In this study, we provide an efficient protocol for a high induction of PDX1+/NKX6.1+ pancreatic progenitors in monolayer culture, critical to the generation of functional β cells. We show that dissociation of a densely formed endoderm and re-plating at low density followed by longer retinoid and FGF10 signaling results in a high yield of pancreatic progenitors expressing key markers for pancreatic β-cell development. This enhanced differentiation is complemented by higher proliferation of PDX1+/NKX6.1+ progenitors during the extended stage 3 treatment as well as being due to impeded alternate hepatic lineage specification.

The significance of cell density and cell-cell contact in promoting pancreatic differentiation has been previously reported, specifically in regulating endocrine differentiation [26]. A previous report showed that cell aggregation and cell-cell contacts stimulate growth of the pancreatic epithelium and increase transcription of pancreatic genes in fetal pancreatic tissue in monolayer culture [25]. In this study, we observed that the PDX1 and NKX6.1 co-expression in non-dissociated protocols is only seen in highly dense regions within the adherent cultures, which is in accordance with the recent findings of Toyoda et al. that PDX1+/NKX6.1+ is enriched in the cell aggregates and that a high density is necessary for NKX6.1 induction [27]. Of note, the re-plated cells of P2-D proliferated to give a high density at the end of stage 4, indicating that a high cell density may be required at later stages during pancreatic progenitor differentiation. On the other hand, we found that re-plating of the dissociated endodermal cells at a lower density induced NKX6.1 expression in both PDX1+/NKX6.1+ and PDX1/NKX6.1+ populations. Our results for NKX6.1 induction at very low (a quarter that of the endoderm) density within the novel PDX1/NKX6.1+ population is in accord with a recent study reporting that NKX6.1+ cells can be produced at low-density culture in the presence of ROCK-NM II inhibitor [28].

Interestingly, we found that the novel PDX1/NKX6.1+ population re-oriented themselves in well-defined, compact, 3D structures. Formation of 3D cell clusters has been shown to contribute to islet function and maturation [32, 33]. Expression of NKX6.1 independent of PDX1 in these clusters highlights the high likelihood of this population differentiating into endocrine cells since few studies have pointed out the disappearance of PDX1 at certain stages of development of endocrine progenitors [34, 35]. Additionally, expression of NKX2.2, a downstream target of NGN3 [35, 36], strongly suggests that these cells may be specified towards the endocrine lineage following loss of transient NGN3 expression, but are not yet endocrine cells as suggested by the absence of chromogranin A (Aigha et al., manuscript submitted for publication). Moreover, these clusters also showed a high proliferative capacity, as determined by the expression of Ki67. The characteristics of this novel population presented here suggest that these may be multipotent progenitors en route to an endocrine specification. Overall, our results from manipulation of re-plating densities indicate that dissociation of the densely formed endodermal cells facilitates re-arrangement and re-establishment of appropriate cell-cell contact of the re-plated cells, which improves the efficiency of inducing PDX1+/NKX6.1+ and PDX1/NKX6.1+ pancreatic progenitors, both of which are proposed to be β-cell precursors.

Nonetheless, re-plating the dissociated endodermal cells on a fresh Matrigel matrix may also have beneficial effects on further differentiation into pancreatic progenitors. The crucial role played by the ECM in directing stem cells towards a specific fate has been previously reported [37, 38]. Matrigel, in particular, along with its core component laminin, was shown to have a pro-endocrine effect since pancreatic ductal epithelial cells upregulated endocrine markers on an overlay with Matrigel more than with other matrices such as collagen [39]. Therefore, re-plating on fresh Matrigel matrix during differentiation enhanced the induction of NKX6.1 and other endocrine markers in our dissociated protocols. Of note, several studies have reported the influence of ECM components on pancreatic development, regulating diverse aspects of the cell’s physical microenvironment [2026]. Taken together, our findings indicate the involvement of factors such as the extracellular microenvironment, in addition to the cell density and soluble molecules, in determining pancreatic cell fate during differentiation.

Importantly, our findings showed that an extended stage 3 treatment of 4 days (retinoid, FGF, hedgehog inhibition, and BMP inhibition) induced the highest NKX6.1 expression following dissociation of endoderm, which was higher than those treated for only 2 days. This is in contrast to the recent study that showed that NKX6.1 is predominantly induced by a shorter stage 3 treatment, and its expression is lowest in extended stage 3 (4 days) induced progenitors [19]. It is noteworthy to highlight that dissociation following endoderm trumps the tendency of these differentiated progenitors (P2-D) with extended stage 3 treatment to prevent NKX6.1 expression and differentiate into PDX1+/NKX6.1 cells (P2-ND, as per Nostro et al. [19]) and instead increases overall NKX6.1 mRNA and protein levels. Although our cytokine and growth factor cocktail for all stages was highly similar to the protocol of Nostro et al. [19], with the exception of the dissociation and re-plating steps, the results for pancreatic gene expression, particularly NKX6.1, were different. This could shed light on the dissociation-induced alteration in the physical environment of the cells, implicating modulation of the extracellular microenvironment and cell-cell contact as key players in pancreatic differentiation enhancement.

Retinoid and FGF signaling are activators for PDX1 in the pancreas [18]; therefore, the upregulation of PDX1 expression in the generated progenitors is as a result of extended retinoic acid and FGF signaling, which was enhanced by endodermal cell dissociation. Furthermore, the enhancement of PDX1 expression following dissociation of the endodermal cells is supported by the previously demonstrated inductive effect of dissociating endoderm before differentiating into pancreatic progenitors in improving PDX1 expression [40]. However, in addition to dissociation, our results also implicate retinoid and FGF signaling in inducing NKX6.1 expression since stage 3 extension (P2-D) showed higher NKX6.1 expression than P1-D, while the duration of nicotinamide and EGF treatment (stage 4), which are the known inducers of NKX6.1 [19], remained the same across all protocols.

Both pancreatic and hepatic progenitors originate from DE cells during development and it has been reported that PDX1+ pancreatic precursors develop at the expense of hepatic progenitors [10, 41]. In vitro pancreatic differentiation protocols employ an inhibitor of hepatic specification (for example, Noggin) to enhance pancreatic fate specification [5, 18, 42]. Our method of dissociating endoderm, in turn, dramatically reduced the expression of the hepatic markers AFP and ALBUMIN compared to the non-dissociated cells, highlighting the enhancement of pancreatic specification by our technique. In agreement with our results, a recent study reported that PDX1 binds and inhibits the expression of hepatic markers in hESC-derived pancreatic progenitors [43]. This finding suggests that in addition to BMP inhibition using soluble molecules, dissociation that alters cell density and cell-cell contact, and the ECM are essential players in inhibiting hepatic differentiation during pancreatic specification.

Expansion of pancreatic progenitors is a necessity for scaling up their production for clinical use. Since pancreatic progenitors are multipotent cells [10, 41] it is important to optimize in vitro protocols to tap into their multipotency to enhance their proliferation. Our data showed that dissociation and re-plating at half the endodermal density increased cell proliferation by promoting G1/S transition of the cell cycle during the early stages of differentiation. While a recent study optimized the self-replicating capacity of PDX1+/SOX9+ progenitors [44], it is crucial to specifically increase the proliferative capacity of NKX6.1+ cells to enhance the efficiency of obtaining mono-hormonal endocrine cells. An increased fraction of dissociated cells entering S phase facilitated the generation of ~90% NKX6.1+/Ki67+ proliferative progenitors at the end of stage 4 in our optimized protocol. Moreover, few studies have reported the essential role of cell proliferation regulators in enhancing pancreatic differentiation [45, 46]. Therefore, an increased co-expression of PDX1 and NKX6.1 (P2-D) may be due to increased proliferation of the progenitor cells during differentiation. Indeed, our optimized method for generating PDX1+/NKX6.1+ proliferative pancreatic progenitors could facilitate their scalable production for transplantation therapy.

PDX1+/NKX6.1+ cells generated by our optimized protocol were able to generate NGN3+/NKX6.1+ endocrine progenitors. The induction of NGN3 expression is essential for pancreatic progenitors to be directed toward the endocrine fate [34, 41]. Specifically, co-localization of NKX6.1 with NGN3 in the same endocrine precursor cell specifically directs the cell towards a pancreatic β lineage [41, 47]. Our endocrine precursors also expressed high levels of CHGA and NKX2.2, validating their potential to differentiate into β cells.

Although the generation of mono-hormonal β cells in vitro has been reported [79], the differentiation efficiency and β-cell functionality requires further improvement. To enhance the differentiation efficiency, protocols attempt to mimic the in vivo microenvironment by determining appropriate combination of soluble factors; however, they do not pay close consideration to the in vitro culture conditions represented by the cell’s physical environment that may potentially contribute to improving the differentiation efficiency. Therefore, here we examined the effect of modulating the factors controlling the cell’s physical microenvironment on pancreatic differentiation. The mechanism underlying our observations is not fully understood; however, our results strongly suggest an orchestrated interaction between soluble molecules and the extracellular microenvironment for enhancing pancreatic differentiation efficiency.