Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galß1-3GlcNAcß1-3Galß1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galß1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Proinflammatory cytokines induce rapid, NO-independent apoptosis, expression of chemotactic mediators and interleukin-32 secretion in human pluripotent stem cell-derived beta cells.

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-ßH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1ß, 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-ßH1 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.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage.

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.

Anterior-Posterior Patterning of Definitive Endoderm Generated from Human Embryonic Stem Cells Depends on the Differential Signaling of Retinoic Acid, Wnt-, and BMP-Signaling.

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-ß-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-ß-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.

Exploring dose-response variability and relative severity assessment in STZ-induced diabetes male NSG mice.

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.

Reversal of diabetes through gene therapy of diabetic rats by hepatic insulin expression via lentiviral transduction.

Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5-7 days after the virus injection of 7 × 10(9) INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50-100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic ß-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach.

Insulin-producing surrogate ß-cells from embryonic stem cells: are we there yet?

Embryonic stem cells (ESCs) harbor the potential to generate every cell type of the body by differentiation. The use of hESCs holds great promise for potential cell replacement therapies for degenerative diseases including diabetes mellitus. The recently discovered induced pluripotent stem cells (iPSCs) exhibit immense potential for regenerative medicine as they allow the generation of autologous cells tailored to the patients' immune system. Research for insulin-producing surrogate cells from ESCs has yielded highly controversial results, because many steps and factors in the differentiation process are currently still unknown. Thus, there is no consensus on common standard protocols. The protocols presently used established the differentiation from pluripotent cells toward pancreatic progenitor cells. However, none of the differentiation protocols reported to date have generated by exclusive in vitro differentiation sufficient numbers of insulin-producing cells meeting all essential criteria of a ß-cell. The cells often lack the crucial function of regulated insulin secretion upon glucose stimulation. This review focuses on past and current approaches to the generation of insulin-producing cells from pluripotent sources, such as ESCs and iPSCs, and critically discusses the hurdles to be taken before insulin-secreting surrogate cells derived from these stem cells will be of clinical use in humans.

New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells.

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-2Kk-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.

Design and Derivation of Multi-Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n-Mediated Homology-Directed Repair.

During the past decade, RNA-guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) has become a powerful tool for gene editing of human pluripotent stem cells (PSCs). Using paired CRISPR/Cas9 nickases (CRISPR/Cas9n) it is furthermore possible to reduce off-target effects that may typically occur with traditional CRISPR/Cas9 systems while maintaining high on-target efficiencies. With this technology and a well-designed homology-directed repair vector (HDR), we are now able to integrate transgenes into specific gene loci of PSCs in an allele conserving way. In this protocol we describe CRISPR/Cas9n design and homology directed repair vector design, transfection of human pluripotent stem cells and selection and expansion of generated cell clones. © 2020 The Authors. Basic Protocol 1: Repair template design and CRISPR/Cas9n construction Basic Protocol 2: Transfection of human pluripotent stem cells by electroporation Basic Protocol 3: Genotyping of generated cell clones.

FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells.

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 (SHH) while suppressing PDX1 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 SHH-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.

Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo.

BACKGROUND: Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. METHODS: ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. RESULTS: In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. CONCLUSIONS: A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation.

Chemically defined and xenogeneic-free differentiation of human pluripotent stem cells into definitive endoderm in 3D culture.

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+/EpCAMlow cell population with reduced DE marker gene expression. These CXCR4+/NCAM+/EpCAMlow cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.

Chemically-Defined, Xeno-Free, Scalable Production of hPSC-Derived Definitive Endoderm Aggregates with Multi-Lineage Differentiation Potential.

For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.

A new experimental protocol for preferential differentiation of mouse embryonic stem cells into insulin-producing cells.

Mouse embryonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with beta-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohormonal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation.

Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting.

Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm-committed cells in high numbers in a 4-day treatment protocol. Second, a method for the purification of CXCR4-positive endoderm cells by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm-derived somatic cells. © 2017 by John Wiley & Sons, Inc.

miRNome Profiling of Purified Endoderm and Mesoderm Differentiated from hESCs Reveals Functions of miR-483-3p and miR-1263 for Cell-Fate Decisions.

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.

Generation and Purification of Definitive Endoderm Cells Generated from Pluripotent Stem Cells.

Differentiation of pluripotent stem cells into cells of the definitive endoderm requires an in vitro gastrulation event. Differentiated somatic cells derived from this germ layer may then be used for cell replacement therapies of degenerative diseases of the liver, lung, and pancreas. Here we describe an endoderm differentiation protocol, which initiates the differentiation from a defined cell number of dispersed single cells and reliably yields in >70-80 % endoderm-committed cells in a short 5-day treatment regimen.

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Gene Transfer into Pluripotent Stem Cells via Lentiviral Transduction.

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes, shRNAs, miRNAs, or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines.