Uriel Barkai - Academia.edu (original) (raw)
Papers by Uriel Barkai
La presente invention porte sur un procede de transplantation de cellules souches hematopoietique... more La presente invention porte sur un procede de transplantation de cellules souches hematopoietiques non differenciees sur un receveur. Le procede consiste a (a) faire se developper les cellules souches hematopoietiques non differenciees pour augmenter le nombre des cellules souches hematopoietiques par (i) la culture dans un bioreacteur a ecoulement piston en phase stationnaire de cellules stromales sous un courant continu d'un milieu de culture etabli sur un substrat biocompatible tridimensionnel afin de generer une culture tridimensionnelle de cellules stromales ; et (ii) l'ensemencement de cellules souches hematopoietiques non differenciees dans le bioreacteur a ecoulement piston en phase stationnaire comprenant la culture tridimensionnelle de cellules stromales et sous un courant continu d'un milieu de culture pour developper les cellules souches hematopoietiques non differenciees et obtenir une culture tridimensionnelle de cellules stromales comprenant un nombre accr...
La presente invention porte sur un procede de transplantation de cellules souches hematopoietique... more La presente invention porte sur un procede de transplantation de cellules souches hematopoietiques non differenciees sur un receveur. Le procede consiste a (a) faire se developper les cellules souches hematopoietiques non differenciees pour augmenter le nombre des cellules souches hematopoietiques par (i) la culture dans un bioreacteur a ecoulement piston en phase stationnaire de cellules stromales sous un courant continu d'un milieu de culture etabli sur un substrat biocompatible tridimensionnel afin de generer une culture tridimensionnelle de cellules stromales ; et (ii) l'ensemencement de cellules souches hematopoietiques non differenciees dans le bioreacteur a ecoulement piston en phase stationnaire comprenant la culture tridimensionnelle de cellules stromales et sous un courant continu d'un milieu de culture pour developper les cellules souches hematopoietiques non differenciees et obtenir une culture tridimensionnelle de cellules stromales comprenant un nombre accr...
American Journal of Transplantation, 2018
Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells wh... more Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem cell-derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The βAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the βAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1-2 βAir devices, each containing 155 000-180 000 islet equivalents (ie, 1800-4600 islet equivalents per kg body weight), and monitored for 3-6 months, followed by the recovery of devices. Implantation of the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C-peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose-stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the βAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).
Proceedings of the National Academy of Sciences of the United States of America, Oct 31, 2017
Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with ... more Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.
PLoS ONE, 2013
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Proceedings of the National Academy of Sciences, 2013
Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destr... more Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies over the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit a widespread application of these strategies. In this paper we present the clinical success of using a bioartificial pancreas for the transplantation of insulin-producing islets without affecting the immune system. In a patient with long-standing type-1 diabetes we could demonstrate persistent graft function and regulated insulin secretion without the need for immune-modulating medication. This strategy opens up avenues for more widespread and safe application of various cell-based therapies.
Journal of Stem Cell and Transplantation Biology, 2017
Islet transplantation effectively treats diabetes but relies on immune suppression and is practic... more Islet transplantation effectively treats diabetes but relies on immune suppression and is practically limited by the number of cadaveric islets available. An alternative cellular source is insulin-producing cells derived from pluripotent cell sources. Three animal cohorts were used in the current study to evaluate whether an oxygen-providing macroencapsulation device, 'βAIR', could function in conjunction with human embryonic stem cells (hESCs) and their derivatives. The first cohort received macro-encapsulated undifferentiated hESCs, a second cohort received hESCs differentiated to a pancreatic progenitor state with limited endocrine differentiation. A reference cohort received human islets. Macro-encapsulation devices were implanted subcutaneously and monitored for up to four months. Undifferentiated pluripotent stem cells did not form teratoma but underwent cell death following implantation. Human C-peptide (hC-peptide) was detectable in host serum one week after implantation for both other cohorts. hC-peptide levels decreasing over time but remained detectable up to the end of the study. Key factors associated with mature endocrine cells were observed in grafts recovered from cohorts containing islets and hESC-derivatives including C-peptide, insulin, glucagon and urocortin 3. We conclude that the 'βAIR' macroencapsulation device is compatible with both human islets and pluripotent derivatives, but has a limited capability of sustaining undifferentiated pluripotent cells.
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2015
Current treatment options for adrenal insufficiency are limited to corticosteroid replacement the... more Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic-pituitary-adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) ...
Abstract. [3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei... more Abstract. [3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.azalR embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in S EVERAL studies support the view that retinoic acid (RA) t promotion of differentiation of em...
Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pig... more Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or organs. To reduce the xenotransplantation-associated risk of infec-tion of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of micro-organisms was examined in Göttingen Minipigs by PCRmethods. Since zoonotic transmis-sion of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly estab...
Biochemical and Biophysical Research Communications, 1998
The rat 20α-hydroxysteroid dehydrogenase (20α-HSD) is an enzyme responsible for the catabolism of... more The rat 20α-hydroxysteroid dehydrogenase (20α-HSD) is an enzyme responsible for the catabolism of progesterone to the inactive 20α-hydroxyprogesterone. We have previously shown that the expression of this enzyme is not regulated by post-translational modification, but at the level of transcription. In this study we have established that the 20α-HSD gene contains nine exons and have isolated a 2.5 kb promoter region. The transcription start site was identified and a TATA box was found. 5′ deletions of this promoter significantly decreased basal promoter activity. Treatment with forskolin led to a dose dependent inhibition of the 2.5kb-20α-HSD-luciferase construct. Computer analysis identified one CRE, two Nur77 response elements, two putative AP1 sites and one progesterone response element half-site. In summary, we have identified and partially characterized the promoter region of the rat ovarian 20α-HSD and demonstrated that the regulatory elements for 20α-HSD are present within a 2...
Apparatus (20) comprising: - a housing (22) configured for insertion into a body of a patient ,; ... more Apparatus (20) comprising: - a housing (22) configured for insertion into a body of a patient ,; - a permeable selective membrane (32) - functional cells (30) immuno by the membrane (32) which prevent the passage therethrough of the cells from the patient and coupled to the housing (22); - an oxygen supply (24); characterized in that the oxygen supply (24) comprises a container comprising oxygen gas, which is configured to deliver gaseous oxygen to the functional cells (30); oxygen supply (24) remaining outside the patient's body and wherein the apparatus (20) further comprises: - a tube (34) coupled transcutaneously oxygen supply (24) to the housing (22 ).
The objective of this study was to characteriae the estrogen action that confers endomethal sensi... more The objective of this study was to characteriae the estrogen action that confers endomethal sensitization to nontraumatic deciduogenic stimuli by use of antiestrogens. Tamoxifen, ethamoxytriphetol, and clomiphene and its two component enantiomers inhibited decidual induction in pseudopregnant rats when administered 17 h before pyrathiazine. Unexpectedly, clomiphene (250 pg/rat) and tamoxifen (25 pg/rat) proved inhibitory at all times up to and including the time of induction. Clomiphene, administered in the hours preceding decidual induction, inhibited the increase of ornithine decarboxylase activity, which nor-mally marks the end of the induction phase. Clomiphene had no inhibitory effect on the availability or receptor binding of progesterone. Clomiphene also inhibited implantation of blastocysts when administered at the time of their adherence to the uterus. The inhibition by antiestrogens of decidual induction could not be explained on the basis of the current understanding of m...
Decidualization of endometrial stroma in the rat induces the expression and secretion of rat deci... more Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (UIII) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, UIII cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor β (ERβ) and α2-macroglobulin (α2-MG), which are known to be PRL-regulated genes. However, when UIII cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 a...
Islet transplantation is a feasible therapeutic alternative for metabolically labile patients wit... more Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the g...
At present, proven clinical treatments but no cures are available for diabetes, a global epidemic... more At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, espe...
PLOS ONE
Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients sh... more Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.
PLOS ONE
Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients sh... more Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.
Xenotransplantation, Jul 1, 2016
Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of... more Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of porcine zoonotic micro-organisms. Hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine endogenous retroviruses (PERVs) are potentially zoonotic micro-organisms which do not show clinical symptoms in pigs and which are due to the low expression level difficult to detect. Göttingen Minipigs (GöMP) are often used for biomedical investigations and they are well characterized concerning the presence of numerous bacteria, fungi, viruses and parasites and therefore may be used for islet cell transplantation. Islet cells derived from three GöMP were transplanted into four healthy, non-diabetic cynomolgus monkeys using a macroencapsulation device. PCR, nested PCR, real-time PCR, real-time RT-PCR and Western blot analyses were used to estimate the presence of PERV, PCMV and HEV in the donors and recipients. Using sensitive detection methods, no HEV was found in the donor pigs and i...
La presente invention porte sur un procede de transplantation de cellules souches hematopoietique... more La presente invention porte sur un procede de transplantation de cellules souches hematopoietiques non differenciees sur un receveur. Le procede consiste a (a) faire se developper les cellules souches hematopoietiques non differenciees pour augmenter le nombre des cellules souches hematopoietiques par (i) la culture dans un bioreacteur a ecoulement piston en phase stationnaire de cellules stromales sous un courant continu d'un milieu de culture etabli sur un substrat biocompatible tridimensionnel afin de generer une culture tridimensionnelle de cellules stromales ; et (ii) l'ensemencement de cellules souches hematopoietiques non differenciees dans le bioreacteur a ecoulement piston en phase stationnaire comprenant la culture tridimensionnelle de cellules stromales et sous un courant continu d'un milieu de culture pour developper les cellules souches hematopoietiques non differenciees et obtenir une culture tridimensionnelle de cellules stromales comprenant un nombre accr...
La presente invention porte sur un procede de transplantation de cellules souches hematopoietique... more La presente invention porte sur un procede de transplantation de cellules souches hematopoietiques non differenciees sur un receveur. Le procede consiste a (a) faire se developper les cellules souches hematopoietiques non differenciees pour augmenter le nombre des cellules souches hematopoietiques par (i) la culture dans un bioreacteur a ecoulement piston en phase stationnaire de cellules stromales sous un courant continu d'un milieu de culture etabli sur un substrat biocompatible tridimensionnel afin de generer une culture tridimensionnelle de cellules stromales ; et (ii) l'ensemencement de cellules souches hematopoietiques non differenciees dans le bioreacteur a ecoulement piston en phase stationnaire comprenant la culture tridimensionnelle de cellules stromales et sous un courant continu d'un milieu de culture pour developper les cellules souches hematopoietiques non differenciees et obtenir une culture tridimensionnelle de cellules stromales comprenant un nombre accr...
American Journal of Transplantation, 2018
Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells wh... more Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem cell-derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The βAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the βAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1-2 βAir devices, each containing 155 000-180 000 islet equivalents (ie, 1800-4600 islet equivalents per kg body weight), and monitored for 3-6 months, followed by the recovery of devices. Implantation of the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C-peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose-stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the βAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).
Proceedings of the National Academy of Sciences of the United States of America, Oct 31, 2017
Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with ... more Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.
PLoS ONE, 2013
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Proceedings of the National Academy of Sciences, 2013
Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destr... more Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies over the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit a widespread application of these strategies. In this paper we present the clinical success of using a bioartificial pancreas for the transplantation of insulin-producing islets without affecting the immune system. In a patient with long-standing type-1 diabetes we could demonstrate persistent graft function and regulated insulin secretion without the need for immune-modulating medication. This strategy opens up avenues for more widespread and safe application of various cell-based therapies.
Journal of Stem Cell and Transplantation Biology, 2017
Islet transplantation effectively treats diabetes but relies on immune suppression and is practic... more Islet transplantation effectively treats diabetes but relies on immune suppression and is practically limited by the number of cadaveric islets available. An alternative cellular source is insulin-producing cells derived from pluripotent cell sources. Three animal cohorts were used in the current study to evaluate whether an oxygen-providing macroencapsulation device, 'βAIR', could function in conjunction with human embryonic stem cells (hESCs) and their derivatives. The first cohort received macro-encapsulated undifferentiated hESCs, a second cohort received hESCs differentiated to a pancreatic progenitor state with limited endocrine differentiation. A reference cohort received human islets. Macro-encapsulation devices were implanted subcutaneously and monitored for up to four months. Undifferentiated pluripotent stem cells did not form teratoma but underwent cell death following implantation. Human C-peptide (hC-peptide) was detectable in host serum one week after implantation for both other cohorts. hC-peptide levels decreasing over time but remained detectable up to the end of the study. Key factors associated with mature endocrine cells were observed in grafts recovered from cohorts containing islets and hESC-derivatives including C-peptide, insulin, glucagon and urocortin 3. We conclude that the 'βAIR' macroencapsulation device is compatible with both human islets and pluripotent derivatives, but has a limited capability of sustaining undifferentiated pluripotent cells.
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2015
Current treatment options for adrenal insufficiency are limited to corticosteroid replacement the... more Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic-pituitary-adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) ...
Abstract. [3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei... more Abstract. [3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.azalR embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in S EVERAL studies support the view that retinoic acid (RA) t promotion of differentiation of em...
Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pig... more Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or organs. To reduce the xenotransplantation-associated risk of infec-tion of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of micro-organisms was examined in Göttingen Minipigs by PCRmethods. Since zoonotic transmis-sion of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly estab...
Biochemical and Biophysical Research Communications, 1998
The rat 20α-hydroxysteroid dehydrogenase (20α-HSD) is an enzyme responsible for the catabolism of... more The rat 20α-hydroxysteroid dehydrogenase (20α-HSD) is an enzyme responsible for the catabolism of progesterone to the inactive 20α-hydroxyprogesterone. We have previously shown that the expression of this enzyme is not regulated by post-translational modification, but at the level of transcription. In this study we have established that the 20α-HSD gene contains nine exons and have isolated a 2.5 kb promoter region. The transcription start site was identified and a TATA box was found. 5′ deletions of this promoter significantly decreased basal promoter activity. Treatment with forskolin led to a dose dependent inhibition of the 2.5kb-20α-HSD-luciferase construct. Computer analysis identified one CRE, two Nur77 response elements, two putative AP1 sites and one progesterone response element half-site. In summary, we have identified and partially characterized the promoter region of the rat ovarian 20α-HSD and demonstrated that the regulatory elements for 20α-HSD are present within a 2...
Apparatus (20) comprising: - a housing (22) configured for insertion into a body of a patient ,; ... more Apparatus (20) comprising: - a housing (22) configured for insertion into a body of a patient ,; - a permeable selective membrane (32) - functional cells (30) immuno by the membrane (32) which prevent the passage therethrough of the cells from the patient and coupled to the housing (22); - an oxygen supply (24); characterized in that the oxygen supply (24) comprises a container comprising oxygen gas, which is configured to deliver gaseous oxygen to the functional cells (30); oxygen supply (24) remaining outside the patient's body and wherein the apparatus (20) further comprises: - a tube (34) coupled transcutaneously oxygen supply (24) to the housing (22 ).
The objective of this study was to characteriae the estrogen action that confers endomethal sensi... more The objective of this study was to characteriae the estrogen action that confers endomethal sensitization to nontraumatic deciduogenic stimuli by use of antiestrogens. Tamoxifen, ethamoxytriphetol, and clomiphene and its two component enantiomers inhibited decidual induction in pseudopregnant rats when administered 17 h before pyrathiazine. Unexpectedly, clomiphene (250 pg/rat) and tamoxifen (25 pg/rat) proved inhibitory at all times up to and including the time of induction. Clomiphene, administered in the hours preceding decidual induction, inhibited the increase of ornithine decarboxylase activity, which nor-mally marks the end of the induction phase. Clomiphene had no inhibitory effect on the availability or receptor binding of progesterone. Clomiphene also inhibited implantation of blastocysts when administered at the time of their adherence to the uterus. The inhibition by antiestrogens of decidual induction could not be explained on the basis of the current understanding of m...
Decidualization of endometrial stroma in the rat induces the expression and secretion of rat deci... more Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (UIII) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, UIII cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor β (ERβ) and α2-macroglobulin (α2-MG), which are known to be PRL-regulated genes. However, when UIII cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 a...
Islet transplantation is a feasible therapeutic alternative for metabolically labile patients wit... more Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the g...
At present, proven clinical treatments but no cures are available for diabetes, a global epidemic... more At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, espe...
PLOS ONE
Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients sh... more Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.
PLOS ONE
Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients sh... more Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.
Xenotransplantation, Jul 1, 2016
Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of... more Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of porcine zoonotic micro-organisms. Hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine endogenous retroviruses (PERVs) are potentially zoonotic micro-organisms which do not show clinical symptoms in pigs and which are due to the low expression level difficult to detect. Göttingen Minipigs (GöMP) are often used for biomedical investigations and they are well characterized concerning the presence of numerous bacteria, fungi, viruses and parasites and therefore may be used for islet cell transplantation. Islet cells derived from three GöMP were transplanted into four healthy, non-diabetic cynomolgus monkeys using a macroencapsulation device. PCR, nested PCR, real-time PCR, real-time RT-PCR and Western blot analyses were used to estimate the presence of PERV, PCMV and HEV in the donors and recipients. Using sensitive detection methods, no HEV was found in the donor pigs and i...