Insulin regulates human pancreatic endocrine cell differentiation in vitro.

OBJECTIVE: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. METHODS: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) ß cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. RESULTS: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-ß cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-ß cell proliferation but had no impact on the expansion of SC-α cells. CONCLUSIONS: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

Automated optimization of endoderm differentiation on chip.

Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues in vitro. Successful engineering of functional cell types and complex organ structures outside the human body requires knowledge of the chemical, temporal, and spatial microenvironment of their in vivo counterparts. Despite an increased understanding of mouse and human embryonic development, screening approaches are still required for the optimization of stem cell differentiation protocols to gain more functional mature cell types. The liver, lung, pancreas, and digestive tract originate from the endoderm germ layer. Optimization and specification of the earliest differentiation step, which is the definitive endoderm (DE), is of central importance for generating cell types of these organs because off-target cell types will propagate during month-long cultivation steps and reduce yields. Here, we developed a microfluidic large-scale integration (mLSI) chip platform for combined automated three-dimensional (3D) cell culturing and high-throughput imaging to investigate anterior/posterior patterns occurring during hiPSC differentiation into DE cells. Integration of 3D cell cultures with a diameter of 150 µm was achieved using a U-shaped pneumatic membrane valve, which was geometrically optimized and fluidically characterized. Upon parallelization of 32 fluidically individually addressable cell culture unit cells with a total of 128 3D cell cultures, complex and long-term DE differentiation protocols could be automated. Real-time bright-field imaging was used to analyze cell growth during DE differentiation, and immunofluorescence imaging on optically cleared 3D cell cultures was used to determine the DE differentiation yield. By systematically alternating transforming growth factor ß (TGF-ß) and WNT signaling agonist concentrations and temporal stimulation, we showed that even under similar DE differentiation yields, there were patterning differences in the 3D cell cultures, indicating possible differentiation differences between established DE protocols. The automated mLSI chip platform with the general analytical workflow for 3D stem cell cultures offers the optimization of in vitro generation of various cell types for cell replacement therapies.

Engineering islets from stem cells for advanced therapies of diabetes.

Diabetes mellitus is a metabolic disorder that affects more than 460 million people worldwide. Type 1 diabetes (T1D) is caused by autoimmune destruction of ß-cells, whereas type 2 diabetes (T2D) is caused by a hostile metabolic environment that leads to ß-cell exhaustion and dysfunction. Currently, first-line medications treat the symptomatic insulin resistance and hyperglycaemia, but do not prevent the progressive decline of ß-cell mass and function. Thus, advanced therapies need to be developed that either protect or regenerate endogenous ß-cell mass early in disease progression or replace lost ß-cells with stem cell-derived ß-like cells or engineered islet-like clusters. In this Review, we discuss the state of the art of stem cell differentiation and islet engineering, reflect on current and future challenges in the area and highlight the potential for cell replacement therapies, disease modelling and drug development using these cells. These efforts in stem cell and regenerative medicine will lay the foundations for future biomedical breakthroughs and potentially curative treatments for diabetes.

Generation of a heterozygous C-peptide-mCherry reporter human iPSC line (HMGUi001-A-8).

The peptide hormone insulin produced by pancreatic ß-cells undergoes post-transcriptional processing before secretion. In particular, C-peptide is cleaved from pro-insulin to generate mature insulin. Here, we introduce a C-peptide-mCherry human iPSC line (HMGUi001-A-8). The line was generated by CRISPR/Cas9 mediated heterozygous insertion of the mCherry sequence into exon 3 of the insulin locus. We demonstrate that the line is pluripotent and efficiently differentiates towards pancreatic ß-like cells, which localize a red fluorescent C-peptide-mCherry fusion protein in insulin containing granules. Hence, the HMGUi001-A-8 line is a valuable resource to purify derived ß-like cells and follow insulin-containing granules in real time.

Generation of an INSULIN-H2B-Cherry reporter human iPSC line.

Differentiating human induced pluripotent stem cells (hiPSCs) into insulin (INS)-producing ß-like cells has potential for diabetes research and therapy. Here, we generated a heterozygous fluorescent hiPSC reporter, labeling INS-producing ß-like cells. We used CRISPR/Cas9 technology to knock-in a T2A-H2B-Cherry cassette to replace the translational INS stop codon, enabling co-transcription and T2A-peptide mediated co-translational cleavage of INS-T2A and H2B-Cherry. The hiPSC-INS-T2A-H2B-Cherry reporter cells were pluripotent and showed multi-lineage differentiation potential. Cells expressing the ß-cell specific hormone INS are identified by nuclear localized H2B-Cherry reporter upon pancreatic endocrine differentiation. Thus, the generated reporter hiPSCs enable live identification of INS hormone-producing ß-like cells.

Pharmacological Targeting of the Actin Cytoskeleton to Drive Endocrinogenesis.

In vitro generation of insulin-secreting beta cells from human pluripotent stem cells (hPSCs) opens new avenues for treating and modeling diabetes. Hogrebe and colleaguesestablished a new 2D differentiation protocol where they targeted the cytoskeleton pharmacologically for controlled endocrine induction and generation of hPSC-derived beta cells with improved function.