72 Immediate Interest
Authors
M. Zhao1, P. Choudhary1, P. Srinivasan2, H. Tang1, N. Heaton2, M. Fung1, A. Barthel3, 4, S. R. Bornstein1, 3, 5, S. A. Amiel1, G. C. Huang1
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ human islet ● ▶ transplantation ● ▶ endothelium cells ● ▶ revascularisation ●
Abstract
▼
Revascularisation of transplanted islets is an essential prerequisite for graft survival and function. However, current islet isolation procedures deprive the islets of endothelial tubulets. This may have a detrimental effect on the revascularisation process of islets following transplantation. We hypothesise that modification of the isolation procedure that preserves islet endothelial vessels may improve the islet revascularisation process following transplantation. Here, we present a modified islet isolation method by which a sub-
Introduction
▼ received 18.08.2014 accepted 22.09.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1390489 Published online: November 5, 2014 Horm Metab Res 2015; 47: 72–77 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence G. C. Huang Department of Diabetes and Endocrinology Division of Diabetes and Nutrients King’s College London 123 Coldharbour Lane London SE5 9NU UK Tel.: + 44/2078/485 498 Fax: + 44/2078/485 498 [email protected]
Human islet transplantation to treat patients with unstable insulin deficient diabetes has been established since more than a decade as an alternative option to whole-organ pancreas transplantation [1–3]. In terms of achieving insulin independence, current long term clinical outcomes of human islet transplantation are less satisfactory than whole organ transplantation [4, 5], indicating that the procedures for islet isolation and transplantation require further optimisation. There are major factors influencing the clinical prospects for human islet transplantation such as the innate immunity of the host against the graft cells, the toxicity necessary of immunosuppressive drugs, and the number of viable islets transplanted. In order to circumvent these problems, several experimental strategies including microencapsulation approaches have been tested [6, 7]. A major technical problem in the context of islet transplantation is that human islets suffer from the loss of vascularisation during the islet isolation procedure, leading to ischemic damage to islets [8]. Successful islet transplantation requires reconnection to the vascular system of the host
Zhao M et al. Endothelial Cells for Revascularisation … Horm Metab Res 2015; 47: 72–77
stantial amount of endothelial cells still attached to the islets could be preserved. The islets with preserved endothelial cells isolated by this method were revascularised within 3 days, not observed in islets isolated by standard methods. Further, we observed that grafts of islets isolated by standard methods had more patches of dead tissue than islet grafts obtained by the modified method, indicating that attached endothelial cells may play an important role in the islet revascularisation process and potentially help to improve the transplantation outcome.
as soon as possible in order to ensure long term graft survival and function. Human islets are endocrine cell clusters that are separated from exocrine cells by collagen based extracellular matrixes [9, 10]. The islets contain a very complex microvascular endothelial system linking the islet cells to the circulation [11]. Both islet and endothelial cells play mutually supporting roles in the development of the endocrine pancreas [12–14] with endothelial cells ensuring the supply of nutrients and oxygen to the islets as well as the transfer of hormones such as insulin and glucagon or metabolic products from the islet cells into the circulation [15–17]. Endothelial cells play a very important role in islet cell survival and function. There is strong evidence that donor endothelial cells may improve the revascularisation of islet grafts following transplantation [18, 19]. The enzymatic dissociation of islets from the exocrine pancreatic tissue is based on the application of a mixture of collagenase and protease, which are mostly derived from bacteria (e. g., Clostridium histolyticum [20]). This method is optimised for the preparation of intact islets, but do not consider the preservation of endothelial vessels, despite the evidence that these may be
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Modification of Human Islet Preparation: An Effective Approach to Improve Graft Outcome After Islet Transplantation?
key to establishing links between the islets with the host vascular system. When the islets are in a state of avascularisation, they are not only under hypoxic and nutrient-deficient conditions, they are also in an inflammatory state, shown by the expression of inflammatory molecules such as tissue factor [21], attracting cells of the host innate immune system. Subsequent activation of the complement system and of the coagulation cascade promotes further damage [22–24]. These conditions expose the isolated islets to prolonged stress resulting in a high rate of irreversibly damaged transplanted cells before they can achieve the revascularisation phase [8]. Furthermore, islets are transplanted into the liver which is a not a favourable site for islet survival and function due to chronic hypoxic conditions [25, 26]. After transplantation, the islets are exposed to the potential toxicity of immunosuppressive drugs, which are essentially required to protect the graft from rejection by host immunity in an allo-graft setting. In order to improve revascularisation, investigators have tested conditions to favour the growth of endothelial cells such as application of VEGF [27, 28]. However, these methods have other disadvantages such as the involvement of gene transfer approaches with related safety issues, which are expensive and time consuming and not very effective. On the other hand, conditions promoting an ideal angiogenic environment are also associated with an improved islet engraftment after transplantation [29]. Based on this background, we hypothesised that preservation of endothelial tubulets still attached to the human islets after isolation, will help to gain rapid access of the graft to the host vascular system thereby promoting sufficient oxygen and nutrient supply from the host circulation, reducing the inflammatory response and consecutive islet damage and promoting long term functional engraftment with an improved clinical outcome of the transplant. Here, we report a method potentially improving the preservation of islet endothelial tubulets. The experiments were conducted with 15 pancreas organs from human organ donors that were not suitable for clinical use. Our data suggest that islets with preserved endothelial cells potentially benefit with regard to revascularisation, survival, and function.
Materials and Methods
▼
Islet isolations were carried out in the King’s College Hospital Cell Isolation Unit. Consent for using human pancreases for research was obtained from donor relatives, and the studies were approved by the Ethical Committee of King’s College Hospital. The pancreata were perfused with UW solution (Du Pont Critical Care, Waukegan, WI, USA) during retrieval and transported to the islet laboratory in UW solution on ice. Islet isolation was performed as described previously [30] with minor modifications. According to the standard method, the pancreata were distended with freshly prepared collagenase [~2 000 U/ 200 ml Perfusion buffer Server NB1) and neutral protease (50 U/5 ml ddw) Server, NP] solution at 37 °C, supplemented to a final concentration of 4 mM serine protease inhibitor (Roche, Germany). In contrast to the standard procedure, digestion of the pancreata by the modified method was performed with freshly prepared collagenase solution only, until the organ was almost fully distended. Then approximate 25 U neutral protease solution (2.5 ml) after extensive titration) were added to the remaining collagenase solution and the distension procedure
continued. Subsequently, the cellular parts were collected from the digestive circuit by centrifugation at 126 g for 1 min at 4 °C. The pellet containing the islets was resuspended in UW solution at 4 °C. In the modified isolation method, UW solution was supplemented with 2.5 % (w/v) of human albumin and 4 mM serine protease inhibitor (Roche, Germany). The digestates were then incubated in UW solution for 1 h at 4 °C and the islets were purified using a cooled 2 991 Cobe machine. Islet equivalents (IEQ) were counted microscopically and expressed as IEQ per gram of pancreas tissue and islet viability was assessed by FDA/PI [fluorescein diacetate (FDA) and propidium iodide (PI)] staining. A proportion of the freshly isolated islets was taken and islets with attached endothelial tubulets were counted under inverted light. The in vitro glucose challenge of the islets was performed as described previously [30–34]. In vivo islet function was assessed using severely combined immunodeficient (SCID) mice. Male SCID mice (20–25 g, C.B-17/Icr), purchased from Charles River Laboratories, Margate, UK, were selected as recipients for islet transplants and maintained in filter cages in the Comparative Biology Centre at King’s College London, according to the guidelines of the Home Office (UK) for Animal Scientific Procedures. Diabetes was induced by a single injection of streptozotocin (STZ; 180 mg/kg i.p.) and confirmed by the presence of hyperglycaemia (as assessed by blood glucose level ≥ 15 mM concentration after overnight fast). The diabetic condition was allowed to stabilise for 3–5 days before transplantation. The diabetic mice were randomly allocated to a transplant recipient group, the surgical procedures and blood glucose monitoring were performed as described earlier [30–34]. The transplanted SCID mice were sacrificed at different time points by cervical dislocation. The pancreas, blood and kidneys were collected. The kidneys were snap frozen in liquid nitrogen and stored at − 80 °C until analyses. The serum was used for the determination of glucose, human insulin, and human C-peptide concentrations. Mouse pancreata were assessed histologically to confirm the loss of beta cells by immunohistochemical staining for insulin. Graft-bearing kidneys were cryosliced in 5 μm sections and immunostained for the presence of insulin expressing cells in the grafts and the expression of endothelial cell markers – CD105 or CD31 – both indicators of the revascularisation status of the islets. The immunohistochemical staining methods for the graft-bearing kidneys were described previously [30–34].
Immunostaining and antibodies
Freshly isolated islet cells were harvested and washed with PBS buffer once. Approximately 50 islets were dried onto SuperFrost Ultra Plus® adhesion slides (VWR International Ltd, Lutterworth UK) at room temperature. Graft-bearing kidneys were cryosliced at 5 μm intervals, placed onto the SuperFrost Ultra Plus® adhesion slides. The sections or cells slides were fixed for 15 min at room temperature in 4 % formaldehyde buffer (pH 7.0), followed by 3 washes with PBS buffer. All slides were blocked for 45 min in blocking buffer (1 % BSA, 5 % normal horse serum in PBS buffer). Primary antibodies were diluted in blocking buffer, and slides were incubated for either 1 h at room temperature or overnight at 4 °C. Slides were washed in PBS and incubated for 1 h with secondary antibodies. Images were obtained using Nikon Eclipse 50i microscope and Nikon digital sight DS-UZ camera. Nikon microscope was equipped with a NIS Elements and software version 3.2. Negative experiments were performed with nonspecific and IgG matched antibodies for each antigen.
Zhao M et al. Endothelial Cells for Revascularisation … Horm Metab Res 2015; 47: 72–77
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Immediate Interest 73
The following antibodies were used for the experiments. Rabbit anti-human insulin antibodies (polyclone, AP7277C, Abgent Europe Ltd, Abingdon, UK, at 1:250 dilution), mouse anti-human CD31 antibody (Clone JC70A, DAKO, UK at 1:500 dilution), and mouse anti-human CD105 antibody (Clone 3H1805, Santa Cruz Biotechnology Europe, at 1:500 dilution). Secondary antibodies: fluorescence conjugated anti mouse IgG (FI2001, Vector Laboratories, Peterborough, UK at 1:100 dilution), Alexa Fluor® 594 Donkey Anti-Rabbit IgG (A21207 Life Technologies Corporation UK, at 1:400 dilution). DNA was stained with DAPI (Vector Laboratories, Peterborough, UK).
Real-time quantitative RT-PCR
Total RNA was isolated using the SV Total RNA Isolation System (Promega UK. Southampton, UK) and quantified photometrically by absorbance at 260 nm. cDNA was synthesised using SuperScript II reverse transcriptase (Invitrogen, Paisley, UK) using 10 ng total RNA. Primers and probes were designed using the Primer 3.0 input program and synthesised by Applied Biosystems (Warrington, UK). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out in an ABI 7900 HT Sequence Detection System using TaqMan master mix in 96-well microtitre plates using a final volume of 25 μl. Amplifications were performed starting with a 10 min template denaturation step at 95 °C, followed by 40 cycles of 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. All data were normalised using endogenous β-actin as a control and expressed as n-fold relative to the amount of actin mRNA.
Statistical analysis
All data are expressed as mean ± standard deviation. Data were analysed with Student’s t-test for paired data. A p-value of
Authors
M. Zhao1, P. Choudhary1, P. Srinivasan2, H. Tang1, N. Heaton2, M. Fung1, A. Barthel3, 4, S. R. Bornstein1, 3, 5, S. A. Amiel1, G. C. Huang1
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ human islet ● ▶ transplantation ● ▶ endothelium cells ● ▶ revascularisation ●
Abstract
▼
Revascularisation of transplanted islets is an essential prerequisite for graft survival and function. However, current islet isolation procedures deprive the islets of endothelial tubulets. This may have a detrimental effect on the revascularisation process of islets following transplantation. We hypothesise that modification of the isolation procedure that preserves islet endothelial vessels may improve the islet revascularisation process following transplantation. Here, we present a modified islet isolation method by which a sub-
Introduction
▼ received 18.08.2014 accepted 22.09.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1390489 Published online: November 5, 2014 Horm Metab Res 2015; 47: 72–77 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence G. C. Huang Department of Diabetes and Endocrinology Division of Diabetes and Nutrients King’s College London 123 Coldharbour Lane London SE5 9NU UK Tel.: + 44/2078/485 498 Fax: + 44/2078/485 498 [email protected]
Human islet transplantation to treat patients with unstable insulin deficient diabetes has been established since more than a decade as an alternative option to whole-organ pancreas transplantation [1–3]. In terms of achieving insulin independence, current long term clinical outcomes of human islet transplantation are less satisfactory than whole organ transplantation [4, 5], indicating that the procedures for islet isolation and transplantation require further optimisation. There are major factors influencing the clinical prospects for human islet transplantation such as the innate immunity of the host against the graft cells, the toxicity necessary of immunosuppressive drugs, and the number of viable islets transplanted. In order to circumvent these problems, several experimental strategies including microencapsulation approaches have been tested [6, 7]. A major technical problem in the context of islet transplantation is that human islets suffer from the loss of vascularisation during the islet isolation procedure, leading to ischemic damage to islets [8]. Successful islet transplantation requires reconnection to the vascular system of the host
Zhao M et al. Endothelial Cells for Revascularisation … Horm Metab Res 2015; 47: 72–77
stantial amount of endothelial cells still attached to the islets could be preserved. The islets with preserved endothelial cells isolated by this method were revascularised within 3 days, not observed in islets isolated by standard methods. Further, we observed that grafts of islets isolated by standard methods had more patches of dead tissue than islet grafts obtained by the modified method, indicating that attached endothelial cells may play an important role in the islet revascularisation process and potentially help to improve the transplantation outcome.
as soon as possible in order to ensure long term graft survival and function. Human islets are endocrine cell clusters that are separated from exocrine cells by collagen based extracellular matrixes [9, 10]. The islets contain a very complex microvascular endothelial system linking the islet cells to the circulation [11]. Both islet and endothelial cells play mutually supporting roles in the development of the endocrine pancreas [12–14] with endothelial cells ensuring the supply of nutrients and oxygen to the islets as well as the transfer of hormones such as insulin and glucagon or metabolic products from the islet cells into the circulation [15–17]. Endothelial cells play a very important role in islet cell survival and function. There is strong evidence that donor endothelial cells may improve the revascularisation of islet grafts following transplantation [18, 19]. The enzymatic dissociation of islets from the exocrine pancreatic tissue is based on the application of a mixture of collagenase and protease, which are mostly derived from bacteria (e. g., Clostridium histolyticum [20]). This method is optimised for the preparation of intact islets, but do not consider the preservation of endothelial vessels, despite the evidence that these may be
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Modification of Human Islet Preparation: An Effective Approach to Improve Graft Outcome After Islet Transplantation?
key to establishing links between the islets with the host vascular system. When the islets are in a state of avascularisation, they are not only under hypoxic and nutrient-deficient conditions, they are also in an inflammatory state, shown by the expression of inflammatory molecules such as tissue factor [21], attracting cells of the host innate immune system. Subsequent activation of the complement system and of the coagulation cascade promotes further damage [22–24]. These conditions expose the isolated islets to prolonged stress resulting in a high rate of irreversibly damaged transplanted cells before they can achieve the revascularisation phase [8]. Furthermore, islets are transplanted into the liver which is a not a favourable site for islet survival and function due to chronic hypoxic conditions [25, 26]. After transplantation, the islets are exposed to the potential toxicity of immunosuppressive drugs, which are essentially required to protect the graft from rejection by host immunity in an allo-graft setting. In order to improve revascularisation, investigators have tested conditions to favour the growth of endothelial cells such as application of VEGF [27, 28]. However, these methods have other disadvantages such as the involvement of gene transfer approaches with related safety issues, which are expensive and time consuming and not very effective. On the other hand, conditions promoting an ideal angiogenic environment are also associated with an improved islet engraftment after transplantation [29]. Based on this background, we hypothesised that preservation of endothelial tubulets still attached to the human islets after isolation, will help to gain rapid access of the graft to the host vascular system thereby promoting sufficient oxygen and nutrient supply from the host circulation, reducing the inflammatory response and consecutive islet damage and promoting long term functional engraftment with an improved clinical outcome of the transplant. Here, we report a method potentially improving the preservation of islet endothelial tubulets. The experiments were conducted with 15 pancreas organs from human organ donors that were not suitable for clinical use. Our data suggest that islets with preserved endothelial cells potentially benefit with regard to revascularisation, survival, and function.
Materials and Methods
▼
Islet isolations were carried out in the King’s College Hospital Cell Isolation Unit. Consent for using human pancreases for research was obtained from donor relatives, and the studies were approved by the Ethical Committee of King’s College Hospital. The pancreata were perfused with UW solution (Du Pont Critical Care, Waukegan, WI, USA) during retrieval and transported to the islet laboratory in UW solution on ice. Islet isolation was performed as described previously [30] with minor modifications. According to the standard method, the pancreata were distended with freshly prepared collagenase [~2 000 U/ 200 ml Perfusion buffer Server NB1) and neutral protease (50 U/5 ml ddw) Server, NP] solution at 37 °C, supplemented to a final concentration of 4 mM serine protease inhibitor (Roche, Germany). In contrast to the standard procedure, digestion of the pancreata by the modified method was performed with freshly prepared collagenase solution only, until the organ was almost fully distended. Then approximate 25 U neutral protease solution (2.5 ml) after extensive titration) were added to the remaining collagenase solution and the distension procedure
continued. Subsequently, the cellular parts were collected from the digestive circuit by centrifugation at 126 g for 1 min at 4 °C. The pellet containing the islets was resuspended in UW solution at 4 °C. In the modified isolation method, UW solution was supplemented with 2.5 % (w/v) of human albumin and 4 mM serine protease inhibitor (Roche, Germany). The digestates were then incubated in UW solution for 1 h at 4 °C and the islets were purified using a cooled 2 991 Cobe machine. Islet equivalents (IEQ) were counted microscopically and expressed as IEQ per gram of pancreas tissue and islet viability was assessed by FDA/PI [fluorescein diacetate (FDA) and propidium iodide (PI)] staining. A proportion of the freshly isolated islets was taken and islets with attached endothelial tubulets were counted under inverted light. The in vitro glucose challenge of the islets was performed as described previously [30–34]. In vivo islet function was assessed using severely combined immunodeficient (SCID) mice. Male SCID mice (20–25 g, C.B-17/Icr), purchased from Charles River Laboratories, Margate, UK, were selected as recipients for islet transplants and maintained in filter cages in the Comparative Biology Centre at King’s College London, according to the guidelines of the Home Office (UK) for Animal Scientific Procedures. Diabetes was induced by a single injection of streptozotocin (STZ; 180 mg/kg i.p.) and confirmed by the presence of hyperglycaemia (as assessed by blood glucose level ≥ 15 mM concentration after overnight fast). The diabetic condition was allowed to stabilise for 3–5 days before transplantation. The diabetic mice were randomly allocated to a transplant recipient group, the surgical procedures and blood glucose monitoring were performed as described earlier [30–34]. The transplanted SCID mice were sacrificed at different time points by cervical dislocation. The pancreas, blood and kidneys were collected. The kidneys were snap frozen in liquid nitrogen and stored at − 80 °C until analyses. The serum was used for the determination of glucose, human insulin, and human C-peptide concentrations. Mouse pancreata were assessed histologically to confirm the loss of beta cells by immunohistochemical staining for insulin. Graft-bearing kidneys were cryosliced in 5 μm sections and immunostained for the presence of insulin expressing cells in the grafts and the expression of endothelial cell markers – CD105 or CD31 – both indicators of the revascularisation status of the islets. The immunohistochemical staining methods for the graft-bearing kidneys were described previously [30–34].
Immunostaining and antibodies
Freshly isolated islet cells were harvested and washed with PBS buffer once. Approximately 50 islets were dried onto SuperFrost Ultra Plus® adhesion slides (VWR International Ltd, Lutterworth UK) at room temperature. Graft-bearing kidneys were cryosliced at 5 μm intervals, placed onto the SuperFrost Ultra Plus® adhesion slides. The sections or cells slides were fixed for 15 min at room temperature in 4 % formaldehyde buffer (pH 7.0), followed by 3 washes with PBS buffer. All slides were blocked for 45 min in blocking buffer (1 % BSA, 5 % normal horse serum in PBS buffer). Primary antibodies were diluted in blocking buffer, and slides were incubated for either 1 h at room temperature or overnight at 4 °C. Slides were washed in PBS and incubated for 1 h with secondary antibodies. Images were obtained using Nikon Eclipse 50i microscope and Nikon digital sight DS-UZ camera. Nikon microscope was equipped with a NIS Elements and software version 3.2. Negative experiments were performed with nonspecific and IgG matched antibodies for each antigen.
Zhao M et al. Endothelial Cells for Revascularisation … Horm Metab Res 2015; 47: 72–77
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Immediate Interest 73
The following antibodies were used for the experiments. Rabbit anti-human insulin antibodies (polyclone, AP7277C, Abgent Europe Ltd, Abingdon, UK, at 1:250 dilution), mouse anti-human CD31 antibody (Clone JC70A, DAKO, UK at 1:500 dilution), and mouse anti-human CD105 antibody (Clone 3H1805, Santa Cruz Biotechnology Europe, at 1:500 dilution). Secondary antibodies: fluorescence conjugated anti mouse IgG (FI2001, Vector Laboratories, Peterborough, UK at 1:100 dilution), Alexa Fluor® 594 Donkey Anti-Rabbit IgG (A21207 Life Technologies Corporation UK, at 1:400 dilution). DNA was stained with DAPI (Vector Laboratories, Peterborough, UK).
Real-time quantitative RT-PCR
Total RNA was isolated using the SV Total RNA Isolation System (Promega UK. Southampton, UK) and quantified photometrically by absorbance at 260 nm. cDNA was synthesised using SuperScript II reverse transcriptase (Invitrogen, Paisley, UK) using 10 ng total RNA. Primers and probes were designed using the Primer 3.0 input program and synthesised by Applied Biosystems (Warrington, UK). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out in an ABI 7900 HT Sequence Detection System using TaqMan master mix in 96-well microtitre plates using a final volume of 25 μl. Amplifications were performed starting with a 10 min template denaturation step at 95 °C, followed by 40 cycles of 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. All data were normalised using endogenous β-actin as a control and expressed as n-fold relative to the amount of actin mRNA.
Statistical analysis
All data are expressed as mean ± standard deviation. Data were analysed with Student’s t-test for paired data. A p-value of
