In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells

Gao, Ru; Ustinov, Jarkko; Korsgren, Olle; Otonkoski, Timo

doi:10.1007/s00125-005-1935-8

Cited by 79 publications

(57 citation statements)

References 32 publications

(53 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the expansion of cells derived from the islet-depleted exocrine fraction, which is usually discarded following isolation, has been shown to give rise to a near homogeneous population of CK19-positive epithelial cells, with minimal fibroblast contamination during the initial phase of culture [21,25]. Although it is not possible to completely eliminate endocrine components from the initial plated material, previous studies have shown that such culture conditions are not conducive either to the adherence or prolonged survival of native islet cells [25] and that any endocrine cells that do exist become de-differentiated within 3-4 days of monolayer culture [24,29]. In our study we found that over 90% of the cells derived from the outgrowth cultures were positive for CK19 at the time of the initiation of the islet-DEC co-cultures.…”

Section: Discussionmentioning

confidence: 99%

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

et al. 2009

View full text Add to dashboard Cite

Aims/hypothesis Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

show abstract

Section: Discussionmentioning

confidence: 99%

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

et al. 2009

View full text Add to dashboard Cite

show abstract

“…We have previously used magnetic cell sorting to obtain non-endocrine CK19-enriched cell populations by eliminating NCAM-positive cells [10]. However, the use of CA19-9 microbeads allows much more efficient purification of ductal cells, as described previously [9,11].…”

Section: Discussionmentioning

confidence: 99%

“…Human islets Islets were isolated as described earlier [10]. Informed consent was obtained from tissue donors and institutional Ethics Committees in Uppsala and Helsinki approved all procedures.…”

Section: Methodsmentioning

confidence: 99%

A simple two-step protocol for the purification of human pancreatic beta cells

Banerjee

Otonkoski

2009

Diabetologia

Self Cite

View full text Add to dashboard Cite

Aims/hypothesis Isolated pure human beta cells would be helpful for a number of research purposes. However, lack of beta cell-specific surface antigens has been a major problem. We aimed to develop a simple method for human beta cell isolation based on the initial elimination of ductal cells by their expression of carbohydrate antigen 19-9 (CA19-9), followed by positive selection of beta cells by their expression of polysialic acid-neural cell adhesion molecule (PSA-NCAM). Methods Cell type-specific expression of CA19-9, NCAM and PSA-NCAM was studied in sections of adult human pancreas and in cultured primary endocrine and exocrine cells. Dispersed human islet cells were purified in two steps, after 4 days of suspension culture, by binding to magnetic microbeads coupled to antibodies against CA19-9 and PSA-NCAM. Results NCAM expression was detected in ducts and islets in the human pancreas. In contrast, PSA-NCAM immunoreactivity was detected only in islets. PSA-NCAM staining in dispersed cells revealed that the marker is expressed in all endocrine cell types, but not in duct cells. Purification of dispersed islet cells using PSA-NCAM microbeads alone did not completely eliminate contaminating duct cells. However, elimination of the duct cells by CA19-9 microbeads followed by positive sorting of the PSA-NCAM-positive cells in five consecutive islet preparations resulted in 90 to 98% pure endocrine cells, of which 89 to 97% were beta cells. Conclusions/interpretation We describe a simple and reproducible method for purification of viable human pancreatic beta cells devoid of exocrine acini and ducts.

show abstract

“…However, in that study, transdifferentiated acinar cells or dedifferentiated ␤-cells could have developed an epithelial (CK19 ϩ ) phenotype during shipment from isolation centers or during the 4-day culture in G418, used to eliminate mesenchymal cells. Gao et al (28) suggested that the insulin ϩ cells may have been derived from the residual ␤-cells in their preparations, since depletion of neural cell adhesion molecule (NCAM) ϩ cells, which were presumed to be only islet cells, resulted in few insulin ϩ cells. However, while NCAM is expressed selectively in rodent islets, we found that in human pancreas, NCAM was also expressed at both the mRNA and protein level in the duct epithelium (S.B.-W., C. Nienaber, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Differentiation of Affinity-Purified Human Pancreatic Duct Cells to β-Cells

et al. 2007

View full text Add to dashboard Cite

To test whether pancreatic duct cells are in vitro progenitors, they were purified from dispersed islet-depleted human pancreatic tissue using CA19-9 antibody. The purified fraction was almost entirely CK19؉ with no insulin ؉ cells, whereas the unpurified cells (crude duct) were 56% CK19 W hereas islet transplantation is an effective and beneficial treatment for type 1 diabetes, its application is limited by the shortage of islets. A possible solution is to generate insulin-producing cells from adult stem/progenitor cells of the pancreas. In vivo new ␤-cells are generated through replication of preexisting ␤-cells and neogenesis, the latter from differentiation of non-hormone-expressing progenitor cells (1-8). Putative adult stem/progenitor cells from mouse pancreas have been expanded clonally and after manipulation were found to express low levels of insulin and other pancreatic markers (9,10). While these findings are provocative, it has not yet been shown that such cells can become fully functional ␤-cells (11).Our group reported that islet-like structures, which secrete insulin in response to glucose, could be generated from islet-depleted pancreatic tissue remaining after human islet isolation (12). These findings were confirmed and extended by Otonkoski and colleagues (13). The cell of origin has been suspected to be ductal in origin but has not been conclusively shown. Additionally, three other groups (14 -16) have reported that putative progenitor cells, which arose from human islet preparations, could be expanded through many passages and then be manipulated to reexpress islet hormones at low levels. Gershengorn et al. (16), Habener and colleagues (14,17) and Efrat and colleagues (18) have suggested that the expanding cells are ␤-cells that have undergone epithelial-mesenchymal transition, no longer express insulin, and have great capacity for expansion. However, even the purest human islet preparations are not pure islet cells but contain many contaminating duct, acinar, and connective tissue cells. Olson and colleagues (15) showed that serpinginous cells expressing vimentin and nestin had no islet hormones during expansion but acquired low levels of islet markers after manipulation of culture conditions. Similar cells were initially suggested to be the preexisting nestinpositive cells found in the islets and in the ductal stroma (17). All of these studies have raised the possibility of generating new islet cells in vitro from human pancreatic tissue, but in each case the cell of origin has not been identified; thus, it is not clear whether ␤-cells actually undergo such a transition.The purpose of this study is to test whether highly purified human adult pancreatic duct cells can differentiate in vitro into insulin-producing cells. To this end, extremely pure duct preparations were obtained following immunomagnetic sorting with CA19-9 antibody. The affinity purification step is highly selective for pancreatic duct epithelial cells and is performed immediately after islet purification. Our method is qu...

show abstract

In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells

Cited by 79 publications

References 32 publications

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

A simple two-step protocol for the purification of human pancreatic beta cells

Differentiation of Affinity-Purified Human Pancreatic Duct Cells to β-Cells

Contact Info

Product

Resources

About