Gene editing, especially using the CRISPR-Cas9 system, represents a key technological breakthrough in recent scientific history – but it isn’t the only important advance. Human-induced pluripotent stem cells (hiPSCs) are another powerful tool that has greatly propelled new research and therapeutic strategies in fields including medicine, molecular biology and genetics. They provide unique opportunities to model human diseases, discover new drugs and develop effective therapies.

Scientists are increasingly looking at combining these two game changing tools to produce gene-edited hiPSCs, opening doors to a novel and improved understanding of human disease.

hiPSCs – pluripotent stem cells made on demand

Pluripotent stem cells are self-renewing cells from which all other types of cells can be generated, thanks to their ability to differentiate into any type under the appropriate instructions. The basic process of generating hiPSCs, which are made by directly reprogramming human somatic cells (any biological, differentiated cell other than an egg or sperm cell), is as follows:

  1. Somatic cells are obtained from a healthy donor or patient for whom researchers wish to study or treat their disease. Cells are typically obtained from a blood sample or a skin biopsy.
  2. The somatic cells are reprogrammed through gene transfer of certain transcription factors (‘reprogramming factors’) that induce pluripotency.
  3. The resulting hiPSCs can then be instructed to differentiate into, for example, muscle cells or blood cells that can be used for applications such as disease or drug modelling and tissue engineering

There are many benefits of using hiPSCs in research, aside from their biomedical and therapeutic promise. They largely replace the requirement to utilize embryonic stem cells for similar types of research, with the latter requiring the destruction of human embryos which is not only ethically controversial but, in many countries, strictly forbidden.

Gene-edited hiPSCs for disease modelling

Using CRISPR-Cas9 to genetically edit hiPSCs enables the fast, cheap and precise modification of these cells to investigate diseases and pathologies in a patient-specific genetic context, in turn aiding translation of this technology into the clinic – and into real-life applications.

By what means can CRISPR-Cas9 edit hiPSCs? The gene expression of hiPSCs can be regulated using either activated or repressed CRISPR systems that target the DNA regions to which proteins bind and initiate transcription, the first step in gene expression. CRISPR-Cas9 can also be used to insert and delete genes or make single base-pair changes to the DNA that either introduce or repair specific mutations.

A significant application of generating gene-edited hiPSCs is the generation of isogenic pairs of hiPSCs that share a genetic background but differ in a specific mutation assumed to be related to or causative of a disease of interest. Isogenic pairs are highly useful for assessing the characteristics of a disease, functionally testing the relation between mutations to these characteristics and determining the disease-causing mutation, which remain merely associative from genome-wide association studies alone. These hiPSCs pairs can also be used to carry out genome-wide screens that can uncover pathological mechanisms and identify targets for drugs, and have helped in the study of neurological and cardiovascular diseases, to name a few.

Gene therapy is another avenue for hiPSCs and CRISPR-Cas9 that can potentially correct disease-causing mutations, such as that for the blood disorder beta-thalassemia. Here, patient-specific and corrected hiPSCs can be differentiated into hematopoietic stem cells and transplanted into the patient as a potential therapeutic strategy. Other gene corrections or substitutions by CRISPR-Cas9 can restore normal expression or regions of the genome, leading to gene therapies for diseases like muscular dystrophy.

In a similar fashion, genetically modified hiPSCs are able to introduce genetic changes that play a key role in drug screening and development. After differentiation into the desired cell type, edited hiPSCs can be tested against different agents to study their effects in cell-specific contexts or be used to identify specific targets for potential drugs, develop compounds and select drug candidates.

Future challenges

The combination of hiPSCs with CRISPR-Cas9 provides newfound possibilities for utilising these innovative technologies in everything from medical research to novel treatments. However, challenges still remain, including:   

  • The efficiency of the reprogramming mechanism of hiPSCs, which could be improved, for example, by avoiding incomplete reprogramming and the risk of new mutations occurring
  • Establishing standardized protocols for gene editing hiPSCs
  • Limiting off-target edits and effects of CRISPR-Cas9, an existing challenge of the technology
  • Improving targeting accuracy to boost success rate in clinical trials
  • Evaluating safety of gene-edited hiPSCs for future human clinical trials

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