Kevin A. D'Amour

Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor—en route to cells that express endocrine hormones. The hES cell–derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal β-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell–derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo

Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human β-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with ∼3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional β-cells, including expression of critical β-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell–derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells.

Efficient differentiation of human embryonic stem cells to definitive endoderm.

The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

In Vivo Fate Analysis Reveals the Multipotent and Self-Renewal Capacities of Sox2+ Neural Stem Cells in the Adult Hippocampus

To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.

Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3‐kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor β family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast‐conditioned medium under feeder‐free conditions, ∼70%–80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1‐II. Microarray and quantitative polymerase chain reaction‐based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin‐like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self‐renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC‐DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self‐renewal and early cell fate decisions.

Cell fusion-independent differentiation of neural stem cells to the endothelial lineage

Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential (referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures. Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation. In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells, with human endothelial cells, which form the lining of blood vessels. We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Using a flow cytometry–based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.

Derivation of insulin-producing cells from human embryonic stem cells.

The potential of pluripotent human cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells, to differentiate into any adult cell type makes them ideally suited for the generation of various somatic cells and tissues in vitro. This remarkable differentiation capacity permits analyzing aspects of human embryonic development in the laboratory, as well as generating specialized adult human cells for screening drugs, and for replacing tissues damaged by injury or degenerative diseases, such as diabetes. Understanding and controlling the fundamental processes that drive the differentiation of specialized cells are the keys to the eventual application of this technology to patients. In this review, we discuss the different protocols developed that are aimed at deriving beta-cells from hESCs. Despite many differences, successful strategies share a general adherence to the normal differentiation pathway through definitive endoderm. Mimicking normal pancreagenesis offers the best strategy for producing glucose-responsive insulin-producing cells in vitro for people with diabetes.

Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo

The PEC‐01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC‐01 candidate product) and transplanted into mice, can mature into glucose‐responsive insulin‐secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC‐01 cells such that 73%–80% of the cell population consisted of PDX1‐positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet‐like cells (ICs) that reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin‐positive cells were single hormone‐positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%–98% endocrine cells and 1%–3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC‐01 candidate product, demonstrating conclusively that in vitro‐produced hESC‐derived insulin‐producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity‐associated markers. Correlating with this, the time to function of ICs was similar to PEC‐01 cells, indicating that ICs required cell‐autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host.

Choline Transporter 1 Maintains Cholinergic Function in Choline Acetyltransferase Haploinsufficiency

Choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine (ACh), is thought to be present in kinetic excess in cholinergic neurons. The rate-limiting factor in ACh production is the provision of choline to ChAT. Cholinergic neurons are relatively unique in their expression of the choline transporter 1 (CHT1), which exhibits high-affinity for choline and catalyzes its uptake from the extracellular space to the neuron. Multiple lines of evidence indicate that the activity of CHT1 is a key determinant of choline supply for ACh synthesis. We examined the interaction of ChAT and ChT activity using mice heterozygous for a null mutation in the Chat gene (Chat+/-). In these mice, brain ChAT activity was reduced by 40-50% relative to the wild type, but brain ACh levels as well as ACh content and depolarization-evoked ACh release in hippocampal slices were normal. However, the amount of choline taken up by CHT1 and ACh synthesized de novo from choline transported by CHT1 in hippocampal slices, as well as levels of CHT1 mRNA in the septum and CHT1 protein in several regions of the CNS, were 50-100% higher in Chat+/- than in Chat+/+ mice. Thus, haploinsufficiency of ChAT leads to an increased expression of CHT1. Increased ChT activity may compensate for the reduced ChAT activity in Chat+/- mice, contributing to the maintenance of apparently normal cholinergic function as reflected by normal performance of these mice in several behavioral assays.