin (CL) in

Cardiac muscle cells can directly interact with various important protein complexes such as

respiratory chain, mitochondrial metabolic carriers, mitochondrial morphology shape proteins, etc.

Since human heart muscle cells are difficult to obtain, this is difficult and costly.

Content of the invention

In order to solve the problems and deficiencies of the existing technology, the present

The invention aims to develop inducible pluripotent stem cells for Barth syndrome and their

To provide manufacturing methods, differentiation culture media and applications to increase sources and application values of inducible pluripotent stem cells.

To achieve the above objectives, the first objective of this invention is to

Providing a method for producing inducible pluripotent stem cells for Barth syndrome, comprising the following steps:

S1: Collection of 150-200 ml urine from a patient with Barth syndrome and collection of

Urine cells by centrifugation;

S2: After cell counting, 3.5-4x1075 cells are seeded into a 6-well culture plate coated with Matrigel and incubated at 37 °C for more than one hour, and after passage, the third generation of urine cells are cultured to 80% confluence, followed by

Digestion and passage with EDTA-trypsin;

S3: The urine cells cultured in step S2 are infected with Sendai viruses that encode the four

contain transcription factors OCT4, SOX2, KLF4 and c-MYC to induce reprogramming, and after 15 days of viral infection, clonal cell groups begin to appear;

S4: Selection and cultivation of the expanded clonal cell groups and transfer to a new Matrigel-coated 24-well culture plate, continued cultivation with ERU506249

Culture medium containing 10 micromoles of Y-27623 only on the first day and daily

Change medium until the clonal cell groups reach 90% confluence after 5-7 days;

SS: Digestion with 0.5 mM EDTA and passage at a ratio of 1:6 to 1:10 to obtain, by multiple passage, a purified cell line of inducible pluripotent stem cells derived from urine cells of patients with Barth syndrome.

The second aim of this invention is to provide a virus for reprogramming and induction to produce inducible pluripotent stem cells for the Barth

Syndrome, where the virus is a Sendai virus that infects the four transcription factors OCT4, SOX2,

KLF4 and c-MYC.

The third aim of this invention is a type of inducible pluripotent stem cells for Barth syndrome, called TAZ-UiPSC.

The fourth object of this invention is to provide an application of the above-mentioned inducible pluripotent stem cells for Barth syndrome in the differentiation culture of cardiomyocytes, wherein said inducible pluripotent stem cells are in a

culture medium and differentiated through several steps to obtain cardiomyocytes.

The further steps include the following: (1) The Barth syndrome inducible pluripotent stem cells are

Matrigel and then cultured in E8 culture medium, with the medium being changed daily for 2-3

days, (2) In the first differentiation phase, the culture medium is changed to RPMI+B27 insulin with 5

Micromol CHIR99021 for 2 days; (3) In the second differentiation phase, the culture medium is changed to RPMI+B27 insulin with 5 micromol IWR-1 for 2 days; (4) In the third differentiation phase, the culture medium is changed to normal RPMI+B27-

Insulin is changed for 2 days; (5) In the fourth differentiation phase, the culture medium is changed to normal

Switch to RPMI+B27+Insulin and continue the culture, changing the medium every two days.

The fifth object of this invention is to provide a differentiation culture medium for the differentiation of cardiomyocytes from said Barth syndrome inducible pluripotent stem cells, comprising:

For the first differentiation phase, a differentiation culture medium with RPMI+B27-

insulin and 5 micromol CHIR99021;

For the second differentiation phase, a differentiation culture medium with RPMI+B27-

insulin and 5 micromol IWR-1;

For the third differentiation phase, an RPMI+B27-insulin differentiation culture medium;

And for the fourth differentiation phase a RPMI+B27+Insulin

Differentiation culture medium.

The advantage of this invention is that it uses a non-invasive method to collect urine cells from the patient, then reprogram them into iPS cells and target them to

Cardiomyocytes, which is of great importance for understanding the pathogenesis of

Barth syndrome and the selection of targeted drugs. The advantages and positive effects of this invention are not limited to this and are described in detail in the

Examples of the specification document are shown. LU506249

Description of the attached drawings

In order to explain the technical solutions in the embodiments of this invention or the existing technology more clearly, a brief introduction to the

Drawings given in the description of the embodiments or the existing

technique. Obviously, the drawings described below are only some embodiments of this invention. It is obvious to those skilled in the art that further drawings based on these drawings without creative

work performance also fall within the scope of this invention.

Figure 1 shows the separation, culture and reprogramming of urine cells (UCs) from

Patients with Barth syndrome, showing (A) primary cultured urine cells (UCs), (B) clonal

Colony-like inducible pluripotent stem cells (iPS) from urine cells by Barth-

syndrome patients and (C) manually selected and cultured inducible pluripotent

Stem cells iPS from urine cells of Barth syndrome patients (TAZ-UiPSC) are shown.

Figure 2 shows the immunofluorescence identification of pluripotency protein expression of

TAZ-UiPSC, where (A) the expression of the pluripotency protein OCT4 in TAZ-UiPSC by

immunofluorescence and (B) the expression of the pluripotency protein SSEA4 in

TAZ-UiPSC is identified by immunofluorescence.

Figure 3 shows real-time quantitative PCR (RT-qPCR) for identifying the expression of pluripotent genes. RT-qPCR measures the expression levels of the pluripotent genes NANOG,

OCT4, SOX2 and SSEA4 in different groups, mean + standard deviation, n = 3, ***P<0.01 means statistically significant differences, n.s> 0.05 no significant

differences;

Figure 4 shows the chromosome karyotype analysis of the chromosome structure of TAZ-

UiPSC;

Figure 5 shows the immunofluorescence identification of the differentiation potential of TAZ-

UIPSC, where (A) the expression of the ectodermal marker NESTIN, (B) the expression of the mesodermal marker SMA and (C) the expression of the endodermal marker AFP by

identified by immunofluorescence;

Figure 6 shows the RT-PCR identification of the differentiation potential of TAZ-UiPSC, with RT-PCR identifying the expression of marker genes for the three germ layers;

Figure 7 shows the results of Sanger sequencing to identify

Gene mutation sites, where (A) the sequencing results of the control group (Control-iPS) and (B) the sequencing results of the experimental group (TAZ-UiPSC) are shown,

Figure 8 shows the immunofluorescence identification of TAZ-UiPSC differentiated

Cardiomyocytes, for the visualization of immunofluorescence identification of cardiomyocytes

Marker proteins cTnT and a-actinin.

Detailed description

In order to make the objects, technical solutions and advantages of this invention clearer, a more detailed description of the invention is given below in conjunction with the accompanying drawings.

Examples 1.Isolation and cultivation of urine cells in patients with Barth syndrome

Strictly following sterile surgical requirements, collect 150-200 ml of medium urine from a patient with Barth syndrome (BTHS), centrifuge it at 2000 revolutions per

Minute for 10 minutes to obtain a cell precipitate from the urine. Wash it twice with PBS containing 1% penicillin/streptomycin. Then resuspend it in 1 ml of special

Urine cell culture medium from Beijing Saibei (plus 2.5 ng/ml bFGF), count the cells and inoculate 3.5-4x 105 cells into a well of a 6-well culture plate coated with Matrigel and incubated at 37 °C for more than one hour. Transfer the cells to a 37 °C, 5%

CO2 cell culture incubator. Change the medium daily with a semi-quantitative

Medium change.

Once the cultured urine cells have become confluent, perform a passage culture with 0.25% EDTA-trypsin. 2. Generation and expansion of inducible pluripotent stem cells (iPS)

Once the third generation urine cells have reached 80% confluence, they are infected with a Sendai virus from Thermo, which contains the four transcription factors OCT4, SOX2, KLF4 and c-MYC, to induce reprogramming. The infected cells are cultured in E6 culture medium from STEMCELL (plus 10 ng/ml FGF2), with the medium being changed daily. 15 days after virus infection, clonal

Cell groups begin to appear, after which the culture medium is changed to E8 from STEMCELL to continue the culture. Once the clonal groups have reached the desired size, they are manually selected and transferred to a new 24-well culture plate incubated with Matrigel. The culture is continued in E8 culture medium containing 10 micromolar

Y-27623 and the medium is changed daily. After 5-7 days, the clonal

Groups 90% confluence, then they are digested with 0.5 mM EDTA and passaged in a ratio of 1:6 to 1:10. 3. Immunofluorescence staining

After the culture of the different cell groups has become confluent, the

Supernatant removed, washed once with PBS, and then 4% paraformaldehyde at

room temperature for 15 minutes. Then wash three times with PBS, 5

minutes. A solution containing 0.1% Triton X-100 and 3% bovine serum albumin (BSA) in PBS is then added and the cells are permeabilized at room temperature for 1 hour.

Then primary antibodies are added without washing: rabbit anti-OCT4 antibody (1:500), mouse anti-SSEA4 antibody (1:200), mouse anti-NESTIN antibody (1:200), mouse

Anti-SMA antibody (1:200), mouse anti-AFP antibody (1:500), rabbit anti-cTnT

Antibody (1:200) and mouse anti-a-actinin antibody (1:200), incubated overnight at 4 °C.

Then wash three times with PBS, each for 5 minutes, followed by the addition of

Secondary antibodies: Alexa Fluor 488-labeled sheep anti-rabbit IgG antibody (1:1000), Alexa Fluor 488-labeled sheep anti-mouse IgG antibody (1:1000) and Alexa

Fluor 594-labeled sheep anti-mouse IgG antibody (1:1000), incubated for 2 hours at room temperature. Then washed three times with PBS, each for 5 minutes, followed by

Add DAPI stain and incubate for 15 minutes at room temperature in the dark.

Finally, wash three times with PBS, 5 minutes each, and then observe and photograph under an inverted fluorescence microscope. 4.Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (RT-qPCR)

analysis

Using Trizol (Invitrogen, USA), the total RNA of the different cell groups is extracted and the OD is measured to ensure that the OD 260/OD 280 ratio of the RNA is between 1.8 and 2.1, which guarantees purity. Then the total RNA is extracted with ah506249

Reverse Transcription Kit (Toyobo, Japan) using the reaction system and program according to the product instructions. A portion of the cDNA is used for

RT-PCR was used, where the primer sequences are:; 5 AFP-F: AGGGAGCGGCTGACATTATT

AFP-R: CAGAGAATGCAGGAGGGACA

GATA6-F: AACTACCACCTTATGGCGCA

GATA6-R: GCTGTAGGTTGTGTTGTGGG

CDHS-F: GGATGCAGACGACCCCACT

CDHS-R: TGTGTACTTGGTCTGGGTGAAG

PECAM-F: GACATGGCAACAAGGCTGTG

PECAM-R: CACTGTCCGACTTTGAGGCT

NKX2-5-F: CCACGCCCTTCTTCAGTCAA

NKX2-5-R: TAGGCACGTGGATAGAAGGC

PAX6-F: GCCAGACCTCCTCATACTCC

PAX6-R: GCCAGACCTCCTCATACTCC

TUBB-F: GGTAACAACTGGGCCAAAGG

TUBB-R: GCTGATAAGGAGAGTGCCCA

ALB-F: TGTTGCATGAGAAAACGCCA

ALB-R: TGCTCTTTGTTGCCTTGGG

GADPH-F: GGCAAATTCCATGGCACCG

GADPH-R: GTTCACACCCATGACGAACA

The reaction system is based on the SYBR kit (Takara, Japan). After preparation, the PCR reaction is carried out at 94 °C for 2 minutes for denaturation, followed by 35 cycles of

reaction (94 °C for 30 seconds, 59 °C for 30 seconds, 72 °C for 30 seconds). 6 uL of

Reaction product is mixed with 2 uL of the 6xLoading Buffer and then using 2%

Agarose gel electrophoresis and photographed. Another part of the cDNA is used for RT-qPCR, where the primer sequences are:

NANOG -F: CAGCCAAATTCTCCTGCCAG

NANOG -R: CACGTCTTCAGGTTGCATGT

OCT4 -F: AATTTGTTCCTGCAGTGCCC

OCT4 -R: CTTCGTTGTGCATAGTCGC

SOX2-F: GACAGTTACGCGCCACATGAA

SOX2-R: GTTCATGTAGGTCTGCGAGC

SSEA4-F: GGACAAGGCGTTCACAGAT

SSEA4-R: CAGGCTAGTTTTCACGCTGG

GADPH-F: GGCAAATTCCATGGCACCG

GADPH-R: GTTCACACCCATGACGAACA

The reaction system is based on the SYBR kit (Takara, Japan). After preparation, the PCR starts with denaturation at 95 °C for 3 minutes, followed by 35 cycles (95 °C for 10 seconds, 60 °C for 30 seconds). After the reaction, statistical analysis based on the Ct values, preparation of a standard curve and quantitative analysis using the Ct-based method (2-AACt) are performed.

S.Embryoid body formation test

The differentiation potential of the reprogrammed iPS cells is determined by

Embryoid body formation assays. Each embryoid body is formed by culturing 5000 iPS cells in 50 μl of E8 culture medium by droplet culture for 3 days.

The embryoid bodies are then cultured in a suspension of 50% E8 culture medium and 50% differentiation medium. This 50% differentiation medium contains DMEM-

F12, 20% fetal calf serum, 1% penicillin/streptomycin, 1x non-essential amino acids (NEAA), 2 mM L-glutamine and 0.1 mM ß-mercaptoethanol. The suspended embryoid bodies are then transferred to a non-adherent culture plate for 2 days. The

Embryoid bodies were transferred to a 24-well culture plate incubated with Matrigel and incubated for 14

days. Some cells are then collected for RNA extraction and RT-PCR analysis, while other cells are subjected to immunofluorescence cytological staining analysis after fixation with 4% paraformaldehyde. 6.Chromosome karyotype analysis

The chromosome stability of the iPS cells is checked by chromosome karyotype analysis. The cells are treated with 10 ug/ml colchicine at 37 °C for 60 minutes.

They are then treated with trypsin to obtain single cells and then with 0.075 M hypotonic potassium chloride solution. They are then incubated with Carnoy's

Fixative (3:1 mixture of methanol and acetic acid). Finally, a

Image analysis of chromosomes in mitosis stage and G-bands performed. 7.Mutation analysis

Genomic DNA of the cells is extracted using Thermo’s genome extraction kit, and the samples are sent to Qingke Biology for Sanger sequencing to determine the

mutation sites. 8.Induction of differentiation of iPS cells from urine cells of patients with Barth

Syndrome in Cardiomyocytes iPS cells are seeded at a density of 5x1075 cells per well in a 6-well culture plate coated with Matrigel and incubated at 37 °C for more than one hour.

Cells are cultured with E8 culture medium, changing the medium daily for 2-3 days until cells reach 70% confluence, at which point differentiation begins. In the first differentiation phase, the culture medium is changed to RPMI+B27 insulin with 5 micromol CHIR99021 for 2 days; in the second differentiation phase, it is changed to

RPMI+B27 insulin with 5 micromol IWR-1 for 2 days; in the third

Differentiation phase, it is changed to normal RPMI+B27 insulin for 2 days; in the fourth differentiation phase, it is changed to normal RPMI+B27+insulin and the

Culture is continued, changing the medium every two days. As soon as beating

When cardiomyocytes appear, they can be digested with Accutase enzyme and passaged, followed by immunofluorescence cytological identification of the cells.

Experimental results 1. Isolation, cultivation and reprogramming of urine cells

The experimental results show that the isolated primary urine cells from patients with Barth syndrome grow well, have a short spindle-shaped morphology and grow adherently, reaching 70% confluence within 5 days (Figure 1A). After the urine cells are fully grown, they are digested with 0.25% EDTA-trypsin and passaged. The third generation cells at 80% confluence are then infected with a commercial Sendai virus that expresses the four transcription factors OCT4, SOX2,

KLF4 and c-MYC for reprogramming. Clonal cell groups begin to appear 15 days after virus infection, and once these clonal groups reach the desired size, they can be clonally selected (Figure 1B). The clonal cells are manually selected and transferred to a new 24-well culture plate incubated with Matrigel.

Culture is continued in E8 culture medium containing 10 micromoles of Y-27623 on the first day only, and the medium is changed daily. After 5-7 days, the clonal groups reach 90% confluence, then they are digested with 0.5 mM EDTA and passaged at a ratio of 1:6 to 1:10 (Figure 1C). 2. Immunofluorescence identification of pluripotency protein expression

To determine the expression of pluripotency proteins in clonal cell groups after several

To identify passages, the immunofluorescence method was used to determine the expression of the pluripotency proteins OCT4 (Figure 2A) and SSEA4 (Figure 2B).

Results show that both pluripotency proteins are positively expressed and the expression overlaps with the position of the nucleus. 3. Real-time quantitative PCR (RT-qPCR) to identify the expression of

Pluripotency genes

To identify the expression of pluripotency genes in clonal cell groups after multiple passages, real-time quantitative PCR (RT-gPCR) was used to determine the

To determine expression levels of the pluripotency genes NANOG, OCT4, SOX2 and SSEA4. The

Results show that the expression level of these pluripotency genes in the reprogrammed iPS cells (TAZ-UiPSC) is comparable to the positive control group (Control-iPSC), without significant differences, and significantly higher than in the negative control group (UC) (Figure 3). 4. Chromosome karyotype analysis

To identify the chromosome structure stability of the reprogrammed cells, a chromosome karyotype analysis was performed on inducible pluripotent stem cells (iPSC) from urine cells of a male patient with Barth syndrome.

Results show that the cell chromosome karyotype is normal (46, XY) (Figure 4). 5. Cellular immunofluorescence to identify the differentiation potential of the

Cells

To identify the differentiation potential of the reprogrammed iPS cells (TAZ-UiPSC), an embryoid body formation assay was used in combination with

Differentiation induction was used to differentiate TAZ-UiPSC into cells of all three germ layers. After immunofluorescence staining of the cells, the results showed that the differentiated TAZ-UiPSC cells positively expressed the ectoderm marker protein NESTIN (Figure 5A), the mesoderm marker protein SMA (Figure 5B) and the endoderm marker protein AFP (Figure 5C). 6. RT-PCR to identify the differentiation potential of the cells

To further identify the differentiation potential of the reprogrammed iPS cells (TAZ-UiPSC), an embryoid body formation assay was used in combination with

Differentiation induction was used to differentiate TAZ-UiPSC into cells of all three germ layers. RT-PCR results show that the differentiated TAZ-UiPSC cells are positive for

Endoderm marker genes AFP and AGTA6, mesoderm marker genes CDH5, PECAM, NKX2-5 and

Ectoderm marker genes PAX6, TUBB, ALB express (Figure 6). 7. Identification of mutation sites

To confirm that the reprogrammed iPS cells (TAZ-UiPSC) contain mutation sites, Sanger sequencing of the reprogrammed iPS cells (TAZ-UiPS&) 506249 was performed. The results show that the control group (Control-iPS) has a normal

gene sequence (Figure 7A), while TAZ-UiPSC contains a deletion mutation (c.517delG) in the TAZ gene from patients with Barth syndrome (Figure 7B). 8. Induction of differentiation of iPS cells from urine cells from patients with

Barth syndrome in cardiomyocytes

With specific differentiation media RPMI+B27-Insulin and RPMI+B27+Insulin and the addition of 5 micromol CHIR99021 and 5 micromol IWR-1 to different

Differentiation is induced at certain time points, and beating cardiomyocytes appear in the fourth differentiation phase. Immunofluorescence cytological identification of differentiated cardiomyocytes shows that iPS cells differentiated from urine cells of patients with Barth syndrome express positively for the cardiomyocyte markers cTnT and a-actinin (Figure 8).

The examples disclosed above are merely preferred embodiments of this

invention and should not be considered as limiting the scope of this invention.

Therefore, equivalent changes based on the claims of this invention still fall within the scope of this invention.

Claims (6)

Claims LU506249 1. A method for producing inducible pluripotent stem cells for Barth syndrome, characterized by the following steps: S1: Collecting 150-200 ml of urine from a patient with Barth syndrome and obtaining urine cells by centrifugation; S2: After cell counting, 3.5-4x1075 cells are inoculated into a 6-well culture plate coated with Matrigel and incubated at 37 °C for more than one hour; After passage, the third generation of urine cells are cultured to 80% confluency, followed by digestion and passage with EDTA-trypsin; S3: The urine cells cultured in step S2 are infected with a Sendai virus containing the four transcription factors OCT4, SOX2, KLF4 and c-MYC to induce reprogramming; 15 days after virus infection, clonal cell groups begin to appear; S4: Selection and cultivation of the expanded clonal cell groups and transfer to a new Matrigel-coated 24-well culture plate; Culture is continued in E8 culture medium containing 10 micromolar Y-27623 only on the first day and the medium is changed daily; After 5-7 days, the clonal groups reach 90% confluence, then they are digested with 0.5 mM EDTA and passaged at a ratio of 1:6 to 1:10; SS: A purified cell line of inducible pluripotent stem cells derived from urine cells of patients with Barth syndrome is obtained by multiple passage. 2. A reprogramming-inducing virus used for the production of inducible pluripotent stem cells for Barth syndrome, characterized in that the virus is a Sendai virus containing the four transcription factors OCT4, SOX2, KLF4 and c-MYC. 3. Inducible pluripotent stem cells for Barth syndrome, produced by the method according to claim 1. 4. An application of the inducible pluripotent stem cells for Barth syndrome prepared according to claim 3 in the differentiation culture of cardiomyocytes, characterized in that said inducible pluripotent stem cells are cultured in a culture medium and differentiated through several steps to obtain cardiomyocytes. 5. The use of the inducible pluripotent stem cells for Barth syndrome prepared according to claim 2 in the differentiation culture of cardiomyocytes, characterized by the following steps: (1) The said inducible pluripotent stem cells for Barth syndrome are incubated in Matrigel and then cultured in E8 culture medium, the medium being changed daily for 2-3 days; (2) In the first differentiation phase, the culture medium is changed to RPMI+B27 insulin with 5 micromoles CHIR99021 for 2 days; (3) In the second differentiation phase, the culture medium is changed to RPMI+B27 insulin with 5 micromoles IWR-1 for 2 days; (4) In the third differentiation phase, the culture medium is changed to normal RPMI+B27 insulin for 2 days; (5) In the fourth differentiation phase, the culture medium is changed to normal RPMI+B27+Insulin and culture continued changing medium every two days 506249. 6. A differentiation culture medium for differentiating cardiomyocytes from inducible pluripotent stem cells for Barth syndrome according to claim 3, characterized in that the differentiation culture medium comprises: a differentiation culture medium for the first differentiation phase with 5 micromoles CHIR99021 in RPMI+B27-insulin; a differentiation culture medium for the second differentiation phase with 5 micromoles IWR-1 in RPMI+B27-insulin; a differentiation culture medium for the third differentiation phase with RPMI+B27-insulin; and a differentiation culture medium for the fourth differentiation phase with RPMI+B27+insulin.