Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 27;10(1):1190.
doi: 10.1038/s41598-020-57787-0.

Controlled Clustering Enhances PDX1 and NKX6.1 Expression in Pancreatic Endoderm Cells Derived From Pluripotent Stem Cells

Affiliations
Free PMC article

Controlled Clustering Enhances PDX1 and NKX6.1 Expression in Pancreatic Endoderm Cells Derived From Pluripotent Stem Cells

Raymond Tran et al. Sci Rep. .
Free PMC article

Abstract

Pluripotent stem cell (PSC)-derived insulin-producing cells are a promising cell source for diabetes cellular therapy. However, the efficiency of the multi-step process required to differentiate PSCs towards pancreatic beta cells is variable between cell lines, batches and even within cultures. In adherent pancreatic differentiation protocols, we observed spontaneous local clustering of cells expressing elevated nuclear expression of pancreatic endocrine transcription factors, PDX1 and NKX6.1. Since aggregation has previously been shown to promote downstream differentiation, this local clustering may contribute to the variability in differentiation efficiencies observed within and between cultures. We therefore hypothesized that controlling and directing the spontaneous clustering process would lead to more efficient and consistent induction of pancreatic endocrine fate. Micropatterning cells in adherent microwells prompted clustering, local cell density increases, and increased nuclear accumulation of PDX1 and NKX6.1. Improved differentiation profiles were associated with distinct filamentous actin architectures, suggesting a previously overlooked role for cell-driven morphogenetic changes in supporting pancreatic differentiation. This work demonstrates that confined differentiation in cell-adhesive micropatterns may provide a facile, scalable, and more reproducible manufacturing route to drive morphogenesis and produce well-differentiated pancreatic cell clusters.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Differentiation of iPSCs into PF cells on tissue culture plastic with timed addition of soluble factors without confinement. (A) The stages of development are mimicked by timed media changes and monitored by expression of key transcription factors. (B) Expression of key pancreatic transcription factors relative to GAPDH based on qPCR. No significant differences were found between β-actin and GAPDH. Each bar represents the average of three separate differentiations (N = 3). (C) Mean nuclear PDX1 intensity at the PF and PE stages of differentiation, showing a small proportion cells with higher PDX1 expression (PDXhigh) emerging at the PE stage. (D) In monolayer cultures, roughly circular aggregates of PDX1high cells were observed sporadically in culture. n.sp > 0.05, *p < 0.05, **p < 0.01, ****p < 0.001 for a one-way ANOVA. Full Tukey multiple comparisons post-hoc test results are shown in Table S1. Scale bars represent 100 µm.
Figure 2
Figure 2
Confined culture of iPSC-derived PF cells in micropatterns promotes clustering of cells in predictable patterns. (A) Process flow to microfabricate agarose microwells to spatially confine iPSC-derived PF cells. (B) Agarose microwells of 150 µm, 300 µm and 500 µm diameters were successfully fabricated with high between-well consistency in diameter. (C) Microwells facilitate confined cell clustering within 72 hours. (D) Areas of 4 concentric circles used for spatial analysis of cell density. (E) Nuclei are concentrated within inner radii of microwells. (F) Local cell density in concentric microwell regions for 150 (n = 21), 300 (n = 13), and 500 (n = 9) µm microwells. (G) Cell densification occurs in 150 µm microwells similar to aggregates in unconfined culture. (H) Bulged morphology observed after 72 hours in 150 µm microwells but not in 300 µm or 500 µm microwells. Representative images of max intensity projection and 3D reconstructions in yz (top) and xy (bottom) plane. ****p < 0.001 for an one-way ANOVA performed on the iPSC-derived cells. Full Tukey multiple comparisons post-hoc test results are shown in Table S2. Scale bars represent 100 µm (B,C,E,G) and 50 μm (H).
Figure 3
Figure 3
Spatially confined differentiation of PF cells promotes the expression of PE markers. (A) Confined culture of iPSC-derived PF cells increases staining intensity of PDX1 and NKX6.1 as shown by immunocytochemistry. Displayed intensity ranges have been matched between confined and unconfined samples to illustrate increased staining intensity (B) Mean N:C ratio in PDX1 (n = 21, 13, 9 for 150, 300, and 500 µm microwells) and NKX6.1 (n = 12, 8, 8 for 150, 300, and 500 µm microwells) immunofluorescence increased when presented with sufficient geometric confinement. Each point represents a data point from a single microwell. n.sp > 0.05, **p < 0.01, ***p < 0.005 for a one-way ANOVA with Tukey post-hoc multiple comparisons. Scale bars: 100 µm.
Figure 4
Figure 4
Controlled cluster culture drives spatially dependent patterns of PDX1 and NKX6.1 within the cluster. (A) Mean nuclear PDX1 (n = 21, 13, 9 for 150, 300, and 500 µm microwells) and NKX6.1 (n = 12, 8, 8 for 150, 300, and 500 µm microwells) intensity varies region-to-region and shows correlation to local cell densities. (B) Confined PF cells show increased PDX1 and NKX6.1 intensity on a single cell level. Dashed lines represent limits of regions defined in Fig. 2D. n.s.p > 0.05, **p < 0.01, ****p < 0.001 for a one-way ANOVA between different regions.
Figure 5
Figure 5
Confined culture promotes temporal changes in F-actin organization during differentiation. (A) Micropatterned iPSC-derived PF cell colonies that show visibly lower PDX1 intensity (red arrows) are correlated with distinct actin cytoskeleton structures concentrated at the microwell periphery. (B) Characteristic actin structures found throughout 72 hours of differentiation in 150 µm microwells. These structures are grouped based on actin intensity analysis (bottom). The reported intensity profile is obtained from an average of 8 intersecting lines (Fig. S6). (C) Proportion of wells with actin structure (classified in (B) at 24 (n = 10), 48 (n = 19), and 72 (n = 32) hours. Distribution of actin structures changes throughout differentiation and aggregates towards the center in 150 µm microwells. (D) PF colonies in 150 µm microwells with central actin distribution (n = 16) showed increased nuclear PDX1 expression compared to those with peripheral actin distribution (n = 5). Addition of Y-26732 abrogated any increase in nuclear PDX1 expression compared to the unconfined control (n = 12). (E) Addition of Y-26732 ROCK inhibitor 24 hours after seeding prevents actin structural reorganization.  *p < 0.05, ****p < 0.001 for the student’s t-test. Scale bars: 100 µm.
Figure 6
Figure 6
Proposed mechanism of action for increased PDX1 expression via collective actin “purse-string” contraction.

Similar articles

See all similar articles

References

    1. Shapiro AMJ, et al. Islet Transplantation in Seven Patients with Type 1 Diabetes Mellitus Using a Glucocorticoid-Free Immunosuppressive Regimen. N. Engl. J. Med. 2000;343:230–238. doi: 10.1056/NEJM200007273430401. - DOI - PubMed
    1. Hering BJ, et al. Phase 3 Trial of Transplantation of Human Islets in Type 1 Diabetes Complicated by Severe Hypoglycemia. Diabetes Care. 2016;39:1230. doi: 10.2337/dc15-1988. - DOI - PMC - PubMed
    1. Nostro MC, et al. Efficient Generation of NKX6-1(+) Pancreatic Progenitors from Multiple Human Pluripotent Stem Cell Lines. Stem Cell Rep. 2015;4:591–604. doi: 10.1016/j.stemcr.2015.02.017. - DOI - PMC - PubMed
    1. Pagliuca FW, et al. Generation of Functional Human Pancreatic β Cells In Vitro. Cell. 2014;159:428–439. doi: 10.1016/j.cell.2014.09.040. - DOI - PMC - PubMed
    1. Rezania A, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotech. 2014;32:1121–1133. doi: 10.1038/nbt.3033. - DOI - PubMed
Feedback