Lawrence Rosenberg

β-Cell Mass Dynamics and Islet Cell Plasticity in Human Type 2 Diabetes

Studies of long-standing type 2 diabetes (T2D) report a deficit in beta-cell mass due to increased apoptosis, whereas neogenesis and replication are unaffected. It is unclear whether these changes are a cause or a consequence of T2D. Moreover, whereas islet morphogenetic plasticity has been demonstrated in vitro, the in situ plasticity of islets, as well as the effect of T2D on endocrine differentiation, is unknown. We compared beta-cell volume, neogenesis, replication, and apoptosis in pancreata from lean and obese (body mass index > or = 27 kg/m(2)) diabetic (5 +/- 2 yr since diagnosis) and nondiabetic cadaveric donors. We also subjected isolated islets from diabetic (3 +/- 1 yr since diagnosis) and nondiabetic donors to an established in vitro model of islet plasticity. Differences in beta-cell volume between diabetic and nondiabetic donors were consistently less pronounced than those reported in long-standing T2D. A compensatory increase in beta-cell neogenesis appeared to mediate this effect. Studies of induced plasticity indicated that islets from diabetic donors were capable of epithelial dedifferentiation but did not demonstrate regenerative potential, as was seen in islets from nondiabetic donors. This deficiency was associated with the overexpression of Notch signaling molecules and a decreased neurogenin-3(+) cell frequency. One interpretation of these results would be that decreased beta-cell volume is a consequence, not a cause, of T2D, mediated by increased apoptosis and attenuated beta-cell (re)generation. However, other explanations are also possible. It remains to be seen whether the morphogenetic plasticity of human islets, deficient in vitro in islets from diabetic donors, is a component of normal beta-cell mass dynamics.

Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells.

BACKGROUND AIMSnEx vivo propagation of sparse populations of human mesenchymal stromal cells (hMSC) is critical for generating numbers sufficient for therapeutic applications. hMSC culture media have typically been supplemented with animal serum and, recently, human-sourced materials. However, these supplements are ill-defined and, thus, undesirable for clinical and research applications. Previously reported efforts to develop defined media for hMSC culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. Therefore a major step forward would be the identification of defined, serum-free culture conditions capable of supporting both the isolation and rapid expansion of hMSC.nnnMETHODSnUsing classical approaches of medium development, we were able to identify a set of growth and attachment factors that allowed the serum-free isolation and expansion of hMSC from bone marrow.nnnRESULTSnHeparin, selenium and platelet-derived growth factor (PDGF)-BB were found to be inhibitory for the growth of hMSC, whereas basic fibroblast growth factor (bFGF) was critical and worked synergistically with transforming growth factor (TGF)-beta1 to allow significant cell expansion. Ascorbic acid, hydrocortisone and fetuin were also found to be important growth and attachment factors that, in conjunction with substrate-coating proteins, allowed the isolation of hMSC from primary culture and their subsequent expansion.nnnCONCLUSIONSnWe report a defined medium formulation (PPRF-msc6), consisting of key recombinant and serum-derived components, for the rapid isolation and expansion of hMSC in the absence of serum. This work represents an important step forward for achieving an ideal, completely defined synthetic medium composition for the safe use of hMSC in clinical settings.

Islet neogenesis : A potential therapeutic tool in type 1 diabetes

Current therapies for type 1 diabetes, including fastidious blood glucose monitoring and multiple daily insulin injections, are not sufficient to prevent complications of the disease. Though pancreas and possibly islet transplantation can prevent the progression of complications, the scarcity of donor organs limits widespread application of these approaches. Understanding the mechanisms of beta-cell mass expansion as well as the means to exploit these pathways has enabled researchers to develop new strategies to expand and maintain islet cell mass. Potential new therapeutic avenues include ex vivo islet expansion and improved viability of islets prior to implantation, as well as the endogenous expansion of beta-cell mass within the diabetic patient. Islet neogenesis, through stem cell activation and/or transdifferentiation of mature fully differentiated cells, has been proposed as a means of beta-cell mass expansion. Finally, any successful new therapy for type 1 diabetes via beta-cell mass expansion will require prevention of beta-cell death and maintenance of long-term endocrine function.

Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review

Human mesenchymal stem cells (hMSCs), also called mesenchymal stromal cells, have been of great interest in regenerative medicine applications because of not only their differentiation potential but also their ability to secrete bioactive factors that can modulate the immune system and promote tissue repair. This potential has initiated many early-phase clinical studies for the treatment of various diseases, disorders, and injuries by using either hMSCs themselves or their secreted products. Currently, hMSCs for clinical use are generated through conventional static adherent cultures in the presence of fetal bovine serum or human-sourced supplements. However, these methods suffer from variable culture conditions (i.e., ill-defined medium components and heterogeneous culture environment) and thus are not ideal procedures to meet the expected future demand of quality-assured hMSCs for human therapeutic use. Optimizing a bioprocess to generate hMSCs or their secreted products (or both) promises to improve the efficacy as well as safety of this stem cell therapy. In this review, current media and methods for hMSC culture are outlined and bioprocess development strategies discussed.

Prospects and Challenges for Islet Regeneration as a Treatment for Diabetes: A Review of Islet Neogenesis Associated Protein

Diabetes mellitus results from inadequate insulin action, which can be viewed as a consequence of the limited ability to restore β cells after they are lost as the result of metabolic exhaustion, autoimmune destruction, or surgical insult. Arguably, a uniformly effective therapeutic pathway to address all forms of diabetes would be to reverse the restrictions on β-cell and islet regeneration. The development from progenitor cells of islets with normal endocrine function does occur in adult humans; it is referred to as islet neogenesis. The induction of islet neogenesis is an important, if not essential, therapeutic approach for curing type 1 diabetes mellitus (T1DM) and could be valuable in the treatment of type 2 diabetes mellitus (T2DM) as well. Islet neogenesis associated protein (INGAP) is the first therapeutic candidate to be identified as the result of a purposeful search for an endogenous molecule with islet neogenic activity. It was found that partial obstruction of the pancreatic duct in hamsters induced islet neogenesis; under this condition, a neogenesis-promoting activity was identified and partially purified from a soluble tissue fraction. A 168-kDa protein product of the cloned gene was found to be responsible for the neogenesis activity. This molecule named INGAP contains an active core sequence of amino acids called INGAP peptide. Results from in vitro, animal, and human studies suggest that INGAP and INGAP peptide are neogenic in at least several vertebrate species, including humans. INGAP has since been found to be a member of the family of Reg proteins, which are found across and in multiple versions within species and are closely associated with embryonic and regenerative processes. Clinical results suggest that INGAP peptide can be a suitable neogenesis therapy, but optimization of the therapy and more data are required to fully access this potential. Understanding of the signaling pathways of INGAP and other related Reg proteins is a promising means of advancing therapeutic development for people with T1DM and T2DM. The quest for the fundamental restorative approach to lost insulin secretion is an enticing target for drug development.

Evidence for the homeostatic regulation of induced beta cell mass expansion

Aims/hypothesisDiabetes results from an insufficient insulin-secreting beta cell mass. Restoration of beta cell mass through pharmaceutically induced endogenous beta cell mass expansion may revolutionise diabetes therapy. However, it remains to be determined whether the induced beta cell mass expansion is under homeostatic regulation.MethodsBeta cell mass expansion rates were derived from three separate studies of continuous stimulation of islet neogenesis, including the partial duct obstruction of euglycaemic Syrian hamsters, administration of a pentadecapeptide with the same amino acid sequence as residues 104–118 of islet neogenesis-associated protein (INGAP104–118) to euglycaemic Syrian hamsters, as well as to euglycaemic CD-1 mice. The incidence of islet neogenesis, average beta cell size, and beta cell replication and apoptotic rates were determined.ResultsPartial duct obstruction led to a ∼2.5-fold increase in endocrine tissue at day 56 (p<0.05). From day 0 to day 7 the average rate of change of islet area was 12.7% per day, and this rate decreased to 5.3% per day from day 7 to day 42, and to 2.8% per day from day 42 to day 56. Administration of INGAP104–118 to adult hamsters led to a 31% increase in total beta cell mass at day 30 (p=0.031). From day 0 to day 10 the average rate of beta cell mass expansion was 148xa0μg/day, whereas from day 10 to day 30 it decreased to 45xa0μg/day. INGAP104–118 administration to adult CD-1 mice resulted in an approximately twofold increase in beta cell mass after 31xa0days (p=0.021). However, at day 90, there was no significant difference vs age-matched control mice (p=0.30), even though the neogenic beta cell mass was approximately fourfold greater (p=0.026). Beta cell replication was decreased by 56% (p<0.048), whereas beta cell apoptosis was fourfold greater (p<0.003) in 90-day INGAP104–118-treated mice compared with age-matched control mice.Conclusions/interpretationThese data indicate that in the presence of ongoing islet neogenesis, homeostatic regulatory mechanisms intervene to regulate beta cell mass according to the prevailing metabolic requirements.

Cellular origins of adult human islet in vitro dedifferentiation

Cultured human islets can be dedifferentiated to duct-like structures composed mainly of cytokeratin+ and nestin+ cells. Given that these structures possess the potential to redifferentiate into islet-like structures, we sought to elucidate their specific cellular origins. Adenoviral vectors were engineered for β-, α-, δ- or PP-cell-specific GFP expression. A double-stranded system was designed whereby cultures were infected with two vectors: one expressed GFP behind the cumate-inducible promoter sequence, and the other expressed the requisite transactivator behind the human insulin, glucagon, somatostatin or pancreatic polypeptide promoter. This system labels hormone+ cells in the islet in a cell-specific manner, allowing these cells to be tracked during the course of transformation from islet to duct-like structure. Post-infection, islets were cultured to induce dedifferentiation. Fluorescence microscopy demonstrated that α-, δ- and PP-cells contributed equally to the cytokeratin+ population, with minimal β-cell contribution, whereas the converse was true for nestin+ cells. Complementary targeted cell ablation studies, using streptozotocin or similar adenoviral expression of the Bax (Bcl2-associated X protein) toxigene, validated these findings and suggested a redundancy between α-, δ- and PP-cells with respect to cytokeratin+ cell derivation. These results call into question the traditional understanding of islet cells as being terminally differentiated and provide support for the concept of adult islet morphogenetic plasticity.

Islet-derived progenitors as a source of in vitro islet regeneration.

Current therapies do not prevent the complications of diabetes. Furthermore, these therapies do not address the underlying pathology; the lack of functional beta-cell mass that occurs in both types 1 and 2 diabetes. While pancreas and islet transplantation do serve to increase beta-cell mass, a lack of donor organs limits the therapeutic potential of these treatments. As such, expansion of beta-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. One potential source of islet progenitors is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. We have developed a tissue culture platform whereby isolated adult human pancreatic islets form proliferative duct-like structures expressing ductal and progenitor markers. Short-term treatment with a peptide fragment of islet neogenesis-associated protein (INGAP) induces these structures to reform islet-like structures that resemble freshly isolated islets with respect to the frequency and distribution of the four endocrine cell types, islet gene expression and hormone production, insulin content, and glucose-responsive insulin secretion. As such, the plasticity of adult human islets has significant implications for islet regeneration.

Pancreatic Cancer: Does Octreotide Offer Any Promise?

The incidence of adenocarcinoma of the pancreas has risen steadily over the past 4 decades. Since pancreatic cancer is diagnosed at an advanced stage, and because of the lack of effective therapies the prognosis of such patients is extremely poor. Despite advances in our understanding of the molecular biology of pancreatic cancer, the systemic treatment of this disease remains unsatisfactory. Systemic chemotherapy and the administration of biologically active molecules such as tumor necrosis factor or interferons have not resulted in significant improvements in response rates or patient survival. New treatment strategies are obviously needed. This paper will discuss current advances in the use of somatostatin analogs in the management of pancreatic cancer.