Methods in Molecular Biology DOI 10.1007/7651_2015_257 © Springer Science+Business Media New York 2015

Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells Emi Shoji, Knut Woltjen, and Hidetoshi Sakurai Abstract Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70–90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening. Keywords: Skeletal muscle cell, Human iPS cells, Myogenic differentiation, MyoD, piggyBac vector, GFP, Doxycycline inducible differentiation

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Introduction Most muscular diseases do not have curative treatment and are limited to palliative care that fails to prevent progression. Additionally, for many myopathies, the pathology remains unclear, although the causative genes have been identified. Thus, there is a requirement to investigate the precise pathology of each myopathy. Human induced pluripotent stem cell (hiPSC) technology has enabled patient-specific models of human disease in vitro [1, 2], since hiPSCs offer indefinite proliferation and multilineage differentiation capacity [3]. Establishing methodology for versatile and practical myogenic differentiation is of particular importance for skeletal muscle cell research, and the identification of therapeutic approaches to overcome inherited muscle diseases. Methods for the differentiation of hiPSCs to skeletal muscle cells have taken several

Emi Shoji et al.

approaches, lending from our knowledge of in vivo development [4–6]. PAX7, a paired box transcription factor that maintains the adult satellite cell [7], and MYOD1, a master transcription factor of skeletal muscle cell differentiation and essential gene in skeletal muscle cell lineage and muscle stem cell function in adult skeletal muscle [8], have both been conditionally expressed in human ES and iPS to induce directed differentiation and obtain large quantities of myogenic precursors [9]. Direct conversion of fibroblasts to muscle cells by MYOD1 was originally demonstrated in 1987 [10]. Reports of skeletal muscle cell differentiation from hiPSC-derived fibroblasts [11] or hiPSCderived mesodermal cells [12] demonstrate the superior potential of MYOD1 for myogenic differentiation. Yet, a universal method effective at delivering MyoD to various hiPSC lines was only recently reported [13]. A piggyBac (PB) transposon vector [14] carrying doxycycline (dox)-inducible MYOD1 (Fig. 1) can be delivered directly into hiPSCs (Tet-MyoD-hiPSCs), excluding the redundant derivation of fibroblasts or mesoderm prior to myogenic differentiation. Moreover, optimized 2D culture conditions promote consistent differentiation through uniform contact of single cells with media and growth factors. Using this approach, mature myocyte differentiation from Tet-MyoD-hiPSCs can be achieved within 2 weeks, and with high efficiency [13]. Myocytes obtained from Tet-MyoD-hiPSCs express skeletal muscle-specific proteins such as Dystrophin, which is only detected in mature skeletal muscle cells. Functionality and maturity of differentiated myocytes are indicated by cell fusion and cell twitching in response to electric stimulation. Moreover, through the reprogramming process from the patient’s fibroblasts, intact matured myocytes without being affected from external factors, such as inflammation or any other signaling molecules, can be produced from Tet-MyoD-hiPSCs. Our PB-delivered MYOD1 approach has successfully induced myocytes differentiation from Miyoshi myopathy patient-derived hiPSCs [13], and is proving invaluable in the derivation of disease models from various myopathic patient hiPSCs (Sakurai, unpublished data). In each case, the delivery of dox-inducible MYOD1 by the PB transposon offered stable and efficient skeletal muscle cell differentiation. Herein, we describe the steps required to derive Tet-MyoD-hiPSCs: from PB transfection, through MYOD1 induction and culture adaptation, and finally the evaluation of differentiated myocytes. This straightforward and tractable technology has great prospects in muscle cell research towards development of new drugs for muscular diseases.

Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells

PB ITR

tetO2

MyoD

IRES

GFP

pA

PB200-MyoD PB ITR

Neo

IRES

rtTA

rEF1α

Fig. 1 The dox-inducible MYOD1-expressing piggyBac vector, PB200-MyoD, derived from PB-TAG-ERN

2

Materials

2.1 Human iPS Cell Culture

1. 201B7 hiPSCs, established from human dermal fibroblasts by retroviral overexpression of the four Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) as previously described [3]. Following the suggested optimization steps, this differentiation protocol should be applicable to all hiPSC lines. 2. Primate ES cell medium (ReproCELL, RCHEMD001) supplemented with 10 ng/mL recombinant human basic fibroblast growth factor (bFGF: Wako, 064-04541). 3. Feeder cells inactivated with mitomycin-C. A feeder line such as SNL (a subclone of the STO mouse fibroblast cell line) that is resistant to neomycin is required to survive drug selection.

2.2 PB200-MyoD Transfection with FuGENE® HD to hiPSCs

1. Plasmids: PB200-MyoD (Fig. 1) and pCAG-PBase (piggyBac transposase). The PB200-MyoD vector is available by MTA upon request to the authors. The parental PB Gateway® Destination vector PB-TAG-ERN (RDB13244) and pCAG-PBase (RDB13241) [15] are available through the RIKEN BRC DNA Bank (dna.brc.riken.jp). 2. FuGENE® HD (Roche, 4709705). 3. OPTI-MEM (GIBCO, 31985). 4. CTK solution: To prepare 50 mL of CTK solution, mix 5 mL of 2.5 % Trypsin (Life technologies, 15090-046), 5 mL of 1 mg/mL collagenase-IV (Life technologies, 17104-019), 500 μL of filtered (0.2 μm) 0.1 M CaCl2, and 10 mL of Knockout Serum Replacement (KSR: Life technologies, 10828-028) in 30 mL of sterile deionized water. 5. Neomycin sulfate (Neo: Nacalai Tesque, 146-08871). 6. 5 mL Polystyrene FACS tubes (BD Falcon™, 352058).

2.3 Differentiation to Skeletal Muscle Cells

1. Primate ES cell medium (ReproCELL, RCHEMD001). 2. 5 % KSR/αMEM: To prepare 50 mL of 5 % KSR/αMEM, add 2.5 mL of KSR and 100 μL of 100 mM 2-Mercaptoethanol (2-ME: Nacalai Tesque, 2143882) to 47.5 mL of penicillin/streptomycin supplemented αMEM medium.

Emi Shoji et al.

3. 2 % horse serum/DMEM: Prepare 50 mL of 2 % horse serum/ DMEM medium by adding 1 mL of horse serum (Sigma, H1138) and 100 mM 2-ME to DMEM basal media. Prepare DMEM basal media by adding 1 % of L-Glutamine and 2.5 mL of penicillin/streptomycin (Nacalai Tesque, 2625384) to 500 mL of DMEM high glucose (GIBCO, 11960069). To complete 2 % horse serum media, add recombinant Human IGF-I (Peprotech, 100-11) before medium change. 4. Matrigel- (Corning, 356231) or collagen-I-coated 6-well plate (AGC, 4810-010). 5. Neomycin sulfate (Neo: Nacalai Tesque, 146-08871). 6. Doxycycline hyclate (Dox: LKT Labs, D5897). 7. Y-27632, ROCK inhibitor (Nacalai Tesque, 08945-84). 8. 0.25 % Trypsin/1 mM EDTA (Nacalai Tesque, 3555464). 9. CTK Solution.

3

Methods

3.1 Transfection of PB200-MyoD into hiPSCs

Transfection of PB200-MyoD into hiPSCs may be carried out by either chemical transfection or electroporation. Here, we describe PB200-MyoD transfection using FuGENE® HD transfection reagent (Roche) and outline a simple approach to optimize hiPSC transfection conditions. For further details, refer to the manufacturer’s protocol for FuGENE® HD. For a detailed protocol on PB electroporation into hiPSCs, refer to Kim et al. [15]. 1. Prepare hiPSCs at approximately 60 % confluency on a 6-well plate with inactivated SNL feeder cells, in primate ES cell medium supplemented with 4 ng/mL bFGF. 2. Change media to primate ES cell medium supplemented with 10 ng/mL of bFGF just before the transfection. 3. Prepare a master mix for 7 wells by mixing 700 μL of OPTIMEM and 7.0 μg each of PB200-MyoD and pCAG-PBase plasmids. Mix the solution thoroughly by pipetting and dispense 100 μL aliquots (total 2.0 μg of plasmid DNA) into each tube. 4. Prepare a concentration series of FuGENE® HD (ranging from 3.0 to 8.0 μL) to each tube (Fig. 2). Optimize transfection parameters by adjusting the concentration of transfection reagent and amount of plasmids for each hiPSC line. For most hiPSCs, a 1:3 ratio is optimum (see Note 1). 5. Mix each tube by vortexing for 1–2 s. 6. Incubate the tubes for 15 min at room temperature.

Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells Dispense 100 µL each

Vol. FuGENE® HD OPTI-MEM: 700 µL Plasmids: 7.0 µg each

15 min incubation

1:1.5

1:2

1:2.5

1:3

1:3.5

1:4

Ratio DNA:FuGENE® HD

3.0 4.0 5.0 6.0 7.0 8.0

Fig. 2 Schematic outline for PB200-MyoD transfection. A concentration range is used to test optimal DNA: FuGENE® HD ratios for each hiPSC line

7. Add each mixture to the indicated well, dropwise (Fig. 2). Swirl to mix. 8. On the following day (~24 h), change media to 10 ng/mL bFGF added primate ES cell medium. 9. Two days after transfection, change media to primate ES cell medium supplemented with 100 μg/mL Neo and bFGF (see Note 2). 10. Maintain Neo selective pressure until colonies emerge (~4–8 days, depending on the hiPSC line and transfection efficiency). 11. At this stage, the cells can be passaged as a population or picked as individual clones. 12. Prepare a dish to test the induction level of MyoD indirectly by GFP in FACS, and directly by MyoD Western blot. 13. Prepare frozen stocks of Tet-MyoD-hiPSCs with appropriate expression. 14. Keep the passage number low, and maintain the cells in Neo until differentiation. 3.2 Skeletal Muscle Cell Differentiation

hiPSCs form tightly packed colonies demonstrating one of the characteristic features of pluripotent stem cells (Fig. 3a). In the absence of dox, Tet-MyoD-hiPSCs that emerged after at least 4 days of Neo drug selection still retain morphological and molecular properties of pluripotency (Fig. 3b, and data not shown). These cells are maintained in a pluripotent state until the addition of dox, which initiates uniform myogenic differentiation. The dox-induced skeletal muscle cell differentiation consists of three phases: induction, differentiation, and maturation (Fig. 4). Skeletal muscle cell maturation is promoted in 2 % horse serum medium [16]. Administration of IGF-I also boosts maturation and enlarges the size of myocytes [17, 18]. Cell density is a critical parameter for efficient differentiation and should be tested empirically using the parameters suggested (see Note 3).

Emi Shoji et al.

Fig. 3 Morphology of pluripotent parental and Tet-MyoD-hiPSCs. (a) hiPSCs generated by four-transcription factor reprogramming form colonies with characteristic morphology. (b) In the absence of dox, Tet-MyoDhiPSCs retain a similar morphology. Scale bars: 200 μm DOX addition Induction

Differentiation

Maturation

Primate ES media

5 % KSR/aMEM 2ME

2 % Horse serum/DMEM 2ME, IGF-I

0 1 2 Days of differentiation

7

14

Fig. 4 Overview of the mature myocyte differentiation procedure. Dox addition initiates myocyte differentiation by overexpressing MYOD1. Differentiation process consists of induction, differentiation, and maturation phases

Dox-treated Tet-MyoD-hiPSCs take on a rounded cell structure and then begin to change to spindle-like structure that is characteristic of skeletal muscle cells (Fig. 5a). Furthermore, successfully differentiated myocytes will undergo cell fusion to form multinucleated myocytes (Fig. 5a, b). Myosin heavy chain (MHC), skeletal muscle actin (SMA), and creatine kinase, muscle type (CKM) are skeletal muscle-specific markers and can be detected in differentiated myocytes at day 9 of differentiation (Fig. 5b), and are used to gauge the efficiency of directed differentiation. 1. Prepare matrigel- or collagen-I-coated 6-well plates. For matrigel coating, dilute matrigel 1:50 in primate ES cell media and apply to the plate at least 2 h prior to starting the differentiation process. 2. To eliminate SNL feeder cells, wash plated cells with PBS and add 1 mL of CTK solution (for 100 mm dish) to the plated cells. Incubate at room temperature for 2–3 min and dislodge feeder cells by washing twice with PBS.

Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells

Fig. 5 Morphological changes through the differentiation process. (a) Rounded cells are observed at day 2 of differentiation and start to form spindle-like structures from day 4 of differentiation. By day 9 of differentiation, myocytes begin to fuse to form multinucleated skeletal muscle cells. Networks of myocytes are observed at day 14 that contract in response to electric stimulation. (b) Immunohistochemistry at day 9 of differentiation also confirms the maturation stage of differentiated myocytes. MHC, SKM, and CKM are skeletal musclespecific markers. Scale bars: 50 μm

3. Detach and dissociate Tet-MyoD-hiPS colonies by adding 1 mL of 0.25 % trypsin and incubating the plate at 37  C for 5 min. 4. Add at least an equal volume of primate ES cell media to neutralize the trypsin and dissociate the culture to a singlecell suspension by pipetting thoroughly. 5. Centrifuge the cell suspension at 280  g for 3 min and aspirate the supernatant. 6. Resuspend Tet-MyoD-hiPSCs with primate ES cell medium supplemented with 10 μM of ROCK inhibitor. 7. Plate 1  105–1  106 cells to prepared matrigel- or collagen-I-coated 6-well plates. For each new hiPSC line and PB200-MyoD transgenic population or clone, carry out a pilot experiment to determine the optimal cell plating density for skeletal muscle cell differentiation (see Note 3).

Emi Shoji et al.

8. Medium for differentiation is adjusted as follows (Fig. 4): l

Differentiation day 1 Primate ES cell medium supplemented with 1 μg/mL of dox.

l

Differentiation day 2–6 5 % KSR/αMEM supplemented with 1 μg/mL of dox.

l

Differentiation day 7–14 2 % horse serum/DMEM supplemented with 1 μg/mL of dox and 10 ng/mL of IGF-1.

9. Observe the cells. As shown in Fig. 5a, cells begin to change morphology after differentiation is initiated with dox. Rounded cell structure gradually changes to spindle-like structure from day 2 to day 7 of differentiation. Cell fusion to form multinucleated myocytes can be noted from day 9 of induction. 10. Evaluate the cells (see Note 4). Differentiated myocytes can be evaluated by immunohistochemistry as shown in Fig. 5b. It is important to confirm with skeletal muscle actin (SMA) antibody that obtained myocytes are actually skeletal myocytes. Yield can be calculated by counting the number of myosin heavy chain (MHC) positive cells per number of DAPI. Refer to Table 1 for primary and secondary antibody details. Further functional evaluation can be done by Western blotting or electric stimulation analysis (see Note 5). 11. Application. Disease modeling using Tet-MyoD-hiPSCderived skeletal muscle cells will require custom assays appropriate to the disease in question. Moreover, in vitro recapitulation of disease pathology may require the application of suitable stresses to initiate disease pathogenesis. For example, in modeling Miyoshi myopathy (MM), we induced membrane injury by laser-irradiation of myocytes differentiated from Table 1 Antibody list First antibody

Source

Clonality

Dilution

Company

MHC

Mouse

Monoclonal

1/200

R&D

Skeletal muscle actin

Mouse IgM

Monoclonal

1/200

Acris

CKM

Rabbit

Polyclonal

1/100

Bioworld Technology

Second antibody

Dilution

Company

Alexa Fluor 488 conjugated goat-anti-mouse IgG

1/500

Invitrogen

Alexa Fluor 488 conjugated goat-anti-rabbit IgG

1/500

Invitrogen

Alexa Fluor 488 conjugated goat-anti-mouse IgM

1/500

Invitrogen

Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells

MM-patient derived hiPSCs [13]. We analyzed the latency time of membrane resealing by FM1-43 dye influx in order to assess the function of membrane repair. Alternatively, methods for inducing stress include electric stimulation, stretching, and chemical treatment.

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Notes 1. If low cell viability is noted following transfection: (a) Refine the purity of plasmid DNA (endotoxin-free). (b) Carry out a preliminary experiment to determine the optimal ratio of transfection reagent to plasmid DNA. (c) Reduce the time for incubation of cells with transfection complexes from 24 h to 4, 8, or 12 h. (d) Transfection efficiencies can be confirmed by GFP expression within 12 h of dox addition. Perform the assay in a replica plate, as cells exposed to dox will express MyoD and may begin to differentiate. 2. How to maintain a pluripotent culture of Tet-MyoD-hiPSCs. Passage cells or subclone to obtain homogenous cell population. Adjusting concentration of Neo may be helpful. 3. Skeletal muscle cell differentiation. (a) If a low efficiency of differentiation is observed. Addition of Neo while maintaining Tet-MyoD-hiPSCs can help to prevent silencing of randomly integrated PB vectors. Determine the appropriate cell plating density for differentiation, as high cell confluency can negatively affect differentiation efficiencies. Subcloning is required to select the appropriate clone that has stable differentiation ability. Optimize the dox concentration appropriate for MyoD induction. (b) Undifferentiated cells remain throughout the differentiation process. Each cell line has different cell plating density for skeletal muscle cell differentiation. It is strongly recommended to carry out a preliminary experiment to determine the appropriate cell plating numbers for each cell line. Ensure that a pure population of Neo-resistant cells is used. Optimize the dox concentration appropriate for MyoD induction.

Emi Shoji et al.

(c) Increased number of dead cells after dox addition. Confirm plating densities and optimize the dox concentration for appropriate MyoD induction. (d) Differentiated cells can be cultured for up to 2 months. However, differentiated cells cannot undergo passage nor freeze/thaw processes. 4. How to evaluate yield and efficiency. (a) Transduction efficiency of MYOD1 in Tet-MyoD-hiPSCs can be evaluated based on GFP expression 1 day after the dox induction. With piggyBac vector, transduction efficiency is expected to be higher than 90 %. (b) Immunohistochemistry will be the most applicable assay to evaluate yield and efficiency of differentiated myocytes. The number of MHC positive cells per DAPI stained nucleus indicates efficiency, mostly ranging 70–90 %. Since cultured cells are in a monolayer, yield can be calculated by directly counting myocytes on the plate. 5. Evaluating functionality of differentiated myocytes. (a) Differentiated myocytes can twitch in response to electric stimulation [13]. (b) It is possible to detect the calcium influx according to the stimulation using Fluo-8, a calcium indicator.

Acknowledgements This work was supported in part by the FIRST Program, Scientific Research Grant No. 22790284 (to H.S.) from JSPS, and a grant from the Leading Project of MEXT (to H.S.). References 1. Kondo T, Asai M, Tsukita K, Kutoku Y, Ohsawa Y, Sunada Y, Imamura K, Egawa N, Yahata N, Okita K, Takahashi K, Asaka I, Aoi T, Watanabe A, Watanabe K, Kadoya C, Nakano R, Watanabe D, Maruyama K, Hori O, Hibino S, Choshi T, Nakahata T, Hioki H, Kaneko T, Naitoh M, Yoshikawa K, Yamawaki S, Suzuki S, Hata R, Ueno S, Seki T, Kobayashi K, Toda T, Murakami K, Irie K, Klein WL, Mori H, Asada T, Takahashi R, Iwata N, Yamanaka S, Inoue H (2013) Modeling Alzheimer’s disease with iPSCs reveals stress phenotypes associated with intracellular Abeta and differential drug responsiveness. Cell Stem Cell 12(4):487–496. doi:10.1016/j.stem. 2013.01.009

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