ioCells ioSkeletal Myocytes

Human iPSC-derived skeletal myocytes

io1002 |  Formerly known as ioMYOCYTES/skeletal, cat no: ea1200

Introducing highly-defined, consistent and reliable human muscle cells for research, disease modelling and high throughput screening across areas such as muscle, neuromuscular, and associated metabolic disorders. ioSkeletal Myocytes, part of our ioCells portofolio, have been reprogrammed from human induced pluripotent stem cells (iPSCs) using our precise reprogramming technology: opti-ox™¹.

The technology allows for human pluripotent stem cells, within days, to convert into skeletal myocytes, providing a high-quality, easy to use, cellular model.

Skeletal myocytes demonstrate robust expression of components of the contractile apparatus and form striated, multinucleated, myocytes by Day 10 post revival, that contract in response to acetylcholine.

Unprecedented batch-to-batch consistency allows for their use in demanding screening applications where small changes need to be detected against background noise.

¹ Pawlowski et al., Stem Cell Report 2017

Product specifications

Advantages

Ready for experimentation within days
Highly characterised and defined
Easy culturing

Applications

– Muscle, neuromuscular and metabolic research
Amenable to high throughput screening
– Contractility assays
– 3D cocultures
– Single cell CRISPR Screening

For more information download our flyer

or email info@bit.bio

ioCells ioSkeletal Myocytes Consistency

Batch to batch reproducibility and homogeneity create a stable human model for the study of muscle, neuromuscular, and associated metabolic disorders.

ioCells ioSkeletal Myocytes Speed

Form striated, multinucleated, myocytes by Day 10 post revival, that contract in response to acetylcholine.

ioCells ioSkeletal Myocytes Scalability

Industrial scale quantities at a price point that allows the cells to be used from research to screening scale.

ioCells ioSkeletal Myocytes Ease Of Use

Cells arrive programmed to rapidly mature upon revival. One medium required in a two-step protocol.

 ioSkeletal Myocytes generated by MYOD1-driven reprogramming of iPSCs using opti-ox™ technology

Video capturing the rapid morphological changes from iPSCs upon induction of MYOD1 expression using opti-ox™ cellular reprogramming. 10 day time course.

 ioSkeletal Myocytes express skeletal myocyte-specific markers

bit.bio ioSkeletal Myocytes Desmin DAPI

A. Desmin (red) / DAPI (blue)

bit.bio ioSkeletal Myocytes Dystrophin DAPI

B. Dystrophin (green) / DAPI (blue)

bit.bio ioSkeletal Myocytes Myogenin MHC

C. Myogenin (green) / MHC (red)

bit.bio ioSkeletal Myocytes Troponin Phalloidin DAPI

D. Troponin (green) / Phalloidin (red) / DAPI (blue)

Immunofluorescence staining at day 10 post revival demonstrates robust expression of components of the contractile apparatus such as Desmin (A), Dystrophin (B), and Myosin Heavy Chain (C), along with the muscle transcription factor Myogenin (C). Cells also demonstrate expression of Troponin with visible striated fibres and multinucleation (D).

Cells demonstrate classical myocyte morphology

ioCells ioSkeletal Mycoytes Day 3
ioCells ioSkeletal Myocytes day 5
ioCells ioSkeletal Myocytes day 10
ioSkeletal Myocytes form elongated multinucleated myocytes over 10 days. Day 1 to 10 post-thawing; 4X magnification; scale bar: 800µm

Cells demonstrate gene expression of key myogenic markers following reprogramming

ioCells ioSkeletal Myocytes gene expression C447
Following reprogramming, ioSkeletal Myocytes downregulate expression of the pluripotency genes (A), whilst demonstrating robust expression of key myogenic markers (B). Gene expression levels assessed by RT-qPCR (data expressed relative to the parental hiPSC, normalised to HMBS). Data represents Day 10 post-revival samples; n=7 biological replicates.

Cells express the insulin regulated glucose transporter GLUT4, critical for metabolic studies

ioCells ioSkeletal Myocytes cells express insulin regulated glucose GLUT4
Figure 5 GLUT4

(A) RT-qPCR, at Day 10 post-revival, demonstrating expression of GLUT4 in the ioSkeletal Myocytes, compared to undifferentiated hiPSCs and ioGlutamatergic Neurons (formerly known as ioNEURONS/glut). (B) Immunocytochemistry, at Day 7 post-revival, demonstrates expression of GLUT4 in peri-nuclear regions, and striations, in the ioSkeletal Myocytes*. (C) Western blotting of differentiated 3T3-L1 adipocytes and maturing ioSkeletal Myocytes demonstrates GLUT4 expression in a time-dependent manner*.

* Dougall Norris & Daniel Fazakerley, Wellcome-MRC Institute of Metabolic Science

 
Cells are suitable for phenotypic based high-throughput screening

 

(A) Human fibroblasts were transduced with lentiviral vectors allowing inducible over-expression of MYOD1 to transdifferentiate them to myocytes in approximately 10 days. Transdifferentiated myotubes were stained for multiple myotube markers to assess the purity and degree of multi-nucleation. (B) ioSkeletal Myocytes generate myocytes within as little as 4 days post-revival with a high-degree of MHC+ cells (>80% purity), suitable for phenotypic based high throughput screens. (C) Comparable total area of MHC positive cells are generated between ioSkeletal Myocytes and transdifferentiated fibroblasts.

Shushant Jain et al, Charles River Laboratories

In vitro human muscle cells suitable for contractility assays

By Day 10 post-revival, cells demonstrate a strong contractile response upon addition of acetylcholine, providing a suitable human muscle model for contractility assays. Spontaneous contraction is also observed during continuous culture (data not shown). Day 10 post-revival skeletal myocytes; 50µM acetylcholine.

 Cells arrive ready to plate

IoCells ioSkeletal Myocytes vials
ioCells ioSkeletal Myocyte ready to plate
ioSkeletal Myocytes are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended medium. The protocol for the generation of these cells is a three-phase process: 1. Induction (carried out at bit.bio) 2. Stabilization for 3 days with Dox 3. Maintenance during which the skeletal myocytes mature.

Available in two vial sizes, tailored to suit your experimental needs with minimal waste

ioCells ioSkeletal Myocytes plating sizes
Recommended seeding density for ioSkeletal Myocytes is 100,000 cells/cm2. One Small vial can plate a minimum of 0.5 x 24-well plate, 0.75 x 96-well plate, or 1 x 384-well plate. One Large vial can plate a minimum of 1 x 24-well plate, 1.5 x 96-well plate, or 2 x 384-well plates.
 

Product specifications


Starting material
Human iPSC line

Donor
Caucasian adult male
(skin fibroblast)

Differentiation method
opti-ox™ cellular reprogramming
 

Karyotype
Normal (46, XY)

Vial size
Small: >2.5 x 106 viable cells
Large: >5 x 106 viable cells

Recommended seeding density
100,000 cells/cm2
 

Seeding compatibility
6 to 384 well plates

Quality control
Sterility, protein expression (IF) and gene expression (RT-qPCR)

User storage
LN2 or -150°C
 

Shipping info
Dry ice

Product use
These cells are for research use only