Raewyn M. Seaberg

Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages

The clonal isolation of putative adult pancreatic precursors has been an elusive goal of researchers seeking to develop cell replacement strategies for diabetes. We report the clonal identification of multipotent precursor cells from the adult mouse pancreas. The application of a serum-free, colony-forming assay to pancreatic cells enabled the identification of precursors from pancreatic islet and ductal populations. These cells proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine β-, α- and δ-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated β-like cells demonstrate glucose-dependent Ca2+ responsiveness and insulin release. Pancreas colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies.

Stem and progenitor cells: the premature desertion of rigorous definitions

A current disturbing trend in stem cell biology is the abandonment of rigorous definitions of stem and progenitor cells in favor of more ambiguous, all-encompassing concepts. However, recent studies suggest that there are consistent, functional differences in the biology of these two cell types. Admittedly, it can be difficult to harmonize the in vivo and in vitro functional differences between stem and progenitor cells. Nonetheless, these distinctions between cell types should be emphasized rather than ignored, as they can be used to test specific hypotheses in neural stem cell biology.

In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord.

Stem cells isolated from the fourth ventricle and spinal cord form neurospheres in vitro in response to basic fibroblast growth factor (FGF2)+heparin (H) or epidermal growth factor (EGF)+FGF2 together. To determine whether these growth factor conditions are sufficient to induce stem cells within the fourth ventricle and spinal cord to proliferate and expand their progeny in vivo, we infused EGF and FGF2, alone or together, with or without H, into the fourth ventricle for 6 days via osmotic minipumps. Animals were injected with bromodeoxyuridine (BrdU) on days 4, 5 and 6 of infusion in order to label cells proliferating in response to the growth factors. Infusions of EGF+FGF2+H into the fourth ventricle resulted in the largest proliferative effect, a 10.8‐fold increase in the number of BrdU+ cells around the fourth ventricle, and a 33.5‐fold increase in the number of BrdU+ cells around the central canal of the spinal cord, as compared to vehicle infused controls. The majority of the cells were nestin+ after 6 days of infusion. Seven weeks post‐infusion, 22 and 30% of the number of BrdU+ cells induced to proliferate after 6 days of EGF+FGF2+H infusions were still detected around the fourth ventricle and central canal of the spinal cord, respectively. Analysis of the fates of the remaining cells showed that a small percentage of BrdU+ cells around the fourth ventricle and in the white matter of the spinal cord differentiated into astrocytes and oligodendrocytes. BrdU+ neurons were not found in the brainstem or in the grey matter of the cervical spinal cord 7 weeks post‐infusion. These results show that endogenous stem cells and progenitors around the fourth ventricle and central canal of the spinal cord proliferate in response to exogenously applied growth factors, but unlike in the lateral ventricle where they generate some new neurons, they only produce new astrocytes and oligodendrocytes at 7 weeks post‐infusion.

The Adult Mouse and Human Pancreas Contain Rare Multipotent Stem Cells that Express Insulin

The search for putative precursor cells within the pancreas has been the focus of extensive research. Previously, we identified rare pancreas-derived multipotent precursor (PMP) cells in the mouse with the intriguing capacity to generate progeny in the pancreatic and neural lineages. Here, we establish the embryonic pancreas as the developmental source of PMPs through lineage-labeling experiments. We also show that PMPs express insulin and can contribute to multiple pancreatic and neural cell types in vivo. In addition, we have isolated PMPs from adult human islet tissue that are also capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types. Finally, both mouse and human PMP-derived cells ameliorated diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin(+) multipotent stem cells and suggest that these cells may provide a promising line of investigation toward potential therapeutic benefit.

Embryonic cortical neural stem cells migrate ventrally and persist as postnatal striatal stem cells

Embryonic cortical neural stem cells apparently have a transient existence, as they do not persist in the adult cortex. We sought to determine the fate of embryonic cortical stem cells by following Emx1IREScre; LacZ/EGFP double-transgenic murine cells from midgestation into adulthood. Lineage tracing in combination with direct cell labeling and time-lapse video microscopy demonstrated that Emx1-lineage embryonic cortical stem cells migrate ventrally into the striatal germinal zone (GZ) perinatally and intermingle with striatal stem cells. Upon integration into the striatal GZ, cortical stem cells down-regulate Emx1 and up-regulate Dlx2, which is a homeobox gene characteristic of the developing striatum and striatal neural stem cells. This demonstrates the existence of a novel dorsal-to-ventral migration of neural stem cells in the perinatal forebrain.

Surfaceome Profiling Reveals Regulators of Neural Stem Cell Function

The composition of cell‐surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry‐based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor‐β signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N‐cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c‐kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain‐derived populations and identifies a host of other candidates that may regulate NSCs. Stem Cells 2014;32:258–268

Adhesion Is Prerequisite, But Alone Insufficient, to Elicit Stem Cell Pluripotency

Primitive mammalian neural stem cells (NSCs), arising during the earliest stages of embryogenesis, possess pluripotency in embryo chimera assays in contrast to definitive NSCs found in the adult. We hypothesized that adhesive differences determine the association of stem cells with embryonic cells in chimera assays and hence their ability to contribute to later tissues. We show that primitive NSCs and definitive NSCs possess adhesive differences, resulting from differential cadherin expression, that lead to a double dissociation in outcomes after introduction into the early- versus midgestation embryo. Primitive NSCs are able to sort with the cells of the inner cell mass and thus contribute to early embryogenesis, in contrast to definitive NSCs, which cannot. Conversely, primitive NSCs sort away from cells of the embryonic day 9.5 telencephalon and are unable to contribute to neural tissues at midembryogenesis, in contrast to definitive NSCs, which can. Overcoming these adhesive differences by E-cadherin overexpression allows some definitive NSCs to integrate into the inner cell mass but is insufficient to allow them to contribute to later development. These adhesive differences suggest an evolving compartmentalization in multipotent NSCs during development and serve to illustrate the importance of cell–cell association for revealing cellular contribution.

Chorda tympani nerve function in children: relationship to otitis media and body mass index.

OBJECTIVE/HYPOTHESIS A relationship between acute otitis media and elevated body mass index has recently been reported. Intriguingly, it was postulated that this relationship may result from altered chorda tympani nerve function impacting taste sensation and eating habits. We sought to test this directly by measuring chorda tympani nerve function in children with and without a previous history of acute otitis media and to determine the relationship to body mass index. STUDY DESIGN Retrospective cohort study. METHODS Institutional research ethics board approval was obtained. Study participants included 142 children (5-18 years of age) who were recruited from an otolaryngology outpatient clinic at a tertiary academic pediatric hospital between May and August 2009. Children were excluded if they were not able to communicate effectively, younger than age 5, or had developmental disabilities. Body mass index was calculated and the history of previous otologic disease carefully elicited from the caregivers. Electrogustometric threshold, a validated measure of chorda tympani function, was obtained bilaterally in each child. Children were divided into cohorts based on the number of acute otitis media episodes, and electrogustometry thresholds were compared between cohorts. RESULTS Electrogustometric thresholds were successfully obtained in all children. There was no significant relationship between chorda tympani nerve function and history of acute otitis media. Similarly, there was no significant association between the history of otitis media and body mass index. CONCLUSION This study did not demonstrate any effect of previous acute otitis media history on chorda tympani nerve function. Furthermore, it did not demonstrate a relationship between acute otitis media and elevated body mass index. This is counter-evidence to the previous hypothesis that increasing acute otitis media is responsible for increasing childhood obesity through alteration in chorda tympani nerve function.

Large intragenic deletion of CDC73 (exons 4–10) in a three-generation hyperparathyroidism-jaw tumor (HPT-JT) syndrome family

BackgroundInactivating mutations of CDC73 cause Hyperparathyroidism-Jaw Tumour syndrome (HPT-JT), Familial Isolated Hyperparathyroidism (FIHP) and sporadic parathyroid carcinoma. We conducted CDC73 mutation analysis in an HPT-JT family and confirm carrier status of the proband’s daughter.MethodsThe proband had primary hyperparathyroidism (parathyroid carcinoma) and uterine leiomyomata. Her father and daughter had hyperparathyroidism (parathyroid adenoma) but no other manifestations of HPT-JT. CDC73 mutation analysis (sequencing of all 17 exons) and whole-genome copy number variation (CNV) analysis was done on leukocyte DNA of the three affecteds as well as the proband’s unaffected sister.ResultsA novel deletion of exons 4 to 10 of CDC73 was detected by CNV analysis in the three affecteds. A novel insertion in the 5’UTR (c.-4_-11insG) that co-segregated with the deletion was identified. By in vitro assay the 5’UTR insertion was shown to significantly impair the expression of the parafibromin protein. Screening for the mutated CDC73 confirmed carrier status in the proband’s daughter and the biochemistry and ultrasonography led to pre-emptive surgery and resolution of the hyperparathyroidism.ConclusionsA novel gross deletion mutation in CDC73 was identified in a three-generation HPT-JT family emphasizing the importance of including screening for large deletions in the molecular diagnostic protocol.

Erratum: Large intragenic deletion of CDC73 (exons 4-10) in a three-generation hyperparathyroidism-jaw tumor (HPT-JT) syndrome family [BMC Med Genet. 2017;18:83] DOI 10.1186/s12881-017-0445-0

Author details Medical Genetics, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy. Department of Otolaryngology Head and Neck Surgery, University of Toronto, Toronto, ON, Canada. Department of Medicine, University of Toronto, Toronto, ON, Canada. Division of Endocrinology, Women’s College Hospital, Toronto, ON, Canada. Department of Otolaryngology, Head & Neck Surgery, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. Departments of Laboratory Medicine and Pathobiology, Medicine and Genetics, University of Toronto, Toronto, ON, Canada. Endocrinology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy. Metabolic Disorders and Complications, McGill University Health Centre-Research Institute, Montreal, QC, Canada. Departments of Medicine, Physiology and Human Genetics, McGill University, Montreal, QC, Canada.