Stem cell research recently celebrated its 60th anniversary. Although the first publications in this field appeared in the early 1960s, it took another 20 years before the first embryonic stem cells could be isolated. Another important milestone was passed in 2006, when Kyoto University’s Kazutoshi Takahashi, PhD, and Shinya Yamanaka, MD, PhD, reported that they had used defined factors to reprogram differentiated cells to an embryonic-like state. This work led to induced pluripotent stem cell (iPSC) technology that made it much easier to conduct stem cell research. This technology not only simplified the procurement of stem cells, but it also made it possible to avoid the ethical concerns that attend the use of human embryonic stem cells. In 2014, the first stem cell therapy for the treatment of limbal stem cell deficiency, a disease in which a regular corneal epithelium is missing, was approved by the European Medicines Agency. At present, according to the Alliance for Regenerative Medicine, 14 cell and gene therapies available in different markets across the world involve stem cells. The intervals between new therapies are becoming shorter and shorter, as the technology has matured to the point where clinical application is possible on a larger scale.

Many established companies are investing heavily because stem cell–derived therapies, along with gene therapies, have great potential to revolutionize medicine. New technologies have also enabled the development of startups. These companies support the optimization of current production processes and the development of new therapeutic products. This article highlights some of the current challenges associated with the development of new stem cell–based therapies and how new technologies are overcoming these challenges.

Mapping neuronal interactions

Stem cell therapy is a promising option for many diseases, particularly those for which cures are lacking. For example, stem cell therapy holds promise for the repair of damaged tissue that has very low regeneration potential, as stem cells can be transformed through targeted differentiation into any adult tissue that would not have regenerated on its own. Tissues that are difficult to replace include lung parenchyma, heart muscle, and neuronal tissue. The regeneration of nerve cells in particular holds great potential for the treatment of diseases associated with brain tissue losses, such as those caused by strokes.

MaxWell Biosystems has developed high-density microelectrode arrays to study the function of neurons derived from iPSCs and other neuronal sources. Two-dimensional and three-dimensional cell culture systems that mimic in vivo conditions to varying degrees, especially when derived from human IPSCs, are a valuable substitute for traditional animal models. Besides providing a crucial starting point for disease modeling, these systems offer significant opportunities for artificial intelligence. The technology provides additional information from network to subcellular resolution allowing the recording of axonal signals at unprecedented resolution and quality compared with conventional patch-clamp methods, particularly the observation of multiple loci in one measurement.

Applying single-cell omics more widely

In general, new approaches to measuring the properties of individual cells rather than cell populations are being explored in the field of stem cell therapy. Some important biological processes are either completely or partially undetectable when tested with standard assays averaged over many cells. However, the latest technologies have enabled researchers to analyze ever smaller systems and even go down to the level of individual cells.

Bulk tissue sequencing has been replaced by multicell analysis, where normal development and disease processes can be studied at single-cell resolution and a link can be made between the cell’s phenotype and its genotype. These single-cell genome, transcriptome, and proteome high-throughput assays provide new and valuable insights into different developmental pathways, gene expression dynamics, and disease progression.

For single-cell omics, one must be able to work with very small volumes (down to the picoliter range) and samples. Cellenion produces instruments for single-cell dispensing and precise reagent dispensing in small volumes.

Cells can be dispensed into individual wells by systems that integrate acoustics, microfluidics, and microscopy. (Indeed, such systems can sort cell types based on fluorescence signals and distinguish between active and dead cells.) The isolation of single cells is necessary to enable multiple applications, including single-cell omics and cell line development.

One example of a single-cell omics application is the characterization of circulating tumor cells (CTCs), which are thought to be the seeds of metastasis in patients with malignant tumors. Like stem cells, these cells have the potential for tissue formation and show genomic instability. Metastasis of a primary tumor is always associated with a significant worsening of prognosis for cancer patients, so it is important to understand how CTCs form metastases.

The analysis of DNA, RNA, protein, methylated DNA, and other constituents of isolated CTCs could inform the development of therapies capable of targeting CTCs for destruction or preventing new metastases. If successful, such therapies could dramatically improve the outcomes for many tumor patients. However, CTCs are extremely rare in peripheral blood (1 in 1011 cells), which makes their isolation from regular blood cells very challenging. After prepurification, CTCs can be isolated with single-cell sorting technologies for further studies in a purity that was previously unattainable.

Ensuring consistent quality

The manufacture of stem cell–based therapies must be carried out under strict quality controls that satisfy regulatory bodies and ensure the safety of potential patients. Such therapies use “living drugs” derived from tissues or cells to treat various diseases. In this cutting-edge field, advances in cell therapies require that large volumes of high-quality cell collections be produced through high-throughput and traceable bioprocessing systems.

As the industry moves from autologous to allogeneic cell therapies, the single-use bioreactor is becoming the preferred tool for manufacturers, who value its ability to improve process control and facilitate scalable production. Besides enabling the shift from autologous to allogeneic products, single-use bioreactors are enabling another kind of shift, namely, the transition from laboratory research to reliable manufacturing. They may prove crucial to realizing the full potential of stem cell therapies.

Single-use bioreactors, such as the bioreactors in Eppendorf’s BioBLU series, offer several advantages. For the consumer, single-use bioreactors eliminate the need for cleaning after each use, and most importantly, eliminate the risk of contamination from previous batches, which greatly increases process safety. Single-use bioreactors also offer noninvasive sensors for temperature, dissolved oxygen, and pH. This avoids direct contact of the sensors with the medium and further reduces the risk of contamination.

Eppendorf has also developed tools for process development in cell and gene therapy. For example, the company offers miniature bioreactor systems. Such systems are intended for statistical design of experiments and the standardization and streamlining of process developments.

Genetic instability is a challenge

In human stem cell research, challenges include genomic abnormalities, cell line verification, and bacterial contamination. A major problem with iPSCs is their inherent genetic instability, which often leads to inconsistent batch quality and represents a major challenge in this field. This problem cannot be solved by simply removing contaminants such as bacteria or cells of previous batches, but rather must be addressed with cell line development and strict quality control mechanisms.

To enhance the quality of stem cell research results and protect the reputation of the stem cell field as a whole, the International Society for Stem Cell Research has issued a guidelines document, Standards for Human Stem Cell Use in Research. When the document appeared in June 2023, Martin Peral, PhD, the editor-in-chief of Stem Cell Reports, offered this comment: “[We] will be introducing the checklist for authors that accompanies the guidelines on a trial basis … My colleagues and I feel this is a major step forward in ensuring rigor and reproducibility in all areas of stem cell research.”

“These new standards put a lot of pressure on existing developers of stem cell therapies,” notes Annick Marcellin, marketing manager, Stem Genomics. The company offers solutions to many challenges in stem cell research, including a range of genomic integrity tests and cell line authentication products. The company also specializes in quality control issues in the development and production of stem cell therapies.

New approaches based on digital PCR enable the detection of the most common genomic abnormalities in iPSCs with a turnaround time of just a few days. In the future, the company aims to bring improved assays into the market that enable the detection of iPSCs at lower detection limits. Stem Genomics’ products also include next-generation sequencing assays that enable a more detailed analysis of cell lines and methods that can screen for common bacterial contaminants such as Mycoplasma species throughout the cell culture life cycle.

Stem Genomics' digital PCR
Digital PCR (dPCR) is the most accurate PCR method for assessing the genetic integrity of stem cells and is ideal for regular in-process controls and fast clone screening. Stem Genomics aims to provide new solutions for common problems in the stem cell field by employing dPCR as analytical tool.

Where is stem cell therapy heading?

Stem cell therapies have reached a point where they can be used clinically to a limited extent for selected diseases. However, the field is expanding rapidly. For example, stem cell therapies show promise against type 1 diabetes, which affects more than 8 million people worldwide. The condition is caused by the destruction of a small but crucial population of cells, specifically, the islet cells in the pancreas. There are only a few of these cells in healthy people, but their destruction can quickly lead to fatal consequences.

The regeneration of these islet cells through stem cell therapy promises to permanently cure this form of diabetes and drastically improve the quality of life of those affected. In many other serious diseases, the regeneration of tissues, or of individual cells in these tissues, is the key to a cure.

The technologies that have been presented in this article reflect the current status of stem cell therapy development. They are also poised to contribute to the continuous progress that has always characterized the field.

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