Control of Adipogenic Differentiation in Mesenchymal Stem Cells via

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Letter

Control of adipogenic differentiation in mesenchymal stem cells via endogenous gene activation using CRISPR-Cas9 Yuichi Furuhata, Yuta Nihongaki, Moritoshi Sato, and Keitaro Yoshimoto ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00246 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017

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ACS Synthetic Biology

Control of adipogenic differentiation in mesenchymal stem cells via endogenous gene activation using CRISPR-Cas9

1 2 3 4 5 6 7

Yuichi Furuhataa, Yuta Nihongakib, Moritoshi Satob*, Keitaro Yoshimotoc, d*

a

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639, Japan

8 9

b

Department of General Systems Studies, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

10 11

c

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

12 13

c

JST, PRESTO, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

14 15

*

Corresponding authors:

16

Moritoshi Sato, Dr. Sci., Department of General Systems Studies, Graduate School of Arts and

17

Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

18

Phone and FAX: +81-3-5454-6579

19

E-mail: [email protected]

20

and

21

Keitaro Yoshimoto, Dr. Sci., Department of Life Sciences, Graduate School of Arts and Sciences,

22

The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

23

Phone and FAX: +81-3-5454-6580

24

E-mail: [email protected]

25 26

1

ACS Paragon Plus Environment


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Abstract:

2

Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to

3

their multilineage differentiation and self-renewal properties. Understanding the in vivo

4

differentiation process is necessary for clinical applications including cell therapy and

5

transplantation. This remains challenging owing to the lack of induction methods that

6

imitate the natural programming process. Endogenous gene regulation of tissue specific

7

transcription factors is therefore desirable. In the present study, we demonstrated

8

endogenous activation of adipogenic genes through the dCas9-based transcription

9

system, and achieved efficient induction of different types of adipocyte-like cells from

10

MSCs. Interestingly, the MSCs converted via single gene activation exhibited

11

morphological and molecular properties of white adipocytes, while beige adipocyte-like

12

cells were induced via multiplex gene activation of three specific transcription factors.

13

These results reveal that the fate of MSCs can be effectively manipulated by direct

14

activation of specific endogenous gene expression using a dCas9-based activator with

15

reduced exogenous additives.

16 17

Keywords:

18

CRISPR-Cas9, Differentiation, Gene activation, Mesenchymal stem cells, White

19

Adipocyte, Beige Adipocyte

20

2


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1

Introduction

2

Mesenchymal stem cells (MSCs) are one of the most attractive stem cell sources in

3

regenerative medicine; in addition to human induced pluripotent stem (iPS) cells and

4

embryonic stem (ES) cells. While MSCs have multilineage differentiation and

5

self-renewal properties, they are a type of somatic stem cell, and can be obtained from

6

various tissues. These features make them an attractive choice for the development of

7

clinical applications, including cell therapy and transplantation

8

avoid ethical issues and tumorigenesis. Previous studies reported that MSCs can

9

differentiate into various cells, including adipocytes, osteocytes, chondrocytes,

10

myocytes, and neurons 4. To regulate their lineage commitment, several induction

11

methods have been proposed, such as chemically modified medium 5, and ectopic

12

expression of tissue specific transcriptional factors 6. Although these induction methods

13

can efficiently control the fate of MSCs, the associated safety concerns present a

14

formidable obstacle to their therapeutic use. Moreover, since cellular differentiation

15

processes are regulated by complex and dynamic gene expression networks, these

16

methods are not suitable to reproduce the in vivo programming process of MSCs.

17

Therefore, there is a need for a novel method to induce programming of MSCs for

18

therapeutic applications.

19

Clustered

regularly

interspaced

short

palindromic

1–4

, since their use can

repeats

(CRISPR)-Cas9

20

(CRISPR-associated protein 9) technology has been at the forefront in the field of

21

biology due to their potential in biotechnological and medical applications. The

22

catalytically inactivated Cas9 protein derived from Streptococcus pyogenes, called

23

nuclease-dead Cas9 (dCas9), is often used to control gene expression 7. dCas9 forms a

24

complex with single chimeric guide RNA (sgRNAs), and binds to DNA that is 3



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8,9

1

complementary to the first 20 nt of the sgRNA

. Several studies have reported that

2

dCas9 fused with the transcriptional activator domains enables targeted endogenous

3

gene activation

4

and manipulation of dynamic gene programs 7.

8,10,11

. This system has accelerated the study of genome-scale screening

5

In addition to above mentioned applications, dCas9-based transcriptional control also

6

enables cell-fate engineering in the native context. Several groups have demonstrated

7

the direct conversion of primary fibroblasts into myogenic and neurogenic lineages 12,13,

8

and the differentiation control of iPSCs and ES cells

9

expression, dCas9-based endogenous gene activation is suitable for multiple gene

10

activation and can remodel the epigenetic state of the target loci towards differentiation

11

13

12

programming processes, which will allow MSCs to be used in clinical applications.

14–17

. Compared to ectopic

. Therefore, cell-fate engineering via endogenous gene activation imitates native

13

In this study, we first demonstrated dCas9-based transcriptional activation of

14

endogenous genes in MSCs. The expression of adipocyte-specific transcription factors

15

was efficiently activated using a dCas9-based activator. We subsequently attempted

16

cell-fate engineering of MSCs via endogenous gene activation with reduced exogenous

17

additives. We successfully controlled the differentiation of MSCs to white

18

adipocyte-like cells and beige adipocyte-like cells, which are important in disease

19

research for obesity and diabetes.

4


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1

Results and Discussion

2

Among all published synthetic transcription factors based on dCas9, synergistic

3

activation mediator (SAM) appeared to be the most potent activator 18,19. Therefore, we

4

decided to use SAM for transcriptional activation and cell-fate engineering of MSCs in

5

this study. In the SAM system, dCas9 protein fused with VP64 (dCas9-VP64) form a

6

complex with sgRNA bearing two MS2-binding stemloops (sgRNA 2.0), which recruits

7

MS2 coat protein fused with two distinct transcriptional activators: p65 and heat shock

8

factor 1 (MS2-p65-HSF1).

9

A fluorescent reporter assay was performed to assess and optimize the efficacy of

10

transcriptional activation via SAM in MSCs. We made use of a lentiviral vector carrying

11

the EGFP reporter under the control of a tetracycline-inducible promoter composed of

12

six copies of sgRNA binding sites and a minimal promoter (Figure 1A). Seven days

13

after transduction with the EGFP reporter, MSCs were further lentivirally transduced

14

with dCas9-VP64 and MS2-p65-HSF1 at the same time, followed by selection with

15

blastcidin S, hygromycin B, and puromycin over twenty-one days (Figure 1B and S1).

16

MSCs strongly expressed EGFP four days after sgRNA 2.0 transduction (Figure 1C).

17

Interestingly, despite high multiplicity of infection (MOI), the fluorescence intensity

18

increased with increasing MOI of sgRNA 2.0, suggesting that the amount of

19

intracellular sgRNA 2.0 is important for efficient transcriptional activation in MSCs

20

(Figure S2). SAM mediated gene activation is functional in MSCs, and we therefore

21

decided to perform transduction of sgRNA 2.0 at an MOI of 20 in subsequent studies.

22

For endogenous gene activation in MSCs, MSCs stably expressing dCas9-VP64 and

23

MS2-p65-HSF1, henceforth referred to as SAM MSCs, were established using

24

lentivirus and antibiotic selection. To determine whether SAM MSCs retained 5



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1

multipotency, osteogenic and adipogenic induction were performed using the relevant

2

induction medium. Alizarin red S staining and Oil red O staining revealed that SAM

3

MSCs grown under osteogenic and adipogenic induction conditions efficiently

4

differentiated into osteocytes and adipocytes, respectively (Figure 2A and B). Moreover,

5

differentiation efficiencies of SAM MSCs were comparable to those of wild-type MSCs.

6

These results indicate that SAM MSCs retain their multipotency.

7

Overexpression of transgenes encoding adipocyte specific transcription factors has 20–22

8

been used to achieve generation of adipocytes from MSCs and fibroblasts

. We

9

hypothesized that the targeted activation of the endogenous genes encoding these

10

factors in their native context could induce adipogenic differentiation of MSCs.

11

Adipogenic lineage commitment of MSCs is mainly regulated by three transcriptional

12

factors; PPARG, CEBPA, and KLF5

13

selected as candidates for the first attempt to induce adipogenesis in MSCs. Since

14

previous studies reported that the level of gene activation by the dCas9-based

15

transcription system can be synergistically enhanced using multiple sgRNAs

16

designed four distinct sgRNAs targeting different positions proximal to the transcription

17

start sites of each gene. Then, we tested whether SAM could activate endogenous genes

18

in MSCs. To activate endogenous gene expression, SAM MSCs were lentivirally

19

transduced with all four sgRNAs for each gene and transduced cells were described as

20

“sgGENE_1-4”. SAM MSCs transduced with sgPPARG_1-4, sgCEBPA_1-4, and

21

sgKLF5_1-4 had 15-, 11110-, and 85-fold upregulation of endogenous gene expression,

22

respectively, compared to cells transduced with control sgRNA (Figure 2C-E).

23

Off-target effect is a general concern to all CRISPR/Cas9-based technology. To validate

24

off-target effects of SAM-mediated gene activation, potential off-target binding sites of

23

. In the present study, these three genes were

10,11

, we

6


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1

each sgRNA were predicted using CCTop software24. Among the predicted candidates,

2

the site upstream of a transcription start site of a protein coding gene were chosen as the

3

off-target binding site for each sgRNA. The sequences of off-target binding sites were

4

shown in Supporting Information. As shown in Figure S4, we found no strong off-target

5

activation of endogenous genes. These results indicate that a dCas9-based activator can

6

efficiently activate targeted endogenous genes in MSCs.

7

Next, we tested whether endogenous gene activation via SAM can induce adipogenic

8

programming of MSCs. The cells were cultured for 2 weeks in adipocyte maintenance

9

medium after transduction of sgRNAs (Figure 3A). To assess adipogenic phenotypes,

10

the production of oil droplets in the treated MSCs was analyzed by Oil red O staining,

11

which is used to detect triacylglycerol accumulation. Though there were no oil droplets

12

observed in mock-, sgControl-, and sgKLF5_1-4-transduced cells (Figure 3B), a

13

number

14

sgCEBPA_1-4-transduced cells (Figure 3C). Surprisingly, these gene-activated cells

15

produced as many droplets as adipocytes induced using adipogenic induction medium

16

(Figure 3B and C). To determine the efficiency of adipogenic differentiation, Oil red O

17

staining intensities were quantified by extracting Oil red O using isopropyl alcohol.

18

Both sgPPARG_1-4- and sgCEBPA_1-4-transduced cells had high Oil red O intensities,

19

similar to those of cells cultured in adipogenic induction medium (Figure 3D). Both

20

sgPPARG_1-4- and sgCEBPA_1-4-transduced cells produced the same number of oil

21

droplets as cells cultured in adipogenic induction medium (Figure 3E).

of

droplets

were

observed

in

both

sgPPARG_1-4-

and

22

For detailed characterization of induced adipocytes, we focused on a panel of

23

adipocyte-associated genes. Real-time RT PCR was carried out to measure the

24

expression levels of adipogenic marker genes, including PPARG, CEBPA, ADIPOQ, 7



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1

FABP4, FASN, and LIPE. Adipocyte marker genes were not expressed in the

2

undifferentiated samples (mock- and sgControl-transduced cells). In contrast, both

3

sgPPARG_1-4- and sgCEBPA_1-4-transduced cells had much higher levels of

4

expression than undifferentiated cells at day 14, which was comparable with that of

5

cells cultured in adipogenic induction medium. These findings suggest that dCas9-based

6

transcriptional activation can induce adipogenic differentiation of MSCs, even in

7

adipocyte maintenance medium. Adipocyte maintenance medium does not contain

8

indomethacin and 3-isobutyl-1-methylxanthine, which act as a PPARγ agonist and an

9

enhancing agent for dexamethasone, respectively. Therefore, we accomplished efficient

10

adipogenic differentiation of MSCs using endogenous gene activation with reduced

11

exogenous additives.

12

Mammalian adipose tissue is divided into white, beige, or brown. White adipocytes,

13

which are specialized for energy storage and cause metabolic disease including obesity

14

and diabetes, have a single, large deposit of lipids. In contrast, beige and brown

15

adipocytes have multiple, small oil droplets in the cytosol, and can counteract metabolic

16

disease through thermogenesis 25. In the human body, the metabolic profile is regulated

17

by the balance of cellular activities in these three adipocytes. Understanding how these

18

adipocytes are induced and characterized in the natural programming process is helpful

19

for the study of disease mechanisms, and could contribute to a cell-based drug screening

20

for metabolic diseases. Ahfeldt et al. reported that overexpression of PPARG transgene

21

alone or combined with CEBPB and PRDM16 transgenes in MSCs derived from iPS

22

cells induced white or brown adipogenesis, respectively 21. On the other hand, the other

23

studies showed that brown adipocytes were derived from skeletal muscle precursor cells

24

rather than white adipocyte precursor cells, while beige adipocytes were come from 8


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1

white adipocyte precursor cells26,27. In the present study, we tested whether SAM can

2

activate at multiple endogenous loci in MSCs and regulate their lineage commitment

3

toward beige or brown adipocytes. Four distinct sgRNAs, each targeting the promoter

4

region of CEBPB and PRDM16, were designed. In a single-gene activation experiment,

5

the endogenous gene activation of each gene was observed (Figure 4A). Next, multiple

6

activation of PPARG, CEBPB, and PRDM16 genes was carried out in SAM MSCs. In

7

the sample transduced with multiple sgRNAs targeting these three genes, we observed

8

successful activation of all target genes, although all genes exhibited a small decrease in

9

the magnitude of upregulation compared to that observed in single gene activation

10

(Figure 4B).

11

To control the adipogenic differentiation among white and beige or brown adipocytes,

12

SAM MSCs were transduced with sgRNAs targeting these three genes independently

13

and together. After culturing in adipocyte maintenance medium for three weeks, cells

14

were stained with Nile red. The cells transduced with multiple sgRNAs targeting three

15

genes (sgMixture) had characteristic beige or brown adipocyte morphology, with a

16

number of small droplets (Figure 4C and D). In contrast, the cells transduced with only

17

sgPPARG_1-4 formed a single large droplet, similar to white adipocytes. Interestingly,

18

there were some beige or brown adipocyte-like cells in SAM MSCs transduced with

19

sgRNAs targeting only PRDM16 (Figure 4C). For characterization of induced

20

adipocytes, real-time RT PCR was carried out to measure the expression levels of beige

21

selective marker genes; TMEM26 and SHOX2, and brown selective marker genes;

22

OPLAH and SLC29A1. As shown in Figure 4E, the gene expression of beige selective

23

markers

24

sgPPARG_1-4-transduced cells. In contrast, that of brown selective markers was not

was

upregulated

in

sgMixture-transduced

cells

compared

to

9



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1

upregulated. These findings suggest that induced adipocytes with a number of small

2

droplets in this study are beige adipocytes rather than brown adipocytes. We have

3

therefore confirmed that the dCas9-based transcription system can simultaneously

4

activate multiple genes in MSCs. Furthermore, we found that it is possible to precisely

5

regulate the cell fate of MSCs between white and beige adipocyte-like cells, through

6

endogenous gene control.

7

In the present study, we established that adipogenic programming of MSCs can be

8

controlled through targeted activation of endogenous genes. A highly efficient

9

CRISPR-Cas9-based transcription activator, named SAM, was introduced into MSCs

10

via lentiviral vectors, and demonstrated robust activation of endogenous adipogenic

11

factors. Individual endogenous activation of both PPARG and CEBPA efficiently

12

induced white adipocyte-like cells from MSCs, which exhibited morphological and

13

molecular properties of adipocytes. Beige adipocyte-like cells were also induced via

14

multiplex activation of endogenous PPARG, CEBPB, and PRDM16, and differentiation

15

control between white and beige adipocyte-like cells was achieved by changing only the

16

sgRNA target sequences. The balance of cellular activities between white and beige

17

adipocytes is closely related to metabolic diseases such as obesity and diabetes. It is still

18

challenging to induce individual homogenous cell populations of white and beige

19

adipocytes using chemically modified medium. Therefore, the cells induced in the

20

present study may provide opportunities for the development of new therapeutic

21

strategies for managing these diseases.

22

In cell fate engineering using ectopic expression of transcriptional factors, the genes

23

targeted are often limited by their large size, which causes difficulty in cDNA cloning

24

and transfection into cells. Since an sgRNA expressing cassette is small and easily 10


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1

introduced into cells, any gene can be activated using dCas9-based transcriptional

2

factors. The sgRNA dependent transcriptional activation also provides temporal

3

regulation of targeted gene expression by control of sgRNA introduction time.

4

Furthermore, the greater part of endogenous gene has several isoforms and the

5

coordinated expressions of them are important for regulating diverse biological

6

phenomena including differentiation. dCas9-based endogenous gene activation enables

7

the induction of all promoter-based products in a similar way as a native transcriptional

8

process, whereas ectopic overexpression allows the induction of only one isoform of

9

interest. These features of dCas9-based transcriptional activation may help to reproduce

10

the complex gene regulatory networks in natural programming processes.

11

Lineage reprogramming has emerged as a promising approach for generating desired

12

cell types and how to obtain desirable cell types is one of the important questions in

13

regenerative medicine. A lot of works have shown that the ectopic expression of

14

multiple transcription factors promotes the differentiation of stem cells into multiple cell

15

types. However, there are difficulties in manipulating multiple gene expression and

16

identifying an optimal combination of transcription factors due to the complexity of the

17

cDNA cloning and ectopic gene introduction. In contrast, dCas9-based endogenous

18

gene activation is suitable for multiple gene activation. The SAM MSC platform shown

19

in this study should be useful for identification of an optimal combination of

20

transcription factors to convert MSCs to various cells such as osteocytes, chondrocytes,

21

myocytes, neurons, and other cells.

22 23 24

Conclusion In conclusion, we demonstrate dCas9-based transcriptional activation in MSCs, 11



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Page 12 of 30

1

which enabled cell-fate engineering of MSCs with reduced exogenous additives. Since

2

endogenous gene activation can induce their differentiation in the native context, this

3

approach improves the usefulness of MSCs as a cell source for clinical applications.

4

Furthermore, it has wide applicability for modulating the cell fate of MSCs into various

5

lineages, and may be useful to determine how MSCs develop into several lineages in

6

vivo.

7 8 9

Methods

10

Cell Culture

11

Cells were cultured at 37 °C under 5% CO2 in all experiment. UE7T-13 (JCRB) cells

12

were used as MSCs in the present study, and were cultured in Dulbecco's modified

13

Eagle medium (Wako Pure Chemical Industries) supplemented with 10% (v/v) fetal

14

bovine serum (FBS, Biosera) and 1% (v/v) Penicillin-Streptomycin-Neomycin (PSN)

15

antibiotic mixture (Thermo Fisher Scientific). HEK293T/17 cells (ATCC) and Lenti-X™

16

293T cells (TAKARA BIO) were cultured in Dulbecco’s Modified Eagle Medium

17

(DMEM,

18

Penicillin-Streptomycin (Thermo Fisher Scientific Inc.).

Sigma-Aldrich)

supplemented

with

10%

FBS

(Hyclone),

1%

19 20

Establishment of stable cell lines

21

To establish stably expressing MSC cell-lines, UE7T-13 cells were plated at

22

approximately 1.0 × 105 cells/dish in 100 mm dishes, and were transduced with the

23

indicated lentiviral vectors with 0.8 µg/ml hexadimethrine bromide. Forty-eight hours

24

after transduction, the culture medium was changed and antibiotics were added. The 12


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1

subsequent duration of antibiotic selection was at least 2 weeks. For antibiotic selection

2

in UE7T-13 cells, antibiotics were used at the following concentrations: blasticidin S

3

(Wako Pure Chemical Industries) 10 µg/ml, hygromycin B (Wako Pure Chemical

4

Industries) 1000 µg/ml, and puromycin 0.5 µg/ml (Wako Pure Chemical Industries).

5 6

Reporter gene activation

7

EGFP reporter SAM MSCs were plated at approximately 2.0 × 103 cells/well in

8

96-well plates and cultured for 24 h. The cells were then transduced with lentiviral

9

vector encoding the indicated sgRNA at the indicated multiplicity of infection (MOI)

10

with 0.8 µg/ml hexadimethrine bromide. The culture medium was changed 48 h after

11

transduction. After incubation for 48 h, samples were washed twice with PBS, stained

12

with 10 µg/ml Hoechst 33258 (Promocell), and observed using a fluorescent microscope

13

(Olympus). For quantification, fluorescent intensities of EGFP were measured using

14

ImageJ, normalized to Hoechst 33258, and then standardized to that of the

15

mock-infected samples.

16 17

Endogenous gene activation

18

SAM MSCs were plated at approximately 2.0 × 103 cells/well in 96-well plates and

19

cultured for 24 h. The cells were then transduced with lentiviral vector encoding the

20

indicated sgRNA with 0.8 µg/ml hexadimethrine bromide. The total MOI was 20. For

21

simultaneous activation of three genes, the ratio of sgRNAs targeting each gene was

22

1:1:1. The culture medium was changed 48 h after transduction. After incubation for 48

23

h, total RNA samples were extracted for quantitative PCR analysis using RNeasy Mini

24

Kit (Qiagen). 13



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Page 14 of 30

1 2

Real-time reverse transcription PCR

3

Reverse transcription reaction was performed using the ReverTra Ace qPCR RT

4

Master Mix with gDNA Remover (Toyobo). Real-time RT PCR was performed using

5

THUNDERBIRD SYBR qPCR Mix (Toyobo) with StepOnePlusTM (Thermo Fisher

6

Scientific). Additional experimental procedures can be found in the Supplemental

7

Experimental Procedures.

8 9

Adipogenic induction using gene activation

10

SAM MSCs were plated at approximately 2.0 × 103 cells/well in 96-well plates and

11

cultured for 24 h at 37 °C in 5% CO2. sgRNA transduction was performed in the same

12

way as Endogenous gene activation. Forty-eight hours after transduction, the medium

13

was changed to adipocyte maintenance medium (DMEM supplemented with 10% (v/v)

14

FBS, 1% (v/v) PSN, 1 µM dexamethasone (Wako Pure Chemical Industries) and 10

15

µg/ml insulin (Wako Pure Chemical Industries)) for two or three weeks for white or

16

beige adipogenesis, respectively. The medium was changed every four or seven days for

17

white adipogenesis or beige adipogenesis, respectively.

18 19

Cell staining

20

For an osteogenic differentiation assay, 4% PFA fixed cells were stained with Alizarin

21

red S (Wako Pure Chemical Industries). For adipogenic differentiation assays, 4% PFA

22

fixed cells were stained with Oil red O (Wako Pure Chemical Industries) or Nile Red

23

(Wako Pure Chemical Industries). Additional experimental procedures can be found in

24

the Supplemental Experimental Procedures. 14


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1 2

Quantification and Statistical Analysis

3

All measurements are presented as mean ± standard error (SE). Statistical

4

significance was evaluated using independent samples Student’s t-test. Differences

5

between groups were defined as statistically significant when *p < 0.05, or **p < 0.01.

6

Sample sizes are indicated in the Figure legends. Statistical details are described in

7

Method Details and Figure legends.

8 9

15



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Page 16 of 30

1

Author contributions

2

Y. F., Y. N., M. S., and K. Y. conceived the project. Y. F., Y. N., M. S., and K. Y

3

designed experiments. Y. F. and Y. N. performed experiments. Y. F. analyzed data. Y. F.

4

and K. Y. wrote the manuscript. All authors edited the manuscript.

5 6 7

Acknowledgements

8

This work was partially supported by the JSPS KAKENHI [grant numbers 15K13721],

9

JST-PRESTO [grant number JPMJPR16FB], JST-CREST [grant number JPMJCR1653],

10

JST-START, and JSPS Research Fellowships for Young Scientists [grant number

11

15J08446 and 15J05897].

12 13 14

Conflict of Interest

15

The authors declare no competing financial interest.

16 17 18

Abbreviations

19

Mesenchymal stem cells (MSCs), Clustered regularly interspaced short palindromic

20

repeats (CRISPR)-Cas9 (CRISPR-associated protein 9), nuclease-dead Cas9 (dCas9),

21

single chimeric guide RNA (sgRNAs), synergistic activation mediator (SAM), dCas9

22

protein fused with VP64 (dCas9-VP64), sgRNA bearing two MS2-binding stemloops

23

(sgRNA 2.0), MS2 coat protein fused with two distinct transcriptional activators: p65

24

and heat shock factor 1 (MS2-p65-HSF1), MOI (multiplicity of infection), proliferator 16


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

activated receptor gamma (PPARG), CCAAT/enhancer binding protein alpha (CEBPA),

2

kruppel like factor 5 (KLF5), adiponectin (ADIPOQ), fatty acid binding protein 4

3

(FABP4), fatty acid synthase (FASN), lipase E, hormone sensitive type (LIPE),

4

CCAAT/enhancer binding protein beta (CEBPB), PR/SET domain 16 (PRDM16),

5

transmembrane protein 26 (TMEM26), short stature homeobox 2 (SHOX2),

6

5-oxoprolinase (ATP-hydrolysing) (OPLAH), solute carrier family 29 member 1

7

(augustine blood group) (SLC29A1), and glyceraldehyde-3-phosphate dehydrogenase

8

(GAPDH)

9 10 11

Supporting Information

12

Supporting Information includes supporting methods, four figures and four tables and

13

can be found with this article online at the publisher’s website.

14

Supporting Methods

15

Figure S1. The lentiviral constructs utilized in this study. Related to Figures 1-4.

16

Figure S2. The relationship between fluorescence intensity and MOI of sgRNA 2.0

17

lentivirus. Related to Figure 1.

18

Figure S3. Maps showing the sgRNA targeting sites. Related to Figures 1-4.

19

Figure S4. Off-target analysis. Related to Figures 2-4.

20

Table S1. Target sequences of sgRNAs. Related to Figures 1-4.

21

Table S2. Primer sequences used in the analysis of mRNA expression by real-time RT

22

PCR. Related to Figures 2-4.

23

Table S3. Medium compositions used in this study. Related to Figures 2-4.

24

Table S4. The sequences of off-target binding sites. Related to Figures 2-4. 17



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Page 18 of 30

Supporting References

18


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Figure Legends

2 3

Fig. 1 Reporter Gene Activation by CRISPR-Cas9-based Transcription System in

4

MSCs.

5

(A) Schematic diagram of the EGFP reporter. (B) Schematic diagram of the

6

experimental

7

CRISPR-Cas9-based transcription system in MSCs. (C) Exogenous EGFP activation in

8

MSCs. MSCs were transduced with indicated sgRNA 2.0 lentivirus at an MOI of 20.

9

Four days post transduction, samples were stained with Hoechst 33258. Scale bar

10

procedure

used

for

reporter

gene

activation

assay

by

a

represents 100 µm. See also Figure S2.

11 12

Fig. 2 Multipotency and Endogenous Gene Activation of SAM MSCs.

13

(A) Alizarin red S staining of osteogenic-induced MSCs. Wild-type and SAM MSCs

14

were cultured using the osteogenic induction medium. Scale bar represents 50 µm. (B)

15

Oil red O staining of adipogenic-induced MSCs. Wild-type and SAM MSCs were

16

cultured using the adipogenic induction medium. Scale bar represents 50 µm. (C-E)

17

Endogenous gene activation of (C) PPARG, (D) CEBPA, and (E) KLF5 in SAM MSCs

18

with targeted activation. SAM MSCs were transduced with indicated sgRNA 2.0

19

lentivirus at an MOI of 20. mRNA levels were measured four days post transduction.

20

The mRNA expression levels were normalized by GAPDH and then standardized to that

21

in the sample of sgControl. Values shown are the mean ± SE of n = 3. *p < 0.05, **p

Control of Adipogenic Differentiation in Mesenchymal Stem Cells via

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