Differentiating functional human islet-like aggregates from pluripotent stem cells

Abstract

We present here a robust and reliable protocol by which to differentiate pancreatic islet-like aggregates (SC-islets) from human pluripotent stem cells. The 7-stage protocol mimics developmental patterning factors that induce endocrine lineage formation and spans monolayer, microwell, and aggregate suspension culture. The SC-islets demonstrate dynamic glucose-sensitive insulin secretion and an endocrine cell composition similar to those of primary human islets. SC-islets generated using this optimized protocol are suitable for in vitro modeling of islet cell pathophysiology and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Balboa et al. (2022).

Keywords: Cell differentiation; Stem cells.

Graphical abstract

Figure 1

Examples of Good and Poor PSC seeding prior to the start of the differentiation Before the start of DE induction PSCs should be at 100% confluency (left panel). Any gaps in the cell layer or drops in confluency due to excessive cell death or under-seeding will negatively affect the differentiation (right panel, white arrow heads). Scale bar represents 200 μm.

Figure 2

Estimating DE induction efficiency A flow cytometry plot of CXCR4-labeling following a successful Stage 1 DE induction. CXCR4+ cells (red), negative control (blue).

Figure 3

Morphological clues of pancreatic progenitor differentiation efficiency Cell layer thickening during Stage 3 and Stage 4 of the protocol (Day 7 to Day 10) and maintenance of confluency is a strong indicator of pancreatic progenitor formation (left panels). A break in the cell layer or the formation of raised “ribbons” of cells is an indicator of poor pancreatic progenitor induction (middle and right panels, white dotted line areas). Pancreatic progenitor marker (PDX1, green), non-pancreatic marker (AFP, red). Scale bars represent 400 μm.

Figure 4

Representative images of microwell plates following anti-adherence treatment Following anti-adherence treatment and centrifugation, microwells should be free of any trapped air/bubbles (left panel). However, some microwells may contain trapped air bubbles that would impede aggregate formation (right panel, red arrow heads) and require further centrifugation (step 26 b).

Figure 5

Overview of correct pipetting technique for media exchanges within AggreWell plates Gentle handling of seeded microwell plates is integral for maintaining aggregate yield. Media exchanges should follow the key principles in the left most panel. Media aspiration also follows the same pattern with slow aspiration of media with a serological pipette. Poor handling of seeded microwells, such as overly fast media addition or aspiration, will cause aggregate loss during aspiration. AggreWell plates should never be tilted for media exchanges as this may also dislodge the seeded aggregates.

Figure 6

Example images of AggreWell seeding at Day 11 and aggregate formation by Day 14 Pancreatic progenitor seeding should appear evenly spread across all microwells and adequately fill each microwell (Day 11). In the following days (Stage 4 to Stage 5 transition) the progenitors should form spherical aggregates with low levels of cell debris. Scale bars represent 400 μm.

Figure 7

Flow cytometry and IHC assessment of pancreatic progenitors generated by Day 11 of this protocol NKX6.1 and PDX1 double-positive cell proportion following a successful induction (left panel), negative control (blue) and antibody-probed cells (red). An even cell layer of double-positive cells can also be seen through IHC assessment of planar cultures prior to dissociation and aggregations (right panel). Scale bar represents 200 μm.

Figure 8

Morphology and predominant endocrine cell populations of SC-islets The expected morphology of SC-islets throughout Stage 6 and Stage 7 (left panel). Flow cytometric analysis of insulin- and glucagon-positive cells from early and late Stage 7 SC-islets (right panels). Antibodies used are shown in the key resources table. Scale bar represents 1,000 μm.

Figure 9

Expected insulin secretion and viability measurements of SC-islets Normalized insulin secretion values of SC-islets in 3 mM glucose (G3) and 17 mM glucose (G17), together with expected total insulin content. Low TUNEL fluorescence indicates high cell viability within SC-islets. Scale bar represents 200 μm.

Figure 10

Expected cell culture morphology throughout the protocol Each panel represents the expected cell confluency/structure at each Stage of the protocol (denoted by the S prefix). Key small molecule modulators of the differentiation step are highlighted in yellow. Approximate time of culture in each stage is also highlighted in white. All scale bars represent 200 μm.

Figure 11

Examples of expected and poor microwell aggregation of PPs Detached and reaggregated PPs of high purity and viability will quickly form rounded aggregates in microwell culture (left panel). Low PP formation or poor handling of Stage 4 cultures during the microwell plating process will result in poor aggregate formation and high cell death (right panel). Scale bar represents 400 μm.