- Email: [email protected]
Mesenchymal Stem Cells in Models of Acute Pancreatitis
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CORRESPONDENCE
of evidence for EMT using both transgenic mouse models (tagging OPNþ biliary and LPC in the adult liver1 or SOX9þ cells at the time of ductal plate formation in the embryo8) decreases the likelihood of such a process in tested models. Coombes and Syn propose that, in our system, a proportion of YFPþ/HNF4–/K19– cells could be mesenchymal in nature. Although it might not be clear from the published pictures, all YFPþ cells carry epithelial markers in diseased and recovery models: If not positive for K19 and SOX9, they are HNF4þ. This model system, however, as explained, does not allow evaluation of mesenchymal to epithelial transition in liver wound healing. Irrespective of experimental findings, murine livers and murine diseases may not reflect the human liver and in particular to decades-long chronic liver diseases. Therefore, further studies are needed to establish whether, to what extent, and under which conditions epithelial cells or progenitors may participate to liver fibrosis or, conversely, whether mesenchymal cells may generate hepatobiliary cells. Finally, to address the possibility that LPC could have a role in regeneration after partial hepatectomy, not only did we demonstrate that biliary cells/LPC do not generate hepatocytes during this process, but we also failed to identify significant change and amplification of the LPC compartment at any time point after partial hepatectomy. The hypothesis proposed by Theise, Dolle, and Kuwahara is very attractive: In disease, LPC could generate hepatocytes with specific features enabling them to reconnect hepatocytes to bile ducts being already well equipped to connect to both cholangiocytes and hepatocytes. We showed that LPC-derived (YFPþ/HNF4þ) hepatocytes are properly polarized and form bile canaliculi with adjacent hepatocytes (native or LPC derived) supporting that those cells can “connect” to hepatocytes. Systematic analysis of the existing material, and time line experiments, are required to evaluate (transient?) contact between LPC-derived hepatocytes and the most proximal branches of the biliary tree during the wound healing process. Transit amplifying cells have a migratory phenotype, a basal membrane and are associated with specialized extracellular matrix and matrix-producing cells, supporting that, although penetrating the parenchyma, they might be “guided” to meet and bind to biliary-disconnected hepatocytes. If verified, the importance of LPC for regeneration would be to preserve/ restore the parenchymal function by providing biliary drainage rather than to increase the number of functional hepatocytes. In support to this view is that injury at the periportal interface, most likely to involve the hepatobiliary connection (eg, choline-deficient ethionine model), is associated with LPC activation, whereas with pericentral damage (eg, CCl4 toxicity) or after partial hepatectomy, conditions under which hepatobiliary connection would remain intact, the LPC compartment is not activated.
GASTROENTEROLOGY Vol. 145, No. 1
We thank the authors of the letters for stimulating the discussion. LPC have not yet revealed all their secrets. Much more experimental work and studies on human material are needed to elucidate the physiologic functions and potential of LPC. REGINA ESPAÑOL–SUÑER Laboratory of Hepato-Gastroenterology Institut de Recherche Expérimentale et Clinique Université catholique de Louvain Brussels, Belgium FRÉDÉRIC P. LEMAIGRE Liver and Pancreas Development Unit de Duve Institute Université catholique de Louvain Brussels, Belgium ISABELLE A. LECLERCQ Laboratory of Hepato-Gastroenterology Institut de Recherche Expérimentale et Clinique Université catholique de Louvain Brussels, Belgium 1. 2. 3. 4. 5. 6. 7. 8.
Espanol-Suner R, et al. Gastroenterology 2012;143:15641575. Uede T. Pathol Int 2011;61:265–280. Taura K, et al. Hepatology 2010;51:1027–1036. Scholten D, et al. Gastroenterology 2010;139:987–998. Chu AS, et al. Hepatology 2011;53:1685–1695. Malato Y, et al. J Clin Invest 2011;121:4850–4860. Le HM, et al. Histochem Cell Biol 2005;123:335–346. Carpentier R, et al. Gastroenterology 2011;141:1432–1438.
Conflicts of interest The author discloses no conflicts. Funding Supported by grants from the Belgian Federal Science Policy Office (Interuniversity Attraction Poles program - networks P6/36-HEPRO and P6/20), the Brussels Capital Region (INNOVIRIS Impulse programmeLife Sciences 2007 and 2011; BruStem project), the D.G. Higher Education and Scientific Research of the French Community of Belgium, the Alphonse and Jean Forton Fund, and the Fund for Scientific Medical Research (Belgium). IL is a FRS-FNRS research associate. http://dx.doi.org/10.1053/j.gastro.2013.05.037
Mesenchymal Stem Cells in Models of Acute Pancreatitis Dear Sir:
I am writing to express concern about interpretation of results presented by Jung et al in their 2011 GASTROENTEROLOGY article on the effect of mesenchymal stem cells (MSC) in models of acute pancreatitis (AP) and discussed in an accompanying commentary in the same issue.1,2 The commentary states that the study “defines for the first time a cell-based therapy for AP that is effective 24 hours after onset of AP.”2 However, the exact timing of administration of MSC in the original article is actually unclear, because the authors state in their Supplementary Methods section that MSC were “infused for 24 hours after the last injection of cerulein and surgery by tail vein,”1 Evidently, this statement does not clearly indicate at which point after induction of AP administration of
July 2013
CORRESPONDENCE 257
MSC was initiated. Moreover, it remains unclear how a 24-hour infusion could have been performed through the tail vein; the exact timing for evaluation of several parameters of disease severity is also not clearly indicated in the original manuscript. These issues raise concern about the interpretation by the authors of the commentary that MSC were administered 24 hours after induction of AP. Indeed, a 2013 time-course study on the effect of umbilical cord MSC in a model of severe AP demonstrates that this intervention is effective when administered up to 6 hours post-induction of AP, but not when cells are injected 12 hours post-AP.3 Although precise indication of experimental procedures and interpretation of results is always essential, these issues are particularly critical in the context of a disease with high morbidity and mortality, such as severe AP, for which no specific treatment is currently available. Cellbased therapies using MSC are receiving a lot of attention as potential therapeutic tools. However, correct interpretation of preclinical studies is essential for safe and effective translation of these and other studies to human trials.4 It is important that the authors clarify the timing and exact mode of MSC cell administration and the extent of injury at the time of cell therapy. At best, it may be that what they describe is a prophylaxis rather than a treatment. GIAMILA FANTUZZI Department of Kinesiology and Nutrition University of Illinois at Chicago Chicago, Illinois 1. 2. 3. 4.
Jung KH, et al. Gastroenterology 2011;140:998–1008. Schneider G, Saur D. Gastroenterology 2011;140:779–782. Yang B, et al. Cytotherapy 2013;15:154–162. Bianco P, et al. Nat Med 2013;19:35–42.
Conflicts of interests The authors disclose no conflicts. http://dx.doi.org/10.1053/j.gastro.2013.04.058
Reply. We thank Dr Fantuzzi for the comments relating
to our 2011 published paper.1 First, we did not describe detailed experimental conditions in the original article because of space limitations. In our Supplementary section, we stated that our human bone marrow-derived clonal mesenchymal stem cells (hcMSCs) were “infused for 24 hours after the last injection of cerulein and surgery by tail vein.”1 The commentary by Schneider et al2 stated that the study “is effective 24 hours after onset of acute pancreatitis (AP).” In fact, we injected hcMSCs at 24 hours (not for 24 hours) after the last injection of cerulein and the rats were humanely killed on day 3 (at 72 hours) after hcMSCs treatment, which was addressed in the Material and Methods as “The rats were killed by decapitation 3 days after hcMSCs infusion.” In this study, we focused on recovery or treatment rather than prevention by hcMSCs in mild and severe AP. Thus, we planned to treat hcMSCs after the induction of
AP models. For the mild model, AP was induced by 3 intraperitoneal injections of cerulein, and hcMSCs were injected at 24 hours after the last injection of cerulein. For the severe model, AP was induced by the administration of 3% sodium taurocholate (TCA) into the pancreatic bile duct.3 In our preliminary study, to determine the TCA concentration necessary to induce pancreatitis-related systemic complications and lethality, dose escalation experiments (2%, 3%, and 5%) were performed. As a result, death rate from AP within 24 hours was 80% of animals receiving the 5% TCA solution, as reported by Yang et al4; all animals receiving the 2% and 3% TCA solution survived. On histologic analysis, however, animals receiving the 2% TCA solution showed slight necrosis and inflammation, whereas animals receiving 3% TCA solution showed strong acinar cell necrosis, fatty tissue necrosis, edema, and inflammation cell infiltration at 24 hours after TCA solution administration to pancreatic duct. Thus, for this study, we induced AP using the 3% TCA solution and then hcMSCs were injected after AP model induction. In the study by Yang et al,4 to investigate the protective effect of umbilical cord blood-derived mesenchymal stem cells (UCMSCs), rats were injected with UCMSCs at 0, 1, 6, or 12 hours after 5% TCA solution administration and then all animals were humanely killed at 48 hours after TCA solution administration. There are 2 major differences between their work and ours, in terms of induction method of animal model and stem cell source and purity. First, they immediately injected UCMSCs time-dependently after 5% TCA solution administration, whereas we injected hcMSCs 24 hours after the induction of AP with 3% TCA to determine the effect of treating or repairing AP. Second, they injected MSCs isolated from umbilical cord blood and we administrated clonal MSCs obtained from human bone marrow using our new protocol, the subfractionation culturing method, which allows highly homogeneous population of clonal MSCs.1 These findings showed that action of stem cells are likely to differ depending on the disease degree, time of hcMSCs treatment and killing, and stem cell source and purity. In stem cell therapy, the important factor is response duration of stem cells in disease rather than starting point of effectiveness after stem cells treatment. Thus, we identified how long the injected hcMSCs are effective in AP in our preliminary study. When rats were killed at 1, 2, 3, 5, or 7 days after hcMSC treatment in AP models, the greatest effectiveness was shown at day 3 in digestive enzyme activity, inflammation and histologic analysis, starting at 1 day after hcMSCs treatment. At days 5 and 7, the effect of hcMSCs slowly started to decrease. Therefore, we decided to kill animals at 3 days after hcMSCs treatment in AP, identifying the duration of hcMSCs effect. Interestingly, and more important, we found in our study that hcMSCs were relatively more effective in severe AP than mild AP. According to the severity of disease, more hcMSCs are likely to migrate to the damaged tissue and respond longer in AP.
CORRESPONDENCE
of evidence for EMT using both transgenic mouse models (tagging OPNþ biliary and LPC in the adult liver1 or SOX9þ cells at the time of ductal plate formation in the embryo8) decreases the likelihood of such a process in tested models. Coombes and Syn propose that, in our system, a proportion of YFPþ/HNF4–/K19– cells could be mesenchymal in nature. Although it might not be clear from the published pictures, all YFPþ cells carry epithelial markers in diseased and recovery models: If not positive for K19 and SOX9, they are HNF4þ. This model system, however, as explained, does not allow evaluation of mesenchymal to epithelial transition in liver wound healing. Irrespective of experimental findings, murine livers and murine diseases may not reflect the human liver and in particular to decades-long chronic liver diseases. Therefore, further studies are needed to establish whether, to what extent, and under which conditions epithelial cells or progenitors may participate to liver fibrosis or, conversely, whether mesenchymal cells may generate hepatobiliary cells. Finally, to address the possibility that LPC could have a role in regeneration after partial hepatectomy, not only did we demonstrate that biliary cells/LPC do not generate hepatocytes during this process, but we also failed to identify significant change and amplification of the LPC compartment at any time point after partial hepatectomy. The hypothesis proposed by Theise, Dolle, and Kuwahara is very attractive: In disease, LPC could generate hepatocytes with specific features enabling them to reconnect hepatocytes to bile ducts being already well equipped to connect to both cholangiocytes and hepatocytes. We showed that LPC-derived (YFPþ/HNF4þ) hepatocytes are properly polarized and form bile canaliculi with adjacent hepatocytes (native or LPC derived) supporting that those cells can “connect” to hepatocytes. Systematic analysis of the existing material, and time line experiments, are required to evaluate (transient?) contact between LPC-derived hepatocytes and the most proximal branches of the biliary tree during the wound healing process. Transit amplifying cells have a migratory phenotype, a basal membrane and are associated with specialized extracellular matrix and matrix-producing cells, supporting that, although penetrating the parenchyma, they might be “guided” to meet and bind to biliary-disconnected hepatocytes. If verified, the importance of LPC for regeneration would be to preserve/ restore the parenchymal function by providing biliary drainage rather than to increase the number of functional hepatocytes. In support to this view is that injury at the periportal interface, most likely to involve the hepatobiliary connection (eg, choline-deficient ethionine model), is associated with LPC activation, whereas with pericentral damage (eg, CCl4 toxicity) or after partial hepatectomy, conditions under which hepatobiliary connection would remain intact, the LPC compartment is not activated.
GASTROENTEROLOGY Vol. 145, No. 1
We thank the authors of the letters for stimulating the discussion. LPC have not yet revealed all their secrets. Much more experimental work and studies on human material are needed to elucidate the physiologic functions and potential of LPC. REGINA ESPAÑOL–SUÑER Laboratory of Hepato-Gastroenterology Institut de Recherche Expérimentale et Clinique Université catholique de Louvain Brussels, Belgium FRÉDÉRIC P. LEMAIGRE Liver and Pancreas Development Unit de Duve Institute Université catholique de Louvain Brussels, Belgium ISABELLE A. LECLERCQ Laboratory of Hepato-Gastroenterology Institut de Recherche Expérimentale et Clinique Université catholique de Louvain Brussels, Belgium 1. 2. 3. 4. 5. 6. 7. 8.
Espanol-Suner R, et al. Gastroenterology 2012;143:15641575. Uede T. Pathol Int 2011;61:265–280. Taura K, et al. Hepatology 2010;51:1027–1036. Scholten D, et al. Gastroenterology 2010;139:987–998. Chu AS, et al. Hepatology 2011;53:1685–1695. Malato Y, et al. J Clin Invest 2011;121:4850–4860. Le HM, et al. Histochem Cell Biol 2005;123:335–346. Carpentier R, et al. Gastroenterology 2011;141:1432–1438.
Conflicts of interest The author discloses no conflicts. Funding Supported by grants from the Belgian Federal Science Policy Office (Interuniversity Attraction Poles program - networks P6/36-HEPRO and P6/20), the Brussels Capital Region (INNOVIRIS Impulse programmeLife Sciences 2007 and 2011; BruStem project), the D.G. Higher Education and Scientific Research of the French Community of Belgium, the Alphonse and Jean Forton Fund, and the Fund for Scientific Medical Research (Belgium). IL is a FRS-FNRS research associate. http://dx.doi.org/10.1053/j.gastro.2013.05.037
Mesenchymal Stem Cells in Models of Acute Pancreatitis Dear Sir:
I am writing to express concern about interpretation of results presented by Jung et al in their 2011 GASTROENTEROLOGY article on the effect of mesenchymal stem cells (MSC) in models of acute pancreatitis (AP) and discussed in an accompanying commentary in the same issue.1,2 The commentary states that the study “defines for the first time a cell-based therapy for AP that is effective 24 hours after onset of AP.”2 However, the exact timing of administration of MSC in the original article is actually unclear, because the authors state in their Supplementary Methods section that MSC were “infused for 24 hours after the last injection of cerulein and surgery by tail vein,”1 Evidently, this statement does not clearly indicate at which point after induction of AP administration of
July 2013
CORRESPONDENCE 257
MSC was initiated. Moreover, it remains unclear how a 24-hour infusion could have been performed through the tail vein; the exact timing for evaluation of several parameters of disease severity is also not clearly indicated in the original manuscript. These issues raise concern about the interpretation by the authors of the commentary that MSC were administered 24 hours after induction of AP. Indeed, a 2013 time-course study on the effect of umbilical cord MSC in a model of severe AP demonstrates that this intervention is effective when administered up to 6 hours post-induction of AP, but not when cells are injected 12 hours post-AP.3 Although precise indication of experimental procedures and interpretation of results is always essential, these issues are particularly critical in the context of a disease with high morbidity and mortality, such as severe AP, for which no specific treatment is currently available. Cellbased therapies using MSC are receiving a lot of attention as potential therapeutic tools. However, correct interpretation of preclinical studies is essential for safe and effective translation of these and other studies to human trials.4 It is important that the authors clarify the timing and exact mode of MSC cell administration and the extent of injury at the time of cell therapy. At best, it may be that what they describe is a prophylaxis rather than a treatment. GIAMILA FANTUZZI Department of Kinesiology and Nutrition University of Illinois at Chicago Chicago, Illinois 1. 2. 3. 4.
Jung KH, et al. Gastroenterology 2011;140:998–1008. Schneider G, Saur D. Gastroenterology 2011;140:779–782. Yang B, et al. Cytotherapy 2013;15:154–162. Bianco P, et al. Nat Med 2013;19:35–42.
Conflicts of interests The authors disclose no conflicts. http://dx.doi.org/10.1053/j.gastro.2013.04.058
Reply. We thank Dr Fantuzzi for the comments relating
to our 2011 published paper.1 First, we did not describe detailed experimental conditions in the original article because of space limitations. In our Supplementary section, we stated that our human bone marrow-derived clonal mesenchymal stem cells (hcMSCs) were “infused for 24 hours after the last injection of cerulein and surgery by tail vein.”1 The commentary by Schneider et al2 stated that the study “is effective 24 hours after onset of acute pancreatitis (AP).” In fact, we injected hcMSCs at 24 hours (not for 24 hours) after the last injection of cerulein and the rats were humanely killed on day 3 (at 72 hours) after hcMSCs treatment, which was addressed in the Material and Methods as “The rats were killed by decapitation 3 days after hcMSCs infusion.” In this study, we focused on recovery or treatment rather than prevention by hcMSCs in mild and severe AP. Thus, we planned to treat hcMSCs after the induction of
AP models. For the mild model, AP was induced by 3 intraperitoneal injections of cerulein, and hcMSCs were injected at 24 hours after the last injection of cerulein. For the severe model, AP was induced by the administration of 3% sodium taurocholate (TCA) into the pancreatic bile duct.3 In our preliminary study, to determine the TCA concentration necessary to induce pancreatitis-related systemic complications and lethality, dose escalation experiments (2%, 3%, and 5%) were performed. As a result, death rate from AP within 24 hours was 80% of animals receiving the 5% TCA solution, as reported by Yang et al4; all animals receiving the 2% and 3% TCA solution survived. On histologic analysis, however, animals receiving the 2% TCA solution showed slight necrosis and inflammation, whereas animals receiving 3% TCA solution showed strong acinar cell necrosis, fatty tissue necrosis, edema, and inflammation cell infiltration at 24 hours after TCA solution administration to pancreatic duct. Thus, for this study, we induced AP using the 3% TCA solution and then hcMSCs were injected after AP model induction. In the study by Yang et al,4 to investigate the protective effect of umbilical cord blood-derived mesenchymal stem cells (UCMSCs), rats were injected with UCMSCs at 0, 1, 6, or 12 hours after 5% TCA solution administration and then all animals were humanely killed at 48 hours after TCA solution administration. There are 2 major differences between their work and ours, in terms of induction method of animal model and stem cell source and purity. First, they immediately injected UCMSCs time-dependently after 5% TCA solution administration, whereas we injected hcMSCs 24 hours after the induction of AP with 3% TCA to determine the effect of treating or repairing AP. Second, they injected MSCs isolated from umbilical cord blood and we administrated clonal MSCs obtained from human bone marrow using our new protocol, the subfractionation culturing method, which allows highly homogeneous population of clonal MSCs.1 These findings showed that action of stem cells are likely to differ depending on the disease degree, time of hcMSCs treatment and killing, and stem cell source and purity. In stem cell therapy, the important factor is response duration of stem cells in disease rather than starting point of effectiveness after stem cells treatment. Thus, we identified how long the injected hcMSCs are effective in AP in our preliminary study. When rats were killed at 1, 2, 3, 5, or 7 days after hcMSC treatment in AP models, the greatest effectiveness was shown at day 3 in digestive enzyme activity, inflammation and histologic analysis, starting at 1 day after hcMSCs treatment. At days 5 and 7, the effect of hcMSCs slowly started to decrease. Therefore, we decided to kill animals at 3 days after hcMSCs treatment in AP, identifying the duration of hcMSCs effect. Interestingly, and more important, we found in our study that hcMSCs were relatively more effective in severe AP than mild AP. According to the severity of disease, more hcMSCs are likely to migrate to the damaged tissue and respond longer in AP.