Replicates in stem cell models-How complicated!

Current complexities in human pluripotent stem cell (hPSC)-based studies. hPSC studies begin with the recruitment of patients harboring disease-associated gene variant(s) that may increase susceptibility to disease development. Somatic reprogramming is then performed to derive patient-specific human induced pluripotent stem cells (hiPSCs), followed by step-wise directed differentiation to a relevant cell type before qualitative/quantitative assays are performed to assess for phenotypic or gene expression differences between the healthy and diseased hiPSCs.

Derivation of human induced pluripotent stem cells from patients with maturity onset diabetes of the young.

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human "stem cell cassette" containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.

Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells.

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic ß cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.

Prioritization of genes associated with type 2 diabetes mellitus for functional studies.

Existing therapies for type 2 diabetes mellitus (T2DM) show limited efficacy or have adverse effects. Numerous genetic variants associated with T2DM have been identified, but progress in translating these findings into potential drug targets has been limited. Here, we describe the tools and platforms available to identify effector genes from T2DM-associated coding and non-coding variants and prioritize them for functional studies. We discuss QSER1 and SLC12A8 as examples of genes that have been identified as possible T2DM candidate genes using these tools and platforms. We suggest further approaches, including the use of sequencing data with increased sample size and ethnic diversity, single-cell omics data for analyses, glycaemic trait associations to predict gene function and, potentially, human induced pluripotent stem cell 'village' cultures, to strengthen current gene functionalization workflows. Effective prioritization of T2DM-associated genes for experimental validation could expedite our understanding of the genetic mechanisms responsible for T2DM to facilitate the use of precision medicine in its treatment.

Emerging use of stem cells in regenerative medicine.

Stem cells represent a unique opportunity for regenerative medicine to cure a broad number of diseases for which current treatment only alleviates symptoms or retards further disease progression. However, the number of stem cells available has speedily increased these past 10 years and their diversity presents new challenges to clinicians and basic scientists who intend to use them in clinics or to study their unique properties. In addition, the recent possibility to derive pluripotent stem cells from somatic cells using epigenetic reprogramming has further increased the clinical interest of stem cells since induced pluripotent stem cells could render personalized cell-based therapy possible. The present review will attempt to summarize the advantages and challenges of each type of stem cell for current and future clinical applications using specific examples.

Cellular stress drives pancreatic plasticity.

Controversy has long surrounded research on pancreatic beta cell regeneration. Some groups have used nonphysiological experimental methodologies to build support for the existence of pancreatic progenitor cells within the adult pancreas that constantly replenish the beta cell pool; others argue strongly against this mode of regeneration. Recent research has reinvigorated enthusiasm for the harnessing of pancreatic plasticity for therapeutic application--for example, the transdifferentiation of human pancreatic exocrine cells into insulin-secreting beta-like cells in vitro; the conversion of mouse pancreatic acinar cells to beta-like cells in vivo via cytokine treatment; and the potential redifferentiation of dedifferentiated mouse beta cells in vivo. Here, we highlight key findings in this provocative field and provide a perspective on possible exploitation of human pancreatic plasticity for therapeutic beta cell regeneration.

Soluble factors secreted by T cells promote ß-cell proliferation.

Type 1 diabetes is characterized by infiltration of pancreatic islets with immune cells, leading to insulin deficiency. Although infiltrating immune cells are traditionally considered to negatively impact ß-cells by promoting their death, their contribution to proliferation is not fully understood. Here we report that islets exhibiting insulitis also manifested proliferation of ß-cells that positively correlated with the extent of lymphocyte infiltration. Adoptive transfer of diabetogenic CD4(+) and CD8(+) T cells, but not B cells, selectively promoted ß-cell proliferation in vivo independent from the effects of blood glucose or circulating insulin or by modulating apoptosis. Complementary to our in vivo approach, coculture of diabetogenic CD4(+) and CD8(+) T cells with NOD.RAG1(-/-) islets in an in vitro transwell system led to a dose-dependent secretion of candidate cytokines/chemokines (interleukin-2 [IL-2], IL-6, IL-10, MIP-1α, and RANTES) that together enhanced ß-cell proliferation. These data suggest that soluble factors secreted from T cells are potential therapeutic candidates to enhance ß-cell proliferation in efforts to prevent and/or delay the onset of type 1 diabetes.