Transformative Models for Research and Drug Discovery

The dawn of stem cell technologies has opened new possibilities in drug discovery and development by providing new research tools for drug screening, target identification and toxicity testing. However, the widespread use of human induced pluripotent stem cell (hiPSC)-derived cells has been restricted by complex and suboptimal differentiation protocols. Now, bit.bio has produced ioSkeletal Myocytes using its optimized inducible overexpression technology (opti-ox™) to reprogram hiPSCs.

Technology Networks spoke to Dr. Mark Kotter, CEO and founder of bit.bio to learn more about opti-ox™ and the development, benefits and applications of ioSkeletal Myocytes.

Kate Robinson (KR): Are there any difficulties with conventional methods of acquiring skeletal myocytes?

Mark Kotter (MK): One of the big challenges facing biomedical research is the lack of a consistent and scalable supply of human cells. For example, scientists studying muscle use human primary skeletal myocytes as the gold standard. However, like with other primary cells, the supply is limited and the validation of new batches of donor cells required prior to experimentation is a time-consuming and costly process.

Many scientists therefore rely mainly on animal models as a much more accessible source of tissue. However, because species differ with regards to their biology, these often cannot replicate relevant human characteristics. These biological differences contribute to the high drug failure rate within clinical trials.

Pluripotent stem cells are another potential source of human cells, including embryonic stem cells, the origin of all cells in an organism, and iPSCs (Induced pluripotent stem cells) that can be generated from e.g., blood or skin samples by a process called Yamanaka reprogramming. However, decades of research have shown how difficult it is to turn stem cells into the desired cell type.

There are various published methods for generating myocytes using a process called directed differentiation where stem cells are coaxed to make a series of cell fate decisions using cytokines or small molecules. This stepwise approach follows the principles of embryogenesis. However, because of the stochastic nature of cell fate decisions, protocols often lead to inconsistencies and due to the long timelines of directed differentiation the process is time consuming and often takes many weeks or even months to generate the desired cell type. As a result, there is high batch-to-batch variability, which reduces the reproducibility of data and the process is difficult to scale e.g., for the use of cells in high-throughput screening applications.

KR: How were ioSkeletal Myocytes developed?

MK: ioSkeletal Myocytes are reprogrammed from human iPSCs by activating the transcriptional network that defines a muscle cell. The concept of cell reprogramming is based on an entirely different paradigm of cell identity. Pioneering work in the 1980s by Harold Weintraub showed that the activation of a transcription factor can turn skin cells into muscle cells. This is the starting point of cellular reprogramming. However, this knowledge was lost for nearly 30 years until Yamanaka developed his protocol for the induction of pluripotent stem cells, for which he received the Nobel Prize in 2012. From then onwards, the field has been rapidly expanding. However, a major challenge remained: scientists were using vectors, such as viruses, to activate the transcription factors in cells. However, this is associated with further stochasticity and hence variability and issues with scale-up. Here is where the technology developed in my academic lab provides a solution.

Our opti-ox™ (optimized inducible overexpression) technology enables transient and precise control of reprogramming factors and results in deterministic and highly synchronized reprogramming of entire hiPSC cultures into the target cell type. ¹ At first, I did not expect that this would be possible, however, over the past five years the technology has been validated and applied to many different iPSC backgrounds and a variety of human cell types. opti-ox reprogramming presents a step change when it comes to reliable human cells for research and drug discovery. It enables manufacturing of precisely defined and functional human cells with consistency and scale. For example, ioSkeletal Myocytes demonstrate robust expression of components of the contractile apparatus and form striated, multinucleated myocytes by Day 10 post revival and the cells contract in response to acetylcholine. opti-ox now forms the basis of bit.bio’s rapidly maturing, highly defined portfolio of ioCells.

KR: What areas of research can ioSkeletal Myocytes be utilized in and do they have any advantages over conventional skeletal myocytes?

MK: About half of our body’s weight is constituted by muscle tissue. Skeletal muscle is organized in fiber bundles and innervated by motor neurons in order to facilitate movement. A wide variety of diseases affect muscle, including rare diseases such as Duchenne’s or Becker’s muscular dystrophy, mitochondrial myopathies and more generalized conditions, such as cachexia, a disease that leads to muscle wastage.

Because our reprogramming technology is highly consistent and scalable, ioSkeletal Myocytes can be used in a broad range of applications from basic research, disease modeling to drug discovery and high throughput screening. Front-end validation and definition of ioSkeletal Myocytes is only required once. This removes the need for continuous validation of new batches as is commonly required for primary cells. This translates into a significant saving in time, effort and cost over the course of extended research projects compared to using conventional muscle cell models. It allows researchers to focus on the research and not complicated cell culture.

KR: Have the model cells been used in any research to date?

MK: ioSkeletal Myocytes are currently being used by researchers studying metabolism, mitochondrial and muscle diseases. I am particularly excited about their use in the more complex co-culture models studying the connection between the spinal cord and muscle tissue.

For example, Amy Rochford at the University of Cambridge studies peripheral nerve injuries with novel therapeutics, specifically through the potential use of transplantable neuroprosthetics.

Amy wanted to develop a functional readout for neuroprosthetics and required skeletal myocytes. She has recently moved away from differentiated muscle stem cells to ioSkeletal Myocytes “an advantage of these cells is their higher population purity compared to other stem cell derived cells. This enables [her group] to achieve higher numbers of functional striated muscles that are capable of contracting under electrical stimulation. This considerably increases the pace at which [they] can test [their] bioelectronics devices.” This improves reproducibility and she is able to fast track the testing of bioelectronic devices. 

Further applications of ioSkeletal Myocytes are within early drug discovery. The functionality and scalability of these muscle cells make them suitable for phenotypic-based high-throughput screening. Dr Shushant Jain, at Charles River thought that “one of the biggest advantages of the ioSkeletal Myocytes is within the early drug discovery phase. You can very quickly screen a large number of molecules in a short amount of time with minimal variability and high reproducibility.”

Reference: Pawlowski M, Ortmann D, Bertero A, et al. Inducible and deterministic forward programming of human pluripotent stem cells into neurons, skeletal myocytes, and oligodendrocytes. Stem Cell Rep. 2017;8(4):803-812. doi: 10.1016/j.stemcr.2017.02.016

Dr. Mark Kotter was speaking to Kate Robinson, Editorial Assistant for Technology Networks.