Figure 5 - available via license: Creative Commons Attribution 3.0 Unported
Content may be subject to copyright.
hFSCs Have the Capacity to Differentiate into Multiple Derivatives of the Foregut (A) Method to differentiate hFSCs into pancreatic cells. (B) hFSCs grown in these culture conditions progressively express pancreatic bud markers (PDX1 and HLXB9) and then endocrine markers (INS and NGN3). F, fetal pancreas; A, adult pancreas. (C) C-peptide and PDX1 expression was confirmed by immunocytochemistry of cells differentiated for 25 days. (legend continued on next page)
Source publication
Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we...
Contexts in source publication
Context 1
... confirm this hypothesis, we tested the ability of hFSCs to differentiate into liver and pancreatic cells using culture systems recently developed by our group to produce these cells types directly from hPSCs ( Cho et al., 2012;Rashid et al., 2010). hFSCs grown in culture conditions inductive for pancreatic specification (Cho et al., 2012) ( Figure 5A) sequentially expressed early pancreatic markers (HLXB9 and PDX1), then endocrine progenitor marker (NGN3), and finally beta cell marker (c-peptide/insulin) ( Figure 5B). As reported previously, more than 90% of cells were PDX1 positive at stage 4 of the pancreatic differentiation protocol (Figures S4B and S4C) ( Cho et al., 2012). ...
Context 2
... confirm this hypothesis, we tested the ability of hFSCs to differentiate into liver and pancreatic cells using culture systems recently developed by our group to produce these cells types directly from hPSCs ( Cho et al., 2012;Rashid et al., 2010). hFSCs grown in culture conditions inductive for pancreatic specification (Cho et al., 2012) ( Figure 5A) sequentially expressed early pancreatic markers (HLXB9 and PDX1), then endocrine progenitor marker (NGN3), and finally beta cell marker (c-peptide/insulin) ( Figure 5B). As reported previously, more than 90% of cells were PDX1 positive at stage 4 of the pancreatic differentiation protocol (Figures S4B and S4C) ( Cho et al., 2012). ...
Context 3
... reported previously, more than 90% of cells were PDX1 positive at stage 4 of the pancreatic differentiation protocol (Figures S4B and S4C) ( Cho et al., 2012). After 25 days of differentiation, cells expressing c-peptide, PDX1, glucagon (GCG), and somatostatin (SST) could be observed by immunocytochemistry and c-peptide release was detected upon glucose stimulation ( Figures 5C and 5D). Impor- tantly, approximately 15%-20% of pancreatic cells were positive for C-peptide and 10%-20% of these C-peptide- positive cells were polyhormonal (i.e., coexpressing both C-peptide and either GCG [20%] (C) Accordingly, the resulting organoids could be grown for 2 months while progressively increasing the expression of gut markers (OLFM4, CHGA, MUC2, Villin, and KRT19). ...
Context 4
... further characterization beyond the scope of the current study will be necessary to validate the real functionality of the insulin-expressing cells gener- ated under our culture conditions. Similar analyses were performed using our liver differentiation protocol and hFSCs described previously ( Hannan et al., 2013;Rashid et al., 2010;Yusa et al., 2011) ( Figure 5E). Under these con- ditions, hFSCs rapidly differentiate into a near-homoge- nous population of hepatocyte-like cells expressing liver markers (AFP, ALB, A1AT, HNF4a, and CK18; Figure 5F and 5G). ...
Context 5
... analyses were performed using our liver differentiation protocol and hFSCs described previously ( Hannan et al., 2013;Rashid et al., 2010;Yusa et al., 2011) ( Figure 5E). Under these con- ditions, hFSCs rapidly differentiate into a near-homoge- nous population of hepatocyte-like cells expressing liver markers (AFP, ALB, A1AT, HNF4a, and CK18; Figure 5F and 5G). In addition, a vast majority of hepatocyte-like cells (90%) coexpressed albumin and ASGPR and alpha-1- antitrypsin as reported previously ( Figures S4H-S4K) (Rashid et al., 2010;Hannan et al., 2013). ...
Context 6
... addition, a vast majority of hepatocyte-like cells (90%) coexpressed albumin and ASGPR and alpha-1- antitrypsin as reported previously ( Figures S4H-S4K) (Rashid et al., 2010;Hannan et al., 2013). Further functional characterization also showed that these cells could uptake low-density lipoprotein ( Figure 5H) and cardiogreen from tissue culture medium ( Figure S6) and secrete AAT and albu- min (data not shown and Figure 5I). Importantly, multiple hFSC lines derived from different hIPSC lines displayed similar differentiation efficiency ( Figures S5 and S6), while frozen/thawed hFSCs could also generate liver and pancre- atic cells. ...
Context 7
... addition, a vast majority of hepatocyte-like cells (90%) coexpressed albumin and ASGPR and alpha-1- antitrypsin as reported previously ( Figures S4H-S4K) (Rashid et al., 2010;Hannan et al., 2013). Further functional characterization also showed that these cells could uptake low-density lipoprotein ( Figure 5H) and cardiogreen from tissue culture medium ( Figure S6) and secrete AAT and albu- min (data not shown and Figure 5I). Importantly, multiple hFSC lines derived from different hIPSC lines displayed similar differentiation efficiency ( Figures S5 and S6), while frozen/thawed hFSCs could also generate liver and pancre- atic cells. ...
Context 8
... functional characterization also showed that these cells could uptake low-density lipoprotein ( Figure 5H) and cardiogreen from tissue culture medium ( Figure S6) and secrete AAT and albu- min (data not shown and Figure 5I). Importantly, multiple hFSC lines derived from different hIPSC lines displayed similar differentiation efficiency ( Figures S5 and S6), while frozen/thawed hFSCs could also generate liver and pancre- atic cells. Indeed, defrosted hFSCs could be expanded for five passages and then differentiate efficiently into cells ex- pressing pancreatic (PDX1 > 90%) and hepatic (AFP > 90%) markers ( Figures S4J-S4M). ...
Context 9
... contaminating cells of nonendodermal origin stopped proliferating and progres- sively disappeared upon passaging. Flow cytometry ana- lyses show that cells grown for five passages homogenously expressed SOX17 and CXCR4 (99%) similarly to hFSCs generated from hIPSCs proficient for endoderm production (BBHX8) ( Figure 5A). Therefore, our culture system selec- tively amplifies hFSCs even when they originate from a heterogeneous population of DE cells. ...
Context 10
... resulting popu- lation of hFSCs was expanded for two additional passages and then transferred into culture conditions inductive for pancreas and liver differentiation. Cells differentiated toward the liver lineage expressed hepatic markers (AAT, ALB, AFP, and HNF4a) at a level similar to hepatocyte-like cells generated from control hFSCs ( Figure 5B). Similarly, cells differentiated toward the pancreatic lineage cells expressed PDX1, INS, and NGN3 ( Figure 5C). ...
Context 11
... differentiated toward the liver lineage expressed hepatic markers (AAT, ALB, AFP, and HNF4a) at a level similar to hepatocyte-like cells generated from control hFSCs ( Figure 5B). Similarly, cells differentiated toward the pancreatic lineage cells expressed PDX1, INS, and NGN3 ( Figure 5C). Together, these results show that hFSCs can be easily generated from hIPSCs with reduced endoderm differentiation capac- ity, enabling the production of hepatic and pancreatic cells. ...
Similar publications
Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchro...
Pluripotent stem cells hold great promise for regenerative medicine since they can differentiate into all somatic cells. MicroRNAs (miRNAs) could be important for the regulation of these cell-fate decisions. Profiling of miRNAs revealed 19 differentially expressed miRNAs in the endoderm and 29 in the mesoderm when analyzing FACS-purified cells deri...
During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in...
Traditional cell culture media do not accurately represent the availability of the nutrients in plasma. They usually contain a supraphysiological concentration of nutrients such as glucose, amino acids, etc. These high nutrients can alter the metabolism of cultured cells and induce metabolic phenotypes that do not reflect in vivo conditions. We dem...
Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will s...
Citations
... The Hippo signaling pathway is essential for regulating pancreatic development, as well as β cell proliferation, differentiation and survival. Overexpression of YAP-S6A in pancreatic progenitor cells reduces the differentiation efficiency into β-like cells, but increases the number of proliferating β-like cells [26,93,[99][100][101][102]. FGF10, fibroblast growth factor 10; EGF, epidermal growth factor; TGFβ, transforming growth factor β; Inh, inhibitor; P, passage; Ha, harmine; LY, LY364947; LIF, leukemia inhibitory factor; DE, definitive endoderm; FG, foregut; PP, pancreatic progenitor; TesR1, mTESR™1; BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; MEF, mouse embryonic fibroblast; HGF, hepatocyte growth factor; OE, overexpression. ...
... can generate functional islet organoids in a mouse model has further encouraged these approaches [96]. The synergistic effect of several drugs based on the new understanding of the mechanism of pancreatic β cell replication has significantly increased human β cell proliferation ( Table 2, Fig. 2) [24][25][26]83,89,92,93,[97][98][99][100][101][102]. ...
In the quest to combat insulin-dependent diabetes mellitus (IDDM), allogenic pancreatic islet cell therapy sourced from deceased donors represents a significant therapeutic advance. However, the applicability of this approach is hampered by donor scarcity and the demand for sustained immunosuppression. Human induced pluripotent stem cells are a game-changing resource for generating synthetic functional insulin-producing β cells. In addition, novel methodologies allow the direct expansion of pancreatic progenitors and mature β cells, thereby circumventing prolonged differentiation. Nevertheless, achieving practical reproducibility and scalability presents a substantial challenge for this technology. As these innovative approaches become more prominent, it is crucial to thoroughly evaluate existing expansion techniques with an emphasis on their optimization and scalability. This manuscript delineates these cutting-edge advancements, offers a critical analysis of the prevailing strategies, and underscores pivotal challenges, including cost-efficiency and logistical issues. Our insights provide a roadmap, elucidating both the promises and the imperatives in harnessing the potential of these cellular therapies for IDDM.
... There has been good progress developing in vitro systems that recapitulate the structure and function of a variety of organs. [1][2][3][4][5][6][7][8][9] Retinal organoids, for example, contain all the major retinal neuronal and glial (M€ uller glia) cell classes and follow the developmental timing and laminar organization of the retina. 10 Importantly, retinal organoids represent an accessible and renewable in vitro system for studies of development 11 and disease progression. ...
With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA sequencing (RNA-seq) multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10× methods produce highly concordant cell-class compositions and then expand sci-Plex to analyze the cell-class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to 6 weeks later. Our data show sci-Plex's potential to dramatically scale up the analysis of treatment conditions on relevant human models.
... Over the past decade, rapid progress has been made towards designing in vitro systems that recapitulate the structure and function of organs. These organs in a dish or "organoids'' now exist for the retina, brain, kidney, intestine and more [1][2][3][4][5][6][7][8][9] . The extent to which retinal organoids recapitulate development of the human fetal retina is nothing short of remarkable. ...
With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA-seq multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10x methods produce highly concordant cell class compositions and then expand sci-Plex to analyze the cell class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to six weeks later. Our data show the potential of sci-Plex to dramatically scale-up the analysis of treatment conditions on relevant human models.
... Our investigation has revealed a variant resulting in decreased ApoB-containing lipoprotein secretion in homozygotes (but not heterozygotes), in contrast to other variants causing abetalipoproteinemia where ApoB is undetectable (Fig. 2, Table 1) 6,7, [23][24][25] . Although other carriage of this variant have been reported, no phenotypic characteristics related to these are previously described 15 , none of the GWAS so far including that using UK biobank 26 and largest cross-ancestry GWAS 27 Our phenotyping studies demonstrated distinct VLDL secretion responses following meal challenge and ApoB levels in the two MTTP-564TT family members: while these biomarkers were substantially low in untreated individual F, these were in the normal range in the liver transplant recipient J ( Figure 2b; Table 1). ...
... All cells were differentiated into hepatocytes as described previously [24]. Briefly, hiPSCs were seeded at a density of 15,000-20,000 cells/cm 2 (dependant on the cell line) onto Matrigel-coated plasticware. ...
Background and aims
Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes.
Methods
Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison to healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis and molecular expression arrays.
Results
We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion compared to wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators and production of reactive oxygen species.
Conclusion
We have identified and characterized a rare causal variant in MTTP and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinemia. Our findings provide insights into mechanisms driving progressive NAFLD.
... Though very exciting data, demonstration of reproducibility and durability of effect is still pending (68). To address the challenge of low cell yield when attempting to differentiate pancreatic progenitors into maturing β cells in vitro, several groups have been able to expand human foregut progenitors in vitro (217), or further, expand pancreatic progenitor cells before continued differentiation (218)(219)(220)(221). Sneddon et al (221) co-cultured progenitor cells with organmatched mesenchyme to enable cellular proliferation and selfrenewal. Trott et al (218) used EGF, retinoic acid, TGFβ inhibitor, and Notch inhibitor-fortified media to support the long-term (> 20 passages) self-renewal of pancreatic progenitors when replated on 3T3-J2 feeder cells but faced challenges of reduced NKX6.1 expression. ...
For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing β-cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing β-cells. Protocols have been developed that mimic the normal in vivo development of human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature β-like cells in vitro that produce insulin but do not yet fully mimic healthy mature β-cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3D bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.
... hPSC-derived progenitor cells can be aggregated to elicit self-organization and subsequent organoid formation (Hannan et al., 2013;Kumar et al., 2019;Takebe et al., 2017). Potentially significant advantages of this approach include (1) bypassing the reliance on the highly variable spontaneous spheroid production and detachment step during the early stages of organoid formation, and (2) the potential for progenitors to be cryopreserved before aggregation and organoid formation, facilitating the production of banks of organoid precursors that can be accessed without maintaining hPSCs in culture. ...
A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.
... During the process of human pluripotent stem cell differentiation, human foregut stem cells were derived and those were maintained in vitro while retaining their capacity to differentiate into pancreatic and hepatic cells [31]. Mou and colleagues reported differentiation into NKX2.1 ? ...
Background:
The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity.
Methods:
By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome.
Results:
These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions.
Conclusion:
Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.
... In recent years, several groups reported methods for culturing human endodermal derivatives at early developmental stages (24)(25)(26). In terms of pancreatic progenitors, Trott et al. (27) developed a culture condition that could expand PDX1-positive pancreatic progenitors, but unfortunately, these progenitors could not maintain the expression of NKX6.1. ...
An unlimited source of human pancreatic β cells is in high demand. Even with recent advances in pancreatic differentiation from human pluripotent stem cells, major hurdles remain in large-scale and cost-effective production of functional β cells. Here, through chemical screening, we demonstrate that the bromodomain and extraterminal domain (BET) inhibitor I-BET151 can robustly promote the expansion of PDX1+NKX6.1+ pancreatic progenitors (PPs). These expandable PPs (ePPs) maintain pancreatic progenitor cell status in the long term and can efficiently differentiate into functional pancreatic β (ePP-β) cells. Notably, transplantation of ePP-β cells rapidly ameliorated diabetes in mice, suggesting strong potential for cell replacement therapy. Mechanistically, I-BET151 activates Notch signaling and promotes the expression of key PP-associated genes, underscoring the importance of epigenetic and transcriptional modulations for lineage-specific progenitor self-renewal. In summary, our studies achieve the long-term goal of robust expansion of PPs and represent a substantial step toward unlimited supplies of functional β cells for biomedical research and regenerative medicine.
... Generation of lineage-committed expandable progenitors is an effective approach for ensuring sufficient quantity and quality of cell sources for various biomedical applications. Recent examples of this concept include generation of foregut stem cells (35) and multipotent endoderm progenitors (36) from hPSCs. The hIEC progenitors also contain proliferating progenitor-like cells that express ISC makers and are suitable for further intestinal differentiation. ...
Advanced technologies are required for generating human intestinal epithelial cells (hIECs) harboring cellular diversity and functionalities to predict oral drug absorption in humans and study normal intestinal epithelial physiology. We developed a reproducible two-step protocol to induce human pluripotent stem cells to differentiate into highly expandable hIEC progenitors and a functional hIEC monolayer exhibiting intestinal molecular features, cell type diversity, and high activities of intestinal transporters and metabolic enzymes such as cytochrome P450 3A4 (CYP3A4). Functional hIECs are more suitable for predicting compounds metabolized by CYP3A4 and absorbed in the intestine than Caco-2 cells. This system is a step toward the transition from three-dimensional (3D) intestinal organoids to 2D hIEC monolayers without compromising cellular diversity and function. A physiologically relevant hIEC model offers a novel platform for creating patient-specific assays and support translational applications, thereby bridging the gap between 3D and 2D culture models of the intestine.
... Previous studies have identified methods to expand definitive endoderm, foregut endoderm, and pancreatic progenitor populations ( Figure 1) (44,46,66,67). While these approaches demonstrated the feasibility to induce self-renewal without impacting the developmental potential of hPSC-derived cells, these methods proved difficult to scale up. ...
Restoring the number of glucose-responsive β-cells in patients living with diabetes is critical for achieving normoglycemia since functional β-cells are lost during the progression of both type 1 and 2 diabetes. Stem cell-derived β-cell replacement therapies offer an unprecedented opportunity to replace the lost β-cell mass, yet differentiation efficiencies and the final yield of insulin-expressing β-like cells are low when using established protocols. Driving cellular proliferation at targeted points during stem cell-derived pancreatic progenitor to β-like cell differentiation can serve as unique means to expand the final cell therapeutic product needed to restore insulin levels. Numerous studies have examined the effects of β-cell replication upon functionality, using primary islets in vitro and mouse models in vivo, yet studies that focus on proliferation in stem cell-derived pancreatic models are only just emerging in the field. This mini review will discuss the current literature on cell proliferation in pancreatic cells, with a focus on the proliferative state of stem cell-derived pancreatic progenitors and β-like cells during their differentiation and maturation. The benefits of inducing proliferation to increase the final number of β-like cells will be compared against limitations associated with driving replication, such as the blunted capacity of proliferating β-like cells to maintain optimal β-cell function. Potential strategies that may bypass the challenges induced by the up-regulation of cell cycle-associated factors during β-cell differentiation will be proposed.
