— The Startup Optimizing Cellular Forward Programming

In the past few decades, research in stem cells has become extremely popular due to the potential stem cells hold in medical research and therapy. However, stem cells are difficult to obtain, and so far, difficult to scale., a cellular reprogramming startup, could have the potential to change that.

Image by Anusorn Nakdee

An Introduction To Stem Cells

Stem cells are cells that have the potential to renew and differentiate themselves; they can become any specific cell type, like a red blood cell. Stem cells can be used for the better understanding of genetic and molecular signals that regulate cellular division, specialization, and differentiation. They can also be used in medical therapies to regenerate and repair solid tissues, and eventually form entire organs that can be used for organ transplants.

Adult stem cells can be found throughout various tissues in the body, but only have the potential to differentiate into a specific category of cell types. For example, an adult skin stem cell may only be able to differentiate into keratinocytes, melanocytes, Merkel cells, etc.. These stem cells act as an internal repair system and are used to maintain and replace adult cells that are lost through wear and tear, injury, or disease.

Pluripotent stem cells are stem cells that have the potential to differentiate into all of the cells of the adult body. These cells would be ideal for stem cell therapies, since they could be used to generate a variety of cells and tissues. However, they are only found in the inner cell mass of mammalian embryos.

Collecting stem cells from human embryos raises a variety of ethical questions, which is why researchers identified methods of reprogramming mature human adult cells into pluripotent stem cells. These cells are known as induced pluripotent stem cells (iPSCs).

iPSCs were created to be differentiated into any type of cell. However, the current process of iPSC differentiation is inefficient, requiring several intermediate states and different culture conditions. In addition, the results of current methods of iPSC differentiation have shown little success, with high costs, immature differentiated cells, high rates of cellular death, and long production times (usually many months). These methods will not allow stem cell research and therapy to become affordable and scalable.

That’s where comes in. — The Startup Optimizing Cellular Forward Programming, founded by Dr. Mark Kotter, a neurosurgeon and stem cell biologist at the University of Cambridge, aims to make stem cells affordable and scalable by optimizing the process of iPSC differentiation. They are dedicated to producing functional human cells for research, drug discovery, and cell therapy.

The original researchers at were able to design a research platform that identifies transcription factor combinations that make up cell type programs. With their new differentiation platform, can skip the traditional intermediate steps of inducing iPSC differentiation, and force iPSC differentiation in just a few days.

OPTi-OX — The Precise Reprogramming of Cells

Current methods for inducing differentiation are lengthy, inefficient, and difficult to reproduce, and ultimately, most differentiated cells don’t reach full maturation. These methods are usually based on the lentiviral transduction of iPSCs, which result in variegated expression or the complete silencing of transgenes. Essentially, transgenes are randomly inserted into the genome, bearing the risk of unwanted interference with the endogenous transcriptional program. As a result, additional purification steps are usually required to achieve the desired cell type.

Forward programming is a method that forces the expression of line-age-specific master regulators, resulting in the direct reprogramming of somatic cell types. By combining the advantages of iPSC differentiation and direct cellular reprogramming, forward programming enables the scalable and rapid generation of human cell types.

To optimize forward programming, Dr. Mark Kotter and his team of researchers created an iPSC forward programming platform known as OPTi-OX (optimized inducible overexpression), which uses the dual targeting of the Tet-ON system to achieve the homogenous and controllable expression of inducible transgenes in iPSCs and their derivatives.

This new platform targets all of the components of the Tet-ON system required for the inducible expression of transcription factors in genomic safe harbors (GSHs). GSHs are sites in the genome that can accommodate the integration of transgenes in a way that ensures that the newly inserted elements function predictably and don’t alter the host genome in a hazardous way.

The Tet-ON system activates gene expression through the administration of doxycycline, and has two primary components.

  • The transcriptional activator protein responsible to doxycycline
  • The inducible promoter regulated by rtTA (Tet-responsible element) that drives the expression of the transgene

Previous forward programming strategies targeted GSHs in the Tet-ON system by introducing both of these components into one GSH of the iPSCs.

Dr. Kotter’s team instead reasoned that targeting the Tet-ON system with the two components into separate GSHs would have several advantages.

For example, the inducible overexpression based on dual-GSH-targeting wouldn’t be affected by promoter interference between two transgenes, as it would be in other forward programming strategies. Multiple GSHs would also allow for a larger capacity of transgenes, which would make transgene design more flexible and enable the insertion of large reprogramming cassettes.

Overall, this optimized approach results in the homogenous, controllable, and extremely high expression of inducible transgenes in iPSCs. This platform could help rapidly develop mature human cell types from iPSCs.

ioCells — Creating Mature Human Cells

By applying their new forward programming platform, was able to create two pure and mature human cell types — excitatory cortical neurons (glutamatergic neurons) and human skeletal myocytes. These cells were made using’s reprogramming platform, OPTi-OX. Their platform allows iPSCs to convert into consistent, mature, and functional cells within just a few days, which could be used to study neurological and skeletal activity.

ioGlutamatergic Neurons developed glutamatergic neurons first to show the capability of OPTi-OX, since these cells could be compared to glutamatergic neurons developed using lentiviral overexpression techniques.

The initial results of Dr. Kotter’s study showed that their differentiated iPSCs developed regular neuronal morphology and expressed pan-neuronal markers after just 7 days. The induced neurons had a strong expression of forebrain markers and glutamatergic neuronal genes, which indicated that they were excitatory cortical neurons.

Immunofluorescent staining on glutamatergic neuron-specific transporters indicates that the iPSCs differentiated into glutamatergic neurons, by

Later on, it was shown that short pulses of treatment from the OPTi-OX for 4 days of longer were enough to allow the iPSCs to differentiate into mature glutamatergic neurons, and they did not observe any reduction in the efficiency of these neurons over extended culture periods.

ioSkeletal Myocytes

The existing methods for differentiating iPSCs into skeletal myocytes were difficult and inefficient, and the yields were generally unsatisfactory. Initially, Dr. Kotter and his team used the same method from the glutamatergic neurons to develop skeletal myocytes, but the differentiated cells would die in 3–5 days, regardless of the culture they used.

Eventually, they found that using the MYOD1 (transcription factor) OPTi-OX alone was not enough to drive myogenesis in iPSCs, which agreed with the postulated existence of epigenetic barriers that prevented forced myogenesis.

To overcome these barriers, their forward programming strategy was enhanced by combining transcription factor overexpression with extra cellular signaling clues. By adding all-trans retinoic acid in conjunction with MYOD1 overexpression, Dr. Kotter’s team was able to achieve the rapid and deterministic conversion of iPSCs into myogenin and myosin heavy chain double positive myocytes after just 5 days of induction.

iPSC derived cells show classical myocyte morphology, by

After further optimization of culture conditions, they achieved highly pure induced skeletal myocytes.

To further demonstrate the purity of these cells, the team found that by adding extremely small concentrations of acetylcholine or selective ACh-receptor agonist carbachol, the cells would demonstrate muscle fiber contraction.

The full report from Dr. Kotter’s team is available here.

Why Is So Remarkable? has received over $50 million in funding from top life sciences investors and entrepreneurs, and it’s because could revolutionize the way we view stem cells forever.’s new technologies have the potential to create stem cells on a massive scale, making them widely available for research and therapy. Using artificially derived human stem cells in comparison to animal bodies would have a plethora of benefits in research and experimentation, and using stem cells in medical therapies would bring immense maintenance and restorative benefits.

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