As known from model organisms, such as frog, fish, mouse, and chicken, the anterior–posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling ...interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all‐trans‐retinoic acid, TGF‐β‐ligands, Wnt‐, and BMP‐inhibitors in the anterior half of the DE sheet. However, it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2‐positive foregut‐like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut, we purified DE‐cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double‐positive cells does not depend on activin A/TGF‐β‐signaling but is mediated by the inhibition of Wnt‐ and BMP‐signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut toward a PDX1‐positive pancreatic duodenal cell type whereas active Wnt/beta‐catenin signaling synergistically with FGF‐2, BMP‐4, and RA induces the formation of CDX2‐positive posterior endoderm. Thus, these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. Stem Cells 2016;34:2635–2647
Differentiation of pluripotent stem cells (PSC) via the definitive endoderm (DE) germ layer into the patterned primitive gut tube (PGT). The anterior–posterior axis of the PGT is controlled by differential signaling of Wnt/beta‐catenin‐, BMP‐, FGF‐, and RA signaling. BMP inhibition (BMPi) and to a lesser Wnt/beta‐catenin inhibition (WNTi) induce a foregut‐like SOX2‐positive state. Active Wnt/beta‐catenin supported by BMPs, FGFs and all‐trans retinoic acid (ATRA) induce a hindgut‐like state. SOX2 is strongly expressed in PSCs, becomes downregulated during endoderm commitment, and reappears in the foregut domain upon A–P patterning, whereas CDX2 is expressed in the hindgut domain. ATRA is able to posteriorize the foregut.
Abstract NSG mice are among the most immunodeficient mouse model being used in various scientific branches. In diabetelogical research diabetic NSG mice are an important asset as a ...xenotransplantation model for human pancreatic islets or pluripotent stem cell-derived islets. The treatment with the beta cell toxin streptozotocin is the standard procedure for triggering a chemically induced diabetes. Surprisingly, little data has been published about the reproducibility, stress and animal suffering in these NSG mice during diabetes induction. The 3R rules, however, are a constant reminder that existing methods can be further refined to minimize suffering. In this pilot study the dose–response relationship of STZ in male NSG mice was investigated and additionally animal suffering was charted by applying the novel ‘Relative Severity Assessment’ algorithm. By this we successfully explored an STZ dose that reliably induced diabetes while reduced stress and pain to the animals to a minimum using evidence-based and objective parameters rather than criteria that might be influenced by human bias.
Aims/hypothesis
The aim of this study was to examine the effects of proinflammatory cytokines on cells of different developmental stages during the generation of stem cell-derived beta cells (SC-beta ...cells) from human pluripotent stem cells (hPSCs). We wanted to find out to what extent human SC-beta cells are suitable as an experimental cellular model and, with regard to a possible therapeutic use, whether SC-beta cells have a comparable vulnerability to cytokines as bona fide beta cells.
Methods
hPSCs were differentiated towards pancreatic organoids (SC-organoids) using a 3D production protocol. SC-beta cells and non-insulin-producing cells were separated by FACS and differential gene expression profiles of purified human SC-beta cells, progenitor stages and the human beta cell line EndoC-βH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1β, TNF-α and IFN-γ via a transcriptome microarray. Furthermore, we investigated apoptosis based on caspase cleavage, the generation of reactive oxygen species and activation of mitogen-activated protein-kinase (MAPK) stress-signalling pathways.
Results
A 24 h exposure of SC-beta cells to proinflammatory cytokines resulted in significant activation of caspase 3/7 and apoptosis via the extrinsic and intrinsic apoptosis signalling pathways. At this time point, SC-beta cells showed a markedly higher sensitivity towards proinflammatory cytokines than non-insulin-producing cells and EndoC-βH1 cells. Furthermore, we were able to demonstrate the generation of reactive oxygen species and rule out the involvement of NO-mediated stress. A transient activation of stress-signalling pathways p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) was already observed after 10 min of cytokine exposure. The transcriptome analysis revealed that the cellular response to proinflammatory cytokines increased with the degree of differentiation of the cells. Cytokines induced the expression of multiple inflammatory mediators including
IL-32
,
CXCL9
and
CXCL10
in SC-beta cells and in non-insulin-producing cells.
Conclusions/interpretation
Our results indicate that human SC-beta cells respond to proinflammatory cytokines very similarly to human islets. Due to the fast and fulminant cellular response of SC-beta cells, we conclude that SC-beta cells represent a suitable model for diabetes research. In light of the immaturity of SC-beta cells, they may be an attractive model for developmentally young beta cells as they are, for example, present in patients with early-onset type 1 diabetes. The secretion of chemotactic signals may promote communication between SC-beta cells and immune cells, and non-insulin-producing cells possibly participate in the overall immune response and are thus capable of amplifying the immune response and further stimulating inflammation. We demonstrated that cytokine-treated SC-organoids secrete IL-32, which is considered a promising candidate for type 1 diabetes onset. This underlines the need to ensure the survival of SC-beta cells in an autoimmune environment such as that found in type 1 diabetes.
Graphical abstract
The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for ...differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.
In vitro differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) represents a key step towards somatic cells of lung, liver and pancreas. For future clinical ...applications, mass production of differentiated cells at chemically defined conditions and free of xenogeneic substances is envisioned. In this study we adapted our previously published two-dimensional (2D) DE induction protocol to three-dimensional (3D) static suspension culture in the absence of the xenogeneic extracellular matrix Matrigel. Next, fetal calf serum and bovine serum albumin present in the standard medium were replaced by a custom-made and xeno-free B-27. This yielded in a chemically defined and xenogeneic-free 3D culture protocol for differentiation of hPSCs into DE at efficiencies similar to standard 2D conditions. This novel protocol successfully worked with different hPSC lines including hESCs and hiPSCs maintained in two different stem cell media prior to differentiation. DE cells obtained by our novel BSA-free 3D protocol could be further differentiated into PDX1- or NKX6.1-expressing pancreatic progenitor cells. Notably, upon DE differentiation, we also identified a CXCR4
/NCAM
/EpCAM
cell population with reduced DE marker gene expression. These CXCR4
/NCAM
/EpCAM
cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.
Alginate as a versatile naturally occurring biomaterial has found widespread use in the biomedical field due to its unique features such as biocompatibility and biodegradability. The ability of its ...semipermeable hydrogels to provide a favourable microenvironment for clinically relevant cells made alginate encapsulation a leading technology for immunoisolation, 3D culture, cryopreservation as well as cell and drug delivery. The aim of this work is the evaluation of structural properties and swelling behaviour of the core-shell capsules for the encapsulation of multipotent stromal cells (MSCs), their 3D culture and cryopreservation using slow freezing. The cells were encapsulated in core-shell capsules using coaxial electrospraying, cultured for 35 days and cryopreserved. Cell viability, metabolic activity and cell-cell interactions were analysed. Cryopreservation of MSCs-laden core-shell capsules was performed according to parameters pre-selected on cell-free capsules. The results suggest that core-shell capsules produced from the low viscosity high-G alginate are superior to high-M ones in terms of stability during in vitro culture, as well as to solid beads in terms of promoting formation of viable self-assembled cellular structures and maintenance of MSCs functionality on a long-term basis. The application of 0.3 M sucrose demonstrated a beneficial effect on the integrity of capsules and viability of formed 3D cell assemblies, as compared to 10% dimethyl sulfoxide (DMSO) alone. The proposed workflow from the preparation of core-shell capsules with self-assembled cellular structures to the cryopreservation appears to be a promising strategy for their off-the-shelf availability.
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 derived from human embryonic stem cells. The mesodermal-enriched miR-483-3p was identified as an important regulator for the generation of mesodermal PDGFRA+ paraxial cells. Repression of its target PGAM1 significantly increased the number of PDGFRA+ cells. Furthermore, miR-483-3p, miR-199a-3p, and miR-214-3p might also have functions for the mesodermal progenitors. The endoderm-specific miR-489-3p and miR-1263 accelerated and increased endoderm differentiation upon overexpression. KLF4 was identified as a target of miR-1263. RNAi-mediated downregulation of KLF4 partially mimicked miR-1263 overexpression. Thus, the effects of this miRNA were mediated by facilitating differentiation through destabilization of pluripotency along with other not yet defined targets.
•miRNome profiling of purified mesoderm and endoderm compared with human ESCs•19 and 29 differently expressed DE-specific and ME-specific miRNAs, respectively•miR-1263 overexpression increased DE differentiation via KLF4 repression•miR-483-3p regulates the development of PDGFRA+ ME cells via PGAM1 repression
In this article, Ishikawa and colleagues show that miRNAs can regulate early cell-fate decisions during differentiation of human ESCs. miRNome profiling in pure endoderm and mesoderm revealed differentially expressed miRNAs. The endoderm-specific miR-489-3p and miR-1263 increased endoderm specification. The miR-1263 effect was mediated by KLF4 repression. The mesoderm-specific miR-483-3p regulated the generation PDGFRA+ paraxial cells via PGAM1 repression.
Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes.
SOX9
plays a crucial role during ...development of the pancreas and particularly in the development of insulin-producing cells as SOX9
+
cells form the source for NEUROG3
+
endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2K
k
-F2A-GFP2 reporter gene into the
SOX9
-locus and a P2A-mCherry reporter gene into the
INS
-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9
+
cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells.
Graphic Abstract
Growth factors are important regulators during organ development. For many vertebrates (but not humans) it is known how they contribute to the formation and expansion of PDX1-positive cells during ...pancreas organogenesis. Here, the effects of the fibroblast growth factors FGF2, FGF7, FGF10, and epidermal growth factor (EGF) on pancreas development in humans were assessed by using human pluripotent stem cells (hPSCs). During this, FGF2 was identified as a potent anti-pancreatic factor whereas FGF7, FGF10, and EGF increased the cell mass while retaining PDX1-positivity. FGF2 increased the expression of the anti-pancreatic factor sonic hedgehog (
) while suppressing
in a dose-dependent manner. Differentiating cells secreted SHH to the medium and we interrogated the cells' secretome during differentiation to globally examine the composition of secreted signaling factors. Members of the TGF-beta-, Wnt-, and FGF-pathways were detected. FGF17 showed a suppressive anti-pancreatic effect comparable to FGF2. By inhibition of specific branches of FGF-receptor signaling, we allocated the
-induction by FGF2 to MEK/ERK-signaling and the anti-pancreatic effect of FGF2 to the receptor variant FGFR1c or 3c. Altogether, we report findings on the paracrine activity of differentiating hPSCs during generation of pancreatic progenitors. These observations suggest a different role for FGF2 in humans compared to animal models of pancreas organogenesis.
To compare the effects of glucocorticoids and thyroid hormones on the regulation of the beta cell mass in the pancreas, the rats were treated and analyzed for cell cycle changes in islet and duct ...cells as a source for beta cell neogenesis.
Different rat pancreases were morphometrically analyzed after immunohistochemical staining for markers of proliferation and apoptosis.
Hydrocortisone increased the beta cell mass of rat pancreases through an increase of proliferation. This effect was counteracted by an increase of apoptosis. In contrast, thyroxine decreased the beta cell mass through an increase of apoptosis. This effect was counteracted by an increased rate of proliferation. Combined treatment with both hormones nullified the antagonistic effects on proliferation, apoptosis, and beta cell mass, thereby contributing to the maintenance of a stable total beta cell volume of the pancreas.
Hydrocortisone and thyroxine induced analogous changes in pancreatic duct cells, which represent a crucial pool for new beta cells through neogenesis. This may explain the positive effects of glucocorticoids in the immunosuppressive therapy regimen after whole pancreas transplantation upon long-term insulin independence, which is not achievable with isolated islets because of the loss of duct cells during the islet process before transplantation.
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