Catarina Limbert

PDX1- and NGN3-mediated in vitro reprogramming of human bone marrow-derived mesenchymal stromal cells into pancreatic endocrine lineages

BACKGROUND AIMS Reprogramming of multipotent adult bone marrow (BM)-derived mesenchymal stromal/stem cells (MSC) (BM-MSC) represents one of several strategies for cell-based therapy of diabetes. However, reprogramming primary BM-MSC into pancreatic endocrine lineages has not yet been consistently demonstrated. METHODS To unravel the role and interaction of key factors governing this process, we used well-characterized telomerase-immortalized human MSC (hMSC-TERT). Pancreatic endocrine differentiation in hMSC-TERT was induced by two major in vitro strategies: (i) endocrine-promoting culture conditions and (ii) ectopic expression of two master regulatory genes of the endocrine lineage, human neurogenin 3 (NGN3) and human pancreatic duodenal homeobox 1 (PDX1). RESULTS Both approaches triggered pancreatic endocrine gene expression, notably insulin, glucose-transporter 2 and somatostatin. Transgenic overexpression of NGN3 and/or PDX1 proteins not only induced direct target genes, such as NEUROD1 and insulin, and but also triggered parts of the gene expression cascade that is involved in pancreatic endocrine differentiation. Notably, ectopic NGN3 alone was sufficient to initiate the expression of specific beta-cell lineage-associated genes, most importantly PDX1 and insulin. This was demonstrated both transcriptionally by mRNA expression and reporter gene analyzes and at a protein level by Western blotting. Such reprogramming of hMSC-TERT cells induced glucose-insensitive insulin biosynthesis and secretion. CONCLUSIONS Our results indicate that establishment of glucose-dependent insulin secretion in partially reprogrammed human MSC may depend on additional maturation factors. Moreover, hMSC-TERT provides a suitable cell model for investigating further the molecular mechanisms of reprogramming and maturation of adult MSC towards pancreatic endocrine lineages.

Functional Signature of Human Islet-Derived Precursor Cells Compared to Bone Marrow-Derived Mesenchymal Stem Cells

Pancreatic islet beta-cell replenishment can be driven by epithelial cells from exocrine pancreas via epithelial-mesenchymal transition (EMT) and the reverse process MET, while specified pancreatic mesenchymal cells control islet cell development and maintenance. The role of human islet-derived precursor cells (hIPCs) in regeneration and support of endocrine islets is under investigation. Here, we analyzed hIPCs as to their immunophenotype, multilineage differentiation capacity, and gene profiling, in comparison to human bone marrow-derived mesenchymal stem cells (hBM-MSCs). hIPCs and hBM-MSCs display a common mesenchymal character and express lineage-specific marker genes upon induction toward pancreatic endocrine and mesenchymal pathways of differentiation. hIPCs can go further along endocrine pathways while lacking some core mesenchymal differentiation attributes. Significance analysis of microarray (SAM) from 5 hBM-MSC and 3 hIPC donors mirrored such differences. Candidate gene cluster analysis disclosed differential expression of key lineage regulators, indicated a HoxA gene-associated positional memory in hIPCs and hBM-MSCs, and showed as well a clear transition state from mesenchyme to epithelium or vice versa in hIPCs. Our findings raise new research platforms to further clarify the potential of hIPCs to undergo complete MET thus contributing to islet cell replenishment, maintenance, and function.

In vitro (re)programming of human bone marrow stromal cells toward insulin-producing phenotypes

Diabetes mellitus is a chronic disease with great social and economical impact. In all its forms, it affects nearly 200 million people worldwide (1). Type 1 diabetes (T1D) represents 10% of all cases. Because of the increase in obesity in developed countries, the prevalence of type 2 diabetes (T2D) has been rising very rapidly, affecting children and adolescents. T1D and advanced T2D are caused by a progressive loss of functional pancreatic β-cell mass within the pancreatic islets. The extraordinary results of the Edmonton protocol have shown that human pancreatic islet transplantation can normalize glycemic control of insulin-dependent diabetic patients (2). Nevertheless, this therapeutic option does not represent a significant clinical benefit for all diabetic patients; the demand of islets for transplantation is very high and human donor pancreas for isolation of islet grafts is limited. Furthermore, transplants do last no longer than 2 yr and are accompanied by significant side effects as a consequence of lifelong aggressive immunosuppression (3). Therefore, intensive search for new sources for β-cell replacement has been undertaken. Regeneration of existing mature β-cells and replacement of insulin-producing cells are current research lines that could possibly solve the problem of islet shortage. Differentiation of embryonic stem (ES) cells has been thoroughly investigated; however, results are not yet satisfactory (4–6). Adult stem/progenitor

A description of clinician reported diagnosis of type 2 diabetes and other non-type 1 diabetes included in a large international multicentered pediatric diabetes registry (SWEET).

Although type 1 diabetes (T1D) remains the most frequent form of diabetes in individuals aged less than 20 years at onset, other forms of diabetes are being increasingly recognized.

Biology of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) are derived from mesodermal precursor and are committed towards mesenchymal differentiation. They are scattered all over the organism, situated in bone, cartilage, adipose tissue and accompany organs for tissue regeneration and structural and functional support. MSC populations are not homogenous, their signature is variable according to their localization. A process called “epithelial mesenchymal transition” is fundamental for the development of mesoderm. Epithelial-mesenchymal interactions specify MSC and this may influence their regeneration potential. Multipotent adult MSC are used for research in tissue regeneration and engineering. Crude mixtures of bone marrow- derived MSC are clinically applied for tissue healing, but complex transplantable tissue engineered constructs are still under development. The role and regeneration potential of MSC in inflammation and ageing organisms remains to be characterized. The establishment of reprogrammed homogenous MSC cultures of high plasticity might allow developing these cells towards multiple cell-based therapeutic strategies. Many applications can be envisioned, e.g. regeneration of bone, cartilage and tendon or engineering of beta cells and neurons. Since homogenous MSC with high plasticity represent a promising tool for the treatment of many diseases, research in this area of adult stem cells should be supported with high priority.

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.