Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

Reviewer #1 (Public Review):

In this manuscript, the authors are developing a new protocol that aims at expanding pancreatic progenitors derived from human pluripotent stem cells under GMP-compliant conditions. The strategy is based on hypothesis-driven experiments that come from knowledge derived from pancreatic developmental biology.

The topic is of major interest in the view of the importance of amplifying human pancreatic progenitors (both for fundamental purposes and for future clinical applications). There is indeed currently a major lack of information on efficient conditions to reach this objective, despite major recurrent efforts by the scientific community.

Using their approach that combines stimulation of specific mitogenic pathways and inhibition of retinoic acid and specific branches of the TGF-beta and Wnt pathways, the authors claim to be able, in a highly robust and reproducible manner) to amplify in 10 passages the number of pancreatic progenitors (PP) by 2,000 folds, which is really an impressive breakthrough.

The work is globally well-performed and quite convincing. I have however some technical comments mainly related to the quantification of pancreatic progenitor amplification and to their differentiation into beta-like cells following amplification.

Reviewer #2 (Public Review):

Summary:

The paper presents a novel approach to expand iPSC-derived pdx1+/nkx6.1+ pancreas progenitors, making them potentially suitable for GMP-compatible protocols. This advancement represents a significant breakthrough for diabetes cell replacement therapies, as one of the current bottlenecks is the inability to expand PP without compromising their differentiation potential. The study employs a robust dataset and state-of-the-art methodology, unveiling crucial signaling pathways (eg TGF, Notch...) responsible for sustaining pancreas progenitors while preserving their differentiation potential in vitro.

Strengths:

This paper has strong data, guided omics technology, clear aims, applicability to current protocols, and beneficial implications for diabetes research. The discussion on challenges adds depth to the study and encourages future research to build upon these important findings.

Weaknesses:

The paper does have some weaknesses that could be addressed to improve its overall clarity and impact. The writing style could benefit from simplification, as certain sections are explained in a convoluted manner and difficult to follow, in some instances, redundancy is evident. Furthermore, the legends accompanying figures should be self-explanatory, ensuring that readers can easily understand the presented data without the need to be checking along the paper for information.

The culture conditions employed in the study might benefit from more systematic organization and documentation, making them easier to follow.

Another important aspect is the functionality of the expanded cells after differentiation. While the study provides valuable insights into the expansion of pancreas progenitors in vitro and does the basic tests to measure their functionality after differentiation the paper could be strengthened by exploring the behavior and efficacy of these cells deeper, and in an in vivo setting.

Quantifications for immunofluorescence (IF) data should be displayed.

Some claims made in the paper may come across as somewhat speculative.

Additionally, while the paper discusses the potential adaptability of the method to GMP-compatible protocols, there is limited elaboration on how this transition would occur practically or any discussion of the challenges it might entail.

Reviewer #3 (Public Review):

Summary:

In this work, Jarc et al. describe a method to decouple the mechanisms supporting progenitor self-renewal and expansion from feed-forward mechanisms promoting their differentiation.

The authors aimed at expanding pancreatic progenitor (PP) cells, strictly characterized as PDX1+/SOX9+/NKX6.1+ cells, for several rounds. This required finding the best cell culture conditions that allow sustaining PP cell proliferation along cell passages, while avoiding their further differentiation. They achieve this by comparing the transcriptome of PP cells that can be expanded for several passages against the transcriptome of unexpanded (just differentiated) PP cells.

The optimized culture conditions enabled the selection of PDX1+/SOX9+/NKX6.1+ PP cells and their consistent, 2000-fold, expansion over ten passages and 40-45 days. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. These optimized culture conditions consisted of substituting the Vitamin A containing B27 supplement with a B27 formulation devoid of vitamin A (to avoid retinoic acid (RA) signaling from an autocrine feed-forward loop), substituting A38-01 with the ALK5 II inhibitor (ALK5i II) that targets primarily ALK5, supplementation of medium with FGF18 (in addition to FGF2) and the canonical Wnt inhibitor IWR-1, and cell culture on vitronectin-N (VTN-N) as a substrate instead of Matrigel.

Strengths:

The strength of this work relies on a clever approach to identify cell culture modifications that allow expansion of PP cells (once differentiated) while maintaining, if not reinforcing, PP cell identity. Along the work, it is emphasized that PP cell identity is associated with the co-expression of PDX1, SOX9, and NKX6.1. The optimized protocol is unique (among the other datasets used in the comparison shown here) in inducing a strong upregulation of GP2, a unique marker of human fetal pancreas progenitors. Importantly GP2+ enriched hPS cell-derived PP cells are more efficiently differentiating into pancreatic endocrine cells (Aghazadeh et al., 2022; Ameri et al., 2017).

The unlimited expansion of PP cells reported here would allow scaling-up the generation of beta cells, for the cell therapy of diabetes, by eliminating a source of variability derived from the number of differentiation procedures to be carried out when starting at the hPS cell stage each time. The approach presented here would allow the selection of the most optimally differentiated PP cell population for subsequent expansion and storage. Among other conditions optimized, the authors report a role for Vitamin A in activating retinoic acid signaling in an autocrine feed-forward loop, and the supplementation with FGF18 to reinforce FGF2 signaling.

This is a relevant topic in the field of research, and some of the cell culture conditions reported here for PP expansion might have important implications in cell therapy approaches. Thus, the approach and results presented in this study could be of interest to researchers working in the field of in vitro pancreatic beta cell differentiation from hPSCs. Table S1 and Table S4 are clearly detailed and extremely instrumental to this aim.

Weaknesses:

The experiments performed and the methods used to evaluate the treatment effects are well-suited and state-of-the-art. However, further details on the characterization or the discussion of some of the results might help to more clearly contextualize their findings, and improve their impact on the field.

The authors strictly define PP cells as PDX1+/SOX9+/NKX6.1+ cells, and this phenotype was convincingly characterized by immunofluorescence, RT-qPCR, and FACS analysis along the work. However, broadly defined PDX1+/SOX9+/NKX6.1+ could include pancreatic multipotent progenitor cells (MPC, defined as PDX1+/SOX9+/NKX6.1+/PTF1A+ cells) or pancreatic bipotent progenitors (BP, defined as PDX1+/SOX9+/NKX6.1+/PTF1A-) cells. It has been indeed reported that Nkx6.1/Nkx6.2 and Ptf1a function as antagonistic lineage determinants in MPC (Schaffer, A.E. et al. PLoS Genet 9, e1003274, 2013), and that the Nkx6/Ptf1a switch only operates during a critical competence window when progenitors are still multipotent and can be uncoupled from cell differentiation. It would be important to define whether culturing PDX1+/SOX9+/NKX6.1+ PP (as defined in this work) in the best conditions allowing cell expansion is reinforcing either an MPC or BP phenotype. Data from Figure S2A (last paragraph of page 7) suggests that PTF1A expression is decreased in C5 culture conditions, thus more homogeneously keeping BP cells in this media composition. However, on page 15, 2nd paragraph it is stated that "the strong upregulation of NKX6.2 in our procedure suggested that our ePP cells may have retracted to an earlier PP stage". Evaluating the co-expression of the previously selected markers with PTF1A (or CPA2), or the more homogeneous expression of novel BP markers described, such as DCDC2A (Scavuzzo et al. Nat Commun 9, 3356, 2018), in the different culture conditions assayed would more shield light into this relevant aspect.

In line with the previous comment, it would be extremely insightful if the authors could characterize or at least discuss a potential role for YAP underlying the mechanistic effects observed after culturing PP in different media compositions. It is well known that the nuclear localization of the co-activator YAP broadly promotes cell proliferation, and it is a key regulator of organ growth during development. Importantly in this context, it has been reported that TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors and disruption of this interaction arrests the growth of the embryonic pancreas (Cebola, I. et al. Nat Cell Biol 17, 615-26, 2015). More recently, it has also been shown that a cell-extrinsic and intrinsic mechanotransduction pathway mediated by YAP acts as gatekeeper in the fate decisions of BP in the developing pancreas, whereby nuclear YAP in BPs allows proliferation in an uncommitted fate, while YAP silencing induces EP commitment (Mamidi, A. et al. Nature 564, 114-118, 2018; Rosado-Olivieri et al. Nature Communications 10, 1464, 2019). This mechanism was further exploited recently to improve the in vitro pancreatic beta cell differentiation protocol (Hogrebe et al., Nature Protocols 16, 4109-4143, 2021; Hogrebe et al, Nature Biotechnology 38, 460-470, 2020). Thus, YAP in the context of the findings described in this work could be a key player underlying the proliferation vs differentiation decisions in PP.

Regarding the improvements made in the PP cell culture medium composition to allow expansion while avoiding differentiation, some of the claims should be better discussed and contextualized with current state-of-the-art differentiation protocols. As an example, the use of ALK5 II inhibitor (ALK5i II) has been reported to induce EP commitment from PP, while RA was used to induce PP commitment from the primitive gut tube cell stage in recently reported in vitro differentiation protocols (Hogrebe et al., Nature Protocols 16, 4109-4143, 2021; Rosado-Olivieri et al. Nature Communications 10, 1464, 2019). In this context, and to the authors' knowledge, is Vitamin A (triggering autocrine RA signaling) usually included in the basal media formulations used in other recently reported state-of-the-art protocols? If so, at which stages? Would it be advisable to remove it?

In this line also, the supplementation of cell culture media with the canonical Wnt inhibitor IWR-1 is used in this work to allow the expansion of PP while avoiding differentiation. A role for Wnt pathway inhibition during endocrine differentiation using IWR1 has been previously reported (Sharon et al. Cell Reports 27, 2281-2291.e5, 2019). In that work, Wnt inhibition in vitro causes an increase in the proportion of differentiated endocrine cells. It would be advisable to discuss these previous findings with the results presented in the current work. Could Wnt inhibition have different effects depending on the differential modulation of the other signaling pathways?