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The regulation of human blood glucose concentration is mainly achieved by the mutual restriction of insulin and glucagon at multiple levels. Among them, insulin produced by islet β cells is responsible for promoting blood glucose absorption and lowering blood glucose concentration. Islet beta cell destruction or dysfunction can lead to diabetes. Unlike long-term injections of insulin, islet transplantation usually keeps patients’ blood glucose levels normal for several years and prevents secondary complications of diabetes. However, the scarcity of donors limits the use of such therapies.

In 2014, in a Cell paper, Dr. Douglas Melton, who led the new study, pioneered a method to convert stem cells into insulin-producing beta cells. However, in the initial study, beta cells accounted for only 30% of the final cell mixture. To improve the proportion of beta cells, the research team wanted to know what other 70% of the cells were. The development of single-cell sequencing technology has helped overcome this problem.

 

In recent years, some studies have shown that the replacement of damaged beta cells in islets with islet beta cells derived from stem cells may bring hope to cure diabetes. On May 8, the latest study published in Nature, a team of scientists led by Dr. Douglas Melton of Harvard University described the molecular steps in the differentiation of stem cells into islet-like cells for future production for transplantation. Islet cells provide important guidance.

 

Human pluripotent stem cells are able to self-renew indefinitely and produce various types of cells in the body. Because many research teams are committed to developing protocols for the differentiation of stem cells into islet cells in vitro. An ideal protocol needs to be implemented to promote differentiation of stem cells into fully mature alpha and beta cells; thereafter, alpha and beta cells can be isolated, purified and reassembled into islet-like structures for transplantation. To achieve this ambitious goal, scientists need a thorough understanding of the differentiation procedures of all islet cells and the way in which islets are formed.

Single cell RNA sequencing of beta cell differentiation in vitro

 

In this new study, Dr. Melton et al analyzed more than 100,000 cells at different time points during differentiation of stem cells into pancreatic progenitor cells and hormone-producing cells (also called endocrine cells). Single-cell RNA sequencing (scRNA-seq) of cells is sampled at each step of the differentiation process and then subjected to computational analysis, which makes it possible to identify cell types and to track cell lineages by time. Dr. Melton et al. used this method to create a beautiful picture of how islet progenitor cells develop different lineages of componentized cells (below).

In vitro Differentiation of Human Islet Cells | This study demonstrates that SC-α and SC-β can be purified and recombined to produce stem-derived islets for cell replacement therapy.

The researchers found that pancreatic progenitor cells derived from stem cells are able to differentiate efficiently, but the combination of signal factors used to treat them is slightly different, affecting the ratio of progenitor cells producing hormone-expressing cells and non-hormone-expressing cells.

 

In this study, the three most abundant hormone-expressing cell types were stem cell-derived alpha cells (SC-alpha), stem cell-derived beta cells (SC-beta), and stem cell-like enterochromaffin cells. Cells (SC-EC).

 

The researchers also found that immature SC-α can express two islet hormones (insulin and glucagon), however, this multi-hormone cell becomes a mono-hormone cell after 5 weeks of culture, ie it becomes only expressed. Glucagon cells.

SC-EC characteristics

 

The discovery of SC-EC is surprising because intestinal chromaffin cells are usually found in the intestine (not present in the pancreas) and produce serotonin (which helps regulate bowel movement and digestion). When the researchers changed the signal factor combination (cocktail) provided to the cells during the differentiation phase, the ratio of SC-EC/(SC-α+SC-β) varied greatly. In addition, different combinations of signal factors also alter the ratio of non-proliferating endocrine cells/proliferative non-endocrine cells (alveolar cells and pancreatic ductal cells).

Purification of SC-β cells by CD49a

 

To create safe and functionally mature stem cell-derived islet equivalents, researchers must isolate and purify the desired endocrine cell subtypes and then recombine them into pseudoislets. In the study, the scientists found that a simple separation and aggregation procedure (a physical separation) removes most proliferative non-endocrine cells from culture. In addition, they determined that CD49a (also known as ITGA1) is a surface molecule for SC-β expression. Combining magnetic cell sorting techniques using CD49a antibodies enables researchers to obtain cultures containing 80% SC-β.

After two steps of enrichment, the cell cluster showed more beta cells (pink).

 

It should be noted that although the study can increase the purity of beta cells in cell mixtures to 80%, scientists say that the higher the purity of beta cells, the better. The ability to control the proportion of beta cells in the mixture is a key finding in this study. Now they will focus on investigating the optimal ratio of cells in the mixture.

 

“Maybe we need more types of cells to help regulate beta cells so they work. We will find out how these cell types interact,” explains Adrian Veres, the first author of the paper.

 

In summary, in this study, Veres et al. reconstructed the lineage relationship between human pancreatic progenitor cells and differentiated endocrine cells, and revealed the sequence in which these cell types appeared, as well as the dynamic molecular changes that occurred with each differentiation trajectory. Provides insight into the mechanisms by which stem cells differentiate into islet cells.

 

So, how far are we from treating diabetes with beta cell replacement therapy? Currently, clinical trials have been conducted to test the safety and efficacy of transplanted stem cell-derived islet progenitor cells into patients with type 1 diabetes. However, transplanted pancreatic progenitor cells still need to differentiate and mature into beta cells to achieve effective insulin secretion triggered by glucose stimulation. But in the body, we can’t control the signal factors that the pancreatic progenitor cells are exposed to. Therefore, a more desirable product is that the cells are capable of responding to glucose-secreting insulin before being transplanted into the patient, that is, immediately after transplantation into the patient, and immediately after transplantation into the patient. In addition, it is worth mentioning that transplantation of SC-β alone is not sufficient to treat type 1 diabetes, as alpha cells are also critical for the strict regulation of hormone secretion in human islets and overall regulation of glucose levels.

 

Nature’s opinion article stated that Veres et al. have made significant progress in solving some key issues. For the first time, they provided a detailed scRNA-seq dataset covering the main steps in islet cell differentiation in vitro. In addition, a key finding of the study was that SC-α and SC-β can be produced together, but intestinal endocrine SC-EC and proliferative non-secretory cells also appear. This suggests that production of safe products requires separation of SC-α and SC-β to high purity, and in order to avoid the risk of cancer, it is also necessary to remove proliferative progenitor cells. Finally, scientists have identified novel surface markers and signaling pathways that are expected to further improve islet cell differentiation and purification through detailed analysis of scRNA-seq data. Therefore, in summary, this study is of great significance, allowing us to be clinically applied from beta cell replacement therapy, thus changing the treatment of diabetes more closely.

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