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Figure 2. Brightfield image of hPSC-derived islet clusters after completion of 35 days of directed differentiation. Photo credit: Dr. Beatrice Ho.

What? 
The pancreatic islets of Langerhans are tissues that produce and secrete endocrine hormones that are required for regulating glucose levels in the body. In particular, the beta cells are a specialized cell type in the islets that secrete insulin, the critical hormone that lowers blood glucose in response to food intake. Diabetes occurs when the beta cells are destroyed, rendering an inability to produce insulin (causing Type 1 diabetes, an autoimmune condition), or when the beta cells are partially defective and unable to secrete sufficient insulin for the body’s needs (causing Type 2 diabetes, the common chronic form of diabetes). In the latter case, insulin resistance is often a major contributing factor in which tissues are unable to respond effectively to insulin. Diabetes inflicts over 530 million people worldwide (1), and patients are being treated with a myriad of drugs and in many cases, insulin therapy. Significant progress has been made in the field of regenerative medicine for diabetes in which pancreatic islet cells are generated in the lab from human pluripotent stem cells (hPSCs) (Figure 1). HPSCs refer to either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), both with the unique ability to differentiate into any mature cell type. The hPSC-derived pancreatic islet cells (Figure 2) have been a valuable resource for studies of human islet developmental biology, disease modeling, and more recently for the development of islet cell replacement therapy. Much like primary human islets, these lab-grown islets comprise different cell types – the insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells, pancreatic polypeptide-expressing gamma cells and ghrelin-expressing epsilon cells. The beta cells, which produce the important hormone insulin that is responsible for keeping blood glucose levels in check, are most often the focus of islet cell differentiation protocols.

Why?
Type 1 diabetes (T1D) is a life-changing, early onset disease (often diagnosed at <20 years of age) in which patients are treated with life-long insulin injections, that often require careful management to combat hyperglycemia as well as reduce the risk of hypoglycemic events. These patients often face a high risk of diabetic complications later in life even with compliance to treatment.

Islet cell replacement therapy is a highly promising curative form of treatment for T1D, particularly for patients with hypoglycemia unawareness. Cell replacement with primary human islets from cadaveric donors is an approved treatment for T1D in several countries, including in Europe Canada, Japan and Australia under organ transplant regulations (2), and most recently in the US under a biologics license (3). Long term followup on primary islet transplantation have demonstrated positive outcomes such as freedom from severe hypoglycemia, and insulin independence for a certain period (4, 5), which is life-changing from a patient’s perspective. However, primary human islet transplant, much like organ transplant, is not a sustainable treatment option due to limited donor availability, lack of accessibility to clinical islet isolation facilities, variable tissue quality and quantity, and the need for life-long immunosuppression (6, 7), thus severely limiting the patient population that can benefit from this treatment. In recent years, hPSC-derived pancreatic islet cells manufactured in the lab present an incredible opportunity as a continuous source of cells for therapeutic use. As these cells are capable of sensing changes in glucose levels and secreting insulin, they have the potential to restore endogenous glucose control in patients. Moreover, this cell therapy may be developed as both autologous therapies (as donor iPSC banks can be made) or allogeneic therapies (from a universal iPSC master cell bank). Although islet cell replacement has not been used to treat T2D patients, it is possible to envision a subset of T2D patients who suffer from severe insulin-deficient diabetes (8) who might benefit from an islet cell therapy product.

Who?
There is global interest in hPSC-islet cells, most actively in North America, Europe and parts of Asia. Only two companies, Viacyte and Vertex Pharmaceuticals, both based in North America, have progressed their hPSC-derived pancreatic cells to first-in-human clinical trials. Previously, hESC-derived pancreatic endoderm cells (progenitor cells that undergo maturation in vivo after transplantation) were developed by Viacyte and tested in Phase I/II clinical trials. However, these cells, though well-tolerated, demonstrated modest levels of cell survival and insulin production, and therefore provided limited clinical benefit to patients at one year of follow-up (9, 10). There were also highly heterogenous outcomes among patients. Nonetheless, the trials represented a bold step forward for the field and since then, the biotech company has been acquired by Vertex Pharmaceuticals in 2022 in a consolidation effort to advance the regenerative medicine product. Vertex Pharmaceuticals began Phase I/II clinical trials this time for their fully differentiated hiPSC-derived pancreatic islet cells and recently released early promising data showing that the first few patients who were dosed achieved improved glycemic control and a significant reduction in insulin use (11). Recent news in China from the lab of Prof. Hongkui Deng at Peking University have also reported initiation of early phase clinical trials for islet cells differentiated from their unique hiPSC lines. Several pre-clinical efforts in developing innovative stem cell-derived cells or technology platforms to treat diabetes are also known to be driven by other industry players such as Sana Biotechnology (12), Novo Nordisk, Orizuru Therapeutics, Seraxis (13), and Sernova (14) in partnership with Evotec.

When? 
The ability to access and utilize hESCs and the groundbreaking technology to generate hiPSCs from somatic cells spurred intensive research on human embryonic development and cellular differentiation. Early studies from 2005 focused on differentiation of hPSCs to definitive endoderm cells, followed by stepwise differentiation to pancreatic endoderm (15, 16) and finally pancreatic islet cells (17, 18). 

These cells have been used to model different diabetes conditions such as monogenic diabetes (19, 20), Wolfram syndrome caused by a WFS1 mutation (21), and T2D in the context of genetic susceptibility variants (22, 23) (disease modelling efforts have been extensively reviewed in (24)). Several reports have also emerged to provide deep characterization of the different cell types in hPSC-islet cells using single cell RNA-Seq (21, 25, 26), shedding light on the heterogeneity even within the beta cell cluster, as well as the existence of other cell types such as progenitor cells or non-endocrine enterochromaffin cells that may not be characteristic of primary human islet cells. Recent work across different labs have sought to functionally characterize the hPSC-islet cells in vitro and in vivo to evaluate their functional capacities and ability to rescue diabetes in animal models, including both rodent models and non-human primates (12, 27, 28). These latest studies are paving the way for the translation of hPSC-islet cells for research into regenerative medicine applications. 

The early clinical trials involving hPSC-pancreatic endoderm cells found that the cells survived with evidence of maturation in vivo, however functional outcomes were limited due to low levels of insulin-producing beta cells and hence low insulin (C-peptide) secretion (9, 10). The more recent reports from clinical trials involving fully-differentiated hiPSC-islet cells appear to provide more promise with high levels of C-peptide secretion detected (11), though it remains to be seen whether the clinically beneficial outcomes can be sustained over time and seen across many T1D patients.

Where?
While there are currently no approved products based on hPSC-derived pancreatic cells, several countries are actively pushing the frontiers in R&D in this space including the US, Europe, Japan, China, and Singapore, with both academic labs and commercial players driving the research efforts on applications of hPSC-derived islet cells.

How?
As a cell product derived from pluripotent stem cells, the source and quality of the starting cell bank are major factors affecting the cell manufacturing process. Clinical grade hESCs that have been established under GMP conditions are available such as from the WiCell Institute or the UK Stem Cell Bank, though the use of hESCs face ethical concerns in certain regulatory environments. As such, clinical grade hiPSCs are gaining more traction as the starting cell source. Services to generate such cells appear to be available through several commercial players such as Lonza, Pluristyx, RoslinCT, REPROCELL, among others. There is a high level of complexity involved due to considerations of the donor somatic cell source, method of reprogramming (viral or non-viral), and usage of a xeno-free culture under GMP conditions. There are currently no regulatory specifications on the required standards for hiPSCs designated for clinical applications. However, as more hiPSC-derived cell products have now advanced into clinical testing, there is some consensus on the key release criteria expected for hiPSC stocks or cell banks. These can cover cell identity, genomic stability or integrity, absence of residual vectors, markers of pluripotency, and sterility testing (29).

hPSCs are conventionally cultured in adherent cultures on attachment matrices (Figure 1A). To facilitate large scale expansion, the cells may be adapted to suspension cultures as 3D cell aggregates (Figure 1B) or microcarrier-based aggregates. For the differentiation of hPSCs to pancreatic islet cells, several research labs begin with adherent cultures during differentiation to pancreatic progenitors before transition to suspension cultures as 3D spheroids for the remainder of the differentiation to islet cells (17, 28, 30). Adherent cultures will require use of multiple flasks or cell factories, which may be challenging to manage in large scale cultures. Other labs utilize suspension spheroid cultures throughout the differentiation process (25, 31, 32), which are more amenable for scale up such as in shaking flasks, spinner flasks or stirred tank bioreactors. Adherent culture systems will present a limitation especially when large scale cell production is needed. Additionally, academic research does not require xeno-free, Good Manufacturing Practice (GMP)-compliant methods of cell production, and therefore substantial changes in culture protocol and re-optimization will be required to generate these cells for clinical use.

Another noteworthy challenge relates to managing the immunogenicity of cells in the case of allogeneic cell therapy. hPSCs provide a cell-based platform that is highly tractable for innovation, such as modification of starting hiPSCs by gene-editing to knock out or express immune genes and/or immune modulatory genes (12, 33), or establishing selective donor-derived hiPSC banks (such as that derived from HLA-homozygous donors) that enable HLA-matching of hiPSC lines for use in cell therapies (34, 35). Successful development in these areas will open up a significant pathway for hiPSC-based cell therapy products to reach larger patient populations.

Did you know that… 
Primary human islets are known to contain 40 – 60% beta cells, 20 – 40% alpha cells, 5 – 15% delta cells and a small abundance of other minor cell types (with cell composition differing across different individuals). Based on prevailing differentiation protocols, hPSC-islet cells largely capture the different cell types within similar ranges of gold standard human islets (based on marker expression), but also contain poly-hormonal cells and non-endocrine cells that may justify further protocol refinement. Much of the spotlight had been thrown on specifically deriving functional insulin-secreting beta cells or enriching for such populations, while far fewer focus on deriving other cell types such as alpha cells, which are also important for maintaining blood glucose levels (36). With the versatility of the hPSC differentiation platform, perhaps it is possible in future to specifically design and manufacture off-the-shelf islet cells with different desired compositions that can recapitulate the function of other islet cell types, providing a system in which different cell types work in concert to achieve precise glucose homeostasis, much like primary human islets.

References

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