Human iPSC-derived Motor Neurons and Skeletal Myotubes Co-culture Enabling Disease ModelingB.C. FREITAS1, L. K. FONG2, J. LIU1, B. MELINE1, C. CHAVEZ1, T. K. FEASTER1, C. MCMAHON1, D. MANN1, W. WANG1, J. ARJOMAND2, U. SCHMIDT2, E. JONES1

1FUJIFILM Cellular Dynamics, Inc, Madison, WI; 2Genea Biocells US Inc., La Jolla, CA

AbstractThe ability to produce motor neurons from induced pluripotent stem cells (iPSCs) provides the means to model several neuromuscular disorders using human cells. It is now possible to explore human-specific drug screening platforms with prospective treatment possibilities using iPSC-derived motor neurons and skeletal muscle for disorders lacking therapeutic options such as amyotrophic lateral sclerosis (ALS). Here we demonstrate the generation of iPSC-derived motor neurons using an optimized differentiation protocol generating a population based on Isl1/2 and Tuj1 positive staining. Furthermore, we used this cell line to generate a TDP-43 mutant line, equipping researchers with a valuable tool for ALS to better understand underlying mechanisms of the disease and drug development. We characterized our cells and co-cultured them with human skeletal muscle that is capable of expressing all components of a neuromuscular junctions in vitro. We are now exploring co-culture conditions with human iPSC-derived skeletal muscle cells based on our published protocol (Caron et al. 2016). Our media-based differentiation method efficiently generates contractile and fused myotubes without the need for any cell sorting or myogenic gene overexpression. Combined with our motor neurons, this co-culture platform will enable ALS drug discovery and facilitate complex disease modeling of other neuromuscular disorders.

ConclusioniPSC-derived differentiated cells from apparently healthy normal and disease phenotype donor backgrounds can be used to develop methods for modeling disease phenotypes in a dish enabling researchers to investigate disease mechanisms and potential drug therapies. A directed differentiation protocol was devised to derive spinal motor neuron populations with cholinergic neuron and motor neuron specifications efficiently from iPSC lines.

Our motor neuron differentiation protocol is robust, consistently producing high purity cells from different clones and multiple donors.

Gene expression analysis shows high expression of motor neuron and cholinergic neuron markers.

In addition, we described the development and characterization of iCell Motor Neurons which can be co-cultured with Genea Biocells skeletal muscle myotubes.

iCell® Motor Neurons: Process Development

iCell Motor Neurons Gene Expression

Q331K

TDP-43 Mutant Lines

Co-Culture Characterization by Staining

Figure 1. Motor Neuron Differentiation. A step-wise approach to the differentiation process was taken by adapting and optimizing techniques previously described in the literature. Directed differentiation was used to neuralize and caudalize induced pluripotent stem cells (iPSCs). Motor neuron specification was initiated after caudalization and differentiated cells were then cryopreserved. Maturation of cells post-thaw was accomplished using treatment with specific growth factors for 1-2 weeks before characterizing the motor neurons.

Figure 2. Motor Neuron gene expression profile. qPCR data at day 14 post-thaw was performed on two different lots of motor neurons differentiated from one iPSC line derived from an apparently healthy donor. Results show high levels of markers specific for cholinergic/motor neurons, namely Isl1, Cht (choline transporter) and FoxP1. Expression of various alpha subunits of the nicotinic acetylcholine receptor were also detected. The glial marker GFAP was not expressed in motor neurons. A RNA from an adult human spinal cord sample was used for comparison.

Genea Biocells Skeletal Muscle

Figure 3. Genea Biocells Skeletal Muscle Differentiation.A) Human pluripotent stem cells (hPSCs) are differentiated in Skeletal Muscle Induction Medium for 10 days. Myogenic progenitors are plated and grown in Myoblast Medium for 8 days then transitioned to Myotube Medium upon confluence. Terminally differentiated, multi-nucleated, and striated myosin heavy chain (MHC)-positive myotubes form within 7 days. B) Genea Biocells myotubes can be cultured in iCell Neural Medium plus supplement and maintain expression of myogenic markers, MYOG and MHC, and an elongated morphology.

M337V

A

B

Figure 4. Immunocytochemistry staining of motor neuron cultures. A) TUJ1 antibody stains the microtubule elements exclusively in neurons; VAChT antibody stains the vesicular acetylcholine transporter located in presynaptic secretory vesicles; Peripherin antibody stains neurofilaments in peripheral neurons. B) Genea Biocells human myotubes are striated, multi-nucleated, and express myosin-heavy chain (MHC). Upon co-culture of Genea Biocells myotubes with iCell Motor Neurons for 5 days, we observe MHC+ myotubes, acetylcholine receptor clusters, and SV2+ motor neurons in close apposition.

Figure 5. TDP-43 (Q331K) mutant line 7 days post-thaw. Phase images and flow data show that the cultured cells are 95% Tuj1 positive and Nestin negative and have an Isl1/2 purity of 64%, with 94% viability.

Figure 6. TDP-43 (M337V) mutant line 7 days post-thaw. Phase images and flow data show that the cultured cells are 97% Tuj1 positive and Nestin negative and have an Isl1/2 purity of 69% with a 97% viability.

A

Hoechst | MYOG | MHC

B

satellite-like cells

myoblasts

myotubes