Ortwin Naujok

Selective Removal of Undifferentiated Embryonic Stem Cells from Differentiation Cultures Through HSV1 Thymidine Kinase and Ganciclovir Treatment

Pluripotent cell lines such as embryonic stem cells are an attractive source for a potential cell replacement therapy. However, transplantation of differentiated cells harbors the risk of teratoma formation, presenting a serious health risk. To overcome this obstacle, a negative selection system was established that permits selective removal of undifferentiated cells during in vitro differentiation. Use of the HSV1 thymidine kinase and eGFP under the control of the Oct4 promoter allowed the destruction of undifferentiated ES cells by ganciclovir treatment; differentiated cells were unharmed. Clonal ES cells remained pluripotent and showed positive staining for a wide range of embryonic markers. Thus, treatment with ganciclovir during in vitro differentiation effectively removed the population of undifferentiated cells and provided a pure population of completely differentiated cells. This approach may pave the way for a safe application of ES cells in regenerative medicine in the future.

Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors.

BackgroundSmall membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized.MethodsCytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated.ResultsCHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T.ConclusionsOut of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.

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 β-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.

The Generation of Definitive Endoderm from Human Embryonic Stem Cells is Initially Independent from Activin A but Requires Canonical Wnt-Signaling

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.

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

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.

A Critical Re-Evaluation of CD24-Positivity of Human Embryonic Stem Cells Differentiated into Pancreatic Progenitors

Differentiation of embryonic stem cells (ESCs) into insulin-producing cells for cell replacement therapy of diabetes mellitus comprises the stepwise recapitulation of in vivo developmental stages of pancreatic organogenesis in an in vitro differentiation protocol. The chemical compounds IDE-1 and (-)-indolactam-V can be used to direct mouse and human ESCs through these stages to form definitive endoderm via an intermediate mesendodermal stage and finally into pancreatic endoderm. Cells of the pancreatic endoderm express the PDX1 transcription factor and contribute to all pancreatic cell types upon further in vitro or in vivo differentiation. Even though this differentiation approach is highly effective and reproducible, it generates heterogeneous populations containing PDX1-expressing pancreatic progenitors amongst other cell types. Thus, a technique to separate PDX1-expressing cells from this mixture is very desirable. Recently it has been reported that PDX1-positive pancreatic progenitors, derived from human embryonic stem cells, express the surface marker CD24. Therefore were subjected mouse and human ESCs to a small molecule differentiation approach and the expression of the surface marker CD24 was monitored in undifferentiated cells, cells committed to the definitive endoderm and cells reminiscent of the pancreatic endoderm. We observed that both mouse and human ESCs expressed CD24 in the pluripotent state, during the whole process of endoderm formation and upon further differentiation towards pancreatic endoderm. Thus CD24 is not a suitable cell surface marker for identification of PDX1-positive progenitor cells.

Beta cell mass regulation in the rat pancreas through glucocorticoids and thyroid hormones.

Objectives: 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. Methods: Different rat pancreases were morphometrically analyzed after immunohistochemical staining for markers of proliferation and apoptosis. Results: 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. Conclusions: 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.

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 β-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.

MicroRNA Target Sites as Genetic Tools to Enhance Promoter-Reporter Specificity for the Purification of Pancreatic Progenitor Cells from Differentiated Embryonic Stem Cells

Pluripotent cells hold great promise for cell replacement therapies in regenerative medicine. All known protocols for directed in vitro differentiation of pluripotent cells did not yield pure populations complicating the characterization of the derived cells. In addition, the risk of tumor formation due to residual undifferentiated cells is a serious unresolved problem. In the present study the tissue-specific mouse Pdx1 promoter was used to control the expression of the reporter gene GFP2 in mouse ES cells in order to purify them via FACS during in vitro differentiation. The background fluorescence of transduced ES cells hampered the purification of Pdx1-positive cells due to a contaminating population of partially undifferentiated cells. MicroRNAs (mir) are important regulators of gene expression and were used to enhance promoter specificity during differentiation towards pancreatic progenitor cells. The mouse mmu-mir-294 was found to be mainly expressed during pluripotency, whereas the expression of the mir-302 cluster was increased during early differentiation. Integration of a microRNA target site for the mmu-mir-294 into the lentiviral vector reduced the background fluorescence specifically during pluripotency and permitted re-occurrence of GFP2 expression upon differentiation. A combination of the microRNA target site with the Pdx1 promoter fragment allowed the purification of pancreatic progenitors from differentiated ES cells. This population reflected an early pancreatic progenitor population without other contaminating cell lineages. In conclusion, microRNA target sites are efficient regulatory elements to control transgene expression and to enhance tissue specificity as presented in this study facilitating the sorting and purification of Pdx1-positive pancreatic progenitor cells.

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‐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