Pancreatic islets transplantation is an established treatment method for type 1 diabetic patients with the hypoglycemia unawareness syndrome in whom a therapy with modern technologies fails. Islet transplantation is most commonly done using an interventional radiology method: a tissue suspension of pancreatic islets is applied into a branch of the portal vein through a percutaneously installed catheter. Although being minimally invasive unlike pancreas organ transplant, this method is associated with many technical difficulties. Possible complications of the procedure include hemorrhage and portal vein thrombosis. Unlike their natural dispersed localization in exocrine pancreas, isolated pancreatic islets are exposed to hypoxia, toxins and immunosuppressive drugs in the liver parenchyma. Direct contact with the recipients blood causes an instant blood mediated inflammatory reaction (IBMIR) resulting in the death of more than half of the pancreatic islets shortly after their application. Therefore the size of the islet graft is often insufficient and a number of transplanted patients require administration of exogenous insulin. All of these are reasons for seeking an alternative transplantation site with more hospitable conditions for long-term islet survival. Various transplantation sites have been tested in experimental and clinical research. The advantages and disadvantages of some of them are summarized in this paper. Currently, transplantation into the greater omentum seems most promising, which has already been used in clinical practice at several institutions.

• Klíčová slova
• Transplantation, epidemiology, hypoglycemia unawareness syndrome, impaired hypoglycaemia awarness, omentum, pancreatic islets, transplantation, type 1 diabetes mellitus,
• MeSH
• diabetes mellitus 1. typu * MeSH
• Langerhansovy ostrůvky * MeSH
• lidé MeSH
• omentum MeSH
• pankreas MeSH
• přežívání štěpu MeSH
• transplantace Langerhansových ostrůvků * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.

• MeSH
• Clostridium MeSH
• kolagenasy * MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kolagenasy * MeSH

Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.6 ± 1.2 % of the total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) needed for the proper function of pancreatic β-cells. Additionally, we have revealed a positive effect of ALK5 inhibitor RepSox on the overall reprogramming efficiency. However, the reprogrammed IPCs possessed only a partial insulin-secretory capacity, as they were not able to respond to the changes in the extracellular glucose concentration by increasing insulin secretion. Based on the achieved results we conclude that due to the incomplete reprogramming, the IPCs have immature character and only partial properties of native human β-cells.

• MeSH
• antigen AC133 metabolismus   MeSH
• beta-buňky cytologie   účinky léků   MeSH
• dospělí MeSH
• genetická transkripce účinky léků   MeSH
• homeoboxový protein Nkx-2.2 MeSH
• homeodoménové proteiny MeSH
• inzulin biosyntéza   MeSH
• jaderné proteiny MeSH
• knihovny malých molekul farmakologie   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• organoidy cytologie   MeSH
• přeprogramování buněk účinky léků   genetika   MeSH
• proliferace buněk MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• transkripční faktory bHLH genetika   metabolismus   MeSH
• transkripční faktory MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigen AC133 MeSH
• homeoboxový protein Nkx-2.2 MeSH
• homeodoménové proteiny MeSH
• inzulin MeSH
• jaderné proteiny MeSH
• knihovny malých molekul MeSH
• messenger RNA MeSH
• NEUROG3 protein, human MeSH Prohlížeč
• NKX2-2 protein, human MeSH Prohlížeč
• proteiny nervové tkáně MeSH
• transkripční faktory bHLH MeSH
• transkripční faktory MeSH

Differentiation of pancreatic β-cells is regulated by a wide range of signalling pathways. The aim of our current work was to evaluate the effect of the Jak/Stat signalling pathway on the differentiation of human non-endocrine pancreatic cells into insulin-producing cells. Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor (LIF) stimulated differentiation of C-peptide-negative human non-endocrine pancreatic cells into insulin-producing cells in 6.3 ± 2.0 % cells (N = 5) and induced expression of pro-endocrine transcription factor neurogenin 3, Notch signalling pathway suppressor HES6 and stimulator of β-cell neogenesis REG3A. The expression of the REG3A gene and increased rate of differentiation into insulin-producing cells (10.2 ± 2.1 %) were further stimulated by a combination of LIF with nicotinamide and dexamethasone. Glucose-stimulated (5 vs. 20 mM) C-peptide secretion confirmed proper insulin secretory function of trans-differentiated insulin-producing cells (0.51 vs. 2.03 pmol C-peptide/μg DNA, P < 0.05). Our results indicate that Jak/Stat signalling critically contributes to trans-differentiation of non-endocrine pancreatic cells into functional insulin-producing cells. The positive effect of the Jak/Stat signalling pathway on trans-differentiation is mediated by the key genes that activate differentiation of pancreatic β-cells.

• MeSH
• beta-buňky cytologie   MeSH
• buněčná diferenciace účinky léků   MeSH
• C-peptid MeSH
• imunohistochemie MeSH
• Janus kinasy genetika   metabolismus   MeSH
• kultivované buňky MeSH
• leukemický inhibiční faktor farmakologie   MeSH
• lidé MeSH
• pankreas cytologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny asociované s pankreatitidou MeSH
• signální transdukce účinky léků   MeSH
• transkripční faktory STAT genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-peptid MeSH
• Janus kinasy MeSH
• leukemický inhibiční faktor MeSH
• proteiny asociované s pankreatitidou MeSH
• REG3A protein, human MeSH Prohlížeč
• transkripční faktory STAT MeSH

Differentiation of pancreatic progenitors into insulin-producing β cells is regulated by various transcription factors. To be expressed the genes coding these transcription factors need to be in accessible DNA. Whether a particular gene is present in a form of active euchromatin structure with accessible DNA or in an inactive heterochromatin structure with inaccessible DNA is determined by various epigenetic modifications. We studied the effect of epigenetic modifiers on differentiation of human nonendocrine cells into insulin-producing cells with the aim to evaluate the effect of epigenetic modifications in that process. Within 3 days of cultivation nonendocrine cells form isletlike cell clusters (ILCCs) containing mainly cytokeratin-19-positive cells. After cultivation with epigenetic modifiers and further differentiation, the highest number of C-peptide-positive cells (10.3% ± 2.9%) as well as glucagon-positive cells (7.2% ± 2.8%) was observed in a sample supplemented with a combination of 5-Aza-2'-deoxycytidine modifiers, BIX01294 and MC1568. In response to glucose stimulation (5 vs 20 mmol/L) these ILCCs secreted increased amounts of C-peptide (0.45 vs 1.05 pmol C-peptide/μg DNA). Control samples treated without any epigenetic modifiers showed significantly lower numbers of C-peptide-positive cells (3.5% ± 1.6%). These results showed that a combination of epigenetic modifiers 5-Aza-2'-deoxycytidine (BIX01294 and MC1568) significantly improved reproducible differentiation of nonendocrine pancreatic cells into insulin-producing cells.

• MeSH
• azacytidin analogy a deriváty   farmakologie   MeSH
• azepiny metabolismus   MeSH
• beta-buňky cytologie   MeSH
• biologické modely MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné kultury metody   MeSH
• C-peptid chemie   MeSH
• chinazoliny metabolismus   MeSH
• decitabin MeSH
• epigeneze genetická * MeSH
• imunohistochemie metody   MeSH
• keratin-19 biosyntéza   MeSH
• kmenové buňky cytologie   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• Langerhansovy ostrůvky MeSH
• lidé MeSH
• modely genetické MeSH
• pankreas metabolismus   MeSH
• pyrroly farmakologie   MeSH
• transkripční faktory MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azacytidin MeSH
• azepiny MeSH
• BIX 01294 MeSH Prohlížeč
• C-peptid MeSH
• chinazoliny MeSH
• decitabin MeSH
• keratin-19 MeSH
• kyseliny hydroxamové MeSH
• MC1568 MeSH Prohlížeč
• pyrroly MeSH
• transkripční faktory MeSH