Questions:
1. What question(s) were the authors asking?
2. What general approach(es) did the authors take in order to answer those question(s)?
3. What conclusions did the authors draw, based on the data generated in the figure?
Answers:
1. The prime query of the authors were to explore the capacity to function of the stem cell derived β cells in vivo after their transplantation. The production of efficient human pancreatic β cells in vitro was the main goal of this research. Thus the question that these In vitro cultured pancreatic β are able to secret insulin in vivo became the prime objective of this research. The authors were also interested to see whether the SC- β cells secrets insulin in response to glucose or not. Figure 5 along with supplementary figure S5 represent the results of those question.
2. The figure 5 showed that the transplanted SC- β cells work sharply in vivo. ELISA measurement of human insulin serum of mice transplanted using comparable amounts of HUES8 SC- β cells (500-1000 IEQ) or PH cells (5 X 106 cells) is showed in the figure 5A. Measurement of human insulin in the blood of a subsection of mice after 2 weeks of post-transplantation were taken before (0 minute) and after (30 minutes) an acute glucose challenging are represented as white bars and black bars consecutively. Immunohistochemistry tests of both SC- β cells and human islet grafts detected the presence C-peptide+ cells and glucagon. The staining with C-peptide+ cells (green) and glucagon (GCG: red) confirmed the existence of grafts (figure 5B and S5).
The general approaches the authors took in order to answer those questions was to transplant the SC- β cells underneath the kidney capsule of immunocompromised mice to check the capability of these cells to function in vivo. Primary human islets (500 IEQ or islet equivalents) were transplanted in those glucose challenged mice. On the other hand, 5 million pancreatic progenitor cells were also transplanted into mice. The authors also experimented whether insulin is evident in the serum of mice transplanted with 5 million SC- β cells earlier than 3-4 months and also the earliest post-transplantation time pointe was tested. The authors injected the mice transplanted with SC- β with glucose subsequently a transitory operating recovery time (2 weeks). Then the serum of those mice was collected and the amount of human insulin in the serum was measured by ELISA. The authors also transplanted the equal quantity of pancreatic progenitors and PH cells as a control. To investigation if the SC- β cells produce insulin in reaction to glucose, the authors measured human insulin in the blood of a subsection of mice both earlier (0 minute) and later (30 minutes) of an acute glucose challenging. Histology of the engrafted kidneys was done after 2 weeks post-transplantation.
3. Based on the data generated in the figure it was confirmed that transplanted SC- β cells functioned rapidly in vivo. Human insulin was identified in the serum of glucose tested mice within 2 weeks after transplantation with primary human islets. But no insulin was detected after 2 weeks when 5 million pancreatic progenitor cells were transplanted into mice. The data of ELISA measurement shown that SC- β cells from both hiPSC and hESC secreted insulin within 2 weeks into mice blood. The controlled pancreatic progenitors and PH cells didn’t secreted noteworthy amount of insulin in vivo within 2 weeks (figure5A). After the glucose challenge, 73% of SC- β transplanted mice (27/37 mice) exhibited amplified human insulin in their blood 2 weeks post-transplantation (figure 5A and tables S2 and S3). In contrast with the previous result, an amplified human insulin secretion was observed subsequent to the glucose challenge in 75% of human islet transplanted mice (9/12 mice). This increased insulin (p= 0.0008) is statistically a vital data. The average ratio of secreted insulin following the glucose challenge compared to the former was 1.9 ± 0.3 for islets transplants and 1.7 ± o.2 for SC- β cell transplants. These in vivo stimulation indices ranged from 0.4 to 4.3 for islets transplants and from 0.5 to 3.8 for SC- β cell transplants (tables S2 and S3). The authors concluded from the data in figure 5 that the production of SC- β cells provided a potentially valuable step in the direction of islet and pancreatic organ generation, in vitro drug discover and treatment of hyperglycaemia.
Two initial questions for discussion:
- How SC- β cells resemble human islet β cells by gene expression and ultrastructure? How SC- β cell transplantation ameliorates hyperglycaemia in mice?
- How might β cells be mass produced to meet future clinical need? How can the β cells made from hPSCs be refined and developed? Will transplantation of human pluripotent stem cell-based β cells provide a cure or therapy?
