Simple derivation of skeletal muscle from human pluripotent stem cells using temperature‐sensitive Sendai virus vector

Abstract Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature‐sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV‐Myod1), a myogenic master transcription factor. SeV‐Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre‐like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell‐targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human‐induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future.

(human iPSCs), into SkM cells have been established, and ESCoriginated SkM cells have become a potential tool to investigate the mechanisms of development and disease in SkM (Chal and Pourquiél, 2017) 2 . To differentiate human ESCs or iPSCs into SkM cells, there are two major approaches: directed differentiation and direct conversion. The directed-differentiation approach recapitulates physiological steps in human embryonic development by sequentially adding small molecules or cytokines during weeks of cultivating duration, and it is appropriate for research of the development of infantile-onset disorders (Chal and Pourquiél, 2017). Conversely, the direct-conversion approach is achieved by the overexpression of myogenic transcriptional factors (TFs) such as myoblast determination protein 1 (MyoD1), paired box gene 3 (PAX3) and paired box gene 7 (PAX7) (Chal and Pourquiél, 2017). The first idea of the direct-conversion system was demonstrated by converting fibroblasts to a muscle fate via increased MyoD1, 3 whose locus is a target of a demethylating agent (5-azacytidine). 4 The drug-inducible expression system, which induces the high expression of MyoD1, offers rapid and robust differentiation methods and opens the way to model SkM-relevant disorders such as Duchenne or Miyoshi muscular dystrophies. [5][6][7][8] To deliver myogenic TFs into human pluripotent stem cells for direct conversion, most of the studies described above employed viral or transposon vectors, which cannot avoid the risk of random integration into the host genome.
In this study, we utilized Sendai virus (SeV) vector to deliver Myod1 into human ESCs and iPSCs for direct conversion. SeV is classified as a non-segmented negative-strand RNA virus belonging to the Paramyxoviridae family. SeV is a cytoplasmic virus, 9 and it has been developed as a gene-delivery tool without genomic integration. Induction of TFs via SeV vector has been demonstrated to convert somatic cells into human iPSCs 10,11 or convert human iPSCs into functional motor neurons. 12 Therefore, we selected temperature sensitive SeV vector for the Myod1-delivery vector without risks of genomic integration. Here, we transfected temperature sensitive SeV vector, which encodes Myod1 (SeV-Myod1) and converts human ESCs and iPSCs into SkM cells. Additionally, cultivation at a temperature higher than 38℃ can eliminate SeV vector from cytosol 13 and enhance the differentiation status unexpectedly. Finally, we eliminated the residual undifferentiated human ESCs and iPSCs by pluripotent stem cell-targeting delivery of cytotoxic compound, and we established a myogenic differentiation method system by using SeV vector encoding Myod1 as a future platform for disease modelling and drug discovery.

| Assay for cell damage and cell survival
We

| Statistical analysis
Immunofluorescence

| Optimized infection of SeV-Myod1 converted human ESCs into SkM cells
Temperature-sensitive F-deficient SeV-Myod1 was constructed

| Removal of SeV-Myod1 after cultivation at high temperature
The SeV vector used in this study has temperature-sensitive mutations, meaning that vectors could be removed after incubation at more than 38°C. 11 To maximize the efficiency of myogenic differentiation by exogenous Myod1, a transient expression was reported to be beneficial. 16 Therefore, we planned to remove SeV-Myod1 by  Figure 2D, E). These results showed that the SeV-Myod1 vector can be eliminated by exposure to high temperature.

| Cultivation at a higher temperature of 38℃ boosted myogenic differentiation
We next examined the myogenic differentiation efficiency after exposure to different temperatures ( Figure 3A). The population of MHC-positive cells was 18.7% at the condition of 37℃ and 20.1% at 38℃ ( Figure 3B). However, the positivity of MHC was significantly decreased to 11.5% at 39℃ and 4.2% at 40℃ after exposure to higher temperatures ( Figure 3A, B). When considering the cell shape on day 8, muscle progenitor cells at the condition of 38℃ had a spindle-like shape like muscle fibre ( Figure 3A) and a larger size in MHC-positive cells ( Figure 3C) compared with the other conditions. In addition, muscle progenitor cells after incubation at 38℃ showed a tendency of higher expression in muscle markers, including MHC ( Figure 3D).

| Non-differentiating pluripotent-state cells were eliminated with pluripotent cell-specific killer compound rBC2LCN-PE38
We converted human ESCs into SkM cells by simply adding SeV-Myod1 and a heat exposure of 38℃. However, the differentiation efficiency remained around 20% due to the residual human ESCs, which had not

| Application of the established method to human iPSC
We applied the established method to human iPSCs, which is a powerful tool for disease modelling and further drug investigations. We utilized three different iPSC clones that originated from healthy individuals. 15 As results similar to those with human ESCs, the infection by SeV-Myod1 followed by the 38℃ condition and rBC2LCN-PE38 treatment successfully converted all three human iPSCs into SkM cells with a similar efficiency to the case of human ESCs ( Figure 5A-C). These results showed that the established differentiation method for SkM cells can be applied to human iPSCs.

| Differentiated SkM cells responded to electrical stimulation
To confirm the functions of the differentiated SkM cells from human ESCs and iPSCs, we used electrical stimulation on human ESC-and F I G U R E 3 Transient heat shock at temperature of 38ºC accelerated myogenic differentiation. (A) Cells were infected twice with SeV-Myod1 at MOI 64 and then incubated at 37ºC, 38ºC, 39ºC or 40ºC from day 3 to day 8, as shown in the schema in Figure 2A.
Myogenic differentiation efficiency was analysed by immunostaining for myosin heavy chain (MHC, green). Nuclei were stained with DAPI (white) to count the total cell number. Scale bar = 100 μm. To construct the assay system, we used the calcium transients that were reported to coincide with twitch and tetanus responses, and that the alteration in the intensity of the calcium indicator, quantified as ΔF/F, was proportional to the magnitude of the applied force. 22 We monitored the time-dependent alteration in fluorescent intensity of the calcium indicator, described as ΔF/F, after adding electric field stimulation (EFS). At first, we used single stimulation, 4-msec pulse width of 10 V, 3 Hz and found the active Ca 2+ influx at the same timing as electrical stimulation ( Figure 6A). Next, we also used high-frequency stimulation, a 4-msec pulse width of 10 -20 V, 2-50 Hz, and found that the high-frequency stimulation could evoke a drastically higher ΔF/F ( Figure 6B), which is widely observed in the tetanic contraction of primary SkM cells or in in vivo analysis. From these results, we also discussed the importance of functional assays and future perspectives of co-culture with motor neurons from human iPSCs to further investigate physiological assays.

| DISCUSS ION
In this study, we established a simple and efficient method to generate SkM cells from human ESCs and iPSCs using SeV encoding Myod1.
This method enables us to obtain a high yield of SkM cells without complicated cell culture processes in directed-differentiation methods using step-wise addition of various cytokines or small compounds, nor with direct-conversion methods that reacquire cell isolation steps and/or sub-cloning steps of human iPSCs. 23 Exogenous Myod1 gene induction is achieved by simply adding SeV-Myod1 to the culture medium, and no other genetic manipulation is required ( Figure 1A). SeV vector has advantages over other viral vectors because SeV vector consists of cytoplasmic RNA, and they express their genes within the cytoplasm without entering the nucleus. Therefore, the SeV vector has no risk of integration into the host genome, thereby providing a potential therapeutic application. In addition, the SeV vector can be applied to both dividing and non-dividing cells, and short-term exposure is enough for efficient transduction. Moreover, SeV vector has been modified to be rapidly eliminated to minimize its effect on the host cells. 9,24 The established direct-conversion method using integration-free SeV vector can be applied for pathological analysis and drug screening assays, which require the use of many different human iPSC lines derived from different patients.
One advantage of our method is the elimination of residual SeV by transient exposure to the cultivation at a treatment temperature of 38℃, as the SeV vector in this study carries a temperature-sensitive mutation. 11 Interestingly, treatment at 38℃ increased both the differentiation efficiency and the number of cells with spindle-like elongated muscle cell morphology.
We speculate that this could be due to increased expressions of specific heat shock proteins. A previous study reported that heat shock proteins (HSPs), including MKBP/HSPB2 and HSPB3, are induced during muscle differentiation under the control of Myod1, suggesting that these HSP oligomers might have an additional system closely related to muscle functions. 25 Others showed that the levels of HSPs, namely HSP25, HSP40, HSP90 and HSP110, were highly elevated in 50% confluent proliferating myoblasts. 26,27 It has also been shown that HSPs play a crucial role in myogenesis. McArdle et al. showed that HSP70 overexpression facilitates muscle regeneration. 28 Barone

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.