Initial Cell Seeding Density Influences Pancreatic Endocrine Development During <i>in vitro</i> Differentiation of Human Embryonic Stem Cells

<div><p>Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date <i>in vitro</i>-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the <i>in vitro</i> formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 10⁴ cells/cm² to 5.3 x 10⁴ cells/cm² followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of <i>MNX1</i>, <i>PTF1a</i>, <i>NGN3</i>, <i>ARX</i>, and <i>PAX4</i> compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (<i>PDX1</i> and <i>MNX1</i>) expression, followed by pancreatic endocrine specification marker expression (<i>BRN4</i>, <i>PAX4</i>, <i>ARX</i>, <i>NEUROD1</i>, <i>NKX6.1</i> and <i>NKX2.2</i>) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols. </p> </div

High Cell Seeding Density Increases Pancreatic Progenitor Differentiation.

<p>(A) hESCs seeded at different densities were differentiated for 14 days and immunostained for PDX1 (green) and DNA (blue). (B) Single-cell quantification of PDX1 positive nuclei as a percentage of total nuclei (C) RT-qPCR of 21 day differentiated cells. Expression is shown relative to isolated human islets. Different superscripts (a, b, c) are significantly different from each other within each graph by one-way ANOVA with Bonferroni post-hoc test. Scale bars are 100 μm.</p

Higher Cell Seeding Density Enhances Pancreatic Endocrine Formation.

<p>(A) Insulin, glucagon, and somatostatin expression were assessed in 21 day differentiated hESCs using RT-qPCR (shown relative to human islets). (B) C-peptide and glucagon release were assayed in static 24 hour media samples take on the indicated culture, or during a sequential glucose and/or potassium chloride (KCl) stimulated hormone release assay performed on day 19 of culture. Following a 2 hour 2 mM glucose wash, cells were treated for 1 hour with 2 mM glucose (2G), 25 mM glucose (25G), then 30 mM KCl (30KCl). Diamonds, squares, triangles, and circles represent 1.3 x 10⁴ cells/cm², 2.6 x 10⁴ cells/cm², 3.9 x 10⁴ cells/cm², and 5.3 x 10⁴ cells/cm² initial seeding density respectively. * represents p<0.05 comparing 5.3 x 10⁴ cells/cm² with other cell densities. # represents p<0.05 comparing KCl stimulation versus other stimuli within the 5.3 x 10⁴ cells/cm² seeding density. (C) hESCs seeded at different densities and differentiated for 21 days were agarose-embedded and immunostained for insulin (blue), glucagon (green), somatostatin (red) and DNA (cyan). Right panel shows hormone staining and left panel shows the same hormone image with DNA. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082076#pone.0082076.s004" target="_blank">Figure S4</a> for single channel images of a larger field of view. (D) Single-cell quantification of hormone population showing the number of cells positive for insulin, glucagon, or somatostatin as a percentage of the total number of nuclei. (E) Single-cell polyhormonal analysis of the hormone positive population in C as a percentage of total hormone positive population. Triple indicates cells scored positive for all three hormones. * represents p<0.05 comparing triple positive populations of 5.3 x 10⁴ cells/cm² vs 3.9 x 10⁴ cells/cm². In panels A and D, different superscripts (a, b) are significantly different from each other within each graph by one-way ANOVA with Bonferroni post-hoc test. Scale bars are 50 μm.</p

Higher Cell Seeding Density Improves Definitive Endoderm Differentiation.

<p>(A) CA1S hESCs were differentiated using a protocol designed to mimic human development in a 21 day, 5 stage process. (B) hESCs were seeded onto matrigel-coated culture plates at the indicated density, yielding 30%-100% confluence as shown at 24 hours after seeding. (C) On day 4 of differentiation, markers of definitive endoderm induction were assessed by flow cytometry (CXCR4 and SOX17 expression) or RT-qPCR (<i>FOXA2</i> and Goosecoid, shown relative to undifferentiated hESC expression levels). (D) Expression of OCT4 (marker of pluripotent cells) was assessed by RT-qPCR and immunofluorescence as a percentage of the total number of nuclei (OCT4 is green, nuclei are blue). * represents significant difference from 1.3 x 10⁴ cells/cm² by one-way ANOVA with Bonferroni post-hoc test. Different superscripts (a, b, c) are significantly different from each other within each graph by one-way ANOVA with Bonferroni post-hoc test. Scale bars are 100 μm. </p

Higher Cell Seeding Density Decreases Cell Cycle Progression.

<p>(A) A representative histogram (left) of low density (1.3 x 10⁴ cells/cm², black line) and high density (5.3 x 10⁴ cells/cm², red line) seeded CA1S hESCs stained for DNA content by propidium iodide to indicate cell cycle state within the depicted gates 24-hours after seeding. (B) Single cells gated for uniform DNA width were assessed in triplicate and quantified as either G0/G1, S or G2/M phases using the gates in (A) as a percentage of the total single cell population. Four cell seeding densities of CA1S cells (1.3, 2.6, 3.9 and 5.3 x 10⁴ cells/cm²) along with 5.3 x 10⁴ cells/cm² seeded cells treated overnight with 2% DMSO to induce cell cycle arrest (2% DMSO) were quantified. * represents significant difference from 1.3 x 10⁴ cells/cm² by one-way ANOVA with Bonferroni post-hoc test within the same cell cycle population. (C) Representative images and quantification of immunocytochemistry of pRb S780 (green, nuclei are blue). pRb S780 positive mitotic cells were quantified as a percentage of the total cell populations in five randomly selected images. Different superscripts (a, b, c) are significantly different from each other by one-way ANOVA with Bonferroni post-hoc test. Scale bars are 100 μm.</p

High Seeding Density Cultures Follow Expected Endocrine Developmental Timeline.

<p>(A) Media samples from multiple (N=12) high hESC cell seeding density differentiations contain reproducibly high levels of C-peptide and glucagon as measured by radioimmunoassay. (B) Over the differentiation time course expression of transcription factors and islet hormones was examined by RT-qPCR relative to adult human islet expression levels. (C) 21 day differentiated hESCs were immunostained as agarose-embedded, paraffinized sections for pancreatic hormones and key transcription factors involved in pancreatic endocrine induction and maturation. * represents p<0.05 comparing day 14 and 21 media content. Different superscripts (a, b, c) are significantly different from each other within each graph by one-way ANOVA with Bonferroni post-hoc test. Scale bars are 50 μm.</p

Decreased amplitude in intracellular Ca²⁺ responses to tolbutamide in <i>Lepr^flox/flox RIP-Cre</i> adult islets.

<p>A: Representative recordings from a control <i>Lepr^flox/flox</i> islet (solid line) and a <i>Lepr^flox/flox RIP-Cre</i> islet (dotted line) in response to tolbutamide. B: Graph plotting ΔFmax-Fmin of each peak in response to tolbutamide in the population of islets that showed two peaks. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test, *** p<0.0001. Responses are representative of 22 islets from 4 mice per group.</p

Transmission electron microscopy reveals autophagy within <i>Lepr^flox/flox RIP-Cre</i> β-cells.

<p>Pancreas sections from <i>Lepr^flox/flox</i> (A) and <i>Lepr^flox/flox RIP-Cre</i> (B and D) mice were analyzed by transmission electron microscopy (magnification 9300X and 11000X) and quantified (C). Multigranular bodies were numerous in <i>Lepr^flox/flox RIP-Cre</i> β-cells compared to <i>Lepr^flox/flox</i> β-cells (white squares). Events of macroautophagy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071075#pone-0071075-g004" target="_blank">Figure 4D</a>, upper inset) and microautophagy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071075#pone-0071075-g004" target="_blank">Figure 4D</a>, bottom inset) were captured in <i>Lepr^flox/flox RIP-Cre</i> β-cells. Scale bar = 2 µm (A and B) and 0.5 µm (D). Micrographs are representative of 3 pancreata analyzed per group. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test, ** p<0.01.</p

Similar glucose metabolic rates in islets from <i>Lepr^flox/flox</i> and <i>Lepr^flox/flox RIP-Cre</i> adult mice.

<p>A: Two [NAD(P)H]_i representative recordings of a <i>Lepr^flox/flox</i> islet (solid line) and a <i>Lepr^flox/flox RIP-Cre</i> islet (dotted line) in response to glucose (G) and sodium azide (NaN₃). B: Graph plotting percentage of AUC/min in response to different glucose concentrations and normalized to the maximum reduction level obtained with 3 mM NaN₃. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test. Graphs are representative of 32–34 islets from 3 mice per group.</p

<i>Lepr^flox/flox RIP-Cre</i> pancreatic β-cells display impaired intracellular Ca²⁺ oscillations in response to glucose.

<p>A: [Ca²⁺]_i recordings of a <i>Lepr^flox/flox</i> islet (left panel) and <i>Lepr^flox/flox RIP-Cre</i> islet (right panel) in response to increasing glucose (G) concentrations and potassium chloride (KCl) from adult mice. B and C: Representative [Ca²⁺]_i recordings showing three different regions per islet of a <i>Lepr^flox/flox</i> islet (left panel) and a <i>Lepr^flox/flox RIP-Cre</i> islet (right panel) from adult (B) and neonatal (C) mice. Graphs are representative of 17–20 islets from 3 neonatal mice per group, and 37–38 islets from 3–4 adult mice per group.</p